Substrate-dependent effect of acetonitrile on human liver microsomal cytochrome P450 2C9 (CYP2C9) activity. Drug Metab Dispos 28:567–572.

This paper is available online at http://www.dmd.org ABSTRACT: Acetonitrile is an organic solvent commonly used to increase the solubility of lipophilic substrates for in vitro studies. In this study, we examined its effect on four reactions (diclofenac hydroxylation, tolbutamide methyl hydroxylation, phenytoin hydroxylation, and celecoxib methyl hydroxylation) catalyzed by human liver microsomes and by the recombinant CYP2C9. In both cases, the effect of acetonitrile on activity was found to be substrate-dependent. Namely, it increased diclofenac 4-hydroxylase and tolbutamide methyl hydroxylase activities, but decreased celecoxib methyl hydroxylase activity in a concentration-dependent manner. By comparison, hydroxylation of phenytoin was resistant to its effect. The presence of acetonitrile (3%, v/v) gave rise to a lower K m and a higher V max for diclofenac hydroxylase in both liver microsomes and recombinant CYP2C9 preparations (87 and 52% increase in V max /K m ratio, respectively). On the other hand, the inhibitory effect of the solvent (1%, v/v) toward celecoxib hydroxylase was characterized by a decrease in V max (human liver microsomes) or a change in both K m and V max (rCYP2C9), leading to 25 and 46% decrease in V max /K m for both systems. The results of this study underscore the need for careful evaluation of solvent effects before initiation of inhibition or cytochrome P450 reaction phenotyping studies

P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4

ABSTRACT The role of P-glycoprotein in secretion of indinavir metabolites produced by CYP3A4 was evaluated in Caco-2 cells expressing CYP3A4. Metabolism of indinavir by CYP3A4 expressing Caco-2 cells grown on filters resulted in the formation of Ndealkylation products (M5 and M6) and hydroxylation of indinavir, which were preferentially secreted into the apical compartment. Apical secretion of the metabolites was inhibited by cyclosporin A (CsA) with all three classes of metabolites showing similar sensitivity to CsA, suggesting that they are all secreted by the same pathway. M6 stimulated P-glycoprotein (Pgp)-ATPase activity in a concentration-dependent manner. This stimulation was inhibited by the Pgp-specific monoclonal antibody C219. A method was developed to specifically inhibit Pgp using the monoclonal antibody UIC2 to determine whether Pgp efflux accounts for a significant proportion of the apical secretion of indinavir metabolites. UIC2 recognizes an extracellular transient conformational epitope that is stabilized by some Pgp substrates or by ATP depletion. Incubation of Caco-2 cells with UIC2 in the presence of 1 M CsA resulted in 50 to 80% inhibition of Pgp-mediated vinblastine efflux, with no significant inhibition observed by UIC2 or CsA alone. Inhibition of Pgp in CYP3A4-expressing Caco-2 cells by UIC2 and 1 M CsA resulted in a significant decrease in the apical secretion of M6, M5, and OH-indinavir and an increase in the amount of the metabolites secreted in the basolateral compartment and retained in the cytosol. These results are consistent with a role of Pgp in elimination of CYP3A4-generated metabolites and indicate that even relatively polar metabolites may be secreted from the cell by Pgp. P-glycoprotein (Pgp) is an ATP-driven efflux pump capable of transporting a wide variety of structurally diverse compounds from the cell interior into the extracellular space In the intestine, Pgp is expressed on the brush-border membrane of enterocytes where it pumps compounds out of the cytosol into the lumen of the intestine. This activity runs countercurrent to the absorptive transport of drugs and has been proposed as a barrier to oral absorption of drugs In recent studies, we addressed the potential for synergy between CYP3A4 and Pgp by studying transport and metabolism of indinavir in Caco-2 cells induced to express CYP3A4 by culturing the cells with di-OH vit D

In vitro and in vivo CYP3A64 induction and inhibition studies in rhesus monkeys: a preclinical approach for CYP3A-mediated drug interaction studies. Drug Metab Dispos 34:1546–1555.

ABBREVIATIONS: MDZ, midazolam; 1'-OH MDZ, 1'-hydroxy midazolam; 4-OH MDZ, 4-hydroxy midazolam; AUC, area under plasma concentration-time curve; C max , peak plasma concentration; CL, plasma clearance; F h , hepatic availability; V dss , volume of distribution at steady-state; t 1/2 , half-life; i.pv., intra-hepatic portal vein; IS, internal standard; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry

vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters. Drug Metab. Dispos

ABSTRACT The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldoketoreductases, was a reversible time-dependent inhibitor (k inact = 0.12 minute 21 , K I = 6.1 mM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC 50 of 18 and 4.9 mM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions