Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes.

CYP2E1 activity is measured in vitro and in vivo via hydroxylation of the Chlorzoxazone (CHZ) producing the 6-hydroxychlorzoxazone (OH-CHZ) further metabolized as a glucuronide excreted in urine. Thus, the quantification of the OH-CHZ following enzymatic hydrolysis of CHZ-derived glucuronide appears to be a reliable assay to measure the CYP2E1 activity without direct detection of this glucuronide. However, OH-CHZ hydrolyzed from urinary glucuronide accounts for less than 80% of the CHZ administrated dose in humans leading to postulate the production of other unidentified metabolites. Moreover, the Uridine 5'-diphospho-glucuronosyltransferase (UGT) involved in the hepatic glucuronidation of OH-CHZ has not yet been identified. In this study, we used recombinant HepG2 cells expressing CYP2E1, metabolically competent HepaRG cells, primary hepatocytes and precision-cut human liver slices to identify metabolites of CHZ (300 µM) by high pressure liquid chromatography-UV and liquid-chromatography-mass spectrometry analyses. Herein, we report the detection of the CHZ-O-glucuronide (CHZ-O-Glc) derived from OH-CHZ in culture media but also in mouse and human urine and we identified a novel CHZ metabolite, the CHZ-N-glucuronide (CHZ-N-Glc), which is resistant to enzymatic hydrolysis and produced independently of CHZ hydroxylation by CYP2E1. Moreover, we demonstrate that UGT1A1, 1A6 and 1A9 proteins catalyze the synthesis of CHZ-O-Glc while CHZ-N-Glc is produced by UGT1A9 specifically. Together, we demonstrated that hydrolysis of CHZ-O-Glc is required to reliably quantify CYP2E1 activity because of the rapid transformation of OH-CHZ into CHZ-O-Glc and identified the CHZ-N-Glc produced independently of the CYP2E1 activity. Our results also raise the questions of the contribution of CHZ-N-Glc in the overall CHZ metabolism and of the quantification of CHZ glucuronides in vitro and in vivo for measuring UGT1A activities.

Differential Pulse Anodic Voltammetric Determination of Chlorzoxazone in Pharmaceutical Formulation using Carbon Paste Electrode.

The electrochemical behavior of chlorzoxazone at the carbon paste electrode was investigated in 0.04 mol/L Britton-Robinson buffer pH 6.50 using cyclic and differential pulse voltammetric techniques. Cyclic voltammetric studies indicated that the oxidation of the drug was irreversible and controlled mainly by diffusion. Experimental and instrumental parameters were optimized (50 mV/s scan rate, 50 mV pulse amplitude, and 0.04 mol/L Britton-Robinson (BR) buffer pH 6.50 as a supporting electrolyte) and a sensitive differential pulse anodic voltammetric method has been developed for the determination of the drug over the concentration range 0.17-1.68 µg/mL chlorzoxazone, with detection and quantitation limits of 0.05 and 0.16 µg/mL, respectively. The proposed voltammetric method was successfully applied to the determination of the drug in its pharmaceutical formulation (Myoflex tablets), and in spiked human urine samples.

Influence of diosmin pretreatment on the pharmacokinetics of chlorzoxazone in healthy male volunteers.

Chlorzoxazone, a centrally acting muscle relaxant, is a probe for cytochrome P450 2E1 (CYP2E1). The first part of the study consisted of oral administration of 250 mg of chlorzoxazone (Paraflex 250 tablet) alone to 12 healthy male volunteers. Blood samples were collected from the antecubital vein at intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours and urine voided during 0-4 and 4-8 hours was collected after the administration of chlorzoxazone. The second part of the study was conducted after a wash-out period of 7 days; 500 mg of diosmin (Venex 500) was administered daily for 9 days. On day 10, 250 mg of chlorzoxazone was administered. Blood and urine samples were obtained as mentioned above. Serum levels of chlorzoxazone were determined by HPLC. Pharmacokinetic parameters were determined based on non-compartmental model analysis using the computer program RAMKIN. Diosmin pretreatment significantly enhanced AUC, C(max) and t1/2 with a concomitant reduction in CL/f. The urinary excretion of 6-hydroxychlorzoxazone was decreased and unchanged chlorzoxazone was increased over 8 hours. Urinary metabolic ratios of 6-hydroxychlorazoxazone and chlorazoxazone were increased. After pretreatment with diosmin, overall excretion (0-8 h) of 6-hydroxychlorazoxazone and chlorazoxazone were decreased. Diosmin might have inhibited the microsomal CYP2E1-mediated hydroxylation of chlorazoxazone.

The CYTONOX trial.

INTRODUCTION: In Denmark, it is estimated that 3-5% of children are obese. Obesity is associated with pathophysiological alterations that may lead to alterations in the pharmacokinetics of drugs. In adults, obesity was found to influence important drug-metabolising enzyme pathways. The impact of obesity-related alterations on drug metabolism and its consequences for drug dosing remains largely unknown in both children and adults. An altered drug metabolism may contribute significantly to therapeutic failure or toxicity. The aim of this trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 substrates in obese versus non-obese children. METHODS: The CYTONOX trial is an open-label explorative pharmacokinetic trial. We intend to include 50 obese and 50 non-obese children. The primary end points are: in vivo clearance of CYP3A4, CYP2E1 and CYP1A2 substrates, which will be defined by using well-tested probes; midazolam, chlorzoxazone and caffeine. Each of the probes will be administered as a single dose. Subsequently, blood and urine samples will be collected at pre-specified times. CONCLUSION: The aim of the CYTONOX trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 in obese and non-obese children. The results are expected to be used in the future as a basis for drug dosing recommendations in obese children. FUNDING: The study was funded by the Danish Regions' "Medicinpuljen". The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TRIAL REGISTRATION: EudraCT: 2014-004554-34.

Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2E1.

Disulfiram and its reduced metabolite diethyldithiocarbamate have been identified previously as selective mechanism-based inhibitors of human liver microsomal cytochrome P450 2E1 in vitro. In animals, a single oral dose of disulfiram has been shown to produce a rapid and selective inactivation of hepatic P450 2E1 content and catalytic activity in vivo. This investigation explored the efficacy of single dose disulfiram as an inhibitor of human P450 2E1 activity in vivo. Clinical P450 2E1 activity was assessed by the 6-hydroxylation of chlorzoxazone, a metabolic pathway catalyzed selectively by P450 2E1. Six healthy volunteers received 750 mg oral chlorzoxazone on two occasions in a crossover design, 10 hours after 500 mg oral disulfiram, or after no pretreatment (control subjects). Disulfiram pretreatment markedly decreased chlorzoxazone elimination clearance to 15% of control values (from 3.28 +/- 1.40 to 0.49 +/- 0.07 ml/kg/min, p < 0.005), prolonged the elimination half-life (from 0.92 +/- 0.32 to 5.1 +/- 0.9 hours, p < 0.001), and caused a twofold increase in peak plasma chlorzoxazone concentrations (20.6 +/- 9.9 versus 38.7 +/- 10.3 micrograms/ml, p < 0.001). Disulfiram also profoundly decreased the formation clearance of 6-hydroxychlorzoxazone, from 2.30 +/- 0.93 to 0.17 +/- 0.05 ml/kg/min (p < 0.005). These findings show that a single dose of disulfiram significantly diminishes the activity of human P450 2E1 in vivo. The efficacy of single-dose disulfiram as an inhibitor of human P450 2E1 suggests that this modality for manipulating clinical P450 2E1 activity may provide a useful probe for delineating P450 2E1 participation in human drug biotransformation or for the treatment of poisoning by P450 2E1-activated toxins.

Impaired 6-hydroxychlorzoxazone elimination in patients with kidney disease: Implication for cytochrome P450 2E1 pharmacogenetic studies.

OBJECTIVE: The purposes of this study were (1) to describe the disposition of chlorzoxazone and 6-hydroxychlorzoxazone in patients with kidney disease, (2) to develop a population pharmacokinetic model including covariates that may influence the pharmacokinetic variability of both compounds, and (3) to examine the effect of covariates on the chlorzoxazone metabolic ratio. METHODS: Twenty-one subjects received a single oral dose of chlorzoxazone, 250 mg, and plasma and urine samples were collected for up to 120 hours. The impact of creatinine clearance (CL(cr)), age, and weight on chlorzoxazone and 6-hydroxychlorzoxazone clearance terms was assessed with NONMEM software (v.5, level 1.1; Globomax LLC, Hanover, Md) by use of a stepwise backward-elimination technique and the likelihood ratio test. RESULTS: A linear model with first-order absorption for chlorzoxazone and first-order formation for 6-hydroxychlorzoxazone simultaneously described the disposition of both compounds. Weight was a significant predictor of 6-hydroxychlorzoxazone formation clearance and other, unaccounted for clearance of chlorzoxazone, whereas CL(cr) was a significant predictor of 6-hydroxychlorzoxazone renal clearance. No relationship between CL(cr) and formation clearance was observed. The 6-hydroxychlorzoxazone area under the plasma concentration-time curve was inversely related to CL(cr), even within the range of normal renal function, resulting in chlorzoxazone metabolic ratio values that were substantially higher in subjects with kidney disease. Both the experimental data and model-based Monte Carlo simulations revealed greatly increased chlorzoxazone metabolic ratio values when CL(cr) was low and weight was high. CONCLUSIONS: Although cytochrome P450 (CYP) 2E1 activity, as estimated by 6-hydroxychlorzoxazone formation clearance, was not affected by kidney disease, the chlorzoxazone metabolic ratio was substantially elevated in these subjects. The results of this study show that the commonly used plasma-based chlorzoxazone metabolic ratio is dependent on renal function and, therefore, does not provide a reliable index of CYP2E1-mediated metabolism.

Urinary excretion of 6-hydroxychlorzoxazone as an index of CYP2E1 activity.

OBJECTIVE: To determine whether the urinary excretion of 6-hydroxychlorzoxazone is an index of CYP2E1 activity in vivo. METHODS: Male volunteers (n = 27; age range, 17 to 36 years) who were abstinent from alcohol were studied. Chlorzoxazone, 500 mg, was given orally and plasma was collected at 31/2, 41/2, 51/2, and 61/2 hours after dosing. Urine was collected for 8 hours. Ten volunteers participated in full kinetic studies to define the absorption phase and plasma area under the concentration-time curve of chlorzoxazone and the urinary kinetics of the 6-hydroxy metabolite. Chlorzoxazone and the 6-hydroxy metabolite were measured by high-performance liquid chromatography. CYP2E1 activity was expressed as a hydroxylation index (HI = mmole oral chlorzoxazone dose/mmole 6-hydroxychlorzoxazone in 8-hour urine). RESULTS: There was a significant positive correlation between plasma elimination rate constant for chlorzoxazone (Ke) and urinary excretion of the metabolite (n = 27, r = 0.42, p < 0.03) and a significant negative correlation between plasma Ke and HI (n = 27, r = -0.41, p < 0.04). The mean absorption rate constant for chlorzoxazone of 3.11 +/- 4.67 hr-1 was fivefold greater than the plasma Ke of 0.57 +/- 0.17 hr-1 for the full kinetic studies. The formation clearance of the 6-hydroxy metabolite was negative between plasma Ke of the parent compound and disposition rate constant for urinary excretion of the 6-hydroxy metabolite (n = 15, r = 0.85, p < 0.0001). CONCLUSIONS: The urinary excretion of 6-hydroxychlorzoxazone is limited by formation rate and may be useful as an in vivo probe of CYP2E1 activity.

The effect of endotoxin administration on the pharmacokinetics of chlorzoxazone in humans.

OBJECTIVE: Inflammation induced by Escherichia coli lipopolysaccharide alters the clearance of several hepatically eliminated drugs. Extensive rat liver research has shown CYP2E1 down-regulation after lipopolysaccharide administration. To further investigate this phenomenon in humans, lipopolysaccharide was administered to healthy male volunteers and chlorzoxazone was used as a CYP2E1 probe drug. METHODS: Twelve healthy men were given 500 mg oral chlorzoxazone after two daily lipopolysaccharide doses (20 endotoxin units/kg/day) and again after administration of saline solution in this balanced crossover study. Serum and urine chlorzoxazone and 6-hydroxychlorzoxazone were quantified, as well as cytokine and C-reactive protein levels. RESULTS: Lipopolysaccharide produced the expected induction of the acute-phase response shown by elevations in tumor necrosis factor, interleukin-6, C-reactive protein, and temperature. Lipopolysaccharide treatment failed to produce a significant change in the chlorzoxazone oral clearance (4.4 +/- 0.9 mL/min/kg for lipopolysaccharide versus 4.2 +/- 1.4 mL/min/kg for control) or the 6-hydroxychlorzoxazone formation clearance (2.8 +/- 0.65 mL/min/kg for lipopolysaccharide versus 2.5 +/- 0.9 mL/min/kg for control). The high intersubject variabilities in oral clearance and formation clearance were not accounted for by changes in protein binding, cytokine, or C-reactive protein values. In contrast, a significant increase in the 6-hydroxychlorzoxazone glucuronide renal clearance was observed (7.5 +/- 1.37 mL/min/kg for lipopolysaccharide versus 6.1 +/- 1.7 mL/min/kg for control). CONCLUSIONS: This study showed that the inflammatory response to lipopolysaccharide (20 endotoxin units/kg/day for 2 days) in humans does not consistently alter chlorzoxazone hepatic metabolism. However, the significant increase in renal clearance of the glucuronidated metabolite suggests that renal tubular secretion may be increased in humans with acute endotoxemia.

Fluorometric determination of chlorzoxazone in tablets and biological fluids.

A fluorometric determination for chlorzoxazone was developed based on the intrinsic drug fluorescence in chloroform using excitation and emission wavelengths of 286 and 310 nm, respectively. A calibration curve for chlorzoxazone in chloroform gave a linear working range of 0.027-2.3 microgram/ml (r = 0.9999) with the minimum detectability at 27 ng/ml. The procedure was applied to chlorzoxazone analysis in spiked plasma and urine samples. Minimum detectable drug levels in these samples were 60 and 130 ng/ml, respectively. Data revealed that chlorzoxazone could be determined in plasma and urine even in the presence of 20-fold molar excesses of its major metabolite, 6-hydroxychlorzoxaxone, and acetaminophen. The method also was applicable to chlorzoxazone analysis in a commercial dosage form containing acetaminophen.

Pharmacokinetics of chlorzoxazone in humans.

Twenty-three normal male subjects received 900 mg of acetaminophen and 750 mg of chlorzoxazone as an oral suspension. Analysis of plasma samples indicated a rapid absorption and rapid elimination of chlorzoxazone. Average values of the elimination half-life and plasma clearance were 1.12 +/- 0.48 hr and 148.0 +/- 39.9 ml/min, respectively. Analysis of urine samples showed that chlorzoxazone was eliminated from the body as the glucuronide conjugate of the intermediate metabolite 6-hydroxychlorzoxazone, to the extent of 74% of the dose. The plasma and the urinary excretion data were fitted to theoretical equations, and excellent fits were obtained using a five-parameter pharmacokinetic model.

Effects of steady-state lasofoxifene on CYP2D6- and CYP2E1-mediated metabolism.

BACKGROUND: Lasofoxifene, a selective estrogen receptor modulator, may be coadministered with other drugs, raising the issue of drug-drug interactions. OBJECTIVE: Using a 7-day, open-label, sequential study to determine whether lasofoxifene at steady-state concentration affects cytochrome P450-mediated drug metabolism. METHODS: Lasofoxifene was tested in 18 postmenopausal women with probe drugs for CYP2E1 and CYP2D6. Changes in CYP2E1 metabolism were measured by the formation clearance of 6-hydroxychlorzoxazone (6-OHCLZ; Cl(f,6-OHCLZ)) following a 250 mg dose of chlorzoxazone in the absence (day 1) and presence (day 6) of lasofoxifene. Changes in the dextromethorphan/dextrorphan urine metabolic ratio (MRDX) measured the effect on CYP2D6 metabolism following a 30 mg dose of dextromethorphan in the absence and presence of lasofoxifene (days 2 and 7). RESULTS: Steady-state lasofoxifene did not affect the formation clearance of 6-OHCLZ or the urinary MRDX. For 6-OHCLZ, the lower boundary (87.12%) of the 90% confidence interval for the ratio (day 6/day 1) of Cl(f,6-OHCLZ) was well above the clinically acceptable ratio of 60%. Both the individual and group mean Cl(f,6-OHCLZ) values were comparable in the absence and presence of lasofoxifene. For MRDX, the upper boundary (129.37%) of the 90% confidence interval for the ratio (day 7/day 2) of MRDX was well below the stipulated ratio of 200%. The individual and mean MRDX values were comparable in the absence and presence of lasofoxifene. Lasofoxifene was well tolerated; adverse events were mild and transient. CONCLUSIONS: Lasofoxifene has no effect on CYP2E1- or CYP2D6-mediated drug metabolism and should not affect drugs metabolized by other cytochrome P450 isoenzymes.

[Identification of chlorzoxazone metabolites in human urine (author's transl)]. / Identifizierung von Chlorzoxazon-Metaboliten im menschlichen Urin.

Besides 6-hydroxychlorzoxazon II, the main metabolite of 5-chlorobenzoxazolin-2-one (chlorzoxazone I, Paraflex) two other compounds were found to be metabolites of chlorzoxazone: 5-chloro-2,4-dihydroxyacetanilide XXII is produced from 6-hydroxychlorzoxazone II by ring cleavage and acetylation of the nitrogen. More interesting is the formation of the 6-hydroxybenzoxazolone XX. This compound is formally produced by substitution of Cl for hydrogen and hydroxylation of a neighbour ring-carbon atom.

Comparison of chlorzoxazone one-sample methods to estimate CYP2E1 activity in humans.

OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl(est) as a percentage of actual mean Cl(fe) was -4.2% at 6 h. Furthermore, regarding Cl(fe), there was a negligible difference ( P=0.56) of bias between Cl(est) at 3 h (MPE=-8.9%) and 6 h (MPE=-4.2%). The best concordance between MR and Cl(fe) was found at 3 h (r=0.74; P<0.001). CONCLUSION: All three single-dose-sample estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.

Dose-dependent pharmacokinetics of zoxazolamine in the rat.

Zoxazolamine (ZX) is a model substrate frequently used in studies on (methylcholanthrene-inducible) hepatic cytochrome P-450 activity. The iv pharmacokinetics of ZX were studied in rats at four dose levels: 5 mg X kg-1 (n = 6), 25 mg X kg-1 (n = 6), 50 mg X kg-1 (n = 5), and 60 mg X kg-1 (n = 4). Concentrations of ZX in blood, as well as the urinary excretion of unchanged ZX and chlorzoxazone, were determined. The apparent systemic clearance (CLs,app) decreased with increasing dose from 52.6 +/- 3.9 at 5 mg X kg-1 to 9.3 +/- 0.4 ml X min-1 X kg-1 at 60 mg X kg-1. The apparent elimination half-life, t1/2,app, increased from 16.1 +/- 0.3 min to 141 +/- 28.5 min. There was only slight concentration dependency of plasma protein binding: 86.0 +/- 0.9% at 4.2 +/- 0.2 micrograms X ml-1 (n = 6) vs. 80.4 +/- 0.4% at 27.1 +/- 1.1 micrograms X ml-1 (n = 6). Since from clearance and protein binding data nonrestrictive clearance of ZX could be inferred, this small change in binding was regarded as irrelevant for the interpretation of pharmacokinetic data of ZX. The blood-plasma concentration ratio was larger than unity: 2.11 +/- 0.09 at 5.4 +/- 0.9 micrograms X ml-1, and 1.85 +/- 0.08 at 47.9 +/- 4.9 micrograms X ml-1 (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Pedigree analysis of a subject with abnormally slow renal elimination of 6-hydroxychlorzoxazone.

The family of an unusual subject was studied. When tested with chlorzoxazone (CX; 250 mg p.o.) on four separate occasions 5 years ago, this subject showed abnormally slow renal elimination of 6-hydroxychlorzoxazone (HCX), the primary CX metabolite. Since rates of CX biotransformation to HCX have served as a probe of the important cytochrome P450 isozyme, CYP 2E1, it was of interest that this unusual subject had normal conversion of CX to HCX. The present study revealed that over the past 5 years this subject accelerated his renal rate of HCX elimination which now lies at the slow end of the curve for normal subjects. His wife and 5 children all had more rapid rates than he for renal HCX elimination.

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography.

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with beta-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C18 column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100-3000 ng/ml and 4-400 micrograms/ml in plasma and urine, respectively. The intra-assay variability was < or = 5.1% and the inter-assay variability was < or = 8.2% for each compound in each matrix. The method presented is applicable to pharmacokinetic and pharmacogenetic studies utilizing chlorzoxazone.

High-performance liquid chromatographic determination of chlorzoxazone and 6-hydroxychlorzoxazone in serum: a tool for indirect evaluation of cytochrome P4502E1 activity in humans.

Chronic alcohol consumption is known to induce the enzyme cytochrome P4502E1 (CYP2E1), which is involved in the toxicity and carcinogenicity of a number of solvents and xenobiotics. It was recently suggested that in vivo chlorzoxazone metabolism could be a potential tool as a non-invasive probe for measuring CYP2E1 activity in humans. Therefore, a simple and sensitive method was developed for the determination of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone in both serum and urine. Biological samples were hydrolysed by Helix pomatia juice, deproteinized with perchloric acid, and then extracted using ethyl acetate. The compounds were separated by high-performance liquid chromatography on an octadecylsilane column with a mobile phase of acetonitrile-0.5% acetic acid in water (30:70, v/v) and detected at 287 nm. The linearity of the method was tested in the concentration range 0.5-20 micrograms/ml, and the limit of detection in biological samples was found to be 0.5 microgram/ml. Within- and between-run precision was below 5% and 10%, respectively, for both compounds at three concentrations (0.5, 10 and 20 micrograms/ml). The accuracy of the procedure was in the range 0.3-6%. Serum levels and urinary excretion of chlorzoxazone and its metabolite were studied in five healthy controls and five alcoholic patients, following oral administration of 500 mg of chlorzoxazone. The concentration ratio 6-hydroxychlorzoxazone/chlorzoxazone in blood was shown to be a valuable tool for the evaluation of CYP2E1 activity in humans.

Diabetes mellitus increases the in vivo activity of cytochrome P450 2E1 in humans.

AIMS: Cytochrome P450 2E1 (CYP2E1) is thought to activate a number of protoxins, and has been implicated in the development of liver disease. Increased hepatic expression of CYP2E1 occurs in rat models of diabetes but it is unclear whether human diabetics display a similar up-regulation. This study was designed to test the hypothesis that human diabetics experience enhanced CYP2E1 expression. METHODS: The pharmacokinetics of a single dose of chlorzoxazone (500 mg), used as an index of hepatic CYP2E1 activity, was determined in healthy subjects (n = 10), volunteers with Type I (n = 13), and Type II (n = 8) diabetes mellitus. Chlorzoxazone and 6-hydroxychlorzoxazone in serum and urine were analysed by high-performance liquid chromatography. The expression of CYP2E1 mRNA in peripheral blood mononuclear cells was quantified by reverse transcriptase-polymerase chain reaction. RESULTS: The mean +/- s.d. (90% confidence interval of the difference) chlorzoxazone area under the plasma concentration-time curve was significantly (P

Variability in the disposition of chlorzoxazone.

Chlorzoxazone is 6-hydroxylated by cytochrome P450 2E1 (CYP 2E1), which bioactivates many toxic and carcinogenic molecules. Seventeen volunteers of varying age, ethnicity, and gender received a 250 mg tablet of chlorzoxazone and their blood and urine were sampled frequently for 8 h. V/F = 42 +/- 21 L and CL/F = 412 +/- 120 mL min-1. Comparison of these values with a study by other investigators using a suspension dosage form suggested that relative Ftablet approximately 0.7. The fraction excreted in the urine as 6-hydroxychlorzoxazone (fe,6-OH) was 0.39 +/- 0.20 and that portion of the total CL accounted for by CYP 2E1-mediated metabolism (CL6-OH) was 163 +/- 95 mL min-1. Thus, while V/F and CL/F varied by factors of less than five, fe,6-OH varied 16-fold and CL6-OH varied 28-fold. These results suggested that there was considerable inter-individual variability in the metabolism of chlorzoxazone to 6-hydroxychlorzoxazone. This variability will significantly affect the construction of physiologically based pharmacokinetic models that use the 6-hydroxylation of chlorzoxazone as a marker for an individual's CYP 2E1 phenotype.