Inhibition of cytochrome P450 enzymes and uridine 5'-diphospho-glucuronosyltransferases by vicagrel in human liver microsomes: A prediction of potential drug-drug interactions.

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 µM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4'-hydroxylation with Ki values of 0.19 µM and 1.2 µM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min-1 and 1.52 µM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 µM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin ß-D-glucuronidation with a Ki value of 5.6 µM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.

The CYP2C19 Intron 2 Branch Point SNP is the Ancestral Polymorphism Contributing to the Poor Metabolizer Phenotype in Livers with CYP2C19*35 and CYP2C19*2 Alleles.

CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.

Prediction of in vivo clearance and associated variability of CYP2C19 substrates by genotypes in populations utilizing a pharmacogenetics-based mechanistic model.

It is important to examine the cytochrome P450 2C19 (CYP2C19) genetic contribution to drug disposition and responses of CYP2C19 substrates during drug development. Design of such clinical trials requires projection of genotype-dependent in vivo clearance and associated variabilities of the investigational drug, which is not generally available during early stages of drug development, but is essential for CYP2C19 substrates with multiple clearance pathways. This study evaluated the utility of pharmacogenetics-based mechanistic modeling in predicting such parameters. Hepatic CYP2C19 activity and variability within genotypes were derived from in vitro S-mephenytoin metabolic activity in genotyped human liver microsomes (N = 128). These data were then used in mechanistic models to predict genotype-dependent disposition of CYP2C19 substrates (i.e., S-mephenytoin, citalopram, pantoprazole, and voriconazole) by incorporating in vivo clearance or pharmacokinetics of wild-type subjects and parameters of other clearance pathways. Relative to the wild-type, the CYP2C19 abundance (coefficient of variation percentage) in CYP2C19*17/*17, *1/*17, *1/*1, *17/null, *1/null, and null/null microsomes was estimated as 1.85 (117%), 1.79 (155%), 1.00 (138%), 0.83 (80%), 0.38 (130%), and 0 (0%), respectively. The subsequent modeling and simulations predicted, within 2-fold of the observed, the means and variabilities of urinary S/R-mephenytoin ratio (36 of 37 genetic groups), the oral clearance of citalopram (9 of 9 genetic groups) and pantoprazole (6 of 6 genetic groups), and voriconazole oral clearance (4 of 4 genetic groups). Thus, relative CYP2C19 genotype-dependent hepatic activity and variability were quantified in vitro and used in a mechanistic model to predict pharmacokinetic variability, thus allowing the design of pharmacogenetics and drug-drug interaction trials for CYP2C19 substrates.

Predicted metabolic drug clearance with increasing adult age.

AIM: To determine the effect of increasing adult age on predicted metabolic drug clearance. METHOD: Predicted metabolic drug clearances (CLPT ) were determined using in vitro-in vivo extrapolation coupled with physiological-based pharmacokinetic modelling and simulation (IVIVE-PBPK) in Simcyp®. Simulations were conducted using CYP-selective 'probe' drugs with subjects in 5 year age groups (20-25 to 90-95 years). CLPT values were compared with human pharmacokinetic data stratified according to age (young = 20-40 years and elderly = 65-85 years) and gender. Age-related changes in the physiological parameters used for IVIVE of CLPT were described. RESULTS: Predicted metabolic drug clearances decreased with increasing adult age to approximately 65-70 years: caffeine from 1.5 to 1.0 ml min(-1) kg(-1) (a 33% decrease), S-warfarin from 0.100 to 0.064 ml min(-1) kg(-1) (36%), S-mephenytoin from 4.1 to 2.5 ml min(-1) kg(-1) (39%), desipramine from 10.6 to 7.3 ml min(-1) kg(-1) (31%) and midazolam from 5.4 to 3.9 ml min(-1) kg(-1) (27%). Except for S-mephenytoin, predictions were within 3.5-fold of clearances from clinical studies when stratified by age and gender. A trend towards higher CLPT was observed in females, but this was only statistically significant in larger virtual trials. Physiological parameters that determine CLPT decreased with increasing adult age: mean microsomal protein g(-1) of liver, liver weight, hepatic blood flow and human serum albumin concentration. CONCLUSION: Decreased metabolic clearance in the elderly was predicted by Simcyp® and was generally consistent with limited clinical data for four out of five drugs studied and the broader literature for drugs metabolized by CYP enzymes. IVIVE-PBPK may be increasingly useful in predicting metabolic drug clearance in the elderly.

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

Evaluation of the effects of 20 nonsynonymous single nucleotide polymorphisms of CYP2C19 on S-mephenytoin 4'-hydroxylation and omeprazole 5'-hydroxylation.

CYP2C19 is a highly polymorphic enzyme that affects the metabolism of a wide range of therapeutic drugs. Almost all the identified alleles of CYP2C19 are derived from nonsynonymous single nucleotide polymorphisms (nsSNPs). The objective of this study was to functionally characterize 20 nsSNPs of CYP2C19, distributed throughout the entire coding region, most of which have not been thoroughly characterized. cDNAs of these variants were constructed and expressed in yeast cells. All variants had similar levels of apoprotein and holoprotein expression, except for CYP2C19.16 and D360N, which had significantly lower holoprotein levels than the wild-type (WT) CYP2C19 enzyme, and CYP2C19.5B, which showed only apoprotein. The activity of the CYP2C19 variants was investigated using two substrates, S-mephenytoin and omeprazole, and six different kinetic parameters were measured. CYP2C19.5B, CYP2C19.6, and CYP2C19.8 were found to be catalytically inactive. The entire dataset of the remaining 17 variants, together with the WT, was analyzed by multivariate analysis. This analysis indicated that CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.18, CYP2C19.19, A161P, W212C, and D360N were substantially altered in catalytic properties in comparison with the WT, with each of these variants exhibiting either dramatically decreased catalytic activities or higher K(m) values. These results not only generally confirmed the function of previously reported variants but also identified additional reduced-function variants. These findings will greatly extend our understanding of CYP2C19 genetic polymorphisms in humans as well as facilitate the structure-function study of the CYP2C19 protein.

A model based assessment of the CYP2B6 and CYP2C19 inductive properties by artemisinin antimalarials: implications for combination regimens.

The study aim was to assess the inductive properties of artemisinin antimalarials using mephenytoin as a probe for CYP2B6 and CYP2C19 enzymatic activity. The population pharmacokinetics of S-mephenytoin and its metabolites S-nirvanol and S-4'-hydroxymephenytoin, including enzyme turn-over models for induction, were described by nonlinear mixed effects modeling. Rich data (8-16 samples/occasion/subject) were collected from 14 healthy volunteers who received mephenytoin before and during ten days of artemisinin administration. Sparse data (3 samples/occasion/subject) were collected from 74 healthy volunteers who received mephenytoin before, during and after five days administration of artemisinin, dihydroartemisinin, arteether, artemether or artesunate. The production rate of CYP2B6 was increased 79.7% by artemisinin, 61.5% by arteether, 76.1% by artemether, 19.9% by dihydroartemisinin and 16.9% by artesunate. The production rate of CYP2C19 increased 51.2% by artemisinin, 14.8% by arteether and 24.9% by artemether. In conclusion, all studied artemisinin derivatives induced CYP2B6. CYP2C19 induction by arteether and artemether as well as CYP2B6 and CYP2C19 induction by artemisinin was confirmed. The inductive capacity is different among the artemisinin drugs, which is of importance when selecting drugs to be used in antimalarial combination therapy such that the potential for drug-drug interactions is minimized.

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.

CYP2C19 inhibition: the impact of substrate probe selection on in vitro inhibition profiles.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical part of the drug discovery process. Factors such as nonspecific binding, atypical kinetics, poor effector solubility, and varying ratios of accessory proteins may alter the kinetic behavior of an enzyme and subsequently confound the extrapolation of in vitro data to the human situation. The architecture of the P450 active site and the presence of multiple binding regions within the active site may also confound in vitro-in vivo extrapolation, as inhibition profiles may be dependent on a specific inhibitor-substrate interaction. In these studies, the inhibition profiles of a set of 24 inhibitors were paneled against the CYP2C19 substrate probes (S)-mephenytoin, (R)-omeprazole, (S)-omeprazole, and (S)-fluoxetine, on the basis of their inclusion in recent U.S. Food and Drug Administration guidance for in vitro drug-drug interactions with CYP2C19. (S)-Mephenytoin was inhibited an average of 5.6-fold more potently than (R)- or (S)-omeprazole and 9.2-fold more potently than (S)-fluoxetine. Hierarchical clustering of the inhibition data suggested three substrate probe groupings, with (S)-mephenytoin exhibiting the largest difference from the rest of the substrate probes, (S)-fluoxetine exhibiting less difference from (S)-mephenytoin and the omeprazoles and (R)- and (S)-omeprazole exhibiting minimal differences from each other. Predictions of in vivo inhibition potency based on the in vitro data suggest that most drug-drug interactions will be identified by either (S)-mephenytoin or omeprazole, although the expected magnitude of the interaction may vary depending on the chosen substrate probe.

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

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

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.

Cynomolgus monkey cytochrome P450 2C43: cDNA cloning, heterologous expression, purification and characterization.

The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.

A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants.

BACKGROUND AND OBJECTIVE: Many drugs, including proton pump inhibitors and certain antidepressants, are metabolized by the polymorphic cytochrome P450 (CYP) 2C19 enzyme. A significant portion of extensive metabolizers do not reach appropriate drug levels, and our objective was to investigate any genetic background. METHODS: The 5'-flanking region of the CYP2C19 gene from subjects with rapid omeprazole metabolism was sequenced, and CYP2C19 phenotype-genotype associations were analyzed in Swedish (n = 107) and Ethiopian (n = 126) extensive metabolizers. The relationship of the metabolic ratio of omeprazole (omeprazole/5-hydroxyomeprazole in plasma 3 hours after drug intake) with the area under the plasma concentration-time curve was used for prediction studies. Electrophoretic mobility shift assays were conducted by use of human nuclear protein extracts. Hepatic reporter vector transfections were carried out in CD1 mice. RESULTS: We identified a novel allele (CYP2C19*17) carrying -806C>T and -3402C>T, with a frequency of 18% in both Swedes and Ethiopians and 4% in Chinese subjects. In Swedes the metabolic ratio of omeprazole was higher in subjects homozygous for CYP2C19*1 (median, 0.50 [interquartile range, 0.37-0.73]) than in those homozygous for CYP2C19*17 (median, 0.25 [interquartile range, 0.15-0.33]) (P = .010). In Ethiopians a similar difference in the S/R-mephenytoin ratio was observed between individuals homozygous for CYP2C19*1 (median, 0.20 [interquartile range, 0.12-0.37]) and those homozygous for CYP2C19*17 (median, 0.05 [interquartile range, 0.03-0.06]) (P = .013). Electrophoretic mobility shift assays showed specific binding of human hepatic nuclear proteins to an element carrying -806T but not -806C. Reporter vector experiments showed an increased transcriptional activity of the CYP2C19*17 allele in vivo in mice. Predictions revealed that CYP2C19*17 homozygotes would attain 35% to 40% lower omeprazole area under the plasma concentration-time curve values than subjects homozygous for CYP2C19*1 taking standard doses of omeprazole. CONCLUSION: CYP2C19*17 is likely to cause therapeutic failures in drug treatment with, for example, proton pump inhibitors and antidepressants.

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.

Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation.

Detailed cytochrome P450 (P450) inhibition profiles are now required for the registration of novel molecular entities. This method uses combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reductases. Kinetic analysis of single substrate with single P450 (sP450) yielded apparent Km values of 25, 2, 20, 9, and 3 microM, for CYP1A2, 2C9, 2C19, 2D6, and 3A4, respectively. Combined substrates with combined P450s (cP450) yielded apparent Km values of 65, 4, 19, 7, and 2 microM. Selectivity of the substrates for each P450 isoform was checked. Phenacetin proved to be the least selective substrate. However, the ratio of the various P450s was modified in the final assay such that metabolism of phenacetin by other enzymes was approximately 20% of the metabolism by CYP1A2. IC50 determinations with alpha-naphthoflavone (0.04 microM), sulfaphenazole (0.26 microM), tranylcypromine (9 microM), quinidine (0.02 microM), and ketoconazole (0.01 microM) were similar for sP450 and cP450 enzymes. The assay was further evaluated with 11 literature compounds and 52 in-house new chemical entities, and the data compared with radiometric/fluorescent values. The overall protein level of the assay was reduced from the original starting point, as this led to some artificially high IC50 measurements when compared with existing lower protein assays (radiometric/fluorometric). This method offers high throughput P450 inhibition profiling with potential advantages over current radiometric or fluorometric methods.

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.

An interaction between the cytochrome P450 probe substrates chlorzoxazone (CYP2E1) and midazolam (CYP3A).

AIMS: The use of multiple probe substrates to evaluate the activity of drug metabolizing enzymes requires that there are no inter-substrate interactions. As part of a series of studies to develop a clinically useful collection of probe substrates that could be given alone or in any combination, we observed an interaction between midazolam (MDZ) and another component of the six-drug cocktail. Published data indicated that the interacting component was likely to be chlorzoxazone. This was investigated as part of a second study. The data relating to the interaction from both studies are reported here. METHODS: Both studies were performed in 16 healthy subjects. All treatments were given orally after an overnight fast. In study 1, which was performed to a four-period, open, crossover design, subjects received on separate occasions MDZ 5 mg, diclofenac 25 mg, a four drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg and chlorzoxazone 250 mg) and a six drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg, chlorzoxazone 250 mg, diclofenac 25 mg and MDZ 5 mg). In study 2, which was performed to a two-period, open, crossover design, subjects received a five drug cocktail (as the six drug cocktail in the first study, but without chlorzoxazone and with diclofenac dose increased to 50 mg) and a six drug cocktail (as five drug cocktail, with chlorzoxazone 250 mg). In both studies, blood samples were taken for measurement of plasma MDZ and 1-hydroxy MDZ (1-OH MDZ) concentrations. In study 1, blood samples were taken up to 12 h post-dose while in study 2 a single sample was taken 2 h after dosing. In study 1, the potential interaction between MDZ and the other components of the six drug cocktail was assessed by comparing AUClast ratios (1-OH MDZ/MDZ) between the two treatments. Additionally, a single sampling timepoint of 2 h post-dose for determination of concentration, rather than AUC, ratios was established. The 2 h plasma concentration ratios from studies 1 and 2 were combined and a pooled analysis performed to compare ratios within each study (to determine the change in ratio when MDZ was dosed with and without chlorzoxazone) and between studies (to determine the consistency of the ratios when MDZ was given either as part of the two six drug cocktails or when given alone and as part of the five drug cocktail). RESULTS: In study 1, both the AUClast ratio and the 2 h post-dose plasma concentration ratio were reduced when MDZ was given as part of the six drug cocktail in comparison with those for MDZ alone. This was the result of an increase in MDZ, rather than decrease in 1-OH MDZ, concentrations and was considered to result from a reduction in first pass metabolism of MDZ. The geometric mean AUClast values (with 95% CI) for MDZ were 95.6 (79.0, 115.7) and 160.4 (133.6, 192.6) microg l(-1) h when given alone and as part of the six drug cocktail, respectively. The corresponding values for 1-OH MDZ were 789.6 (697.6, 893.6) and 791.4 (701.7, 892.6) microg l(-1) h. The ratio of adjusted geometric mean AUClast ratios for the two treatments was 1.82 (90% CI 1.48, 2.23, P < 0.001). The pooled plasma 1-OH MDZ/MDZ ratio data from both studies showed that the differences in MDZ metabolism observed in study 1 were replicated in study 2. The adjusted geometric mean 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the six drug cocktail were 7.79 and 4.59, respectively, for study 1 (ratio 1.70, 95% CI 1.36, 2.11, P < 0.001) and 7.64 and 4.60 for study 2 (ratio 1.66, 95% CI 1.34, 2.06, P < 0.001). These data indicate that when given orally chlorzoxazone interacts with MDZ, increasing plasma MDZ concentrations. In contrast, there was no difference between the plasma 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the five drug cocktail indicating that there were no interactions between MDZ and any of the other components of that cocktail. CONCLUSIONS: Chlorzoxazone appears to significantly influence the pharmacokinetics of oral MDZ, probably through inhibition of first pass metabolism by CYP3A in the GI tract. Data from these studies and literature evidence showing a further interaction between chlorzoxazone and CYP1A2 substrates and questions concerning the specificity of chlorzoxazone as a probe substrate for CYP2E1, indicate that the use of chlorzoxazone in multisubstrate probe cocktails should be avoided.

Pharmacokinetic drug interaction potential of risperidone with cytochrome p450 isozymes as assessed by the dextromethorphan, the caffeine, and the mephenytoin test.

Two published case reports showed that addition of risperidone (1 and 2 mg/d) to a clozapine treatment resulted in a strong increase of clozapine plasma levels. As clozapine is metabolized by cytochrome P450 isozymes, a study was initiated to assess the in vivo interaction potential of risperidone on various cytochrome P450 isozymes. Eight patients were phenotyped with dextromethorphan (CYP2D6), mephenytoin (CYP2C19), and caffeine (CYP1A2) before and after the introduction of risperidone. Before risperidone, all eight patients were phenotyped as being extensive metabolizers of CYP2D6 and CYP2C19. Risperidone at dosages between 2 and 6 mg/d does not appear to significantly inhibit CYP1A2 and CYP2C19 in vivo (median plasma paraxanthine/caffeine ratios before and after risperidone: 0.65, 0.69; p = 0.89; median urinary (S)/(R) mephenytoin ratios before and after risperidone:0.11, 0.12; p = 0.75). Although dextromethorphan metabolic ratio is significantly increased by risperidone (median urinary dextromethorphan/dextrorphan ratios before and after risperidone: 0.010, 0.018; p = 0.042), risperidone can be considered a weak in vivo CYP2D6 inhibitor, as this increase is modest and none of the eight patients was changed from an extensive to a poor metabolizer. The reported increase of clozapine concentrations by risperidone can therefore not be explained by an inhibition of CYP1A2, CYP2D6, CYP2C19 or by any combination of the three.

P-hydroxylation of phenobarbital: relationship to (S)-mephenytoin hydroxylation (CYP2C19) polymorphism.

The aim of the current study was to compare the pharmacokinetics of phenobarbital (PB) in extensive metabolizers (EMs) and poor metabolizers (PMs) of S-mephenytoin. Ten healthy volunteers (5 EMs and 5 PMs) were given 30 mg PB daily for 14 days. PB and p-hydroxyphenobarbital (p-OHPB) in serum and urine were measured by high-performance liquid chromatography (HPLC). Urinary excretion (12.5% versus 7.7%) and formation clearance (29.8 versus 21.1 mL/h) of p-OHPB, one of the main metabolites of PB, were significantly lower (p < .05) in PMs than in EMs. However, area under the serum concentration-time curve (153.3 in the EMs versus 122.9 microg x h/mL in the PMs), total (210.8 versus 254.9 mL/h) and renal clearance (53.1 versus 66.1 mL/h) of PB were identical between the two groups. To compare the inducibility of CYP2C19, mephenytoin was also given prior to and on the last day of PB treatment. The urinary level of 4'-hydroxymephenytoin was analyzed by a validated gas chromatograpy/mass spectrometry (GC/MS) method. The mephenytoin hydroxylation index did not change in either EMs (1.42 versus 1.42) or PMs (341.4 versus 403.5), showing that CYP2C19 was not induced by treatment with PB. These results indicated that the p-hydroxylation pathway of PB co-segregates with the CYP2C19 metabolic polymorphism. However, the overall disposition kinetics of PB were not different between EMs and PMs, and therefore polymorphic CYP2C19 seems have no major clinical implications.

Genetic polymorphism of CYP2D6 and CYP2C19 metabolism determined by phenotyping Israeli ethnic groups.

Genetic polymorphism of the cytochrome P450 isoenzymes CYP2D6 and CYP2C19 was determined by phenotyping four ethnic groups of the Israeli population. The groups consisted of Ethiopian subjects, Yemenite subjects, and Russian subjects representing first-generation new immigrants and an Israeli Arab group. Dextromethorphan was used as the probe for CYP2D6 activity and mephenytoin was used for CYP2C19 activity. The two drugs were administered simultaneously and urine samples were collected over a period of 8 hours. The CYP2D6 phenotype was determined from the ratio of dextromethorphan conversion to dextrorphan and the CYP2C19 phenotype from the ratio of S-mephenytoin and R-mephenytoin. The used liquid chromatographic method was able to completely separate dextrorphan and dextromethorphan. Fluorescence detection allowed dextromethorphan quantification at 1 ng/mL. Mephenytoin enantiomers were completely separated in high-performance liquid chromatography and the respective fractions were collected and analyzed using a gas chromatography/mass spectrometry system with selective ion monitoring. The prevalence of poor metabolizer phenotype of dextromethorphan (CYP2D6) in the Yemenite (0%) and Ethiopian groups (0%) was significantly different from the prevalence in the Russian (17%) and Israeli Arab (9%) groups. A significant difference was also found in the distribution of the metabolic ratio of the extensive metabolizer phenotype between the Ethiopian group and the Russian and Yemenite groups. No significant difference was found in the prevalence of poor mephenytoin metabolizer phenotype (CYP2C19) between the Yemenite (8%), Ethiopian (6%), Russian (9%), and Israeli Arab (8%) groups. No difference was observed in the distribution of metabolic ratio within the extensive metabolizer phenotype subgroups of the four ethnic groups.