Abstract
Time dependent inhibition (TDI) represents a more severe form of inhibition for cytochrome P450 (CYP) enzymes relative to reversible inhibition because inactivated enzymes have to be regenerated to restore their function by de novo synthesis. Moreover, it has been observed that many in vitro TDI exhibiting drugs do not exhibit significant TDI in vivo. The overall goal of this project is to improve TDI predictions with in vitro data. To this end, in vivo TDI data were generated in rats for future in vitro‐in vivo correlation analysis.Drugs exhibiting TDI in vitro (podophyllotoxin (PPT), verapamil, paroxetine) and probe substrate for specific rat cyps (midazolam for cyp3a and desipramine for cyp2d1) were selected for pharmacokinetic (PK) studies. PK studies were conducted in singular jugular cannulated Sprague Dawley (SD) rats with selected CYP substrates. Rats were divided in two groups, control and inactivator treated group. Treated group was initially administered the inactivator and subsequently administered the probe substrate. Blood samples were withdrawn and analyzed for substrate concentration by LC‐MS/MS. Area under the curve (AUC(0‐∞)) ratios were compared between the two groups. Equilibrium dialysis was conducted to find the fraction unbound in plasma (fu) of substrate and the inactivator in five different matrices. PK studies were additionally conducted in SD rats for inactivator alone.The AUC(0‐∞) ratios of two groups were calculated by both non‐compartmental analysis and compartmental analysis. The midazolam AUC ratio for PPT treated vs control groups was found to be 1.18 ± 0.24. The midazolam AUC ratio for verapamil treated vs control groups was 1.26 ± 0.43. The fu for midazolam was 0.04, 0.016, 0.78, 0.43, 0.76 in rat plasma, human plasma, human liver microsome (HLM), rat liver microsome (RLM) and rat hepatocytes respectively.The AUC ratios indicate that the inactivators might not have a significant effect in vivo in rats. However an AUC ratio higher than 1 indicates some enzyme inactivation. In vitro studies are currently underway to conduct in vitro‐in vivo correlation of TDI.Support or Funding InformationThis work was supported by NIH grant GM1R01GM114369 to Ken Korzekwa and Swati Nagar.
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