(-)-N-3-Benzylphenobarbital Is Superior to Omeprazole and (+)-N-3-Benzylnirvanol as a CYP2C19 Inhibitor in Suspended Human Hepatocytes.

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.

Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

Confirmation that cytochrome P450 2C8 (CYP2C8) plays a minor role in (S)-(+)- and (R)-(-)-ibuprofen hydroxylation in vitro.

Various groups have sought to determine the impact of CYP2C8 genotype (and CYP2C8 inhibition) on the pharmacokinetics (PK) of ibuprofen (IBU) enantiomers. However, the contribution of cytochrome P450 2C8 (CYP2C8) in human liver microsomes (HLMs) has not been reported. Therefore, in vitro cytochrome P450 (P450) reaction phenotyping was conducted with selective inhibitors of cytochrome P450 2C9 (CYP2C9) and CYP2C8. In the presence of HLMs, sulfaphenazole (CYP2C9 inhibitor), and anti-CYP2C9 monoclonal antibodies (mAbs) inhibited (73-100%) the 2- and 3-hydroxylation of both IBU enantiomers (1 and 20 microM). At a higher IBU concentration (500 microM), the same inhibitors were less able to inhibit the 2-hydroxylation of (S)-(+)-IBU (32-52%) and (R)-(-)-IBU (30-64%), whereas the 3-hydroxylation of (S)-(+)-IBU and (R)-(-)-IBU was inhibited 66 to 83 and 70 to 89%, respectively. In contrast, less inhibition was observed with montelukast (CYP2C8 inhibitor, < or =35%) and anti-CYP2C8 mAbs (< or =24%) at all concentrations of IBU. When (S)-(+)-IBU and (R)-(-)-IBU (1 microM) were incubated with a panel of recombinant human P450s, only CYP2C9 formed appreciable amounts of the hydroxy metabolites. At a higher IBU enantiomer concentration (500 microM), additional P450s catalyzed 2-hydroxylation (CYP3A4, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP2B6) and 3-hydroxylation (CYP2C19). When the P450 reaction phenotype and additional clearance pathways are considered (e.g., direct glucuronidation and chiral inversion), it is concluded that CYP2C8 plays a minor role in (R)-(-)-IBU (<10%) and (S)-(+)-IBU ( approximately 13%) clearance. By extension, one would not expect CYP2C8 inhibition (and genotype) to greatly affect the pharmacokinetic profile of either enantiomer. On the other hand, CYP2C9 inhibition and genotype are expected to have an impact on the PK of (S)-(+)-IBU.

Liver disease selectively modulates cytochrome P450--mediated metabolism.

BACKGROUND: The liver plays a significant role in drug metabolism; thus it would be expected that liver disease may have a detrimental effect on the activity of cytochrome P450 (CYP) enzymes. The extent to which the presence and severity of liver disease affect the activity of different individual drug-metabolizing enzymes is still not well characterized. The purpose of this study was to assess the effect of liver disease on multiple CYP enzymes by use of a validated cocktail approach. METHODS: The participants in this investigation were 20 patients with different etiologies and severity of liver disease and 20 age-, sex-, and weight-matched healthy volunteers. Liver disease severity was categorized by use of the Child-Pugh score. All participants received a cocktail of 4 oral drugs simultaneously, caffeine, mephenytoin, debrisoquin (INN, debrisoquine), and chlorzoxazone, as in vivo probes of the drug-metabolizing enzymes CYP1A2, CYP2C19, CYP2D6, and CYP2E1, respectively. The primary end points were measurements of specific CYP metabolism indexes for each enzyme. RESULTS: Mephenytoin metabolism was significantly decreased in both patients with mild liver disease (Child-Pugh score of 5/6) (-63% [95% confidence interval (CI), -86% to -40%]; P = .0003) and patients with moderate to severe liver disease (Child-Pugh score >6) (-80% [95% CI, -95% to -64%]; P = .0003). In comparison with control subjects, the caffeine metabolic ratio was 69% lower (95% CI, -85% to -54%; median, 0.14 versus 0.62; P = .0003), the debrisoquin recovery ratio was 71% lower (95% CI, -96% to -47%; median, 0.10 versus 0.65; P = .012), and the chlorzoxazone metabolic ratio was 60% lower (95% CI, -91% to -29%; median, 0.21 versus 0.83; P = .0111) in patients with moderate to severe liver disease. All 4 drugs showed significant negative relationships with the Child-Pugh score. CONCLUSIONS: CYP enzyme activity is differentially affected by the presence of liver disease. We propose that the data can be explained by the "sequential progressive model of hepatic dysfunction," whereby liver disease severity has a differential effect on the metabolic activity of specific CYP enzymes.

In vitro metabolism of tributyltin and triphenyltin by human cytochrome P-450 isoforms.

The metabolic fate of tributyltin and triphenyltin may contribute to the toxicity of these chemicals. We used human hepatic cytochrome P-450 (CYP) systems to confirm the specific CYP(s) involved in the in vitro metabolism of tributyltin and triphenyltin. There were no significant sex differences in the metabolic pattern of tributyltin or triphenyltin, indicating that the CYP(s) responsible for the metabolism of these chemicals in humans is/are not sex-specific form(s). Six major drug-metabolizing isoforms of cDNA-expressed human CYPs and the CYP2C subfamily were tested to determine their metabolic capacities for tributyltin and triphenyltin. CYP2C9, 2C18, 2C19, and 3A4 significantly mediated both dealkylation and dearylation of these triorganotins. Furthermore, the metabolism of tributyltin and triphenyltin was significantly inhibited in vitro by pretreatment with selective inhibitors, azamulin for CYP3A4 and N-3-benzylnirvanol for CYP2C19. Since the CYP2C18 content of hepatic microsomes in humans is relatively low, CYP2C9, 2C19, and 3A4 might be the main isoforms of CYP that are responsible for tributyltin and triphenyltin metabolism in the human liver.

P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats.

Male F344/NCr rats, 6 wk old, were fed 500 ppm of phenobarbital (PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or 5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufin O-dealkylase and aminopyrine N-demethylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective. To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 rats were given a single i.p. injection of 75 mg N-nitrosodiethylamine/kg body weight. Beginning 2 wk later, they were placed either on normal diet or diet containing 500 ppm of PB or equimolar doses of EPH or EEH for the remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificed at either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk and hepatocellular carcinomas at 78 wk in N-nitrosodiethylamine-initiated rats. Neither the incidence of hepatocellular neoplasms nor the number and size of hepatocellular foci was significantly increased by EEH. At 78 wk, both PB and EPH enhanced the development of thyroid follicular cell neoplasms in N-nitrosodiethylamine-initiated rats while no such enhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticonvulsant, like the structurally similar PB, promoted hepatocellular and thyroid follicular cell carcinogenesis and induced the PB-inducible form(s) of cytochrome P-450 (P-450b) in rats. In contrast, EEH unlike barbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochrome P-450 in rats.

Mephenytoin hydroxylation deficiency in Caucasians: frequency of a new oxidative drug metabolism polymorphism.

The ability of normal subjects to hydroxylate mephenytoin (100 mg) or debrisoquine (10 mg) after oral dosing was investigated in 156 unrelated Caucasians living in middle Tennessee. Urinary recovery of 4-hydroxymephenytoin (4-OH-M) and the urinary S:R enantiomeric ratio of mephenytoin measured in an 8-hr urine sample were investigated as phenotypic traits for mephenytoin, and the urinary metabolic ratio of debrisoquine was used to determine the debrisoquine hydroxylase phenotype. Both urinary 4-OH-M and the S:R ratio of mephenytoin discriminated between extensive (EM) and poor (PM) metabolizers of mephenytoin. The frequencies of PMs for mephenytoin and debrisoquine hydroxylation activity were 2.6% and 7.0%. These two defects in oxidative metabolism were not observed in the same subjects, which suggests that 4-hydroxylation of mephenytoin is a new polymorphism independent of that for debrisoquine.

Stereoselective mephobarbital hydroxylation cosegregates with mephenytoin hydroxylation.

The 8-hour urinary recovery of 4-hydroxy-mephobarbital has been measured after oral administration of racemic mephobarbital (90 mg) in 17 extensive (EM) and six poor (PM) metabolizer phenotypes of mephenytoin. The recovery of this metabolite was measurable in every EM and ranged from 2.5% to 48% (10.9% +/- 1.9% of dose), but was not detected in any PM (less than 1% of dose). In EMs, the 8-hour urine recovery of 4-OH-mephobarbital after mephobarbital was approximately half that of 4-OH-mephenytoin over the same time after mephenytoin administration. One EM received similar doses of R- and S-mephobarbital on separate occasions. Urinary recovery of 4-OH-mephobarbital was 33% and less than 1%, respectively. These results suggest that mephobarbital is stereoselectively hydroxylated by the same drug metabolizing enzyme that is responsible for the stereoselective aromatic hydroxylation of mephenytoin.

Incidence of S-mephenytoin hydroxylation deficiency in a Korean population and the interphenotypic differences in diazepam pharmacokinetics.

We studied the genetically determined hydroxylation polymorphism of S-mephenytoin in a Korean population (N = 206) and the pharmacokinetics of diazepam and demethyldiazepam after an oral 8 mg dose of diazepam administered to the nine extensive metabolizers and eight poor metabolizers recruited from the population. The log10 percentage of 4-hydroxymephenytoin excreted in the urine 8 hours after administration showed a bimodal distribution with an antimode of 0.3. The frequency of occurrence of the poor metabolizers was 12.6% in the population. In the panel study of diazepam in relation to the mephenytoin phenotype, there was a significant correlation between the oral clearance of diazepam and log10 urinary excretion of 4-hydroxymephenytoin (rs = 0.777, p less than 0.01). The plasma half-life of diazepam in the poor metabolizers was longer than that in the extensive metabolizers (mean +/- SEM, 91.0 +/- 5.6 and 59.7 +/- 5.4 hours, p less than 0.005), and the poor metabolizers had the lower clearance of diazepam than the extensive metabolizers (9.4 +/- 0.5 and 17.0 +/- 1.4 ml/min, p less than 0.001). In addition, the plasma half-life of demethyldiazepam showed a statistically significant (p less than 0.001) difference between the extensive metabolizers (95.9 +/- 11.3 hours) and poor metabolizers (213.1 +/- 10.7 hours), and correlated with the log10 urinary excretion of 4-hydroxymephenytoin (rs = -0.615, p less than 0.01). The findings indicate that the Korean subjects have a greater incidence of poor metabolizer phenotype of mephenytoin hydroxylation compared with that reported from white subjects and that the metabolism of diazepam and demethyldiazepam is related to the genetically determined mephenytoin hydroxylation polymorphism in Korean subjects.

S-mephenytoin hydroxylation phenotypes in a Jordanian population.

We tested the ability of 194 unrelated, healthy Jordanian volunteers to metabolize S-mephenytoin. Mephenytoin (100 mg) was coadministered with debrisoquin (10 mg) orally and urine was collected for 8 hours. Mephenytoin metabolism was tested according to three measures: the amount of 4-hydroxymephenytoin, the S/R enantiomeric ratio, and the presence of a polar, acid-labile metabolite in urine collected for 8 hours after the dose. The S/R ratio and the presence of the acid-labile metabolite were determined in the urine of 16 patients who had low amounts of 4-hydroxymephenytoin (log hydroxylation index > or = 1). On examination of these three parameters of oxidation status, nine subjects were found to be poor metabolizers of mephenytoin by all three parameters. Thus 4.6% (95% confidence interval of 1.6% to 7.6%) of Jordanian subjects studied were poor metabolizers of mephenytoin. According to the Hardy-Weinberg Law, the frequency of the recessive autosomal gene controlling the poor metabolizer status of mephenytoin was predicted to be 0.215% (95% confidence interval of 0.146% to 0.283%). These results are on the same order of magnitude as those determined in European white populations and constitute the first report in Arab populations.

Metabolic disposition of pantoprazole, a proton pump inhibitor, in relation to S-mephenytoin 4'-hydroxylation phenotype and genotype.

OBJECTIVES: To assess the possible relationship between the metabolic disposition of pantoprazole and genetically determined S-mephenytoin 4'-hydroxylation phenotype and genotype. METHODS: The pharmacokinetic disposition of pantoprazole was investigated in 14 Japanese male volunteers (seven extensive and seven poor metabolizers of S-mephenytoin). All subjects received a single 40 mg oral dose of pantoprazole as the enteric-coated formulation. RESULTS: An interphenotypic difference in the metabolic disposition of pantoprazole was observed: the mean values for area under the concentration-time curve (AUC), elimination half-life (t1/2), and apparent oral clearance were significantly (p < 0.01) greater, longer, and lower, respectively, in the poor metabolizers than in the extensive metabolizers. The mean AUC of pantoprazole sulfone was greater (p < 0.01) in the poor metabolizers than in the extensive metabolizers, whereas the mean AUC of the main demethylated metabolite (M2) was lower (p < 0.01) in the poor metabolizers than in the extensive metabolizers. A significant negative correlation was observed between the individual values for log10% urinary excretion of 4'-hydroxymephenytoin and AUC of pantoprazole (rs = -0.816; p < 0.005). The CYP2C19 genotyping test results were found to be in a complete accordance with the phenotypes. CONCLUSION: These data indicated that the metabolic disposition of pantoprazole is under the pharmacogenetic control of S-mephenytoin 4'-hydroxylase (CYP2C19).

Isozyme-specific induction of low-dose aspirin on cytochrome P450 in healthy subjects.

OBJECTIVE: This study was designed to define the effect of low-dose aspirin administration on the activity of cytochrome P450 (CYP) in normal human subjects. METHODS: Aspirin, 50 mg daily, was given for 14 days to 18 nonsmoking healthy male volunteers. A modified 5-drug cocktail procedure consisting of caffeine, mephenytoin, metoprolol, chlorzoxazone, and midazolam was performed to simultaneously assess in vivo activity of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A, respectively. The activities were assessed on 4 occasions including at baseline, after 7 and 14 daily doses of aspirin, and at 7 days after discontinuation of aspirin. Concentrations of parent drugs and corresponding metabolites in biologic samples were assayed by reversed-phase HPLC. RESULTS: Both 7-day and 14-day aspirin intake increased the activity of CYP2C19 significantly, as indicated by 4-hydroxymephenytoin urinary recovery (P <.001). Induction of low-dose aspirin on CYP2C19 was time-dependent. CYP3A activity indices increased moderately but significantly by both 7-day and 14-day aspirin treatment (P <.05), but the percentage changes in CYP3A activity indices were not significant. Low-dose aspirin had no effect on CYP1A2, CYP2D6, and CYP2E1 in vivo activity by either 7-day or 14-day treatment. CONCLUSIONS: The effect of low-dose aspirin on CYPs was enzyme-specific. Both 7-day and 14-day low-dose aspirin induced the in vivo activities of CYP2C19 but did not affect the activities of CYP1A2, CYP2D6, and CYP2E1. The effect of low-dose aspirin on CYP3A activity awaits further confirmation. When low-dose aspirin is used in combination with drugs that are substrates of CYP2C19, doses of the latter should be adjusted to ensure their efficacy.

Selective effect of liver disease on the activities of specific metabolizing enzymes: investigation of cytochromes P450 2C19 and 2D6.

BACKGROUND AND OBJECTIVES: Drug metabolism is influenced by liver disease because of the central role that the liver plays in metabolic activities in the body. However, it is still unclear how activities of specific drug-metabolizing enzymes are influenced by the presence and severity of liver disease. As a consequence, alteration in metabolism of specific drugs cannot be easily predicted or appropriate dosage adjustment recommendations made. METHODS: The activities of cytochromes P450 (CYP) 2C19 and 2D6 were investigated in a group of patients with mild or moderate liver disease (n = 18) and a group of healthy control subjects (n = 10). The disposition of racemic mephenytoin for CYP2C19 and debrisoquin for CYP2D6 were characterized in plasma and urine samples collected over 192 hours. RESULTS: The elimination of S-mephenytoin was severely reduced among patients with liver disease, resulting in a 79% decrease in plasma clearance for all patients combined. This reduction was related to the severity of disease, patients with moderate disease being affected more severely than patients with mild disease. Similar differences were observed in the urinary excretion of 4'-hydroxymephenytoin metabolite. By contrast, there was no effect on the disposition of R-mephenytoin or debrisoquin. CONCLUSION: These results show selectivity in the effect of liver disease on activities of specific metabolizing enzymes, CYP2C19 being more sensitive than CYP2D6. They suggest that recommendations for modification in drug dosage in the presence of liver disease should be based on knowledge of the particular enzyme involved in metabolism of the drug. The results emphasize the need for further studies of each specific drug-metabolizing enzyme in the presence of liver disease.

Disposition of mephenytoin and its metabolite, nirvanol, in epileptic patients.

We investigated the conversion of mephenytoin to nirvanol in five patients with uncontrolled complex partial seizures. After a 50-mg single oral dose, mean peak mephenytoin level was 0.48 microgram/ml and nirvanol 0.37 microgram/ml. After 400 mg, peak mephenytoin level was 3.9 micrograms/ml and nirvanol 2.5 micrograms/ml. On 400 mg daily, mephenytoin reached a mean steady-state level of 1.5 micrograms/ml. Nirvanol mean steady-state level was 18 micrograms/ml. Mean plasma half-life was 17 hours for mephenytoin and 114 hours for nirvanol. Two patients had reduced seizures during mephenytoin therapy and one a transient increase during drug withdrawal. No toxicity was seen, but mephenytoin was not more effective than phenytoin.

A markedly diminished pleiotropic response to phenobarbital and structurally-related xenobiotics in Zucker rats in comparison with F344/NCr or DA rats.

Phenobarbital (PB) and certain structurally-related compounds induce a variety of hepatic drug-metabolizing enzymes in many strains of rats. Thus, following administration of PB (300, 500 ppm), barbital (BB, 1500 ppm) or 5-ethyl-5-phenylhydantoin (EPH, 500 ppm), CYP2B1-mediated benzyloxyresorufin O-dealkylase activity and epoxide hydrolase activity were profoundly induced in female DA and F344/NCr rats. In contrast, outbred female lean and obese Zucker rats showed markedly reduced CYP2B1 responses (less than 15% and less than 5% of those observed in the female DA or F344/NCr rat) to PB (doses less than or equal to 300 ppm), BB (1500 ppm) or EPH (500 ppm). In parallel studies, profound increases in RNA levels coding for CYP2B1, glutathione S-transferases Ya/Yc (alpha subclass), or epoxide hydrolase were detected in the female F344/NCr rat following treatment with PB (300 ppm), BB (1500 ppm) or EPH (500 ppm). In contrast, lean Zucker rats showed a strong response only to the highest dose of PB (500 ppm), implying that the diminished response in the Zucker rats may occur at some pretranslational level. Similar studies with lower doses of PB, EPH or BB in male lean Zucker rats showed a decreased response, relative to that in male F344/NCr rats. However, this insensitivity was not as profound as that observed in the female Zucker rats. In fact, the response to PB-type inducers in male or female Zucker rats is probably most clearly explained as a shift of the dose-response curve sharply to the right (decreased responsiveness, compared to F344/NCr or DA rats of the same sex). This decreased responsiveness of female lean Zucker rats to induction of CYP2B1, relative to that of F344/NCr rats, was also observed with the structurally-diverse PB-type inducers clonazepam, clotrimazole and 2-hexanone. In contrast, the female Zucker rat (obese or lean) displayed a pronounced response to induction of CYP1A-mediated ethoxyresorufin O-deethylase activity by beta-naphthoflavone, a prototype inducer of CYP1A1 and CYP1A2. The Zucker rat would thus appear to represent a potentially exploitable genetic model for examining the mechanism of enzyme induction by the myriad xenobiotics which induce a PB-type response.

The influence of St John's Wort on CYP2C19 activity with respect to genotype.

Induction of cytochrome p450 isozymes is the major cause for clinical drug interactions of St. John's wort. The relationships of St. John's wort to cytochrome p450 isoforms have been fully investigated, but its effect on CYP2C19 is lacking. Thus, the aim of the present study was to observe the effect of St. John's wort on CYP2C19 activity using CYP1A2 as a control. Twelve healthy adult men-6 extensive metabolizers of CYP2C19 (2C19(*)1/2C19(*)1) and 6 poor metabolizers (4 2C19(*)2/2C19(*)2 and 2 2C19(*)2/2C19(*)3)-were enrolled in a two-phase, randomized, crossover manner. All subjects took a 300-mg St. John's wort tablet or placebo three times daily for 14 days, and then the activities of CYP2C19 and CYP1A2 were measured using mephenytoin and caffeine. It was found that St. John's wort treatment significantly increased CYP2C19 activity in CYP2C19 wild-genotype subjects, with urinary 4'-hydroxymephenytoin excretion raised by 151.5% +/- 91.9% (p = 0.0156), whereas no significant alteration was observed for CYP2C19 poor metabolizers. Repeated St. John's wort administration did not affect the CYP1A2 phenotypic ratio for both CYP2C19 genotype subjects. In conclusion, St. John's wort is an inducer to the human CYP2C19, and clinicians should pay great attention when St. John's wort is added to or withdrawn from an existing drug regimen containing substrates for such enzymes.

Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection.

Dextromethorphan, a constituent of many over-the-counter cough syrups, is used as a probe drug for phenotyping subjects for their cytochrome P450 2D6 (CYP2D6) enzyme activity and for measuring CYP2D6 activity of preparations such as microsomes. In such studies, formation of the metabolite dextrorphan is used as indicator of the activity of this CYP enzyme. The present report describes an electron-capture gas chromatographic procedure developed for detection and quantification of dextrorphan in human liver microsomal preparations in vitro. After basification of the incubation mixture, dextrorphan was derivatized with pentafluorobenzoyl chloride under aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column, an electron capture detector, and a printer-integrator. Para-hydroxymephenytoin was carried through the procedure as internal standard. The procedure, which involves the derivatization of dextrorphan under aqueous conditions, is rapid and involves the use of the relatively economical procedure of electron-capture gas chromatography. The derivative is stable and possesses excellent chromatographic properties.

A rapid electron-capture gas chromatographic procedure for the analysis of p-hydroxymephenytoin.

An electron-capture gas chromatographic procedure was developed for detection and quantification of p-hydroxymephenytoin (OHMEP), a metabolite of S-mephenytoin, in human liver microsomal preparations. OHMEP was derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. Dextrorophan was carried through the procedure as internal standard. The structure of the PFB derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). The procedure is rapid and reproducible and produces a stable derivative that has excellent chromatographic properties. The limit of detection was less than 5 ng/ml, and the method was applied to extracts of human liver microsomes, which had been incubated with S-mephenytoin [a probe substrate for cytochrome P450 (CYP) 2C19].

Carbamazepine and oxcarbazepine decrease phenytoin metabolism through inhibition of CYP2C19.

Multiple studies suggest that phenytoin concentrations increase with CBZ co-medication. This study evaluated the hypothesis that CBZ and/or its major metabolite (CBZE) inhibit CYP2C19-mediated phenytoin metabolism using human liver microsomes and cDNA-expressed CYP2C19. Oxcarbazepine (OXC), and its 10-monohydroxy metabolite (MHD) were also evaluated. CBZ and MHD inhibited CYP2C19-mediated phenytoin metabolism at therapeutic concentrations. Thus, administration of CBZ and OXC with CYP2C19 substrates with narrow therapeutic ranges should be done cautiously.