Development and Validation of a Rapid LC-MS/MS Method for Quantifying Alvocidib: In Silico and In Vitro Metabolic Stability Estimation in Human Liver Microsomes

Abstract

Alvocidib (AVC; flavopiridol) is a potent cyclin-dependent kinase inhibitor used in patients with acute myeloid leukemia (AML). The FDA has approved orphan drug designation to AVC for treating patients with AML. In the current work, the in silico calculation of AVC metabolic lability was done using the P450 metabolism module of the StarDrop software package, that is expressed as a composite site lability (CSL). This was followed by establishing an LC-MS/MS analytical method for AVC estimation in human liver microsomes (HLMs) to assess metabolic stability. AVC and glasdegib (GSB), used as internal standards (IS), were separated utilizing a C18 column (reversed chromatography) with an isocratic mobile phase. The lower limit of quantification (LLOQ) was 5.0 ng/mL, revealing the sensitivity of the established LC-MS/MS analytical method that exhibited a linearity in the range 5-500 ng/mL in the HLMs matrix with correlation coefficient (R2 = 0.9995). The interday and intraday accuracy and precision of the established LC-MS/MS analytical method were -1.4% to 6.7% and -0.8% to 6.4%, respectively, confirming the reproducibility of the LC-MS/MS analytical method. The calculated metabolic stability parameters were intrinsic clearance (CLint) and in vitro half-life (t1/2) of AVC at 26.9 µL/min/mg and 25.8 min, respectively. The in silico results from the P450 metabolism model matched the results generated from in vitro metabolic incubations; therefore, the in silico software can be used to predict the metabolic stability of the drugs, saving time and resources. AVC exhibits a moderate extraction ratio, indicating reasonable in vivo bioavailability. The established chromatographic methodology was the first LC-MS/MS method designed for AVC estimation in HLMs matrix that was applied for AVC metabolic stability estimation.