Inhibition of CYP2E 1 by chlormethiazole as measured by chlorzoiazone pharmacokinetics in patients with alcoholism and in healthy volunteers

Backpound: Chlormethiazole has been shown in in vitro studies, with use of rat and human liver micro- somes, to specifically inhibit cytochrome P4502El (CYP2El)-mediated activity by inhibition of the rate of CYP2El gene transcription. It is known that CYPZEl is involved in the activation of many low-molecular- weight toxins and carcinogens and may be involved in the development of alcohol-induced liver disease. Methods: The pharmacokinetics of a single oral dose of 250 mg chlorzoxazone, a marker of the activity of CYP2E1, were measured in five healthy drug-free volunteers and in 16 patients with alcoholism receiving 1.2 gm or 2.4 gm chlormethiazole per day for 1, 2, or 3 days. The patients were starting an alcohol- withdrawal program and were supposed to have an induced CYP2El activity. Resz&: The results suggest that chlormethiazole strongly decreased chlorzoxazone clearance in the patients with alcoholism compared with clearance in the control subjects (3.98 + 1.8 L/hr versus 12.7 + 5.6 L/hr, p < O.OOS), prolonged the elimination half-life (3.91 + 1.23 hours versus 1.12 f 0.34 hours; p < O.OOl), and caused a threefold increase in the area under the concentration versus time curve of chlorzoxazone (73.0 2 35.5 mg . hr/L versus 21.3 + 13.7 mg . hr/L; p < 0.005). They also suggest that chlormethia- zole significantly decreased the area under the concentration versus time curve of the metabolite 6-hydroxy- chlorzoxazone (4.56 2 1.27 mg . hr/L versus 7.1 2 1.84 mg . hr/L; p < 0.05). Conclusion: Chlormethiazole administration seems to result in a marked reduction of CYP2El activity in subjects with high CYP2El activity and could at least partially explain the claimed hepatoprotective action of this drug. (Clin Pharmacol Ther 1998;64:52-7.)

Chin B. Eap, PhD, Chris&me Schnyder, MD, Jaques Besson, MD, Line Savary, and Thierry Buclin, MD Prilly and Lausanne, Switzerland

Cytochrome P4502El (CYP2El) is involved in the bioactivation of many low-molecular-weight toxins and carcinogens, including benzenes, N-nitrosamine, styrenes, and some halocarbons. A recent study has shown a strong reduction of benzene metabolism and toxicity in knock-out mice that lack CYP2El expres- sion.* CYP2El is also involved in the metabolism of some drugs or substances such as acetaminophen (INN,

From the Departement Universitaire de Psychiatric Adulte, HBpital de Cery, Prilly, and the Division de Pharmacologic Clinique, Cen- tre Hospitalier Universitaire Vaudois, Lausanne.

Received for publication Dec. 17, 1997; accepted March 3, 1998. Reprint requests: C. B. Eap, PhD, Unite de Biochimie et Psychophar-

macologie Clinique, Hopital de Cery, CH-1008 Prilly-Lausanne, Switzerland. E-mail: Chin.Eap@inst.hospvd.ch

Copyright 0 1998 by Mosby, Inc. 0009-9236/98/$5.00 + 0 13/l/90052

paracetamol),j fluorinated volatile anesthetics such as enflurane, sevoflurane, methoxyflurane, and isoflurane,J and ethanol.5 The hepatotoxicity of acetaminophen is thought to be mediated by the CYP2El-catalyzed for- mation of a hepatotoxic metabolite, namely, the N-acetyl-p-benzoquinone imine. CYP2E 1 -catalyzed formation of reactive acyl halide from enflurane, a fluo- rinated ether, leads to the production of free fluoride ions by the reaction of the halide with water, and high concentrations of fluoride ions are associated with volatile anesthetic-induced nephrotoxicity.4 CYP2El activity is higher in alcoholic and obese subjects,5 and it has been proposed that CYP2El might be involved in the development of alcohol-induced liver disease, pos- sibly through increasing liver peroxidation.

Chlorzoxazone is a skeletal-muscle relaxant used for the treatment of painful muscle spasms. Its main

52

CLINICAL PHARMA COLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Eap et al. 53

metabolite is 6-hydroxychlorzoxazone, and this meta- bolic pathway is a useful marker of the activity of CYP2E1.7 An increase of chlorzoxazone metabolism has been shown in patients with alcoholism compared with that in nonalcoholic control subjects.8 An induc- tion of chlorzoxazone biotransformation has also been measured after the administration of isoniazid, a CYP2El inducer, to healthy volunteers.9 Finally, seri- ous obesity, which appears to have a selective effect on CYP2E1, is associated with increased 6-hydroxylation of chlorzoxazone in humans.lO The involvement of other enzymes, namely, cytochrome P4501A1,” cytochrome P4501A2,12 and cytochrome P4503A4,13 has also been suggested in the biotransformation of chlorzoxazone. However, the participation of cytochrome P4501Al and cytochrome P4501A2 in chlorzoxazone metabolism can be assumed to be neg- ligible. Indeed, CYPlAl is not constitutively expressed in human liver and, although smoking induces CYPlA2, no differences in chlorzoxazone metabolism were found between smokers and nonsmokers.8 Finally, furafylline, a selective CYPlA inhibitor, showed little inhibition of chlorzoxazone 6-hydroxylase activity.13

Because of the role of CYP2El in enhancing the tox- icity of various substances, compounds that can block or reduce its activity are of interest. It has been shown that disulfiram and its metabolite formed in vivo, namely, diethyldithiocarbamate, are potent inhibitors of CYP2El.4J4 It has also recently been shown in human healthy volunteers that ingestion of watercress, a veg- etable rich in gluconasturtiin, a precursor of phenethyl isothiocyanate, which is an effective inhibitor of CYP2E1, causes a decrease in the levels of oxidative metabolites of acetaminophen.15 Chlormethiazole is a hypnotic-sedative-anxiolytic-anticonvulsant drug used for the management of alcohol withdrawal syndrome. It has been shown that chlormethiazole is an efficient inhibitor of CYP2El expression in rat liver.16 These results confirm the results of an in vitro study that found a decreased rate of O-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin, a biotransformation step for which CYP2El is an efficient catalyst, in liver micro- somes from patients with alcoholism also receiving a chlormethiazole treatment compared with the rate in those not receiving treatment.17 Interestingly, some hepatoprotective action by chlormethiazole against toxic oxidation products in the liver of subjects with alco- holism has been claimed.is In our psychiatry depart- ment, chlormethiazole is given to patients with alco- holism entering an alcohol-withdrawal program as a routine treatment. We report here the pharmacokinetics of chlorzoxazone as measured in 16 patients with alco-

holism receiving chlormethiazole and in five healthy drug-free volunteers who served as control subjects.

MATERIAL AND METHODS Material and chlorzoxazone determinations.

Chlorzoxazone tablets (Escoflex, 250 mg tablets) were obtained from G. Streuli & Co. (Uznach, Switzerland). Determinations of chlorzoxazone and 6-hydroxychlorzoxazone plasma concentrations were done in duplicate by gas chromatography-mass spec- trometry, as extensively described elsewhere. l9 In summary, intraday and interday coefficients of varia- tion were always less than 9% and the limit of quan- titation of the method was found to be 5 rig/ml for the two compounds.

Human study. Five healthy volunteers, who were occasional drinkers and who did not drink any alcohol within 1 week before the study, and 21 patients admit- ted to our clinic for treatment in an alcohol-withdrawal program gave their written informed consent to partic- ipate in the study, which was approved by the institu- tional Ethics Committee of the University Department of Psychiatry (Lausanne, Switzerland). Patients with known or suspected hepatic cirrhosis and those with ALT and AST values higher than four times the upper limit were excluded from the study.

Chlormethiazole is given routinely to every patient entering the alcohol-withdrawal program at a usual starting dose of 2.4 gm per day (two tablets of 300 mg four times a day) for 1 week, with gradual tapering of the dose over 1 week during the second week. Patients are admitted to the program at various times of the day, but the majority of admissions are made in the after- noon. Various doses of chlormethiazole are thus given on the day of program admission (day 0). The study took place on day 2 (after 1 full day of chlormethia- zole treatment), day 3, or day 4 after program admis- sion. Because of the very short half-life of chlormethi- azole (3 to 5 hours), ls a full day of treatment is con- sidered sufficient to reach steady-state conditions. On the day of the study, at approximately 7 AM, an intra- venous catheter was inserted into a forearm vein and a blood sample (5 ml) was taken into a heparinized tube (time 0). After ingestion of 250 mg chlorzoxazone, blood samples were taken at %, 1, l%, 2, 3, 4, and 6 hours. Patients were asked to refrain from walking for 90 minutes and were given a standard breakfast 90 minutes after ingestion of chlorzoxazone. It should be mentioned that no further extended times were selected because of the difficulty of obtaining blood samples from patients over long periods. In fact, 5 of 21 sub- jects did not even complete the 6-hour study. After col-

54 Eap et al. CLINICAL. P HARMACOLOGY & THERAPEUTICS

JULY 1998

0 Chlorzoxazone - Patients (with chlormethiazole) 0 6-hydroxychlorroxazone - Patients (with chlormethiazole) A Chlorzoxazone - Controls (drug-free) A 6-hydroxychlorzoxazone - Controls (drug-free)

Fig 1. Plasma concentration-time profile of chlorzoxazone and 6-hydroxychlorzoxazone (mean f SD) after a single oral dose of 250 mg in five healthy drug-free subjects and in 16 patients with alcoholism entering an ethanol withdrawal program and receiving 1.2 or 2.4 gm/day chlomrethiazole for 1, 2, or 3 days.

lection, blood samples were centrifuged and the plasma samples stored at -20” C until analysis. The same blood collection scheme was used for the healthy volunteers.

The data from the five patients who did not complete the 6-hour blood sampling period were removed from subsequent analysis. Among the remaining 16 patients (14 men and 2 women; mean age f SD, 41 f 11 years; age range, 23 to 58 years; mean weight f SD, 71 f 12 kg; weight range, 49 to 89 kg), 14 subjects (12 men) received 600 mg chlormethiazole four times a day (total daily dose, 2.4 gm) and two patients received 300 mg four times a day (total daily dose, 1.2 gm) until the day of the study. All but two patients (one man and one woman) were smokers. They were all heavy drinkers and on admission to the alcohol-withdrawal program all but two patients had values of at least one of AST, ALT, and y-glutamyltransferase above the upper limit of the range (but still lower than four times the upper limit, as noted previously). The chlorzoxazone pharmacokinetic study took place after 1 day (two patients), 2 days (eight patients), or 3 days (six patients) of chlormethiazole treatment. There were no statistically significant differ- ences in pharmacokinetic parameters between patients receiving 1.2 or 2.4 gm chlormethiazole per day or between those for whom the study took place after 1, 2, or 3 days of chlormethiazole treatment (data not shown). All patients received vitamin supplements and 50 mg/day intravenous clorazepate, and one or several doses of tri- azolam and oxazepam were given when needed to nine

and four patients, respectively. The five healthy volun- teers (four men and one woman; mean age, 37 years; age range, 26 to 50 years; mean weight, 80 kg; weight range, 55 to 87 kg; two smokers) were free of any medication. None of the patients or the healthy volunteers reported any subjective effects from chlorzoxazone.

Phamacokinetic and statistical analysis. The chlor- zoxazone concentrations were considered to follow monocompartmental kinetics with first-order absorp- tion. The model was fitted to each individual set of time- concentration points by means of classic nonlinear regression, with a l/y2 weighting scheme. This approach provided estimates of the elimination rate constant, absorption rate constant, and apparent volume of distri- bution, from which the elimination and absorption half- lives and the apparent total clearance could be derived. The area under the curve (AUC) was also calculated with the trapezoidal rule. Although the 6-hydroxychlor- zoxazone concentrations seemed to follow a similar pro- file, no compartmental fitting could be applied in the absence of information about the fraction of the chlor- zoxazone dose transformed into 6-hydroxychlorzoxa- zone, which made it impossible to derive meaningful volume or clearance values. Thus only the AUC of 6- hydroxychlorzoxazone was calculated, with extrapola- tion toward infinity on the basis of the elimination rate constant value of chlorzoxazone, which appeared to be the limiting factor governing the terminal phase of 6- hydroxychlorzoxazone kinetics. The maximum plasma concentration was determined by data inspection. All

CLINICAL P HARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Eap et al. 55

Table I. Chlorzoxazone and 6-hydroxychlorzoxazone pharmacokinetic parameters in five drug- and alcohol-free healthy volunteers and in 16 patients with alcoholism receiving chlormethiazole

Healthy volunteers Patients with alcoholism Percent of

Mean Median Range Mean Median Range control p Value

k, a-9 0.87 + 0.17 Absorption tX (hr) 0.82 + 0.14 Chlorzoxazone C,, (mg/L) 6.84 k 3.31 k, WI 0.66 k 0.18 Elimination tK (hr) 1.12 + 0.34 CL/F (L/hr) 12.7 + 5.6 v/F (L) 20* 10 Chlorzoxazone AUC 21.3 f 13.7 (mg . hW 6-Hydroxychlorzoxazone 7.11 + 1.84 AUC (mg . hr/L)

0.83 0.69-1.15 1.22 + 0.87 0.86 0.33-3.04 140 0.84 0.61-1.01 0.91 f 0.62 0.81 0.23-2.12 111 8.48 2.48-9.66 9.13 + 2.75 8.65 5.17-16.1 133 0.69 0.42-0.83 0.19 + 0.06 0.19 0.1 l-0.30 29 1.01 0.84-1.63 3.91 + 1.23 3.68 2.28-6.09 349 12.0 5.6-18.6 4.0 f 1.8 3.5 1.6-8.2 31 14 11-35 20*5 19 12-31 100

17.1 11.5-44.4 73.0 + 35.5 68.0 28.7-164.7 343

7.11 4.11-8.82 4.56 zi 1.27 4.73 2.37-6.48 64

NS NS NS

< 0.001 < 0.001 < 0.005

NS < 0.005

< 0.05

k,, Absorption rate constant; NS, not significant; tz, half-life; C,,, maximum plasma concentration; k,, elimination rate constant; CL/F, apparent total clearance; V/F, apparent volume of distribution

the parameters estimated are presented both as mean values f SD and medians with ranges. The values were compared between patients and control subjects by means of a Wilcoxon-Mann-Whitney test.

RESULTS The mean plasma concentration-time profiles of

chlorzoxazone and 6-hydroxychlorzoxazone in the 16 patients receiving chlormethiazole and in the five healthy drug-free volunteers are shown in Fig. 1. Val- ues of the absorption rate constant, the absorption half- life, the maximum plasma concentration, the elimina- tion rate constant, the elimination half-life, the appar- ent total clearance, and the apparent volume of distri- bution as measured in both groups are provided in Table I. There were no significant differences in the absorp- tion rate constant (1.22 f 0.87 hr-1 versus 0.87 2 0.17 hr-l), the absorption half-life (0.91 rf: 0.62 hour versus 0.82 f 0.14 hour), the maximum plasma concentration (9.13 f 2.75 mg/L versus 6.84 + 3.31 mg/L), or the apparent volume of distribution (20 + 5 L versus 20 + 10 L) between chlormethiazole-treated patients with alcoholism and healthy drug-free volunteers. On the other hand, the elimination half-life was about 3.5fold higher in patients with alcoholism receiving chlorme- thiazole (3.91 f 1.23 hours) than in healthy drug-free volunteers (1.12 f 0.34 hours; p < O.OOl), and the apparent total clearance was about 3.2 times lower (4.0 f 1.8 L/hr versus 12.7 + 5.6 L/hr; p < 0.005).

The values of the AUC for chlorzoxazone and its metabolite (6-hydroxychlorzoxazone) as measured in both groups are also provided in Table I. The AUC of chlorzoxazone was about 3.4 times higher (73 + 35.5

mg . hr/L versus 21.3 +- 13.7 mg . hr/L; p < 0.005) and the AUC of 6-hydroxychlorzoxazone was 36% lower (4.56 + 1.27 mg . hr/L versus 7.11 f 1.84 mg . hr/L; p < 0.05) in patients with alcoholism receiving chlormethiazole than in healthy volunteers. The rate constant of the terminal phase of 6-hydroxychlorzoxa- zone disposition was similar to that of chlorzoxazone, suggesting that the appearance of 6-hydroxychlorzoxa- zone is the limiting step in its kinetics.

DISCUSSION Pharmacokinetic parameters in healthy volunteers as

calculated in this study are in agreement with those published previously.10~20 Because of the limited blood sampling times, the conditions presented in this study for determining pharmacokinetic parameters in subjects with alcoholism are far from ideal, and the data can only be considered as estimates. However, in these patients, the administration of chlormethiazole for 1, 2, or 3 days lead to an approximately 3.5-fold higher value for the elimination half-life and a 3.2-fold lower value for the clearance as compared with values in healthy volunteers. Accordingly, a lower (36%) AUC was mea- sured for the metabolite (6-hydroxychlorzoxazone). As previously mentioned, because of the very short half- life of chlormethiazole (3 to 5 hours),‘8 a full day of chlormethiazole treatment is considered to be sufficient to reach chlormethiazole steady-state conditions, and the inclusion of patients after either 1, 2, or 3 days would not theoretically change the influence of chlormethiazole on chlorzoxazone pharmacokinetics. However, CYP2El is known to be induced in subjects with alcoholism and the plasma clearance of chlorzox-

56 Eap et al.

azone was found to be enhanced by 13% in patients with alcoholism.* Because of the short half-life of CYP2El (2% days), a rapid decrease of CYP2El activ- ity can be measured in patients after ethanol with- drawal, with values returning to control values after 8 days of alcohol abstinence.21 No data are available concerning the rate of decrease of CYP2El activity dur- ing the first 3 days of abstinence but, as previously men- tioned, no statistically significant differences were noted in the pharmacokinetic parameters in this study between patients included after 1, 2, or 3 days of chlormethiazole treatment, that is, after approximately the same period of abstinence from alcohol.

Just before this article was submitted, another study was published on the effects of chlormethiazole on CYP2El activity in subjects with alcoholism and in control subjects, although the effects were only assessed by a single-point determination (6-hydroxy- chlorzoxazonelchlorzoxazone blood concentration ratio measured 2 hours after the oral intake of 500 mg chlor- zoxazone).** These investigators’ results are in com- plete agreement with ours, because chlormethiazole was found to strongly decrease 6-hydroxychlorzoxa- zone/chlorzoxazone ratios in chlormethiazole-treated control and alcoholic subjects compared with results in chlormethiazole-free control and alcoholic subjects. Interestingly, chlorazepate was found to have no influ- ence on CYP2El activity.**

The CYP2El gene is constitutively expressed and is regulated both at the transcriptional and at the posttran- scriptional levels. Ethanol has been shown to increase the rate of CYP2EI gene transcription and to stabilize CYP2E 1 messenger ribonucleic acid. l6 It has been pos- tulated that different transcriptional factors control the CYP2El expression under constitutive and under inducible conditions.16 Our results show that the clear- ance of chlorzoxazone is lowered by chlormethiazole in patients with alcoholism at the beginning of the with- drawal phase, that is, in subjects with induced CYP2El activities. The inhibition is such that the mean calculated clearance is lower than the value found in control sub- jects, which suggests that chlormethiazole acts both with the constitutive and the inducible systems. In an in vitro system under noninduced conditions, a lowering of 40% of rat CYP2El activity by chlormethiazole, although not significant, was measured. 16 In another study that used rat microsomes, the hepatoprotective effect of 2- (allylthio)pyrazine, an organosulfur compound, was examined and found to be effective in suppressing both constitutive and inducible CYP2El expression.23

Because of the potential toxicity of several sub- stances or drugs through activation by CYP2E1, com-

CLINICAL P HARMACOLOGY & THERAPEUTICS IULY 1998

pounds that can block or reduce its activity are of inter- est. Several compounds, such as disulfiram and diethyldithiocarbamate,’ chlormethiazole,l6 diallylsul- fide, allylmercaptan, allylmethylsulfide, and 2- (allylthio)pyrazine, 23 have been shown in vitro to inhibit CYP2El-mediated activity, but few studies have been done in vivo with human subjects. In a study with six human volunteers, a single 500 mg dose of disulfi- ram markedly decreased the chlorzoxazone elimination clearance (by 85%) and raised its elimination half-life (from 0.92 to 5.1 hours).14 In another study, disulfiram completely abolished the rise of plasma fluoride con- centrations in patients undergoing elective operations receiving enflurane in oxygen for 3 hours.4 Unlike disulfiram and diethyldithiocarbamate, which act as inhibitors of CYP2El-dependent catalytic activities, chlormethiazole specifically inhibits the elevation of CYP2El messenger ribonucleic acid by inhibition of the rate of the gene transcription. l6 Studies have used disulfiram and diethyldithiocarbamate to protect ani- mals against known CYP2El-mediated protoxin acti- vation, and studies with human subjects have confirmed the potential interest of these two compounds.14 It has been postulated, in in vitro studies, that chlormethia- zole could constitute one of the most nontoxic CYP2El inhibitors available for in vivo studies.16 In the present study, a marked reduction of CYP2El activity, as assessed by chlorzoxazone pharmacokinetics, was mea- sured in patients with alcoholism entering a withdrawal program, that is, in patients with induced CYP2El activity. This is an argument that supports the claimed hepatoprotective action of chlormethiazole against toxic oxidation products in the liver of patients with alcoholism.l*

We gratefully acknowledge the editorial assistance of Mrs. C. Bertschi and the bibliographic help of Mrs. M. Gobin, Mrs. J. Rosse- let, and Mrs. T. Bocquet.

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