Introduction

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Cytochrome P450 (CYP)¹ enzymes play a key role in the metabolism of many exogenous and endogenous substances (Porter and Coon, 1991), with CYP3A being one of the most important CYP subfamilies for drug metabolism (Wright and Paine, 1994). Several drugs have been investigated as probes of CYP3A activity, including midazolam (Thummel et al., 1994), erythromycin (Lown et al., 1992), nifedipine, dapsone, lidocaine (Watkins, 1994), and quinidine (Guengerich et al., 1986). Cortisol 6-hydroxylation has also been employed as a CYP3A expression marker (Horsmans et al., 1992).

Ethosuximide is an anticonvulsant that has been investigated as an alternative probe for CYP3A activity (Bachmann et al., 1992; Bachmann and Jauregui, 1993). Ethosuximide is well suited for use as a probe molecule in that it can be given orally, its clearance can be estimated from a single plasma or saliva sample (Bachmann and Jauregui, 1993), and it is not significantly bound by plasma proteins. In vivo studies have indicated that ethosuximide is principally oxidized by CYP3A in rats (Bachmann et al., 1992) and humans (Bachmann and Jauregui, 1993). The primary ethosuximide metabolite has been shown to have an -hydroxy group on the ethyl side chain, 2-(1-hydroxyethyl)-2-methylsuccinimide (Horning et al., 1973; Pettersen, 1980; Maurer, 1990; Millership et al., 1993; Millership et al., 1995; Pisani et al., 1995), accounting for 40-60% of the administered drug excreted into the urine in rats and humans (Horning et al., 1973; Millership et al., 1993; Pisani et al., 1995). Other forms of the drug excreted in urine include unchanged drug (about 10% of administered drug), glucuronide conjugates (20-40%), a ring-hydroxylated form (2-ethyl-3-hydroxy-2-methylsuccinimide, near 7%), a form with a -hydroxy group on the ethyl side chain (2-3%), and dihydroxy and carboxylic acid forms detected at lower levels (Horning et al., 1973; Pisani et al., 1995).

The present study was undertaken to determine how in vitro ethosuximide biotransformation and ethosuximide metabolite formation are affected when ethosuximide is incubated with isolated rat microsomes enriched with different CYP subfamily enzymes. An HPLC method developed in our laboratory was used to measure the levels of ethosuximide and its primary metabolite after in vitro incubation of ethosuximide with isolated rat liver microsomes enriched in selected CYP isoforms. Incubation experiments included time series measurements with a selected subset of the isoform-enriched microsomes, 60-min incubations with each type of isoform-enriched microsomes considered in this study, and inhibition measurements made with specific CYP subfamily antibodies.

	Materials and Methods

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Chemicals. NADP and 3,3-dimethylglutarimide were purchased from Aldrich Chemical Company (Milwaukee, WI); methyl cellulose 1500 USP from Ruger Chemical Company (New York, NY); rabbit anti-rat CYP2B and CYP3A2 antisera from Gentest Corporation (Woburn, MA); Bio-Rad protein assay reagent from Bio-Rad Laboratories (Hercules, CA); and HPLC grade acetonitrile from Fisher Scientific (Pittsburgh, PA); all other chemicals were the highest grade available from Sigma Chemical Company (St. Louis, MO). HPLC grade water was generated on a Millipore MilliQ system (Bedford, MA).

Preparation of Liver Microsomes. Fifty male Sprague-Dawley 230-270 g rats from Harlan Sprague-Dawley (Indianapolis, IN) were divided randomly into seven groups. Inducing agents suspended in 1% methyl cellulose (MC) were administered by single daily gavage for 3 days. Seven control animals received only MC suspension, eight animals received BNF (a CYP1A inducer), eight received PB (a CYP2B/2C/3A inducer), seven received CTZ (a CYP3A inducer), six received INH (a CYP2E1 inducer), seven received CLO, a CYP4A inducer), and seven received CDD3543, a CTZ-analog and CYP3A inducer). Doses of 100 mg/kg/day were used for BNF, CTZ, and INH, while 50 mg/kg/day was used with PB, CLO, and CDD3543. Livers were excised 24 hr after the last dose except in imidazole-treated animals (CTZ and CDD3543) which were excised 48 hr after the last dose (Ritter and Franklin, 1987). Animals were sacrificed under CO₂ anesthesia after an overnight fast. The liver was quickly removed, perfused with 1.15 g/ml potassium chloride solution, homogenized in four volumes of ice-cold 1.15 g/ml potassium chloride solution, centrifuged at 10,000 × g for 20 min at 4°C, and the supernatent was then further ultracentrifuged at 105,000 × g for 60 min at 4°C. The microsomal pellets were resuspended in 10 ml of 100 mM pH 7.4 potassium phosphate buffer containing 0.1 mM EDTA. Protein and CYP levels were measured by standard methods (Bradford, 1976; Omura and Sato, 1964). There were no significant differences in initial or final animal weights or final liver weights between any of the animal treatment groups. CYP levels per mg protein were significantly higher for all treated animal groups vs. controls, with nmol CYP/mg protein levels of 0.40 ± 0.22, 1.38 ± 0.28, 1.95 ± 0.22, 1.00 ± 0.22, 1.15 ± 0.17, 2.29 ± 0.51, and 3.54 ± 0.68, respectively, for microsomes from control, BNF, PB, INH, CLO, CTZ, and CDD3453 treated animals. Microsomal suspensions were stored at 80°C until use.

Incubation Time Series. Incubations with microsomes from control, BNF, INH, CTZ, and CLO treated animals were carried out aerobically at 37°C in glass culture tubes. NADPH generating system was prepared by adding 5.4 mg NADP, 27.2 mg glucose-6-phosphate, and 1.0 unit glucose-6-phosphate dehydrogenase to 1.0 ml of 60 mM magnesium chloride and preincubating 5 min at 37°C. Microsome suspensions were thawed and diluted with 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA to a final concentration of 10 mg protein/ml. Seven hundred microliters of 1.0 M 7.4 pH potassium phosphate buffer, 100 µl of 10 mM aqueous ethosuximide solution, 100 µl of 10 mg protein/ml microsomal suspension, and 100 µl of NADPH generating system solution were then added to each culture tube and incubated at 37°C with shaking for 0, 10, 30, 60, or 120 min. Metabolism was stopped at the end of the incubation period by placing the tube on ice and adding 500 µl of ice-cold 0.2 g/ml trichloroacetic acid (TCA) solution. Five hundred microliters of an aqueous 200 µg/ml 3,3-dimethylglutarimide solution was then added to each tube as an HPLC internal standard. Each tube was centrifuged at 9600 × g for 10 min, and the supernatent was filtered through a 0.2 µm nylon syringe filter. Samples were found to be stable for at least one month when stored at 4°C. Three tubes were prepared at each time period for each inducing agent using microsomes from a single animal source for each agent, with duplicate HPLC assays performed on 0 and 60 min samples and single sample assays for other incubation time samples.

60-Minute Incubations. Sixty-minute incubations were carried out with all microsome groups using the procedures described for the time series incubations. Microsomes from three different animals were used for each treatment group, with three incubations performed for each animal source, yielding nine samples per treatment group with a single HPLC assay performed on each sample.

Antibody Inhibition. Microsomes from CTZ treated animals were incubated after exposure to anti-CYP3A2 antibodies, whereas microsomes from PB treated animals were tested after exposure to either anti-CYP2B1 or anti-CYP3A2 antibodies. Microsomal suspensions were diluted to 2 mg protein/ml with 100 mM phosphate buffer containing 0.1 mM EDTA to match supplier suggested antibody conditions. Fifty microliters of this microsomal suspension were then preincubated at room temperature for 30 min with either 0, 10, 20, or 50 µl of anti-CYP rabbit antiserum. An appropriate volume of normal rabbit serum was added to keep the total rabbit serum volume in each tube at 50 µl. After preincubation, 340 µl of 0.1 M pH 7.4 phosphate buffer, 10 µl of 10 mM ethosuximide solution, and 50 µl of NADPH generating solution was added to each tube and incubated at 37°C for 120 min. After incubation, tubes were placed on ice, 250 µl of ice-cold 0.2 g/ml TCA solution was added, 250 µl of HPLC internal standard solution was added, and samples were prepared as described previously. Triplicate HPLC assays were performed on all samples.

HPLC Assay Procedure. Samples were assayed by reversed phase HPLC using a 20-µl injection onto a Waters HPLC system containing two Model 501 pumps, a Model U6K injector with 2.0 ml sample loop, a Model 484 UV detector set at 195 nm detection, and a NovaPak 3.9 mm × 15 cm C18 column. Mobile phase consisted of water as phase A and acetonitrile as phase B flowing at a rate of 2.0 ml/min, with a gradient elution profile of 3.0-35.0%B linear ramp from 0-6.4 min, 35.0-3.0%B linear ramp from 6.4-7.0 min, followed by a 9-min washout period. Standards were prepared on ice with the same reagents as the incubation samples, but the TCA solution was added first and the ethosuximide solution was adjusted to give equivalent incubation media ethosuximide concentrations of 2, 20, 200, 1000, and 2000 nmol/ml. Control samples at 2.0, 200, and 2000 nmol/ml were prepared similarly by separate weighing. A two-parameter quadratic equation with zero intercept was used to produce a calibration curve for the ethosuximide to internal standard peak area ratio. As there was no source of the primary ethosuximide metabolite, the metabolite was assumed to have the same peak area response per mole as ethosuximide and the ethosuximide calibration curve was used to quantify the metabolite level. Relative error and within/between-day coefficients of variation were determined by multiple measurements on control samples and on samples produced by incubation with CTZ microsomes for 0, 30, and 120 min. To identify the ethosuximide metabolite structure, Electrospray Ionization-Liquid Chromatography-Mass Spectrometry (ESI-LCMS) analysis was performed on a 240 min CTZ microsome incubation sample using a similar gradient elution profile with 0.1% ammonium hydroxide post-column ionization.

Statistical Analysis. Results are expressed as mean ± SD. Differences between groups were assessed by one-way ANOVA with Tukey's post-hoc analysis using p < 0.05 as the criterion for statistical significance. Linear regression was employed to evaluate correlations between continuous variables.

	Results and Discussion

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HPLC Assay Method Validation. Because it is difficult to detect the small changes in ethosuximide levels that occur during in vitro microsomal incubations, it became necessary to measure the corresponding increase in ethosuximide metabolite levels. To this end, a novel HPLC method was developed to quantify the levels of ethosuximide and its primary metabolite in incubation samples. This is the first reported HPLC method for analysis of an ethosuximide metabolite, as all previously published ethosuximide metabolite measurements have been made using GC-MS (Horning et al., 1973; Pettersen, 1980; Millership et al., 1993; Millership et al., 1995). An example chromatogram illustrating clear separation of ethosuximide, primary metabolite, and internal standard peaks is provided in fig. 1A. A smaller peak apparently resulting from a less prominent ethosuximide metabolite appears near 4.5 min elution but was too small to consistently quantify. Fig. 1B illustrates the metabolite peak increases in size as the period of CTZ microsome incubation increases. Ethosuximide measurement accuracy ranged from 5.2% to + 3.7% over the entire 2.0-2000 nmol/ml range, while within- and between-day variation over these concentrations ranged from 0.6-6.3%. Primary metabolite within- and between-day variation ranged from 0.7%-6.3% over the measured concentration range of 3.5-153 nmol/ml, with the exception of a 13.7% within-day variation at the lowest measured concentration.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   HPLC chromatographs of microsome incubation samples. Graphs represent relative absorbance at 195 nm vs. the elution time after sample injection. Peaks are: M, presumed minor metabolite; p, primary metabolite; I, internal standard, E, ethosuximide. Plot A is a chromatograph for a 120 min CTZ microsome incubation sample. Plot B shows a series of chromatograms for CTZ samples incubated for different periods of time. Some peaks in Plots B have been graphically truncated for clarity.

Microsome Incubations. Fig. 2A shows only CTZ microsomes produce detectable drops in ethosuximide levels in the time series experiments, with ethosuximide concentrations for CTZ microsomes significantly lower at 60 and 120 min compared with those for each of the other microsome treatment groups. Linear regression analysis of the ethosuximide concentration vs. time for the 0-60 min samples of each microsome group shows only CTZ microsome values correlate strongly with incubation time (r = 0.9965). Other microsome groups exhibit weak correlations and yield essentially horizontal fitted lines. Fig. 2B shows the CTZ microsomes produce significantly higher metabolite levels than all other microsome treatment groups at each incubation time period. At 120 min incubation, the metabolite level for CTZ microsomes was 36.5-fold that of control microsomes. Metabolite levels for all microsomes tested correlated strongly with incubation time over the first 60 min.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Changes in ethosuximide and primary metabolite levels with microsome incubation time. CTZ, clotrimazole; INH, isoniazid; and BNF, -naphthoflavone. c, i, b indicate a significant difference vs. corresponding values at the same incubation time for control, INH, and BNF microsome incubations, respectively. Linear regression analyses were carried out on the 0-60 min samples for each inducing agent treatment group.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Ethosuximide levels and primary metabolite formation rates after 60-min incubations. CTZ, clotrimazole; INH, isoniazid; and BNF, -naphthoflavone; CDD, CDD3543; CLO, clofibrate; PB, phenobarbital. Lower case abbreviations above bars indicate significant differences vs. the listed inducing agent treatment groups.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Inhibition of ethosuximide metabolite formation by antibodies to specific CYP isoforms. Plot A shows the inhibition of phenobarbital-induced microsomes after incubation with antibodies to CYP3A2 (solid symbols) and CYP2B1 (open symbols). Plot B shows the inhibition of clotrimazole-induced microsomes after incubation with anti-CYP3A2 antibodies. Numbers next to symbols indicate a statistically significant difference vs. the corresponding metabolite level at the antiserum volume listed.