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.     

Role of polymorphic CYP2E1 and CYP2D6 genes in NNK-induced chromosome aberrations in cultured human lymphocytes

Polymorphisms in genes of xenobiotic-metabolizing enzymes are largely responsible for interindividual differences in ability to activate and detoxify genotoxic agents and therefore may influence individual susceptibility to environmental cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), requires metabolic activation by cytochrome P450 (CYP) enzymes to generate DNA-reactive intermediates that induce mutations and cancer. In the current study, we investigated the role of the polymorphic CYP2E1 and CYP2D6 genes in the genotoxicity of NNK using the tandem-probe fluorescence in-situ hybridization (FISH) chromosome aberration assay as a marker. Our results, using whole blood cultures from 39 volunteers, indicated that NNK (0.12, 0.24 or 0.72 mM) induced a concentration-dependent increase in the frequency of chromosome aberration. The potential role of CYP2E1 and CYP2D6 in NNK-induced genetic damage in cultured human lymphocytes was characterized using specific CYP inhibitors. Treatment of blood cultures with 25 microM diethyldithiocarbamate (DDC), a specific CYP2E1 inhibitor, or 0.5 microM quinidine, a specific CYP2D6 inhibitor, simultaneously with NNK, significantly decreased NNK-induced chromosome aberration. We also studied the role of CYP2E1 and CYP2D6 allelic variants on NNK-induced chromosome aberration. Our results indicate that NNK induced a significantly higher level of chromosome aberration in cells with the CYP2E1 WT/*5B genotype compared to cells with the CYP2E1 WT/WT. In contrast, no difference in NNK-induced chromosome aberration was observed between cells with the CYP2D6 extensive metabolizers compared to cells with the CYP2D6 poor metabolizer genotypes. These results underscore the important role of polymorphic metabolizing genes in influencing the genotoxic responses to environmental mutagens and provide support to the reported findings linking CYP2E1 polymorphism to smoking-related lung cancer.     

Inhibition of human cytochrome P450 2D6 (CYP2D6) by methadone.

1. In microsomes prepared from three human livers, methadone competitively inhibited the O-demethylation of dextromethorphan, a marker substrate for CYP2D6. The apparent Ki value of methadone ranged from 2.5 to 5 microM. 2. Two hundred and fifty-two (252) white Caucasians, including 210 unrelated healthy volunteers and 42 opiate abusers undergoing treatment with methadone were phenotyped using dextromethorphan as the marker drug. Although the frequency of poor metabolizers was similar in both groups, the extensive metabolizers among the opiate abusers tended to have higher O-demethylation metabolic ratios and to excrete less of the dose as dextromethorphan metabolites than control extensive metabolizer subjects. These data suggest inhibition of CYP2D6 by methadone in vivo as well. 3. Because methadone is widely used in the treatment of opiate abuse, inhibition of CYP2D6 activity in these patients might contribute to exaggerated response or unexpected toxicity from drugs that are substrates of this enzyme.     

CYP2D6 inhibition by selective serotonin reuptake inhibitors: analysis of achievable steady-state plasma concentrations and the effect of ultrarapid metabolism at CYP2D6

STUDY OBJECTIVE: To assess the correlation between plasma concentrations of four commonly administered selective serotonin reuptake inhibitors (SSRIs) and the magnitude of cytochrome P450 (CYP) 2D6 inhibition. DESIGN: Prospective analysis. SETTING: University-affiliated research laboratory. PATIENTS: Thirty-two healthy, drug-free volunteers. INTERVENTION: Subjects were randomized to four groups and received daily administration of either fluoxetine 60 mg (as a loading dose), fluvoxamine 100 mg, paroxetine 20 mg, or sertraline 100 mg for 8 days. MEASUREMENTS AND MAIN RESULTS: The urinary concentration ratio of dextromethorphan:dextrorphan (interpreted as an in vivo index of CYP2D6 activity) was determined for each subject before and after the 8 days of receiving SSRIs. Plasma SSRI trough concentrations were measured on days 6-9. The CYP2D6 genotype was determined in a subject with an undetectable paroxetine concentration. Inhibition of CYP2D6 correlated significantly with plasma concentrations of paroxetine and fluoxetine. In contrast, no significant correlations emerged between CYP2D6 inhibition and plasma concentrations of sertraline or fluvoxamine. The subject with an undetectable paroxetine concentration was found to carry at least three functional CYP2D6 genes. CONCLUSIONS: For paroxetine and fluoxetine, plasma concentrations and dosage strongly influence the magnitude of enzyme inhibition. The potential of paroxetine (a CYP2D6 substrate) as an inhibitor may be affected by the genotypes and metabolic capacities of individual 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.     

Inhibition of CYP2D6 activity by bupropion

The purpose of this study was to assess the effect of bupropion on cytochrome P450 2D6 (CYP2D6) activity. Twenty-one subjects completed this repeated-measures study in which dextromethorphan (30-mg oral dose) was administered to smokers at baseline and after 17 days of treatment with either bupropion sustained-release (150 mg twice daily) or matching placebo. Subjects quit smoking 3 days before the second dextromethorphan administration. To assess CYP2D6 activity, urinary dextromethorphan/dextrorphan metabolic ratios were calculated after an 8-hour urine collection. Thirteen subjects received bupropion, and 8 received placebo. In those receiving active medication, the dextromethorphan/dextrorphan ratio increased significantly at the second assessment relative to the first (0.012 +/- 0.012 vs. 0.418 +/- 0.302; P < 0.0004). No such change was observed in those randomized to placebo (0.009 +/- 0.010 vs. 0.017 +/- 0.015; P = NS). At baseline, all subjects were phenotypically extensive CYP2D6 metabolizers (metabolic ratio <0.3); after treatment, 6 of 13 subjects receiving bupropion, but none of those receiving placebo, had metabolic ratios consistent with poor CYP2D6 metabolizers. Bupropion is therefore a potent inhibitor of CYP2D6 activity, and care should be exercised when initiating or discontinuing bupropion use in patients taking drugs metabolized by CYP2D6.     

The effect of selective serotonin re-uptake inhibitors on cytochrome P4502D6 (CYP2D6) activity in human liver microsomes.

Inhibition of human cytochrome P4502D6 (CYP2D6)-catalysed metabolism can lead to clinically significant alterations in pharmacokinetics. Since there is evidence that the selective serotonin reuptake inhibitor (SSRI) class of antidepressant drugs might inhibit CYP2D6, the effects of five SSRIs on human liver microsomal CYP2D6 activity were compared with each other and with three tricyclic antidepressant drugs. On a molar basis, paroxetine was the most potent of the SSRIs at inhibiting the CYP2D6-catalysed oxidation of sparteine (Ki = 0.15 microM), although fluoxetine (0.60 microM) and sertaline (0.70 microM) had Ki values in the same range. Fluvoxamine (8.2 microM) and citalopram (5.1 microM) also inhibited CYP2D6 activity. The major circulating metabolites of paroxetine in man produced negligible inhibition. In contrast, norfluoxetine the active metabolite of fluoxetine, was a potent CYP2D6 inhibitor (0.43 microM). CYP2D6 activity was also diminished by the tricyclic antidepressant drugs clomipramine (2.2 microM), desipramine (2.3 microM) and amitriptyline (4.0 microM). These findings suggest that compounds with SSRI activity are likely to interact with human CYP2D6 in vivo with the potential of causing drug interactions.     

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.     

Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

Aim  The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. Method  The main polymorphisms on the cytochrome P450 (CYP) genes, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, and the multi-drug resistance 1 gene (MDR1) were genotyped in 78 healthy Vietnamese subjects. Pharmacokinetic metrics were available for CYP2A6 (coumarin), CYP2C19 (mephenytoin), CYP2D6 (metoprolol) and CYP3As (midazolam), allowing correlations with the determined genotype. Results  In the CYP2 family, we detected alleles CYP2A6*4 (12%) and *5 (15%); CYP2B6*4 (8%), *6 (27%); CYP2C19*2 (31%) and *3 (6%); CYP2D6*4, *5, *10 (1, 8 and 44%, respectively). In the CYP3A family, CYP3A4*1B was detected at a low frequency (2%), whereas CYP3A5 *3 was detected at a frequency of 67%. The MDR1 3435T allele was present with a prevalence of 40%. Allele proportions in our cohort were compared with those reported for other Asian populations. CYP2C19 genotypes were associated to the S-4′-OH-mephenytoin/S-mephenytoin ratio quantified in plasma 4 h after intake of 100 mg mephenytoin. While CYP2D6 genotypes were partially reflected by the α-OH-metroprolol/metoprolol ratio in plasma 4 h after dosing, no correlation existed between midazolam plasma concentrations 4 h post-dose and CYP3A genotypes. Conclusions  The Vietnamese subjects of our study cohort presented allele prevalences in drug-metabolising enzymes that were generally comparable with those reported in other Asian populations. Deviations were found for CYP2A6*4 compared to a Chinese population (12 vs. 5%, respectively; P = 0.023), CYP2A6*5 compared with a Korean population (15 vs. <1%, respectively; P < 0.0001), a Malaysian population (1%; P < 0.0001) and a Chinese population (1%; P < 0.0001); CYP2B6*6 compared with a Korean population (27 vs. 12%; P = 0.002) and a Japanese population (16%; P = 0.021). Pharmacokinetic metrics versus genotype analysis reinforces the view that the predictive value of certain globally common variants (e.g. CYP2D6 single nucleotide polymorphisms) should be evaluated in a population-specific manner.     

Interactions of bupranolol with the polymorphic debrisoquine/sparteine monooxygenase (CYP2D6)

Summary The -adrenoceptor blocker bupranolol turned out to be a competitive inhibitor of the polymorphic cytochrome P450 CYP2D6 of which sparteine is a substrate. There was stereo-selectivity of bupranolol involved: (–)-bupranolol was the weakest inhibitor with an apparent K_i value of 1.32 M, (+)-bupranolol was the most potent with an apparent Ki value of 0.55 M, while the therapeutically used racemic bupranolol had an intermediate value of 0.88 M. A 10 min pre-incubation of 5 M bupranolol with the enzyme preparation prior to the addition of substrate, reduced the inhibition of sparteine metabolism from 52 to about 25%.This suggests that — during these inhibition studies — bupranolol was much more rapidly metabolized than was sparteine, so that the measured K_i values must represent overestimates. The enzyme catalysing bupranolol metabolism was CYP2D6: microsomes from a liver with the genetic enzyme deficiency did not metabolize bupranolol; in microsomes from livers containing the enzyme and 10 M bupranolol, 5 M quinidine caused a 72% inhibition of bupranolol metabolism.Although our methods were not sufficiently sensitive to measure the K_m of bupranolol directly, it is undoubtedly the -adrenoceptor blocker with the highest-known apparent affinity for CYP2D6. High affinity and rapid metabolism are infrequent combinations in enzymology.     

The use of genetically engineered cells for assessing CYP2D6-related polymorphic effects.

As an example of advanced testing in the field of metabolism in an industrial environment, the introduction of some novel approaches, including the use of genetically engineered cell lines for assessing CYP 2D6-related polymorphic effects is illustrated. In this paper, it is demonstrated that novel in vitro test systems can be developed by using these genetically engineered cell lines for evaluating the potential risks associated with proprietary drugs (especially if their metabolism depends to a high extent on CYP 2D6). Moreover, it is demonstrated that, by the use of these in vitro methods, issues such as polymorphism, for which no animal models are available, can be assessed in such a way that predictions can be made on adverse effects which, up to now, could only be detected during clinical trials. Through the use of these new biotechnological in vitro metabolism models, clinically relevant data can be obtained for a scientifically-based human risk assessment, and animal use can be reduced.     

Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters K_m, V_max, and intrinsic clearance (V_max/K_m) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.     

Mechanism-based inactivation of CYP2D6 by methylenedioxymethamphetamine.

The potency of methylenedioxymethamphetamine (MDMA) as a mechanism-based inhibitor of CYP2D6 has been defined using microsomes prepared from yeast expressing the enzyme and from three human livers. The inhibitory effect was increased by preincubation through formation of a metabolic intermediate complex. Inactivation parameters (kinact and KI), defined with respect to the O-demethylation of dextromethorphan, were 0.29 +/- 0.03 (S.E.) min(-1) and 12.9 +/- 3.6 (S.E.) microM for yeast-expressed CYP2D6, and 0.26 +/- 0.02 min(-1) and 14.4 +/- 2.5 microM, 0.15 +/- 0.01 min(-1) and 8.8 +/- 2.6 microM, and 0.12 +/- 0.05 min(-1) and 45.3 +/- 32.1 microM for the liver microsomal preparations. The rate of inactivation of CYP2D6 by MDMA decreased when quinidine, a competitive inhibitor of CYP2D6, was added to the primary incubation mixture. However, inactivation was unaffected by the addition of glutathione. The results indicate that MDMA is a potent mechanism-based inhibitor of CYP2D6, with implications for understanding its in vivo disposition and drug interaction potential.     

CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.