Vanderlan da Silva Bolzani

IQAr, Araraquara-SP

bolzaniv@iq.unesp.br

Mesa Redonda - UNIVALI Itajaí-SC, 24 a 27 de Outubro de 2016

Current art and science of traditional medicine, example of TCM

Tentative Brazilian Bioprospecting Programs

Desenvolvimento Industrial do Brasil

DEPID-FAPESP, an example

Conclusões

SUMMARY OF THE PRESENTATION

CURRENT ART AND SCIENCE OF TRADITIONAL MEDICINE

Science 347 : 6219 Suppl. (2015)

Multidisciplinary Approaches for Studying Traditional Medicine

HERBAL GENOMICS: EXAMIMING THE BIOLOGY OF MEDICINES

Traditional herbal medicines, such as plant- and fungi-based remedies, have been used for more than

5,000 years. However, the genetic background, the agricultural traits, and the medicinal quality of most

traditional herbs are poorly understood;

With rapid advances in high throughput sequencing technologies and greatly reduced costs, a new

discipline called “herbal genomics” has emerged;

Effective model based on the studies of the biosynthetic pathways of secondary metabolites in

medicinal and fungal species have facilitated;

Genomic information, together with transcriptomic, proteomic, and metabolomic data, can therefore be

used to predict secondary metabolite biosynthetic pathways and their regulation, triggering a revolution

in discovery-based research aiming to understand the genetics and biology of herbs.

Herbal genomics provides an effective platform to sup- port the chemical and biological analyses of

complex herbal products that may contain more than one active component;

Mundial Market of pharmaceuticals in 2015 US$ 320 billions; Herbal Medicines US$ 20 billions;

Recent advances in genome editing

have provided feasible approaches

by introducing or altering specific

alleles; hence, genetic control over

metabolites can be investigated;

The elucidation of biosynthetic

pathways is one of their most

appealing features - model herbs

can also provide information on

perennial habits, development

patterns, cultivation requirements,

and resistance to environmental or

biological insult;

Ganoderma lucidum or “mushroom

of immortality”) commonly used

herbs has its genomes already been

sequenced;

S. Chen et al., Nat. Commun. 3, 913 (2012); S. J. Gagne et al., Proc. Natl. Acad. Sci. U.S.A. 109, 12811 (2012); Q. Shan, Y. Wang, J. Li, C.

Gao, Nat. Protoc. 9, 2395 (2014); P. R. Hirsch, T. H. Mauchline, Nat. Biotechnol. 30, 961 (2012);

A Robust analytical tools will be able to provide the chemical fingerprint of each individual component of a complex preparation; ICMs is necessary to monitor quality and biological consistency; Combining both chemical and biological analyses provides an effective strategy for revealing all active components; A multi-level biological approach are used to integrated pharmacology, molecular biology, and systems biology; This technique was similarly applied to a number of TCM herbs: Salvia miltiorrhiza, Ganoderma lucidum, Glycyrrhiza uralensis, and Rheum palmatum

MEDICINAL PLANTS CONSTITUTE AN IMPORTANT SOURCE FOR NEW DRUGS;

To facilitate botanical drug development, the Center for Drug Evaluation and Research

(CDER) of the U.S. Food and Drug Administration (FDA) established the Botanical Review

Team in 2003 and published its first Guidance for Industry: Botanical Drug Products in

2004;

This guidance represents FDA’s thinking and provides recommendations on quality,

nonclinical, clinical, and other unique aspects associated with herbal medicine new drug

development through the investigational new drug (IND) and new drug application (NDA)

processes.

From 2004 to 2013, CDER received over 400 botanical IND applications and pre-IND meeting requests

R=H or OH Range n = 5.5

Fulyzaq (crofelemer) is the first FDA-approved drug for

symptomatic relief of noninfectious diarrhea in patients

with HIV/AIDS on antiretroviral therapy;

It is available as a delayed-release tablet containing 125

mg crofelemer, a botanical drug substance derived from

the red latex of Croton lechleri Mull. Arg.

(Euphorbiaceae);

Crofelemer is an oligomeric proanthocyanidin mixture

primarily composed of (+)-catechin, (-)-epicatechin, (+)-

gallocatechin, and (-)-epi-gallocatechin monomer units

linked in random sequence;

The red latex, commonly known as Dragon’s Blood, has

been commonly used in South America as an herbal

medicine for treating diarrhea;

Veregen (sinecatechins) ointment was approved for

topical treatment of external genital and perianal warts; It

contains 15% (w/w) sinecatechins, partially purified

fraction of the water extract of green tea leaves from

Camellia sinensis (L.) Kuntze (Theaceae);

Sinecatechins is a mixture of catechins (85% to 95% of

the total drug substance) and other green tea

components.

These catechins include more than 55% of

epigallocatechin gallate and other catechin derivatives;

S.T. Chen et al., Nat. Biotechnol. 26, 1077 (2008).

3. FDA Guidance for Industry-Botanical Drug Products,

RegulatoryInformation/Guidances/ucm070491.pdf, accessed on June 28, 2014.

4. Approved Labeling for Veregen (NDA 021902) http://www.

accessdata.fda.gov/drugsatfda_docs/nda/2006/021902s000_ prntlbl.pdf, accessed

on June 28, 2014.

Natural products and drug discovery: a survey of stakeholders in industry and academia Vafa Amirkia and Michael Heinrich *

Aim: We seek to understand this

phenomenon through insights from

highly experienced natural product

experts in industry and academia.

Method: We conducted a survey

including a series of qualitative and

quantitative questions related to

current insights and prospective

developments in natural product drug

development. The survey was

completed by a cross-section of 52

respondents in industry and

academia.

The pharmaceutical industry and natural

products: historical status and new trends

Bruno David, Jean-Luc Wolfender, Daniel A. Dias

Selection of representative publications on the outlook of NPs as drug leads in modern drug

discovery programs and their overall levels of optimism

Natural Products as Source of Molecular Diversity

Consequences:

*To rationally approach quality of natural products vs. synthetics

*Selection of most promising producer organism

The potential role of Chinese herbal medicines

in cancer management

Science 16 Jan 2015: Vol. 347, Issue 6219, pp. 337 DOI: 10.1126/science.347.6219.337-c

Possible targets of Chinese herbal medicines (CHMs) in experimental systems and their implications

Science 16 Jan 2015: Vol. 347, Issue 6219, pp. 337 DOI: 10.1126/science.347.6219.337-c

O

O

H3CO

O

H

O

CH3

CH3

O

O

H3CO

O

H

CH3

CH3

RO

artemeter; R = CH3

arteéter; R = C2H5

artemisina (quinghaosu)

artesunato de sódio; R = COCH2CH2CO2Na

Na década de 90, foram descobertos outros derivados antimaláricos,

entre eles a artemisinina, isolada de Artemisia annua L. (Asteraceae)

Artemisia annua - usada na China há mais de 2000 anos como

antipirético e no tratamento de malária

artemisinina marcou uma nova geração de antimaláricos e associou

a pesquisa sobre plantas de uso tradicional/química de produtos

naturais/química medicinal

(Woerdenberg, et al., Pharmaceutical World Science, 16, 169, 1994)

Production of the antimalarial drug precursor artemisinic acid in

engineered yeast

Key Steps

• Engineer farnesyl PP pathway to

increase

FPP production. • Block conversion to squalene. • Introduce gene producing

amorphadiene

synthase from A. annua. • Clone novel cytochrome P450 for

conversion of amorphadiene to

artemisinic

acid from A. annua and express in

diene

producer • Artemsinic acid transported out of

engineered

yeast and purified • Yield up to 100mg/L

Dae-Kyun Ro, Jay D. Keasling, et al., Nature, 2006, 440, 940-943

18

19

All New Approved Drugs 1981 - 2014; n = 1562

All Small-molecule Approved Drug 1981 - 2014s; n = 1211

Total Small Molecules/Year

Percentage of N* by Year, 1981 – 2014

Newman, J. D. & Cragg, G.M.: J. Nat. Prod., 2016, 79 (3), pp 629–661

Natural Products as Sources of New Drugs from 1981 to 2014

BIODIVERSIDADE DO BRASIL

……………..CONTINUAREMOS COM UMA ENORME BIODIVERSIDADE ADORMECIDA EM BERÇO

ESPLENDIDO?

Cienc. Cult. vol.68 no.1 São Paulo jan./mar. 2016

FANTASTIC BRAZILIAN BIODIVERSITY

Simon M F et al. PNAS

2009;106:20359-20364

✓ Brazil is a country with a unique terrestrial biodiversity…….. ✓ 70% of all species, including animals and plants on Earth; ✓ 15-20% of the World’s biodiversity – huge endemism; ✓ ~ 7% of the total terrestrial plant species are endemics ✓ ~ 18.000 species occur on in Brazilian territory (Lewinsonhn,T. M. 2002)

Amazonian

“Cerrado”

Pantanal (Wetland)

Caatinga

Atlantic Forest

Subtropical Grassland

A wonderful place for the development of natural

products chemistry research aimed at biodiscovery?

Próximo Texto | Índice

Ilusão de um paraíso biotecnológico

Esperança do Brasil de ganhar dinheiro explorando os

recursos genéticos da Amazônia esbarra em um excesso

de diversidade biológica que nada tem de especial para a

indústria farmacêutica

Se a Amazônia fosse o Pulmão Molecular do planeta, como

explicar que permaneça vazio o elefante branco no Distrito

Industrial de Manaus?

DO COLUNISTA DA FOLHA (Marcelo Leite)

DOMINGO, 27 DE MAIO DE 2007

DIRETOR DE REDAÇÃO: OTAVIO FRIAS FILHO ANO 87 Nº 28.543 EDIÇÃO SÃO PAULO

FOLHAONLINE

Industry

Saldo Comercial crescentemente dependente de setores intensivos em recursos naturais

Brasil, país que detém uma biodiversidade única, abriga 70% de todas as espécies, incluindo plantas e animais o que equivale a 15-20% da biodiversidade da Terra

Fonte de

Matérias Primas

Produtos de alto valor agregado

Produtos de baixo

valor agregado

Tentative Brazilian Bioprospecting Programs

Ethnomedical

Claims

Phytochemical

knowledge Random

1997-PROBEM;

1998-presente Extracta

Amazônia

1971- CEME

Rain Forest, Cerrado,

Caatinga, Atlantic Forest

2003- RENORBIO

Cerrado, Caatinga

1998-BIOTA

FAPESP

2003-

BIOprospeTA

São Paulo State

1. tomo- Indústria extrativista

2. tomo- Indústria agrícola

3. tomo – Indústria de transporte

e fabril

1907-1909 – publicação da primeira monografia sobre o Centro Industrial do Brasil

Processo histórico

Desenvolvimento Industrial do Brasil

Desenvolvimento Industrial do Brasil

Passado

- Processo recente ( ~ 50 anos)

- Cultura dominante – compra de pacotes

tecnológicos

- Indústria básica nacional – necessidade

de exploração dos recursos naturais

para gerar produtos de valor agregado

(siderurgias, petróleo)

- Novo Paradigma

- Necessidade de

- desenvolvimento para

competir

- Ambiente acadêmico e industrial favorável

- Inovação em produtos de maior valor agregado

- Política de desenvolvimento

industrial do governo federal

- Bioeconomia

Desafios do presente

Fonte: Abiquifi

Fonte: Ministério de desenvolvimento, Indústria e Comércio (MDIC) – ALICEWEB (2014)

Evolução do Comércio exterior – de Plantas Medicinais in natura e suas partes (Valores em Mil US$ FOB)

(6,6%)

40,5%

(325,8%)

O saldo da balança comercial saiu de um déficit de

733 mil dólares para 3,6 milhões de dólares

2005 – The antiinflammatory herbal medicine

mixture according international protocol

Aché produz Fitoterápico da biodiversidade

Sintocalmy - usa princípios ativos da passiflora

(patente internacional para a empresa, que investe em

pesquisas da biodiversidade nacional)

Notícia Jornal Brasil Econômico, 10-03-2010

FITOSCAR – lançado em 2007, é uma mistura de taninos

constituinte do extrato seco de Stryphnodendron

adstringens (Mart.) Coville a 50% 60 mg

Luis Marques, et al, 2012

CENTER FOR INNOVATION IN BIODIVERSITY & DRUG DISCOVERY

São Carlos Headquarter

IFSC-USP

Ribeirão Preto FCFRP-USP

Araraquara IQ-UNESP

Campinas IQ-UNICAMP

São Carlos DQ-UFSCar

Director Vice-Director

Coordinator of Technology Transfer

Coordinator of Education &

science dissemination

NATURAL PRODUCTS

NuBBE Design Model for CIBFar A robust analytic platform

HPLC, LC, GC, MS, NMR

Identification known and unknowns

secondary metabolites and peptides

Map metabolic

network

Biosynthesis

Biological

activity Metabolome

data information

*Role

In plant

*Regulation,

genes,

proteins

Bolzani, V. et al. Pure App. Chem. 2012

MEDICINES AND COSMETICS LEAD COMPOUNDS

Erythrinian type Akaloids from Erythrina Mulungu

Amer, M.E J. Nat. Prod., 54, 329-363, 199; Bolzani, Flausino, Souza, J. Nat. Prod.2006

Flausino, Bolzani, Souza, Biol. & Pharm. Bull. v. 30, p. 375-378, 2007

Erythrina - have been used in traditional medicines as anti-bacterial, anti-inflammatory, analgesic, sleep inducing and anti-depression agents;

In Brazil - E. mulungu is one of the active principles of Maracujina® e Ritmoneuran. ®

N

CH3O

CH3O

CH3O

N

CH3O

HO

CH3O

N

CH3O

HO

CH3O

N

HO

CH3O

CH3O

N

HO

HO

CH3O

(+)-Erysopine

(+)-Erisodine

(+)-Erysoline(+)-Erysotrine

N

CH3O

CH3O

HO

(+)-Erythrvine

N

CH3O

CH3O

CH3O

OH

(+)-Erythetine

N

CH3O

CH3O

CH3O

O-

(+)-Erysotrine N-oxide

N

O

O

HO

N

CH3O

CH3O

HO

OH

(+)-11a-hydroxylerythravine

N

CH3O

CH3O

CH3O

OH

(+)-a-hydroxyerysotrine

New derivative

conformations for compound (+)-a-hydroxyerysotrine

Erythrina mulungu (Fabaceae)

Erythrina mulungu árvore-de-coral, mulungu-coral,

capa-homem, suiná-suiná

http://verdeemfolha.blogspot.com.br

medicinal species

Metabolomic studies

of 3 Erythrina species

Metabolomic fingerprint of Erythrina mulungu harvested in three different

places using NMR and Multivariate data Analysis;

Comparison of the profile with other species of Erythrina;

Scale up of an anxiolytic herbal medicine ®Apsen

Erythrina mulungu Preclinical/clinical studies

2012 - Ongoing process at ANVISA to release a herbal medicine from E. mulungu (a standardized fraction of erythrinian alkaloids) 2014 – DISCONTINUED BY THE COMPANY

NMR methodology used for Quality Control

São Paulo State University

Institute of Chemistry

Araraquara -Brazil

Jean-Luc Wolfender

Supervisor

Emerson Queiroz

Evaluation of the health benefits of Brazilian fruits and vegetables for the development of safe and valuable functional foods

Study of uncommon fruits and vegetables consumed in Brazil with limited local use

and estimation of their potential as a functional food and cosmetics to improve

health, and chemopreventive activity of these fruits; and vegetables on the

prevention of chronic diseases;

Discovery of new lead compounds of natural origin with a chemopreventive activity

using state-of-the-art methodologies; Patent issued; articles in preparations;

Safety assessment by screening for known toxic compounds that might represent a

risk for the local population in the most interesting edible plants;

Vanderlan Bolzani Ian Castro Dulce H. S. Silva Supervisor

2011-2014 INTERNATIONAL COLABORATION

Muriel Cuendet

Isolated Compounds

DPPH˙ ABTS˙+

ORAC (Oxygen Radical

Absorbance Capacity )

AChE

Biological Assays

Structural elucidation: NMR

and HR-MS

Transfer analytical

HPLC-UV conditions to

preparative MPLC-UV

Fruit Extract

MPLC/UV

Col 26 ACN

Time0.50 1.00 1.50 2.00 2.50 3.00

%

0

100

130309_SC_MLZ_MPLC_STPM_POS 1: TOF MS ES+ BPI

3.11e4

0.71721.2340

0.79510.2267 1.54

303.0862

UHPLC/TOF/MS

A series of phenolic compounds were identified

Patent protocol at the INPI-Brazil and International PCT

2015 Kurt Pulitzer Award - ABIQUIM

CIEnp Pre-clinical

studies

Vantagens para o desenvolvimento de fitoderivados no Brasil

• Grupos de pesquisa qualificados com reconhecida competência nas áreas de

botânica, agronomia, química, farmacologia toxicologia, química farmacêutica

(essenciais para a pesquisa de plantas medicinais);

• Mercado favorável para os produtos da biodiversidade e de plantas de uso

medicinal;

• Setor industrial farmacêutico nacional entende que o mercado de bioprodutos é

promissor;

• Maior interação entre a academia e o setor empresarial – Lei de inovação

favorece as parcerias;

• Lei do Bem contribui – empresários e capital de risco longe da pesquisa de risco;

• Tradição de uso popular de inúmeras espécies sem qualquer estudo –

possibilidade de pesquisa inovadora com espécies da biodiversidade;

• Regulamentação do registro de plantas medicinais pela ANVISA

• Mercado internacional de fito-derivados em plena expansão

Conclusion Remarks

Brazilian biodiversity, potential source for finding new lead for

drug development; Chemistry is base science to understand and

explore biodiversity;

A tool complement useful for the understanding of biological

systems – metabolome versus proteome (Systems Biology);

Raw material for Medicinal/combinatorial chemistry/parallel

synthesis using natural product or n. p.-like scaffolds;

Long-term research programs like CEPIDS-Fapesp and

International collaboration are essential for exploration our plants

aimed at innovation on new medicines; food supplements;

Building an expertise of excelence in the Universities in

Association with PHARMACEUTICAL SECTORS can be important for

innovation in bioproducts from Brazilian Biodiversity

Maria Cláudia Mrax Young (IBot –SMA)

Luce Maria Brandrão (IBot –SMA)

Celia R. Garcia (IB-USP, São Paulo)

Noberto P. Lopes (FCF-USP, RP)

Paulo Cezar Vieira (UFSCAR)

Adriano Andricopulo (IF-USP, SC)

Maria José Giannini (FCF-UNESP)

Marcia Graminha (FCF, UNESP)

Sandro Valentini (FCF, UNESP)

Glaucius Oliva (IF-USP, Scarlos)

Rafael Guido (IF-USP, Scarlos)

João B. Calixto (UFSC)

Edilberto R. Silveira (UFC)

Leticia Lotufo (USP)

Claudia Pessoa (UFC)

Luce Torres (IB, SP)

International Collaboration Arthur Edison (University of Florida, USA);

Leslie Gunatilaka (Arizona University)

Christian W. Gruber

Medical University of Austria

Jean-Luc Wolfender, Emerson Queiroz

(Ecole de Pharmacie Genève-Lausanne,

Université de Genève

Simon Gibbons (School of Pharmacy,

University of London)

David Craik (Queensland University,

Australia) – Starting collaboration on

peptides

Michele Reboud-Ravoux, UPMC, Paris VI

Sylvie Michel, René Descartes, Paris

Christian W. Gruber – Center for Physiology and

Pharmacology, Medical University of Vienna -

Austria.

National Colaboration

ACKNOWLEDGEMENTS FOR GRANT SUPPORT TO NuBBE

2011-2014 - ChemBioFight

International Research Staff

Exchange Program FP7-

PEOPLE-2010-IRSES

Brazilian Switzerland Joint Research Program

(BSJRP-#590128/2011-6)

WELCOME TO IUPAC 2017 IN SÃO PAULO, BRAZIL

É missão da Universidade gerar conhecimento de vanguarda

Boa ciência gera tecnologia e desenvolvimento

OBRIGADA PELA

ATENÇÃO