ABSTRACT:In this study, induction and inhibition of rhesus monkey CYP3A64 versus human CYP3A4 were characterized in vitro, and the corresponding pharmacokinetic consequences were evaluated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced CYP3A64 mRNA (EC 50 ‫؍‬ 0.5 M; E max ‫؍‬ 6-fold) and midazolam (MDZ) 1-hydroxylase activity (EC 50

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ABSTRACT:Effects of rifampin on in vitro oxidative metabolism and in vivo pharmacokinetics of diclofenac (DF), a prototypic CYP2C9 marker substrate, were investigated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced DF 4-hydroxylase activity, with values for EC 50 of 0.2 to 0.4 M and E max of 2-to 5-fold over control. However, pretreatment with rifampin did not alter the pharmacokinetics of DF obtained after either i.v. or intrahepatic portal vein (i.pv.) administration of DF to monkeys. At the dose studied, plasma concentrations of rifampin reached 10 M, far exceeding the in vitro EC 50 values. Under similar treatment conditions, rifampin was previously shown to induce midazolam (MDZ) 1-hydroxylation in rhesus monkey hepatocytes (EC 50 and E max values ϳ0.2 M and ϳ2-to 3-fold, respectively), and markedly affected the in vivo pharmacokinetics of MDZ (>10-fold decreases in the i.pv. MDZ systemic exposure and its hepatic availability, F h ) in this animal species. In monkey liver microsomes, DF underwent, predominantly, glucuronidation, and, modestly, oxidation; the intrinsic clearance (CL int ‫؍‬ V max /K m ) value for the glucuronidation pathway accounted for >95% (versus about 75% in human liver microsomes) of the total (glucuronidation ؉ hydroxylation) intrinsic clearance value. In monkey hepatocytes, the hydroxylation also was a minor component (<10%) relative to the glucuronidation, supporting the liver microsomal finding. Collectively, our results suggest that the oxidative metabolism is not the major in vivo clearance mechanism of DF in either untreated or rifampin-treated monkeys and, conceivably, also in humans, raising a question about the utility of DF as an in vivo CYP2C9 probe.Induction of drug-metabolizing enzymes, especially the cytochrome P450 (P450) superfamily, by some drug molecules is a well known phenomenon and generally is undesirable since it can cause profound clinical effects, either by reducing therapeutic efficacy of drugs or enhancing toxicity from toxic or reactive metabolites (Thummel and Wilkinson, 1998). Accordingly, the potential for new chemical entities to cause P450 induction is usually assessed during lead optimization and identification in early drug discovery processes (Weaver, 2001;Worboys and Carlile, 2001). Currently, measurement of enzyme activities in cultured hepatocytes is the accepted and recommended method for studying P450 induction (LeCluyse, 2001;Bjornsson et al., 2003). However, systematic and quantitative extrapolations of such in vitro enzyme induction data to in vivo situations have not been extensively studied, and studies to date, including our recent investigation on in vitro-in vivo drug interactions in rhesus monkeys (Prueksaritanont et al., 2006), have been limited to CYP3A, the most abundant of all the human isoforms.In a quest to expand the database, we subsequently evaluated a relationship between in vitro-in vivo induction of CYP2C9 activity by rifampin, using diclofenac (DF) as a functional probe and the rhesus monkey as an anima...

After oral treatment (once daily) for 4 weeks with the potent bradykinin B 1 receptor antagonist methyl 3-chloro-3Ј-fluoro-4Ј-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1Ј-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was Ն2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.Cytochromes P450 (P450s) are a superfamily of enzymes involved in the oxidative metabolism of a variety of chemical xenobiotics, including pharmaceuticals, carcinogens, and environmental pollutants (Wrighton and Stevens, 1992). Altered catalytic activity or capacity of the enzymes, by inhibition or induction, is the major source of metabolism-based drug-drug interactions (Tanaka, 1998). The past decades have witnessed the advance in prediction of drug-drug interactions due to P450 inhibition (Brown et al., 2006), but pre-