© M. Wyszogrodzka

„The Magic Bullet“

„The magic bullet“

Paul Ehlrich vision(begining of 20th century)

Nobel in Medicine - 1908

mAbs as a carriers of drugs

© M. Wyszogrodzka

Ringsdorf Model

Ringsdorf Model (1975)

Chemistry of bioconjugationEg. PK2, CT-2130 etc.

© M. Wyszogrodzka

Drug Classes & Modes of Action

Targeting the tumor cell cytostatic drug

Targeting the blood supply of the tumor anti-angiogenetic drug

Inhibition of cell growth and proliferation

Inhibition of angiogenesis (the growth of new blood vessels)

Principles of chemotherapeutic treatment

Paclitaxel

Doxorubicine

N

O

O

NHO

O

(S)-Thalidomide

© M. Wyszogrodzka

Low Molecular Weight Drugs

• most drugs are small molecules (MW ~ 500 g/mol)

• short half life in blood circulation

• fast diffusion into healthy regions

• fast clearance from body

• low selectivity for target tissue causing side effects

• low amounts of drug reach target tissue

• no constant levels of drug concentration

• many side effects

• multiresistance

• poor selectivity

• poor water solubility

• high toxicity

Pharmacokinetic profile of Interferon and Pegasys -Interferon conjugated to branched PEG 40 kDa

Circulation life-time difference between unmodified drug and polymer-drug conjugate

© M. Wyszogrodzka

Low Molecular Therapeutics vs. Macromolecular Therapeutics

Polymers in Drug Delivery, Taylor, 2006, Uchegbu, p 159.

Slubility problems Cell entry by diffision

No preferential accumulation Targeting (prodrug) possible

Fast elimination Non-specific toxicity

Improved water slubility Cell entry by endocytosis

(abbility to overcome multidrug resistance) Preferentia accumulation in solid tumors Effective targeting

- cell surface receptors- specific subcellular organelles Slower elimination Decreased non-specific toxicity

Low Molecular Therapeutics Macromolecular Therapeutics

© M. Wyszogrodzka

Requirements for Polymeric Drug Carriers

Polymers in Drug Delivery, Taylor, 2006, Uchegbu, p 161.

1. Well characterized (reproducibility, PDI, heavy metal traces)

2. Hydrophilic for intravenous drug delivery.

3. Biocompatible (non-toxic and non-immunogenic), also metabolic products

4. Multivalent (for conjugation of drug, targeting and/or imaging moiety)

5. Stable in circulation (drug linkage)

6. Formulation (stabile under convenient administration)

7. Easly eliminated from the body (whole polymer or metabolites)

Polymeric drug carriers have to be:

Cancer Therapy

Higher Selectivity to Cancer Cells

Passive Targeting Active Targeting

Antigene

Growth Factors

Peptide receptors

Carbohydrate receptors

Vaccins receptors

speciffic phisiological differences:

True for tumors bigger than 2-3 mm

Based on Tumor Vasculature

Solution: Tumor specific delivery

Based on Tumor Cell-Surface Receptor

© M. Wyszogrodzka

Passive Targeting

Enahnced Permeability and Retention Effect (Maeda, 1986, SMANCS)

Tumor size: 2-3 mm cell clusters inducing angiogenesisPore size: 100 – 1200 nm (depending on tumor type)

Uncharged or negatively chargedmacromolecular carriers that larger than 40 kDa efficiently escape renal clearance.

Intravenous administration of macromoleculartherapeutics

Size-dependent selectivity of drugs entry

Structural features of macormolecules: molecular weight (> 40kDa) charge (negative or neutral) shape (globular, linear, branched) hydrodynamic radius

Selectivity Passive targeting

© M. Wyszogrodzka

Demonstration of the EPR-Effect

tumor tumor tumor

Indotricarbocyanin (unbound dye)

after 24 h

PEG 40kDa-Indocarbo-cyanin-conjugate

after 24 h

Problem: Tumor specificity only with nanoparticles >5 nm

K. Licha, F. Kratz

© M. Wyszogrodzka

Active Targeting

Endocytotic pathway for the cellular uptake of macromolecules and nanocarriers for drug delivery

Selectivity Active targeting

© M. Wyszogrodzka

Dendritic Polymers in Biomedical Applications -From Synthesis to Clinical Use

© M. Wyszogrodzka

Degree of Branching

© M. Wyszogrodzka

Synthesis of Dendrimers

Convergent Growth Approach

Divergent Growth Approach

from shell to the core

from core to the shell

© M. Wyszogrodzka

Synthesis of Polyglycerol Dendrons

O

OO

RO OR

RO

RO

O

OO

ORRO

OR

OR

X

Divergentapproach

Convergentapproach

dihydroxylation

O

OO

R1O OR1

R1O

R1O

O

OO

OR1R1O

OR1

OR1

O

OO

O

OO

X

1. oxidation2. reduction

[G2.0]-X-OR

R = R1 or R = R1

Cl

ClClWiliamson's

ether formationWiliamson's

ether formation

O O OHX OHOH

HO O O OR1

OH OR1OR1R1O

O O OHOH OHOH

HO

TRIGLYCEROL

© M. Wyszogrodzka

Properties of Dendrimers

highly branched highly reactive three-dimentional high structural purity (low PDI) monodispercity (single MW) globular shape large number of „peripheral“ functionalities bifunctionality multivalency

A. W. Bosman, H. M. Janssen, E. W. Meijer Chem. Rev. 1999, 99, 1665-1688.

Advantages:

step-by-step synthesis (tedious) high costs of production „mistakes“ in the structure

Disadvantages:

© M. Wyszogrodzka

Dendrimers in Cancer Therapy and Diagnostic

© M. Wyszogrodzka

Dendrimers in Cancer Therapy and Diagnostic

© M. Wyszogrodzka

First Dendrimers

NN

NH

NH

HN

HN

O

O O

O

N

N

N

N

NHNHN

N

NH N

NH

N

HN NNH

N

NHN

HN

N

O

O

O

O

NH HNO O

N N

NHO

HNO

NN

HN

HN O

O

N

N HN

O

NHO

N

N

O

ONH

NH

HNNHO O

O

O

NN

N

N

O

ONH

O

HNO

OHNNH

O

NN

N

N

HNNH

HN

NH

HN

HN

HN

HN

HN

HN

NH

HN NH HN NHHN

NH

HN

NH

HN

NH

NH

NH

NH

HN

NH

HN

NHHNNHHNNH

OO OO

OO

O

O

O

O

O

O

O

O

O

O

O

OO

OOOOOO

OO

O

O

O

O

O

NH2H2NNH2H2NNH2

NH2

H2N

H2N

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

H2NNH2

H2NNH2H2NNH2H2N

NH2

H2N

H2N

H2N

H2N

H2N

[G4.0] PAMAM - Poly(amido amine)

1985 – Don Tomalia

Starburst® (Dendritech)

NN

N

N

N

NN

NN

N

N

NN

N

N

N

N

N

N

N

NN N

N

N

N

N

N

NN

N

NN N N N

N

N

N

N

N

N

N

N

N

N

NN

N

N

N

N

N

N

N

N

N

N

N N N

N

H2N

H2NH2N

H2NH2N H2N NH2 H2N NH2 H2N NH2 NH2 NH2

NH2NH2

NH2

NH2

NH2NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2NH2

NH2NH2

NH2NH2NH2NH2NH2NH2H2NNH2H2NNH2H2N

H2NH2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

H2N

[G5.0] PPI* - Poly(propylene imine)

Astramol® (DSM Fine Chemicals)

* known also as POPAM, or DAB with DiAminoButan

1978 – F. Vögtle1993 – E. W. ´ Bert´ Meijerand indepedently G. Mülthaupt

© M. Wyszogrodzka

Drug-Loaded Dendrimers

N

HNO

RNH

OO

NH

OR

NHO

O

N

N

HN O

R NH

O O

NHO

RNH

OO

N

N

HN O

R NH

O O

NH O

R NH

O O

N

N

HNO

RNH

OO

NH O

R NH

O O

N

N

HNO

RNH

OO

NHO

RNH

OO

NN

HN

O

R

NH

O

ONHO

RNH

OO

N

N HN

O

R

NH

O

O

NH

O

R

NH

O

O

N

NHN

O

R

NH

O

O

NH

O

R

NH

O

O

NHN

O

R

NH

O

O

NH

O

R

NH

O

O

N

N

HN

O

R

NH

O

O

NH

O

R

NH

O

O

N

N

NH

O

R

HNO

O

NH

O

R

HN

O

O

N

N

NH

O

R

HN O

O

NH

O

R

HNO

O

N

N

NH

OR

HN O

O

NH

O

R

HNO

O

N

N

NH

O R

HN O

O

NH

OR

HN O

O

N

N

NH

OR

HNO

O

HN

O R

HNO

O

N

N

NHO

RHN

OO

HN

OR

HNO

O

N

N

NHO

RHN

OO

HN

OR

HNO

O

N

N

NHO

RHN

OO

HNO

RHN

OO

N

N

NHO

RHN

OO

HNO

RHN

OO

N

N

NHO

RHN

OO

HNO

RHN

OO

N

N

NHO

RHN

OO

HN

O

RHN

OO

NN

NH

O

R

HN

O

O HN

O

RHN

OO

N

NNH

O

R

HN

O

O

HN

O

R

HN

O

O

N

N

NH

O

R

HN

O

O

NH

O

R

HN

O

O

N

NH

O

R

HN

O

O

NH

O

R

HN

O

O

N

N

NH

O

R

HN

O

O

NH

O

R

HN

O

O

N

N

HN

O

R

NHO

O

NH

O

R

NH

O

O

N

N

HN

O

R

NHO

O

NH

O

R

NHO

O

N

N

HN

OR

NHO

O

NH

O

R

NHO

O

N

N

HN

OR

NHO

O

NH

OR

NHO

O

N

N

HN

OR

NHO

O

NH

OR

NHO

O

N

N

HNO

RNH

OO

NH

OR

NHO

O

N

N

H2NNH2

N

N

H2NNH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NN

NH2

NH2

N

N NH2

NH2

N

N

NH2

NH2

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2H2N

N

N

NH2 H2N

N

N

NH2H2N

N

N

NH2 H2N

N

N

H2NH2N

N

N

H2N

H2N

N

N

H2N

H2N

NN

H2N

H2N

N

NH2N

H2N

N

N

H2N

H2N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2NNH2

N

N

H2NNH2

N

N

H2NNH2

N

N

O

O

O

O HN O

O

95% HCOOH

N

HNO

RNH2

NH

OR

NH2

N

N

HN O

R NH2

NHO

RNH2

N

N

HN O

R NH2

NH O

R NH2

N

N

HNO

RNH2NH O

R NH2

N

N

HNO

RNH2NH

O

RNH2

NN

HN

O

R

NH2

NHO

RNH2

N

N HN

O

R

NH2

NH

O

R

NH2

N

NHN

O

R

NH2

NH

O

R

NH2

NHN

O

R

NH2

NH

O

R

NH2

N

N

HN

O

R

NH2

NH

O

R

NH2

N

N

NH

O

R

NH2

NH

O

R

NH2

N

N

NH

OR

NH2

NH

O

R

NH2

N

N

NH

OR

NH2

NH

O

R

NH2

N

N

NH

O R

H2N

NH

OR

NH2

N

N

NH

OR

H2N

HN

O R

H2N

N

N

NHO

RH2N

HN

OR

H2N

N

N

NHO

RH2N

HN

OR

H2N

N

N

NHO

RH2N

HNO

RH2N

N

N

NHO

RH2N

HNO

RH2N

N

N

NHO

RH2N

HNO

RH2N

N

N

NHO

RH2N

HN

O

RH2N

NN

NH

O

R

H2N HN

O

RH2N

N

NNH

O

R

H2N

HN

O

R

H2N

N

N

NH

O

R

H2N

NH

O

R

H2N

N

NH

O

R

H2N

NH

O

R

H2N

N

N

NH

O

R

H2N

NH

O

R

H2N

N

N

HN

O

R

H2N

NH

O

R

H2N

N

N

HN

O

R

H2N

NH

O

R

H2N

N

N

HN

OR

H2N

NH

O

R

H2N

N

N

HN

OR

NH2

NH

OR

H2N

N

N

HN

OR

NH2

NH

OR

NH2

N

N

HNO

RNH2

NHO

R

NH2

N

12 N HCl

N

H2NNH2

N

N

H2NNH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NN

NH2

NH2

N

N NH2

NH2

N

N

NH2

NH2

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2

NH2

N

N

NH2H2N

N

N

NH2 H2N

N

N

NH2H2N

N

N

NH2 H2N

N

N

H2NH2N

N

N

H2N

H2N

N

N

H2N

H2N

NN

H2N

H2N

N

NH2N

H2N

N

N

H2N

H2N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2N

H2N

N

N

H2NNH2

N

N

H2NNH2

N

N

H2NNH2

N

Shape-Selective Release of Guest

© M. Wyszogrodzka

Drug-Loaded Dendrimers

[G5.0]

[G4.0]

[G3.0]

PEG400

PTX

10.000 fold better solubilization than in H2O

Solubilization of Paclitaxel by PG Dendrimers

© M. Wyszogrodzka

Dendrimers for MRI

GdCore

*

Gd

*Gd

*Gd

* GdLys Lys

Lys

*

Gd*

Gd

*

Gd

*

Gd

Lys

LysLys

**

Gd*

Gd *Gd

LysLys

Lys

*

Gd

*

Gd

*Gd

*GdLys

Lys

Lys

*

Gd

* Gd*

Gd

* Gd

Lys

Lys

Lys

*

Gd

*

Gd

*

Gd

*

Gd

Lys

Lys Lys

Gd

Gadomer®-17 (Schering AG)

DTPA

© M. Wyszogrodzka

(Bio)Polymers in Biomedical Applications -From Lab to Clinical Use

© M. Wyszogrodzka

Dendrimers for MRI

Gadomer®-17 (Gadomer-24)

• Polylysine dendrimer carrying 24 Gadolium complexes on the surface• MW = 17 kDa• blood pool contrast agent• high in vivo stability

© M. Wyszogrodzka

Polymer-Drug Conjugates

© M. Wyszogrodzka

Folate-Targeted Cancer Therapeutic

EC145 (Leamon, 2006)

Vinca alkaloid desacetylvinblastine monohydrazide

(DAVLBH, a depolymerization inhibitor)

Water soluble spacer - arginine and aspartic acid

Release by disulfide reduction

MTD ~ 0.8 mg/kg DAVLBH-eq.

Phase II clinical trial (EC145)

© M. Wyszogrodzka

Polymer-Protein Conjugate

SMANCS -Poly(Styrene-co-Maleic Acid-half-n-butylate)-conjugated NeoCarzinoStatin

NCS - proteinaceous antitumor antibiotic (Streptomyces carzinostaticus)

Apoprotein (113 aminoacids) + tightly, non-covalently bound labile Chromophore

Chromophor

Cytotoxicity (0.01 g/ml) against:- mammalian cells- gram (+) bacteriaShort half-timet0.5 = 1.9 min

SMA – copolymer of styrene-maleic acid-half-n-butylate

SMANCS-Lipiodol - approved in 1993 (Japan) for treatment of liver cancer

© M. Wyszogrodzka

Biopolymer-Drug Conjugates

Doxo-EMCH – in clinical studies (Phase I and II)

© M. Wyszogrodzka

Biopolymer-Drug Conjugates

Doxo-EMCH

Acid-labile Hydrazone

Reactive maleimid

Blood protein albumin (HSA)as polymeric drug carrier

F. Kratz F, J. Contr. Release, 2000, 132, 171-183.

Direct injection of Doxo-maleimid (Pro-Drug) Ultimate coupling to Cys-34 of HSA (70% free thiol) In situ generation of polymeric pro-drug Longer circulation, cellular uptake, higher therapeutic window

© M. Wyszogrodzka

Biopolymer-Drug ConjugatesDoxo-EMCH

© M. Wyszogrodzka

Immunoconjugates

Senter et. al. Nature Biotechnol. 2005, 23, 1137-1146,www.seattlegenetics.com

Structure: chimeric mAbs BR96 – targeting anti-LewisY

~ 8 DOX molecules per mAb

BR96-DOX

Phase I clinical study66 patients, intravenous administration over 21dDose: 66 – 875 mg/m2 (2-25 mg/m2 free DOX)

Phase II clinical study29 patients Dose: 700 mg/m2 (20 mg/m2 free DOX)

cross-reactivity of BR96-DOX with normal gastrointestinal tissue BR96-DOX demonstrated synergistic effects with paclitaxel and docetaxel(Taxotere®)

© M. Wyszogrodzka

Polymer-Drug Conjugate

HPMA - N-(2-hydroxypropyl)methacrylamideDeveloped in Czechoslovakia as a plasma expander - Kopecek and Bazilova, 1973

• hydrophilic• non toxic in rats• non-biodegradable

x/y = 90/10

© M. Wyszogrodzka

Polymer-Drug Conjugates

Diblock Co-Polymer-Doxorubicin enzyme-cleavable peptide structure

N-(2-Hydroxypropyl)methacrylamide (HPMA) Copolymer-Doxorubicin

Doxorrubicin content: ~ 8,5 wt % Tetrapeptide Linker EPR targeting Cleavage by lysosomal Cathepsin B

> selective release of Doxo MW: 28 kDa > Excretion via kidney

(barrier: 30-50 KDa) maximal tolerated dose: 320 mg/m2 (10 mg/kg) Currently Phase II clinical trial Problem: not optimal tumor accumulation of PK1

PK 1 (FCE28068) - 1994

© M. Wyszogrodzka

Polymer-Drug Conjugates

Diblock Co-Polymer-Doxorubicin enzyme-cleavable peptide structure

PK 2 (FCE28069) - 2002

HPMAcopolymer-Doxorubicin / Galactosamine

Doxorubicin content: ~ 8,5 wt % Tetrapeptide linker EPR Targeting

+ targeting via galactosamine (asialglycoprotein receptor) Cleavage by lysosomal cathepsin B MW: 25 kDa maximal tolerated Dose: 320 mg/m2 (10 mg/kg) Currently Phase II clinical trial

© M. Wyszogrodzka

Polymer-Drug Conjugates

Diblock Co-Polymer-Taxol pH-cleavable release of drug

PG-TXL (CT2103) - 1992Polyglutamate-Paclitaxel

Mixture of connection via two different

OH-groups in Paclitaxel (ester bonds)

Drug content ~ 37%

Improved water solubility >20mg/kg

Release by ester hydrolysis

PG backbone is biodegradable

(in vitro and in vivo -clevage by Cathepsin B)

MTD ~ 200 mg/m2 Taxol-eq.

Phase III clinical trial (CT2103, Cell Ther.)

© M. Wyszogrodzka

Polymer-Drug Conjugates

H3CO

O NH

O HN

H

NH

O

NOH

OH

OHO

O

OH3C

OH

O

O

OHNH2

H3C

n

m

Schematic picture of block copolymer and chemical structure of PEG-b-p(Asp-HYD-DOXO)

PEG-b-p(Asp-HYD-DOXO)

© M. Wyszogrodzka

PEG-b-p(Asp-HYD-DOXO)

© M. Wyszogrodzka

Polymeric Micelle in Cancer Therapy

NK911 • Doxorubicin-loaded PEG-b-PAsp copolymer micelle formulation (Kataoka)• drug physically entrapped in the micelle core and chemically conjugated to the aspartic acid side chains of the core-forming block via amide linkages• phase II clinical trial evaluation for efficacy and toxicity profiles

Phase I (2001):• longer circulation half-life (50nm)• a larger area under the curve (AUC)• reduced toxicities in comparison to the conventional formulation of Dox

© M. Wyszogrodzka

Polymeric Vesicles in Cancer Therapy

DOXIL (doxorubicin HCl liposome injection) is doxorubicin hydrochloride (HCl) encapsulated in STEALTH® liposomes for intravenous administration

Compositions of the STEALTH® liposome carriers

MPEG-DSPEHSPC

© M. Wyszogrodzka

Polymeric Vesicles in Cancer Therapy

DOXIL, Caelyx (Doxorubicin-Liposomal)• Encapsulation of Dox through active loading that is based on an

ammonium sulfate gradient• 15000 Doxorubicin molecules per Vesikel (100 nm)• Slow tissue clearance after injection PEGylation• Long circulation in bloodstream• slow, targeted release of the drug• 6-fold higher effectivity in comparison to the free Dox