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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% S
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Time (months)
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 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®
RGD conjugate design and effect on activity
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
Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.
Cilengitide® Mode of Action
Inhibit angiogenesis
Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. Acc. Chem. Res. 2009, 42, 969-980.
Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.
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
RGD conjugate design and effect on activity
Future directions
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
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
PCLA-PEG-PCLA
cRGD Immobilization on Hydrophilic Site
PCLA-PEG-PCLA
PCLA-(RGD)-PEG-PCLA
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
cRGD Peptide Immobilization Sites
RGD–cell interaction
PEG blocks
PCLA blocks
RGD peptides
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
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
ll a
dh
es
ion
(%
)
No RGD
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
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.
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 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
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
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
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
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
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
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.
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
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)
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
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Days after Tumor inoculation
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
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
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Days after tumor innoculation
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
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Sensitive cells
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ice
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Days after tumor innoculation
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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Days after Tumor inoculation
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
Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Model of Redirected Drug Delivery
Multidrug resistant cells
Drug efflux
p-gp
Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Outline
RGD definition
RGD-Integrin receptors interaction
Cilengitide®
RGD conjugate design and effect on activity
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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