Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Polymorphic drug oxidation in humans

Genetic polymorphisms in the oxidative metabolism of debrisoquine, mephenytoin, phenformin, sparteine, and tolbutamide have been discovered during recent years. Among these pharmacogenetic conditions, polymorphic oxidation of debrisoquine and sparteine has been intensively studied. Two phenotypes, the extensive (EM) and the poor (PM) metabolizers, have been observed in all populations so far investigated. The PM phenotype exhibits a grossly impaired or nearly absent capacity to metabolize these drugs. The incidence of the PM phenotype in European populations ranges from 5 to 9%. Pronounced variations in the incidence of the PM phenotype have been demonstrated among different ethnic groups. The metabolism of debrisoquine and sparteine is determined by two alleles at a single gene locus; PMs are homozygous for an autosomal recessive gene. Because of markedly impaired metabolism, the PM phenotype develops side effects if normal doses of debrisoquine and sparteine are administered. Defective metabolism in the PM phenotype is not restricted to debrisoquine and sparteine. Impaired metabolism of guanoxan , phenformin, perhexiline, methoxyamphetamine, phenacetin, encainide, metoprolol, alprenolol, bufuralol, nortriptyline, and desipramine have been described. As a consequence of impaired metabolism of these drugs, toxicity and therapeutic failure are observed in the PMs. With regard to molecular mechanisms, studies with microsomes from human liver provide evidence that in the PM phenotype a cytochrome P-450 isozyme is either missing or functionally inadequate.     

The AmpliChip CYP450 genotyping test: Integrating a new clinical tool

The AmpliChip CYP450 Test, which analyzes patient genotypes for cytochrome P450 (CYP) genes CYP2D6 and CYP2C19, is a major step toward introducing personalized prescribing into the clinical environment. Interest in adverse drug reactions (ADRs), the genetic revolution, and pharmacogenetics have converged with the introduction of this tool, which is anticipated to be the first of a new wave of such tools to follow over the next 5-10 years. The AmpliChip CYP450 Test is based on microarray technology, which combines hybridization in precise locations on a glass microarray and a fluorescent labeling system. It classifies individuals into two CYP2C19 phenotypes (extensive metabolizers [EMs] and poor metabolizers [PMs]) by testing three alleles, and into four CYP2D6 phenotypes (ultrarapid metabolizers [UMs], EMs, intermediate metabolizers [IMs], and PMs) by testing 27 alleles, including seven duplications. CYP2D6 is a metabolic enzyme with four activity levels (or phenotypes): UMs with unusually high activity; normal subjects, known as EMs; IMs with low activity; and PMs with no CYP2D6 activity (7% of Caucasians and 1-3% in other ethnic groups). Levels of evidence for the association between CYP2D6 PMs and ADRs are relatively reasonable and include systematic reviews of case-control studies of some typical antipsychotics and tricyclic antidepressants (TCAs). Evidence for other phenotypes is considerably more limited. The CYP2D6 PM phenotype may be associated with risperidone ADRs and discontinuation due to ADRs. Venlafaxine, aripiprazole, duloxetine, and atomoxetine are newer drugs metabolized by CYP2D6 but studies of the clinical relevance of CYP2D6 genotypes are needed. Non-psychiatric drugs metabolized by CYP2D6 include metoprolol, tamoxifen, and codeine-like drugs. CYP2C19 PMs (3-4% of Caucasians and African Americans, and 14-21% of Asians) may require dose adjustment for some TCAs, moclobemide, and citalopram. Other drugs metabolized by CYP2C19 are diazepam and omeprazole. The future of pharmacogenetics depends on the ability to overcome serious obstacles, including the difficulties of conducting and publishing studies in light of resistance from grant agencies, pharmaceutical companies, and some scientific reviewers. Assuming more studies are published, pharmacogenetic clinical applications may be compromised by economic factors and the lack of physician education. The combination of a US FDA-approved test, such as the AmpliChip CYP450 Test, and an FDA definition of CYP2D6 as a 'valid biomarker' makes CYP2D6 genotyping a prime candidate to be the first successful pharmacogenetic test in the clinical environment. One can use microarray technology to test for hundreds of single nucleotide polymorphisms (SNPs) but, taking into account the difficulties for single gene approaches such as CYP2D6, it is unlikely that very complex pharmacogenetic approaches will reach the clinical market in the next 5-10 years.     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene.

The debrisoquine-4-hydroxylase polymorphism is a genetic variation in oxidative drug metabolism characterized by two phenotypes, the extensive metabolizer (EM) and poor metabolizer (PM). Of the Caucasian populations of Europe and North America, 5%-10% are of the PM phenotype and are unable to metabolize debrisoquine and numerous other drugs. The defect is caused by several mutant alleles of the CYP2D6 gene, two of which are detected in about 70% of PMs. We have constructed a genomic library from lymphocyte DNA of an EM positively identified by pedigree analysis to be homozygous for the normal CYP2D6 allele. The normal CYP2D6 gene was isolated; was completely sequenced, including 1,531 and 3,522 bp of 5' and 3' flanking DNA, respectively; and was found to contain nine exons within 4,378 bp. Two other genes, designated CYP2D7 and CYP2D8P, were also cloned and sequenced. CYP2D8P contains several gene-disrupting insertions, deletions, and termination codons within its exons, indicating that this is a pseudogene. CYP2D7, which is just downstream of CYP2D8P, is apparently normal, except for the presence, in the first exon, of an insertion that disrupts the reading frame. A hypothesis is presented that the presence of a pseudogene within the CYP2D subfamily transfers detrimental mutations via gene conversions into the CYP2D6 gene, thus accounting for the high frequency of mutations observed in the CYP2D6 gene in humans.     

DNA haplotype dependency of debrisoquine 4-hydroxylase (CYP2D6) expression among extensive metabolisers

Deficient debrisoquine/sparteine type oxidation is inherited as an autosomal recessive trait. Of all Caucasians, 5–10% are poor metabolisers, due to the absence of cytochrome P4502D6. Extensive metabolisers (EMs) exhibit highly variable metabolic activity. We investigated the relationship between CYP2D6 activity and genotypes of the CYP2D locus in a large set of French Caucasian families. Genotypes concern both common mutations affecting the enzyme activity and linkedBamHI polymorphisms of the locus. We found, like other authors, that in EMs part of the heterogeneity is explained by a subgroup of individuals heterozygous for a mutant allele. However, a second level of heterogeneity was detected among individuals not carrying mutations, and this was related to a polymorphicBamHI-defined DNA haplotype. Different combinations of haplotypes are associated with differences in CYP2D6 metabolic activity. This finding might help to clarify the conflicting data on the relation between CYP2D6 activity and susceptibility to lung cancer.     

Genetic variations of S-mephenytoin 4'-hydroxylase (CYP2C19) in the Chinese population

Most of phenotyping studies have shown that Chinese populations have a higher incidence of poor metabolizers (PMs) of S-mephenytoin 4'-hydroxylation compared with populations of African and European descent. The present study was aimed at defining an exact population frequency of the genetic defect of S-mephenytoin 4'-hydroxylase (CYP2C19) in native and overseas Chinese healthy populations. All the related data were systematically summarized and re-analyzed using meta-analysis method, and consistency between phenotypic and genotypic frequencies of the PM was tested. A statistically significant homogeneity was across all 11 phenotyping studies (chi2 = 15.17, d.f. = 10; P > 0.05) and also across the remaining 4 genotyping studies (chi2 = 2.61, d.f. = 3; P > 0.05) except for a non-randomly selected population analysis. An approximate estimate of the PM phenotypic and genotypic frequencies was 13.6% (212 of 1555; 95% CI: 11.9%-15.3%) and 13.8% (79 of 573; 95%CI: 11.0%-16.6%), respectively. There was a good consistency between phenotyped and genotyped PM frequencies. The half of all genotyped EMs (50.3%, 276 of 549) were heterozygotes. The data estimate that 14% of Chinese would be homozygotes of CYP2C19 defective alleles, and that 176 million Chinese would be slow metabolizers of CYP2C19 substrates.     

Effect of clarithromycin on the enantioselective disposition of lansoprazole in relation to CYP2C19 genotypes

The aim of this study was to examine the effect of clarithromycin, a CYP3A4 inhibitor, on the enantioselective disposition of lansoprazole among three different CYP2C19 genotype groups in healthy Japanese subjects. These subjects included 6 each of homozygous extensive metabolizers (homEMs), heterozygous extensive metabolizers (hetEMs), and poor metabolizers (PMs). In the EMs of CYP2C19, clarithromycin markedly increased Cmax and the AUC0-infinity of (S)-lansoprazole and (S)-hydroxylansoprazole compared with those of the corresponding (R)-enantiomers. Clarithromycin significantly increased Cmax and the AUC0-infinity of (S)-lansoprazole in the homEMs by 110% and 115%, respectively, and in the hetEMs by 105% and 103%, respectively, compared with placebo. Furthermore, clarithromycin slightly prolonged the elimination half-life of (R)-lansoprazole in the homEMs and hetEMs but did not alter that of (S)-lansoprazole. In the of PMs CYP2C19, clarithromycin significantly increased Cmax and the AUC0-infinity and significantly prolonged the elimination half-lives of (R)- and (S)-lansoprazole by 51% and 49%, respectively. The present study suggests that there are significant drug interactions between (R)- or (S)-lansoprazole and clarithromycin in EMs by inhibiting the CYP3A4-catalyzed sulfoxidation primarily during the first pass, whereas in PMs, the overall metabolism of lansoprazole is inhibited.     

Polymorphic Cytochromes P450 and Drugs Used in Psychiatry

1. The cytochrome P450 monooxygenases, CYP2D6, CYP2C19, and CYP2C9, display polymorphism. CYP2D6 and CYP2C19 have been studied extensively, and despite their low abundance in the liver, they catalyze the metabolism of many drugs.2. CYP2D6 has numerous allelic variants, whereas CYP2C19 has only two. Most variants are translated into inactive, truncated protein or fail to express protein.3. CYP2C9 is expressed as the wild-type enzyme and has two variants, in each of which one amino acid residue has been replaced.4. The nucleotide base sequences of the cDNAs of the three polymorphic genes and their variants have been determined, and the proteins derived from these genes have been characterized.5. An absence of CYP2D6 and/or CYP2C19 in an individual produces a poor metabolizer (PM) of drugs that are substrates of these enzymes.6. When two drugs that are substrates for a polymorphic CYP enzyme are administered concomitantly, each will compete for that enzyme and competitively inhibit the metabolism of the other substrate. This can result in toxicity.7. Patients can be readily phenotyped or genotyped to determine their CYP2D6 or CYP2C19 enzymatic status. Poor metabolizers (PMs), extensive metabolizers (EMs), and ultrarapid metabolizers (URMs) can be identified.8. Numerous substrates and inhibitors of CYP2D6, CYP2C19, and CYP2C9 are identified.9. An individual's diet and age can influence CYP enzyme activity.10. CYP2D6 polymorphism has been associated with the risk of onset of various illnesses, including cancer, schizophrenia, Parkinson's disease, Alzheimer's disease, and epilepsy.     

Phenotype-genotype analysis of CYP2C19 in Colombian mestizo individuals

Background  

Omeprazole is metabolized by the hepatic cytochrome P450 (CYP) 2C19 enzyme to 5-hydroxyomeprazole. CYP2C19 exhibits genetic polymorphisms responsible for the presence of poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers (EMs). The defective mutations of the enzyme and their frequencies change between different ethnic groups; however, the polymorphism of the CYP2C19 gene has not been studied in Colombian mestizos. The aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population.     

Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA) and Its Phase I and II Metabolites in Humans

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (C_max) and area under the plasma concentration-time curve from 0 to 24 h (AUC₂₄) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the C_max and AUC₂₄ of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism.     

Miotic action of tramadol is determined by CYP2D6 genotype

Polymorphic CYP2D6 is the enzyme that activates the opioid analgesic tramadol by O-demethylation to its active metabolite O-demethyltramadol (M1). Our objective was to determine the opioid effects measured by pupillary response to tramadol of CYP2D6 genotyped volunteers in relation to the disposition of tramadol and M1 in plasma. Tramadol displayed phenotypic pharmacokinetics and it was possible to identify poor metabolizers (PM) with >99% confidence from the metabolic ratio (MR) in a single blood sample taken between 2.5 and 24 h post-dose. Homozygous extensive metabolizers (EM) differed from PM subjects by an almost threefold greater (P=0.0014) maximal pupillary constriction (Emax). Significant correlations between the AUC and Cmax values of M1 versus pupillary constriction were found. The corresponding correlations of pharmacokinetic parameters for tramadol itself were weaker and negative. The strongest correlations were for the single-point metabolic ratios at all sampling intervals versus the effects, with rs ranging from 0.85 to 0.89 (p<0.01). It is concluded that the concept of dual opioid/non-opioid action of the drug, though considerably stronger in EMs, is valid for both EM and PM subjects. This is the theoretical basis for the frequent use and satisfactory efficacy of tramadol in clinical practice when given to genetically non-selected population.     

Pharmacogenetic basis for therapeutic optimization in Alzheimer's disease

Alzheimer's disease is a major health problem in developed countries. Approximately 10-15% of direct costs in dementia are attributed to pharmacological treatment, and only 10-20% of the patients are moderate responders to conventional antidementia drugs, with questionable cost effectiveness. The phenotypic expression of Alzheimer's disease is characterized by amyloid deposition in brain tissue and vessels (amyloid angiopathy), intracellular neurofibrillary tangle formation, synaptic and dendritic loss, and premature neuronal death. Primary pathogenic events underlying this neurodegenerative process include genetic factors involving more than 200 different genes distributed across the human genome, accompanied by progressive cerebrovascular dysfunction, and diverse environmental factors. Mutations in genes directly associated with the amyloid cascade (APP, PSEN1, PSEN2) are present in less than 5% of the Alzheimer's disease population; however, the presence of the epsilon4 allele of the apolipoprotein E gene (APOE) represents a major risk factor for more than 40% of patients with dementia. Genotype-phenotype correlation studies and functional genomics studies have revealed the association of specific mutations in primary loci and/or APOE-related polymorphic variants with the phenotypic expression of biological traits. It is estimated that genetics accounts for between 20% and 95% of the variability in drug disposition and pharmacodynamics. Recent studies indicate that the therapeutic response in Alzheimer's disease is genotype specific, depending on genes associated with Alzheimer's disease pathogenesis and/or genes responsible for drug metabolism (e.g. cytochrome P450 [CYP] genes). In monogenic studies, APOEepsilon4/epsilon4 genotype carriers are the worst responders to conventional treatments. Some cholinesterase inhibitors currently being use in the treatment of Alzheimer's disease are metabolized via CYP-related enzymes. These drugs can interact with many other drugs that are substrates, inhibitors or inducers of the CYP system, this interaction eliciting liver toxicity and other adverse drug reactions. CYP2D6 enzyme isoforms are involved in the metabolism of more than 20% of drugs used in CNS disorders. The distribution of the CYP2D6 genotypes in the European population of the Iberian peninsula differentiates four major categories of CYP2D6-related metabolizer types: (i) extensive metabolizers (EM) [51.61%]; (ii) intermediate metabolizers (IM) [32.26%]; (iii) poor metabolizers (PM) [9.03%]; and (iv) ultra-rapid metabolizers (UM) [7.10%]. PMs and UMs tend to show higher transaminase activity than EMs and IMs. EMs and IMs are the best responders, and PMs and UMs are the worst responders to pharmacologic treatments in Alzheimer's disease. At this early stage of the development of pharmacogenomic/pharmacogenetic procedures in Alzheimer's disease therapeutics, it seems very plausible that the pharmacogenetic response in Alzheimer's disease depends on the interaction of genes involved in drug metabolism and genes associated with Alzheimer's disease pathogenesis.     

104名藏族志愿者中细胞色素P450 2C19m1的基因多态性

细胞色素P450 2C19（CYP2C19）参与临床上许多重要药物的代谢。根据其代谢S－美芬妥英或其他CYP2C19底物的能力不同,有强代谢者（EMs）和弱代谢者（PMs）之分。PMs表型的频发率存在明显的种族差异。在本文中,我们主要报道了细胞色素P450 2C19 m1在中国藏族人群中的多态性分布。在104例无血缘关系藏族人群中,49人（47.1%）为CYP2C19野生型纯合子（wt/wt）,46人（44.2%）为CYP2C19m1杂合子（wt/m1）,9人（8.7%）为CYP2C19m1突变型纯合子（m1/m1）。CYP2C19野生型等位基因频率为0.692,CYP2C19m1等位基因频率为0.308。该结果与国内外报道的中国其余民族的CYP2C19m1等位基因频率相比具有一定可比性。 关键词：细胞色素P450 2C19;基因型;藏族;聚合酶链反应;限制性内切核酸酶片段长度多态（RFLP）Abstract: Cytochrome P 450 2C19（CYP2C19） is involved in the metabolism of a number of clinically used drugs. Individuals can be characterized as extensive metabolizers （EMs）or poor metabolizers（PMs）, according to the drugs-metabolized ability of CYP2C19 in population studies. The incidence of poor metabolizer phenotype shows marked interracial differences. In this article we report the gene polymorphism of CYP2C19 in Zang population. There were 49 wild-type homozygotes（wt/wt）, 46 were heterozygotes（wt/m1） and 9 were homozygotes（m1/m1） among 104 unrelated Zang subjects. The frequency of CYP2C19m1 allele was 0.308, which was in agreement with that in other published data.     

Development of a PCR-based strategy for CYP2D6 genotyping including gene multiplication of worldwide potential use

There is growing consensus on the potential use of pharmacogenetics in clinical practice, and hopes have been expressed for application to the improvement of global health. However, two major challenges may lead to widening the "biotechnological gap" between the developing and the industrial world;first the unaffordability of some current technologies for poorer countries, and second the necessity of analyzing all described alleles for every clinical case due to the inability to predict the ethnic group of a given patient. Because of its role in the metabolism of a number of drugs, cytochrome P450 2D6 (CYP2D6) is an excellent candidate for use in the optimization of drug therapy. CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles, and subjects can be classified as poor metabolizers (PM), extensive metabolizers (EM), or ultrarapid metabolizers (UM) of a given CYP2D6 substrate. Several strategies and methods for CYP2D6 genotyping exist. Some, however, are expensive and laborious. The aim of this study was to design a PCR-based genotyping methodology to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes for routine clinical use, independent of the individual's ethnic group. CYP2D6 is amplified in initial extra long PCRs (XL-PCRs), which subsequently undergo fragment-length polymorphism analysis for the determination of carriers of CYP2D6 allelic variants. The same XL-PCRs are also used for the determination of CYP2D6 multiplication and 2D6*5 allele (abolished activity). The application of this new strategy for the detection of CYP2D6 mutated alleles and multiplications to routine clinical analysis will enable the PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals at most locations.     

Enantioselectivity of debrisoquine 4-hydroxylation in Brazilian Caucasian hypertensive patients phenotyped as extensive metabolizers

Debrisoquine (D), an antihypertensive drug metabolized to 4-hydroxydebrisoquine (4-OHD) by CYP2D6, is commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as β-adrenergic blockers, tricyclic antidepressants, and class 1C antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-OHD or S-(+) and R-(−)-4-OHD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-(−)- and S-(+)-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-OHD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-OHD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-OHD/% of dose excreted as D+4-OHD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5–acetonitrile (9:1, v/v) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-OHD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-(+)-4-OHD and R-(−)-4-OHD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5–acetonitrile–methanol (85:12:3, v/v/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter-day RSDs were less than 10% for each enantiomer. A high degree of enantioselectivity in the 4-hydroxylation of D favouring the S-(+) enantiomer was observed, resulting in R-(−)-4-OHD not detected in the urine of the EM hypertensive patients studied.     

Frequency of single nucleotide polymorphisms of CYP2D6 in the Czech population

CYP2D6 is a member of cytochrome P450 enzymes that metabolise over 25% of commonly used drugs. Genetic polymorphisms can cause insufficient drug efficacy at usually administered doses or can be the cause of adverse drug reaction. CYP2D6 genotyping can be used to predict CYP2D6 phenotype and thereby explain some abnormalities in drug response and thus optimize pharmacotherapy. The aim of this study was to investigate the frequency of functionally important variant alleles of the CYP2D6 gene throughout the Czech population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes. The DNA of 223 unrelated, healthy volunteers was analysed to detect the presence of CYP2D6*6, *5, *4, *3 and gene duplication. The variant allele frequencies in our population were 0.22%, 3.14%, 22.87%, 1.12% and 3.14% for CYP2D6*6, CYP2D6*5, CYP2D6*4, CYP2D6*3 and CYP2D6*MxN, respectively. Fifteen subjects carried two variant alleles leading to predicted poor type of metabolism, 84 subjects were heterozygous extensive metabolizers (het-EM). The full-text contains detailed comparison with European white populations. The distribution of variant alleles complies with the Hardy-Weinberg equilibrium. The frequencies of functional variant alleles of CYP2D6 in Czech population are in concordance with other Caucasian populations.     

Paroxetine-induced conversion of cytochrome P450 2D6 phenotype and occurence of adverse effects

The paper is focused on a comparison of the distribution of side effects of treatment with paroxetine within a group of 30 patients genotyped and phenotyped for their CYP 2D6 metabolic status. Genotyping procedure showed that the patient group did not include any individual with poor metabolizer (PM) genotype; on the other hand, most patients (24) were classified as PMs by virtue of their phenotype, which suggests that a conversion to the poor metabolic phenotype ("phenocopy") occurred, probably as a consequence of a long-term administration of the strong CYP 2D6 inhibitor paroxetine. As to the occurence of common adverse effects, no marked difference between subjects converted into the PM group and those who had no history of such conversion was found. A significantly higher incidence of sexual dysfunction (p < 0.05) was, nevertheless, recorded in patients with the PM phenotype. The results of the study may provide evidence that it is the metabolic phenotype status, rather than the genetically given enzyme capacity (CYP 2D6 genotype), that is relevant for the actual toleration of treatment with CYP 2D6 inhibitors.