Characterization of the selectivity and mechanism of human cytochrome P450 inhibition by the human immunodeficiency virusprotease inhibitor nelfinavir mesylate. Drug Metab. Dispos

This paper is available online at http://www.dmd.org ABSTRACT: In vitro studies with human liver microsomes and P450 probe substrates were performed to characterize selectivity and mechanism of cytochrome P450 inhibition by nelfinavir mesylate. At therapeutic concentrations (steady-state plasma concentrations Ϸ4 M), nelfinavir was found to be a competitive inhibitor of only testosterone 6␤-hydroxylase (CYP3A4) with a K i concentration of 4.8 M. At supratherapeutic concentrations, nelfinavir competitively inhibited dextromethorphan O-demethylase (CYP2D6), S-mephenytoin 4-hydroxylase (CYP2C19), and phenacetin O-deethylase (CYP1A2) with K i concentrations of 68, 126, and 190 M, respectively. Nelfinavir did not appreciably inhibit tolbutamide 4-hydroxylase (CYP2C9), paclitaxel 6␣-hydroxylase (CYP2C8), or chlorzoxaxone 6␤-hydroxylase (CYP2E1) activities. The inhibitory potency of nelfinavir toward CYP3A4 suggested the possibility of in vivo inhibition of this isoform, whereas in vivo inhibition of other P450s was considered unlikely. In a one-sequence crossover study in 12 healthy volunteers, nelfinavir inhibited the elimination of the CYP3A substrate terfenadine and the carboxylate metabolite of terfenadine. The 24-hr urinary recoveries of 6␤-hydroxycortisol were reduced by an average of 27% during nelfinavir treatment, consistent with CYP3A inhibition by nelfinavir. Inhibition of CYP3A4 by nelfinavir in vitro was NADPH-dependent requiring the catalytic formation of a metabolite or a metabolic intermediate. The catechol metabolite of nelfinavir (M3) was considered unlikely to be responsible for inhibition as the addition of catechol O-methyl transferase, S-adenosyl methionine, and ascorbic acid to the preincubation mixture did not protect against the loss of testosterone 6␤-hydroxylase activity. Also, the addition of M3 to human liver microsomes did not inhibit CYP3A4. Although incubations with nelfinavir showed a time-and concentration-dependent loss of CYP3A4 activity, the partial or complete recovery of enzyme activity upon dialysis indicated that inhibition was reversible. Microsomal incubations with nelfinavir and NADPH did not result in a loss of spectral P450 content compared with the NADPH control. Glutathione, N-acetylcysteine, and catalase did not attenuate CYP3A4 inhibition by nelfinavir. Collectively, these results suggest that the probable mechanism for CYP3A4 inhibition by nelfinavir is a transient metabolic intermediate or stable metabolite that coordinates tightly but reversibly to the heme moiety of the P450. Nelfinavir mesylate is a potent, orally active HIV protease inhibitor (PI) 1 approved for the treatment of HIV infection. Optimal drug therapy for suppression of HIV viral replication is currently considered to be chronic drug treatment involving the combination of two reverse transcriptase inhibitors and a potent HIV-PI Because of the pivotal role of cytochrome P450 in general drug metabolism, significant inhibition of P450 and particularly the major human hepatic and intestinal CYP3A4 isoforms could result in adverse drug reactions and potentially life-threatening drug-drug interactions. Among HIV-PIs, ritonavir is recognized clinically as a broad spectrum P450 inhibitor and a very potent CYP3A4 inhibitor 609 potently inhibited by nelfinavir; and 3) to gain insight into the mechanism of inhibition for the P450 most potently inhibited by nelfinavir mesylate. Materials and Methods Chemicals. Testosterone, troleandomycin, diethyldithiocarbamic acid, retinoic acid, 6␤-hydroxytestosterone, 11-␣-hydroxyprogesterone, chloropropamide, pentoxifylline, glutathione, N-acetylcysteine, acetaminophen, phenacetin, quinidine, sulfaphenazole, 7,8-benzoflavone, catalase, catechol O-methyl transferase, S-adenosyl methionine, NADPH, ascorbic acid, EDTA, and midazolam were purchased from the Sigma Chemical Company (St. Louis, MO). 1-Aminobenzotriazole and 4-hydroxy-3-(␣-iminobenzyl)-1-methyl-6-phenypryridin-2(1H)-one (which is used as an internal standard and is referred to in this paper as ALD25033-3) were purchased from Aldrich (Milwaukee, WI). Paclitaxel, 4-hydroxy-S-mephenytoin, tolbutamide, 4-hydroxytolbutamide, dextrorphan D-tartrate, dextromethorphan hydrobromide, chlorzoxazone, 6␤-hydroxychlorzoxazone, and ketoconazole were purchased from Research Biochemicals International (Natick, MA). S-Mephenytoin was obtained from Cedra Corp. (Austin, TX). Magnesium chloride was obtained from GIBCO BRL (Gaithersburg, MD). Seldane™ was purchased from Marion Merrell Dow (Kansas City, MO). Human liver tissue and pooled human liver microsomes were purchased from the Pennsylvania Regional Skin Bank (Exton, PA). Nelfinavir mesylate, 3-methoxy-4-hydroxy nelfinavir (M1), 3,4-dihydroxy nelfinavir (M3), and nelfinavir hydroxy-t-butylamide (M8) were synthesized and indinavir, ritonavir, and saquinavir were isolated at Agouron Pharmaceuticals Inc. (La Jolla, CA). All reagents used in the extraction and analysis were HPLC grade (Fisher Scientific). Microsomal Incubations. The concentrations of nelfinavir selected for the in vitro studies were based on steady-state total (free plus bound) C max plasma concentrations of nelfinavir that averaged approximately 5.3 to 7.0 M after a multiple oral dosing regimen of 750 mg t.i.d. (Agouron Pharmaceuticals Inc., 1997). For incubation studies designed to determine the inhibition constant (K i ) of nelfinavir inhibition toward various P450s, the preincubation mixture contained 1.0 mg/ml microsomal protein (except for chlorzoxazone 6␤-hydroxylase, a CYP2E1 probe substrate, where 1 nmol/ml P450 was used), 1.0 mM NADPH, nelfinavir (0.35 to 100 M), and various P450 isoform-specific probe substrates (for specific concentrations, see P450 probe substrate assays) in 100 mM phosphate buffer, pH 7.4, in a final volume ranging from 0.25 to 0.5 ml at 37 o C in a shaking water bath. CYP3A4-related K i determination studies involving M1, M3, M8, the other marketed HIV-PIs, and ketoconazole were conducted in a similar manner as described above for nelfinavir except that the range of concentrations was adjusted as follows: M1, 0.1 to 1.0 M; M3, 0.5 to 5.0 M; M8, 1 to 25 M; indinavir, 0.1 to 5 M; ritonavir, 0.025 to 0.250 M; saquinavir, 2.5 to 25 M; and ketoconazole, 0.05 to 2 M. For the time dependency studies, a 400-l incubate consisted of microsomes (1 mg/ml), 1 mM NADPH, and varying concentrations of nelfinavir (1, 3, 5, or 10 M), and midazolam (10 M). The effect of preincubating different concentrations of nelfinavir over time on CYP3A4 activity was examined in pooled human liver microsomes. Time-dependent studies with nelfinavir were also conducted in the presence of modifiers such as GSH, ascorbic acid, N-acetylcysteine, and catechol-O-methyltransferase/S-adenosyl methionine (COMT/SAM). The final concentrations of these modifiers in the incubation mixture were as follows: 1.0 and 5.0 mM GSH, 500 M ascorbic acid, 1 mM N-acetylcysteine; and 200 units/ml COMT (which included 2 mM SAM and 1 mM MgCl 2 ). Time dependency studies were conducted according to the following method: aliquots (25 l) of the preincubate were removed at 5-min intervals, from 0 to 20 min, and added to test tubes containing 200 M testosterone and 1.0 mM NADPH in 100 mM phosphate buffer, pH 7.4, in a final volume of 0.5 ml. The preincubation mixture was diluted 20 times in this process. P450 Probe Substrate Assays. Phenacetin O-deethylation, paclitaxel 6␣-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6␤-hydroxylation, and testosterone 6␤-hydroxylation were determined in human liver microsomes for human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, respectively. For the determination of nelfinavir K i values toward various P450 enzymes, the following probe substrate concentrations were used: 40, 80, and 200 M for S-mephenytoin, tolbutamide, phenacetin, and testosterone; 4, 8, and 20 M for dextromethorphan; 2.5, 5, and 20 M for paclitaxel; and 20, 60, and 120 M for chlorzoxazone. Known P450 isoform inhibitors were incubated with each probe substrate as positive controls; the mechanism-based inhibitors [100 M troleandomycin Quenched incubation samples for phenacetin, paclitaxel, and dextromethorphan were vortexed for 10 min on an SP Multitube Vortexer (Baxter, McGaw Park, IL) and centrifuged at 2,500g for 15 min on an IEC Centra-8R (Damon, Needham Heights, MA). The organic layer was removed and evaporated on a Dri-Block sample concentrator (Techne, Princeton, NJ) under nitrogen at 40 o C. Quenched incubation samples for tolbutamide, S-mephenytoin, chlorzoxazone, and testosterone were spiked with internal standards of chloropropamide (400 ng), ALD25033-3 (200 ng), pentoxifylline (1 g), 11␣-hydroxyprogesterone (250 ng), respectively, and then vortexed and centrifuged as described above. Similarly, the organic layer was removed and evaporated under nitrogen at 40 o C. HPLC Analysis. Chromatography was performed using a Hewlett Packard 1050 system and monitored using either a Hewlett Packard multiwavelength UV or fluorescence detector. The standard curves were linear over their respective ranges, and interday and intraday coefficients of variation for the slopes of the standard curves were less than 10%. The probe substrate metabolites were analyzed as described in the literature LILLIBRIDGE ET AL. 75/25% 25 mM ammonium phosphate, pH 4.5/ACN (v/v) at a flow rate of 1.0 ml/min. Dextrorphan was monitored by fluorescence detection (excitation 230, emission 315 nm). Retention times for dextrorphan and dextromethorphan were 4.4 and 18 min, respectively. The mobile phase for chlorzoxazone 6␤-hydroxylation was 80/20% 0.15% (v/v) glacial acetic acid, pH 4.7/ACN (v/v) at a constant flow rate of 1.2 ml/min. 6␤-Hydroxychlorozoxazone was monitored by UV absorption at 282 nm. Retention times for 6␤-hydroxychlorzoxazone, pentoxifylline, and chlorzoxazone were 5.4, 6.6, and 17.2 min, respectively. 6␤-Hydroxytestosterone was eluted by a gradient mobile phase consisting of methanol/ACN/water under the following time course of: 0 min, 58/0/48%; 0 -15 min, 62/5/33%; 15-24 min, 62/25/13%; 24 -30 min, 58/0/ 42% delivered at a constant flow rate of 1.0 ml/min. The 6␤-hydroxytestosterone metabolite was monitored by UV absorption at 254 nm. The retention times for 6␤-hydroxytestosterone, 11␣-hydroxyprogesterone, and testosterone were 4.4, 9.4, and 13.5 min, respectively. Measurement of Cytochrome P450 Content. Pooled human liver microsomes (1.0 nmol of P450/ml at a final volume of 1.5 ml in 100 mM phosphate buffer, pH 7.4) were incubated in triplicate at 37 o C in a shaking water bath to examine the effects of nelfinavir on P450 content. P450 content was determined in samples containing human liver microsomes only, human liver microsomes plus NADPH (1.0 mM) or nelfinavir (10 M), and human liver microsomes plus NADPH (1.0 mM) and nelfinavir (10 M) or ABT (100 M). At 0, 0.5, 5, 10, and 20 min, a 300-l aliquot of each incubation mixture was transferred to a test tube containing 300 l of ice-cold phosphate buffer and kept on ice until analysis (Յ30 min). The zero time point was the baseline condition prior to the addition of components to microsomes. P450 content was determined by the method of Dialysis Experiment. Pooled human liver microsomes were incubated with NADPH in the presence or absence of nelfinavir (1.0, 3.0, 5.0, and 10.0 M) for 20 min. Samples were immediately placed in 6,000 -8,000 molecular weight cutoff Spectra/Por dialysis tubing (Spectrum Medical Industries Inc., Houston, TX) and dialyzed for 18 hr at 4 o C against 1000 ml of 100 mM potassium phosphate buffer, pH 7.4, containing 5 mM EDTA. Dialysis buffer was changed once after 6 hr. Protein content was subsequently measured, and 20-min incubation studies were conducted to assess testosterone 6␤-hydroxylase activity as described above. Binding Spectra. The P450 substrate binding spectra were obtained using a Varian Cary 3E dual beam spectrophotometer equipped with a temperature controller, which maintained the samples at 37°C. Both reference and sample cuvettes contained 2 nmol/ml human P450 and 100 mM phosphate buffer, pH 7.4, with or without 1 mM NADPH. Nelfinavir (100 M) was added to the test cuvette, and scans were recorded every 5 min for 35 min. Pharmacokinetic Studies. Twelve healthy male volunteers, 18 to 36 years of age, body weight within 15% of ideal, gave informed consent to participate in a one sequence (1 ϫ 2) crossover study. On the morning of day 1, a 60-mg dose of terfenadine (60-mg Seldane™ tablet) was administered 10 min after completion of a standard breakfast in the absence of nelfinavir. Serial plasma samples were collected at predose (0 hr) and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hr post-dose for assay of terfenadine carboxylate and unchanged terfenadine. On days 6 through 12, 750 mg of nelfinavir (3 ϫ 250-mg Viracept tablets) was administered every 8 hr (the standard clinical dose of Viracept is 750 mg t.i.d.). Each dose of nelfinavir was ingested within 10 min after eating a meal or light snack. On the morning of day 10 (5th day of nelfinavir treatment), a predose plasma sample was drawn from each subject for assay of nelfinavir trough concentration, and a 60-mg dose of terfenadine was administered concomitantly with the morning dose of nelfinavir at 10 min following the standard breakfast. Serial plasma samples were collected on day 10 from predose through 72 hr post-dose (same times as listed above) for assay of terfenadine carboxylate and unchanged terfenadine. Total 24-hr urine collections were performed on day 5 (before nelfinavir treatment) and day 12 (7th day of nelfinavir) for measurement of 6␤-hydroxycortisol. In two other healthy volunteer studies not described here in detail, 24-hr urine collections for measurement of 6␤-hydroxycortisol were performed before and on the 5th day of treatment with 600 mg rifampin daily (in combination with 750 mg nelfinavir every 8 hr) or 400 mg ketoconazole daily (in combination with 500 mg nelfinavir every 8 hr). Bioanalytical Methods for Pharmacokinetic Studies. Plasma concentrations of nelfinavir were measured by a validated HPLC method with ultraviolet detection . The calibration curve for nelfinavir (0.25 ml plasma volume) over the range of 0.05 to 10.0 g/ml yielded a correlation coefficient (r) Ͼ0.998 with precision based on quality control samples within 2.9% and accuracy expressed as per cent of nominal within 96.4 -100.2%. Plasma concentrations of terfenadine and terfenadine carboxylate and urinary concentrations of 6␤-hydroxycortisol were measured by validated HPLC methods with fluorescence detection Pharmacokinetic and Statistical Analysis. The maximal plasma concentration (C max ) and time of maximal concentration (t max ) for terfenadine were estimated by inspection of individual subject plasma concentration-time profiles. The elimination rate constant (K el ) for terfenadine carboxylate was estimated by least-squares regression of the terminal log-linear portion of the plasma concentration-time profile. Terminal half-life for the carboxylate was estimated as the ratio of the natural logarithm of 2 divided by K el . The area under the plasma concentration-time curve for the carboxylate metabolite from time of terfenadine dosing to infinity (AUC ϱ ) was estimated by the trapezoidal method to the time of last measurable concentration with extrapolation to infinity by addition of the quantity C last /K el , where C last represents the last measurable concentration of the carboxylate. Twenty-four hour urinary recoveries of 6␤-hydroxycortisol were estimated as the product of urine volume and concentration of 6␤-hydroxycortisol for a 24-hr pooled urine collection. When the concentration of 6␤-hydroxycortisol was below the lower limit of quantitation (which occurred in 4 of 12 subjects treated with ketoconazole), the concentration was assumed to be equal to the lower limit of quantitation for the purpose of estimating 24-hr recovery (in which case the 24-hr recovery represents an upper limit, potentially resulting in an underestimate of ketoconazole inhibitory effect). Paired t test analyses were used to statistically compare terfenadine carboxylate terminal half-life and AUC ϱ in the absence vs. presence of nelfinavir and 24-hr urinary recoveries of 6␤-hydroxycortisol in the absence vs. presence of drug treatment (nelfinavir, ketoconazole, rifampin). K i values were determined with PCNONLIN software (SCI Software, Lexington, KY). Raw data were fitted to a Michaelis-Menten competitive inhibition model described by the equation: Results Selectivity of Nelfinavir Mesylate on the Inhibition of P450 Isoforms. The inhibition of specific P450 isoforms by nelfinavir mesylate was investigated using various P450 isoform-specific probe substrates. Among the various positive control inhibitors, extent of inhibition was at least 60% at the lowest probe substrate concentration. K i values for the inhibition of various P450s were determined when the criterion of Ն10% decrease in probe substrate activity was observed with up to 100 M nelfinavir in preliminary studies. This criterion was met for CYP3A4, CYP2C19, CYP2D6, and CYP1A2 (data not shown), and further studies were conducted to evaluate the K i of nelfinavir for these specific P450 isoforms. The results are summarized in table 1. Nelfinavir did not significantly inhibit the CYP2E1-, CYP2C8-, or CYP2C9-mediated reactions, and consequently K i values were not determined. The HIV-PIs ritonavir, indinavir, and saquinavir have been shown to inhibit CYP3A4 (Abbott Laboratories, 1996; Time Course of Inhibition and the Effect of Dialysis on Catalytic Activity. Nelfinavir (10 M) added to human liver microsomes in the absence of preincubation with NADPH did not display timedependent inhibition of CYP3A4-mediated testosterone 6␤-hydroxylation ( Effect of Modifiers on the Inhibition of CYP3A4. Several modifiers were chosen to investigate the potential for a reactive metabolite to leave the active site and to inhibit CYP3A4. A supratherapeutic concentration of nelfinavir (10 M) was selected to increase the generation of metabolite levels to assess the effects of the modifiers. The addition of highly reactive nucleophiles such as glutathione (1 and 5 mM) and N-acetylcysteine (1 mM) did not alter the timedependent loss of CYP3A4 activity ( Effect of Nelfinavir on P450 Content in Human Liver Microsomes. The results of spectral studies that assessed the effect o