(-)-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.

Assessment of urinary mephenytoin metrics to phenotype for CYP2C19 and CYP2B6 activity.

OBJECTIVES: (S)-Mephenytoin is selectively metabolised to (S)-4'-hydroxymephenytoin by CYP2C19. The urinary excretion of 4'-hydroxymephenytoin reflects the activity of individual enzymes. We evaluated fractioned urinary collection and beta-glucuronidase pre-treatment in order to determine the optimal CYP2C19 metrics. We also assessed whether urinary excretion of N-desmethylmephenytoin (nirvanol) might be a useful CYP2B6 metric in in vivo studies. METHODS: A 50-mg dose of mephenytoin was administered to 52 volunteers as a component of phenotyping cocktails in four separate studies. Urine was collected up to 166 h post-dose. Urinary excretion of 4'-hydroxymephenytoin and nirvanol was quantified by liquid chromatography-tandem mass spectrometry, and common CYP2C19 and CYP2B6 genotypes were determined. RESULTS: Cumulative excretion of 4'-hydroxymephenytoin in urine with beta-glucuronidase treatment collected from before mephenytoin administration up to 12-16 h thereafter showed the greatest difference between CYP2C19 genotypes and the lowest intra-individual variability (7%). Renal elimination of nirvanol was highest for a *4/*4 individual and lowest for individuals carrying the *5/*5 and *1/*7 genotype, but lasted for several weeks, thus making its use in cross-over studies difficult. CONCLUSION: Cumulative urinary excretion of 4'-hydroxymephenytoin 0-12 h post-administration is a sensitive and reproducible metric of CYP2C19 activity, enabling the effect of a drug on CYP2C19 to be assessed in a small sample size of n=6 volunteers. While nirvanol excretion may reflect CYP2B6 activity in vivo, it is not useful for CYP2B6 phenotyping.

Evaluation of 3-O-methylfluorescein as a selective fluorometric substrate for CYP2C19 in human liver microsomes.

Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K(m) and V(max) values of 1.14 +/- 0.90 microM and 11.3 +/- 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 +/- 6.4 microM and 258 +/- 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations < or =1 microM. At substrate concentrations > or =10 microM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 microM OMF and 2 microM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC(50) data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.

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.

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.

Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry.

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate.

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL(int)) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CL(int) of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL(int) of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL(int) of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors.

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K(i) towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(-)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K(i) values of < 250nM--at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.

Quantification of mephenytoin and its metabolites 4'-hydroxymephenytoin and nirvanol in human urine using a simple sample processing method.

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method without the use of sample extraction was developed for the simultaneous quantification of urinary concentrations of mephenytoin, a standard phenotyping substrate for the cytochrome P450 enzyme CYP2C19, and its phase I metabolites 4'-hydroxymephenytoin and nirvanol. Fifty microL of urine were diluted with a buffered beta-glucuronidase solution and incubated at 37 degrees C for 6 h followed by addition of methanol, containing the internal standard 4'-methoxymephenytoin. The chromatographic separation was achieved using a 100 x 3 mm, 5 micro Thermo Electron Aquasil C18 column with a gradient flow, increasing the organic fraction (acetonitrile/methanol 50:50) of the mobile phase from 10 to 90%. Quantification by triple-stage mass spectrometry (TSQ Quantum, Thermo Electron) was accomplished by negative electrospray ionization in the selected reaction monitoring mode. Linearity was observed for all substances in the concentration range 15-10 000 ng/mL. The lower limit of quantification (LLOQ) was 20 ng/mL for 4'-hydroxymephenytoin and 30 ng/mL for nirvanol and mephenytoin, respectively. Intra- and inter-day inaccuracy did not exceed 9.5% for all substances from LLOQ to 10 000 ng/mL. Intra- and inter-day precision were in the range of 0.8-10.5%. The method was validated according to international ICH and FDA guidelines and successfully applied for phenotyping of Caucasian male volunteers who received an oral dose of 50 mg mephenytoin.

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.

Hepatic CYP2B6 expression: gender and ethnic differences and relationship to CYP2B6 genotype and CAR (constitutive androstane receptor) expression.

CYP2B6 metabolizes many drugs, and its expression varies greatly. CYP2B6 genotype-phenotype associations were determined using human livers that were biochemically phenotyped for CYP2B6 (mRNA, protein, and CYP2B6 activity), and genotyped for CYP2B6 coding and 5'-flanking regions. CYP2B6 expression differed significantly between sexes. Females had higher amounts of CYP2B6 mRNA (3.9-fold, P < 0.001), protein (1.7-fold, P < 0.009), and activity (1.6-fold, P < 0.05) than did male subjects. Furthermore, 7.1% of females and 20% of males were poor CYP2B6 metabolizers. Striking differences among different ethnic groups were observed: CYP2B6 activity was 3.6- and 5.0-fold higher in Hispanic females than in Caucasian (P < 0.022) or African-American females (P < 0.038). Ten single nucleotide polymorphisms (SNPs) in the CYP2B6 promoter and seven in the coding region were found, including a newly identified 13072A>G substitution that resulted in an Lys139Glu change. Many CYP2B6 splice variants (SV) were observed, and the most common variant lacked exons 4 to 6. A nonsynonymous SNP in exon 4 (15631G>T), which disrupted an exonic splicing enhancer, and a SNP 15582C>T in an intron-3 branch site were correlated with this SV. The extent to which CYP2B6 variation was a predictor of CYP2B6 activity varied according to sex and ethnicity. The 1459C>T SNP, which resulted in the Arg487Cys substitution, was associated with the lowest level of CYP2B6 activity in livers of females. The intron-3 15582C>T SNP (in significant linkage disequilibrium with a SNP in a putative hepatic nuclear factor 4 (HNF4) binding site) was correlated with lower CYP2B6 expression in females. In conclusion, we found several common SNPs that are associated with polymorphic CYP2B6 expression.

Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma by liquid chromatography.

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <+/-20%). The method was validated for human plasma in the concentration range 10-2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.

Comparison of in vitro and in vivo inhibition potencies of fluvoxamine toward CYP2C19.

A previous study suggested that fluvoxamine inhibition potency toward CYP1A2 is 10 times greater in vivo than in vitro. The present study was designed to determine whether the same gap exists for CYP2C19, another isozyme inhibited by fluvoxamine. In vitro studies examined the effect of nonspecific binding on the determination of inhibition constant (K(i)) values of fluvoxamine toward CYP2C19 in human liver microsomes and in a cDNA-expressed microsomal (Supersomes) system using (S)-mephenytoin as a CYP2C19 probe. K(i) values based on total added fluvoxamine concentration (K(i,total)) and unbound fluvoxamine concentration (K(i,ub)) were calculated, and interindividual variability in K(i) values was examined in six nonfatty livers. K(i,total) values varied with microsomal protein concentration, whereas the corresponding K(i,ub) values were within a narrow range (70-80 nM). In vivo inhibition constants (K(i)iv) were obtained from a study of the disposition of a single oral dose (100 mg) of the CYP2C19 probe (S)-mephenytoin in 12 healthy volunteers receiving fluvoxamine at 0, 37.5, 62.6, and 87.5 mg/day to steady state. In this population, the ratio of (S)-4-hydroxy-mephenytoin formation clearances (uninhibited/inhibited) was positively correlated with fluvoxamine average steady-state concentration with an intercept of 0.85 (r(2) = 0.88, p < 0.001). The mean (+/-S.D.) values of K(i)iv based on total and unbound plasma concentrations were 13.5 +/- 5.6 and 1.9 +/- 1.1 nM, respectively. Comparison of in vitro and in vivo K(i) values, based on unbound fluvoxamine concentrations, suggests that fluvoxamine inhibition potency is roughly 40 times greater in vivo than in vitro.

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates.

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S-warfarin (CYP2C9 in humans) by dog liver microsomes (mean +/- SD from 12 (six male and six female) dogs = 10.8 +/- 1.9 fmol mg(-1) protein min(-1)) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S-mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 +/-1.5 pmol mg(-1) protein min(-1)) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R-enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K(m) or K(0.5) 15-22 micro M, V(max) 35-59 pmol mg(-1) protein min(-1), n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S-mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K(m) 42-64 micro M, V(max) 22-46 pmol mg(-1) protein min(-1), n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol mg(-1) protein min(-1) (p < 0.02); S-warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol mg(-1) protein min(-1) (p < 0.02); R-mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol mg(-1) protein min(-1) (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol mg(-1) protein min(-1) (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S-warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.

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.

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.