Experimental Procedures

Materials.

Diclofenac sodium salt, dimethyl sulfoxide, reduced glutathione (GSH), mefenamic acid, NADPH, quinidine, quinidine gluconate, and troleandomycin were purchased from Sigma Chemical Co. (St. Louis, MO). Trifluoroacetic acid (TFA) and polyethylene glycol 400 were obtained from Fisher Scientific (Fair Lawn, NJ). Ketoconazole was purchased from Janssen Biotech (Olen, Belgium). All other chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI). BondElut C₁₈ solid-phase extraction cartridge columns were obtained from Varian Chromatography Systems (Walnut Creek, CA).

L-754,394 (N-[2-hydroxy-1-indanyl]-5-[2-[((1,1-dimethylethyl)amino) carbonyl]-4-[(furo[2,3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4-hydroxy-2-(phenylmethyl)pentanamide) was synthesized at Merck Research Laboratories. [carbonyl-¹⁴C]Diclofenac (44.4 mCi/mmol, 99% radiochemical purity) was synthesized according to the published methodology (Horio et al., 1985). The synthesis of 4′-hydroxydiclofenac, 5-hydroxydiclofenac, 4′-hydroxy-3′-(glutathion-S-yl)diclofenac (4′-OH-3′-GS-diclofenac), 5-hydroxy-4-(glutathion-S-yl)diclofenac (5-OH-4-GS-diclofenac), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (5-OH-6-GS-diclofenac) was reported previously (Tang et al., 1999a).

An antipeptide antibody against human hepatic CYP3A4 was prepared in rabbits by immunization with a synthetic peptide that was identical with residues 253 to 273 of CYP3A4 and was coupled to keyhole limpet hemocyanin (Wang et al., 1997). A monoclonal antibody against human hepatic CYP3A4 and monkey hepatic CYP3A was the product of hybridomas derived from the fusion of myeloma cells and spleen cells from mice immunized with baculovirus-expressed CYP3A4 (Mei et al., 1999).

Instrumentation and Analytical Methods.

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) was carried out on a Perkin-Elmer SCIEX API III⁺ tandem mass spectrometer (Toronto, Canada) interfaced to an HPLC system consisting of two Shimadzu LC-10A pumps and a static-bed mixer (Kyoto, Japan). LC/MS/MS experiments were performed using either an IonSpray interface or an atmospheric pressure chemical ionization interface with positive ion detection.

With the IonSpray interface, the ionization voltage was set at 5 kV, orifice potential at 65 V, collision energy at 30 eV, and collision gas (argon) at a thickness of 1.3 × 10¹⁴atoms/cm². Chromatography was performed on a DuPont Zorbax (Wilmington, DE) Rx-C8 column (4.6 × 250 mm, 5 μm), and samples were delivered at a flow rate of 1 ml/min with 1:25 split. The mobile phase consisted of aqueous acetonitrile containing 10% methanol and 0.05% TFA; a linear gradient was used in which the acetonitrile content increased from 10 to 70% during a 30-min period.

With the atmospheric pressure chemical ionization interface, the heated nebulizer was set at 500°C, Corona discharge at 4.9 μA, orifice potential at 46 V, collision energy at 30 eV, and collision gas (argon) at a thickness of 2.0 × 10¹⁴atoms/cm². Chromatography was performed on a Keystone Scientific (Wilmington, DE) Betasil C₈column (4.6 × 50 mm, 5 μm), and samples were delivered at a flow rate of 1 ml/min. The mobile phase consisted of 90% aqueous acetonitrile containing 1 mM ammonium acetate and 0.1% TFA.

Animal Experiments.

Experiments were performed according to procedures approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.

Three male rhesus monkeys (weight 5–6 kg) were treated with diclofenac (1 mg/kg) in an aqueous solution via i.v. injection. They were restrained in metabolism chairs for 6 h after dosing and then put into metabolism cages for the remainder of the study. Plasma was prepared from blood collected from peripheral veins at predosing and at various time points after dosing for 24 h.

Three male rhesus monkeys (weight 8–10 kg) with catheters surgically implanted in their portal vein were treated with diclofenac (0.055 mg/kg/h) or a mixture of diclofenac (0.055 mg/kg/h) and quinidine (0.25 mg/kg/h) in aqueous solutions by portal infusion for 3.5 h. The monkeys were restrained in metabolism chairs during dosing and for 60 min after dosing. Plasma was prepared from blood collected from peripheral veins at before dosing and at various time points during and after the infusion.

The three portal vein-cannulated monkeys were treated with L-754,394 (10 mg/kg) in ethanol/polyethylene glycol 400/water (10:40:50, v/v/v) by i.v. injection. After 10 min, they were dosed with diclofenac (0.055 mg/kg/h) in aqueous solutions via portal infusion for 3.5 h. The monkeys were restrained in metabolism chairs during dosing and for 60 min after dosing. Plasma was prepared from blood collected from peripheral veins before dosing and at various time points during and after the infusion.

All monkeys had a 3-week washout period between treatments.

Incubations with Monkey Hepatocytes.

Hepatocytes were isolated from an adult male rhesus monkey through the use of a two-step perfusion procedure (Pang et al., 1997). On isolation, hepatocytes that exhibited a viability of greater than 80%, as determined with the trypan blue exclusion test, were suspended in Krebs-bicarbonate buffer (pH 7.4).

Diclofenac in aqueous solution was added to the suspension to provide final drug concentrations of 100 and 200 μM. Quinidine, dissolved in methanol, was added to provide a final concentration of 100 μM. The concentration of methanol was 0.2% (v/v). Controls contained no quinidine. After incubation for 3 h, the suspension medium was acidified with 10% aqueous TFA.

Incubations with Monkey Liver Microsomes.

Liver microsomes were isolated by differential centrifugation (Raucy and Lasker, 1991) and pooled from four male rhesus monkeys.

Diclofenac and GSH in phosphate buffer (pH 7.4) and quinidine in methanol were added to monkey liver microsomes (0.5–2.0 nmol CYP/ml) suspended in phosphate buffer (0.1 M, pH 7.4) containing EDTA (1 mM). Diclofenac concentrations ranged from 10 to 500 μM, and quinidine concentrations ranged from 0 to 200 μM. The concentration of GSH was 5 mM, and the methanol content was 0.2% (v/v). Controls contained no quinidine but the same amount of methanol (0.2%). The mixture was incubated at 37°C for 5 min, and then NADPH in phosphate buffer was added to give a final concentration of 1 mg/ml. After an additional 10-min incubation, the reaction was quenched with 10% aqueous TFA.

In experiments involving CYP3A inhibitors, microsomes were preincubated with ketoconazole for 10 min and preincubated with troleandomycin or L-754,394 in the presence of NADPH for 15 min at 37°C. The inhibitors were dissolved in methanol, and their final concentrations were 2 μM (ketoconazole), 10 μM (L-754,394), and 40 μM (troleandomycin). The same amount of solvent was added into control incubations at a final concentration of 0.2% (v/v). Reactions were quenched 10 min after the addition of the substrate by adding 10% aqueous TFA (60 μl).

In immunoinhibition experiments, microsomes (0.5 nmol CYP/ml) were preincubated with antibodies against CYP3A (5 mg IgG/nmol CYP) for 30 min at room temperature. Control incubations contained IgG from untreated animals. Diclofenac, quinidine, GSH, and NADPH were added thereafter, and the incubations were performed in a manner similar to that described above.

Detection and Quantification of Diclofenac and Its Metabolites.

Aliquots of plasma (200 μl) were mixed with mefenamic acid (internal standard, 20 ng) and 4 M urea (1 ml) and applied to a 96-well plate solid-phase extraction cartridge that was prewashed with methanol and water. The cartridge was washed consecutively with water and 90% aqueous acetonitrile (400 μl) containing 0.1% TFA. The acetonitrile eluate was analyzed by LC/MS/MS (heated nebulizer interface) through multiple reaction monitoring of mass transitions m/z 296→214 (diclofenac) andm/z 242→180 (internal standard). Standard curves were generated over a range of 0.5 to 1000 ng.

Samples from microsomal incubations or from hepatocyte cultures were applied to a C₁₈ extraction cartridge column that was prewashed with methanol and water. The column was then washed consecutively with water and methanol. The methanol eluate was evaporated to dryness under a stream of nitrogen, the residue reconstituted in 60% aqueous acetonitrile (300 μl) containing 0.05% TFA, and the resulting sample was analyzed by LC/MS/MS (IonSpray interface). Identification of diclofenac metabolites was based on multiple reaction monitoring detection of four transitions, namelym/z 617→542, 617→488, 617→342, and 617→324 (Tang et al., 1999b). Data are expressed relative to controls.

Determination of Blood/Plasma Ratio and Plasma Protein Binding of Diclofenac.

Monkey blood (1 ml), preincubated at 37°C for 15 min, was treated with [¹⁴C]diclofenac in aqueous solution (pH 7.4) and quinidine in methanol. The final concentrations of diclofenac were 10, 50, 100, 150, and 200 ng/ml, all containing 0.0014 μCi of radiolabeled drug. The concentration of quinidine was 100 μM, and the methanol content was 0.2% (v/v). Controls contained no quinidine but the same amount of methanol (0.2%). All samples were incubated at 37°C for an additional 20 min. Plasma was then prepared, and radioactivity in the resulting samples (100 μl) was determined. Blood/plasma concentration ratios were estimated based on comparison of the radioactivity in plasma prepared from the treated blood samples with that in control plasma containing 0.0014 μCi of radiolabeled drug.

Monkey plasma (0.5 ml) in dialysis cells (Bel-Art Products, Pequannock, NJ) was spiked with [¹⁴C]diclofenac in aqueous solution (pH 7.4) and quinidine in methanol. The final concentrations of diclofenac were 10, 50, 100, and 200 ng/ml, all containing 0.0014 μCi of radiolabeled drug. The concentration of quinidine was 100 μM, and that of methanol was 0.2% (v/v). Controls contained no quinidine but the same amount of methanol (0.2%). The dialysis membrane (Sigma Chemical Co., St. Louis, MO) had a molecular mass cutoff of 12.4 kDa. The resulting plasma samples then were subjected to dialysis against isotonic phosphate buffer for 3 h. The protein-bound fraction was determined through a comparison of the differences between radioactivity in the plasma and that in the buffer solution.

Pharmacokinetic Calculations.

Pharmacokinetic parameters of diclofenac in monkeys were calculated based on a noncompartment model. Values for the plasma clearance and volume of distribution are presented as means ± S.D.

Results

Pharmacokinetics of Diclofenac in Monkeys.

The pharmacokinetic parameters of diclofenac were determined in three male rhesus monkeys after i.v. dosing at 1 mg/kg. The plasma clearance was 8.7 ± 0.7 ml/min/kg, and the volume of distribution, extrapolated to steady state, was 0.17 ± 0.04 l/kg. The terminal phase half-life was estimated at 0.51 ± 0.17 h.

Studies on the interaction of diclofenac and quinidine were carried out in three male rhesus monkeys with catheters surgically implanted in their portal vein. The experiment was designed such that two monkeys were treated with diclofenac, and the third received diclofenac plus quinidine. Three weeks later, the same monkeys were treated with diclofenac or diclofenac plus quinidine in a crossover manner. Systemic plasma diclofenac concentrations reached steady-state at approximately 105 min (∼3 times the terminal half-life) after the start of the portal vein infusion (0.055 mg/kg/h); these steady-state concentrations were measured as the mean values of six time-points during 120 to 195 min of infusion. Thus, after a dose of diclofenac (control), plasma drug concentrations at steady-state in the three monkeys were 87, 104, and 32 ng/ml, respectively. When diclofenac was coadministered (0.055 mg/kg/h) with quinidine (0.25 mg/kg/h) via portal vein infusion, the corresponding plasma diclofenac concentrations were 50, 59, and 18 ng/ml, which represent 56% of the control values (Fig.2).

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Figure 2

The steady-state systemic plasma concentration-time profiles of diclofenac in a monkey treated with diclofenac plus L-754,394 (A), diclofenac only (B), and diclofenac plus quinidine (C). Diclofenac and quinidine were dosed by portal vein infusion at 0.055 and 0.25 mg/kg/h, respectively, whereas L-754,394 was dosed by i.v. injection at 10 mg/kg. Similar profiles were observed with two other monkeys.

Systemic diclofenac concentrations also were determined in the same three monkeys after pretreatment with L-754,394 (10 mg/kg i.v.), a potent CYP3A inhibitor (Chiba et al., 1995). After portal vein infusion of diclofenac at 0.055 mg/kg/h, plasma drug concentrations at steady state were 136, 141, and 82 ng/ml, respectively, a 1.4- to 2.5-fold increase compared with control values (Fig. 2). The more profound effect on plasma diclofenac concentrations observed in the third monkey after L-754,394 dosing probably is associated with a relatively higher metabolic capacity in that monkey. Because L-754,394 is a mechanism-based inhibitor of CYP3A, more rapid turnover of the inhibitor would result in less active enzyme.

Metabolism of Diclofenac in Monkey Hepatocytes and Liver Microsomes.

The detection of diclofenac metabolites in hepatocyte suspensions or in incubations with liver microsomes was based on LC/MS multiple reaction monitoring coupled with HPLC separation (Tang et al., 1999a). Because the monohydroxylated metabolites of diclofenac in these in vitro systems have been shown to undergo further biotransformation to form reactive intermediates (Tang et al., 1999a,b), diclofenac metabolism in this study was evaluated by monitoring the formation of the GSH adducts of hydroxydiclofenac. Thus, when 50 μM diclofenac was incubated with monkey hepatocytes, two 5-hydroxydiclofenac derivatives, namely 5-OH-4-GS-diclofenac and 5-OH-6-GS-diclofenac, were the major metabolites, whereas their 4′-hydroxy analog, 4′-OH-3′-GS-diclofenac, was a minor product (Fig. 3). Compared with controls, a 2-fold increase in the formation of 5-hydroxydiclofenac derivatives was observed in suspensions containing quinidine.

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Figure 3

LC/MS/MS detection of 5-OH-4-GS-diclofenac (M1), 4′-OH-3′-GS-diclofenac (M2), and 5-OH-6-GS-diclofenac (M3) in monkey hepatocyte suspensions treated with diclofenac. Four mass transitions were used as criteria for metabolite identification, namelym/z 617→524, 617→488, 617→342, and 617→324. A similar profile was observed when incubations were performed in the presence of quinidine except that the formation of 5-hydroxy derivatives increased 2.5-fold.

The 5-hydroxylated derivatives of diclofenac also were the predominant products in incubations with monkey liver microsomes over a substrate concentration range of 10 to 400 μM. At 50 μM diclofenac, the formation of 5-OH-4-GS-diclofenac and 5-OH-6-GS-diclofenac increased with increasing quinidine concentrations, reaching a plateau at ∼100 μM quinidine (Fig. 4). This quinidine concentration subsequently was used in kinetic studies to evaluate the effect of quinidine on diclofenac metabolism.

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Figure 4

Formation of the 5-hydroxylated derivatives in incubations of diclofenac with monkey liver microsomes containing quinidine. The diclofenac concentration was 50 μM. Data are expressed relative to controls.

Kinetic studies were performed in incubations of diclofenac with monkey liver microsomes in the presence or absence of quinidine. Double-reciprocal plots of 1/V versus 1/S were linear (Fig.5). Linearity also was obtained with Eadie-Scatchard plots (data not shown). TheK_m values were estimated at 59 μM for incubations containing quinidine and 75 μM in its absence. TheV_max, expressed as a ratio of the values generated from incubations containing quinidine versus those lacking quinidine, was 2.5. Because the oxidation of 5-hydroxydiclofenac to the corresponding 2,5-benzoquinone imine, which is trapped by GSH conjugation, does not require enzymatic catalysis (Fig. 1; Tang et al., 1999b), the apparent K_m determined by monitoring the formation of 5-OH-4-GS-diclofenac and 5-OH-6-GS-diclofenac actually may reflect theK_m of a single-step reaction from diclofenac to 5-hydroxydiclofenac.

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Figure 5

Double-reciprocal plots for diclofenac metabolism in incubations with monkey liver microsomes in the presence or absence of quinidine. The quinidine concentration was 100 μM.

Inhibition of CYP-Mediated Metabolism of Diclofenac.

Diclofenac metabolism in incubations with monkey liver microsomes was inhibited by CYP3A inhibitors, namely troleandomycin, ketoconazole, and L-754,394 (Table 1). The inhibition also was observed when microsomes were treated with antibodies against CYP3A (Table 1). With the monoclonal antibodies, nearly complete inhibition was achieved. The concentrations of the inhibitors and the antibodies against CYP3A were selected based on literature values generated with human liver microsomes (Newton et al., 1995; Mei et al., 1999; Tang et al., 1999b).

Diclofenac metabolism also was inhibited by antibodies against CYP3A in incubations containing quinidine (Table 1). Interestingly, the formation of 5-hydroxydiclofenac derivatives in incubations containing both the monoclonal antibody and quinidine was similar to that in incubations containing the antibody alone (Table 1), indicating that quinidine had no effect on diclofenac metabolism when CYP3A was completely inhibited.

Blood/Plasma Ratio and Plasma Protein Bindings of Diclofenac.

The blood-to-plasma concentration ratio of diclofenac in monkeys was determined in vitro over a concentration range of 10 to 200 ng/ml. The ratio was estimated at 0.7. When quinidine was coadministered at the final concentration of 100 μM, the blood/plasma concentration ratio of diclofenac remained unchanged.

The plasma protein binding of diclofenac in monkeys was investigated in vitro over a drug concentration range of 10 to 200 ng/ml. The binding was estimated to be greater than 99%. In the presence of 100 μM quinidine, the plasma protein binding of diclofenac remained greater than 99%.

Discussion

The oxidative metabolism of diclofenac in humans mainly involves CYP2C9 and 3A4 (Fig. 1; Leemann et al., 1993; Shen et al., 1999; Tang et al., 1999b). The CYP3A4-catalyzed formation of 5-hydroxydiclofenac in incubations with human liver microsomes was reported to be stimulated by the presence of quinidine (Tang et al., 1999b). To investigate whether the observed effect of quinidine on diclofenac metabolism also occurs in vivo, our initial efforts focused on choosing an appropriate animal model.

The clearance of diclofenac is known to be due largely to metabolism, but the mechanism of clearance differs considerably among species in terms of partition between phase I and phase II pathways. For example, glucuronidation and taurine conjugation contribute nearly 90% to the elimination of diclofenac in dogs, whereas CYP-catalyzed hydroxylation is the primary route of clearance in rats and monkeys (Riess et al., 1978; Stierlin and Faigle, 1979). The stimulatory effect of quinidine, however, was not observed in incubations of diclofenac with rat liver microsomes (data not shown). This is probably due to the participation in rats of several CYP enzymes (CYP2B, 2C and 3A subfamilies) in diclofenac metabolism where CYP3A is not a predominant contributor (Tang et al., 1999a).

On the other hand, high CYP3A activities are observed in monkey liver (Sharer et al., 1995). In our study with CYP inhibitors and inhibitory antibodies, monkey hepatic CYP3A was demonstrated to catalyze the conversion of diclofenac to 5-hydroxydiclofenac derivatives that were the major metabolites in rhesus monkeys dosed with the drug (Riess et al., 1978; Stierlin and Faigle, 1979). More importantly, the formation of 5-hydroxy derivatives increased 3-fold when incubations of diclofenac with monkey liver microsomes were performed in the presence of quinidine. This phenomenon is similar to that observed in incubations with human liver microsomes (Tang et al., 1999b). Consequently, the effect of quinidine on diclofenac metabolism was investigated in vivo in the rhesus monkey.

In rhesus monkeys, diclofenac appeared to be a low clearance compound with blood clearance estimated at 12 ml/min/kg, approximately one fourth of hepatic blood flow in this species (44 ml/min/kg; Davies and Morris, 1993). This estimate was made based on two parameters determined in this study: the plasma clearance of 8.7 ml/min/kg and the blood-to-plasma ratio of 0.7. The systemic plasma diclofenac concentration reached steady state after portal vein infusion for 105 min. This route of administration provides the advantages of maximizing the first-pass effect and controlling exposure quantitatively.

Subsequent in vivo studies of the effect of quinidine consisted of two experiments. In the first, monkeys were administered diclofenac together with quinidine via portal vein infusion. Coadministration of quinidine was associated with lower steady-state plasma diclofenac concentrations that occurred in a time frame too short for the de novo synthesis of CYP proteins (Conney, 1967, Haugen et al., 1976). In the second experiment, monkeys were treated with L-754,394, an inhibitor of CYP3A, followed by a dose of diclofenac via portal vein infusion. In this study, steady-state plasma diclofenac concentrations increased 1.4- to 2.5-fold relative to controls. Further investigation indicated that neither diclofenac plasma protein binding nor blood/plasma ratio was influenced by the presence of quinidine. Taken together, these data suggest that the hepatic clearance of diclofenac, measured on the basis of the portal infusion rate and steady-state plasma drug concentrations, is increased on quinidine coadministration. This increase in the clearance of diclofenac most likely results from the stimulation of oxidative metabolism catalyzed by CYP3A. Consistent with this proposed mechanism, a 2-fold increase in the formation of 5-hydroxydiclofenac derivatives was observed in monkey hepatocyte suspensions containing diclofenac and quinidine. Moreover, the stimulative effect of quinidine on diclofenac metabolism was diminished when monkey liver microsomes were pretreated with antibodies against CYP3A. Therefore, it may be concluded that the stimulation is mediated through the interaction of quinidine with CYP3A.

Quinidine is an antiarrhythmic drug (Grace and Camm, 1998). In a research setting, however, quinidine has been used extensively as an inhibitor of CYP2D6 (Newton et al., 1995; Bourrie et al., 1996). Several reports indicate that quinidine also is an inhibitor of CYP3A4, albeit a weak one (Schellens et al., 1991; Bowles et al., 1993). The inhibitory effect on CYP3A4 presumably is competitive in nature because quinidine metabolism is catalyzed by that enzyme (Guengerich et al., 1986). In contrast, our studies suggest that CYP3A-mediated metabolism of diclofenac is enhanced by quinidine. In the presence of quinidine, the K_m value for monkey hepatic CYP-mediated conversion of diclofenac to its 5-hydroxy derivatives was little changed, whereas the V_max increased 2.5-fold. It is tempting to speculate, therefore, that quinidine is a modulator of CYP3A activity and that its effect may be either inhibitory or stimulatory depending on the nature of the CYP3A substrate. Studies are under way to investigate this hypothesis.

In summary, the CYP3A-dependent oxidative metabolism of diclofenac is stimulated by quinidine. In monkeys, quinidine coadministration leads to increased hepatic clearance of diclofenac. This case appears to be a rare example in which stimulation of CYP-mediated xenobiotic metabolism is observed in vivo. Whether a similar drug-drug interaction occurs in humans remains to be established.