Marine Drugs and Cosmeceuticals - THP FPIK IPBthp.fpik.ipb.ac.id/Download/Prof. Ulrike_Marine Drugs...
71
Marine Drugs and Cosmeceuticals: From Lab to Market Bogor, September 2019 Prof. (em.) Ulrike Lindequist University Greifswald Institute for Pharmacy [email protected] 1
Transcript of Marine Drugs and Cosmeceuticals - THP FPIK IPBthp.fpik.ipb.ac.id/Download/Prof. Ulrike_Marine Drugs...
Marine Drugs and Cosmeceuticals From Lab to Market
Bogor September 2019
Prof (em) Ulrike Lindequist
University Greifswald
Institute for Pharmacy
lindequiuni-greifswaldde
1
Content
bull Introduction
bull Approved marine drugs
bull The potential of seaweeds
bull The potential of marine fungi
bull What is to do to explore the potential
Specialities of Marine Environment
WaterTemperature - 15 - 350 o CPressure 1 - gt 1000 atmSaltLightNumber of speciesClose communities of different species
This means Marine organisms need special mechanismsfor adaption to their environment They produceother secondary metabolites than terrestric organisms
Number of Marine Species
Estimated 3 Mio till 500 MioKnown Plants 40000 species
3900 red algae1500 brown algae900 green algae45 flowering plants
Animals 33 of 34 known Phyla (15 only in the Sea)157000 benthic
5000 sponges9000 cnidarian8000 echinoderms
Microorganisms
Marine natural products
By organism By region
(Source Blunt et al Nat Prod Rep 2009 26 170ndash244)
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Content
bull Introduction
bull Approved marine drugs
bull The potential of seaweeds
bull The potential of marine fungi
bull What is to do to explore the potential
Specialities of Marine Environment
WaterTemperature - 15 - 350 o CPressure 1 - gt 1000 atmSaltLightNumber of speciesClose communities of different species
This means Marine organisms need special mechanismsfor adaption to their environment They produceother secondary metabolites than terrestric organisms
Number of Marine Species
Estimated 3 Mio till 500 MioKnown Plants 40000 species
3900 red algae1500 brown algae900 green algae45 flowering plants
Animals 33 of 34 known Phyla (15 only in the Sea)157000 benthic
5000 sponges9000 cnidarian8000 echinoderms
Microorganisms
Marine natural products
By organism By region
(Source Blunt et al Nat Prod Rep 2009 26 170ndash244)
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Specialities of Marine Environment
WaterTemperature - 15 - 350 o CPressure 1 - gt 1000 atmSaltLightNumber of speciesClose communities of different species
This means Marine organisms need special mechanismsfor adaption to their environment They produceother secondary metabolites than terrestric organisms
Number of Marine Species
Estimated 3 Mio till 500 MioKnown Plants 40000 species
3900 red algae1500 brown algae900 green algae45 flowering plants
Animals 33 of 34 known Phyla (15 only in the Sea)157000 benthic
5000 sponges9000 cnidarian8000 echinoderms
Microorganisms
Marine natural products
By organism By region
(Source Blunt et al Nat Prod Rep 2009 26 170ndash244)
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Number of Marine Species
Estimated 3 Mio till 500 MioKnown Plants 40000 species
3900 red algae1500 brown algae900 green algae45 flowering plants
Animals 33 of 34 known Phyla (15 only in the Sea)157000 benthic
5000 sponges9000 cnidarian8000 echinoderms
Microorganisms
Marine natural products
By organism By region
(Source Blunt et al Nat Prod Rep 2009 26 170ndash244)
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Marine natural products
By organism By region
(Source Blunt et al Nat Prod Rep 2009 26 170ndash244)
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Comparison Marine (MNP) ndash Terrestrial Natural Products (TNP) by Cheminformatics (Shang et al 2018)
MNP TNP
Lower solubility in water Higher solubility in water
Often larger
Contain often ester bonds connected Contain often more stable ringto 10-membered rings systems and bond types
More nitrogen and halogen atomsFewer oxygen atoms
More diverse biosynthetic pathways (Shang J et al J Chem Inf Mod 58(6) 1182-1193 2018
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Types of Products
bull Pharmaceutical product = pharmaceutical = medicinal product = medication = medicine = drug
a drug used to diagnose cure treat or prevent disease
bull Food supplement any food the purpose of which is to supplement the normal dietand which is concentrated source of a vitamin or mineral or other substances with a nutritional or physiological effect alone or in combination and is sold in dose form (EU law) between food and drugs
bull Food
bull Cosmetic a preparation applied to the body especially the face to improve itsappearance
8
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Types of Products
Cosmeceutical- bdquohybridldquo product between a cosmetic and a pharmaceutical
- to provide desired esthetical effects (immediate) and
- to treat dermatological conditions (prolonged)
- anti-aging anti-acne solar-protective wound healing skin whitheningactivities
Regulation in most countries not a separate category from cosmetics(exceptions Japan Korea = functional cosmetics)
But Safety and efficacy have to be ensured
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Clinical Phase I 10
Clinical Phase II 14
Clinical Phase III 7
FDA-approved 9
Pipeline of marine Drugs 2019
1 only in Australia (Aplidin) httpmarine pharmacologymidwesternedu
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Approved Marine Drugs
Virostatika Vidarabin Vidarabin 3 Thilo reg
Cancerostatics Cytarabin Alexanreg ARA-Cell reg etc
Trabectedin YondelisregEribulin Mesylat HalavenregBrentuximab Vedotin AdcetrisregPolatuzumab Vedotin Polivy regPlitidepsin Aplidinreg Australia
Analgetics Ziconotid Prialtreg
Dietetics Omega-3-acid-ethyl esters
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Nucleosides from Sponges
Spongouridin
Tethya crypta (Karibik)
Arabinose instead of Ribose
Spongothymidin Models for Vidarabin and Cytarabin
O N NH
O
HO
HO OH
O
O N NH
O
HO
HO OH
O wwwspongeguideorg
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Vidarabin
Mode of action Transformation into Arabinosidtriphosphate
Inhibition of virale (HSV) DNA-Polymerase
and DNA-Synthesis
Indication as eye ointment (3) for treatment of acute
Keratoconjunctivitis and recurring
epithelial Keratitis caused by
HSV I oder II
Today mostly replaced by Acyclovir
O
HO
OH
HO N
N
N
N
NH2
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cytarabin Ara-C Alexanreg ARA-cellreg
Cytosinarabinosid
Transformation to Cytosinarabinosidtriphosphate
Mode of action Inhibition of DNA Polymerase bycompetition with the physiological substrateDeoxycytidintriphosphate
Indication Acute lymphatic and non-lymphatic leukemia
Chronic myeloic leukemia
O
HO
OH
HON
N
O
NH2
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Trabectedin Yondelis reg
Tetrahydroisochinolinalkaloid = ET 743
Produced by partial synthesis starting substance Cyanosafracin B (fromPseudomonas fluorescens)
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Trabectedin Yondelis reg
bull Occurence in Tunicata
bull Animals with a mantle (tunica) as a
coat outside the epidermis
bull Benthic
bull about 2000 species
Ecteinascidia turbinata Living on corals in the Mediterranean looks like grapes
Photo Courtesy PharmaMar
Bertelsmann Lexikon
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Trabectedin Yondelis reg
Mode of action Binding at small groove of DNA
Inhibition of cell proliferation
Indication
Advanced soft tissue sarcom
Ovarian carcinom (recurrent platin sensitive)
Approval 2007 (2001 as orphan drug)
Producer PharmaMar
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
bull Randomisied study with 266 patients iiposarkom or leiomyosarkom Progress of disease though pretreatment with anthracyclins and ifosamid
15 mgm2 body surface 24 h iv each 3 weeks
after 1 Jahr survival of gt 60 of patients
average survival time 14 month
bull Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
Trabectedin Yondelis reg
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Synthetic Analogon of Halichondrin B
Makrolid Polyketid
Eribulin Mesylat Halaven reg
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Halichondrin B
Halichondria okadai
(Pacific)
Lyssodendoryx sp
HO
HO
HO
O
O
O
O
O
O
OO
O
OO
O
O
O
O
O
H
H
H
H
HH
H
H
H
H H
H
httpvitalsignsmeorgobservationspecies-
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Eribulin Mesylat Halaven reg
Mode of action Reaction with Mikrotubuli (other bindingplace than taxanes and Vinca alkaloids)
Indication metastasized breast cancer after two earlier ineffective chemotherapies
Approval 2011
Producer Eisai Co Japan
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Brentuximab Vedotin Adcetris reg
Conjugat consisting of
bull Antibody against CD30
bull Linking molecule and
bull the cytostatic Auristatin
( Monomethyl-Auristatin E MMAE)
Conjugat stable in blood
After internalisation into
CD30 carrying tumour cells release of the
cytostatic Auristatin
22
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Auristatin
O
N
H3C
CH3
CH3 O
HN
O
N
H3C
O
N
O
H3CO NH
CH3
H
Synthetic derivate of Dolastatin 10 isolated from Dolabella auricularia(Mollusc Gastropoda) Produced by Cyanobacteria genus SymplocaPeptid Because of high toxicity direct application not possible
TeuscherLindequist Biogene Gifte
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Brentuximab Vedotin Adcetrisreg
Mode of action Attack at Tubulin
Indication Advanced Morbus Hodgkin
(Marker CD30)
recurrent anaplastic lymphom
Approval November 2012
Producer Takeda Japan
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Polatuzumab Vedotin Polivyreg
The same principle like Brentuximab Vedotin
Antibody against CD79b ndash Linker ndash Monomethylauristatin E
Indication therapy resistent or recurrent diffuse large cellular B cell lymphom
Approval June 2019 (FDA)
Producer Roche
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Ziconotid PrialtE
R
G
C
A
C
D
E
K
C
C
C
g
S
S
C
g
C
R
N P
T
L
G
R
S
G
S
S
A
P
Q
P
G
P
P
P L g L = -Hydroxyprolin = -Carboxy- -Glutaminsaumlureg
P
R
C
P
g
G
K
N
R
K
Q
S
C
H
C
g
Y
K
L
T
P
S
D
I
S
Q
C
R
R
Y
C
Q
g
N
C
C
K
C
M
K
S
C
G
P
N
R
L
Q
S
R
R
C
C
C
N
G
C A
Y
G
T
N
K
P
R
Q
C
K
Y
C I G A H g D V
_
_
_
_
_
NH2
NH2
NH2
NH2
NH2
10
10
10
10
10
20
20 30
20
a-Conotoxin GI
m-Conotoxin GIIIA
v-Conotoxin GVIA
Conotoxin GS
Sleeper-Peptid
(intrachenare S-S-Bruumlcken vorhanden)
Peptid consisting of 25 amino acids with 3 disulfide bridges basicModel Ω-Conotoxin
reg
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Ziconotid Prialt
Mebs bdquoGifttiere 1992
Conus specCone snails
reg
TeuscherLindequist Biogene Gifte
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
- Snail hidden in the soil
- Fishes recognize the redpharynx tube of the snail asbdquofoodldquo
- poisonous file comes out and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfedby the snail
Olivera BM (1985) Science 2301338-
1334
Hunting strategy of cone snails
LD 50 082 mgkg iv
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Ziconotid Prialtreg Mode of Action
Aus Putzier und Frings 2002
-Selective Inhibition of N Typ-Ca-channels
-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons
-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels
-Prevention of transmissionof pain signal
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Ziconotid Prialtreg Application
Long lasting infusion through intrathekal catheterby implanted infusion pump
Treatment of nozizeptive and neuropathic pain
1000x stronger active than morphine
No dependance
Tolerance development unprobably
Increased risk for suicide
108 patients with malign255 with non malign painTreatment for 10-12 d
Wissenschaftliche Broschuumlre Ziconotid
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Recently approved Pegvaliase (Palynzig reg)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacteriumAnabaena variabilis
- Recombinant produced in Escherichia coli
- Pegylated
- Metabolizes the amino acid phenylalanine
- As Orphan Drug for patients with phenylketonuria (PKU cannot metabolizephenylalanine neurological cognitive and other disturbances)
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Further marine substances with medicinal importance
Auxiliary products Agar Carrageenan Alginic acid Chitin Chitosan
Diagnostics LAL GFP Phycoerythrin Taq Polymerase
Food PUFAs Iod
Cosmetics Compatible Solutes Pseudopterosins Sun protectingAgents
Wellness Thalasso
H C
H C
3
3
COOH
COOH
Docosahexaensaumlure (Omega-3-Fettsaumlure)
Eicosapentaensaumlure (Omega - 3 - Fettsaumlure)
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds
bull = Macroalgae
bull Macroscopic organisms in the marine ecosystem
bull Three main phyla associated with different pigments
Chlorophyta (green algae) ndash chlorophyll
Rhodophyta (red algae) ndash phycobilins
Phaeophyta (brown algae) ndash fucoxanthin
bull Good source of carbohydrates dietary fiber proteins vitamins
PUFAs minerals
33
Enteromorpha compressa
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds Biopolymers
bull Rhodophyceae Agar Carrageenan
bull Phaeophyceae Alginic acid
bull Wound healing materials againstgastritis as dietary agent as gellingagent to stop bleeding
HO
H H
H H
H HH H
H H
O O
CH OR CH 2 2
O O
H H
H HCH CH OSO H2 2 3
O HO
H HO O
H H
HO HO
OH
O
OH
H
O
O
CH3
C O
COOH
D-Galaktose (R=H) 36-Anhydro 46-0-(1-Carb- L-Galaktose -6-0-Methyl-D-galaktose(R=CH )3
L-galaktose oxyethyliden)D-galaktose
6-sulfat
Agar
-
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds Cosmeceuticals
bull Photo-protective compounds
-mycosporine-like amino acids
-carotinoids
-photolyase
bull Skin care and protection
35
La mer medCouperose Creme
HO
O
O
Astaxanthin
OH
OCH3
NH
COOH
HO
HO
O
Mycosporine glycin
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds Pharmaceuticals (research)
bull Along the last five decades more than 3000 NPs have been discovered from algae most of them with cytotoxic activities (Review Alves C et al Frontiers in Pharmacol August 2018)
bull Cancer Isoprenoids eg halomon (halogenated monoterpene) dictyolactone (diterpene) dimericsesquiterpenes of the cycloaurane-type fucoxanthin alkaloids eg caulerpin
bull Cardio-vascular diseases Polyphenols (green and red algae bromophenols phenolic acids flavonoids brown algae phlorotannins)
bull Disturbances in blood coagulation Sulfated polysaccharides
bull Inflammation Polyphenols
bull Diabetes Polyphenols
bull Infectious diseases (human and animal eg aquaculture) Sesquiterpenes
bull Prebioticdietetics Dietary fibers (Review OacuteSullivan L et al Mar Drugs 8 2038-2069 2010)
36
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds Pharmaceuticals (examples)
37
Eckol a phlorotannin from Halomon a monoterpen from Chamigranepoxid a sesquiterpenEcklonia spec Portieria hornemannii from Laurencia glomerataantioxidative cytotoxic cytotoxic
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Seaweeds Pharmaceuticals (examples)
38
Br
HO
Cl
Br
HO
Br
Halogenated sesquiterpenes from Laurencia chondrioides with activityagainst human and fish pathogenic bacteria
Bansemir A et al Chem Biodiv 1 463-467 2004
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Marine Fungi
bull Ecological diverse group which belong to different phyla mainly Ascomycota
bull Grow on numerous substrata such as decaying wood and leaves algae corals etc
bull Found in sand muds soils sediments
bull Play a substantial components role in nutrient cycling (wwwmarinefungiorg)
bull Up to 10000 species of maine fungi estimated
bull gt 1100 species are documented (Jones MD et al Nature 474 200-203 2011)
bull Occur often as endophytes
bull Among the 272 new compounds discovered from marine fungi till 2002 85 ofthem are produced by epiendophytes (Zhang Y et al Mar Drugs 7 97-112 2009)
39
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Marine Fungi Cosmeceuticals
bull Photo-protective compounds Mycosporine-glutaminol-glucoside
bull Anti-aging products Polysaccharides EPS PUFAs
bull Antioxidant compounds Mycosporine like amino acids (MAAs) carotenoids diketopiperazine alkaloids dioxopiperazine alkaloids
bull Skin-whitening products kojic acid and derivatives pyron derivatives thalassothalicacids etc
bull Additives (preservatives surfactants emulsifier thickener stabilizers moistourizing) polysaccharides (chitin chitosan and derivatives) glycolipids lipopeptides
(Review Espinosa-Leal CA et al Planta med 85 535-551 2019)
40
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Marine Fungi Pharmaceuticals (research)
bull Antimicrobial about 50 of test extracts contain antimicrobial activity peptides alkaloids pyridines diketopiperazines steroids terpenoids polyketides (Review Xu L et al Mar Drugs 13 3479-3513 2015)
bull Cytotoxic (anticancer) Alkaloids (Review Gomes NGM et al Mar Drugs 13 3950-3991 2015)
bull First cephalosporin Cephalosporin C from Acremonium chrysogenum
Cephalosporin C Helicascolide C from a fungus (KT32) isolated from a Gracilaria spec
active against phytophathogenic fungi
Tarman K et alPlanta med 76 1246 2010 41
H ON S
O H
O
O
OO
OO
N H 2
H
O
HO
O
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Marine Fungi Pharmaceuticals (Examples)
42
Neoechinulin B isolated from Eurotium rubrum prenylated indole diketopiperazine alkaloidinhibits H1N1 virus and drug-resistant influenzaclinical isolates
(Abdelmohsen et al Lancet Infect Dis 17(2) E30-E41 2017)
Stachybotrin D isolated from Stachybotryschartarum a phenylspirodrimaninhibits NNRTI-resistant HIV strains
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
What is to do to explore this potential
Discoverybull Collection identification and if possible cultivation of organisms
bull Screening
bull Isolation and identification of active compounds
Product developmentbull Establishment of sustainable manufacturing processes
bull Development of suitable application forms (Formulation)
bull Preclinical and clinical assays
Authorization and Marketingbull Authorization and commerzialization as pharmaceutical
bull Commerzialization as cosmeticcosmeceutical
bull Commerzialization as foodfood supplement
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Phases of Drug Development(Example Antibiotic)
44
Basic research -Screening for antibiotic materials(mushrooms plants synthetic compoundshellip)
-Identification of antibiotic material-Production in small amounts
Small company
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Screening strategiesbull Biologically guided
Search for selected biological activities (antibacterial cytostatic enzyme inhibiting etc) of an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk compounds are already known
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Screening strategies
bull Chemically guided
Search for unknown chemical structures in an extract by NMR MS etc
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk compounds without interesting biological activities
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Screening strategies
bull Genetically guided (bdquoGenome miningldquo)
Search for genes and gene clusters coding for interesting biosynthetic pathways
eg polyketide synthases
Activation of silent genes
Risk long way high risk
OSMAC concept (One Strain ndash Many Compounds)
Changes in cultivation conditions (composition of growth media temperature aeration co-cultivation etc)
Expression of silent genes
Production of different (novel) compounds
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Main criteria for a pharmaceutical product
bull Pharmaceutical Quality
bull Safety
bull Efficacy
48
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Pharmaceutical quality Identity
49
Is it really the declared material
bull Taxonomy strain differences
bull Confusion due to the similar popular names use ofscientific names
bull Voucher specimen
bull Content of substrate or other residues
Methods
Taxonomic chemotaxonomic macroscopic organoleptic microscopic chromatographic DNA analysis
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Pharmaceutical quality Purity
50
Are there any unallowed impurities adulterations etc
bull Microbiological quality
bull Limits for residues of pesticides fumigation agents toxicmetals radioactivity possibly toxic components etc
bull Adulterations by foreign drugs pure drugs (eg corticosteroids hormoneshellip) cheap material (starchhellip) etc
bull Contents of ash water
Methods
Visuell microscopic microbiologic chromatographic NMR AAS specific methods
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Pharmaceutical quality Content
51
Contains the product the active components or analytical leadcompounds in the right amount
bull Assays of constituents with known therapeutic activity responsiblefor the main effect of the product
bull Assays of marker substances (where responsible active substancesare not known)
bull Content must be indicated the lowest possible tolerance
Methods
HPLC physico-chemical methods chemical methods
determination of biological value
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Efficacy and Safety
52
bull Pharmacodynamics Study of pharmacological actions on living systems including the reactions with and binding to cellconstituents and the biochemical and physiologicalconsequences of these actions (IUPAC)
bull Pharmacokinetics Process of the uptake of drugs by thebody the biotransformation they undergo the distribution ofthe drugs and their metabolites in the tissues and theelimination of the drugs and their metabolites from the bodyover a period of time (IUPAC)
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Preclinical studies Efficacy
53
bull In vitro assays
spectrum of activities
determination of main activity
mode of action
bull Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Preclinical studies Safety
54
bull In vitro assays + animal assays (one rodent + one non rodentspecies)
bull Acute subacute and chronic toxicity
bull Genotoxicity (mutagenic potential carcinogenic potential)
bull Reproductive Toxicity
bull Observations during clinical studies and further application
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Clinical Studies
55
bull First stage (I)
small group of healthy adult people (10-50)
safety and pharmakokinetics
bull Second stage (II)
small group of patients with the relevant indication (100-300)
efficacy and pharmacokinetics
bull Third stage (III)
controlled randomized and multicentric study withhigher number of patients
bull Fourth stage (IV)
observation of treated patients after licensation
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Criteria for a Good Clinical Study
56
bull Good Clinical Practice (GCP)
bull Allowance and supervision by appropriate regulatoryauthorities
bull Clinical trial protocol
bull Statistical power
The larger the number of participants the greater the
statistical power and the greater the costs
bull Informed consent
bull Ethical aspects
bull Regarding the types of studies interventional studies arehigher evaluated than observational studies and case reports
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Authorization of pharmaceuticals
57
bull The regulation varies greatly between countries and global regions
European Union
bull National authorization procedure
bull Centralized
bull Decentralized (mutual recognition procedure)
Authorization as well established drugs or as traditional drugs
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Alternatives
58
The development of a pharmaceutical for EBM is very stronglyregulated very expensive and needs a lot of time In the case of natural products patent protection is often impossible
A way out can be food supplements Marine derived food supplementscan represent an easier way to enable the consumer access topromising products
Another way out can be cosmetics cosmeceuticals
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
CosmeticsCosmeceuticals
-Global market for cosmetics and cosmeceuticals is forecasted to grow at a rate of 43 by 2022 with a value of USD 430 billion
-Anti-aging market is expected to grow at 75 from 2016 to 2021
-Photo-protective skin-care and hair-care products drive this trend in increasing demand
-Consumersacutedemand is turning to natural products given health concerns andpopular trends (Corinaldesi et al 2017)
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cosmeceuticals Development
Fig 1 Espinosa
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cosmeceuticals Bioactivity analysis
Anti-agingAnti-collagenase testAnti-elastase testHyaluronidase activityVitality and proliferation of cultivated fibroblasts and keratinocytesAnti-oxidative tests
Anti-acneAgar disk-diffusion methodMinimum inhibitory concentrationAnti-inflammatory activity
Solar protectionSPF measurementUVA-PF
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cosmeceuticals Bioactivity analysis
Skin whiteningTyrosinase inhibition
Wound healingin vitro scratch assay co-culture of keratinocytes and fibroblasts
in vivo rodents (skin differs strongly from human skin) rabbitsexcision wound model measurement of wound area andhydroxyproline content
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cosmeceuticals Safety
Acute toxicity QSAR predict based on chemical structure
Skin corrosion or irritation Cell models
Eye irritation Cell models
Skin sensitization Cell models
Genotoxicity Micronucleus test
Carcinogenicity Cell models
Phototoxicity Cell models
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Cosmeceuticals New trends
bull Formulation Use of delivery systems liposomes microsomes transferosomes lipid nanoparticles polymeric microparticles nanoparticlesto facilitate penetration of the skin barrier by the active ingredients to reachappropriate sites
bull Eco-friendly use of less polluting solvents or solvent-free processes like microwave irradiation for extraction of active ingredients
bull Restriction of in vivo tests creation of new in vitro methods (safety cellmodels for skin penetration)
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Example Maresomereg
bull A preparation consisting of microparticles of a defined cyanobacterial strain
bull Protects human skin against colonization by MRSA
bull Developed at the University Greifswald in collaboration with the IMaB eV
bull Patent protection
bull Licensation for use as cosmetics to a company
bull In the company
Scale up of production
Formulation
Packing
Commercialisation as cosmetic
0
100
200
300
400
500
600
700
800
900
Number of
colonies
Control Donator Acceptor
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Example Maresome reg
Can we be satisfied with the present situation No
This product deserves application in hospitals nursing homes etc
Necessary are
Licensation of the patent for use in hospital hygiene
Broader clinical trials
Licensation as medical product
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Problems open questions
bull Not enough starting material
bull Real producer of the active metabolites
bull Often only in vitro assays
bull Often high toxicity
bull Lack of good clinical assays
bull High risk
bull High financial need
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
What can be done if the organisms cannot be cultivated
bull Collection in greater amounts (sustainability)
bull Synthesis of responsible biologically active compounds
bull Partial synthesis of responsible biologically active compounds
bull Isolation of genetic information and expression in
cellsorganisms which can be cultivated more easily
(increasing importance of metagenomic assays)
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
What is necessary in general
bull Expanding the knowledge on marine life and ecological relationships
bull Sustainable production in accordance with ecology biotechnology gentechnology synthesis
bull Improvement of teaching and education
bull Close interactions between basic and applied research and between research and industry
bull Development of intelligent management and application strategies patent protection
bull Finances
Thank you very much for your attention
Greifswald Germany
Greifswald
Thank you very much for your attention
Greifswald Germany
Greifswald
Greifswald Germany
Greifswald
