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SHORT COMMUNICATION

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

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania

(Received July 2, 2006; accepted August 22, 2006)

	Abstract

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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 µM 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 µM, 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 E_max values 0.2 µM and 2- 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.

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

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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 µM) 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 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 2- to 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 µg/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 µM 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 x 10⁶ cells/ml), incubated with 5 µM DF, in 10 mM HEPES buffer with a final incubation volume of 0.2 ml. The concentration of 5 µM is below K_m 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 x 50 mm, 4 µm; Phenomenex, Torrance, CA) for DF or a Betasil C₁₈ column (2.1 x 50 mm, 5 µm; 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).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Effect of various concentrations of rifampin on DF 4'-hydroxylase activity in rhesus monkey hepatocytes from three different individuals. Results are mean of triplicate determinations. DF 4'-hydroxylase activities were determined using 250 µM DF. Rhesus monkey hepatocytes were treated with vehicle (dimethyl sulfoxide, control) or rifampin for 48 h before enzyme activity determinations.

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 (AUC_0-last) was calculated from time 0 to the last detectable sampling time using the linear trapezoidal rule. The apparent terminal half-life (t_1/2) 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 (AUC_0-inf).

Hepatic availability (F_h) was estimated by dividing AUC obtained after i.pv. administration to that obtained by i.v. administration. Volume of distribution at steady state (V_dss) 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 (AUC_0-inf)². The peak plasma concentration (C_max) was determined by observation.

	Results

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In Vitro Induction Studies. In rhesus monkey hepatocytes, rifampin markedly induced the formation of 4'-OH DF in hepatocyte preparations from three individual monkeys; values for EC₅₀ ranged from 0.2 to 0.4 µM, whereas those for E_max were about 2- to 5-fold over control values (Fig. 1). A comparable magnitude of induction (EC₅₀ and E_max values of 0.2 µM and 2- to 3-fold over control values, respectively) was observed with MDZ 1'-hydroxylation in two of the same subjects (Prueksaritanont et al., 2006). In three additional subjects, approximately 2- to 3-fold increases in both enzyme activities also were obtained with 10 µM rifampin (data not shown).

In Vivo Studies. Upon i.v. administration of DF (0.1 mg/kg/h), plasma concentrations of DF appeared to reach a steady-state level of about 0.8 µM at about 2 h postdose and declined relatively quickly after the infusion stopped (Fig. 2A), with t_1/2 of about 1 to 2 h (Table 1). The CL of DF was moderate, with values of 10 ml/min/kg (Table 1). Treatment with rifampin (18 mg/kg) for 5 days did not significantly affect the i.v. plasma profiles (Fig. 2A) or pharmacokinetic parameters of DF; values for AUC, V_dss, CL, and t_1/2 of DF were comparable between the control and rifampin-treated animals (Table 1). Effects of rifampin also were determined after i.pv. administration of DF to monkeys; this route of administration provides advantages of maximizing the magnitude of interactions resulting from changes in both hepatic first pass and systemic clearance (Wilkinson, 1987; Prueksaritanont et al., 2006). As shown in Fig. 2B and Table 1, rifampin also minimally affected the i.pv. plasma levels of DF and its corresponding pharmacokinetic parameters; plasma levels of DF, although significantly lower than those observed after i.v. administration, were comparable between the control and rifampin-treated animals. The hepatic availability, F_h, of DF was estimated to be about 30% in monkeys with or without rifampin pretreatment (Table 1).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Mean plasma concentration-time profiles of DF after i.v. (A) or i.pv. (B) administration of DF (0.1 mg/kg/h for 4 h) to rhesus monkeys pretreated with vehicle or rifampin (18 mg/kg p.o. once daily) for 5 days. Results are mean ± S.D., n = 4.

In rifampin-pretreated monkeys, rifampin plasma concentrations were maintained above 5 µM over a period of 6 h after the last dose (5th dosing day); values for C_max were about 16 and 10 µM, and AUC_0-last values were 67 and 42 µM · h in the animals given i.v. and i.pv. administration of DF, respectively (Table 1).

In Vitro Metabolism Studies. Liver microsomal studies showed that DF underwent extensive glucuronidation in monkeys (Fig. 3A), with values for V_max, K_m, and CL_int (V_max/K_m) of 7.5 nmol/min/mg, 12.2 µM, and 613 µl/min/mg, respectively. In contrast, the formation of 4'-OH DF was relatively minor (Fig. 3A); the V_max, K_m, and CL_int values were 1.2 nmol/min/mg, 72.2 µM, and 16 µl/min/mg, respectively. Accordingly, in monkey liver microsomes, the glucuronidation pathway represented about 95% of the total intrinsic clearances. In a parallel study using human liver microsomes, the glucuronidation pathway accounted for about 75% of the total intrinsic clearance. Values for the V_max, K_m, and CL_int were 11.1 nmol/min/mg, 21.5 µM, and 517 µl/min/mg for the glucuronidation, and 1.9 nmol/min/mg, 10.3 µM, and 184 µl/min/mg for the oxidation pathways, respectively. These human liver microsomal results were in a range comparable to those reported previously by Kumar et al. (2002).

View larger version (15K): [in this window] [in a new window]   FIG. 3. Kinetics of 4'-OH DF and DF acyl glucuronide formation in rhesus monkey liver microsomes (A), and time profiles of DF metabolism (DF disappearance and formation of its metabolites) in rhesus monkey hepatocytes (B). Results shown in A are mean ± S.D. of triplicate determinations. Formation of 4'-OH DF and DF acyl glucuronide was determined after a 5-min (for glucuronidation) or 20-min (for oxidation) incubation with various concentrations of DF and liver microsomes (0.2 mg/ml for oxidation and 0.05 mg/ml for glucuronidation) in the presence of 1 mM NADPH and 5 mM UDPGA, respectively. Results shown in B are the mean of two individual subjects, each determined in triplicate, using 5 µM DF and rhesus monkey hepatocytes (0.5 x106 cells/ml).

	Discussion

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In this study, we examined the in vivo consequence of CYP2C9 induction observed in vitro by rifampin, using DF as a functional probe. Comparative in vitro metabolism studies of DF in rhesus monkey and human liver microsomes, including immunoinhibition, chemical inhibition, and activities of recombinant P450s, consistently suggested the major contribution of CYP2C to the major oxidative pathway 4'-hydroxylation, and of CYP3A to the minor 5-hydroxylation in both species (Leemann et al., 1993; Shen et al., 1999; Tang et al., 2005). However, we observed an apparent disconnect between the in vitro and in vivo induction of DF metabolism in rhesus monkeys. In vitro, rifampin was a potent inducer of rhesus CYP2C; both EC₅₀ and E_max values for the induction of 4'-OH DF formation in rhesus monkey hepatocytes were comparable to the respective values observed in this species for MDZ 1'-hydroxylase activity, a CYP3A marker (Prueksaritanont et al., 2006). In contrast to this in vitro observation, subsequent pharmacokinetic interaction studies in rhesus monkeys revealed that rifampin was not an inducer of DF clearance in vivo.

This apparent lack of in vivo effect of rifampin on the DF pharmacokinetics in the present monkey study is not likely due to an inadequate systemic exposure to rifampin. At the dose used in our in vivo studies, C_max values for rifampin were similar to those reported after a therapeutic dose in humans (Sanofi-Aventis, 2003) and well exceeded its in vitro EC₅₀ estimate for the induction of DF 4'-hydroxylase activity in monkey hepatocytes. In addition, rifampin, at comparable plasma levels, has been shown to cause a marked decrease (>10-fold) in the MDZ systemic exposure after i.pv. administration of MDZ to monkeys (Prueksaritanont et al., 2006), and after its oral administration to humans (Niemi et al., 2003). Rifampin is equally potent as an inducer of CYP3A-mediated MDZ 1'-hydroxylase activity in both rhesus monkey and human hepatocytes (Prueksaritanont et al., 2006).

Not only the present negative in vivo interaction results between DF and rifampin, but also the earlier positive observations between rifampin and MDZ ruled out the possibility that the CYP3A-mediated oxidative metabolism is a major determinant of the in vivo clearance of DF in monkeys, as suggested by Tang et al. (1999). In contrast to earlier in vivo metabolism data which seemed to suggest that the oxidative metabolism of DF might account for the majority of in vivo clearance (Stierlin and Faigle, 1979; Stierlin et al., 1979), Kumar et al. (2002) have recently proposed that the direct glucuronidation of DF is a more important component to the in vivo clearance than the oxidation pathway in rats, dogs, and humans. This proposal was made based primarily on 1) in vitro liver microsomal metabolism data, which showed much higher microsomal intrinsic clearance values for the acyl glucuronide (70–90%) than the 4'-hydroxylation pathways, 2) a good agreement between in vitro and in vivo clearance data, in untreated subjects, when both the in vitro oxidation and direct glucuronidation were considered, and 3) in vitro liver microsomal data, which showed that the glucuronide of DF could be further metabolized via oxidation, presumably leading to the in vivo formation of its 4'-hydroxy derivative, which is eventually excreted to bile and urine. However, this proposal has not been corroborated by clinical drug interaction studies between DF and CYP2C9 inducers or inhibitors. Before our studies, there was no report on the glucuronidation of DF in monkeys.

Our in vitro metabolism findings in both the liver microsomal and hepatocyte systems revealed that direct glucuronidation was indeed the major (90%), and hydroxylation, the minor (5%), metabolic pathway of DF in control rhesus monkeys. If the findings in vitro are scalable to in vivo, one would anticipate that the pharmacokinetics of DF in monkeys would not be affected appreciably by changes in its hydroxylation rate. This suggestion, which was in complete agreement with the present in vivo rifampin-DF interaction observations, was derived based on our preliminary study, which showed a modest increase (<30%) in DF glucuronide formation in rhesus monkey hepatocytes treated with 10 µM rifampin (data not shown), and an assumption that the intrinsic clearance value for the hydroxylation pathway was increased by 2- to 5-fold with rifampin treatment (i.e., from 5 of 95 units to 10 of 105 units to 25 of 120 units), based on the present in vitro E_max value of 2- to 5-fold obtained for the 4'-OH DF formation in monkey hepatocytes.

In humans, the oxidative metabolism of DF, albeit more substantial and catalyzed by relatively higher affinity enzyme than in monkeys, also is probably a minor contributor (25% based on the liver microsomal data) to its in vivo clearance. Similarly, it is expected that any changes in the DF hydroxylation rate, yielding <50% of its contributions to the total metabolic rate, would not have substantial impacts on the in vivo clearance of DF in humans. In this regard, there have been no clinical reports on the pharmacokinetic interactions of DF with known potent CYP2C9 inhibitors or inducers, including rifampin. In human hepatocytes, rifampin is an inducer of DF 4'-hydroxylase activity; a mean increase in the activity of about 2-fold over control has been reported in the presence of 20 µM rifampin (Parkinson et al., 2004), and a comparable magnitude of increase also was obtained with 10 µM rifampin in our preliminary study (data not shown). By analogy, rifampin may also elicit modest effects on the DF pharmacokinetics, via induction of CYP2C9, in humans, as was the case in monkeys. It is worth noting that possible differences in the magnitude of drug interaction between DF and other potent enzyme inhibitors/inducers exist between monkeys and humans, based on the quantitative differences in the kinetics of DF metabolism observed in this study between the two species, and considering potential species differences, in general, in enzyme induction and inhibition reported in the literature.

In conclusion, using the rhesus monkey as an animal model, we showed that in contrast to significant induction of the in vitro oxidative metabolism, the in vivo pharmacokinetics of DF were not sensitive to rifampin treatment. Subsequent in vitro metabolism studies suggested that this apparent in vitro-in vivo discrepancy was due probably to the fact that the CYP2C-mediated oxidative metabolism of DF is not the major determinant for its in vivo clearance in monkeys, both untreated and pretreated with rifampin. Our data also substantiated a previous suggestion made based on an in vitro finding in rat, dog, and human liver microsomes (Kumar et al., 2002) that the direct glucuronidation of DF instead is a more important determinant for the in vivo clearance of DF in both animals and humans, thus raising a question on the utility of DF as a useful probe for measuring CYP2C9 activity in vivo.

	Acknowledgments

 
We thank J. Brunner, K. Michel, and J. Adelsburger for animal experiments.

	Footnotes

 
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; 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., intrahepatic portal vein; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; UDPGA, UDP-glucuronic acid.

Address correspondence to: Dr. Thomayant Prueksaritanont, Department of Drug Metabolism, WP 75-100, Merck Research Laboratories, West Point, PA 19486. E-mail: thomayant_prueksaritanont{at}merck.com

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.011643v1 34/11/1806    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Prueksaritanont, T. Articles by Carr, B. Search for Related Content PubMed PubMed Citation Articles by Prueksaritanont, T. Articles by Carr, B.