Arg-Gly-Asp (RGD) Tumor Targeting Conjugates · Arg-Gly-Asp (RGD) Tumor Targeting Conjugates Ruth...

Arg-Gly-Asp (RGD) Tumor Targeting Conjugates

Ruth Njeri Muchiri

Michigan State University

Department of Chemistry

Normal cell division

DNA damage

( No repair)

Cell suicide (apoptosis)

Uncontrolled cell growth, invasion and metastases

Cancer

Cancer cell division

Tumor

Damaged cell

http://www.cancer.gov/

Causes & Types of Cancer

Main Causes

Radiation

Age-related causes

Chemicals

Types

Solid cancers

Examples: lung, breast, bowel cancer

Non-solid cancers

Examples: leukemia, lymphoma, myeloma

http://www.immunotherapyforcancer.info

Cost of Cancer

2007

11.7 million

2008

$895 billion(1.5% World’s GDP)

2010

1,529,560569,490

USA population with

cancer

World’s economic loss

New cancer cases in USA

New deaths in USA

http://www.cancer.org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-and-figures-2010

Attempts Towards Cancer Treatment

Chemotherapy RadiotherapySurgery

.

http://www.immunotherapyforcancer.info

http://www.medindia.net/index.htm

Agent Examples

Alkylating agents Cyclophosphamide, Oxaliplatin, Carboplatin, Chlorambucil, Mechloethamine, Melphalan

Antimetabolite agents Folate antagonists, Methotrexate, Fludarabine, Cytarabine

Plant alkaloids and terpenoids

Doxorubicin, Vinca alkaloids, Taxanes, Camptothecin

Antitumor antibiotics Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin,Mitoxantrone

Conventional Chemotherapy Treatment

Pros

Less mutilating

Neo-adjuvant treatment

Preferred in metastasis

cases

Cons

Non specific

Toxic

Multiple drug resistance

http://www.cancer.gov/

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Patients with extensive lung tumor

Patients with limited lung tumor

Development on Chemotherapy

One leg good, two legs better: Cocktail therapy

Pavlidis, A. N.; Tolis, T. F.; Briassoulis, E. C.; Sowla, A.; Klouvas, G. D. Lung Cancer 1991, 1, 279-283.

Kindler, H. L. et al. J. Clin. Oncol. 2005, 23, 8033-8040.

VAC (Etoposide, Doxorubicin, Cyclophosphamide)

CVM (Cisplatin, Vincristine, Methotrexate)

MCP (Mitomycin-C, CCNU, Procarbazine)

High toxicity observed

Bevacizumab + Gemcitabine

in advanced pancreatic cancer

Drug analogues

Development on Chemotherapy

Improved binding but low specificityRapoport, N. Y.; Herron, J. N.; Pitt, W. G.; Pitina, L. J. Controlled Release 1999, 58, 153–162.

Bigioni, M.; Salvatorea, C. Biochemical Pharmacology 2001, 62, 63–70.

Targeted Cancer Therapy

Reduced toxicity

Specific

Maximization of drug effect

Picture from: http://www.isncc.org/files/100-Wed_Ball_1-3_0830.2_Ouwerkerk.pdf.

Tumor Targeting Mechanism

Cancer Cell

Ojima, I. Acc. Chem. Res. 2008, 41,108–119.

Tumor Targeting Molecules

c(Arg-Gly-Asp-x-y) αvβ3 integrinEndothelial cells

Epithelial

Glioblastoma cells

Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I. Bioorg. Chem. 2005, 13, 5043-5054.

Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem. Int. Ed. 1997, 36, 1374-1389.

Antibodies

Diverse Diverse

CD44

Layilin

HARE

CDC37

Epithelial

Ovarian

Colon

Stomach

SSTR1-5 Gastro-intestinal cells

Ligand Target Target location

Hyaluronic acid

Somastatin analogues

Outline

RGD definition

RGD–integrin receptors interaction

Cilengitide® (Merck)

RGD conjugate design and effect on activity

Future directions

Arg-Gly-Asp (RGD)

Pierschbacher, M. D.; Ruoslahti. Nature 1984, 309, 30-33

Proteins Sequence

Fibronectin (450 kDa) AVTGRGDSPASSK

Fibrinogen α-chain (72 kDa)

TSYNRGDSTFESK

λ-receptor on E.coli(47.7 kDa)

GSFGRGDSDEWTF

Sindbis coat protein (15.5 kDa)

GVGGRGDSGRPIM

α-lytic protease ACMGRGDSGGSWI

Testis specific basic protein

KSRKRGDSADRNY

Natural RGD containing proteins

R

G

D

RGD Receptor- αvβ3 Integrin

αv

β3

RGD-peptide

Xiong, J. P. et al. Science 2002, 296,151-155.

Function

Control angiogenesis,

cell proliferation & migration

Mn2+

PDB1JV2.

Cyclic RGD

Natural peptide – Fibronectin (450 kDa)

AVTGRGDSPASSK

Leahy, D. J.; Aukhil, I.; Erickson, H.P. Cell 1996, 84, 155–164.

Peishoff, E. C. et al. J. Med. Chem. 1992, 35, 3962-3969.

RGDxy

Arg-Gly-Asp-f-(N-Me)Val

1FNF

RGD - αvβ3 Integrin Interaction

Mn2+

H-bonding

Metal ion co-ordination

Xiong, J. P. et al. Science 2002, 296,151-155.

PDB1JV2

Advantages of RGD

Selective binding - ligand for tumor marker, αvβ3 integrin

Has better cellular uptake

Easy to synthesize - solid phase synthesis

Application in tumor imaging

Aguzzi, M.S. et al. Blood, 2004, 103, 4180-4187..

Liu, S. Bioconjugate Chem. 2009, 20, 2199-2213.

Advantages of RGD

Liu, S. Bioconjugate Chem. 2009, 20, 2199-2213.

Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.

Outline

RGD definition

RGD –integrin receptors interaction

Cilengitide®


Future directions

Cilengitide®

Phase III clinical trial (Merck Germany)

Active against glioblastoma (brain tumor)

Induce apoptosis in tumors cells

http://www.chemblink.com/products/188968-51-6.htm


Cilengitide® Mode of Action

Inhibit angiogenesis

Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. Acc. Chem. Res. 2009, 42, 969-980.


Proof of Principle

Tagai, T. et al. Int. J. Cancer 2002, 98, 690-697.

Reardon, A. D. et al. J. Clin. Oncol. 2008, 26, 5610-5617.

In vivo Studies

Brain tumor cells inoculated in

nude mice

E1 - Control

E2 - After Cilengitide treatment

Blood vessels in brain tumor

A. Before treatment

B. After treatment

Phase II clinical trials

Radiographic response to

Cilengitide in glioblastoma patient

Brain tumorBA

cRGD Conjugates:Old Chemotherapy in New Form

Pozzo, A. D.et al. Bioorg & Med. Chem. 2010, 18, 64–72.

cRGD Conjugates

Cilengitide modification

Vachutinsky, Y. et al. J. Control. Release 2010, doi:10.1016/j.jconrel.2010.02.002.

Outline

RGD definition

RGD –integrin receptors interaction

Cilengitide


Future directions

Immobilization

Sites of RGD Multimerization Linker Bond

Effect of RGD Conjugate Design on Activity

cRGD Peptide Immobilization Sites

PCLA -PEG- PCLA

RGD-PCLA -PEG- PCLA-RGD

PCLA –(RGD)-PEG- PCLA

Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.

Liu, C. B.; et al. J. Biomed. Mater. Res. B Appl. Biomater. 2007, 165–175.

PCLA: poly(ε-caprolactone-co-lactide)

PEG: poly(ethylene glycol)Approved by FDA

RGD-PCLA-PEG-PCLA-RGD

cRGD Immobilization on Hydrophobic Site

n=6 to 32x=3 to 7y=4 to 9


PCLA-PEG-PCLA

cRGD Immobilization on Hydrophilic Site

PCLA-PEG-PCLA

PCLA-(RGD)-PEG-PCLA


cRGD Peptide Immobilization Sites

RGD–cell interaction

PEG blocks

PCLA blocks

RGD peptides


Effect of cRGD Immobilization Sites

Thiazolyl blue assay

Cell line used: Chondrocytes +

fetal bovine serum

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RGD(-PEG) RGD(-PCLA) Unmodified

Ce

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dh

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No RGD


cRGD Conjugate Design and Effect on Activity

Multimerization

Immobilization

of RGD Sites

Linker Bond Hydrophilic site

Enhance RGD-

integrin interaction

cRGD Conjugate Multimerization

Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181.

Effective cRGD Conjugate Multimerization

Antitumor

agent

Antitumor

agent

Shuang, L. Bioconjugate Chem. 2009, 20, 2199-2213

60-90 Å

αvβ3 Integrin

60 Å

Effective cRGD Conjugate Multimerization

Xiong, J. P. et al. Science 2002, 296,151-155PDB1JV2.

Scaffolds in RGD Conjugate Multimerization

Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181.

Multimerization Effect on RGD Conjugate Binding

U87MG -Human glioblastoma cell line

No. of RGD Ki [nM] No. of RGD Ki [nM]

c(RDG)1 32 ± 6 c(RGD)1 1627 ± 199

c(RGD)2 13 ± 2 c(RGD)2 522 ± 57

c(RGD)4 1.30 ± 0.10 c(RGD)4 248 ± 48

c(RGD)8 0.32 ± 0.04 c(RGD)8 46 ± 5

c(RGD)16 0.26 ± 0.04 c(RGD)16 10 ± 1

Competitive binding assay against 125I-echistatin

Immobilized αvβ3 U87MG cells

Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168-2181.

RGD Conjugate Design and Effect on Activity

Linker Bond

Multimerization Immobilization

of RGD Sites

Increase affinity Hydrophilic site

enhance RGD-

integrin interaction

Linker Bond Stability

Amide vs hydrazone

Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787.

Hydrolyzes slowly Hydrolyzes fast

Amide bond

Hydrazone bond

Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787.

More toxic

Less toxic

Cellular Cleavage of Linker Bonds

Amide Linker Bond

Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.

Hydrazone Linker Bond


Amide vs Hydrazone Bond Linker Stability

Entry pH 7.4, 37 oCt1/2, h

pH 5, 37 oCt1/2, h

1 >72 >72

2a 6 25

2b 5.8 <2 min


In Vivo Evaluation of Amide Linker Bond Stability

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Amide bond linked conjugate in plasma Free Drug in plasma of Mice treated with 1Amide bond linked conjugate in tumorFree Drug in tumor of Mice treated with 1

A2780 -Human ovarian cancer cell line Amide bond linked conjugates

- Less cytotoxic


Two Linker Bonds in RGD Conjugate System

Bond cleavage Bond cleavage

Drug DrugRGD RGD

Bond stability at pH ~ 5

Drug-Y < Y-X

X-Z < Z-Drug

Drug derivative

X

Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.

Free drug

Two Linker Bonds in RGD Conjugate System

Application in multidrug resistance (MDR)

X – Ester

Y – Hydrazone

Z - Amide


Targets cell nucleus - Prone to Phospho-glycoprotein efflux

Two Linker Bonds in Redirected Drug Delivery

Salerno, M.; Przewloka, T.; Fokt, I.; Priebe, W.; Garnier-Suillerot, A. Biochem. Pharmacol. 2002,

63, 1471-1479.

Targets Mitochondria -No Interaction with Phospho-glycoprotein (p-gp)

Two Linker Bonds in Redirected Drug Delivery

Salerno, M.; Przewloka, T.; Fokt, I.; Priebe, W.; Garnier-Suillerot, A. Biochem. Pharmacol. 2002,

63, 1471-1479.


In Vitro Assay -Linker Bond Effect on MDR

Construct Sensitive

cells

Multi-drug

resistant cells

Free DOX 0.84 g/mL 11.60 μg/mL

Acetal-PEO-b-P(CL-Hyd-DOX) 1.36 μg/mL -

RGD4C-PEO-b-P(CL-Hyd-DOX) 0.14 μg/mL 7.92 μg/mL

Acetal-PEO-b-P(CL-Ami-DOX) - 7.24 μg/mL

RGD4C-PEO-b-P(CL-Ami-DOX) 0.73 g/mL 0.092 μg/mL

IC50 Values

MDA-435 /LCC6WT & MDA-435 /LCC6MDR (Breast tumor cell line)


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RGD-PEO-b-P(CL-Hyd-DOX) Control

Free DOX Acetal- PEO-b-P(CL-Hyd-DOX)

Doxorubicin sensitive breast tumor cell line

In Vivo Studies: Targeted Doxorubicin


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RGD-PEO-b-P(CL-Ami-DOX) Control

Free DOX Acetal- PEO-b-P(CL-Ami-DOX)

Multidrug resistant breast tumor cell line

In Vivo Studies: Targeted Doxorubicin


Sensitive cells

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RGD-PEO-b-P(CL-Ami-DOX) ControlFree DOXAcetal- PEO-b-P(CL-Ami-DOX)

Multidrug resistant cells

Targeted DOX in Breast Tumor Cell Line

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RGD-PEO-b-P(CL-Hyd-DOX)

Control

Free DOX

Acetal- PEO-b-P(CL-Hyd-DOX)

Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.

Model of Redirected Drug Delivery

Doxorubicin sensitive cells


Model of Redirected Drug Delivery

Multidrug resistant cells

Drug efflux

p-gp


Outline

RGD definition

RGD-Integrin receptors interaction

Cilengitide®


SummaryChecklist

Future directions

Checklist

Criteria Non-Targeted Drug

delivery

cRGD cRGD-

Conjugates

Specificity

Less-Toxic

Low dosage

Summary

RGD Tumor Targeting is dependent on the design of the Conjugate;

Immobilization site-Binding enhanced on hydrophilic sites

Multimerization increase binding affinity

The linker bond stability - key to reduced toxicity

Mechanistic studies to explain RGD-conjugate mediated

antitumor agent redirection in MDR and effect of amide linked

RGD conjugate on the functional properties of mitochondria.

Designing new RGD conjugates that are more soluble by

incorporating a sugar moiety.

Future Directions

• Dr. Walker, Dr. Maleczka, Dr. Huang, Dr. Borhan

• Lab members-

Chelsea, Danielle, Dilini, Getrude, Irosha, Mark,

Udayanga.

• YCC Panel

• Friends-Camille, David, Philip, Salinda, Washington

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