Abstract

Time-dependent inhibition (TDI) of the cytochrome P450 (P450) family of enzymes is usually studied in human liver microsomes (HLM) by investigating whether the inhibitory potency is increased with increased incubation times. The presented work was initiated after a discrepancy was observed for the TDI of an important P450 enzyme, CYP3A4, during early studies of the investigational drug compound AZD3839 [(S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate]; TDI was detected using a regulatory method but not with an early screening method. We show here that the different solvents present in the respective studies, dimethyl sulfoxide (DMSO, screening method) versus methanol or water (regulatory method), were responsible for the different TDI results. We further demonstrate why DMSO, present at the levels of 0.2% and 0.5% in the incubations, masked the TDI effect. In addition to the TDI experiments performed in HLM, TDI studies with AZD3839 were performed in pooled human hepatocytes (Hhep) from different suppliers, using DMSO, methanol, or water. The results from these experiments show no TDI or attenuated TDI effect, depending on the supplier. Metabolite identification of the compound dissolved in DMSO, methanol, or water shows different profiles after incubations with the different systems (HLM or Hhep), which may explain the differences in the TDI outcomes. Thorough investigations of the biotransformation of AZD3839 have been performed to find the reactive pathway causing the TDI of CYP3A4, and are presented here. Our findings show that the in vitro risk profile for drug-drug interactions potential of AZD3839 is very much dependent on the chosen test system and the experimental conditions used.