Involvement of multiple cytochrome P450 isoenzymes in drug interactions between ritonavir and direct oral anticoagulants.

Herein, we aimed to determine the significance of drug interactions (DIs) between ritonavir and direct oral anticoagulants (DOACs) and identify the involved cytochrome P450 (CYP) isoenzymes. Using an in vitro cocktail method with human liver microsomes (HLM), we observed that ritonavir strongly inhibited CYPs in the following order: CYP3A, CYP2C8, CYP2D6, CYP2C9, CYP2C19, CYP2B6, and CYP2J2 (IC50: 0.023-6.79 µM). The degree of CYP2J2 inhibition was inconclusive, given the substantial discrepancy between the HLM and human expression system. Selective inhibition of CYP3A decreased the O-demethylation of apixaban by only 13.4%, and the involvement of multiple CYP isoenzymes was suggested, all of which were inhibited by ritonavir. Multiple CYP isoenzymes contributed also to the metabolism of rivaroxaban. Replacement of the incubation medium with phosphate buffer instead of HEPES enhanced apixaban hydroxylation. On surveying the FDA Adverse Event Reporting System, we detected that the signal of the proportional reporting ratio of "death" and found increase for "hemoglobin decreased" (12.5-fold) and "procedural hemorrhage" (201.9-fold) on administering apixaban with ritonavir; these were far less significant for other CYP3A inhibitors. Overall, these findings suggest that co-administration of ritonavir-boosted drugs with DOACs may induce serious DIs owing to the simultaneous inhibition of multiple CYP isoenzymes.

Integrated Use of In Vitro and In Vivo Information for Comprehensive Prediction of Drug Interactions Due to Inhibition of Multiple CYP Isoenzymes.

BACKGROUND: Mechanistic static pharmacokinetic (MSPK) models are simple, have fewer data requirements, and have broader applicability; however, they cannot use in vitro information and cannot distinguish the contributions of multiple cytochrome P450 (CYP) isoenzymes and the hepatic and intestinal first-pass effects appropriately. We aimed to establish a new MSPK analysis framework for the comprehensive prediction of drug interactions (DIs) to overcome these disadvantages. METHODS: Drug interactions that occurred by inhibiting CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A in the liver and CYP3A in the intestine were simultaneously analyzed for 59 substrates and 35 inhibitors. As in vivo information, the observed changes in the area under the concentration-time curve (AUC) and elimination half-life (t1/2), hepatic availability, and urinary excretion ratio were used. As in vitro information, the fraction metabolized (fm) and the inhibition constant (Ki) were used. The contribution ratio (CR) and inhibition ratio (IR) for multiple clearance pathways and hypothetical volume (VHyp) were inferred using the Markov Chain Monte Carlo (MCMC) method. RESULT: Using in vivo information from 239 combinations and in vitro 172 fm and 344 Ki values, changes in AUC, and t1/2 were estimated for all 2065 combinations, wherein the AUC was estimated to be more than doubled for 602 combinations. Intake-dependent selective intestinal CYP3A inhibition by grapefruit juice has been suggested. By separating the intestinal contributions, DIs after intravenous dosing were also appropriately inferred. CONCLUSION: This framework would be a powerful tool for the reasonable management of various DIs based on all available in vitro and in vivo information.

Significance of Basal Membrane Permeability of Epithelial Cells in Predicting Intestinal Drug Absorption.

Drug absorption from the gastrointestinal tract is often restricted by efflux transport by P-glycoprotein (P-gp) and metabolism by CYP3A4. Both localize in the epithelial cells, and thus, their activities are directly affected by the intracellular drug concentration, which should be regulated by the ratio of permeability between apical (A) and basal (B) membranes. In this study, using Caco-2 cells with forced expression of CYP3A4, we assessed the transcellular permeation of A-to-B and B-to-A directions and the efflux from the preloaded cells to both sides of 12 representative P-gp or CYP3A4 substrate drugs and obtained the parameters for permeabilities, transport, metabolism, and unbound fraction in the enterocytes (fent) using simultaneous and dynamic model analysis. The membrane permeability ratios for B to A (RBA) and fent varied by 8.8-fold and by more than 3000-fold, respectively, among the drugs. The RBA values for digoxin, repaglinide, fexofenadine, and atorvastatin were greater than 1.0 (3.44, 2.39, 2.27, and 1.90, respectively) in the presence of a P-gp inhibitor, thus suggesting the potential involvement of transporters in the B membrane. The Michaelis constant for quinidine for P-gp transport was 0.077 µM for the intracellular unbound concentration. These parameters were used to predict overall intestinal availability (FAFG) by applying an intestinal pharmacokinetic model, advanced translocation model (ATOM), in which permeability of A and B membranes accounted separately. The model predicted changes in the absorption location for P-gp substrates according to its inhibition, and FAFG values of 10 of 12 drugs, including quinidine at varying doses, were explained appropriately. SIGNIFICANCE STATEMENT: Pharmacokinetics has improved predictability by identifying the molecular entities of metabolism and transport and by using mathematical models to appropriately describe drug concentrations at the locations where they act. However, analyses of intestinal absorption so far have not been able to accurately consider the concentrations in the epithelial cells where P-glycoprotein and CYP3A4 exert effects. In this study, the limitation was removed by measuring the apical and basal membrane permeability separately and then analyzing these values using new appropriate models.

Plausible drug interaction between cyclophosphamide and voriconazole via inhibition of CYP2B6.

The inhibitory activities of eight cytochrome P450 (CYP) isoenzymes for representative or suspected inhibitors of CYPs, including pesticides, were evaluated simultaneously using an in vitro cocktail incubation method to demonstrate the importance of systematic evaluation of CYP inhibitory risks in drug interaction (DI). Potent inhibition of CYP2B6 was noticeable for some azoles, including voriconazole. When voriconazole and cyclophosphamide were co-administered in mice, cyclophosphamide-induced alopecia and leukopenia were significantly suppressed by approximately 50% with increased blood concentrations of cyclophosphamide. The formation of an active metabolite of cyclophosphamide was suppressed effectively by voriconazole in the mouse liver microsomes. Surveys of adverse event reporting databases in Japan (JADER) and the U.S. (FAERS) showed that the proportional reporting ratios of neutropenia, hemorrhagic cystitis, and alopecia for cyclophosphamide, which is principally activated by CYP2B6 in humans, were mostly reduced, or tended to be reduced when azoles, including voriconazole, were prescribed in combination. It is highly likely that DIs between cyclophosphamide and azoles occur in the clinical setting. This study also suggests that more proper consideration of CYP2B6-mediated DIs is warranted. The combination of the in vitro cocktail method and a survey of adverse event reporting databases was a useful method to comprehensively detect pharmacokinetic DIs.