The effect of quercetin on the pharmacokinetics of chlorzoxazone, a CYP2E1 substrate, in healthy subjects.

PURPOSE: Previous in vitro studies have demonstrated that quercetin inhibits CYP2E1 enzyme, but there are no available data to indicate that quercetin inhibits CYP2E1 enzyme in humans. The purpose of the present study was to assess the effect of quercetin on CYP2E1 enzyme activity in healthy subjects using chlorzoxazone (CHZ) as a CYP2E1 substrate. METHODS: An open-label, two-period, sequential study was conducted in 12 healthy subjects. A single dose of CHZ 250 mg was given to subjects during control phase and after treatment phases. Quercetin at a dose of 500 mg was given to subjects twice daily for a period of 10 days. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed to determine the concentrations of CHZ and 6-hydroxychlorzoxazone (6-OHCHZ). RESULTS: Treatment with quercetin significantly enhanced the maximum plasma concentration (C max), area under the curve (AUC), and half-life (t 1/2) by 47.8, 69.3, and 36.4%, respectively, while significantly decreased the elimination rate constant (k el) and apparent oral clearance (CL/F) of CHZ by 25.1 and 41.6%, respectively, in comparison with the control. On the other hand, C max and AUC of 6-OHCHZ were decreased by 30.1 and 32.6%, respectively, after quercetin treatment when compared to control. In addition, geometric mean ratios and 90% confidence intervals for C max and AUC of CHZ and 6-OHCHZ were both out of the no-effect boundaries of 0.80-1.25, which indicates a significant pharmacokinetic interaction present between CHZ and quercetin. Furthermore, treatment with quercetin significantly decreased the metabolic ratios of C max and AUC by 57.1 and 60.1%, respectively, as compared to control suggesting that reduced formation of CHZ to 6-OHCHZ. CONCLUSIONS: The results suggest that altered pharmacokinetics of CHZ might be attributed to quercetin-mediated inhibition of CYP2E1 enzyme. Further, the inhibition of CYP2E1 by quercetin may represent a novel therapeutic approach for minimizing the ethanol-induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.

Liquid chromatography-tandem MS/MS method for simultaneous quantification of paracetamol, chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study.

A facile, fast and specific method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid-liquid extraction. The analysis was performed on a reversed-phase C18 HPLC column (5 µm, 4.6 × 50 mm) using acetonitrile-10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 µL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple-reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03-30.0, 0.015-15.00 and 0.15-15.00 µg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers.

Pharmacokinetics of a cytochrome P450 2E1 probe, chlorzoxazone, and its 6-hydroxy metabolite in poloxamer 407-induced hyperlipidemic rats.

PURPOSE: To evaluate the possible changes in CYP2E1 expression and activity in hyperlipidemia (HL), we evaluated the pharmacokinetics of chlorzoxazone (CZX) as a CYP2E1 probe in rats with HL induced by poloxamer 407 (HL rats). METHODS: The pharmacokinetics of CZX and its 6-hydroxy metabolite (OH-CZX) were evaluated after intravenous administration of 20 mg/kg CZX to both control and HL rats. We also examined changes in the expression of CYP2E1 and its in vitro metabolic activity in hepatic microsomal fractions from HL rats. RESULTS: The total area under the plasma concentration-time curve (AUC) of CZX in the HL rats after its intravenous administration was comparable with that in the controls due to unchanged non-renal clearance (CLNR). The AUC of OH-CZX and AUCOH-CZX/AUCCZX ratios in HL rats also remained unchanged. This was primarily due to the comparable hepatic CLint for metabolism of CZX to OH-CZX via CYP2E1 between the control and HL rats as a result of unchanged expression of CYP2E1 in HL rats. CONCLUSIONS: This is the first study to evaluate CYP2E1 expression and activity in HL rats and their effects on the pharmacokinetics of a CYP2E1 probe drug. These findings have potential therapeutic implications assuming that the HL rat model qualitatively reflects similar changes in patients with HL.

An LC-MS/MS Validated Method for Quantification of Chlorzoxazone in Human Plasma and Its Application to a Bioequivalence Study.

A simple, sensitive, specific, accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determination of chlorzoxazone in human plasma was developed and validated to evaluate the pharmacokinetic characteristics of chlorzoxazone test or reference formulation. Sample preparation was achieved by one step protein precipitation and dilution with acetontrile. The chromatographic separation was performed at 40°C with a gradient mobile phase (0.3 mL/min) and a Shimadzu VP-ODS C18 analytical column (column size: 150 × 2.0 mm). TSQ quantum access triple-quadrapole MS/MS detection was operated in a negative mode by multiple reaction monitoring. Ion transitions at m/z 168.0→132.1 for chlorzoxazone and m/z 451.3→379.3 for repaglinide (internal standard) were used for the LC-MS/MS analysis. The calibration was linear (r ≥ 0.995) over the tested concentration range of 0.2-20 µg/mL for chlorzoxazone in plasma. Precision, accuracy, recovery, matrix effect and stability for chlorzoxazone were evaluated and were excellent within the range of tested concentrations. This method was successfully applied to a bioequivalence study in 20 healthy Chinese volunteers. This method could also contribute to the personalized medication and therapeutic drug monitoring of chlorzoxazone.

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 influence of a newly developed quinolone: antofloxacin, on CYP activity in rats.

To investigate a newly developed quinolone antibiotics, the effect of antofloxacin hydrochloride on cytochrome P450 isoforms in rats was examined. A cocktail approach was adopted. Theophylline (CYP1A2), midazolam (CYP3A), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), omeprazole (CYP2C19) and diclofenac (CYP2C9) were used as probes in the study, and own control was adopted. In Protocol 1, probes were given to rats simultaneously by co-administration with antofloxacin. The blood samples were obtained at designated time, and plasma concentrations of the six probes were determined by LC-MS. The pharmacokinetic parameters were calculated and compared in experimental groups in the absence and presence of antofloxacin. The result showed that the presence of antofloxacin resulted in a significant increase in theophylline values of AUC0-T and t1/2 (PAUC0-T = 0.0004 vs control Pt1/2 = 0.005 vs control), indicating that antofloxacin delayed the clearance of theophylline. In Protocol 2, the probes' pharmacokinetic parameters were compared in rats that received six probes before and after 14.5 days of consecutive administration of antofloxacin (15 mg x kg(-1), given orally, twice daily). The results suggested that the AUC0-T of chlorzoxazone was significantly decreased (P = 0.024), while that of dextromethorphan was significantly increased (P = 0.027). In conclusion, these results indicated that antofloxacin may inhibit the activity of CYP1A2, thus delaying the clearance of its substrates, and may have a slight inhibitory effect on CYP2D6 as well as an inductive effect on CYP2E1 following chronic administration.

Hepatic cytochrome P450 2E1 activity in nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a worldwide phenomenon spanning all the continents. The pathogenesis of NAFLD has not been completely elucidated. One hypothesis is that hepatic cytochrome P450 2E1 (CYP2E1) plays an important role in increasing the lipid peroxidation and oxidative stress in NAFLD. OBJECTIVE: The aim of the present study was to examine hepatic CYP2E1 activity in patients with NAFLD. MATERIAL AND METHOD: Healthy subjects were included. After an overnight fasting, the subjects were orally administered 400 mg chlorzoxazone (CHZ) and serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 8 hours after dosing. For patients with NAFLD, plasma samples were collected at 0 (predose), 1.5, 2, 2.5 and 3 hours after dosing. Plasma CHZ and 6-hydroxychlorzoxazone (6-OH-CHZ) was assayed by reversed-phase high-performance liquid chromatography (HPLC) with UV detector. Hepatic CYP2E1 activity was calculated by using concentration ratio of 6-OH-CHZ / CHZ. RESULTS: High concentration levels of CHZ and 6-OH-CHZ in healthy subjects were found between 1.5 to 3 hours after the dose. At 1.5 to 3 hours, the concentration ratio of 6-OH-CHZ / CHZ of patients with NAFLD seemed to be more than of healthy subjects. The time point which showed most different was 2.5 hours. (0.40 +/- 0.27 vs. 0.25 +/- 0.12 microg/ml, respectively, p = 0.10). CONCLUSION: Although significant difference of the concentration ratio of 6-OH-CHZ / CHZ between the two groups was not exhibited, the data demonstrated the possibility of the increasing hepatic CYP2E1 activity in NAFLD. The concentration ratio of 6-OH-CHZ / CHZ at the point 2.5 hours may be the best index for measuring hepatic CYP2E1 activity in NAFLD.

Effect of Gambogenic Acid on Cytochrome P450 1A2, 2B1 and 2E1, and Constitutive Androstane Receptor in Rats.

BACKGROUND AND OBJECTIVES: Gambogenic acid (GNA), which possesses diverse antitumor activities both in vitro and in vivo, is regarded as a potential anticancer compound. Cytochrome P450 (CYP) enzymes play an important role in the metabolism of most xenobiotics; constitutive androstane receptor (CAR), a nuclear receptor that might be activated by xenobiotics and associated with the expression of some CYPs. In this study, we determined the effect of GNA on multiple rat liver CYP isoforms (CYP1A2, 2B1, and 2E1) and CAR as well as the potential of GNA to interact with co-administered drugs. METHODS: Male SD rats were randomly divided into the control, and the low (5 mg/kg)-, medium (25 mg/kg)-, and high- (100 mg/kg) dose GNA groups. After the intragastric administration of GNA for 14 consecutive days, a cocktail method was adopted to evaluate the activities of CYP1A2, 2B1, and 2E1. The liver expression of CYP1A2, 2B1, and 2E1 and CAR was analyzed by Western blotting (WB) and quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). RESULTS: The 14-day administration of GNA significantly increased both the mRNA and protein expressions and the activity of CYP2E1. Additionally, the mRNA and protein expressions of CYP1A2 were clearly induced, while only the high GNA dose increased the activity of liver CYP1A2. Moreover, the mRNA expression levels of CYP2B1 and CAR were increased, but their protein levels and the activity parameters of CYP2B1 did not show significant changes. CONCLUSIONS: The obtained results suggest that the CYP1A2 and CYP2E1 enzymes could be induced in rats after treatment with GNA. Therefore, when GNA is administrated with other drugs, potential drug-drug interactions (DDI) mediated by CYP1A2 and CYP2E1 induction should be taken into consideration.

Artemisinin antimalarials moderately affect cytochrome P450 enzyme activity in healthy subjects.

The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity. Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for 5 days (days 1-5). A six-drug cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered orally on days -6, 1, 5 and 10 to assess the activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Four-hour plasma concentrations of parent drugs and corresponding metabolites and 7-hydroxycoumarin urine concentrations were quantified by liquid chromatography-tandem mass spectrometry. The 1-hydroxymidazolam/midazolam 4-h plasma concentration ratio (CYP3A) was increased on day 5 by artemisinin [2.66-fold (98.75% CI: 2.10-3.36)], artemether [1.54 (1.14-2.09)] and dihydroartemisinin [1.25 (1.06-1.47)] compared with day -6. The S-4'-hydroxymephenytoin/S-mephenytoin ratio (CYP2C19) was increased on day 5 by artemisinin [1.69 (1.47-1.94)] and arteether [1.33 (1.15-1.55)] compared with day -6. The paraxanthine/caffeine ratio (CYP1A2) was decreased on day 1 after administration of artemisinin [0.27 (0.18-0.39)], arteether [0.70 (0.55-0.89)] and dihydroartemisinin [0.73 (0.59-0.90)] compared with day -6. The alpha-hydroxymetoprolol/metoprolol ratio (CYP2D6) was lower on day 1 compared with day -6 in the artemisinin [0.82 (0.70-0.96)] and dihydroartemisinin [0.83 (0.71-0.96)] groups, respectively. In the artemisinin-treated subjects this decrease was followed by a 1.34-fold (1.14-1.58) increase from day 1 to day 5. These results show that intake of artemisinin antimalarials affect the activities of several principal human drug metabolizing CYP450 enzymes. Even though not significant in all treatment groups, changes in the individual metrics were of the same direction for all the artemisinin drugs, suggesting a class effect that needs to be considered in the development of new artemisinin derivatives and combination treatments of malaria.

A study on the pharmacokinetics of chlorzoxazone in healthy Thai volunteers.

BACKGROUND: Chlorzoxazone (CHZ), a centrally acting skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone (6-OH-CHZ) by CYP2E1. CHZ can be used as an in vivo probe of CYP2E1 activity in patients with liver diseases. Pharmacokinetics of CHZ in Thai subjects should be studied for application to Thai patients. OBJECTIVE: The purpose of the present study was to determine clinical pharmacokinetics of CHZ and 6-OH-CHZ. MATERIAL AND METHOD: Ten healthy Thai volunteers were included. After an overnight fasting, the volunteers were orally administered 400 mg CHZ and serial blood samples were collected at 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 hours after dosing. Plasma CHZ and 6-OH-CHZ were assayed by reversed-phase high-performance liquid chromatography (HPLC) with UV detector. The pharmacokinetic parameters including maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the concentration-time curve (AUC0-8 and AUC0-infinity), elimination half-life (t1/2), elimination rate constant (K(el)), oral clearance (Cl), and volume of distribution (Vd) were determined. RESULTS: CHZ was absorbed into systemic circulation with time to reach maximum concentration (Tmax) of 2.00 +/- 0.82 hrs and maximum concentration (Cmax) of 7.15 +/- 2.09 microg/ml. It was metabolized to 6-OH-CHZ with Tmax of 3.05 +/- 1.17 hrs and Cmax of 1.77 +/- 0.50 microg/ml. The extent of CHZ absorption (area under the concentration-time curve, AUC) was 25.47 +/- 7.11 and 27.52 +/- 8.05 microg x hr/ml for AUC0-8 and AUC0-infinity respectively. The AUC0-8 and AUC0-infinity of 6-OH-CHZ were 7.32 +/- 2.21 and 8.50 +/- 2.78 microg x hr/ml, respectively. The elimination rate constant (K(el)) was 0.48 +/- 0.10 and 0.40 +/- 0.13 hr-1 for CHZ and 6-OH-CHZ, respectively The elimination half-life (t1/2) was 1.49 +/- 0.32 and 1.95 +/- 0.73 hours for CHZ and 6-OH-CHZ, respectively. Oral clearance (Cl) and volume of distribution (Vd) of CHZ was found to be 15.77 +/- 4.81 (L/hr) and 33.13 +/- 9.75 L, respectively. CONCLUSION: An oral dose of 400 mg CHZ was used to probe for the pharmacokinetic characteristics of this drug in Thai volunteers. Those parameters reflected absorption, distribution, and elimination of CHZ in healthy Thai volunteers.

The Effects of Formaldehyde on Cytochrome P450 Isoform Activity in Rats.

Formaldehyde (FA) is an occupational and indoor pollutant. Long-term exposure to FA can irritate the respiratory mucosa, with potential carcinogenic effects on the airways. The effects of acute FA poisoning on the activities of CYP450 isoforms CYP1A2, CYP2C11, CYP2E1, and CYP3A2 were assessed by determining changes in the pharmacokinetic parameters of the probe drugs phenacetin, tolbutamide, chlorzoxazone, and testosterone, respectively. Rats were randomly divided into three groups: control, low FA dose (exposure to 110 ppm for 2 h for 3 days), and high FA dose (exposure to 220 ppm for 2 h for 3 days). A mixture of the four probe drugs was injected into rats and blood samples were taken at a series of time points. Plasma concentrations of the probe drugs were measured by HPLC. The pharmacokinetic parameters t1/2, AUC(0-t), and Cmax of tolbutamide, chlorzoxazone, and testosterone increased significantly in the high dose versus control group (P < 0.05), whereas the CL of chlorzoxazone and testosterone decreased significantly (P < 0.05). However, t1/2, AUC(0-t), and Cmax of phenacetin decreased significantly (P < 0.05), whereas the CL of phenacetin increased significantly (P < 0.05) compared to controls. Thus, acute FA poisoning suppressed the activities of CYP2C11, CYP2E1, and CYP3A2 and induced the activity of CYP1A2 in rats. And the change of CYP450 activity caused by acute FA poisoning may be associated with FA potential carcinogenic effects on the airways.

Chlorzoxazone inhibits contraction of rat thoracic aorta.

Chlorzoxazone has been reported to activate the intermediate-conductance, Ca(2+)-activated K(+) channels in aortic endothelial cells and to relax the artery. The aim of the present study was to characterize the chlorzoxazone-induced relaxation of rat thoracic artery. Chlorzoxazone did not affect the tension of the thoracic artery rings at rest, but relaxed the precontraction induced by 1 muM noradrenaline in an endothelium independent manner. Preincubation with chlorzoxazone also antagonized the contraction induced by 1 microM noradrenaline or 25 mM KCl. The chlorzoxazone-induced relaxation of the thoracic artery pre-contracted by noradrenaline was suppressed by 5 mM tetraethylammonium, 75 mM ethanol and 2 microM paxilline, but not by 2 microM clotrimazole. Chlorzoxazone relaxed the 4-aminopyridine-induced contraction. The pattern of chlorzoxazone-induced relaxation was different from that of verapamil, the L-type Ca(2+) channel blocker. The inhibition of the noradrenaline-induced contraction by chlorzoxazone was attenuated when chlorzoxazone treatment was prolonged to 4 h. No difference in the contraction-relaxation was found between the artery rings from normal rats and those from rats that received 100 mg/kg chlorzoxazone for 7 days. We conclude that chlorzoxazone abolishes the contraction of rat thoracic artery induced by noradrenaline and that the effect of chlorzoxazone is endothelium independent and also not mediated by intermediate-conductance, Ca(2+)-activated K(+) channels.

Phenobarbital induces monkey brain CYP2E1 protein but not hepatic CYP2E1, in vitro or in vivo chlorzoxazone metabolism.

Cytochrome P450 2E1 (CYP2E1) is expressed in the brain and liver, and can metabolize clinical drugs and activate toxins. The effect of phenobarbital on hepatic and brain CYP2E1 is unclear. We investigated the effect of chronic phenobarbital treatment on in vivo chlorzoxazone disposition (a CYP2E1 probe drug), in vitro chlorzoxazone metabolism, and hepatic and brain CYP2E1 protein levels in African Green monkeys (Cercopithecus aethiops). Monkeys were given oral saccharine or saccharine supplemented with 20 mg/kg phenobarbital (N = 6/group) for 22 days. Phenobarbital did not induce in vivo chlorzoxazone disposition, in vitro chlorzoxazone metabolism or hepatic CYP2E1 protein levels (all P > 0.05). However, phenobarbital induced brain CYP2E1 protein levels, using immunoblotting, by 1.26-fold in the cerebellum (P = 0.01) and 1.46-fold in the putamen (P = 0.04). Phenobarbital also increased cell-specific CYP2E1 expression, for example in the frontal cortical pyramidal neurons and cerebellar Purkinje cells. This data indicates that phenobarbital does not alter hepatic metabolism, but may alter metabolism of CYP2E1 substrates within the brain.

Pharmacokinetics of chlorzoxazone in rats with diabetes: Induction of CYP2E1 on 6-hydroxychlorzoxazone formation.

Pharmacokinetic parameters of chlorzoxazone (CZX) and its main metabolite, 6-hydroxychlorzoxazone (OH-CZX), were compared after intravenous (20 mg/kg) and oral (50 mg/kg) administration of CZX in rat model of diabetes induced by alloxan (DMIA) or streptozotocin (DMIS), and their respective control rats. In both rat models of diabetes, the expression and mRNA level of CYP2E1 increased, and CZX was metabolized to OH-CZX via CYP2E1 in rats. Hence, it could be expected that formation of OH-CZX increased in both rat models of diabetes. As expected, after intravenous (80.5% and 74.4% increase in rat models of DMIA and DMIS, respectively) and oral (55.6% and 70.5% increase, respectively) administration of CZX, the AUC of OH-CZX was significantly greater than their respective control rats. Since, CZX is an intermediate hepatic extraction ratio drug, the greater AUC values of OH-CZX (the significantly faster CL(NR) of CZX) in both rat models of diabetes could be supported by significantly faster CL(int) for the formation of OH-CZX (75.9% and 129% increase for rat models of DMIA and DMIS, respectively) and significantly greater free fractions of CZX in plasma (51.9% and 58.9% increase, respectively). Also it was reported that hepatic blood flow rate was faster in male Wister rat model of DMIS.

Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats.

Earlier data showed that men fasted for 38 h had a reduced rate of chlorzoxazone metabolism, suggesting a decreased level of cytochrome P450 2E1 (CYP2E1). In contrast, the level of CYP2E1 in fasted rats had been shown to be elevated. In this study, we have investigated whether chlorzoxazone metabolism in fasted rats was changed by determining the pharmacokinetics of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone (6-OHCZ), as a CYP2E1 probe, and by measuring liver CYP2E1 using immunoblot techniques. Chlorzoxazone was administered by gavage (50 mg kg(-1)) or intravenously (25 mg kg(-1)) to control (nine for oral and three for intravenous) and 24 h-fasted (nine for oral and four for intravenous) male Sprague-Dawley rats. Following sampling of blood through a jugular vein cannula, chlorzoxazone and 6-OHCZ plasma concentrations were measured by HPLC with UV detection. Pharmacokinetic parameters for chlorzoxazone and 6-OHCZ in each treatment group were determined by model fitting and non-compartmental analysis. In parallel with the increased liver CYP2E1 level, the elimination of chlorzoxazone and 6-OHCZ was significantly increased in fasted rats in the oral and the intravenous study. A multiple analysis of variance covariance analysis and a multiple regression analysis revealed a significant correlation between 1/t(1/2) and CYP2E1 level and aniline hydroxylase activity. However, the correlation between 1/t(1/2) and pentoxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase and erythromycin N-demethylase was not significant. Therefore the contribution of other P450s to chlorzoxazone metabolism seemed to be minor in the concentration range that we tested. In conclusion, fasting rats for 24 h caused a measurable induction of CYP2E1, which produced a significant increase in the rate of chlorzoxazone metabolism and elimination.

Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing.

Chlorzoxazone is a probe drug to assess cytochrome P450 (CYP) 2E1 activity (phenotyping). If the pharmacokinetics of the probe drug is linear, pharmacologically ineffective doses are sufficient for the purpose of phenotyping and adverse effects can thus be avoided. For this reason, we developed and validated an assay for the ultrasensitive quantification of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma. Plasma (0.5mL) and liquid/liquid partitioning were used for sample preparation. Extraction recoveries ranged between 76 and 93% for both analytes. Extracts were separated within 3min on a Waters BEH C18 Shield 1.7µm UPLC column with a fast gradient consisting of aqueous formic acid and acetonitrile. Quantification was achieved using internal standards labeled with deuterium or (13)C and tandem mass spectrometry in the multiple reaction monitoring mode using negative electrospray ionization, which yielded lower limits of quantification of 2.5pgmL(-1), while maintaining a precision always below 15%. The calibrated concentration ranges were linear for both analytes (2.5-1000pgmL(-1)) with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <15% and <11% and plasma matrix effects always were below 50%. The assay was successfully applied to assess the pharmacokinetics of chlorzoxazone in two human volunteers after administration of single oral doses (2.5-5000µg). This ultrasensitive assay allowed the determination of chlorzoxazone pharmacokinetics for 8h after microdosing of 25µg chlorzoxazone.

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.

Clinical assessment of effects of botanical supplementation on cytochrome P450 phenotypes in the elderly: St John's wort, garlic oil, Panax ginseng and Ginkgo biloba.

OBJECTIVES: Elderly patients are more likely to ingest prescription medications concurrently with botanical supplements, and may therefore be vulnerable to herb-drug interactions. Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Some evidence suggests that CYP activity may decrease in the elderly. If so, herb-mediated changes in CYP activity may take on greater clinical relevance in this population. In this study, single timepoint, phenotypic metabolic ratios were used to determine whether long-term supplementation of St John's wort, garlic oil, Panax ginseng, and Ginkgo biloba affected CYP1A2, CYP2D6, CYP2E1 or CYP3A4 activity in elderly subjects. METHODS: Twelve healthy volunteers between the ages of 60 and 76 years (mean age 67 years) were randomly assigned to receive each botanical supplement for 28 days followed by a 30-day washout period. Probe drug cocktails of midazolam, caffeine, chlorzoxazone and debrisoquine were administered before and at the end of supplementation. Pre- and post-supplementation phenotypic ratios were determined for CYP3A4, CYP1A2, CYP2E1 and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hour), paraxanthine/caffeine serum ratios (6-hour), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour) and debrisoquine urinary recovery ratios (8-hour), respectively. The content of purported 'active' phytochemicals was determined for each supplement. RESULTS: Comparisons of pre- and post-St John's wort phenotypic ratios revealed significant induction of CYP3A4 (approximately 140%) and CYP2E1 activity (approximately 28%). Garlic oil inhibited CYP2E1 activity by approximately 22%. P. ginseng inhibition of CYP2D6 was statistically significant, but the magnitude of the effect (approximately 7%) did not appear to be clinically relevant. None of the supplements tested in this study appeared to affect CYP1A2 activity. CONCLUSIONS: Elderly subjects, like their younger counterparts, are susceptible to herb-mediated changes in CYP activity, especially those involving St John's wort. Pharmacokinetic herb-drug interactions stemming from alterations in CYP activity may adversely affect drug efficacy and/or toxicity. When compared with earlier studies that employed young subjects, the data suggest that some age-related changes in CYP responsivity to botanical supplementation may exist. Concomitant ingestion of botanical supplements with prescription medications, therefore, should be strongly discouraged in the elderly.

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.

Induction of CYP2E1 activity in liver transplant patients as measured by chlorzoxazone 6-hydroxylation.

OBJECTIVE: To examine the phenotypic expression of CYP2E1 in liver transplant patients, as measured by the in vivo probe chlorzoxazone, and to evaluate CYP2E1 activity over time after transplantation. METHODS: Thirty-three stable liver transplant patients were given 250 mg chlorzoxazone within 1 year after transplantation as part of a multiprobe CYP cocktail; urine and blood were collected for 8 hours. Chlorzoxazone and 6-hydroxychlorzoxazone concentrations were determined by HPLC. Twenty-eight healthy control subjects, eight patients with moderate to severe liver disease, and four patients who had not received liver transplants were also studied for comparison. The chlorzoxazone metabolic ratio, calculated as the plasma concentration of 6-hydroxychlorzoxazone/chlorzoxazone at 4 hours after chlorzoxazone administration, was used as the phenotypic index. In a subgroup of patients and control subjects, additional blood samples were obtained to allow for the calculation of chlorzoxazone pharmacokinetic parameters by noncompartmental methods. RESULTS: The chlorzoxazone metabolic ratio for the liver transplant patients in the first month after transplantation (mean +/- SD, 6.4 +/- 5.1) was significantly higher than that after 1 month after surgery (2.1 +/- 2.0), when the chlorzoxazone metabolic ratio was not different from control subjects (0.8 +/- 0.5). The chlorzoxazone metabolic ratios in the patients who had not received liver transplants (1.1 +/- 0.7) were equivalent to those of healthy control subjects. The maximum observed 6-hydroxychlorzoxazone plasma concentration was 3046 +/- 1848 ng/ml in seven liver transplant patients in the first month after surgery compared with 1618 +/- 320 ng/ml in 16 healthy control subjects (p < 0.05). The maximum observed concentration of chlorzoxazone, the chlorzoxazone apparent oral clearance, and the formation clearance of 6-hydroxychlorzoxazone were also significantly different between the groups. CONCLUSIONS: We conclude that significant induction of CYP2E1, as indicated by the chlorzoxazone metabolic ratio, occurs in the first month after surgery in liver transplant patients and that drugs that are substrates for CYP2E1 may require dosage alteration during that period. Contrary to expectations, drug metabolism is not uniformly depressed after liver transplantation.