CPX-351, a Dual-Drug Liposomal Formulation, Alleviates the Cardiotoxicity of Daunorubicin and Cytarabine to Induced Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Introduction: CPX-351 (US: Vyxeos ®; EU: Vyxeos ® Liposomal), a dual-drug liposomal encapsulation of daunorubicin + cytarabine at a synergistic 1:5 molar ratio, is a standard of care for therapy-related acute myeloid leukemia (AML) or AML with myelodysplastic-related changes. Daunorubicin, an anthracycline, is a known cardiotoxicant. While liposomal formulations have shown promise in mitigating these effects (O'Brien, et al. Ann Oncol 2004), this potential advantage is difficult to assess clinically due to numerous confounding factors. Here, we sought to develop and employ an in vitro model to study the relative toxicity of CPX-351 versus free daunorubicin + cytarabine applied as a combination at the same concentrations.Model development: The hiPSC-derived cardiomyocytes used in this study were derived from fibroblasts obtained from a single adult Caucasian female donor with no known diseases, induced into a pluripotent state, and then reprogrammed into cardiomyocytes (CDI Datasheet 2018). They present many of the characteristics of healthy human cardiac muscle cells, including gene and protein expression and rhythmic beating. During model development, the prototypical compounds doxorubicin and liposomal doxorubicin were used as proof-of-concept to establish the translational value of the model because clinical data regarding their relative cardiotoxicity were available (O'Brien, et al. Ann Oncol 2004). The model recapitulated the cumulative toxicity of free doxorubicin and differentiated between liposomal and free drug.The in vitro model was then applied to compare the relative cardiotoxicity of CPX-351 versus the combination of free daunorubicin + cytarabine (1:5), which were applied to the cardiomyocytes for 24 hours on Days 1, 3, and 5 at concentrations ranging from 0 to 1,000 ng/mL daunorubicin (0 to 2,273 ng/mL cytarabine). Bright-field microscopy imaging, lactate dehydrogenase (LDH) release (reflects cell membrane integrity), ATP content (indicates metabolic activity), cell beat rate (indicates cardiomyocyte function), and cardiac biomarkers (FABP3, cardiac troponin I, NT-proBNP, and BNP) were evaluated on Days 2, 4, 6, and 8.Results: Qualitative review of the microscopic images suggested single dose- and cumulative dose-dependent cytotoxicity of free daunorubicin + cytarabine, especially at the highest doses on Day 8. These observations were confirmed by dose-dependent increases in single-day or cumulative LDH activity in the cell media and decreases in ATP content starting on Day 4. Specifically, after a single 24-hour exposure, LDH activity (a measure of plasma membrane damage) was comparable to that of saline-treated cardiomyocytes for both the free-drug combination and CPX-351. However, after repeated exposure to the free-drug combination, increasing drug concentration was associated with increasing LDH activity in the media, peaking at levels ~12 times the saline control on Day 8. In contrast, the LDH activity following repeated equivalent doses of CPX-351 was only ~3 times the saline control. Conversely, ATP content (a measure of cellular metabolic fitness) gradually decreased between Day 2 and Day 8. The ATP depletion (Day 8 vs Day 2) was more profound in cardiomyocytes exposed to the free-drug combination (−98.1%) than in those exposed to equivalent concentrations of CPX-351 (−38.5%). Following repeated exposure to the free-drug combination, the cell beat rate demonstrated a biphasic response consisting of an initial increase followed by a significant slowing and sometimes arrest of beating, demonstrating significant injury; this effect was not observed following repeated exposure to CPX-351. Finally, the cardiac biomarkers FABP3 and cardiac troponin I were significantly released from cardiomyocytes exposed to the free-drug combination, but not from those exposed to CPX-351, even after 3 repeated exposures.Conclusions: Overall, at equivalent concentrations administered on the same schedule, CPX-351 was considerably less toxic to hiPSC-derived cardiomyocytes than the free-drug combination of daunorubicin + cytarabine, as measured by viability (imaging, LDH release), metabolic health (ATP content), function (beating rate), and the cardiac biomarkers FABP3 and cardiac troponin I. Clinical data are needed to confirm the reduced cardiotoxicity observed with CPX-351 versus free drugs in this in vitro model. DisclosuresFortin: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. LaCroix: StemoniX: Current Employment; Jazz Pharmaceuticals: Consultancy. Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cheung: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.