Short Communication Rifampin Induces the in Vitro Oxidative Metabolism, but Not the in Vivo Clearance of Diclofenac in Rhesus Monkeys

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 EC50 of 0.2 to 0.4 M and Emax of 2to 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 EC50 values. Under similar treatment conditions, rifampin was previously shown to induce midazolam (MDZ) 1 -hydroxylation in rhesus monkey hepatocytes (EC50 and Emax values 0.2 M and 2to 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, Fh) in this animal species. In monkey liver microsomes, DF underwent, predominantly, glucuronidation, and, modestly, oxidation; the intrinsic clearance (CLint Vmax/Km) 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 animal model. Rifampin is a known human CYP2C9 inducer (Bjornsson et al., 2003; Parkinson et al., 2004). DF has been commonly used as a probe substrate for measuring in vitro and in vivo activity of CYP2C9 in humans (Tucker et al., 2001; Bjornsson et al., 2003). The rhesus monkey, which has recently been demonstrated to be a good animal model for studying CYP3A-mediated interactions in humans (Prueksaritanont et al., 2006), was selected as an animal model, based on several similarities between rhesus and human CYP3A and 2C isoforms (Tang et al., 2005). This article describes apparently conflicting in vitro-in vivo results obtained from these studies, as well as results obtained subsequently, to help explain the observed discrepancies. The latter studies included in vitro metabolism of DF in monkey liver microsomes and hepatocytes. Materials and Methods Materials. DF, 4 -hydroxy (4 -OH) DF, midazolam (MDZ), 1 -hydroxy midazolam, diazepam, and rifampin were obtained from Sigma (St. Louis, MO). All other reagents were of analytical or HPLC grade. Rhesus monkey and human liver microsomes (pooled from 10–20 individuals) were purchased from Xenotech (Kansas City, KS). Fresh rhesus monkey hepatocytes were prepared in-house according to the method of Moldeús et al. (1978). In Vitro Induction Studies. The study was conducted using rhesus monkey hepatocytes (n 3) as described previously (Prueksaritanont et al., 2005, 2006). In brief, hepatocyte cultures were treated, in triplicate for each treatment, for 2 days with culture media containing various concentrations of rifampin or vehicle control (dimethyl sulfoxide, 0.1% v/v). At the end of the treatment (48 h), DF 4 -hydroxylase activities were measured, in triplicate, by incubating DF (250 M) with rhesus hepatocytes in 10 mM HEPES buffer, at 37°C, 95% humidity, and 5% CO2, for 20 min. Samples from each well were transferred to a 96-well plate containing an equal volume of acetonitrile, and Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.011643. ABBREVIATIONS: DF, diclofenac; 4 -OH DF, 4 -hydroxy diclofenac; MDZ, midazolam; AUC, area under the plasma concentration-time curve; Cmax, peak plasma concentration; CL, plasma clearance; Fh, hepatic availability; Vdss, volume of distribution at steady state; t1/2, half-life; i.pv., intrahepatic portal vein; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; UDPGA, UDP-glucuronic acid. 0090-9556/06/3411-1806–1810$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 11 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 11643/3150133 DMD 34:1806–1810, 2006 Printed in U.S.A. 1806 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from stored at 4°C until analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for DF and 4 -OH DF (Kumar et al., 2002). In Vivo Studies. All studies were reviewed and approved by the Merck Research Laboratories Institutional Animal Care and Use Committee. The in vivo studies were carried out in a crossover fashion, with at least a 2to 3-week washout period. Male rhesus monkeys (n 4, body weight 4–7 kg) were pretreated orally with either vehicle (PEG400) or rifampin (18 mg/kg, in PEG400), once daily for 5 days. On the morning of day 5, DF was administered via a cannulated intrahepatic portal vein (i.pv.) at 0.1 mg/kg/h for 4 h to monkeys, and blood samples were collected via a saphenous or femoral vein at predose, and at 60, 120, 150, 180, 200, 220, 240 (end of i.pv. infusion), 270, 300, 360, and 420 min after DF administration. Plasma samples were separated immediately at 10°C and kept frozen at 20°C. Additional studies also were conducted by i.v. administration of DF (0.1 mg/kg/h for 4 h) via a cephalic vein on day 5 in animals pretreated with the vehicle PEG400 and rifampin (18 mg/kg orally in PEG400) for 5 days. In Vitro Metabolism Studies. Studies to investigate the acyl glucuronidation pathway were conducted using rhesus monkey liver microsomes incubated with various concentrations of DF in the presence of UDPGA, as described previously (Kumar et al., 2002). For comparison purpose, a parallel study also was conducted using human liver microsomes. In brief, incubations (0.2-ml final volume) consisted of liver microsomes (0.05–0.1 mg/ml) previously preincubated with alamethicin (25 g/mg of microsomal protein) for 15 min, potassium phosphate buffer (100 mM pH 7.4), MgCl2 (10 mM), UDPGA (2 mM), and DF (0.5–150 M final concentrations). After an incubation time of 5 to 10 min, the reaction was quenched with 0.4 ml of acetonitrile containing 3% formic acid, and the supernatant was separated for analysis by LC-MS/MS (Kumar et al., 2002). Studies on kinetics of the oxidative metabolism of DF also were conducted in monkey and human liver microsomes, using conditions described previously by Kumar et al. (2002). Subsequent metabolism studies were conducted using rhesus monkey hepatocytes (0.5 10 cells/ml), incubated with 5 M DF, in 10 mM HEPES buffer with a final incubation volume of 0.2 ml. The concentration of 5 M is below Km values for both the glucuronidation and hydroxylation of DF estimated from the aforementioned liver microsomal studies. After incubation at 37°C, 95% humidity, and 5% CO2, the reaction was quenched, at various incubation times, with 0.2 ml of acetonitrile containing 3% formic acid, and the supernatant was separated for analysis by LC-MS/MS (Kumar et al., 2002). Analytical Procedures. Concentrations of DF and rifampin in plasma were analyzed using LC-MS/MS. Plasma samples were spiked with the respective internal standard (tolbutamide for DF and diazepam for rifampin), and proteins were precipitated with acetonitrile (acetonitrile/sample 2:1 v/v). After centrifugation, the supernatants were subjected directly to LC-MS/MS analysis, and the analytes were quantitated by LC-MS/MS in selective reaction monitoring mode using an AB/MDS SCIEX API 3000 tandem mass spectrometer (MDS Sciex, Concord, ON, Canada) interfaced with a SCIEX Turbo IonSpray source to a PerkinElmer Series 200 liquid chromatography system (PerkinElmer Life and Analytical Sciences, Boston, MA). Chromatography was accomplished on a Synergi Fusion-RP column (2.0 50 mm, 4 m; Phenomenex, Torrance, CA) for DF or a Betasil C18 column (2.1 50 mm, 5 m; Keystone, Bellefonte, PA) for rifampin. The mobile phase consisted of 90% acetonitrile in water (solvent B) and 10% acetonitrile in 0.02% acetic acid (pH 4.5; solvent A), and was delivered at a flow rate of 0.5 ml/min. The elution of DF was achieved by a linear increase of solvent B from 5% to 25% over 0.5 min, from 25% to 53% over 3.5 min, and 53% to 80% over 0.5 min. Equilibration was allowed for an additional 1.5 min, giving a total chromatographic run time of 6.0 min. The elution of rifampin was accomplished through a linear increase of solvent B from 0% to 90% over 0.6 min and held at that value for an additional 1.9 min. Equilibration was allowed for an additional 1.5 min, giving a total chromatographic run time of 4.0 min. Selective reaction monitoring experiments in the positive ionization mode were performed using a dwell time of 150 ms per transition to detect ion pairs at m/z 296/215 (DF), 271/155 (tolbutamide), 823/399 (rifampin), and 285/195 (diazepam). Calibration curves (5–5000 ng/ml) were prepared by plotting the appropriate peak area ratios against the concentrations of analyte in plasma using a weighted (1/x) quadratic regression. The concentration of the analyte in the unknown samples was determined by interpolation from the standard curve. For each analyte, standard curves showed satisfactory linearity and precision ( 15% coefficient of variation). Data Analysis. For the liver microsomal studies, apparent Km and Vmax values were estimated using a nonlinear regression program (Enfit from Biosoft, Ferguson, MO). The intrinsic clearance (CLint) values were estimated by dividing Vmax by Km. For the hepatocyte studies, the CLint values were calculated by dividing initial metabolite formation rates obtained during the first 10-min incubation (nmol/min/10 cells) by the substrate concentration used (5 M), which is below the Km values determined for the glucuronidation and oxidation reactions. The concentration of rifampin producing a 50% increase in DF 4 hydroxylase or MDZ 1 -hydroxylase activity (EC50) was determined using nonlinear regression analysis (PCNONLIN; Scientific Consulting, Cary, NC), as described previously (Prueksaritanont et al., 2006). The area under the plasma concentration-time profile (AUC0-last) was calculated from time 0 to the last detectable sampling time using the linear trapezoidal rule. The apparent terminal half-life (t1/2) was estimated by dividing 0.693 by the elimination rate constant determined using least-squares regression analysis of the log-linear portion of the DF plasma concentrationtime data. Plasma clearance (CL) values for DF were calculated as the i.v. dose divided by their corresponding AUC from time 0 to infinity (AUC0-inf). Hepatic availability (Fh) was estimated by dividing AUC obtained after i.pv. administration to that obtained by i.v. administration. Volume of distribution at steady state (Vdss) values were estimated by conventional moment analysis as i.v. dose multiplied by the first moment of the plasma concentration-time profile (AUMC) and divided by (AUC0-inf) . The peak plasma concentration (Cmax) was determined by observation.