ABSTRACT
Type II transmembrane serine proteases represent pharmacological targets for blocking entry and spread of influenza or coronaviruses. In this study, the depletion rates of the 3-amidinophenylalanine (3-APhA)-derived matriptase/TMPRSS2 inhibitors MI-463, MI-472, MI-485 or MI-1900 were determined by LC-MS/MS measurements over a period of 300 min using suspensions of rat, dog and cynomolgus monkey primary hepatocytes. From these in vitro pharmacokinetic (PK) experiments, intrinsic clearance values (Clint) were evaluated, and in vivo pharmacokinetic parameters (hepatic clearance, hepatic extraction ratio and bioavailability) were predicted. It was found that rat hepatocytes were the most active in the metabolism of 3-APhA derivatives (Clint 31.9-37.8 mL/min/kg), whereas dog and monkey cells displayed somewhat lower clearance of these compounds (Clint 6.6-26.7 mL/min/kg). These data support elucidation of important PK properties of anti-TMPRSS2/anti-matriptase 3-APhAs using mammalian hepatocyte models and thus contribute to the optimization of lead compounds.
ABSTRACT
Introduction: Hydroxychloroquine is used for the treatment of malaria, rheumatoid arthritis and lupus erythematosus. In 2020, hydroxychloroquine was also repurposed for the treatment of COVID-19. Although current evidence does not encourage the use of hydroxychloroquine to treat COVID-19, its therapeutic and prophylactic use against COVID-19 is still investigated in clinical trials. Despite being in clinical use for more than 60 years, its clinical pharmacology is not well understood. Hydroxychloroquine is metabolized into three active metabolites, but the key metabolizing enzymes have not been unambiguously identified. Moreover, little is known about the inhibitory effects of hydroxychloroquine on cytochrome P450 (CYP) enzymes. Objectives: This study aimed to investigate the CYP metabolic and inhibitory profile of hydroxychloroquine and its three metabolites in vitro. Methods: Hydroxychloroquine metabolism was studied in human liver microsomes (HLM) and recombinant CYP enzymes using substrate depletion and CYP-selective inhibitors. The inhibitory effects of hydroxychloroquine and its metabolites on nine CYP enzymes were also determined in HLM, using automated probe substrate cocktail assays. Results: Based on screening experiments, CYP3A4, CYP2D6 and CYP2C8 were the key enzymes involved in hydroxychloroquine metabolism in vitro. Although the intrinsic clearance (CLint) value of hydroxychloroquine depletion by recombinant CYP2D6 (0.87 μl/min/pmol) was more than 10-fold higher than that by CYP3A4 (0.075 μl/min/pmol), scaling of the recombinant data to HLM level resulted in similar CLint values for CYP2D6 and CYP3A4 (11 and 14 μl/min/mg) because of the much greater abundancy of CYP3A4 than that of CYP2D6. The scaled HLM CLint of CYP2C8 was 5.7 μl/min/mg. Data in HLM with CYPselective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in hydroxychloroquine metabolism, and a smaller contribution for CYP2C8. In CYP inhibition experiments, hydroxychloroquine and its three metabolites were direct CYP2D6 inhibitors (50% inhibitory concentration IC50 18-135 μM), while all metabolites were CYP3A timedependent inhibitors (IC50 12-117 μM, IC50 shift 2.2-3.4-fold). CYP2D6 inhibition explains the reported clinical drug-drug interaction between hydroxychloroquine and the CYP2D6 substrate metoprolol. The present data, together with the inhibitors' estimated intracellular hepatocyte concentrations, were successfully used in a static model to predict the fold increase in metoprolol AUC (predicted: 2.3- 2.8-fold, observed: 1.65-fold). Conclusion: The present study unambiguously demonstrates that hydroxychloroquine is metabolized mainly by CYP2D6, CYP3A4 and CYP2C8 in vitro. Moreover, hydroxychloroquine and its three metabolites are CYP2D6 reversible inhibitors, and hydroxychloroquine metabolites are CYP3A time-dependent inhibitors. The current data can be used in static and physiologically-based pharmacokinetic models to predict hydroxychloroquine drug-drug interaction potential, as shown with the successful prediction of hydroxychloroquine - metoprolol drug-drug interaction. (Table Presented).