Effects of CYP2C19 variants on methadone metabolism in vitro

CYP2C19 is an important member of the cytochrome P450 (CYP450) enzyme super family and is responsible for clearing approximately 10% of commonly used clinical drugs that undergo phase I metabolism. Genetic polymorphisms of CYP2C19 significantly influence the efficacy and safety of some drugs, which might cause undesirable adverse effects or cure failure at standard dosages. The aim of this study was to clarify the catalytic activities of 31 CYP2C19 alleles on the oxidative in vitro metabolism of methadone. Insect microsomes expressing the CYP2C19 alleles were incubated with 50–2000 μM methadone for 30 min at 37 °C and terminated by cooling to ‐80 °C immediately. Methadone and its metabolite EDDP were analyzed by an ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) system. Of the 31 tested CYP2C19 allelies variants, CYP2C19*1 is the wild‐type. Compared with CYP2C19*1, two CYP2C19 variants (CYP2C19*3 and *35FS) had no detectable enzyme activity, one variant L16F exhibited slightly increased intrinsic clearance values, and one variant N277K showed no significant difference. In addition, 26 variants exhibited significantly decreased values (from 1.48% to 80.40%). These findings suggest that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2C19 alleles. Copyright © 2016 John Wiley & Sons, Ltd.     

In Vitro Assessment of 36 CYP2C9 Allelic Isoforms Found in the Chinese Population on the Metabolism of Glimepiride

Of the 57 reported CYP2C9 alleles, to date, 36 of them have been identified in the Chinese population. The aim of this study was to assess the catalytic characteristics of these allelic isoforms and their effects on the metabolism of glimepiride in vitro. Baculovirus‐mediated expressing system was used to highly express wild‐type and the 35 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using glimepiride as the substrate. Reactions were performed at 37°C with the insect microsomes and 0.125–10 μM glimepiride for 40 min. After termination, the products were extracted and used for signal collection by LC‐MS/MS. Of the 36 tested CYP2C9 allelic isoforms, only four variants (CYP2C9.40, CYP2C9.47, CYP2C9.51 and CYP2C9.54) exhibited similar relative clearance values to that of wild‐type CYP2C9.1. In addition, one variant (CYP2C9.36) showed a higher intrinsic clearance value than the wild‐type protein, while the remaining 30 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.1% to 87.2%) compared to CYP2C9.1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used antidiabetic drug, glimepiride. Our results indicate that most of the tested rare alleles significantly decrease the catalytic activity of CYP2C9 variants towards glimepiride hydroxylation in vitro.     

Frequency distribution of high risk alleles of CYP2C19, CYP2E1, CYP3A4 genes in Haryana population

The genotype of an individual can significantly influence the disposition of a chemical, and determine their susceptibility to its toxicity. Many enzymes involved in either activation or detoxification of chemical carcinogen metabolism are polymorphically expressed, with the alleles presenting different enzymatic activities and some of them having been associated with susceptibility to cancer. Cytochrome P450 (P450 or CYP) constitutes the most important phase I enzyme group responsible for the metabolism of endogenous and exogenous (xenobiotics) substances. The present study was aimed to analyze the frequencies of commonly known polymorphisms of human xenobiotic metabolizing genes (XMG) in the Haryana State population of North India. The study was conducted in 308 healthy Haryana volunteers. DNA was extracted from leucocytes and the genetic polymorphisms in CYP2C19*2, CYP2C19*3, CYP2E1*5B and CYP3A4*1B were determined by digesting the PCR Product with restriction enzymes BamHI, SmaI, PstI and PstI respectively. The genotype frequencies of CYP2C19*2, CYP2C19*3, CYP2E1*5B, and CYP3A4*1B were found to be 22.0%, 0.0%, 2.11% and 2.0% respectively. The North Indian population which is known to be Caucasoid Aryans is ethnically different from South Indians known as Caucasoid Dravidians but no significant difference in genetic polymorphism was found.     

Cyp2c19 genotype and omeprazole hydroxylation phenotype in Chinese Li population

1. CYP2C19 is a polymorphism of cytochrome P450, which is responsible for the metabolism of many drugs. The genetic polymorphism shows interethnic variation and it has been demonstrated that the frequency of poor metabolizers (PM) and the distribution of alleles of CYP2C19 vary among Chinese ethnic nationalities. The aim of the present study was to investigate the incidence of CYP2C19 polymorphism in the Chinese Li population. 2. One hundred and sixty-five unrelated healthy Li subjects were identified with respect to CYP2C19 by genotype and phenotype analysis. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping. The plasma concentrations of omeprazole and 5-hydroxyomeprazole were assayed by reversed-phase high-performance liquid chromatography and the omeprazole hydroxylation index (HI) was determined. 3. The frequency distribution of omeprazole HI is bimodal and the antimode for HI was estimated to be 5.6. The prevalence of phenotypic PM in the Li population was 16.6% (13.7-19.5; 95% CI). Genotype analysis revealed that the frequencies of the CYP2C19*1, *2 and *3 alleles in the Li population were 0.617 (0.590-0.644; 95% CI), 0.353 (0.327-0.379; 95% CI) and 0.031 (0.021-0.041; 95% CI), respectively. The frequency of genotypic PM was 14.7% (11.9-17.5; 95% CI), which almost agreed with the frequency of phenotypic PM. Omeprazole HI was significantly different among the different genotype groups (P < 0.05). 4. The present study revealed that the incidence of the CYP2C19*1, *2 and *3 alleles in Chinese Li population is different to that in other ethnic populations of China. There was an obvious relationship between CYP2C19 genotype and omeprazole hydroxylation phenotype, and about 90% of phenotypic PM can be explained by the CYP2C19*2 and *3 alleles.     

Effect of co-administered inducer or inhibitor on omeprazole pharmacokinetics based on CYP2C19 genotype

Polymorphisms of cytochrome P450 (CYP) enzymes can affect enzymatic activity, drug metabolism and drug interactions. Although the potential for drug interactions is especially important when co-administering drugs with strong inductive or inhibitory potential towards drug-metabolizing enzymes, the relationship between CYP genotypes and the extent of the inductive or inhibitory effects remain poorly understood. We investigated the effects of rifampicin (inductive) and fluvoxamine (inhibitory) on metabolism of omeprazole and CYP2C19 enzymatic activity in 19 healthy Japanese subjects. Pharmacokinetic analyses of the CYP2C19 probe drug, omeprazole, were performed before and after rifampicin or fluvoxamine administration. The allele frequencies of the CYP2C19*1, CYP2C19*2 and CYP2C19*3 genotypes were 65.8%, 26.3% and 7.9%, respectively. Subjects with the CYP2C19*1 allele displayed higher levels of omeprazole metabolism than those without the CYP2C19*1 allele. Rifampicin increased omeprazole metabolism in all subjects irrespective of genotype, which suggested that CYP2C19 enzymatic activity was induced by rifampicin administration for all genotypes. Conversely, while fluvoxamine reduced omeprazole metabolism in subjects carrying the CYP2C19*1 allele, it had no impact on omeprazole pharmacokinetics in subjects without this allele. The genotyping of CYP2C19 may be useful for predicting drug interactions with metabolic inhibitors.     

In vitro assessment of 24 CYP2D6 allelic isoforms on the metabolism of methadone

CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme super family, with at least 100 CYP2D6 alleles being previously identified. Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to clarify the catalytic activities of 24 CYP2D6 alleles on the oxidative in vitro metabolism of methadone. Reactions were incubated with 50–2000 µM methadone for 30 min at 37 °C and terminated by cooling to ‐80 °C immediately. Methadone and the major metabolite EDDP were analyzed by an ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) system. Compared with wild‐type CYP2D6*1, most variants showed significantly altered values in V_max and intrinsic clearance (V_max/K_m). Only three variants (CYP2D6*88, *91 and E215K) exhibited markedly increased intrinsic clearance values, and one variant CYP2D6*94 showed no significant difference. On the other hand, the kinetic parameters of two CYP2D6 variants (CYP2D6*92 and *96) could not be determined because they had no detectable enzyme activity, whereas 18 variants exhibited significantly decreased values. To sum up, this study demonstrated that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2D6 alleles. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of the effects of 18 non-synonymous single-nucleotide polymorphisms of CYP450 2C19 on in vitro drug inhibition potential by a fluorescence-based high-throughput assay

To comprehensively understand the effects of CYP2C19 genetic polymorphisms on inhibition-based drug-drug interactions (DDIs), 18 human CYP2C19 non-synonymous single-nucleotide polymorphic variants and the wild-type isoform (CYP2C19.1A) were expressed in yeast cells. Using a fluorescence-based high-throughput method, the kinetic constants of these variants, as well as the inhibition constants for 10 drugs, were determined. CYP2C19.5B and CYP2C19.6 showed no activity towards CEC (3-cyano-7-ethoxycoumarin) O-deethylation. CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.19, E122A and A161P* (an allele containing both A161P and I331V) exhibited significantly reduced catalytic activities compared with CYP2C19.1A. The inhibition assay showed that the CYP2C19 genotype significantly affected the in vitro drug inhibition potential. Although the effect on drug inhibition potential is genotype- and inhibitor-dependent, there was an obvious trend: drugs tended to exhibit higher IC?? values (i.e. decreased inhibition potential) towards variants with reduced activity compared with variants with normal activity. This indicated that patients with reduced-function alleles may be less susceptible to CYP2C19-related DDIs. In this study, we provided the first in vitro evidence of CYP2C19 genotype-dependent effects on drug inhibition potential. This work greatly extends our understanding of the functional consequences of CYP2C19 genetic polymorphisms, and thus should prove valuable for CYP2C19 genotype-based therapy.     

Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phenprocoumon in healthy volunteers

CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon. Plasma and 12 h urine concentrations of both enantiomers and their monohydroxylated metabolites were measured by high-performance liquid chromatography with mass spectrometry detection. No significant effect of the CYP2C9 variants *2 and *3 on R-phenprocoumon pharmacokinetic parameters was detected, but S-phenprocoumon clearance tended to decrease with increasing number of CYP2C9*2 and *3 alleles. The ratios of S- to R-phenprocoumon plasma clearances were higher with a median of 0.95 in carriers of *1/*1 versus 0.65 in *3/*3 (P < 0.001 for trend). Plasma and urine concentrations of 4'-, 6- and 7-hydroxyphenprocoumon were significantly lower in homozygous carriers of the CYP2C9*2 and *3 variants compared to CYP2C9*1/*1. Carriers of CYP2C9*3/*3 had a median AUC of (R,S) 7-OH-phenprocoumon of only approximately 25% compared to the wild-type genotype. The AUC of (R,S) 6-OH-phenprocoumon was only approximately 50% in CYP2C9*3/*3 compared to the homozygous wild-type genotype. In conclusion, carriers of CYP2C9*2 and *3 alleles had a lower metabolic capacity regarding phenprocoumon hydroxylation than those with CYP2C9*1/*1. However, regarding phenprocoumon hydroxylation CYP2C9 genotypes had only marginal effects on S- and R-phenprocoumon total clearance in healthy volunteers.     

Influence of CYP2C19*18 and CYP2C19*19 alleles on omeprazole 5-hydroxylation: in vitro functional analysis of recombinant enzymes expressed in Saccharomyces cerevisiae

Omeprazole is one of the most widely used proton pump inhibitors for the treatment of gastric acid-related disorders. The major metabolic pathway of omeprazole is 5-hydroxylation, which is catalysed by CYP2C19. In this study, the effect of CYP2C19*18 and CYP2C19*19 alleles on omeprazole 5-hydroxylation was studied using recombinant CYP2C19 enzymes of wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 having Arg329His/Ile331Val and CYP2C19.19 Ser51Gly/Ile331Val) expressed in yeast cells. The K(m) value for omeprazole 5-hydroxylation of CYP2C19.1B was 1.46 microM. The K(m) value of CYP2C19.19 was significantly higher (1.5-fold) than that of CYP2C19.1B. V(max) and V(max)/K(m) values for omeprazole 5-hydroxylation of CYP2C19.1B on the basis of cytochrome P450 protein level were 8.09 pmol/min./pmol CYP and 5.45 microl/min./pmol CYP, respectively. The V(max) value of CYP2C19.19 was significantly higher (1.8-fold) than that of CYP2C19.1B, whereas the V(max)/K(m) value was comparable to that of CYP2C19.1B. In contrast, K(m), V(max) and V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that CYP2C19*19 allele decreases the affinity between CYP2C19 enzyme and the substrate in omeprazole metabolism.     

Celecoxib is a substrate of CYP2D6: Impact on celecoxib metabolism in individuals with CYP2C9*3 variants

Celecoxib was characterized as a substrate of human cytochrome P450 (CYP) 2D6 in vitro. In recombinant CYP2D6, celecoxib hydroxylation showed atypical substrate inhibition kinetics with apparent K_m, K_i, and V_max of 67.2 μM, 12.6 μM, and 1.33 μM/min, respectively. In human liver microsomes (HLMs), a concentration-dependent inhibition of celecoxib hydroxylation by quinidine was observed after CYP2C9 and CYP3A4 were inhibited. In individual HLMs with variable CYP2D6 activities, a significant correlation was observed between celecoxib hydroxylation and CYP2D6-selective dextromethorphan O-demethylation when CYP2C9 and CYP3A4 activities were suppressed (r = 0.97, P < 0.0001). Molecular modeling showed two predominant docking modes of celecoxib with CYP2D6, resulting in either a substrate or an inhibitor. A second allosteric binding antechamber, which stabilized the inhibition mode, was revealed. Modeling results were consistent with the observed substrate inhibition kinetics. Using HLMs from individual donors, the relative contribution of CYP2D6 to celecoxib metabolism was found to be highly variable and dependent on CYP2C9 genotypes, ranging from no contribution in extensive metabolizers with CYP2C9*1*1 genotype to approximately 30% in slow metabolizers with allelic variants CYP2C9*1*3 and CYP2C9*3*3. These results demonstrate that celecoxib may become a potential victim of CYP2D6-associated drug-drug interactions, particularly in individuals with reduced CYP2C9 activity.     

Polymorphisms in NAT2, CYP2D6, CYP2C19 and GSTP1 and their association with prostate cancer

The development of prostate cancer is dependent on heredity, androgenic influences, and exposure to environmental agents. A high intake of dietary fat is associated with an increased risk of prostate cancer, either through influence on steroid hormone profiles or through production of carcinogenic compounds that require biotransformation by enzymes. The polymorphic glutathione S-transferase (GST), N-acetyltransferase (NAT), and cytochrome P450 (CYP) enzymes are of particular interest in prostate cancer susceptibility because of their ability to metabolize both endogenous and exogenous compounds, including dietary constituents. Association between different NAT2, CYP2D6, CYP2C19 and GSTP1 genotypes and prostate cancer was studied in a Swedish and Danish case-control study comprising 850 individuals. The combined Swedish and Danish study population was analysed by polymerase chain reaction for the NAT2 alleles *4, *5A, *5B, *5C, *6 and *7, and for the CYP2D6 alleles *l, *3 and *4. The Swedish subjects were also analysed for the CYP2C19 alleles *1 and *2, and the GSTP1 alleles *A, *B and *C. No association was found between prostate cancer and polymorphisms in NAT2, CYP2D6, CYP2C19 or GSTP1. An association between CYP2D6 poor metabolism and prostate cancer was seen among smoking Danes; odds ratio 3.10 (95% confidence interval 1.07; 8.93), P = 0.03, but not among smoking Swedes; odds ratio 1.19 (95% confidence interval 0.41; 3.42), P = 0.75. Smoking is not a known risk factor for prostate cancer, and the association between CYP2D6 poor metabolism and prostate cancer in Danish smokers may have arisen by chance.     

Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.     

Metabolism of 7‐ethoxycoumarin,flavanone and steroids by cytochrome P450 2C9 variants

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild‐type CYP2C9 and mutants were coexpressed with NADPH‐cytochrome P450 reductase in Escherichia coli . The hydroxylase activities toward 7‐ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO‐difference spectra. CYP2C9.38 had the highest 7‐ethoxycoumarin de‐ethylase activity. All the CYP2C9 variants showed lower flavanone 6‐hydroxylation activities than CYP2C9.1 (the wild‐type). CYP2C9.38 showed higher activities in testosterone 6β‐hydroxylation, progesterone 6β−/16α‐hydroxylation, estrone 11α‐hydroxylation and estradiol 6α‐hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17‐oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α‐hydroxylase activity and CYP2C9.12 showed higher estrone 11α‐hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7‐ethoxycoumarin and steroids.     

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.     

Linkage disequilibrium between the CYP2C19*17 allele and wildtype CYP2C8 and CYP2C9 alleles: identification of CYP2C haplotypes in healthy Nordic populations

Purpose

To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium.

Methods

A total of 896 healthy subjects from three Nordic populations (Danish, Faroese, and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3, and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population.

Results

Ten CYP2C haplotypes were inferred, the most frequent of which (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1, and CYP2C19*1. The second most frequent haplotype (19%) is composed of CYP2C19*17, CYP2C8*1, and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects.

Conclusion

CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wildtype CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting an efficient CYP2C substrate metabolism.     

Functional Characterization of CYP2C8.13 and CYP2C8.14: Catalytic Activities toward Paclitaxel

Abstract: Cytochrome P450 2C8 (CYP2C8) plays important roles in the metabolism of various drugs, including the anti‐cancer drug, paclitaxel. We recently identified two novel CYP2C8 alleles (CYP2C8*13 and CYP2C8*14; wild‐type, CYP2C8*1A) with non‐synonymous single nucleotide polymorphisms in a Japanese population. To precisely investigate the effect of amino acid substitutions (CYP2C8*13, Ile223Met; CYP2C8*14, Ala238Pro) on CYP2C8 function, CYP2C8 proteins of the wild‐type (CYP2C8.1) and variants (CYP2C8.13 and CYP2C8.14) were heterologously expressed in yeast cells, and their paclitaxel 6α‐hydroxylation activities were determined. The K_m, V_max and CL_int values for paclitaxel 6α‐hydroxylation of CYP2C8.1 were 2.3 μM, 4.1 pmol/min./pmol CYP and 1.7 μl/min./pmol CYP, respectively. The K_m value of CYP2C8.14 was significantly higher (2.9‐fold) than that of CYP2C8.1. The V_max value of CYP2C8.14 was comparable to that of CYP2C8.1 and the CL_int value was reduced to 46% of CYP2C8.1. In contrast, the K_m, V_max and CL_int values of CYP2C8.13 were similar to those of CYP2C8.1. These results suggest that Ala238Pro substitution in CYP2C8.14 decreases the affinity toward paclitaxel of the CYP2C8 enzyme, and that the genetic polymorphism of the CYP2C8*14 allele may influence the clinical response to drugs metabolized mainly by CYP2C8.     

Evaluation of the effects of 18 non-synonymous single-nucleotide polymorphisms of CYP450 2C19 on in vitro drug inhibition potential by a fluorescence-based high-throughput assay

1. To comprehensively understand the effects of CYP2C19 genetic polymorphisms on inhibition-based drug–drug interactions (DDIs), 18 human CYP2C19 non-synonymous single-nucleotide polymorphic variants and the wild-type isoform (CYP2C19.1A) were expressed in yeast cells. Using a fluorescence-based high-throughput method, the kinetic constants of these variants, as well as the inhibition constants for 10 drugs, were determined.

2. CYP2C19.5B and CYP2C19.6 showed no activity towards CEC (3-cyano-7-ethoxycoumarin) O-deethylation. CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.19, E122A and A161P* (an allele containing both A161P and I331V) exhibited significantly reduced catalytic activities compared with CYP2C19.1A. The inhibition assay showed that the CYP2C19 genotype significantly affected the in vitro drug inhibition potential. Although the effect on drug inhibition potential is genotype- and inhibitor-dependent, there was an obvious trend: drugs tended to exhibit higher IC₅₀ values (i.e. decreased inhibition potential) towards variants with reduced activity compared with variants with normal activity. This indicated that patients with reduced-function alleles may be less susceptible to CYP2C19-related DDIs.

3. In this study, we provided the first in vitro evidence of CYP2C19 genotype-dependent effects on drug inhibition potential. This work greatly extends our understanding of the functional consequences of CYP2C19 genetic polymorphisms, and thus should prove valuable for CYP2C19 genotype-based therapy.

     

Allele and genotype frequencies of polymorphic cytochromes P450 (CYP2C9, CYP2C19, CYP2E1) and dihydropyrimidine dehydrogenase (DPYD) in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), c2 variant of CYP2E1 and DPYD alleles (*2 A-*6 ) was carried out in a total of 247 unrelated Egyptian subjects. An allele-specific fluorogenic 5' nuclease chain reaction assay was applied for detection of CYP2C9 and CYP2C19 variants. Other variants of the CYP2E1 and DPYD genes were determined using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR based assays. RESULTS: CYP2C9 allele frequencies in 247 Egyptian subjects were 0.820 for CYP2C9*1, 0.120 for CYP2C9*2 and 0.060 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.888, 0.110 and 0.002, respectively. CYP2C19*3, which is considered an Asian mutation, was detected in one subject (0.40%) who was heterozygous (*1/*3). Two subjects (0.80%) were homozygous for *2/*2, while no compound heterozygotes (*2/*3) or homozygotes for *3 were detected. For CYP2E1, only four subjects (1.70%) had the rare c2 variant, expressed heterozygously, giving an allele frequency of 0.009. Five variants of DPYD were analysed, with no splice sites (*2 A) or DeltaC1897 (*3) found in this population. The frequencies of other variants were 0.028, 0.115 and 0.090 for *4, *5 and *6, respectively. CONCLUSIONS: Comparing our data with that obtained in several Caucasian, African-American and Asian populations, we found that Egyptians resemble Caucasians with regard to allelic frequencies of the tested variants of CYP2C9, CYP2C19, CYP2E1 and DPYD. Our results may help in better understanding the molecular basis underlying ethnic differences in drug response, and contribute to improved individualization of drug therapy in the Egyptian population.     

CYP1A1 alleles in women with focal nodular hyperplasia of the liver (FNH)

OBJECTIVE: Disorders of steroid hormone metabolism might be related to the etiology of focal nodular hyperplasia of the liver (FNH), a benign tumor, especially prevalent in women. The cytochrome P450 1A1 (CYP1A1) enzyme is implicated in the bioactivation of multiple precarcinogens as well as in the metabolism of steroids. Genetic polymorphisms of CYP1A1 have been associated with altered catalytic activity in the hydroxylation of sex hormones and this may account for interindividual variability in exposure to hormone-mediated cell proliferation signals and reactive steroid metabolites. In the study at hand, we aimed to evaluate a possible association between CYP1A1*1, *2A, *2B, and *4 alleles and FNH. METHOD: Genotyping of 26 affected female patients of Caucasian origin was carried out using PCR/RFLP. RESULTS: Allele frequencies for the CYP1A1 variants *2A, *2B and *4 in 26 female patients with FNH were 0.058, 0.019 and 0.058, respectively. Crude odds ratios for the individual alleles were 0.75 (95% CI 0.23-2.44), 0.72 (95% CI 0.10-5.34) and 1.96 (95% CI 0.59-6.50), respectively. There were no significant differences between these values and corresponding allele frequencies obtained in a large German sample of unaffected Caucasian women. CONCLUSION: The present data do not suggest a relevant association between CYP1A1 polymorphisms and focal nodular hyperplasia of the liver in female Caucasians.     

Hydroxylation index of omeprazole in relation to CYP2C19 polymorphism and sex in a healthy Iranian population

Background

Polymorphism of CYP2C19 gene is one of the important factors in pharmacokinetics of CYP2C19 substrates. Omeprazole is a proton pump inhibitor which is mainly metabolized by cytochrome P450 2C19 (CYP2C19). The aim of present study was to assess omeprazole hydroxylation index as a measure of CYP2C19 activity considering new variant allele (CYP2C19*17) in Iranian population and also to see if this activity is sex dependent.

Methods

One hundred and eighty healthy unrelated Iranian individuals attended in this study. Blood samples for genotyping and phenotyping were collected 3 hours after administration of 20 mg omeprazole orally. Genotyping of 2C19 variant alleles *2, *3 and *17 was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and semi-nested PCR methods. Plasma concentrations of omeprazole and hydroxyomeprazole were determined by high performance liquid chromatography (HPLC) technique and hydxroxylation index (HI) (omeprazole/ hydroxyomeprazole) was calculated.

Results

The CYP2C19*17 was the most common variant allele in the studied population (21.6%). Genotype frequencies of CYP2C19*17*17, *1*17, and *2*17 were 5.5%, 28.8% and 3.3% respectively. The lowest and the highest median omeprazole HI was observed in *17*17 and *2*2 genotypes respectively (0.36 vs. 13.09). The median HI of omeprazole in subjects homozygous for CYP2C19*1 was 2.16-fold higher than individuals homozygous for CYP2C19*17 (P < 0.001) and the median HI of CYP2C19*1*17 genotype was 1.98-fold higher than CYP2C19 *17*17 subjects (P < 0.001). However, subjects with CYP2C19*2*17 (median HI: 1.74) and CYP2C19*1*2 (median HI: 1.98) genotypes and also CYP2C19*1*17 (median HI: 0.71) and CYP2C19*1*1 (mean HI: 0.78) did not show any significantly different enzyme activity. In addition, no statistically significant difference was found between women and men in distribution of CYP2C19 genotypes. Furthermore, the hydroxylation index of Omeprazole was not different between women and men in the studied population.

Conclusion

Our data point out the importance of CYP2C19*2 and CYP2C19*17 variant alleles in metabolism of omeprazole and therefore CYP2C19 activity. Regarding the high frequency of CYP2C19*17 in Iranian population, the importance of this new variant allele in metabolism of CYP2C19 substrates shall be considered.