Cytochrome P450 2D6 Variants Research Articles

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.

Variation in the inhibitory potency of terbinafine among genetic variants of CYP2D6

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that is involved in the metabolism of many drugs. Terbinafine (TER) is a CYP2D6 inhibitor and causes persistent drug interactions in the clinical setting; however, its inhibitory mechanism and the differences in its inhibitory potency among genetic variants of CYP2D6 remain to be investigated. This study aimed to investigate the inhibitory mechanism of TER and the differences in its inhibitory potency among three CYP2D6 variants, CYP2D6.1, CYP2D6.2, and CYP2D6.10.In a competitive inhibition study, the metabolic activity of the CYP2D6 was assessed based on their demethylation of dextromethorphan in the presence or absence of TER, and the time-dependency of the inhibitory effects were examined by preincubating the enzymes with TER.TER had weaker inhibitory effects on CYP2D6.2 and CYP2D6.10 than on CYP2D6.1; i.e., TER exhibited Ki values (the concentration of inhibitor that results in half-maximal inhibition) of 0.0525, 0.355, and 1.85 μM for CYP2D6.1, CYP2D6.2, and CYP2D6.10, respectively. The inhibitory effects of TER were not time-dependent.Since TER's Ki value for CYP2D6.10 was 35.2-fold higher than its Ki value for CYP2D6.1, the CYP2D6 genotype of subjects should be taken into account when estimating the severity of drug interactions involving TER.

In vitro functional assessment of 22 newly identified CYP2D6 allelic variants in the Chinese population.

Cytochrome P450 2D6 (CYP2D6) is one of the most widely investigated CYPs related to genetic polymorphisms and is responsible for one-quarter of the currently used clinical drugs. We previously detected 22 novel, non-synonymous, mutated sites in the Chinese population, but nothing is known about the functional effects of these mutations in terms of specific CYP2D6 substrates. In this study, wild-type CYP2D6, two common allelic variants and 22 newly reported CYP2D6 isoforms were transiently expressed in 293FT cells, and the enzymatic activities of these variants were systematically assessed using dextromethorphan and bufuralol as the probing substrates. Consequently, 19 and 21 allelic variants were found to exhibit significantly decreased enzymatic activities for dextromethorphan and bufuralol, respectively. Of 22 novel CYP2D6 variants, six allelic isoforms (CYP2D6.89, CYP2D6.92, CYP2D6.93, CYP2D6.96, E215K and R440C) exhibited absent or extremely reduced metabolic activities compared with those observed for the wild-type enzyme. Our in vitro functional data can be useful for CYP2D6 phenotype prediction and provide valuable information for the study of clinical impact of these newly found CYP2D6 variants in China.

Functional Characterization of Wild-type and 49 CYP2D6 Allelic Variants for N-Desmethyltamoxifen 4-Hydroxylation Activity

Genetic variations in cytochrome P450 2D6 (CYP2D6) contribute to interindividual variability in the metabolism of clinically used drugs, e.g., tamoxifen. CYP2D6 is genetically polymorphic and is associated with large interindividual variations in therapeutic efficacy and drug toxicity. In this study, we performed an in vitro analysis of 50 allelic variants of CYP2D6 proteins. Wild-type CYP2D6.1 and 49 variants were transiently expressed in COS-7 cells, and the enzymatic activities of the CYP2D6 variants were characterized using N-desmethyltamoxifen as a substrate. The kinetic parameters K(m), V(max), and intrinsic clearance (V(max)/K(m)) of N-desmethyltamoxifen 4-hydroxylation were determined. Among the 50 CYP2D6 variants, the kinetic parameters for N-desmethyltamoxifen 4-hydroxylation were determined for 20 CYP2D6 variants. On the other hand, the kinetic parameters of 30 CYP2D6 variants could not be determined because the amount of metabolite produced was at or below the detection limit at the lower substrate concentrations. Among them, 8 variants, i.e., CYP2D6.2, .9, .26, .28, .32, .43, .45, and .70, showed decreased intrinsic clearance at <50% of CYP2D6.1. The comprehensive in vitro assessment of CYP2D6 variants provides novel insights into allele-specific activity towards tamoxifen and may be valuable when interpreting in vivo studies.

Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism.

CYP2D6 expression is regulated by polymorphisms that affect splicing and trancription: new biomarkers for CYP2D6 activity

Cytochrome P450 2D6 (CYP2D6) metabolizes ~25% of clinical used drugs. CYP2D6 variants are currently used as biomarkers to predict CYP2D6 activity, but the relationship between genotype and phenotype is still ambiguous in most individuals except in poor metabolizers. In this study, we showed common SNP located in exon 6 (rs16947, *2) affects exon 6 splicing, resulting in 2 fold reduction in total CYP2D6 mRNA expression via nonsense‐mediated RNA decay. In addition we identified a novel SNP (E‐SNP) located in an enhancer region over 100 kb downstream of CYP2D6 that increases CYP2D6 transcription 2 fold. Testing in a pediatric cohort of 160 individuals with metabolic activity measured as the ratio of dextromethorphan/metabolites (DM/DX), SNP rs16947 alone is associated with reduced metabolic activity, while haplotype containing both rs16947 and E‐SNP has similar metabolic activity as reference haplotype, indicating gain of function E‐SNP can compensate for the loss of function effect of rs16947. Our data also indicate that the key SNP in *41 rs28371725 does not have independent effect on CYP2D6 mRNA level, the in vivo reduced activity effects associated with *41 was resulted from the lack of E‐SNP in *41 haplotype which also contain rs16947. These results suggest that the combination of rs16947 and E‐SNP may become a new biomarker for predicting CYP2D6 activity.Supported by NIH grant U01 GM092655.

Genetic variants in the cytochrome P450 2D6 gene in the Sri Lankan population

INTRODUCTION:Cytochrome P450 2D6 (CYP2D6) enzymes are involved in the metabolism of a large number of commonly prescribed drugs such as antidepressants and cardiovascular drugs. The CYP2D6 *3, *4 and *14 variants associated with the loss of enzyme function; CYP2D6 *10 and *17 variants with reduced enzyme function; and CYP2D6 *2 variant with no effect on enzyme function. Establishing the frequency of these variant alleles in Sri Lankan population would be useful for optimizing pharmacotherapy with CYP2D6-substrate drugs.OBJECTIVE:The objective of this study was to determine the prevalence of CYP2D6 *2, *3, *4, *10, *14 and *17 variants in the main ethnic groups in the Sri Lankan population.MATERIALS AND METHODS:A total of 90 deoxyribonucleic acid (DNA) samples (30 each from Sinhalese, Tamils and Moors) were selected from a DNA resource at the Human Genetic Unit, Faculty of Medicine, University of Colombo. This collection had been made for population genetic studies from a random population based volunteers. Genotyping was performed using published polymerase chain reaction/restriction fragment length polymorphism methods.RESULTS:The prevalence of the CYP2D6 variants in Sinhalese, Sri Lankan Tamils and Moors respectively were CYP2D6 *2: 37%, 41.6% and 37.9%; CYP2D6 *3: 60.3%, 45% and 30%; CYP2D6 *4: 21.6%, 6.6% and 8.3%; CYP2D6 *10: 40%, 35% and 44%. CYP2D6 *14 and *17 variants were not identified.CONCLUSION:CYP2D6*3, *4 and *10 variants, which are associated with reduced or loss of CYP2D6 enzyme function were found in our population in significant frequencies. CYP2D6*4, which is reported to be a Caucasian variant was also found in all three ethnic groups.

P4-01-04: Effects of CYP2D6 Phenotype and Drug Adherence on Tamoxifen Metabolite Levels.

Abstract Background Although the activity of cytochrome P450 2D6 (CYP2D6), the enzyme responsible for conversion of tamoxifen (TAM) to its most important active metabolite endoxifen, varies significantly with genotype. Routine genotype testing in patients on TAM has recently been discouraged. Conflicting results in publications regarding the prognostic utility of this test remain unexplained. Confounding factors could be lack of predicted correlation between CYP2D6 genotype and TAM active metabolites, or variability of patient compliance Methods: Consecutive breast cancer patients on TAM were asked to enroll in a study to examine the relationship between CYP 2D6 phenotype, patient-reported treatment adherence, and TAM metabolites levels. Patients were genotyped for CYP2D6 polymorphisms using long-range PCR allele-specific amplification and single-nucleotide primer extension assay. From the genotypes, four phenotype groups were defined: Ultra rapid Metabolizer (UM), Extensive Metabolizer (EM), Intermediate Metabolizer (IM) and Poor Metabolizer (PM). Plasma was collected after at least 6 weeks of TAM (20 mg daily). The parent drug TAM, as well as 4-hydroxy N-desmethyl tamoxifen (endoxifen), 4 hydroxy tamoxifen (4OHtam) and N-desmethyl-tamoxifen (NDtam), were determined by Liquid Chromatography tandem mass-spectrometry (LC-MS/MS). Patients also completed a questionnaire about ethinicity, side effects, concurrent medications and tamoxifen adherence. Correlation between metabolite/TAM ratio and phenotype was tested by Spearman correlation test. Relationship between metabolite levels and adherence was tested by Wilcoxon rank sum test. Chi square test was used to compare proportions. Results: Of the 100 patients enrolled there were 62 Caucasians, 25 Asians, 4 Africans and 6 Unknown. We found a strong correlation between ratio of endoxifen/TAM and phenotype (p &amp;lt;.0001) (Table 1) Over a 2 week period 68 never missed a TAM dose, 25 missed 1–2 times, 2 missed 3–5 times and 2 &amp;gt; 5times (2 missing data). In EM group we found significantly lower levels of TAM (p &amp;lt;.0001), NDtam (p=.008), 4OHtam (p=.003) in less adherent patients. A trend to decreased levels was also shown for endoxifen (p=.081). No associations were found between adherence or phenotype activity and side effects. Conclusions: Our data suggests the predicted association between endoxifen levels and genotype. However, non-adherence may have a significant confounding effect. Prospective studies to evaluate the prognostic impact of CYP2D6 variants for patients on adjuvant tamoxifen should be done but results could be confounded by variable drug adherence if this is not measured concurrently. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-04.

Lack of any association between functionally significant CYP2D6 polymorphisms and clinical outcomes in early breast cancer patients receiving adjuvant tamoxifen treatment

Active metabolites of tamoxifen are formed mainly by the action of cytochrome P450 2D6 (CYP2D6). Since there are controversies regarding associations between CYP2D6 polymorphisms and outcomes among women with early breast cancer (EBC) treated with tamoxifen, the present evaluation of links with clinical outcomes was conducted. We analyzed a total of 716 patients treated with tamoxifen for hormone receptor positive EBC between 2001 and 2005 at the National Cancer Center, Korea. All patients received tamoxifen 20 mg/day for more than 6 months. DNA obtained from whole blood samples was genotyped for CYP2D6 variants associated with reduced (*10, *41) and absent (*5) activity. Of the total of 716 patients, 558 (77.9%) received adjuvant or neoadjuvant chemotherapy prior to the tamoxifen therapy. From the genotyping of CYP2D6, 152 (21.2%) patients were classified as having the wild type (W/W), 376 (52.7%) one variant allele (W/V), and 188 (26.1%) two variant alleles (V/V). Seventy (9.8%) patients experienced disease recurrence with a median follow-up of 5.6 (range, 0.6-10.3) years. Although known prognostic factors, including tumor size, nodal status, Ki67, PgR negativity, and HER2 positivity showed strong associations with the recurrence free survival (RFS) in this population, no significant association with any of the CYP2D6 genetic variants was evident (P = 0.61; hazard ratio [HR] = 1.14; 95% CI 0.68-1.92). This remained the case after subgroup analysis according to different adjuvant treatments. Polymorphisms of CYP2D6 were not associated with clinical outcomes in EBC patients receiving adjuvant tamoxifen treatment.

Abstract P4-02-01: Correlations between Genetic Variants in CYP2D6 and UGT2B7 and Survival in Breast Cancer Patients Treated with or without Tamoxifen: Results from a Large Cohort Study

Abstract Background. A large number of studies testing the hypothesis that genetic variants in drug metabolizing enzymes can influence breast cancer patient response to the selective estrogen receptor modulator tamoxifen have provided equivocal and conflicting data. Possible confounding factors in all studies to date include the relatively small number of patients, lack of comprehensive genotyping analysis (in particular regarding the cytochrome P450 2D6 (CYP2D6) enzyme), use of selective serotonin reuptake inhibitors (SSRIs) (antidepressant medication), and lack of untreated (i.e. no tamoxifen treatment) control groups. Currently, it is not clear whether genotype determinations can predict which patients will likely benefit from tamoxifen therapy. Methods: We conducted a large community-based cohort study to test for associations between clinical outcomes and variants in CYP2D6, one of the key enzymes responsible for the conversion of tamoxifen to its potent anti-estrogenic metabolite endoxifen, as well as variants in UDP-glucuronosyltransferase 2B7 (UGT2B7), an enzyme responsible for the elimination of endoxifen. The majority of patients in this cohort began their tamoxifen treatment more than twenty ago, at a time when SSRIs (a possible confounding factor in CYP2D6 studies) were not widely used. Five hundred patients with ER+ early breast cancer treated adjuvantly with tamoxifen monotherapy and 500 who did not receive any adjuvant therapy were genotyped and assigned a CYP2D6 “score” based on known specific allele activities from 0 (no activity) to 2 (high activity) and also grouped as either low, intermediate and high UGT2B7 activity based on the *2 allele. Results: Univariately, Kaplan-Meier estimates in conjunction with the log-rank test did not reveal any significant associations between either CYP2D6 or UGT2B7 score and time to relapse (TTR). In tamoxifen treated cases, after adjustment for tumor size, nodal status and PgR, Cox regression found increased CYP2D6 score, reflecting increased conversion of tamoxifen to endoxifen, was significantly associated with worse time to relapse (HR=1.53, p=0.02). This was not seen in the patients without endocrine treatment (HR=0.73, p=0.10). Patients with high UGT2B7 activity, reflecting increased elimination of endoxifen had better survival rates (HR= 0.75, p = 0.04) in the tamoxifen treated group, while again no effect of UGT2B7 was seen in the untreated group (HR=1.0, p=0.78). Finally, CYP2D6 and UGT2B7 genotypes were not associated with any special tumor characteristics in either patient group. Conclusion: In our study, CYP2D6 and UGT2B7 genotypes associated with increased endoxifen levels were associated with worse outcome in tamoxifen treated (but not untreated) breast cancer patients. Thus, the data from this cohort study do not support the hypothesis that breast cancer patients with low CYP2D6 are resistant to tamoxifen. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-01.

CYP2D6 gene variants: association with breast cancer specific survival in a cohort of breast cancer patients from the United Kingdom treated with adjuvant tamoxifen

IntroductionTamoxifen is one of the most effective adjuvant breast cancer therapies available. Its metabolism involves the phase I enzyme, cytochrome P4502D6 (CYP2D6), encoded by the highly polymorphic CYP2D6 gene. CYP2D6 variants resulting in poor metabolism of tamoxifen are hypothesised to reduce its efficacy. An FDA-approved pre-treatment CYP2D6 gene testing assay is available. However, evidence from published studies evaluating CYP2D6 variants as predictive factors of tamoxifen efficacy and clinical outcome are conflicting, querying the clinical utility of CYP2D6 testing. We investigated the association of CYP2D6 variants with breast cancer specific survival (BCSS) in breast cancer patients receiving tamoxifen.MethodsThis was a population based case-cohort study. We genotyped known functional variants (n = 7; minor allele frequency (MAF) > 0.01) and single nucleotide polymorphisms (SNPs) (n = 5; MAF > 0.05) tagging all known common variants (tagSNPs), in CYP2D6 in 6640 DNA samples from patients with invasive breast cancer from SEARCH (Studies of Epidemiology and Risk factors in Cancer Heredity); 3155 cases had received tamoxifen therapy. There were 312 deaths from breast cancer, in the tamoxifen treated patients, with over 18000 years of cumulative follow-up. The association between genotype and BCSS was evaluated using Cox proportional hazards regression analysis.ResultsIn tamoxifen treated patients, there was weak evidence that the poor-metaboliser variant, CYP2D6*6 (MAF = 0.01), was associated with decreased BCSS (P = 0.02; HR = 1.95; 95% CI = 1.12-3.40). No other variants, including CYP2D6*4 (MAF = 0.20), previously reported to be associated with poorer clinical outcomes, were associated with differences in BCSS, in either the tamoxifen or non-tamoxifen groups.ConclusionsCYP2D6*6 may affect BCSS in tamoxifen-treated patients. However, the absence of an association with survival in more frequent variants, including CYP2D6*4, questions the validity of the reported association between CYP2D6 genotype and treatment response in breast cancer. Until larger, prospective studies confirming any associations are available, routine CYP2D6 genetic testing should not be used in the clinical setting.

Association Between CYP2D6 Polymorphisms and Outcomes Among Women With Early Stage Breast Cancer Treated With Tamoxifen

The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen. Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n=609), heterozygous extensive/intermediate (n=637), or poor (n=79) CYP2D6 metabolism. Time to recurrence, event-free survival, disease-free survival, and overall survival. Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04-1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10-3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06-1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03-1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88-1.51). Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes.

Cytochrome P450 2D6 variants and their association with breast cancer survival.

Abstract Abstract #3089 Background: Cytochrome P450 2D6 (CYP2D6), a phase I drug metabolising enzyme, is involved in the metabolism of up to 25% of all drugs. The CYP2D6 gene is highly polymorphic with over 60 reported variant forms. Many of these have been classified by their effect on drug metaboliser status (poor, intermediate, extensive and ultra). Many variants have low minor allele frequencies (MAF) and metaboliser classification is not always certain. There is conflicting evidence for the potential predictive value of CYP2D6 genotype in patients treated with tamoxifen. In this study we investigate the association of the CYP2D6 gene variants with breast cancer survival in patients treated with tamoxifen.&amp;#x2028; Methods: Invasive breast cancer cases (n=6510), came from SEARCH (East Anglian, UK population-based study), 3155 of these cases had received Tamoxifen therapy. CYP2D6 single nucleotide polymorphisms (SNPs) for genotyping were selected using 2 approaches: (i) Known CYP2D6 functional variants (n=13; MAF&amp;gt;0.01), (ii) tag SNPs , that efficiently tagged all known CYP2D6 common variants on the HapMap database (n=5; MAF&amp;gt;0.05), such that known common SNPs had an rp2 of &amp;gt;0.8 with at least one tagSNP. (Pairwise correlation coefficient (rp2) is a measure of how well one SNP tags another.) The combination of these two approaches aims to capture all known functional variants as well as any, as yet undiscovered variants that may be tagged by the common tagSNPs. Genotyping was carried out using Taqman Assays by Design.&amp;#x2028; Results: Preliminary results, from this ongoing study, indicate that in patients treated with tamoxifen, one rare functional variant (carrier frequency = 0.02), CYP2D6*6, is associated with decreased breast cancer survival (P=0.007; HR=2.03; 95%CI=1.21-3.40), in comparison to patients who have not received hormone therapy (P=0.359; HR=0.52; 95%CI=0.13-2.10). None of the common tagSNPs (MAF&amp;gt;0.05) were associated with differences in survival (at P&amp;lt;/=0.05 level).&amp;#x2028; Conclusion: We conclude that at least one variant of the CYP2D6 gene may affect breast cancer survival in patients treated with tamoxifen. CYP2D6*6 is associated with poor metaboliser status, resulting in impaired tamoxifen metabolism. Consequently genotyping for this CYP2D6 variant may help to determine which patients are unlikely to benefit from standard tamoxifen treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3089.

Functional Characterization of 17CYP2D6Allelic Variants (CYP2D6.2, 10, 14A–B, 18, 27, 36, 39, 47–51, 53–55, and 57)

Cytochrome P450 2D6 (CYP2D6) is an enzyme of potential importance for the metabolism of drugs used clinically, and it exhibits genetic polymorphism with interindividual differences in metabolic activity. To date, 21 CYP2D6 allelic variants have been identified in the Japanese population. The aim of this study was to investigate the functional characterization of CYP2D6 variants identified in Japanese subjects. Wild-type CYP2D6 and its variants, namely, CYP2D6.2, CYP2D6.10, CYP2D6.14A, CYP2D6.14B, CYP2D6.18, CYP2D6.27, CYP2D6.36, CYP2D6.39, CYP2D6.47, CYP2D6.48, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.53, CYP2D6.54, CYP2D6.55, and CYP2D6.57 were transiently expressed in COS-7 cells, and enzymatic activities of the CYP2D6 variant proteins were characterized using bufuralol and dextromethorphan. Functional characterization of 17 CYP2D6 variants revealed an absence of enzyme activity in four (CYP2D6.14A, CYP2D6.36, CYP2D6.47, and CYP2D6.57), low activity in eight (CYP2D6.10, CYP2D6.14B, CYP2D6.18, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.54, and CYP2D6.55), and high activity in one (CYP2D6.53) compared with the wild type. Analysis of CYP2D6 variant proteins can be useful for predicting CYP2D6 phenotypes and could be applied to personalized drug therapy.

Development and characterization of a rapid and comprehensive genotyping assay to detect the most common variants in cytochrome P450 2D6.

Development and characterization of a rapid and comprehensive genotyping assay to detect the most common variants in cytochrome P450 2D6.

Molecular modelling of cytochrome P4502D6 (CYP2D6) based on an alignment with CYP102 : structural studies on specific CYP2D6substrate metabolism

1. A molecular model of CYP2D6 has been constructed from the bacterial form CYP102 via a homology alignment between the CYP2D subfamily and CYP102 protein sequences. 2. A number of typical CYP2D6 substrates are shown to fit the putative active site of the enzyme, as can the specific inhibitor quinidine. 3. Some of the allelic variants in CYP2D6, which give rise to genetic polymorphisms in 2D6-mediated metabolism, can be rationalized in terms of their position within the active site region. 4. The results of site-directed mutagenesis experiments are consistent with the CYP2D6 model generated from the CYP102 crystal structure. 5. The possibility of an alternative orientation within the active site may explain the CYP2D6-mediated metabolism of relatively large-sized substrates.