Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus‐mediated expression system was used to express wild‐type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high‐performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1′‐hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O‐demethylation than those of the wild‐type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild‐type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.     

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.     

Functional Characterization of 22 CYP3A4 Protein Variants to Metabolize Ibrutinib In Vitro

Cytochrome P450 3A4 (CYP3A4) is quantitatively the most important P450 enzyme in adults. It is suggested that CYP3A4 genetic polymorphisms may influence the rate of the metabolism and elimination of CYP3A4 substrates in human beings. Ibrutinib is an anticancer drug and primarily metabolized by CYP3A4. The aim of this study was to systematically investigate the effects of 22 CYP3A4 protein variants on the metabolism of ibrutinib in vitro. When compared with wild‐type CYP3A4.1, two variants (CYP3A4.17 and CYP3A4.24) had no detectable enzyme activity; five variants (CYP3A4.10, .11, .18, .23 and .33) exhibited no significant differences; another five variants (CYP3A4.3, .4, .9, .19 and .34) showed increased intrinsic clearance values, while the remaining nine variants (CYP3A4.2, .5, .14, .15, .16, .28, .29, .31 and .32) displayed decreased enzymatic activities in different degrees. As the first study of 22 CYP3A4 protein variants in ibrutinib metabolism, these comprehensive data may help in the clinical assessment of the metabolism and elimination of ibrutinib and also offer a reference to the personalized treatment of ibrutinib in clinic.     

Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population

AIM: The human cytochrome P450 2D6 (CYP2D6) gene copy number variation, involving CYP2D6 gene deletion (CYP2D6*5) and duplication or multiduplication (CYP2D6*xN), can result in reduced or increased metabolism of many clinically used drugs. The identification of CYP2D6*5 and CYP2D6*xN and the investigation of their allelic distributions in ethnic populations can be important in determining the right drug and dosage for each patient. METHODS: The CYP2D6*5 and CYP2D6 genes, and CYP2D6 gene duplication were identified by 2 modified long PCR, respectively. To determine duplicated alleles, a novel long PCR was developed to amplify the entire duplicated CYP2D6 gene which was used as template for subsequent PCR amplification. A total of 363 unrelated Eastern Han Chinese individuals were analyzed for CYP2D6 gene copy number variation. RESULTS: The frequency of CYP2D6*5 and CYP2D6*xN were 4.82% (n=35) and 0.69% (n=5) in the Eastern Han Chinese population, respectively. Of the 5 duplicated alleles, 3 were CYP2D6*1xN and 2 were CYP2D6*10xN. One individual was a carrier of both CYP2D6*5 and CYP2D6*1xN. Taken together, the CYP2D6 gene rearrangements were present in 10.74% of subjects. CONCLUSION: Allelic distributions of the CYP2D6 gene copy number variation differ among Chinese from different regions, indicating ethnic variety in Chinese. Long PCR are convenient, cost effective, specific and semiquantitative for the detection of the CYP2D6 gene copy number variation, and amplification of the entire duplicated CYP2D6 gene is necessary for the accurate identification of duplicated alleles.     

The Role of Human CYP2C8 and CYP2C9 Variants in Pioglitazone Metabolism In Vitro

Abstract: The cytochrome P450 enzyme CYP2C8 appears to have a major role in pioglitazone metabolism. The present study was conducted to further clarify the role of individual CYPs and of the CYP2C8/9 polymorphisms in the primary metabolism of pioglitazone in vitro. Pioglitazone (2–400 μM) was incubated with isolated cytochrome P450 enzymes or human liver microsomes, some of them carrying either the CYP2C8*3/*3 genotype (and also the CYP2C9*2/*2 genotype) or the CYP2C8*1/*1 genotype (five samples each). The formation of the primary pioglitazone metabolite M‐IV was monitored by HPLC. Enzyme kinetics were estimated assuming a single binding site. Mean intrinsic clearance of pioglitazone to the metabolite M‐IV was highest for CYP2C8 and CYP1A2 with 58 pmol M‐IV/min/nmol CYP P450/μM pioglitazone each, 53 for CYP2D6*1, 40 for CYP2C19*1, and 34 for CYP2C9*2, respectively. CYP2A6, CYP2B6, CYP2C9*1, CYP2C9*3, CYP2E1, CYP3A4 and CYP3A5 did not form quantifiable amounts of M‐IV. CYP2C8*1/*1 microsomes (25 ± 4 pmol M‐IV/min/mg protein/μM pioglitazone) showed lower intrinsic clearance of pioglitazone than CYP2C8*3/*3 microsomes (35 ± 9, p = 0.04). In all samples, metabolite formation showed substrate inhibition, while pioglitazone did not inhibit CYP2C8‐mediated paclitaxel metabolism. CYP2C8, CYP1A2 and CYP2D6 are major CYPs forming M‐IV in vitro. The higher activity of CYP2C8*3/CYP2C9*2 microsomes may result from a contribution of CYP2C9*2, or from differences in CYP2C8 expression. The evidence for substrate‐specific inhibitory effects of pioglitazone on CYP2C‐mediated metabolism needs to be tested in further studies.     

High Frequency of CYP2D6 Ultrarapid Metabolizer Genotype in the Finnish Population

CYP2D6 participates in the biotransformation of many commonly used drugs. Large genetic variability in CYP2D6 results in a wide interindividual variability in the response to CYP2D6 substrate drugs. Previous studies have assessed the phenotype and genotype distributions of CYP2D6 in relatively small Finnish population samples. The aim of our study was to investigate the frequencies of CYP2D6 genotypes in a larger Finnish population cohort of 857 healthy volunteers. The volunteers were genotyped for 10 CYP2D6 genetic variants (*2, *3, *4, *5, *6, *9, *10, *17, *39, *41) and copy number variation performed with TaqMan genotyping assays and copy number assay targeting exon 9. CYP2D6 phenotypes were inferred from the genotype data with the classical and activity score methods. According to the classical method, a large majority of the study cases were extensive metabolizers (EM; 87.3%; 95% confidence interval 84.9–89.3) and the second largest group was ultrarapid metabolizers (UM; 7.2%; 5.7–9.2%). Intermediate (IM) and poor metabolizers (PM) were in clear minority (3.0%; 2.1–4.4% and 2.3%; 1.5–3.6%, respectively). The activity score method yielded similar phenotype predictions. These results show that the frequency of UM genotype is higher and that of PM and IM genotype is lower in the Finnish population than in other North European populations. Accordingly, CYP2D6 genetic profile of the Finnish population differs from its geographically close neighbours, which has implications for the effective and safe use of drugs metabolized by CYP2D6.     

In vivo inhibition of CYP2C19 but not CYP2D6 by fluvoxamine

Studies were performed in eight healthy extensive metabolizers of mephenytoin and debrisoquine to determine the effect of fluvoxamine on the activities of S-mephenytoin 4'-hydroxylase (CYP2C19) and metoprolol α-hydroxylase (CYP2D6). Therapeutic dosing with fluvoxamine (100  mg day⁻¹) for 2 weeks caused a significant increase in the 0–8  h urinary S/R ratio of mephenytoin from 0.16 to 0.55 (95% confidence interval for difference between means: 0.28–0.50; P <0.01), accompanied by a 54% reduction in the 0–8  h urinary recovery of 4'-hydroxymephenytoin (95% confidence interval for difference between means: 3.64–16.24  mg; P <0.05). However, this did not alter the assigned phenotype of any of the subjects based on the established antimode of 0.95 (S/R-mephenytoin ratio). Two weeks after fluvoxamine was discontinued, both metabolic indices returned to their pre-study values. By contrast, fluvoxamine had no effect on either 0–8  h urinary metoprolol/α-hydroxymetoprolol ratio (95% confidence interval for difference between means: −0.38–0.46; P >0.05) or the 0–8  h urinary recovery of α-hydroxymetoprolol (95% confidence interval for difference between means: −0.61–0.70  mg; P >0.05). These results indicated fluvoxamine has a modest inhibitory effect on the activity of CYP2C19, but no effect on that of CYP2D6 in vivo .     

CYP2D6*11 and challenges in clinical genotyping of the highly polymorphic CYP2D6 gene

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.     

CYP2D6 polymorphism in a Gabonese population: contribution of the CYP2D6*2 and CYP2D6*17 alleles to the high prevalence of the intermediate metabolic phenotype

AIMS: To determine the molecular basis of the intermediate extensive metaboliser (EM) CYP2D6 phenotype in healthy Gabonese subjects. METHODS: The CYP2D6 phenotype of 154 healthy Gabonese subjects was assessed by giving the subject a single dose of 30 mg dextromethorphan, and collecting their urine for the next 8 h. The CYP2D6 genotype was determined for 50 individuals of the EM phenotypic group by Southern blotting and various PCR-based procedures aimed at identifying different CYP2D6 alleles. RESULTS: We found that in the studied Gabonese population, as compared with a French population, there is significantly higher frequency of intermediate EM phenotype having lower frequency of CYP2D6 PM alleles. To clarify this discrepancy phenotype-genotype relationship was studied. We found that the CYP2D6*17 and CYP2D6*2 alleles, prevalent in this black population, are characterised by their low capacity for dextromethorphan demethylation. Our data also show that the CYP2D6*1 allele is associated with the highest in vivo activity followed by the CYP2D6*2 allele and then the CYP2D6*17 allele. CONCLUSIONS: The higher frequencies of the CYP2D6*2 and CYP2D6*17 alleles than the CYP2D6*1 allele account for the high frequency of the intermediate EM phenotype in this black population. The polymorphism of the CYP2D6 enzyme activity in African populations could have important implications for use of drugs that are substrates for CYP2D6 and have a narrow therapeutic window.     

Genotypes for the cytochrome P450 enzymes CYP2D6 and CYP2C19 in human longevity Role of CYP2D6 and CYP2C19 in longevity

Objective: To test whether some genotypes for CYP2D6 or CYP2C19 could contribute to longevity, we genotyped 241 Danish nonagenarians and centenarians for CYP2D6 and CYP2C19. Methods: For CYP2D6 we identified the alleles CYP2D6*1, CYP2D6*3 and CYP2D6*4 with allele-specific polymerase chain reaction (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. Results: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism was slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75–2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26–5.75)]. Conclusion: CYP2D6 could play a role in human longevity due to the lack of Hardy-Weinberg proportions. If CYP2D6 only plays a role in longevity by protecting the poor metabolizers from cancer, we should expect a rise in the frequency in these genotypes in Denmark from 7.7% among young adults to 10–11% among very old people. We found a frequency of poor metabolism of 10.2% in the very old group. CYP2C19 is – due to the occurrence of Hardy-Weinberg proportions and the expected number of poor metabolizers – unlikely to contribute to human longevity.     

Debrisoquin and S-Mephenytoin Hydroxylation Phenotypes and CYP2D6 Genotypes in an Estonian Population

Abstract: The polymorphisms of debrisoquin (CYP2D6) and S-mephenytoin (CYP2C19) hydroxylation were studied in 210 unrelated healthy native Estonians by coadministration of mephenytoin and debrisoquin or dextromethorphan. Among the 210 volunteers 21 (10%) were poor metabolizers of debrisoquin/dextromethorphan and two (0.95%) were poor metabolizers of S-mephenytoin. By pooling these data with an earlier study on 156 Estonians, the prevalences of poor metabolizers of debrisoquin/dextromethorphan and poor metabolizers of S-mephenytoin were 7.6% and 2.2%, respectively. The CYP2D6 genotype of 151 subjects was analysed by allele-specific PCR amplification for the defect alleles CYP2D6A and CYP2D6B. All poor metabolizers of debrisoquin carried two defect CYP2D6-alleles. The phenotype (extensive or poor metabolizer) was in all subjects correctly predicted by the genotype. The frequencies of the defect alleles CYP2D6B and CYP2D6A among these 151 subjects (including 14 poor metabolizers - 9.3%) were 21.5% and 2.3%, respectively. DNA from 6 subjects with very high CYP2D6 activity (debrisoquin MR<0.1) was analysed by EcoRI RFLP to identify duplicated or amplified CYP2D6-genes. Two of the subjects were found to carry a duplicated CYP2D6L-gene. In conclusion, the distribution of genetically determined metabolic capacities of CYP2D6 and CYP2C19 in Estonian unrelated subjects did not differ significantly from that in other Caucasian populations. The CYP2D6 phenotype was predicted by PCR-based amplification for the CYP2D6A and CYP2D6B-aellels.     

Impact of CYP2D6 polymorphisms on the pharmacokinetics of lovastatin in Chinese subjects

Purpose

Although CYP3A4/5 enzymes play the predominant role in the metabolism of simvastatin and lovastatin, polymorphisms in CYP2D6 were reported to be associated with the cholesterol-lowering effect and/or tolerability of simvastatin. This study was performed to examine whether common CYP2D6 polymorphisms affect the pharmacokinetics of lovastatin, which is taken as the inactive prodrug lovastatin lactone and converted to active lovastatin acid.

Methods

A single-dose pharmacokinetic study was performed with lovastatin in 23 Chinese healthy male subjects. Plasma concentrations of lovastatin lactone and acid were determined by an LC-MS-MS method in samples collected over 24?h after single oral doses of 40-mg lovastatin.

Results

Compared with the CYP2D6 wt/wt group, the area under the plasma concentration–time curve (AUC_0-∞) values for lovastatin lactone increased (P?wt/*10, *10/*10, *10/*5, and *5/*5 groups, and the values of lovastatin lactone plasma clearance (CL/F) were reduced on average (95% CI) by 40.4% (10.2–60.5%), 53.1% (29.3–68.9%), 63.8% (40.2–78.1%) and 84.2% (72.1–91.1%) in these genotype groups respectively. The pharmacokinetics of lovastatin acid did not differ among the genotype groups.

Conclusion

This study demonstrates that CYP2D6 polymorphisms appeared to influence the disposition of lovastatin lactone in these subjects.     

Novel structure of the CYP2D6 gene that confuses genotyping for the CYP2D6*5 allele

We encountered DNA samples which showed a positive product using a long PCR-based method for the detection of CYP2D6*5, indicating deletion of the entire CYP2D6 gene, but the samples did not show a band related to CYP2D6*5 in either XbaI- or EcoRI-RFLP analysis. To achieve genotyping with accuracy, we performed a further genetic analysis to clarify the discrepancy. An unknown 1.6-kb insert was identified in a region downstream from the CYP2D6 stop codon where a specific primer was designed for long-PCR analysis for CYP2D6*5 genotyping. This finding suggested that the CYP2D6 gene might not be deleted in the samples even if a positive product was detected by the long-PCR method. Furthermore, the allelic frequency of this type was found to be approximately 0.3% (4 heterozygous/771 samples) in a Japanese population. In conclusion, we found a novel structure of the CYP2D6 gene, which might lead to incorrect genotyping for CYP2D6*5. Although the long PCR-based strategy for the detection of CYP2D6*5 has been widely used due to its usefulness and convenience, we recommend caution when adopting this method and propose re-evaluating the method for detecting CYP2D6*5.