Arg-Gly-Asp (RGD) Tumor Targeting Conjugates. Ruth Njeri Muchiri Michigan State University Department of Chemistry

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1 Arg-Gly-Asp (RGD) Tumor Targeting Conjugates Ruth Njeri Muchiri Michigan State University Department of Chemistry

2 Normal cell division Cancer Uncontrolled cell growth, invasion and metastases Cancer cell division DNA damage ( No repair) Cell suicide (apoptosis) Damaged cell Tumor

3 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

4 Cost of Cancer million USA population with cancer 2008 $895 billion (1.5% World s GDP) World s economic loss ,529, ,490 New cancer cases in USA New deaths in USA

5 Attempts Towards Cancer Treatment Surgery Chemotherapy Radiotherapy Agent Examples Alkylating agents Antimetabolite agents. Plant alkaloids and terpenoids Cyclophosphamide, Oxaliplatin, Carboplatin, Chlorambucil, Mechloethamine, Melphalan Folate antagonists, Methotrexate, Fludarabine, Cytarabine Doxorubicin, Vinca alkaloids, Taxanes, Camptothecin Antitumor antibiotics Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Mitoxantrone

6 Conventional Chemotherapy Treatment Pros Less mutilating Cons Non specific Neo-adjuvant treatment Toxic Preferred in metastasis cases Multiple drug resistance

7 % Survival Development on Chemotherapy One leg good, two legs better: Cocktail therapy VAC (Etoposide, Doxorubicin, Cyclophosphamide) CVM (Cisplatin, Vincristine, Methotrexate) MCP (Mitomycin-C, CCNU, Procarbazine) Patients with extensive lung tumor Patients with limited lung tumor High toxicity observed Bevacizumab + Gemcitabine in advanced pancreatic cancer Time (months) Pavlidis, A. N.; Tolis, T. F.; Briassoulis, E. C.; Sowla, A.; Klouvas, G. D. Lung Cancer 1991, 1, Kindler, H. L. et al. J. Clin. Oncol. 2005, 23,

8 Drug analogues Development on Chemotherapy Improved binding but low specificity Rapoport, N. Y.; Herron, J. N.; Pitt, W. G.; Pitina, L. J. Controlled Release 1999, 58, Bigioni, M.; Salvatorea, C. Biochemical Pharmacology 2001, 62,

9 Targeted Cancer Therapy Specific Reduced toxicity Maximization of drug effect Picture from:

10 Tumor Targeting Mechanism Cancer Cell Ojima, I. Acc. Chem. Res. 2008, 41,

11 Tumor Targeting Molecules Ligand Target Target location Diverse Diverse Antibodies Hyaluronic acid CD44 Layilin HARE CDC37 Epithelial Ovarian Colon Stomach Somastatin analogues SSTR1-5 Gastro-intestinal cells c(arg-gly-asp-x-y) α v β 3 integrin Endothelial cells Epithelial Glioblastoma cells Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I. Bioorg. Chem. 2005, 13, Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem. Int. Ed. 1997, 36,

12 Outline RGD definition RGD integrin receptors interaction Cilengitide (Merck) RGD conjugate design and effect on activity Future directions

13 Arg-Gly-Asp (RGD) G Natural RGD containing proteins Proteins Sequence Fibronectin (450 kda) AVTGRGDSPASSK Fibrinogen α-chain (72 kda) TSYNRGDSTFESK R D λ-receptor on E.coli (47.7 kda) Sindbis coat protein (15.5 kda) GSFGRGDSDEWTF GVGGRGDSGRPIM α-lytic protease ACMGRGDSGGSWI Testis specific basic protein KSRKRGDSADRNY Pierschbacher, M. D.; Ruoslahti. Nature 1984, 309, 30-33

14 RGD Receptor- α v β 3 Integrin Function Control angiogenesis, cell proliferation & migration PDB1JV2. α v β 3 RGD-peptide Mn 2+ Xiong, J. P. et al. Science 2002, 296,

15 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, Peishoff, E. C. et al. J. Med. Chem. 1992, 35,

16 RGD - α v β 3 Integrin Interaction Mn 2+ H-bonding Metal ion co-ordination PDB1JV2 Xiong, J. P. et al. Science 2002, 296,

17 Advantages of RGD Selective binding - ligand for tumor marker, α v β 3 integrin Has better cellular uptake Easy to synthesize - solid phase synthesis Application in tumor imaging Aguzzi, M.S. et al. Blood, 2004, 103, Liu, S. Bioconjugate Chem. 2009, 20,

18 Advantages of RGD Liu, S. Bioconjugate Chem. 2009, 20, Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25,

19 Outline RGD definition RGD integrin receptors interaction Cilengitide RGD conjugate design and effect on activity Future directions

20 Cilengitide Phase III clinical trial (Merck Germany) Active against glioblastoma (brain tumor) Induce apoptosis in tumors cells Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25,

21 Cilengitide Mode of Action Inhibit angiogenesis Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. Acc. Chem. Res. 2009, 42, Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25,

22 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, Reardon, A. D. et al. J. Clin. Oncol. 2008, 26,

23 crgd Conjugates:Old Chemotherapy in New Form Pozzo, A. D.et al. Bioorg & Med. Chem. 2010, 18,

24 crgd Conjugates Cilengitide modification Vachutinsky, Y. et al. J. Control. Release 2010, doi: /j.jconrel

25 Outline RGD definition RGD integrin receptors interaction Cilengitide RGD conjugate design and effect on activity Future directions

26 Effect of RGD Conjugate Design on Activity Multimerization Immobilization Sites of RGD Linker Bond

27 crgd Peptide Immobilization Sites PCLA (RGD)-PEG- PCLA PCLA -PEG- PCLA PCLA: poly(ε-caprolactone-co-lactide) PEG: poly(ethylene glycol) RGD-PCLA -PEG- PCLA-RGD Approved by FDA Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, Liu, C. B.; et al. J. Biomed. Mater. Res. B Appl. Biomater. 2007,

28 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,

29 crgd Immobilization on Hydrophilic Site PCLA-PEG-PCLA PCLA-(RGD)-PEG-PCLA Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31,

30 crgd Peptide Immobilization Sites RGD cell interaction PEG blocks PCLA blocks RGD peptides Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31,

31 Cell adhesion (%) Effect of crgd Immobilization Sites Thiazolyl blue assay Cell line used: Chondrocytes + fetal bovine serum 5 0 RGD(-PEG) RGD(-PCLA) Unmodified No RGD Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31,

32 crgd Conjugate Design and Effect on Activity Multimerization Immobilization of RGD Sites Hydrophilic site Enhance RGDintegrin interaction Linker Bond

33 crgd Conjugate Multimerization Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11,

34 Effective crgd Conjugate Multimerization Antitumor agent Antitumor agent Å Shuang, L. Bioconjugate Chem. 2009, 20,

35 Effective crgd Conjugate Multimerization α v β 3 Integrin 60 Å Xiong, J. P. et al. Science 2002, 296, PDB1JV2.

36 Scaffolds in RGD Conjugate Multimerization Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11,

37 Scaffolds in RGD Conjugate Multimerization Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11,

38 Multimerization Effect on RGD Conjugate Binding Competitive binding assay against 125 I-echistatin Immobilized α v β 3 U87MG cells No. of RGD K i [nm] No. of RGD K i [nm] c(rdg) 1 32 ± 6 c(rgd) ± 199 c(rgd) 2 13 ± 2 c(rgd) ± 57 c(rgd) ± 0.10 c(rgd) ± 48 c(rgd) ± 0.04 c(rgd) 8 46 ± 5 c(rgd) ± 0.04 c(rgd) ± 1 U87MG -Human glioblastoma cell line Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11,

39 RGD Conjugate Design and Effect on Activity Multimerization Increase affinity Immobilization of RGD Sites Hydrophilic site enhance RGDintegrin interaction Linker Bond

40 Linker Bond Stability Amide vs hydrazone Hydrolyzes slowly Hydrolyzes fast Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21,

41 Cellular Cleavage of Linker Bonds Amide bond Hydrazone bond Less toxic More toxic Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21,

42 Amide Linker Bond Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18,

43 Hydrazone Linker Bond Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18,

44 Amide vs Hydrazone Bond Linker Stability Entry ph 7.4, 37 o C t 1/2, h 1 >72 >72 2a 6 25 ph 5, 37 o C t 1/2, h 2b 5.8 <2 min Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18,

45 Plasma & Tumor drug content ng/ml plasma & ng/g tissue 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 Time (h) A2780 -Human ovarian cancer cell line Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, Amide bond linked conjugates - Less cytotoxic

46 Two Linker Bonds in RGD Conjugate System Drug RGD Drug RGD Bond cleavage Bond cleavage Free drug X Drug derivative 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,

47 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,

48 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,

49 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, Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31,

50 In Vitro Assay -Linker Bond Effect on MDR Construct IC 50 Values Sensitive cells Multi-drug resistant cells Free DOX 0.84 g/ml 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) μg/ml RGD4C-PEO-b-P(CL-Ami-DOX) 0.73 g/ml μg/ml MDA-435 /LCC6 WT & MDA-435 /LCC6 MDR (Breast tumor cell line) Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31,

51 Mice Survival % In Vivo Studies: Targeted Doxorubicin Doxorubicin sensitive breast tumor cell line 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,

52 Mice Survival % In Vivo Studies: Targeted Doxorubicin Multidrug resistant breast tumor cell line Days after tumor innoculation RGD-PEO-b-P(CL-Ami-DOX) Free DOX Control Acetal- PEO-b-P(CL-Ami-DOX) Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31,

53 Mice Survival % Mice Survival % Targeted DOX in Breast Tumor Cell Line Sensitive cells Multidrug resistant cells Days after Tumor inoculation RGD-PEO-b-P(CL-Hyd-DOX) Control Free DOX Acetal- PEO-b-P(CL-Hyd-DOX) 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,

54 Model of Redirected Drug Delivery Doxorubicin sensitive cells Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31,

55 Model of Redirected Drug Delivery Multidrug resistant cells Drug efflux p-gp Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31,

56 Outline RGD definition RGD-Integrin receptors interaction Cilengitide RGD conjugate design and effect on activity Summary Checklist Future directions

57 Checklist Criteria Non-Targeted Drug delivery crgd crgd- Conjugates Specificity Less-Toxic Low dosage

58 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

59 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.

60 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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