Chlorzoxazone Research Articles

Chemometric Approach to Simultaneous Chromatographic Determination of Paracetamol and Chlorzoxazone in Tablets and Spiked Human Plasma

New chemometric approaches were introduced into the high performance liquid chromatographic (HPLC) determination of chlorzoxazone (CZX) and paracetamol (PAR) in the tablets and spiked human plasma. These chemometric approaches contain the application of classical least squares (CLS), principle component regression (PCR), and partial least squares (PLS) calibrations to the multiwavelength HPLC peak area obtained by plotting the chromatograms at the five wavelengths. The multichromatograms were obtained by using the photodiode array detector at 225 (A), 240 (B), 255 (C), 270 (D), and 285 (E) nm. The algorithms of CLS, PCR, and PLS were applied to the multichromatogram data to construct the HPLC‐CLS, HPLC‐PCR, and HPLC‐PLS calibrations. A mixture of acetonitrile and 0.1 M ammonium carbonate (60:40, v/v) on a Waters Symmetry® C18 Column 5 µm 4.6×250 mm at a flow rate of 0.8 mL/min was used as a mobile phase to separate and determine CZX and PAR in samples. Hydrochlorothiazide (IS) was used as an internal standard in this chromatographic separation. The HPLC chemometric calibrations were successfully applied to the quantitative analysis of the investigated drugs in commercial pharmaceutical preparation and spiked human plasma samples with high precision and accuracy.

Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h

It was reported that in rats with water deprivation for 72 h with food (dehydration rat model), the expression of CYP2E1 was 3-fold induced with an increase in mRNA level and glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the CYP2E1 induction in dehydration rat model. It was also reported that chlorzoxazone (CZX) is metabolized to 6-hydroxychlorzoxazone (OH–CZX) mainly via CYP2E1 in rats. Hence, the effects of glucose supplementation on the pharmacokinetics of CZX and OH–CZX were investigated after intravenous administration of CZX at a dose of 25 mg/kg to control male Sprague–Dawley rats and dehydration rat model and dehydration rat model with glucose supplementation. Based on the above mentioned results of CYP2E1, it could be expected that increased formation of OH–CZX in dehydration rat model could decrease in dehydration rat model with glucose supplementation. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH–CZX was significantly smaller (1900 versus 1050 μg min/ml), AUC OH–CZX/AUC CZX ratio was considerably smaller (105 versus 34.3%), C max was significantly lower (20.6 versus 8.08 μg/ml), total amount excreted in 24-h urine as unchanged OH–CZX was significantly smaller (62.3 versus 42.7% of intravenous dose of CZX), and in vitro V max (2.18 versus 1.20 nmol/min/mg protein) and CL int (0.0285 versus 0.0171 ml/min/mg protein) were significantly slower than those in dehydration rat model.

Hepatic Disposition of the Cytochrome P450 2E1 Marker Chlorzoxazone and its Hydroxylated Metabolite in Isolated Perfused Rat Livers

The steady-state disposition of chlorzoxazone (CZX) and its hydroxylated metabolite 6-hydroxychlorzoxazone (HCZX) was determined in a single-pass isolated perfused rat liver (IPRL) model using constant CZX concentrations of 10-200 microM. The concentrations of CZX, HCZX, and/or HCZX glucuronide in the perfusate, bile, and liver tissues were measured and kinetic parameters calculated. Upon an increase in CZX inlet concentrations from 10 to 200 microM, its extraction ratio sharply declined from 0.681 to 0.087. This was associated with a saturable formation of HCZX, which was rapidly and completely metabolized to its glucuronide conjugate. Whereas the biliary excretion of CZX was negligible, that of HCZX was substantial (up to 40% of the generated metabolite). Overall, 79-93% of the CZX dose (10-200 microM) was recovered in our model as CZX and HCZX. Additionally, HCZX accounted for 56% (200 microM) to 71% (10 microM) of the extracted CZX dose. Further, a preliminary study using the preformed HCZX showed a complete (100%) recovery of the metabolite as its conjugate. Therefore, the unrecovered portion of CZX dose in our study (7-21% of the administered dose or 29-44% of the extracted dose at inlet CZX concentrations of 10-200 microM) is most likely due to parallel metabolism of CZX to other metabolites.

In Vivo and in Vitro Characterization of Chlorzoxazone Metabolism and Hepatic CYP2E1 Levels in African Green Monkeys: Induction by Chronic Nicotine Treatment

CYP2E1 metabolizes compounds, including clinical drugs, organic solvents, and tobacco-specific carcinogens. Chlorzoxazone (CZN) is a probe drug used to phenotype for CYP2E1 activity. Smokers have increased CZN clearance during smoking compared with nonsmoking periods; however, it is unclear which cigarette smoke component is causing the increased activity. The relationships between in vivo CZN disposition, in vitro CZN metabolism, and hepatic CYP2E1 have not been investigated in a within-animal design. In control-treated monkeys (Cercopithecus aethiops), the in vivo CZN area under the curve extrapolated to infinity (AUC(inf)) was 19.7 +/- 4.5 microg x h/ml, t1/2 was 0.57 +/- 0.07 h, and terminal disposition rate constant calculated from last three to four points on the log-linear end of the concentration versus time curve was 1.2 +/- 0.2 /h. In vitro, the apparent Vmax was 3.48 +/- 0.02 pmol/min/mug microsomal protein, and the Km was 95.4 +/- 1.8 microM. Chronic nicotine treatment increased in vivo CZN disposition, as indicated by a 52% decrease in AUC(inf) (p < 0.01) and 52% decrease in Tmax (p < 0.05) compared with control-treated monkeys. The log metabolic ratios at 0.5, 1, 2, and 4 h significantly negatively correlated with CZN AUC(inf) (p = 0.01-0.0001). Monkey hepatic CYP2E1 levels significantly correlated with both in vivo AUC(inf) (p = 0.03) and in vitro (p = 0.004) CZN metabolism. Together, the data indicated that nicotine induction of in vivo CZN disposition is related to the rates of in vitro CZN metabolism and hepatic microsomal CYP2E1 protein levels. Nicotine is one component in cigarette smoke that can increase in vivo CZN metabolism via induction of hepatic CYP2E1 levels. Thus, nicotine exposure may affect the metabolism of CYP2E1 substrates such as acetaminophen, ethanol, and benzene.

SELECTIVE EFFECTS OF NITRIC OXIDE ON THE DISPOSITION OF CHLORZOXAZONE AND DEXTROMETHORPHAN IN ISOLATED PERFUSED RAT LIVERS

The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50-75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways.

Characterisation of the xenobiotic-metabolizing Cytochrome P450 expression pattern in human lung tissue by immunochemical and activity determination

The lung represents an important target for the toxic effects of chemicals. Many of the chemicals require enzymatic activation to exert their adverse effects, which is mostly catalysed by Cytochrome P450 (CYP) enzymes. Although there is considerable evidence that individual members of the xenobiotic-metabolizing P450 family are expressed in human lung tissue at the mRNA level, there is conflicting evidence concerning the following issues: (I) the qualitative expression pattern of CYP isoenzymes; (II) CYP expression at the protein and/or activity level; and (III) interindividual variability of CYP enzymes in human lung. The latter can be the basis for individual susceptibility towards the adverse effects of lung toxicants. In preparing for studying factors to explain interindividual variability of CYP expression in lung tissue, we investigated the qualitative pulmonary expression pattern of xenobiotic-metabolizing CYP enzymes and elaborated the optimal conditions for quantification at the protein and activity level. By using either individual human lung samples or pooled microsomes from different individuals, immunoreactive bands specific for the following CYP enzymes could be determined by Western blotting: CYP1A1, CYP1A2, CYP2E1 and CYP3A5. Western blotting experiments were also supportive of the presence of CYP2A, CYP2B6, CYP2D6 and CYP3A4 in human lung. By using antibodies specific for CYP2C enzymes and CYP1B1, respectively, immunoreactive bands, which differed slightly in mobility from corresponding standards, were detectable. In addition, we measured methoxy- and ethoxyresorufin dealkylase activities and chlorzoxazone (CLX)-hydroxylase activity in human lung and confirmed the specifities of the latter two activities by inhibition experiments. In summary, we have established methodologies to quantify a panel of CYP enzymes in human lung samples among which there are CYP enzymes whose expression at the protein and activity level has not been evidenced so far.

Relationship between in vivo chlorzoxazone hydroxylation, hepatic cytochrome P450 2E1 content and liver injury in obese non-alcoholic fatty liver disease patients

The aim of the present study was to test the hypothesis that induction of cytochrome P450 2E1 (CYP2E1) in the liver of patients with non-alcoholic fatty liver disease (NAFLD) is correlated both with the in vivo activity of the cytochrome and with the development of liver injury. For this purpose, the liver content of CYP2E1 was determined by Western blot and the CYP2E1 activity by the in vivo hydroxylation of chlorzoxazone (CLZ). The study groups were obese women with an average body mass index (BMI) of 40.3kg/m(2), who underwent therapeutic gastroplasty or gastrectomy with a gastro-jejunal anastomosis. Further, the hepatic histology was determined to establish the pathological score grouping the subjects into three categories: control, steatosis and steatohepatitis. The liver CYP2E1 content and the CLZ hydroxylation of obese patients with steatosis and, particularly, with steatohepatitis were significantly higher than controls and correlated positively with both the severity of the liver damage. These data provide evidence that CYP2E1 would be involved in the mechanism of liver injury found in obese NAFLD patients. Also, the correlation between liver CYP2E1 content and in vivo CLZ hydroxylation would validate the latter as a reliable indicator of liver injury in NAFLD, thus providing a simple and not invasive method to study these patients.

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons.

Effect of 1-aminobenzotriazole on the in vitro metabolism and single-dose pharmacokinetics of chlorzoxazone, a selective CYP2E1 substrate in Wistar rats

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics (Vmax and Km) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486 pmol mg protein−1 min−1 and 345 µM), human liver microsomes (3194 pmol mg protein−1 min−1 and 335 µM) and pure isozyme (3423 pmol mg protein−1 min−1 and 403 µM), but KI and Kinact values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 µM and 0.12 min−1; human: 27.19 µM and 0.14 min−1) and pure isozyme (3.18 µM and 0.29 min−1). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was ∼71% when dosed alone and reached 100% under pre-treatment with ABT. The t1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h post-treatment.

Activity of CYP2E1 and CYP3A enzymes in adults with moderate alcohol consumption

Alcohol consumption is known to induce hepatic CYP2E1 activity, but its effect on hepatic and intestinal CYP3A in humans is not known. We have conducted a study to compare the CYP2E1 and CYP3A activities in 20 individuals with moderate alcohol consumption and 20 gender-, race-. and body mass index (BMI)-matched nonalcoholics. Intravenous and oral midazolam (MDZ) clearances were used to measure the in vivo CYP3A activity, and chlorzoxazone (CHZ) oral clearance was used to assess in vivo CYP2E1 activity. Furthermore, we assessed the relationship between hepatic CYP2E1 and CYP3A activities and their messenger RNA (mRNA) expression in the peripheral lymphocytes. The systemic clearance (CL) of MDZ was not different between alcoholics (36.9 +/- 12 L/hr) and nonalcoholics (36.6 +/- 14.1; P = .9). The oral availability of MDZ was significantly lower in alcoholics than in the nonalcoholics (0.28 +/- .09 vs. 0.38 +/- .17, respectively, P = .03). The maximum serum concentration after oral midazolam dosing was significantly different between the 2 groups. CHZ CL was significantly higher in alcoholics than in nonalcoholics (31.5 +/- 11.9 vs. 23.4 +/- 8.7 L/hr, P < 0.05). CYP3A4 and CYP2E1 mRNA levels were not significantly different between the groups, and no correlation was observed between lymphocyte CYP mRNA and in vivo CYP activity. In conclusion, in individuals with moderate alcohol consumption, there was no alteration in the hepatic CYP3A activity, but the reduced midazolam oral bioavailability suggests that moderate alcohol consumption may cause intestinal CYP3A induction. Lymphocyte CYP2E1 and CYP3A4 mRNA levels did not correlate with CYP2E1 and CYP3A activities.

Comparison of cytochrome P4502E1 (CYP2E1) activity and hepatic and lymphocyte mRNA expression in patients with chronic hepatitis C

The induction of cytochrome P4502E1 (CYP2E1) is believed to play a role in the development of fibrosis in hepatitis C patients. However, information about CYP2E1 activity in chronic hepatitis C patients is fragmentary and the relationship between CYP2E1 activity and mRNA expression is unknown in this disease. The purpose of this study was (a) to characterise CYP2E1 activity in those patients and (b) to analyse its relationship with CYP2E1 mRNA expression in the liver and in peripheral blood lymphocytes (PBLs), previously proposed as a surrogate to assess changes in CYP2E1 activity. Fourteen chronic hepatitis C patients were submitted to a routine transcutaneous liver biopsy. CYP2E1 activity was assessed by using chlorzoxazone (CZX) pharmacokinetic parameters and hepatic and PBLs CYP2E1 mRNA expression was measured by real-time RT-PCR. The mean oral clearance of CZX (CL T: 21.5 ± 10.1 L/h) was within the normal range and the chlorzoxazone metabolic ratio (CMR) at t = 2 h was closely related to other CZX pharmacokinetic parameters. None of the pharmacokinetic parameters did significantly correlate with CYP2E1 mRNA, neither in the liver nor in PBLs. Furthermore, there was no significant relationship between CYP2E1 mRNA levels in paired liver and PBL samples. Our data indicate that early stages of chronic hepatitis C are not associated with CYP2E1 induction. In this disease, the determination of the CMR at t = 2 h represents a reliable index to assess CYP2E1 activity. The measurement of CYP2E1 expression, at the mRNA level, in PBLs or in liver is not useful for that purpose.

Bioactivation of 1,1-dichloroethylene by CYP2E1 and CYP2F2 in murine lung.

1,1-Dichloroethylene (DCE) exposure evokes lung toxicity with selective damage to bronchiolar Clara cells. Recent in vitro studies have implicated CYP2E1 and CYP2F2 in the bioactivation of DCE to 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. An objective of this study was to test the hypothesis that bioactivation of DCE is catalyzed by both CYP2E1 and CYP2F2 in murine lung. Western blot analysis of lung microsomal proteins from DCE-treated CD-1 mice showed time-dependent loss of immunodetectable CYP2F2 and CYP2E1 protein. Dose-dependent formation of conjugate [C] was observed in the lungs of CD-1 mice treated with DCE (75-225 mg/kg), but it was not detected after pretreatment with 5-phenyl-1-pentyne (5-PIP). Treatment of mice with 5-PIP and also with diallyl sulfone (DASO2) significantly inhibited hydroxylation of p-nitrophenol (PNP) and chlorzoxazone (CHZX). Incubation of recombinant CYP2F3 (a surrogate for CYP2F2) and recombinant CYP2E1 with PNP and CHZX confirmed that they are substrates for both of the recombinant enzymes. Incubation of the recombinant enzymes with DASO2 or 5-PIP significantly inhibited hydroxylation of both PNP and CHZX. Bronchiolar injury was elicited in CD-1 mice treated with DCE (75 mg/kg), but it was abrogated with 5-PIP pretreatment. Bronchiolar toxicity also was manifested in the lungs of CYP2E1-null and wild-type mice treated with DCE (75 mg/kg), but protection ensued after pretreatment with 5-PIP or DASO2. These results showed that bioactivation of DCE in murine lung occurred via the catalytic activities of both CYP2E1 and CYP2F2 and that bioactivation by these enzymes mediated the lung toxicity.

Lipopolysaccharide Induces CYP2E1 in Astrocytes through MAP Kinase Kinase-3 and C/EBPβ and -δ

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.

Lack of correlation between lymphocytic mrna expression and activity of CYP3A4 and CYP2E1 In-vivo in alcoholics and non-alcoholics

Lymphocyte mRNA levels may serve as a surrogate for activity of drug metabolizing enzymes. We conducted a study to examine the relationship between CYP2E1 and CYP3A4 lymphocyte mRNA levels and the clearances (CL) of oral chlorzoxazone (CHZ) and oral and IV midazolam (MDZ), respectively. 20 alcoholics (ALC) (> 140gm of alcohol/wk) and 20 non-alcoholics (N-ALC) (20 African-Americans 20 Caucasians) enrolled in the study. MDZ was given IV (0.05mg/kg over 30 min) and orally (5mg) on consecutive days. CHZ (500mg) was administered orally 8 hr after IV MDZ. mRNA was isolated from blood lymphocytes, CYP3A4 and 2E1 mRNA was quantitated using real time PCR. Serum CHZ and MDZ concentrations were determined by HPLC-UV or LC-MS. There was no significant difference (p>0.05) between ALC and N-ALC for CYP3A4 mRNA, MDZ CLORAL (149.4±87.8 vs 120.3±84.1 L/hr) and CLIV (36.9±12 vs 36.6±14.1 L/hr). No correlation was observed between lymphocytic CYP3A4 mRNA and CYP3A activity. CYP2E1 mRNA levels were not significantly different between ALC and N-ALC. In contrast, CHZ CLORAL was significantly greater (p<0.05) in ALC (31.5±11.9) compared to N-ALC (23.4±8.7) but no correlation was observed between CYP2E1 activity and mRNA. No significant race based differences were observed for either CYP mRNA or activity. In conclusion, lymphocyte CYP2E1 and CYP3A4 mRNA levels did not reflect hepatic CYP2E1 activity nor hepatic and intestinal CYP3A activity. Clinical Pharmacology & Therapeutics (2004) 75, P36–P36; doi: 10.1016/j.clpt.2003.11.137

Application of a map Bayesian method for CYP2E1 phenotyping

Purpose The CYP2E1 probe drug chlorzoxazone (CZ) is extensively metabolized to 6-OHchlorzoxazone (HCZ). The HCZ/CZ ratio is used as an index of CYP2E1 activity, but it is not valid in patients with kidney disease. Individual estimates of HCZ formation clearance (CLf) obtained via MAP Bayesian estimation (MBE) could be an alternative to full profile (FP) PK studies. Methods: A population PK model incorporating covariate effects, creatinine clearance (CLcr) on CL and weight on V, was developed based on FP data from 21 subjects (CLcr 9 to 100 ml/min). MBE of HCZ CLf were determined for several single-point (HCZ and CZ) sampling times using the FP model as a prior. The bias (%MPE = mean % prediction error) and precision (%MAPE = mean % absolute PE) of single-point CLf estimates vs. FP were determined. Results (See Table) Conclusion The HCZ/CZ ratio is highly dependent on renal function, but MBE of HCZ CLf were reasonably precise and accurate and estimation performance was independent of CLcr. These results suggest that MBE can provide reliable estimation of HCZ CLf (CYP2E1 phenotype). Prospective validation is warranted. Clinical Pharmacology & Therapeutics (2004) 75, P41–P41; doi: 10.1016/j.clpt.2003.11.156 Table 1. Time (hr) 0.5 1 2 4 6 8 %MPE 6.6 3.2 −0.86 −0.21 3.5 4.0 %MAPE 16.4 14.3 11.7 11.3 18.4 15.1

Effects of Glucose on the Pharmacokinetics of Intravenous Chlorzoxazone in Rats with Acute Renal Failure Induced by Uranyl Nitrate

The effects of glucose on CYP2E1 expression in rats with acute renal failure induced by uranyl nitrate (U-ARF) have been reported. CYP2E1 was significantly induced (2.3-fold) in rats with U-ARF compared with that in control rats. In contrast, CYP2E1 expression was significantly decreased in rats with U-ARF supplied with glucose (dissolved in tap water to make 10%, w/v) in their drinking water for 5 days (U-ARFG) compared with that in rats with U-ARF. However, CYP2E1 in rats with U-ARFG was significantly greater than that in control rats. Chlorzoxazone (CZX) primarily undergoes hydroxylation, catalyzed mainly by CYP2E1, to form 6-hydroxychlorzoxazone (OH-CZX) rats. Hence, it could be expected that in rats with U-ARFG, formation of OH-CZX could significantly decrease and increase compared with those in rats with U-ARF and control rats, respectively. This expectation is proven by the following results of a study of intravenous administration of CZX at a dose 20 mg/kg to control rats and rats with U-ARF and U-ARFG. First, the total area under the plasma concentration-time curve from time zero to 8 h (AUC(0-8 h)) of OH-CZX in rats with U-ARFG (8730 microg x min/mL) was significantly greater than that in control rats (414 microg x min/mL) and significantly smaller than that in rats with U-ARF (11500 microg x min/mL). Second, the AUC(0-8 h, OH-CZX)/AUC(CZX) ratio in rats with U-ARFG (10.0) was significantly greater than that in control rats (0.252) and significantly smaller than that in rats with U-ARF (17.5). Finally, the in vitro intrinsic OH-CZX formation clearance (CL(int)) in rats with U-ARFG (27.9 mL/min/mg protein) was significantly slower than that in rats with U-ARF (36.7 mL/min/mg protein) and significantly faster than that in control rats (17.7 mL/min/mg protein).

CYP2E1 activity before and after weight loss in morbidly obese subjects with nonalcoholic fatty liver disease

Previous studies suggest that hepatic cytochrome P450 2E1 (CYP2E1) activity is increased in individuals with chronic alcoholism, nonalcoholic steatohepatitis (NASH), and morbid obesity, and may contribute to liver disease. We studied 16 morbidly obese subjects with varying degrees of hepatic steatosis and 16 normal-weight controls. Obese subjects were evaluated at baseline, 6 weeks, and 1 year after gastroplasty, a procedure that leads to weight loss. Hepatic CYP2E1 activity was assessed by determination of the clearance of chlorzoxazone (CLZ), an in vivo CYP2E1-selective probe. Liver biopsy tissue was obtained during surgery for histopathology. Both the total and unbound oral CLZ clearance (Cl u/F) was elevated approximately threefold in morbidly obese subjects compared with controls ( P < .001). The Cl u/F was significantly higher among subjects with steatosis involving >50% of hepatocytes, compared with those with steatosis in ≤50% of hepatocytes ( P = .02). At postoperative week 6 and year 1, the median body mass index (BMI) of subjects who underwent gastroplasty decreased by 11% and 33%, total oral CLZ clearance declined by 16% ( P < .01) and 46% ( P < .05), and Cl u/F decreased by 18% ( P < .05) and 35% ( P = .16), respectively. Moreover, those subjects with a year 1 BMI <30 kg/m 2 exhibited a median Cl u/F that was 63% lower ( P = .02) than the respective clearance for all other subjects. In conclusion, hepatic CYP2E1 activity is up-regulated in morbidly obese subjects. A positive association between the degree of steatosis and CYP2E1 activity preoperatively and between the extent of obesity and CYP2E1 activity postoperatively, suggests that CYP2E1 induction is related to or caused by hepatic pathology that results from morbid obesity. (H epatology 2003;38:428-435.)

Effects of acute renal failure on the pharmacokinetics of chlorzoxazone in rats.

The purpose of this study is to report the changes of CYP2E1, CYP1A2, CYP2B1/2, CYP2C11, CYP3A23, and CYP3A2 expression and pharmacokinetics and tissue distribution of chlorzoxazone (CZX) and 6-hydroxychlorzoxazone (OH-CZX) in rats with acute renal failure induced by uranyl nitrate (U-ARF), and the role of CYP3A23 and CYP3A2 in the formation of OH-CZX in rats with U-ARF. In rats with U-ARF, CYP2C11 decreased to 20% of control, whereas CYP2E1 and CYP3A23 increased 2.3 and 4 times, respectively, compared with control. But expression of CYP1A2 and CYP2B1/2 was not changed by U-ARF. After i.v. administration of CZX at a dose of 20 mg/kg to rats with U-ARF, the areas under the plasma concentration-time curve from time 0 to time infinity (AUCs) of CZX and OH-CZX were significantly smaller and greater, respectively, than those in control rats. In rats with U-ARF, CZX was below the detection limit at 120 min in all rat tissues studied, whereas it was detected in all tissues of control rats at both 30 and 120 min. However, in control rats, OH-CZX was below the detection limit at both 30 and 120 min in all rat tissues except kidney, whereas it was detected in all tissues of rats with U-ARF at both 30 and 120 min. Based on results from supporting experiments with DDT and 2,2-bis(4-chlorophenyl)1,1-dichloroethylene treatment of rats, the contribution of CYP3A23 and CYP3A2 to the enhanced formation of OH-CZX in rats with U-ARF is likely to be negligible.

Effects of recombinant human growth hormone on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate

It has been reported from our laboratories that expression of CYP2E1 significantly increased and decreased in rats with acute renal failure induced by uranyl nitrate (U-ARF) treated with recombinant human growth hormone (rGH) for one day (U-ARF1) compared with those in control rats and rats with U-ARF, respectively. Chlorzoxazone (CZX) primarily undergoes hydroxylation to form 6-hydroxychlorzoxazone (OH-CZX) catalyzed mainly by CYP2E1 in rats. Hence, the effects of rGH on the pharmacokinetics of intravenous CZX (20 mg/kg) were investigated in rats with U-ARF. Based on CYP2E1 expression, it could be expected that in rats with U-ARF1, the formation of OH-CZX significantly increased and decreased compared with those in control rats and rats with U-ARF, respectively. This was proven in the following results. First, the total area under the plasma concentration-time curve from time zero to 8 hr (AUC 0→8 hr) of OH-CZX in rats with U-ARF1 (36100 μg min/ml) was significantly greater and smaller than those in control rats (1040 μg min/ml) and rats with U-ARF (50300 μg min/ml), respectively. Second, the AUC 0→8 hr, OH-CZX/AUC CZX ratio in rats with U-ARF1 (28.9) was significantly greater and smaller than those in control rats (0.468) and rats with U-ARF (72.6), respectively.

Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis

Cytochrome P450 2E1 (CYP2E1) plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH) in animal models, but its role in the pathogenesis of human NASH is unclear. Therefore, we measured hepatic CYP2E1 activity and its correlates in a cohort of nondiabetic patients with NASH (NDN) and controls to explore its role in the pathogenesis of human NASH. Hepatic CYP2E1 activity was assessed using the oral clearance (CL PO) of chlorzoxazone (CHZ) in 20 NDN and 17 age, gender, and body mass index (BMI)-matched controls. The relationship between hepatic CYP2E1 activity and demographic and anthropometric variables; fasting levels of insulin, glucose, lipids, and β-OH butyrate; insulin resistance; and nocturnal hypoxemia was assessed. Furthermore, expression of CYP2E1 in the peripheral lymphocytes was assessed using reverse transcription-polymerase chain reaction (RT-PCR). The CL PO of CHZ was significantly ( P = .03) greater in NDN (41 ± 12 L/h) compared with controls (33 ± 16 L/h). Lymphocyte CYP2E1 messenger RNA was significantly higher in NDN compared with controls (11.5 × 10 3 ± 10 × 10 3 vs. 2.6 × 10 3 ± 1.2 × 10 3 molecules/μg total RNA, respectively, P < .001). On univariate analysis, BMI, respiratory quotient, high-density lipoprotein, triglycerides, insulin, insulin resistance, hypoxemia, and β-OH butyrate significantly correlated with hepatic CYP2E1 activity. However, on stepwise regression analysis, only nocturnal hypoxemia ( r = 0.50, P = .009) and β-OH butyrate ( r = 0.37, P = .04) were independent predictors of hepatic CYP2E1 activity. In conclusion, hepatic CYP2E1 activity and lymphocyte CYP2E1 expression are enhanced in NDN. The significant correlations noted between CYP2E1 and hypoxemia and β-OH butyrate suggest that these factors play a role in increased CYP2E1 activity that is seen in patients with NASH. (H EPATOLOGY 2003;37:544-550.)