Leukemia Research 34 (2010) 1214–1223 Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts Wah-Seng Lim, Paul G. Tardi, Nancy Dos Santos, Xiaowei Xie, Mannie Fan, Barry D. Liboiron, Xiaoping Huang, Troy O. Harasym, David Bermudes, Lawrence D. Mayer ∗ Celator Pharmaceuticals Corporation, 1779W. 75th Avenue, Vancouver, BC V6P 6P2, Canada article info Article history: Received 4 August 2009 Received in revised form 12 January 2010 Accepted 17 January 2010 Available online 6 February 2010 Keywords: Drug delivery Efficacy Synergy Pharmacodynamics Acute leukemia abstract The objective of this study was to examine the pharmacodynamic basis for the potent preclinical and clin- ical anti-leukemic activity of CPX-351, a nano-scale liposome formulation of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio. A bone marrow-engrafting CCRF-CEM leukemia model in Rag2-M mice was utilized to correlate the therapeutic and myelosuppressive properties of CPX-351 with bone marrow delivery and drug uptake in leukemia cells relative to normal bone marrow cell populations. When administered to mice bearing CCRF-CEM human leukemia xenografts, CPX-351 ablated bone mar- row (BM) leukemic cells to below detectable levels for multiple weeks, whereas the free-drug cocktail only transiently suppressed leukemia growth. In contrast to the activity against leukemia cells, CPX-351 and free-drug cocktail induced similar myelosuppression in non-tumor-bearing BM. In leukemia-laden BM, drug concentrations were markedly elevated for CPX-351 over free-drug cocktail and the first dose of CPX-351, but not free-drug cocktail, potentiated BM drug accumulation for subsequent doses. Con- focal fluorescence microscopy revealed that CPX-351 liposomes are taken up by CCRF-CEM cells and subsequently release drugs intracellularly. The improved in vivo efficacy of CPX-351 appears related to increased and prolonged exposure of synergistic cytarabine:daunorubicin ratios in BM, and the selective killing of leukemia may arise from direct liposome-leukemia cell interactions. These features may also have broader applicability in the treatment of other haematological malignancies. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Combination chemotherapy consisting of cytarabine and anthracycline (daunorubicin or idarubicin) has been one of the most effective induction treatments for acute myelogenous leukemia (AML) for over three decades [1]. Both drugs have also shown activity against acute lymphocytic leukemia (ALL) [2,3]. In a previous report we identified the presence of drug ratio-dependent synergy and antagonism for combinations of cytarabine and daunorubicin [5]. A liposomal formulation was developed to maintain the synergistic 5:1 molar drug ratio in vivo (referred to hereafter as CPX-351), and superior antitumor activity was observed for this formulation compared to other liposome-formulated drug ratios as well as free-drug cocktail in a wide range of leukemia models [4]. The rationale for develop- ing CPX-351 was based on the principle of ratiometric dosing, where nano-scale drug delivery vehicles such as liposomes could be utilized to maintain the desired drug:drug ratio in vivo for combinations that exhibit drug ratio-dependent synergy in vitro ∗ Corresponding author. Tel.: +1 604 675 2103; fax: +1 604 708 5858. E-mail address: lmayer@celatorpharma.com (L.D. Mayer). [5,6]. This approach facilitates the exposure of tumor cells to syn- ergistic drug ratios and ensures that antagonistic ratios which could compromise efficacy are avoided after injection, a feature not attainable with conventional free-drug formulations due to the independent and dissimilar pharmacokinetics of the individual agents [5,6]. Encouraging data from a Phase I clinical study in relapsed and refractory leukemia patients showed that CPX-351 treatment produced a significant number of complete remissions (CR) with very limited non-hematological toxicity including patients exhibit- ing high-risk cytogenetics as well as a lack of response to prior conventional 7 + 3 caytarabine and daunorubicin treatment [7]. Furthermore, analysis of plasma drug concentrations following CPX-351 treatment in the Phase I patients demonstrated that the cytarabine:daunorubicin ratio was maintained near the synergis- tic 5:1 molar ratio for 48 h. These clinical results were consistent with observations made in the preclinical evaluations that provided the basis for testing CPX-351 in a clinical setting [4,5]. CPX-351 is currently being tested against standard of care therapy, including conventional “7 + 3” cytarabine plus daunorubicin treatment in two 120-patient randomized Phase II clinical trials, one in newly diag- nosed elderly AML [38] and the other in ≤60 years old AML patients in first relapse. 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2010.01.015