Log Cell Kill 0 1 2 3 4 5 B Day Post Tumor Cell Inoculation 10 15 20 25 30 35 40 45 50 55 60 Tumor Weight (mg) 0 200 400 600 800 1000 1200 Saline Irino:Cis Cocktail 7:1 (34:2.1 mg/kg) Q4Dx3 X2 Irino:Cis Cocktail 7:1 (47:3 mg/kg) D1 (I:C) ; D5/D9 (I) X 2 CPX-571 34:2.1 mg/kg Q4Dx3 X2 Irinotecan and Cisplatin were encapsulated in liposomes that maintain the optimal ratio following systemic administration Recent in vitro and in vivo studies have indicated that drug ratios can have profound effects on synergistic and antagonistic interactions (Mayer et al., Mol Cancer Ther 2006 5: 1854-63). Dissimilar drug pharmacokinetics creates disparate drug exposure rates and therefore even if the drugs are injected at a synergistic ratio they are unlikely to remain at that ratio for a significant amount of time. Thus, dissimilar pharmacokinetics likely contributes to the inability to achieve drug synergy in vivo, and may even result in drug antagonism. Dual drug formulations with coordinated drug release is a newly developed application of liposomal delivery technology developed in order to deliver synergistic drug combinations at fixed drug ratios and avoid tumor exposure to antagonistic ratios (Tardi et al., Biochim Biophys Acta 2007 1768: 678- 87). Co-delivery and coordinated release of synergistic drug combinations facilitates maximum efficacy by maintaining synergy throughout the pharmacodynamic distribution. In this study, we systematically evaluated drug ratio-dependent synergy of irinotecan and cisplatin against a panel of 20 human and murine tumor cell lines in vitro. The tumor cell panel was enriched in lung tumor carcinomas and exhibited a range of irinotecan and cisplatin sensitivities. Following dose-ratio evaluations, we translated our in vitro findings in vivo by co-formulation of irinotecan and cisplatin in liposomes capable of maintaining the combination at a synergistic 7:1 molar ratio (CPX-571) following intravenous (IV) administration. The efficacy of the CPX-571 dual drug formulation was compared to that of the free drug cocktail as well as each individual liposome-encapsulated drug in a range of human tumor xenograft models implanted in nude mice. The liposomal dual drug formulation showed a high degree of antitumor activity and significantly improved efficacy over the unencapsulated cocktail as well as the individual liposomal drugs in all tumor models tested. Abstract C114 Drug Ratio-Dependent Antitumor Activity of Irinotecan and Cisplatin Combinations In Vitro and In Vivo P. G. Tardi, T. O. Harasym, S. A. Johnstone, N. Zisman, L. D. Mayer and D. Bermudes Celator Pharmaceuticals Corp., Vancouver, B.C., Canada 1 2 Median effect analysis of irinotecan:cisplatin molar ratios for synergy 1 CONCLUSIONS In vitro screening of irinotecan and cisplatin for median-effect combination indices (CI; Chou and Talalay 1984, Adv. Enzyme Reg. 22:27-55) based on molar drug ratios. CI screening a human tumor cell line panel results in a fixed-ratio matrix “heat map” shown for drug concentrations resulting in an 80% fraction of cells affected (ED = 80). Green (CI< 1), synergy; yellow (CI ~ 1), additivity; red (CI > 1), antagonism. Plasma levels of irinotecan and cisplatin were determined at five time points after i.v. administration in female CD-1 nude mice. A. Following injection of CPX-571 at an irinotecan dose of 60 μmoles/kg (41 mg/kg) and a cisplatin dose of 8.6 μmoles/kg (2.6 mg/kg), mouse plasma was isolated at the indicated times and assayed for irinotecan (●) and cisplatin (○) by HPLC. B. Based on the plasma drug levels, the molar ratio of irinotecan:cisplatin was calculated for each time point. All data points represent the mean values obtained from three mice per time point, and the error bars represent the SE. 2 3 Irinotecan:cisplatin combinations display strong drug ratio-dependent synergy in vitro Liposomal formulations of irinotecan:cisplatin maintained a synergistic ratio in plasma for at least 24 hours following systemic administration CPX-571 is superior when compared to free drug cocktails of irinotecan:cisplatin in a range of solid tumor xenograft models Log cell kill analysis indicates that CPX-571 exhibits greater than additive antitumor activity CPX-571 is a promising candidate for clinical evaluation INTRODUCTION DEVELOPMENT OF CPX-571 RESULTS Irinotecan:cisplatin formulations are also more efficacious than the free drug cocktail in HT29 human colon tumor and Capan-1 human pancreatic tumor xenograft models. CD-1 nude mice were inoculated s.c. with 2X10 6 H460 human NSCLC tumor cells. Mice (n=6) were treated with MTD doses of free drug combinations optimized for efficacy consisting of either two courses of alternating irinotecan:cisplatin (I:C) on day one followed by irinotecan only (I) on days 5 and 9, or two courses of co-administration of irinotecan and cisplatin on a Q4Dx3 schedule. The free drug schedules were compared with two courses of MTD doses of CPX-571 on a Q4Dx3 schedule. A. mean tumor growth curves with error bars representing the SE. B. log cell kill calculated from the mean tumor volume using the formula log 10 cell kill = (T - C)/ (3.32) (Td) where T – C (tumor growth delay) = days to reach a defined mass for the treated animals – days to reach the same mass for control animals; 3.32 is the log 10 unit constant; Td is the doubling time for the tumor in days determined using LabCat® (Innovative Programming Associates, Inc., Princeton, NJ). CPX-571 is more effective than free drug cocktails against H460 NSCLC CPX-571 has greater than additive antitumor activity against H69 SCLC Hours After Injection 0 5 10 15 20 25 Plasma Drug Levels (nmoles/ml) 0 200 400 600 800 1000 1200 Irinotecan Cisplatin Hours After Injection 0 5 10 15 20 25 Irinotecan to Cisplatin Ratio 0.1 1 10 100 A B Days Post Tumor Cell Inoculation 10 20 30 40 50 60 70 80 90 100 Tumor Weight (mg) 100 200 300 400 500 600 700 800 900 1000 4.0 304% 78 39:2.5 CPX-571 0.72 55% 14 2.5 Lipo- Cisplatin 1.95 149% 38 39 Lipo- Irinotecan Log 10 cell kill (LCK) % Growth Delay %(T-C)/C Growth Delay (Days) Dose (mg/kg) Treatment Free Drug D1 I:C D5,9 I X2 47:3 mg/kg Free Drug Q4Dx3 X2 34:2.1 mg/kg CPX-571 Q4Dx3 X2 34:2.1 mg/kg H69 human SCLC tumor cells (1X10 7 ) in growth factor reduced matrigel were implanted in female Foxn1 nude mice. Mice (n=6) were treated with MTD doses of CPX-571 on a Q7Dx3 schedule. The mean tumor growth curves (left) were used to estimate the log cell kill values shown in the table to the right. Mice received injections of (●) saline, (▲) 8.3 μmoles/kg (2.5 mg/kg) liposomal cisplatin, (■) 58 μmoles/kg (39 mg/kg) liposomal irinotecan or (▼) CPX-571 containing 58 and 8.3 μmoles/kg irinotecan and cisplatin. Both individual liposomal agents were therapeutically active and contributed to the overall antitumor activity. CPX-571 showed greater than additive antitumor activity when compared to the sum of dose matched individual agents. Day Post Tumor Cell Inoculation 20 30 40 50 60 70 Tumor Weight (mg) 100 1000 Day Post Tumor Cell Inoculation 20 40 60 80 100 Tumor Weight (mg) 1 10 100 1000 Effect of treatment on human tumor xenografts implanted in female Foxn1 nude mice (n=6) and staged until tumors reached 50 to 100 mg. A. HT29 human colon tumor xenograft model. B. Capan-1 human pancreatic xenograft model. Mice received Q7Dx3 injections of -●- saline, -■- free drug cocktail of irinotecan:cisplatin 42:3.4 mg/kg, or -▲- liposomal irinotecan:cisplatin 28:2.5 mg/kg. A B A Cell Lines Screened 1:64 1:32 1:16 1:8 1:4 1:2 1:1 2:1 4:1 8:1 16:1 32:1 64:1 LCC6 Breast 0.69 0.58 0.71 0.89 0.83 1.01 1.57 1.03 0.85 0.63 0.52 0.54 0.76 MCF-7 Breast 0.71 0.67 0.86 0.88 0.95 1.15 0.71 0.82 0.70 1.00 0.87 0.83 0.88 MB 231 Breast 1.02 1.01 0.90 0.90 0.70 0.87 1.54 1.34 0.90 1.10 0.76 1.40 0.61 HCT-116 Colon 0.38 0.36 0.37 0.33 0.62 0.84 1.50 1.73 9.40 0.71 0.76 0.82 1.06 Colon-26 Colon 1.22 1.67 1.40 1.16 1.00 1.21 1.04 1.38 1.10 1.03 1.31 1.25 0.82 HT-29 Colon 1.05 0.88 0.90 0.86 1.21 1.09 1.57 1.17 0.90 0.76 0.82 0.97 1.05 A549 Lung 0.67 0.61 0.67 0.56 0.44 0.39 0.44 1.65 1.31 0.49 0.37 0.41 0.49 H460 Lung 0.92 0.81 0.84 0.80 0.75 0.73 0.94 1.14 2.03 0.53 0.36 0.80 0.48 H322 Lung 0.47 0.52 0.68 0.96 0.57 1.28 1.24 0.95 0.73 0.54 0.44 0.52 0.54 H1299 Lung 1.07 1.08 1.12 1.09 0.76 0.98 3.01 2.57 1.83 0.65 0.64 1.49 0.82 H522 Lung 1.15 0.62 1.09 0.84 0.82 0.92 2.26 1.80 0.89 0.48 0.39 0.73 0.47 Ovcar-3 Ovarian 1.33 1.35 1.08 1.10 0.80 0.78 0.89 0.89 1.84 0.53 0.28 0.33 0.35 Ovcar-5 Ovarian 1.29 1.64 1.55 1.34 1.30 1.42 1.51 1.30 1.15 0.80 1.05 0.99 1.17 SK-OV-3 Ovarian 1.33 1.26 1.30 1.51 1.49 1.70 1.75 1.28 1.17 0.55 0.70 0.79 0.76 IGROV-1 Ovarian 1.16 1.16 1.16 1.00 0.95 0.95 1.23 1.20 1.16 0.77 0.78 0.71 0.75 A2780 Ovarian 0.93 0.94 0.81 0.75 0.80 0.81 0.87 0.70 0.81 0.81 1.20 1.03 1.25 Capan-1 Pancreatic 1.46 1.22 1.11 1.25 0.86 0.89 1.12 1.17 0.69 0.89 0.83 0.71 0.99 BXPC-3 Pancreatic 1.00 1.02 0.91 1.10 0.81 0.99 1.04 0.88 0.70 0.60 0.49 0.64 0.61 N87 Gastric 1.76 1.87 1.35 1.55 1.65 1.09 1.05 0.68 0.51 0.18 0.11 0.06 0.04 A253 H&N 0.85 0.84 0.81 0.92 0.76 0.63 0.79 0.78 0.86 0.83 0.90 0.82 0.84 Tumor CI @ ED80 3/6 CRs View publication stats View publication stats