APPLICAZIONI CLINICHE DEI FARMACI ANTIANGIOGENETICI Giampaolo Tortora Cattedra di Oncologia Medica UOC e Laboratori di Terapia molecolare dei tumori Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica Università di Napoli Federico II

Strategies to inhibit VEGF signalling Include monoclonal antibodies targeting VEGF-A (a) or the VEGF receptors (b, c). d,Chimaeric soluble receptors such as the ‘VEGF-trap’ (domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to a Fc fragment of an antibody) are also undergoing clinical development. e, Additional extracellular inhibitors are aptamers that bind the heparin-binding domain of VEGF165 (pegaptanib). A variety of small-molecule VEGF RTK inhibitors that inhibit ligand-dependent receptor autophosphorylation of VEGFR-1 and VEGFR-2 are being tested. Additional strategies to inhibit VEGF signalling include antisense and siRNA targeting VEGF-A or its receptors. Ferrara & Kerbel Nature 438: 967–974, 2005.

VEGF and VEGFR inhibitors under investigation Hicklin and Ellis, JCO 2005

VEGF isoforms recognised by hypervariable murine antibody fragment Bevacizumab VEGF isoforms recognised by hypervariable murine antibody fragment 93% human, 7% murine Recognizes all isoforms of VEGF (Kd = 8x10–10M) Terminal half-life 17–21 days No DLT as single agent inhibits all functions of the VEGF ligand: on vascular endothelial cells on non-endothelial cells (dendritic cells, monocytes) In preparation for cliical testing, the murine Ab used in the animal studies was humanized to minimize immunogenicity. As you can see from this cartoon, the entire Ab has been humanized with the exception of a small fraction in the variable region. The antibody binds all isoforms of VEGF A with high affinity and has terminal half life of 2-3 wks Human IgG-1

Conseguenze della iperespressione e ipersecrezione di VEGF Migrazione e proliferazione endoteliale Distorsione dell’architettura vascolare Aumento della permeabilità vascolare e della PIF Modulazione della risposta immune

Bevacizumab: effetti sulla vascolarizzazione tumorale Normale Anormale Normalizzata Riduce la pressione del liquido interstiziale e la densità microvascolare Incrementa il rilascio intratumorale dei farmaci La vascolarizzazione del tumore è immatura e dipende da VEGF per la sopravvivenza. Il blocco del VEGF dà luogo a un processo di normalizzazione nel quale la vascolarizzazione viene “sfrondata” dei vasi inefficienti mediante apoptosi delle cellule endoteliali, con il risultato di una maggiore organizzazione. L’inibizione del VEGF determina inoltre una riduzione della permeabilità vascolare, fa diminuire la pressione del liquido interstiziale e aumentare la pressione dell’ossigeno. In questo modo si migliora il rilascio delle sostanze citotossiche a livello del tumore ed è possibile che si aumenti l’efficacia della radioterapia. Una rapida riduzione della vascolarizzazione del tumore causata dalla morte delle cellule endoteliali può fare sì che la vascolarizzazione risulti inadeguata a sostenere la crescita neoplastica determinando, in definitiva, la quiescenza o la regressione del tumore. Modificato da Jain RK. Nat Med 2001; 7:987–9 Willett CG. et al. Nat Med 2004; 10:145–7 Tong R. et al. Cancer Res 2004; 64:3731–6$

Evidenza diretta degli effetti di Bevacizumab sulla vascolatura tumorale umana: studio fase I Patienti con ca rettale primario e non metastatico (n=6) Avastin 5 mg/kg + 5-FU i.c. + Radioterapia Avastin 5 mg/kg Chirurgia 2 settimane 3 volte ogni 2 settimane  perfusione sanguigna tumorale (40–44%, p<0.05)  volume sanguigno tumorale (16–39%, p<0.05)  densità microvascolare (25–59%, p<0.05)  pressione interstiziale (da 15.0 ± 2.0 a 4.0 ± 2.2 mm Hg, p<0.01) Fase 1 con dimostrazione dell’effetto di avastin sulla vascolarizzazione tumorale. Sangue/urine TAC funzionale/PET Endoscopia Biopsia tumorale Willett CG, et al. Nat Med 2004;10:145–7

Avastin aumenta la sopravvivenza in diversi tumori Ca colorettale 1a linea (NEJM 2004) Ca colorettale 2a linea (ASCO 2005) Ca polmonare 1a linea (ASCO 2005) Ca mammario 1a linea (ASCO 2005) Impatto di avastin in diversi setting e patologie neoplastiche. Il meccanismo d’azione è indipendente dal regime chemioterapico utilizzato?

E3200: progression-free survival 50% Incremento netto Giantonio BJ, et al. J Clin Oncol 2005;23(June 1 Suppl.):1s (Abstract 2)

E3200: overall survival Giantonio BJ, et al. J Clin Oncol 2005;23(June 1 Suppl.):1s (Abstract 2)

Planned trials in adjuvant colon cancer Studio n Tumore Stadio Trattamento BO17920 (Avant) 3450 Colon II (alto rischio) III FOLFOX4 vs FOLFOX4 + Avastin XELOX + Avastin NSABP C - 08 2700 FOLFOX6 ± Avastin E5205 3125 rischio QUAS AR 2 1900 III Xeloda ± Avastin Based on the activity of VEGF and its role in angiogenesis from the earliest stages of cancer development, the greatest effects of Avastin might be expected when it is used to treat patients with small tumours or minimal residual disease. By blocking further vascularisation of such tumours, Avastin would inhibit further growth and metastasis, improving disease control. In addition to the trials in the metastatic setting, plans to investigate the effectiveness of Avastin as adjuvant therapy for colon cancer are well underway. These trials incorporate a control arm of FOLFOX as the new standard adjuvant therapy. >11.000

Binding del VEGF ai recettori VEGFR-3/Flt-4 VEGFR-1/Flt-1 VEGFR-2/KDR LYMPHANGIOGENESIS ANGIOGENESIS

Key receptor tyrosine kinases and selective inhibitors EGFR gefitinib (IRESSA™) erlotinib (Tarceva™) ZD6474 AEE788 VEGFR vatalanib AZD2171 SU11248 sorafenib Bcr-Abl imatinib (Glivec™) c-Kit AZD2171 imatinib SU11248 PDGFR sorafenib

Agents affecting all VEGFRs AZD2171 LY317615 (Enzastaurin) CEP7055 GW786024

AZD2171 AZD2171 VEGFR3 (Flt-4) VEGFR1 (Flt-1) VEGFR2 (KDR) AZD2171 is an oral therapy with potential application in multiple tumor types AZD2171 has activity against VEGF receptors 1, 2 & 3- No activity on EGFR Phase I clinical studies in refractory solid tumors underway Manageable toxicity in early phase I

PKC-b and the Proposed Action of Enzastaurin on Angiogenesis and Apoptosis Acyclic indolylmaleimide competing with the ATP binding site VEGF ENZASTAURIN Receptor DAG Tumor invasion Angiogenesis IP3/Ca2+ Apoptosis PKC-b AKT COX2 GSK3b Caspase 9 IL-8 IL-6 Protein translation Activation mRNA

Combination of 2 selective inhibitors approach

Combined blockade of EGFR and VEGF Erlotinib Cetuximab, etc Bevacizumab etc. EGFR Endothelial cells Cancer cells VEGF Angiogenesis Cell Proliferation Tortora et al. 2004

RCC (59 pts.): 47% Responses (including MR) and 39% SD; 3 Phase II studies of Bevacizumab and Erlotinib in Patients with Renal Carcinoma (RCC), Breast cancer and NSCLC RCC (59 pts.): 47% Responses (including MR) and 39% SD; 76% 1 year Overall survival (Hainsworth et al., ASCO 2004; Spigel et al., ASCO 2005). NSCLC: 85% Disease control (including 20% PR); 52% 1 year Overall survival and 7 mo. PFS (Sandler et al., ASCO 2005). Breast Cancer: 33% Disease control (including PR) in heavily pretreated patients. Early data (Rugo et al., ASCO 2005). Hainsworth JD, et al. J Clin Oncol 2004;22(July 15 Suppl.): Abstract 4502 Sandler AB, et al. J Clin Oncol 2004;22(July 15 Suppl.) Abstract 2000 Rugo H et al., J Clin Oncol 2004;22(July 15 Suppl.)

Indirect Comparison of the Efficacy Results in BOND-1 and BOND-2   BOND-1 C225 BOND-2 C225 + BEV BOND-1 C225 + CPT BOND-2 C225 + CPT + BEV No. patients 111 35 218 39 Prev oxal (%) 64 89 62 87 RR (%) 11 23 38 TTP (months) 1.5 6.9 4.1 8.5 Median OS (months) -- 8.6 While Modified from Saltz et al., ASCO 2005

Multitargeted agents approach

Multitargeted agents affecting VEGF-Rs and EGFR, PDGF-Rs etc. ZD6474 (VEGF-R2 + EGFR + RET) AE778 (VEGF-R2 + EGFR) SU11248 (VEGFRs + PDGF-Rs+ c-Kit) Sorafenib (VEGFRs + PDGF-Rs + MAPK + Erk + c-Kit) PTK787 (VEGFRs + PDGF-Rs)

ZD6474 inhibits KDR and EGFR TGF EGFR ZD6474 KDR RET Endothelial cells Cancer cells VEGF Angiogenesis Tortora & Ciardiello 2003 Carlomagno et al, Cancer Res. 2002 Ciardiello et al., Clin Cancer Res. 2003 Ciardiello et al., Clin Cancer Res. 2005 Damiano et al., Clin Cancer Res 2005 Cell Proliferation

Progression-free survival (days) Randomized Phase II trial of ZD6474 plus docetaxel in patients with NSCLC. Primary endpoint : PFS 1.00 0.75 0.50 0.25 Progression-free survival (days) 50 100 150 200 250 300 350 400 Probability of remaining progression-free Median progression-free survival: Placebo + docetaxel = 12.0 weeks ZD6474 100 mg + docetaxel = 18.7 weeks ZD6474 300 mg + docetaxel = 17.0 weeks Objective responses Placebo+D = 12% ZD 100 +D = 26% ZD 300 +D = 18% DATI ANCORA IMMATURI PER OS

Targeting VEGF and EGFR: AEE788 Phase I trial dose-dependent inhibition of EGFR signalling in skin and tumour observed most common adverse events were diarrhea (67%), fatigue/asthenia (51%), anorexia (49%), rash (43%), nausea (42%) and vomiting (28%) DLT (diarrhea) dose levels defined at 500 and 550 mg 2Baselga J, et al. J Clin Oncol 2005;23(June 1 Suppl.):198s (Abstract 3028) 3Martinelli E, et al. J Clin Oncol 2005;23(June 1 Suppl.):201s (Abstract 3039)

The endothelial cell-pericyte network of signals Pericytes protect endothelial cells from apoptosis and overexpress PDGF-R PDGF-R is overexpressed in many tumors PDGF-R and VEGF cooperate Nature Review Cancer

SU11248 is active in different tumors Activity observed in leukemia Active in sarcomas Active in GIST (including those resistant to imatinib)

SU11248 in mRCC Activity Versus Other Second-Line Agents No. ORR, % TTP, mo SU11248 63 33 8.3 Interleukin-2* 65 5 NA Interferon-a* 48 2 Avastin (high dose)† 39 10 4.8 Placebo† 40 2.5 Multiple agents in phase II trials‡ 137 3 2.9 As illustrated in this slide, few patients with mRCC respond to second-line therapy, and time to tumor progression is short. Compared with these agents, SU11248 produced responses in one third of the patients, and the time to tumor progression was prolonged (8.3 month median). *Escudier et al. J Clin Oncol. 1999;17:2039-2043; †Yang et al. N Engl J Med. 2003;349:427-434; ‡Motzer et al. J Clin Oncol. 2004; 22:454-463.

Phase II study of SU11248 in MBC 6 weeks cycle (50 mg/day for 28 d. and 2 weeks rest). 64 pts enrolled (80% HER-2 negative/unknown). The majority with multiple visceral sites. Heavily pretreated (several previous CT regimens, also in adjuvant setting). Asthenia and diarrhea major grade 2 toxicities. 40% grade 3 neutropenia. 51 evaluable for responses. PR: 7 (14%); SD > 6 mo: 1 (2%). Study is ongoing. No obvious correlation between response and ER or HER-2 status. Miller et al., ASCO 2005

PTK/ZK: A multi-VEGF receptor tyrosine kinase inhibitor (Vatalanib) Formula: C24H21N4Cl MW = 346.82 Complete inhibitor of the VEGF receptor tyrosine kinases VEGFR1(FLT-1), VEGFR2 (KDR) and VEGFR3 (FLT-4). It also inhibits PDGF-R. Well tolerated up to 1250 mg/day (phase III dose, used up to 15 mo) Rapidly absorbed (1 to 2.5 hours), T1/2: 3-6 hrs Renal metabolism The purpose of this slide is to introduce PTK/ZK as a multi-VEGF receptor inhibitor PTK/ZK is multi-VEGF receptor inhibitor that completely inhibits the activation of VEGF receptors: VEGFR-1, VEGFR-2, and VEGFR-31,2 PTK/ZK also inhibits the PDGF receptor which plays a role in blood vessel stabilization PTK/ZK is being codeveloped by Novartis Pharmaceuticals Corporation AG and Schering AG Berlin, Germany 1. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 2000;60:2178-2189. 2. Bold G, Altmann KH, Frei J, et al. New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis. J Med Chem. 2000;43(12):2310-23.

Over 700 pts treated up to date ONGOING AND PLANNED PHASE IIICONFIRM STUDIES Chemonaive CRC FOLFOX + PTK Over 700 pts treated up to date in combination with CT CPT-11/FU resistant CRC FOLFOX + PTK

BAY 43-9006 (Sorafenib) Bisaryl urea, multiple targeted inhibitor. Cl F 3 C N H O Bisaryl urea, multiple targeted inhibitor. Inhibits B-Raf-1 kinase (including the mutated form) with IC50 of 6 nM, MAPK, ERK. Inhibits also endothelial cells and VEGFR2, VEGFR-3, FLT-3, PDGFR, c-Kit. Raf is probably important in endothelial cells and double targeting (Raf and VEGFR-2) may be critical. BAY 43-9006 has clear activity in several tumor types and is going on to phase II studies.