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Arg-Gly-Asp (RGD) Tumor Targeting Conjugates Ruth Njeri Muchiri Michigan State University Department of Chemistry Cancer Uncontrolled cell growth, invasion and metastases Normal cell division DNA damage ( No repair) Cell suicide (apoptosis) Cancer cell division Damaged cell Tumor 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 USA population with cancer 2008 $895 billion (1.5% World’s GDP) World’s economic loss 2010 1,529,560 569,490 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 Surgery Chemotherapy Radiotherapy 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 http://www.immunotherapyforcancer.info http://www.medindia.net/index.htm Conventional Chemotherapy Treatment Pros Cons Less mutilating Non specific Neo-adjuvant treatment Toxic Preferred in metastasis cases Multiple drug resistance http://www.cancer.gov/ Development on Chemotherapy One leg good, two legs better: Cocktail therapy VAC (Etoposide, Doxorubicin, Cyclophosphamide) CVM (Cisplatin, Vincristine, Methotrexate) MCP (Mitomycin-C, CCNU, Procarbazine) 100 Patients with extensive lung tumor 90 80 High toxicity observed % Survival 70 Patients with limited lung tumor 60 50 40 30 Bevacizumab + Gemcitabine in advanced pancreatic cancer 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (months) 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. Development on Chemotherapy Drug analogues Improved binding but low specificity Rapoport, 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 Specific Reduced toxicity 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 Ligand Target Target location Diverse Diverse CD44 Layilin HARE CDC37 Epithelial Ovarian Colon Stomach Antibodies Hyaluronic acid Somastatin analogues c(Arg-Gly-Asp-x-y) SSTR1-5 αvβ3 integrin Gastro-intestinal cells Endothelial 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. Outline RGD definition RGD–integrin receptors interaction Cilengitide® (Merck) RGD conjugate design and effect on activity Future directions Arg-Gly-Asp (RGD) Natural RGD containing proteins G R D 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 Pierschbacher, M. D.; Ruoslahti. Nature 1984, 309, 30-33 RGD Receptor- αvβ3 Integrin Function Control angiogenesis, cell proliferation & migration PDB1JV2. αv β3 RGD-peptide Xiong, J. P. et al. Science 2002, 296,151-155. Mn2+ Cyclic RGD Arg-Gly-Asp-f-(N-Me)Val RGDxy 1FNF 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. RGD - αvβ3 Integrin Interaction Mn2+ H-bonding Metal ion co-ordination PDB1JV2 Xiong, J. P. et al. Science 2002, 296,151-155. 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 In vivo Studies Blood vessels in brain tumor Brain tumor cells inoculated in nude mice E1 - Control E2 - After Cilengitide treatment A Brain tumor B Phase II clinical trials Radiographic response to Cilengitide in glioblastoma patient A. Before treatment B. After treatment Tagai, T. et al. Int. J. Cancer 2002, 98, 690-697. Reardon, A. D. et al. J. Clin. Oncol. 2008, 26, 5610-5617. 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 Effect of RGD Conjugate Design on Activity Multimerization Immobilization Sites of RGD Linker Bond cRGD Peptide Immobilization Sites PCLA –(RGD)-PEG- PCLA PCLA -PEG- PCLA RGD-PCLA -PEG- PCLA-RGD PCLA: poly(ε-caprolactone-co-lactide) PEG: poly(ethylene glycol) Approved by FDA 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. cRGD Immobilization on Hydrophobic Site n=6 to 32 x=3 to 7 y=4 to 9 PCLA-PEG-PCLA RGD-PCLA-PEG-PCLA-RGD Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882. 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 30 Cell adhesion (%) 25 20 15 10 5 Thiazolyl blue assay Cell line used: Chondrocytes + fetal bovine serum 0 RGD(-PEG) RGD(-PCLA) Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882. Unmodified No RGD cRGD Conjugate Design and Effect on Activity Immobilization of RGD Sites Multimerization Hydrophilic site Enhance RGDintegrin interaction Linker Bond cRGD Conjugate Multimerization Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181. Effective cRGD Conjugate Multimerization Antitumor agent Antitumor agent 60-90 Å Shuang, L. Bioconjugate Chem. 2009, 20, 2199-2213 Effective cRGD Conjugate Multimerization αvβ3 Integrin 60 Å Xiong, J. P. et al. Science 2002, 296,151-155 PDB1JV2. 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 Competitive binding assay against 125I-echistatin Immobilized αvβ3 U87MG cells 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 U87MG -Human glioblastoma cell line Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168-2181. RGD Conjugate Design and Effect on Activity Multimerization Increase affinity Immobilization of RGD Sites Hydrophilic site enhance RGDintegrin interaction Linker Bond Linker Bond Stability Amide vs hydrazone Hydrolyzes slowly Hydrolyzes fast Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787. Cellular Cleavage of Linker Bonds Amide bond Less toxic Hydrazone bond More toxic Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787. Amide Linker Bond Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72. Hydrazone Linker Bond Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72. Amide vs Hydrazone Bond Linker Stability Entry pH 7.4, 37 oC t1/2, h pH 5, 37 oC t1/2, 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 Amide bond linked conjugate in plasma Free Drug in plasma of Mice treated with 1 Amide bond linked conjugate in tumor Free Drug in tumor of Mice treated with 1 Plasma & Tumor drug content ng/mL plasma & ng/g tissue 100000 10000 1000 100 10 1 0 5 10 15 20 25 30 Time (h) 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 Drug RGD Bond cleavage Drug RGD Bond cleavage Free drug Drug derivative X Bond stability at pH ~ 5 Drug-Y < Y-X X-Z < Z-Drug Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768. Two Linker Bonds in RGD Conjugate System X – Ester Y – Hydrazone Z - Amide Application in multidrug resistance (MDR) Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768. Two Linker Bonds in Redirected Drug Delivery Targets cell nucleus - Prone to Phospho-glycoprotein efflux Salerno, M.; Przewloka, T.; Fokt, I.; Priebe, W.; Garnier-Suillerot, A. Biochem. Pharmacol. 2002, 63, 1471-1479. Two Linker Bonds in Redirected Drug Delivery Targets Mitochondria -No Interaction with Phospho-glycoprotein (p-gp) 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 IC50 Values 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) RGD4C-PEO-b-P(CL-Ami-DOX) 0.73 g/mL 7.24 μg/mL 0.092 μg/mL MDA-435 /LCC6WT & MDA-435 /LCC6MDR (Breast tumor cell line) Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768. In Vivo Studies: Targeted Doxorubicin Doxorubicin sensitive breast tumor cell line Mice Survival % 100 80 60 40 20 0 0 10 20 30 40 50 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. In Vivo Studies: Targeted Doxorubicin Multidrug resistant breast tumor cell line Mice Survival % 100 80 60 40 20 0 0 10 20 30 40 Days after tumor innoculation RGD-PEO-b-P(CL-Ami-DOX) Control Free DOX Acetal- PEO-b-P(CL-Ami-DOX) Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768. Targeted DOX in Breast Tumor Cell Line Sensitive cells Multidrug resistant cells 100 80 Mice Survival % Mice Survival % 100 60 40 20 0 0 10 20 30 40 Days after Tumor inoculation RGD-PEO-b-P(CL-Hyd-DOX) Control Free DOX Acetal- PEO-b-P(CL-Hyd-DOX) 50 80 60 40 20 0 0 10 20 30 40 Days after tumor innoculation RGD-PEO-b-P(CL-Ami-DOX) Control Free DOX Acetal- PEO-b-P(CL-Ami-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 Summary Checklist Future directions Checklist Criteria Specificity Less-Toxic Low dosage Non-Targeted Drug cRGD cRGDdelivery Conjugates 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 Future Directions 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. • Dr. Walker, Dr. Maleczka, Dr. Huang, Dr. Borhan • Lab membersChelsea, Danielle, Dilini, Getrude, Irosha, Mark, Udayanga. • YCC Panel • Friends-Camille, David, Philip, Salinda, Washington