Allele and genotype frequencies of cytochrome P450 2B6 gene in a Mongolian population.

CYP2B6 plays an important role in metabolizing various drugs in common clinical use. Increasing interest in CYP2B6 genetic polymorphism was stimulated by revelations of a specific CYP2B6 genotype significantly affecting the metabolism of efavirenz, an anti-HIV type-1 agent. The present study determined the CYP2B6 haplotype in 100 healthy unrelated Mongolian volunteers by analyzing the genotypes of nine single nucleotide polymorphism (SNP) positions (-82T>C, 64C>T, 499C>T, 516G>T, 777C>A, 785A>G, 983T>C, 1375A>G, and 1459C>T) in the CYP2B6 gene. The CYP2B6 *1 allele was the most frequent in the Mongolian population tested at 64.5%, higher than the equivalent frequency in African-Americans and Ghanaians. The second most frequent allele was CYP2B6 *6 (21.0%), although this allele was less frequent than that in Ghanaians. Only one CYP2B6 *5 allele was identified in our Mongolian subjects (0.5%), although it is the third most frequent allele in white and African-American populations. These CYP2B6 genotypes revealed seven slow efavirenz metabolizers in 100 Mongolians, which is significantly fewer than the same group among Ghanaians. Overall, the Mongolian CYP2B6 allele distribution was comparable with that in Japanese, Koreans, and Han Chinese. This is the first report of CYP2B6 genotype frequency in a Mongolian population, and it could provide clinically useful information on drug metabolism in this population group.

Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.

BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation.

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasian populations, but at higher frequencies (18-23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.

Psychotropic drug interactions with valproate.

Valproate is a well-established anticonvulsant that is increasingly being employed, often in combination with other psychotropics, for its mood-stabilizing properties. This compound is metabolized by conjugation, beta-oxidation, and cytochrome P450 oxidation (CYP2C9, CYP2C19, and CYP2A6) and also acts as a broad-spectrum inhibitor of a variety of hepatic enzymes including glucoronyltransferase, epoxide hydrolase, and the CYP2C enzymes. In addition, it exhibits saturable protein binding and competes with many drugs for protein binding sites. It is therefore not surprising that valproate has been noted to interact with psychotropic medications of all classes. This article provides an overview of the noted pharmacokinetic psychotropic interactions with valproate, with a particular focus on the mechanisms of these interactions and potential clinical consequences.

Defining relationships between the known members of the cytochrome P450 3A subfamily, including five putative chimpanzee members.

An analysis of the cytochrome P450 3A subfamily (CYP3A) was undertaken in order to define relationships across species among subfamily members. Some members were excluded due to incomplete sequences, while others were held in abeyance because of their almost complete homology. This is the first publication of five chimpanzee CYP3A genes-CYP3A4, CYP3A5, CYP3A7, CYP3A43, and CYP3A67. This project utilized two approaches for characterizing possible relationships-phylogenetic analysis and genomic structure. For the phylogenetic analysis, both nucleotide and amino acid sequences were aligned in silico using the CLUSTAL algorithm, and then visually inspected for accuracy. Three different computer software packages were utilized: MEGA 2.1, TREECON 1.3b, and PHYLIP 3.5. Multiple methods were used: neighbor-joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML). The resulting topologies were compared against each other to define the consensus topology. In addition, the chimpanzee, human, mouse, and rat genome databases were searched for intron/exon information pertaining to the included genes. Both methods suggest the same conclusion, defining orthologs is plausible between similar species (i.e., mouse and rat), but is less useful between species of different orders (i.e., primate and rodent) or classes (i.e., mammal and avian).

In vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor alpha/gamma agonist. I. Role of cytochrome P450, methyltransferases, flavin monooxygenases, and esterases.

The metabolism of MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide, a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in liver microsomes and hepatocytes from humans and rat, dog, and rhesus monkey, to characterize the enzyme(s) involved in its metabolism. The major site of metabolism is the TZD ring, which underwent opening catalyzed by CYP3A4 to give the mercapto derivative, M22. Other metabolites formed in NADPH-fortified liver microsomes included the TZD-5-OH derivative (M24), also catalyzed by CYP3A4, and the O-desmethyl derivative (M28), whose formation was catalyzed by CYP2C9 and CYP2C19. Metabolite profiles from hepatocyte incubations were different from those generated with NADPH-fortified microsomal incubations. In addition to M22, M24, and M28, hepatocytes generated several S-methylated metabolites, including the methyl mercapto (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) metabolites. Addition of the methyl donor, S-adenosyl methionine, in addition to NADPH, to microsomal incubations enhanced the turnover and resulted in metabolite profiles similar to those in hepatocyte incubations. Collectively, these results indicated that methyltransferases played a major role in the metabolism of MK-0767. Using enzyme-specific inhibitors, it was concluded that microsomal thiol methyltransferases play a more important role than the cytosolic thiopurine methyltransferase. Baculovirus-expressed human flavin-containing monooxygenase 3, as well as CYP3A4, oxidized M25 to M16, whereas further oxidation of M16 to M20 was catalyzed mainly by CYP3A4. Esterases were involved in the formation of the methyl sulfone carboxylic acids, minor metabolites detected in hepatocytes.

Development and evaluation of a rapid and reliable method for cytochrome P450 2C8 genotyping.

The analysis of gene polymorphisms has started to become an interesting field of new services to be provided by clinical laboratories for both, clinical research and routine determinations. To expand the use of these methods and to increase the benefit for patients and the society, cheap and reliable methods for genotyping also allowing for high-turnover analysis need to be established in clinical laboratories. The presented investigation was performed to develop and evaluate a fast real-time PCR method for the detection of three different allele mutations of Cytochrome P450 isoenzyme 2C8 (CYP2C8*2, *3, and *4), which have been demonstrated to influence drug metabolism (and thus the efficacy) of a variety of drugs. The DNA of 122 Caucasian subjects (56 male, 66 female, age (mean +/- STD): 50 +/- 16 years) was analyzed for these mutations by means of classical RFLP-PCR and a new protocol developed for the LightCycler real-time PCR method. The polymorphisms within the CYP2C8 gene were detected by use of two primer pairs and three different pairs of hybridization probes. The results of both methods were perfectly concordant and comparable to results published in the literature (allele frequencies: CYP2C8*2: 0.016, *3: 0.140, *4: 0.074). A subsequent analysis of the related costs revealed comparable resource requirements but substantially longer time needs for RFLP-PCR, resulting in higher overall analysis costs for the older method. In conclusion, the elaborated protocol for real-time PCR analysis of gene mutations CYP2C8*2, *3, and *4 is reliable and cost effective, and thus, suitable for routine laboratory use.

In vitro induction of cytochrome P450 enzymes in hepatocytes isolated from the regenerating rat liver.

Partial hepatectomy results in the loss of cytochrome P450 enzymes. During regeneration, the levels of cytochrome P450 activities, apoproteins and mRNA are reduced. Our present study investigated CYP1A, CYP2E1 and CYP3A induction in the cells of rat liver regenerating for 1, 3, 7, or 14 days. Hepatocytes were isolated from regenerating liver of hepatectomized rats and treated with enzyme inducers: 3-methylcholanthrene, imidazole and dexamethasone. CYP1A enzymes of the cells isolated from regenerating liver were inducible by 3-methylcholanthrene. The rate of induction of the cells from 3-day regenerating liver by 3-methylcholanthrene was three times higher than that of the hepatocytes of sham-operated rats. Dexamethasone caused about two- or three-fold stronger elevation of CYP3A in the cells of 1-, 3- and 7-day regenerating liver than in hepatocytes of sham-operated animals. However, the degree of CYP2E1 induction by imidazole was the same (about 2.5-fold) at each regenerating time as it was detected in the hepatocytes of sham-operated animals. In conclusion, the inducibility of the cells was retained at each regenerating time, but the degree of induction showed some differences.

Cloning CYP2D21 and CYP3A22 cDNAs from liver of miniature pigs.

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1'-hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts.

Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling.

A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.

Predominantly neuronal expression of cytochrome P450 isoforms CYP3A11 and CYP3A13 in mouse brain.

Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippocampus) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain's plasticity.

Functional characterization of cytochrome P450 2B6 allelic variants.

Cytochrome P450 (P450) 2B6 is a hepatic enzyme of potential importance for the metabolism of clinically used drugs and environmental or abused toxicants. Genetic polymorphisms of CYP2B6 (CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6 and CYP2B6*7; wild-type, CYP2B6*1) were found previously in white and Japanese populations. In the present study, the goal was to investigate the effects of amino acid substitutions on CYP2B6 function. Wild-type (CYP2B6.1) and all of the known variants of CYP2B6 (CYP2B6.2, CYP2B6.3, CYP2B6.4, CYP2B6.5, CYP2B6.6, and CYP2B6.7) were transiently expressed in COS-1 cells, and their 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activities were determined. The levels of the variant CYP2B6 proteins were relatively low compared with that of CYP2B6.1, although the differences were not significant. The activities of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation on the basis of the CYP2B6 protein level at low (0.5 microM) and high (50 microM) substrate concentrations varied among wild-type and variant CYP2B6 proteins. All CYP2B6 enzymes showed typical Michaelis-Menten kinetics. The K(m) value of CYP2B6.6 was significantly higher than that of CYP2B6.1. Those CYP2B6 variants having a Lys262Arg substitution (CYP2B6.4, CYP2B6.6, and CYP2B6.7) showed increased values for V(max) and V(max)/K(m), whereas the kinetic parameters of CYP2B6.2 and CYP2B6.3 were not affected by the corresponding amino acid substitution. These results may mean that Lys262 in combination with other amino acid residues such as Gln172 and Arg487 is associated with the CYP2B6 function and that the genetic polymorphism of CYP2B6 leads to interindividual differences in xenobiotic metabolism.

Polymorphic CYP2C19 and N-acetylation: human variability in kinetics and pathway-related uncertainty factors.

CYP2C19-mediated oxidation and N-acetylation constitute major phase I and phase II polymorphic pathways of xenobiotic metabolism in humans. Analysis of human variability in kinetics for these pathways has been carried out for compounds metabolised extensively (>60%) by these routes. Data for minor substrates for CYP2C19 metabolism (10-60%) have also been analysed. Published pharmacokinetic studies (after oral and intravenous dosing) in CYP2C19 non-phenotyped healthy adults (NPs), and phenotyped extensive (EMs), slow-extensive (SEMs) and poor metabolisers (PMs) have been analysed using data for parameters that relate primarily to chronic exposure (metabolic and total clearances, area under the plasma concentration-time curve) and primarily to acute exposure (peak concentration). Similar analyses were performed for the N-acetylation pathway using data for fast acetylators (FA) and slow acetylators (SA). Interindividual variability in the kinetics of CYP2C19 substrates after oral dosage was greater in EMs than in NPs (60 vs 43% for clearances and 54 vs 45% for Cmax). Lower variability was found for N-acetylation for both phenotypes (32 and 22% for FA and SA, respectively). The internal dose of CYP2C19 substrates in PM subjects would be 31-fold higher than in EMs, while for N-acetylated substrates there was a three-fold difference between SA and FA subjects. Pathway-related uncertainty factors were above the default safety factor of 3.16 for most subgroups and values of 52 and 5.2 would be necessary to cover to the 99th centile of the poor metaboliser phenotype for CYP2C19 and N-acetylation, respectively. An exponential relationship (R(2)=0.86) was found between the extent of CYP2C19 metabolism and the difference in internal dose between EMs and PMs. The kinetic default factor (3.16) would cover PMs for substrates for which CYP2C19 was responsible for up to 20-30% of the metabolism in EMs.