Original Paper

Pharmacology 1992;45:121-128

Department of Pharmacology, University of Toledo, College of Pharmacy, Toledo, Ohio, USA

Key Words Ethosuximide Clearance Cytochromes P450 Cytochrome P450IIIA Substrate

In vivo Evidence that Ethosuximide Is a Substrate for Cytochrome P450IIIA

Abstract The role of various subfamilies of rat hepatic cyto­ chrome P450 in the oxidation of ethosuximide was evaluated by comparing ethosuximide clearance in control rats and those pretreated with relatively selective P450 inducers and/or inhibitors. Clotrimazole pretreatment increased ethosuximide clearance threefold (p < 0.005). Dexamethasone increased ethosuximide clearance twofold (p < 0.001), and the dexa­ methasone effect was completely abolished by a single dose of triacetyloleandomycin. These results suggest a prominent role for cytochrome P450I1IA in ethosuximide metabolism in the rat. Isoniazid increased ethosuximide clearance twofold (p < 0.001), and this effect was abolished by a single dose of diallylsulfide, suggesting that ethosuximide is also processed by cyto­ chrome P450IIE1 in rats. Phénobarbital pretreatment in­ creased ethosuximide clearance 2-2.7 fold (p < 0.001); an effect that was only partially reversed by orphenadrine, an inhibitor of cytochrome P450IIB/IIC enzymes. This suggests a quantitatively less important role for the IIB/IIC subfamilies in processing ethosuximide, since phénobarbital is an inducer of P450 subfamilies 1IB, IIC, IIE, and IIIA. Neither the cyto­ chrome P450IA inducer, P-naphthoflavone, nor the inhibitor, a-naphthoflavone altered ethosuximide clearance. Ajmaline, an inhibitor of cytochrome P450IID, had no effect on etho­ suximide clearance. Together, these findings suggest that etho­ suximide is principally oxidized by cytochrome P450IIIA, and that cytochrome P450IIE may play an important role. Cytochromes P450IIB/C play less prominent roles in ethosux­ imide oxidation, and neither cytochrome P450IA nor cyto­ chrome P450IID is involved.

Received: August 27. 1991 Accepted: December 2,1991

Kenneth Bachmann, PhD, FCP Director, Center for Applied Pharmacology Professor of Pharmacology The Univcnity of Toledo College of Pharmacy 2801 W. Bancroft. Toledo. OH 43606 (USA)

© 1992 S. Karger AG. Basel 0031-7 0 12/92/ 0453-012152.75/0

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Kenneth Bachmann Chang An Chu Vincent Greear

Table 1. Sequence of inducer/inhibitor pretreatments and ethosuximide treatment Day 1

2

3

4

1

vehicle

vehicle

vehicle

ethosuximide

2

inducer

inducer

inducer

ethosuximide

3

inducer

inducer

inducer

inhibitor/ethosuximide

4

vehicle

vehicle

vehicle

inhibitor/ethosuximide

Introduction The hepatic mixed function oxidases (MFOs) play an important role in the detoxi­ fication and activation of numerous endoand xenobiotics [1, 2]. In humans and other vertebrates it is generally held that the activi­ ties of the cytochrome P450I. II, and III fami­ lies account for nearly all of the P450-dependent oxidation of xenobiotics [3]. Being able to gauge the activities of the cyto­ chromes P450 in vivo, particularly with re­ gard to host-factor influences on them, has obvious predictive therapeutic and toxico­ logic value. Methods for probing in vivo MFO activity have included the measure­ ment of drug concentrations in plasma [4]; estimation of drug half-lives [5]; CCF breath tests [6, 7]; urinary 6(3-hydroxycortisol levels and urinary D-glucaric acid levels [8], and uri­ nary drug/metabolite ratios [9], Though such methods vary in predictive value they gener­ ally all suffer either from a lack of specificity or generalizability. Our approach to characterizing xenobiotic-induced effects on human MFOs in vivo addresses those difficulties, at least partially, through the use of multiple probes. Each probe is selected taking account of its phar­ macokinetic characteristics, safety, ease of

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measurement in biological fluids, and relative substrate specificity. Probe clearances are es­ timated from single plasma concentration measurements after single doses [10]. We have used quinidine as a probe for P450IIIA activity; others have used erythromycin [7], However, concerns about the safety of quini­ dine and also its substantial nonmetabolic clearance and the necessity of administering radioisotopically labeled erythromycin intra­ venously have prompted us to search for other suitable P450IIIA probes. We describe in vivo experiments in rats aimed at determining whether ethosuximide is a substrate for P450IIIA in rats as a prece­ dent step in ascertaining the suitability of ethosuximide as a P450IIIA probe in hu­ mans. Methods Animals Male Sprague-Dawley rats ranging in weight from 180 to 332 g (mean. 222 g) were used in groups of 4-6. All rats had free access to standard chow and water. Animals were pretreated with either an active inducer, an inducer followed by an inhibitor, vehicle followed by an inhibitor, or with vehicle alone (1 % mcthylcellulose).

Bach mann/Chu/Greear

Ethosuximide Metabolism by Cytochrome P450IIIA

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Group

Inducer Pretreaimenis (i-Naphthoflavone (BNF) was used as an inducer of cytochrome P450IA activity: phénobarbital (PB) was used as an inducer of cytochrome P450IIB activ­ ity; isoniazide (INH) was used as an inducer of cyto­ chrome P450IIE activity; dexamethasone (DEX) and clotrimazole (CTZ) were used as inducers of P450IIIA activity. PB. BNF. INH. and CTZ were administered in doses of I00mg/kg/day for 3 days, and DEX was administered in a dose of 50 mg/kg/ day for 3 days. Inhibitor Pretreatments The following inhibitors were used: a-naphthoflavone (ANF). orphenadrine. diallylsulfide (DAS), and triacetyloleandomycin (TAO) were used as inhibitors of P450IA, IIB. HE and IIIA activity, respectively. ANF (100 mg/kg), orphenadrine (100. 250. and 500 mg/kg). DAS (500 mg/kg). and TAO (500 mg/kg) were given in single doses 24 h following the last pre­ treatment dose of BNF. PB, and DEX, respectively. Ajmalinc (500 mg/kg) was used as an inhibitor of cyto­ chrome P450IID1, and was administered in a single dose to animals that had received only vehicle pre­ treatment (c.g. group 3 only in table 1). Ethosuximide Administration and Measurement Ethosuximide was prepared as a 3.5% (w/v) solu­ tion dissolved in normal saline. Ethosuximide was administered in a dose of 35 mg/kg via a tail vein either 24 h after the third inducer pretreatment dose, or 2 h after the single inhibitor dose. Animals were anesthetized with urethane ( 1.2 g/kg) approximately 15 min prior to ethosuximide infusion. Plasma was separated from heparinized whole blood at room temperature using a bench top centri­ fuge for5 min. Plasma was stored at - 20 °C for up to 5 days. Concentrations of ethosuximide were measured in plasma by fluorescence polarization immunoassay (fpia) using a TDx analyzer (Abbott Laboratories, Ir­ ving, Tex.) and commercially available reagents. The assay has a coefficient of variation of < 5 % , and a detection limit of 0.5 mg/1.

Ethosuximide clearance (CL) was estimated from plasma concentration measurements in samples ac­ quired from blood drawn 24 h after ethosuximide administration according to the following expression: CL = [In(D/V) - InCt] X (V/t), where D is the dose of ethosuximide (35 mg/kg), t is the sampling time (24 h). C, is the concentration of ethosuximide. and V is the volume of distribution of ethosuximide which was set at 0.7 liters/kg [11], Statistics A one-way ANOVA and a post hoc Tukey test were used to test for differences between ethosuximide clearances for vehide-pretreated. inducer-pretreated and inducer/inhibitor pretreated groups. An unpaired t test was used to test for differences in ethosuximide clearances between vehicle control animals and those administered ajmaline. Differences were considered significant at p < 0.05.

Results Ethosuximide CL was neither enhanced by multiple-dose BNF pretreatment (0.024 liters/h/kg cf 0.022 liters/h/kg), nor inhibited by single-dose ANF pretreatment either fol­ lowing (0.024 liters/h/kg) or absent (0.024 li­ ters/h/kg) BNF pretreatment (table 2), sug­ gesting an insignificant role for the cyto­ chrome P450IA subfamily in processing etho­ suximide in rats. The absence of a significant inhibitory effect of a high dose of ajmaline (0.031 liters/h/kg cf 0.036 liters/h/kg for con­ trol animals: table 2) likewise suggests a mi­ nor role, if any, for the debrisoquine/sparteinc isoform (cytochrome P450IID1) in the nor­ mal oxidation of ethosuximide in rats. PB pretreatment elevated ethosux­ imide CL 2.0- to 2.7-fold (table 2; p < 0.05). Flowever, orphenadrine in single doses of 100-500 mg/kg failed to restore ethosux­ imide CL values back to control levels, sug­ gesting that the PB-induced increase in etho­ suximide CL arose only partially - if at all -

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Pretreatnient Schedules Animals were randomly allocated to pretreatment groups illustrated in table 1. Each pretreatment was administered in a single dose per day. and each pretreatment dose followed the previous one by 24 h. All pretreatments were administered by gavage in a 1 % suspension of mcthylcellulose.

Table 2. Effects of inducer or inducer/inhibitor pretreatments on ethosuximide clearances in rats Pretreatm ent1

Experimental ethosuximide CL liters/h/kg

Control ethosuximide CL liters/h/kg

P

BNF

(IA)

0.022 ±0.002

0.024 ±0.001

NS

BNF/ANF (IA)

0.024 ±0.001

0.024 ±0.002

NS

PB

(IIB)

0.077 ±0.003

0.029 ±0.002

In vivo evidence that ethosuximide is a substrate for cytochrome P450IIIA.

The role of various subfamilies of rat hepatic cytochrome P450 in the oxidation of ethosuximide was evaluated by comparing ethosuximide clearance in c...
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