We have previously demonstrated that the in vitro and in vivo activity of anticancer drug combinations is dependent on the drug:drug ratio, where some ratios act synergistically while others can interact additively or antagonistically. We examined here the influence of drug ratio on the in vitro and in vivo anti-tumor effects of Cyt:Daun, a drug combination used for the treatment of acute myeloid leukemia. The median-effect analysis method (Chou and Talalay, Adv Enzyme Regul, 22: 27-55, 1984) provided a quantitative measure of synergy for MTT cytotoxicity assays with Cyt:Daun molar ratios ranging from 10:1 to 1:10. This combination exhibited drug ratio and concentration dependent synergy when tested against P388 and L1210 leukemia cells in vitro. Subsequently, Cyt:Daun was encapsulation inside liposomes engineered to maintain the 5:1 molar ratio in the plasma compartment over 24h post i.v. injection. When administered i.v. to mice bearing ascitic P388 tumors, this fixed ratio liposome formulation (hereafter referred to as CPX-351) provided 90% long-term survivors (60 day), whereas the free drug cocktail at its MTD achieved only a modest increase in life span (22 day median survival time). CPX-351 was also therapeutically superior to individual free drugs and individual liposomal drugs administered at their respective MTDs. Similar results were obtained in the L1210 leukemia model. Quantitative comparisons of in vivo tumor cell kill for CPX-351 vs. individual liposomal drugs were performed based on tumor doubling time values and treatment-induced increases in survival. This analysis demonstrated that CPX-351 therapeutic activity was approximately 500-fold greater than predicted based on additive efficacy contributions of the individual liposomal drugs in the P388 tumor model, indicating strong in vivo synergy. These results demonstrate that fixing synergistic drug:drug ratios in carriers such as liposomes can lead to dramatic improvements of in vivo therapeutic activity. Furthermore, this approach provides an avenue whereby in vitro information on drug:drug interactions can be directly translated in vivo, thus facilitating the optimization of drug combinations based on efficacy in preclinical development.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]