CPX-351 exhibits potent and direct ex vivo cytotoxicity against AML blasts with enhanced efficacy for cells harboring the FLT3-ITD mutation

Highlights

• CPX-351 is a liposome formulation of cytarabine and daunorubicin in 5:1 molar ratio.

• CPX-351 has ex vivo activity against a broad range of hematologic malignancies.

• Consistent with clinical data, CPX-351 activity is retained in high-risk AML blasts.

• Ex vivo analysis of AML blasts with FLT3-ITD shows increased sensitivity to CPX-351.

• Ex vivo analysis identifies AML subgroups warranting further clinical investigation.

Abstract

Purpose

Identify AML patients most likely to respond to CPX-351, a nano-scale liposome formulation containing cytarabine and daunorubicin co-encapsulated at a 5:1 molar ratio.

Methods

We examined the ex vivo cytotoxic activity of CPX-351 against leukemic cells isolated from 53 AML patients and an additional 127 samples including acute lymphoblastic leukemia, myelodysplastic syndrome/myeloproliferative neoplasms, or chronic lymphocytic leukemia/lymphoma. We assessed activity with respect to common molecular lesions and used flow cytometry to assess CPX-351 cellular uptake.

Results

AML specimen sensitivity to CPX-351 was similar across conventional risk groups. FLT3-ITD cases were five-fold more sensitive to CPX-351. CPX-351 was active across other indications with nearly all cases exhibiting IC₅₀ values markedly lower than reported 72-h plasma drug concentration in patients receiving CPX-351. The range and distribution of CPX-351 IC₅₀ values were comparable for AML, CLL, and ALL, whereas MDS/MPN cases were less sensitive. CPX-351 uptake analysis revealed a correlation between uptake of CPX-351 and cytotoxic potency.

Conclusions

Our findings are consistent with clinical data, in which CPX-351 activity is retained in high-risk AML patients. Ex vivo analysis of cytotoxic potency may provide a means to identify specific AML subsets, such as FLT3-ITD, that benefit most from CPX-351 and warrant additional clinical evaluation.

Introduction

CPX-351 is a nano-scale (100 nm diameter) low-cholesterol liposome formulation containing cytarabine and daunorubicin co-encapsulated at a 5:1 molar ratio shown to be optimally synergistic both ex vivo and in vivo. Improvements in efficacy over the conventional free drug combination were observed in several preclinical studies [1], [2], [3], [4]. In a randomized controlled Phase 2 clinical trial of newly diagnosed acute myeloid leukemia (AML) in the elderly, CPX-351 produced superior response rates, and in secondary AML, superior event free survival (EFS) and overall survival (OS) [5]. A randomized phase 2 trial of CPX-351 vs investigator’s choice after first relapse demonstrated superior response rates, EFS and overall survival in patients with poor risk disease treated with CPX-351 [6]. Consequently, the aim of CPX-351 clinical development is to replace conventional cytarabine plus anthracycline therapy in several AML patient populations where it is considered standard of care.

The basis of the efficacy improvements observed with CPX-351 are attributable to 1) elevated cytarabine:daunorubicin concentrations that are maintained in the circulation at a synergistic ratio for prolonged periods of time (hence avoidance of antagonistic ratios [2], [7], [8], 2) the increased accumulation and persistence of CPX-351 in bone marrow [3], and 3) the selective accumulation and cytotoxicity of intact CPX-351 liposomes by leukemia cells compared to normal cells in the bone marrow [3], [9]. CPX-351 has been shown to be very effective at rapidly eliminating leukemia cells from the circulation and bone marrow in a high proportion of AML patients, including those who had failed to respond to standard 7 + 3 cytarabine:daunorubicin treatment just prior to CPX-351 therapy, as well as in advanced adult ALL and MDS patients [5], [7], [10].

Short-term, ex vivo cytotoxicity assays using freshly isolated cancer cells from patients with hematologic malignancies can be useful to provide an indication of the spectrum of activity of emerging therapeutic agents against hematological malignancies [11]. In this context, we have established a process of collecting and purifying circulating blast cells from freshly obtained blood samples in patients with a wide array of hematological malignancies, including AML, acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), and chronic lymphocytic leukemia (CLL) or other types of lymphoma for the purpose of testing anti-neoplastic activity [12], [13]. Using this approach, we have established a profile of anti-proliferative/cytotoxic activity for many new investigational agents, categorized not only based on the type of hematological malignancy, but also specific phenotypic profiles that are hallmarks of patient sub-types within individual malignancies [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Fifty and ninety percent growth inhibition concentrations (IC50 and IC90, respectively) can also be used to project whether treatment sensitivities reflect drug concentrations that are achievable in a clinical setting and if so, what proportion of samples within a given class of patients appear to be sufficiently sensitive to warrant testing in a clinical trial setting. Furthermore, correlations can be made between drug sensitivity and certain sub-population phenotypes in an attempt to identify biomarkers that could further enrich patient selection to enhance the likelihood of a clinical response.

Historically the evaluation of nano-particulate (e.g. liposome and nanoparticle) drug formulations has not been performed extensively ex vivo for their cytotoxicity against cancer cells. This is due to the fact that the prevailing design for such formulations has been for the delivery vehicles to act as an in situ drug infusion reservoir where the vehicles accumulate preferentially in sites of cancer growth and once there, slowly release free drug, which is then taken up by the cancer cells. Since drug release rates ex vivo are normally much slower than observed in vivo, the cytotoxic potency of encapsulated anticancer drugs are often orders of magnitude lower than observed for their free drug counterparts [24]. However, in the case of CPX-351, we have demonstrated both ex vivo and in vivo that human leukemia cells take up cytarabine and daunorubicin in liposome-encapsulated form via an energy dependent mechanism [3], [9]. Once taken up into vacuoles within the cytoplasm, processing of the liposomes generates bioavailable drug, leading to cell killing activity. This not only ensures delivery of the synergistic 5:1 molar ratio of cytarabine:daunorubicin but also leads to CPX-351 ex vivo cytotoxic potencies (based on IC50 values) that are comparable, and in some cases more potent, than those for the free drugs [9]. Consequently, examination of ex vivo cytotoxicity against fresh patient samples would be expected to provide relevant insight into the potential anti-neoplastic activity of CPX-351 in various patient populations.

Here we describe the ex vivo assessment of CPX-351 cytotoxicity against a wide range of blast types freshly harvested from hematological malignancy patients. We examine the range of CPX-351 cytotoxic potencies within major hematological malignancy groups such as AML and CLL as well as across different disease sub-types and correlate the results with plasma concentrations observed in patients and patient outcomes following conventional treatments.