Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats

In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (V _max and CL_int) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC₅₀ values in microsomes from toluene-treated rats were between 3 and 5 μM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r=0.88 and r=0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CL_int: V _max/K _m) in vitro and total body clearance (CL_tot) of CZX hydroxylation and the elimination half-life (t _1/2) of CZX in vivo in toluene-treated rats were high (r=0.784, P < 0.001; r=−0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CL_int (V _max/K _m) in vitro (r=−0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats. Received: 28 September 1999 / Accepted: 10 January 2000     

Chlorzoxazone: a probe drug the metabolism of which can be used to monitor one-point blood sampling in the carbon tetrachloride-intoxicated rat.

In this study, we have carried out an investigation to determine if chlorzoxazone (CZX) is a suitable probe drug for predicting hepatic injury in carbon tetrachloride (CCl4)-intoxicated rats. The animals received oral doses of CCl4 (0.25, 0.5 and 1 ml/kg) 24 h prior to intraperitoneal administration of CZX. The total CYP and CYP2E1 content, as well as the aniline and CZX hydroxylase activity (Vmax and CLint), was reduced depending on the dose of CCl4 administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from CCl4-treated rats was reduced by about 85%. The IC50 value in microsomes from CCl4-treated rats was between 3 and 5 microM. The production of HCZX and the activity of aniline hydroxylase in CCl4-treated rats correlated with the amount of rat CYP2E1 protein (r=0.881, P<0.001 and r=0.822, P<0.001, respectively). The elimination of CZX by CCl4-treated rats was reduced and the HCXZ production in the CCl4-treated group was less than that in the olive oil-treated control group. The correlations between the intrinsic clearance [CLint: Vmax/Km) in vitro and the total body clearance (CLtot) of CZX hydroxylation and the elimination half-life (t1/2) of CZX in vivo in CCl4-treated rats were high (r=0.839, P<0.001 and r= -0.828, P<0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and, 2 h after CZX administration in vivo, there was also a high correlation with the CLint (Vmax/Km) in vitro (r= -0.909, P<0.01). In conclusion, these results from this study demonstrate that CZX is a good probe for monitoring the inhibition of metabolism in rats due to CCl4 treatment.     

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.     

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.     

The effects of alcohol and diallyl sulphide on CYP2E1 activity in humans: a phenotyping study using chlorzoxazone.

The effects of acute administration of dietary levels of ethanol and the garlic oil extract, diallyl sulphide (DAS), on cytochrome P450 2E1 (CYP2E1) activity in volunteers were studied using the selective probe substrate, chlorzoxazone (CZX). The ratio of the CZX metabolite 6- hydroxychlorzoxazone (6-OHCZX) to CZX was taken to indicate CYP2E1 activity. The mean differences between the baseline and DAS-treated (0.2 mg/kg) CYP2E1 activities were significantly different (two-tailed p value = 0.0242, n = 8). Likewise, the mean differences between the baseline and ethanol-treated (0.8 g/kg) CYP2E1 activities were also significantly different (two-tailed p value = 0.0005, n = 7). The reduction in in vivo CYP2E1 activity by DAS is consistent with reported inhibition observed in vitro. The marked reduction in CYP2E1 activity following acute ingestion of ethanol is consistent with a competitive inhibition mechanism of CZX metabolism. The inhibitory effect of DAS maybe additive with daily consumption of Allium vegetables in particular. This may explain the lower 6-OHCZX/CZX metabolic ratios measured in various European and Mexican cohorts and is consistent with the lower incidence of stomach, liver and colon cancers observed in southern Europeans.     

Pharmacokinetics of intravenous chlorzoxazone in rats with dehydration and rehydration: effects of food intakes

The following results were obtained recently from our laboratories; in rats with 72-h water deprivation (rats with dehydration), the hepatic cytochrome P450 2E1 (CYP2E1) was three-fold induced with an increase in the mRNA. Rehydration of 48-h water-deprived rats for the next 24 h with free access of food (rats with rehydration) restored CYP2E1 level to that of control. However, rehydration of 48-h water-deprived rats for the next 24 h with limited food supply (20% of control) failed to restore the CYP2E1 level to that of control. Hence, the CYP2E1 changes in rats with dehydration and rehydration resulted from differences in food intakes but not from dehydration or rehydration per'se. Chlorzoxazone (CZX) is metabolized to 6-hydroxychlorzoxazone (OH-CZX) mainly by CYP2E1 in rats. Therefore, the pharmacokinetics of CZX and OH-CZX were compared after intravenous administration of CZX, 25 mg/kg, to control rats and rats with dehydration and rehydration with free access of food. In rats with dehydration, the amount of 24-h urinary excretion of free OH-CZX plus its glucuronide conjugates (Ae (OH-CZX, 0-24 h,) expressed in terms of intravenous dose of CZX) was significantly greater (45.6 compared with 35.6%) and area under the plasma concentration-time curve from time zero to time infinity (AUC) of CZX was significantly smaller (2190 compared with 3200 micro g min/ml) than those in control rats. The above data indicated that the formation of OH-CZX increased significantly in rats with dehydration due to 3-fold induction of CYP2E1. In rats with rehydration with free access of food, the Ae (OH-CZX, 0-24 h) (39.0 compared with 35.6%) and AUC of CZX (2870 compared with 3200 micro g min/ml) were restored (comparable) to control levels since the expression of CYP2E1 in rats with dehydration returned to control level by rehydration. The above data indicate that CZX could be used as a chemical probe to assess the activity of CYP2E1 in rats with dehydration and rehydration.     

Down-regulation of cytochrome P450 proteins and its activities by Shiga-like toxin II from Escherichia coli O157:H7

Escherichia coli O157:H7 infection frequently induces clinical complications such as hemolytic uremic syndromes and intestinal dysfunctions. These changes could alter the disposition of drugs, consequently changing their efficacy. However, the possible changes of drug-metabolizing activities by E. coli O157:H7 infection have not been addressed. Thus, we have investigated the effect of Shiga-like toxin type II (SLT-II), derived from E. coli O157:H7, on the hepatic cytochrome P450 (CYP) content and its activity in rats. SLT-II (2microg per animal, i.v.) time-dependently decreased total CYP content and the contents of CYP2C11 and CYP3A2 in hepatic microsomal preparations up to 24hr following injection. Consistently, SLT-II time-dependently decreased CYP activity in vivo, as represented by systemic clearance of antipyrine. An inhibitor of inducible nitric oxide synthase, S-methylisothiourea, restored the decreased systemic clearance of antipyrine by SLT-II, suggesting the involvement of the overproduction of nitric oxide by SLT-II. Moreover, dexamethasone restored the decreased systemic clearance of antipyrine by SLT-II. In the hepatic microsomal preparation, dexamethasone restored the SLT-II-induced decrease of CYP3A2 whereas S-methylisothiourea did not affect both CYP subtypes. Taken together, these results suggest that SLT-II might alter hepatic drug-metabolizing function during E. coli O157 infection and that more than one cytokines induced by SLT-II, including nitric oxide, might make a critical contribution to the decrease of CYP content and its activity.     

Effect of hypoxia on cytochrome P450 activity and expression

In the last three decades, numerous reports have shown that patients with chronic pulmonary disease and with heart failure with hypoxemia cleared drugs at a lower rate than healthy volunteers. As a consequence decreased clearance, drug toxicity is frequent in these patients. The reduction in drug clearance is due to a decrease in activity of cytochrome P450 isoforms, partly associated to the hypoxemia. With in vivo animal models, acute moderate hypoxia (PaO2 of around 35-50 mm Hg) reduces the clearance of drugs biotransformed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2E1, although hypoxia does not affect the clearance of drugs biotransformed by CYP3A6. Ex vivo and in vitro experiments demonstrate that hypoxia down-regulates CYP1A1, CYP1A2, CYP2B6, CYP2C9 and CYP2C19, decrease preceded by a reduction in activity. On the other hand, acute moderate hypoxia up-regulates CYP3A6. The changes in protein expression are preceded by modifications in the mRNA coding for the proteins. The effect of hypoxia on hepatic cytochrome P450 is carried out by serum mediators, e.g. interferon-gamma, interleukin-1beta, and interleukin-2 are responsible for the decrease in activity and in expression of cytochrome P450 isoforms, and erythropoietin accounts for the increase in CYP3A6. Probably several mechanisms underlie and contribute to the decrease in activity and down-regulation of cytochrome P450 isoforms by hypoxia, e.g. reducing potentiation factors, inducing repressor elements and activating negative regulatory elements. The up-regulation of CYP3A6 implies a PTK- and p42/44MAPK-dependent stabilization/activation, nuclear translocation of HIF-1 and AP-1, binding to CYP3A6 promoter, and transactivation of the gene to induce CYP3A6 expression.     

Effect of 4-(4-chlorobenzyl)pyridine on rat hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in vivo and in vitro

The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12), and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.     

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.     

Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity.

1. The interindividual variation and enantioselectivity of the in vitro styrene oxidation by cytochrome P450 have been investigated in 20 human microsomal liver samples. Liver samples were genotyped for the CYP2E1*6 and CYP2E1*5B alleles. 2. Kinetic analysis indicated the presence of at least two forms of styrene-metabolizing cytochrome P450. The enzyme constants for the high-affinity component were subject to appreciable interindividual variation, i.e. Vmax1 ranged from 0.39 to 3.20 nmol mg protein(-1) min(-1) (0.96+/-0.63) and Km1 ranged from 0.005 to 0.03 mM (0.011+/-0.006). Inhibition studies with chemical inhibitors of CYP2E1, CYP1A2, CYP2C8/9 and CYP3A4 demonstrated that CYP2E1 was the primary enzyme involved in the high-affinity component of styrene oxidation. No relationship between the interindividual variation in Vmax1 and Km1 and the genetic polymorphisms of the CYP2E1 gene was found. 3. Cytochrome P450-mediated oxidation of styrene demonstrated a moderate enantioselectivity, with an enantiomeric excess (ee) of (S)-styrene oxide of 15% (range 4-27%) at low styrene concentration and an ee of (R)-styrene oxide of 7% (range -11 to +22%) at high styrene concentration. This points towards the involvement of at least two cytochrome P450, with different enantioselectivities. 4. The data indicate that cytochrome P450-mediated styrene oxidation is subject to considerable interindividual variation, but only to a moderate product enantioselectivity.     

Decreased in vivo metabolism of drugs in chronic renal failure.

Chronic renal failure (CRF) is associated with a decrease in renal excretion of drugs, but its effects on the liver metabolism of xenobiotics are poorly defined. The objectives of this study were to determine the effects of CRF on hepatic cytochrome P450 (CYP450) and its repercussions on in vivo hepatic metabolism of drugs. Two groups of rats were studied: control paired-fed and CRF. CRF was induced by subtotal nephrectomy. Total CYP450 activity and protein expression of several CYP450 isoforms (CYP1A2, CYP2C11, CYP3A1, CYP3A2) were assessed in liver microsomes. In vivo cytochrome P450 activity was evaluated with breath tests using substrates for different isoenzymes: caffeine (CYP1A2), aminopyrine (CYP2C11), and erythromycin (CYP3A2). Creatinine clearance was reduced by 60% (P <. 01) in rats with CRF. Compared with control paired-fed rats, total CYP450 activity was reduced by 40% in rats with CRF. Protein expression of CYP2C11, CYP3A1, and CYP3A2 was considerably reduced (more than 45%, P <.001) in rats with CRF, whereas the levels of CYP1A2 were unchanged. In rats with CRF, there was a 35% reduction in the aminopyrine (CYP2C11) and the erythromycin (CYP3A2) breath tests compared with control animals (P <.001). The caffeine (CYP1A2) breath tests remained comparable to controls. Creatinine clearance correlated with the aminopyrine and erythromycin breath tests (r(2) = 0.73 and r(2) = 0.81, respectively, P <.001). In conclusion, CRF is associated with a decrease in total liver CYP450 activity in rats (mainly in CYP2C11, CYP3A1, and CYP3A2), which leads to a significant decrease in the metabolism of drugs.     

Effects of cysteine on the pharmacokinetics of intravenous chlorzoxazone in rats with protein-calorie malnutrition

The effects of cysteine on the pharmacokinetics of chlorzoxazone (CZX) and one of its metabolites, 6-hydroxychlorzoxazone (OH-CZX), were investigated after intravenous administration of CZX, 25 mg/kg, to control rats (4-week fed on 23% casein diet) and rats with PCM (4-week fed on 5% casein diet) and PCMC (PCM with oral cysteine supplementation, 250 mg/kg, twice daily during the fourth week). In rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity (AUC) of OH-CZX (436 compared with 972 microgmin/ml) and the percentages of intravenous dose of CZX excreted in 8-h urine as OH-CZX (20.2 compared with 38.5%) were significantly smaller than those in control rats. The above data indicated that the formation of OH-CZX from CZX decreased significantly in rats with PCM due to a significant decrease in chlorzoxazone-6-hydroxylase activity (328 compared with 895 pmol/min/mg protein) in the rats. The results were expected since in rats with PCM, hepatic CYP2E1 expression and its mRNA levels decreased significantly as compared to control, and CZX was metabolized to OH-CZX primarily by CYP2E1 in rats. By cysteine supplementation (rats with PCMC), some pharmacokinetic parameters restored fully (hepatic microsomal chlorzoxazone 6-hydroxylation activity based on both mg protein and nmol CYP450) or partially (total body clearance and apparent volume of distribution at steady state of CZX, and AUC, terminal half-life and 8-h urinary excretion of OH-CZX) to control levels.     

Human cytochrome P450 maximal activities in pediatric versus adult liver.

Drug clearance is often higher in children than in adults, particularly when normalized to body weight. We previously showed that liver volume normalized to body weight was inversely related to age, but that the systemic clearance of a nonspecific cytochrome P450 (CYP) substrate (antipyrine) was higher in young children compared with adults even when normalized per liver volume. Our purpose herein was to evaluate whether P450 catalytic activities, expressed as maximal catalytic rates per milligram of microsomal protein, differed in up to 37 normal livers from subjects <10 (range 0.5-9 years of age), >10 but <60 years of age (range 10-59 years), and >60 year (range 63-93 years of age). There were no age-related differences in the oxidation of ethoxyresorufin (P =.83) (CYP1A2), ethoxycoumarin (P =.52) (CYP2E1 and other P450s), teniposide (P =. 58), midazolam (P =.47) (CYP3A4/3A5), or paclitaxel (P =.24) (at the 17alpha position, CYP2C8). Tolbutamide hydroxylation tended to be lower in children versus adults (P =.047) (CYP2C9), but did not reach statistical significance after correcting for multiple comparisons. No relationship was found to exist between age and microsomal recovery (P =.98); thus, recovery did not account for the lack of age-related differences in catalytic activity. We conclude that increased intrinsic cytochrome P450 activity is unlikely to account for increased clearance of most P450 drug substrates in children.     

Preclinical and in vitro assessment of the potential of D0870, an antifungal agent, for producing clinical drug interactions.

1. D0870, an azole antifungal agent, produced dose-related increases in total cytochrome P450 and aldrin epoxidase when administered as 14 daily oral doses (0, 0.5, 2.5 and 12.5 mg/kg/day) to the male rat. Administered as single doses, D0870 increased pentobarbitone-sleeping time in a dose-related manner. 2. In human hepatic microsomal incubations, D0870 produced pronounced inhibition of CYP2C9 (tolbutamide hydroxylase) and, to a lesser degree, CYP3A4 (testosterone 6beta-hydroxylase), but had more limited effects on CYP1A2, 2C19 and 2D6 activity. In comparison with ketoconazole, itraconazole and fluconazole, D0870 was the most potent inhibitor of CYP2C9 activity. It is predicted that D0870 may inhibit the in vivo clearance of CYP2C9 substrates by approximately 58%, thereby increasing their steady-state concentrations by 2.4 times, which would be of clinical significance for some compounds. 3. During incubation of [14C]-D0870 with cultured human hepatocytes for up to 72 h, two discrete metabolites (A and B) were formed. Formation of metabolite A was abolished by both quinidine and ketoconazole and is probably CYP3A4-mediated, whereas generation of metabolite B did not appear to be dependent on cytochrome P450. 4. D0870 has potential to produce both induction and inhibition of cytochrome P450 enzymes in man.     

Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

AIMS: The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. METHODS: The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. RESULTS: The CYP1A2 inhibitor furafylline variably inhibited (0-65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by approximately 55-60%, 35-55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. CONCLUSIONS: Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans.     

Effect of the acute-phase response on the pharmacokinetics of chlorzoxazone and cytochrome P-450 2E1 in vitro activity in rats.

The acute-phase response is known to produce alterations in hepatic cytochrome P-450 (CYP) expression. Lipopolysaccharide (LPS), a well known inducer of acute-phase response decreases hepatic CYP2E1 in vitro activity in rats. This study was designed to determine if LPS administration produced alterations in the pharmacokinetics of chlorzoxazone (CZN), a marker for CYP2E1 expression. Sprague-Dawley rats were administered a single i.p. injection of LPS (5 mg/kg) or saline control approximately 24 h before a single i.v. bolus dose of CZN (15 mg/kg). Serial blood samples were collected over a 120-min period to quantitate CZN plasma concentrations and protein binding. In addition, livers were removed and processed for evaluating in vitro CYP2E1 protein concentrations and activity. Systemic clearance decreased by 35% in LPS-treated rats, whereas half-life and steady-state volume of distribution increased by 167 and 66%, respectively. The plasma free-fraction of CZN increased 2-fold after LPS treatment. The CZN intrinsic clearance decreased in LPS rats by 71% compared with control values. The CYP2E1 liver microsomal activity decreased between 55 and 75% along with a 41% decrease in CYP2E1 protein concentration. The CZN intrinsic clearance was significantly correlated with both the CZN and p-nitrophenol liver microsomal activity (r = 0.97 and r = 0.91, respectively). This study demonstrated that LPS administration produced expected reductions in the in vivo intrinsic clearance of CZN, and these changes were highly correlated with in vitro activity studies. In addition, LPS produced significant increases in the steady-state volume of distribution of CZN secondary to reductions in its plasma protein binding.     

Pulmonary bioactivation of trichloroethylene to chloral hydrate: relative contributions of CYP2E1, CYP2F, and CYP2B1.

Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-dependent bioactivation to reactive metabolites. In this investigation, studies were undertaken to test the hypothesis that TCE metabolism to chloral hydrate (CH) is mediated by cytochrome P450 enzymes, including CYP2E1, CYP2F, and CYP2B1. Recombinant rat CYP2E1 catalyzed TCE metabolism to CH with greater affinity than did the recombinant P450 enzymes, rat CYP2F4, mouse CYP2F2, rat CYP2B1, and human CYP2E1. The catalytic efficiencies of recombinant rat CYP2E1 (V(max)/K(m) = 0.79) for generating CH was greater than those of recombinant CYP2F4 (V(max)/K(m) = 0.27), recombinant mouse CYP2F2 (V(max)/K(m) = 0.11), recombinant rat CYP2B1 (V(max)/K(m) = 0.07), or recombinant human CYP2E1 (V(max)/K(m) = 0.02). Decreases in lung microsomal immunoreactive CYP2E1, CYP2F2, and CYP2B1 were manifested at varying time points after TCE treatment. The loss of immunoreactive CYP2F2 occurred before the loss of immunoreactive CYP2E1 and CYP2B1. These protein decreases coincided with marked reduction of lung microsomal p-nitrophenol hydroxylation and pentoxyresorufin O-dealkylation. Rates of CH formation in the microsomal incubations were time-dependent and were incremental from 5 to 45 min. The production of CH was also determined in human lung microsomal incubations. The rates were low and were detected in only three of eight subjects. These results showed that, although CYP2E1, CYP2F, and CYP2B1 are all capable of generating CH, TCE metabolism is mediated with greater affinity by recombinant rat CYP2E1 than by recombinant CYP2F, CYP2B1, or human CYP2E1. Moreover, the rates of CH production were substantially higher in murine than in human lung.     

Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole

To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.     

Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.