Validation of an LC-MS/MS Method for the Simultaneous Intracellular Quantification of the CDK4/6 Inhibitor Abemaciclib and the EZH2 Inhibitors GSK126 and Tazemetostat

<b>Background:</b> Inhibitors of cyclin-dependent kinases (CDKs) and epigenetic modifier enhancer of zeste homolog 2 (EZH2) have emerged as promising options in the pharmacotherapy of malignant tumors. Recently, we demonstrated synergistic antitumor effects of the CDK4/6 inhibitor abemaciclib and the EZH2 inhibitors GSK126 or tazemetostat in patient-derived glioblastoma (GBM) models. Importantly, all three drugs are substrates of the two most important plasma membrane multidrug transporters ABCB1 and ABCG2, with abemaciclib and tazemetostat also being inhibitors of these proteins. <b>Methods:</b> To investigate whether increased intracellular accumulation of either of the two drugs used in combination could have contributed to corresponding synergisms, we developed a simple LC-MS/MS method for simultaneous detection of the three substances in cell culture lysates. The method was validated in accordance with the current International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline M10 on bioanalytical method validation and study sample analysis. <b>Results:</b> All acceptance criteria were met. Subsequent analysis of intracellular drug concentrations confirmed increased cellular uptake of tazemetostat in the presence of abemaciclib in both GBM cell lines studied compared to single agent treatment. A comparable pattern was also observed for GSK126, but in only one of the two cell lines used. <b>Conclusions:</b> In conclusion, the observed synergistic antitumor effect could be partly due to increased intracellular accumulation, although this alone is certainly not sufficient to explain it. Overall, the developed method provides a valuable approach for characterizing interactions at the transport level and for predicting the efficiency of both anticancer substance classes in different cell lines.