Poor Metabolizer Phenotype Research Articles

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.

CYP2D6 genotype and tamoxifen response

We read with interest the article by Wegman and coworkers [1] entitled 'Genotype of metabolic enzymes and the benefit of tamoxifen in postmenopausal breast cancer patients'. The authors conducted a retrospective analysis of a tamoxifen adjuvant clinical trial and reported that a small subset of patients with estrogen receptor (ER)-positive breast cancer treated with tamoxifen who carried the CYP2D6*4 variant allele did significantly better than patients with the same variants not treated with tamoxifen. We believe that these data are actually not in contrast to data presented by us and others, as the authors suggest. Rather, we believe that alternative interpretations of the data may shed a different light on their conclusions. First, it is important to understand the patients included in this study. They began with a subset of patients (226/679 [33%]) treated in an adjuvant tamoxifen trial designed to compare the effects of tamoxifen (40 mg/day for 2 years) alone or with chemotherapy, and with or without radiation. Of these, only 112 patients received tamoxifen and only 71 were ER positive. Thus, only 10.5% of patients in the overall trial (71/679) were included in this small substudy, meaning that there is great potential for selection bias. Initially, the authors did not find a difference in outcome by CYP2D6*4 genotype, and therefore they compared the outcomes in patients with at least one CYP2D6*4 allele who received tamoxifen (n = 24) versus no tamoxifen (n = 23). They found that patients with the CYP2D6*4 genotype did significantly better than patients not treated with tamoxifen and observed a similar trend in women with the SULT1A1*1 homozygous genotype. They concluded that genotyping of these enzymes may be useful in predicting clinical outcomes and that their findings were in contrast to the current hypotheses on the role of metabolic enzymes in tamoxifen activation and inactivation. We believe the data presented are consistent with our results, which suggest that CYP2D6 poor metabolizers may experience less benefit from tamoxifen treatment [2]. It is expected that ER-positive women treated with tamoxifen will have better outcomes than ER-positive women treated without tamoxifen, even if they have one CYP2D6*4 allele, because this group would not completely eliminate the benefits of tamoxifen. Indeed, it appears that the data presented by Wegman and coworkers are consistent with this hypothesis, albeit the numbers are very small because of the considerable subsetting and selection. Our studies have shown that the serum concentration of an active metabolite of tamoxifen, namely endoxifen, is effected by CYP2D6 status in a gene dose dependent manner, with low, intermediate and high concentrations in homozygous variant, heterozygous and homozygous wild-type patients, respectively [3,4]. Furthermore, data from a large randomized trial of postmenopausal women receiving adjuvant tamoxifen for 5 years [2] indicate that women who are homozygous (but not heterozygous) for the CYP2D6*4 allele have significantly worse relapse-free survival and disease-free survivals than do women who are heterozygous or homozygous for the common allele. These findings suggest that the outcome of women with 'intermediate' concentrations of endoxifen, as represented by the heterozygous CYP2D6*4, are no different than women who carry two wild-type CYP2D6 alleles. This suggests that the clinical effect of tamoxifen is due to multiple metabolites, and only when women carry two null CYP2D6 alleles, corresponding to the CYP2D6 poor metabolizer phenotype, do outcomes deteriorate.

CYP2D6 and CYP2C19 genotypes and amitriptyline metabolite ratios in a series of medicolegal autopsies

In a series of 202 postmortem toxicology cases, the CYP2D6 and CYP2C19 genes were genotyped, and the concentrations of amitriptyline (AT) and six metabolites were analyzed. The polymorphic CYP2D6 and CYP2C19 genes encode enzymes participating in the metabolism of several potentially toxic drugs, and mutations in these genes may lead to adverse drug reactions, possibly even intoxications. AT was chosen as the substrate of interest because it is mainly metabolized by these enzymes, is considered relatively toxic, and ranks among the major causes of fatal drug poisoning in Finland. Our objective was to evaluate genetically determined interindividual variation in conjunction with metabolite ratios of drugs found in toxicological analysis in a series of medicolegal autopsies. Positive correlations were found between the proportion of trans-hydroxylated metabolites and the number of functional copies of CYP2D6 and between the proportion of demethylated metabolites and the number of functional copies of CYP2C19. None of the accidental or undetermined AT poisonings coincided with the CYP2D6 or CYP2C19 genotype which predicts a poor metabolizer phenotype. However, an unusually high femoral blood concentration of AT, 60 mg/l, was found in one suicide case with no functional CYP2D6 genes. Our study shows a concordance of AT metabolite patterns with CYP2D6 and CYP2C19 genotypes in the presence of confounding factors typical for postmortem material. This result demonstrates the feasibility of postmortem pharmacogenetic analysis and supports the dominant role of genes in drug metabolism.

Direct analysis of unphased SNP genotype data in population-based association studies via Bayesian partition modelling of haplotypes

We describe a novel method for assessing the strength of disease association with single nucleotide polymorphisms (SNPs) in a candidate gene or small candidate region, and for estimating the corresponding haplotype relative risks of disease, using unphased genotype data directly. We begin by estimating the relative frequencies of haplotypes consistent with observed SNP genotypes. Under the Bayesian partition model, we specify cluster centres from this set of consistent SNP haplotypes. The remaining haplotypes are then assigned to the cluster with the "nearest" centre, where distance is defined in terms of SNP allele matches. Within a logistic regression modelling framework, each haplotype within a cluster is assigned the same disease risk, reducing the number of parameters required. Uncertainty in phase assignment is addressed by considering all possible haplotype configurations consistent with each unphased genotype, weighted in the logistic regression likelihood by their probabilities, calculated according to the estimated relative haplotype frequencies. We develop a Markov chain Monte Carlo algorithm to sample over the space of haplotype clusters and corresponding disease risks, allowing for covariates that might include environmental risk factors or polygenic effects. Application of the algorithm to SNP genotype data in an 890-kb region flanking the CYP2D6 gene illustrates that we can identify clusters of haplotypes with similar risk of poor drug metaboliser (PDM) phenotype, and can distinguish PDM cases carrying different high-risk variants. Further, the results of a detailed simulation study suggest that we can identify positive evidence of association for moderate relative disease risks with a sample of 1,000 cases and 1,000 controls.

Identification and characterization of novel sequence variations in the cytochrome P4502D6 (CYP2D6) gene in African Americans

Cytochrome P4502D6 (CYP2D6) genotyping reliably predicts poor metabolizer phenotype in Caucasians, but is less accurate in African Americans. To evaluate discordance we have observed in phenotype to genotype correlation studies, select African American subjects were chosen for complete resequencing of the CYP2D6 gene including 4.2 kb of the CYP2D7-2D6 intergenic region. Comparisons were made to a CYP2D6(*)1 reference sequence revealing novel SNPs in the upstream, coding and intervening sequences. These sequence variations, defining four functional alleles (CYP2D6(*)41B, (*)45A and B and (*)46), were characterized for their ability to influence splice site strength, transcription level or catalytic protein activity. Furthermore, their frequency was determined in a population of 251 African Americans. A -692(TGTG) deletion (CYP2D6(*)45B) did not significantly decrease gene expression, nor could any other upstream SNP explain a genotype-discordant case. CYP2D6(*)45 and (*)46 have a combined frequency of 4% and can be identified by a common SNP. Carriers are predicted to exhibit an extensive or intermediate CYP2D6 phenotype.

Evidence for a combined genetic effect of the 5-HT1A receptor and serotonin transporter genes in the clinical outcome of major depressive patients treated with citalopram

In the context of a long-term follow-up study, we analysed the possible implication of the 5-HT(1A) receptor gene (HTR1A) -1018C/G polymorphism in the clinical outcome of major depressive patients treated with citalopram. We had previously reported an association between variation on the SERT gene (SLC6A4) and clinical remission after citalopram treatment. In the present 12-week follow-up study, the combined effect of HTR1A and SLC6A4 genes in clinical outcome and response to citalopram was also evaluated. The sample consisted of 130 patients, all of Spanish origin, who were diagnosed as having a current major depressive episode according to DSM-IV criteria. A 21-item Hamilton Depression Rating Scale was used to assess severity of symptoms at the beginning and during the follow-up to determine the outcome and remission status at week 12. Patients were genotyped for HTR1A gene and, in addition, for two polymorphisms at the CYP2C19 gene, which together account for the 87% of the Caucasian poor metabolizer phenotype. Data were analysed adjusting for the effect of poor metabolizers in clinical response. No independent effect was found for the 5-HT(1A) receptor gene in relation to clinical outcome or remission after citalopram treatment. However, a combined genetic effect of HTR1A and SLC6A4 genes was found to influence the clinical outcome of patients [F(4,102) = 2.89, p= 0.02]. When considering the remission status, an increase of patients carrying the risk genotype combination (S/S-G/G) was found among those subjects who did not reach remission (Fisher's exact test = 0.009). Our results suggest that the combined effect of the serotonin transporter and the 5-HT(1A) receptor genes could be related to the clinical outcome of depressive patients treated with citalopram.

Functional analysis of CYP2D6.31 variant: Homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.

The effect of the CYP2C19 genotype on the hydroxylation index of omeprazole in South Indians

To investigate the relationship between CYP2C19 genotypes and the hydroxylation index (HI) of omeprazole in the South Indian population. Healthy unrelated South Indian subjects (n=300) were separated into three groups based on their CYP2C19 genotypes. They were administered a single oral dose of 20 mg omeprazole, and venous blood was collected 3 h later. Plasma was assayed using reversed-phase high-performance liquid chromatography, and the omeprazole HI was calculated. The means of HIs in individuals with CYP2C19*1/*1 (n=124), *1/*2 (n=129) and *2/*2,*2/*3 (n=47) were 2.4, 5.3 and 22.5, respectively, and were found to be significantly different between any two groups (P<0.0001). A good correlation was established between CYP2C19 genotype and omeprazole HI (r=0.54, 95% CI 0.45-0.62; P<0.0001). Of the 300 subjects, 42 (14.0%; 95% CI 10.1-17.9) were phenotypic poor metabolizers (PMs), but only 33 of them had two mutant alleles and the remaining 9 PMs had at least one wild-type allele. Among the 258 extensive metabolizers, 14 had two mutant alleles. The prevalence of PMs in the South Indian population was 14.0%, which is similar to that in North Indians and Orientals but significantly higher than in Caucasians and Africans. A genotype-phenotype relationship was established between the CYP2C19 genotype and HI of omeprazole, but 7.7% of subjects deviated from expected genotype-phenotype associations. This could be due to an additional mutation, either in the exons/introns or in the 5'-regulatory region of the CYP2C19 gene.

The CYP2D6 Poor Metabolizer Phenotype May Be Associated With Risperidone Adverse Drug Reactions and Discontinuation

The cytochrome P450 2D6 (CYP2D6) enzyme metabolizes risperidone. CYP2D6 poor metabolizers have no CYP2D6 activity (7% of whites and 1%-2% of other races). This study tested whether the CYP2D6 poor metabolizer phenotype was associated with adverse drug reactions (ADRs) and discontinuation due to ADRs. Adult inpatients and outpatients were recruited from July 2000 to March 2003 including (1) 325 who were stabilized on risperidone therapy and classified as either expressing moderate-to-marked ADRs (22%, 73/325) or not (78%, 252/325) and (2) 212 who discontinued risperidone and were classified as discontinued due to ADRs (38%, 81/212) or for other reasons (62%, 131/212). Genetic tests were performed by allele-specific polymerase chain reaction and/or by the AmpliChip CYP450 microarray system for up to 34 separate CYP2D6 alleles. Two logistic regression models with dependent variables (moderate-to-marked ADRs while taking risperidone and risperidone discontinuation due to ADRs) were evaluated with respect to the CYP2D6 phenotype. The odds ratios (ORs) and 95% confidence intervals (CIs) for the CYP2D6 poor metabolizer phenotype in the univariate analyses and after correcting for clinical variables were (1) OR = 3.1 (CI = 1.4 to 7.0) and 3.4 (CI = 1.5 to 8.0) for moderate-to-marked ADRs on risperidone and (2) OR = 3.0 (CI = 0.85 to 10.6) and 6.0 (CI = 1.4 to 25.4) for discontinuation due to ADRs. The CYP2D6 poor metabolizer phenotype appears to be associated with risperidone ADRs and discontinuation due to ADRs; however, this finding requires further study in larger patient populations. The CYP3A5 and p-glycoprotein exon 21 and 26 genotypes were not significantly associated with risperidone response.

Pharmacokinetics of Escalating Doses of Intravenous Repinotan in Healthy Male Volunteers

To investigate the pharmacokinetics of intravenous (IV) repinotan, a potent selective full serotonin (5-HT(1A)) receptor agonist, after administration of escalating doses/infusion rates to healthy volunteers with extensive or poor metaboliser phenotype, and to compare weight-adjusted (mg/kg) to fixed (mg/day) dosing regimens. SUBJECTS AND M ethods: The pharmacokinetic profile of IV repinotan was evaluated in healthy male volunteers. In eight studies, extensive metabolisers (EMs) and poor metabolisers (PMs) identified by sparteine phenotyping were given IV repinotan at doses of 5-100microg over 1 hour or 200-1000microg over 4 hours. In two additional studies of EMs and PMs, IV repinotan was administered as a weight-adjusted dose of 1 microg/kg/h for 24 hours. Repinotan plasma concentrations were measured for determination of non-compartmental pharmacokinetic parameters. Sixty-five male subjects (54 EMs and 11 PMs) were valid for pharmacokinetic evaluation. During continuous IV infusion, a steady-state repinotan plasma level was reached within 4-5 hours in EMs, and the elimination half-life was 1 hour. Pharmacokinetics were linear in EMs across a wide range of doses (5-2250microg) and infusion rates (5-250 microg/h). Repinotan clearance was correlated with the log metabolic ratio (MR) of sparteine as an indicator for cytochrome P450 (CYP)2D6 phenotype, and subjects phenotyped as PMs had reduced clearance, resulting in longer elimination half-lives (up to a mean value of 11 hours) and increased peak plasma concentrations and area under the concentration-time curve values. Bodyweight was not a significant covariable for clearance and the interindividual variability in clearance was not reduced after normalisation to bodyweight. Repinotan was well tolerated at infusion rates up to 150 microg/h (EMs), and no clinically relevant safety issues were reported. Repinotan demonstrates linear pharmacokinetics in EMs for CYP2D6, who constitute about 93% of the Caucasian population. Repinotan clearance is correlated with CYP2D6 phenotype and reduced in PMs. Bodyweight does not appear to be a major determinant of its pharmacokinetic variability and a bodyweight-adjusted dosing regimen is not warranted. Further studies should be performed in patients to investigate whether the tolerability profile of repinotan is comparable to that observed in healthy volunteers.

A new simple diagnostic assay for the identification of the major CYP2D6 genotypes by DNA sequencing analysis

To establish a method suitable for diagnostic genotyping of CYP2D6 alleles by DNA sequencing. Initial PCR reactions were performed to specifically amplify exons 3, 4, 5 and 6 of the CYP2D6 gene using primers previously published. New primers were used to identify *2, *3, *4, *6, *7, *8, *9 and *41 in 2 sequencing reactions. Additional primers were designed for reverse sequencing in samples with 1 or 3 b.p. deletions. Previously published assays were used to detect *5, *10 and *16 alleles to complete genotype assignment. We reliably detected the nonfunctional alleles, *3, *4, *6, *7 and *8, which are associated with the poor metabolizer phenotype, and 2 important alleles associated with decreased enzyme activity, *9 and *41. Observed allele frequencies were comparable to those found previously in Caucasian populations. CYP2D6 genotype has been shown in previous clinical studies to be a good predictor of CYP2D6 phenotype and, therefore, related to therapeutic response and the risk of drug toxicity. This genotyping method is simple and reliable, and, therefore, can be routinely performed on an isolated patient sample, providing a relatively quick turnaround time needed for clinical practice. In addition, the simultaneous drawing of blood with the commencement of drug therapy will allow dosage adjustment on the basis of the CYP2D6 genotype to reduce the risk of adverse drug reactions.

Assessment of In Vivo CYP2D6 Activity: Differential Sensitivity of Commonly Used Probes to Urine pH

Drug/metabolite ratios (MRs) are used as in vivo markers of enzyme activity. The ratios are potentially confounded by the renal clearance of the drug (urine-based MRs) or metabolite (plasma-based MRs). The authors have investigated the relative sensitivity of urinary MR of 3 in vivo probe substrates of CYP2D6 debrisoquine (DB), dextromethorphan (DM), and metoprolol (MP) to changes in urine pH. Three groups of healthy volunteers each comprising 12 individuals were given DB (10 mg), DM (25 mg), or MP (100 mg) on 3 occasions. In 1 study arm, urine was acidified by the oral intake of ammonium chloride; in another, it was alkalinized by intake of sodium bicarbonate; and in the third, urine pH was uncontrolled. Urinary MP/alpha-hydroxy-MP, DM/dextrorphan, and DB/4-hydroxy-DB ratios were calculated. The mean(geo) MR for DB was not significantly different in any of the study arms, whereas those for MP and DM were significantly different under acidified and alkalinized urine conditions compared to uncontrolled urine pH (P < .01) and were correlated with urine pH (P < .001). Without control of urine pH, in vivo estimates of CYP2D6 metabolic activity are likely to be less precise using DM or MP as probe substrates compared to DB. Although this is unlikely to cause any problem in distinguishing the large functional differences in CYP2D6 in poor metabolizer (PM) and extensive metabolizer (EM) phenotypes, this may contribute to difficulties in differentiating in vivo metabolic activity among allelic variants within the overall CYP2D6 EM phenotype using MP or DM. However, because DB is not available in many countries (eg, United States), alternative in vivo markers of CYP2D6 with low sensitivity to urine pH should be sought.

Identification of non-functional allelic variant of CYP1A2 in dogs

Recently, we reported that AC-3933, a novel cognitive enhancer, is polymorphically hydroxylated in beagle dogs and that dogs could be phenotyped as extensive metabolizers (EM) or poor metabolizers (PM). AC-3933 polymorphic hydroxylation is caused by polymorphic expression of CYP1A2 protein in dog liver. In order to clarify the mechanism of polymorphic expression of CYP1A2 protein in beagle dogs, we investigated, in this study, the sequence of CYP1A2 cDNA in EM and PM dogs. In PM dogs CYP1A2 gene, we discovered a nonsense mutation (C1117T) that induces a premature termination, and is associated with PM phenotype for AC-3933 hydroxylation. All PM dogs studied were homozygote of the mutant allele (m/m) and seemed to be CYP1A2-null phenotype as they lacked the heme-binding region in CYP1A2. These results indicate that the polymorphic expression of CYP1A2 protein observed in our previous study is caused by a single nucleotide polymorphism on CYP1A2 coding region. Furthermore, we developed a genotyping method for the mutant allele using a mismatch PCR-restriction fragment length polymorphism, and carried out frequency analysis in 149 beagle dogs. Our results indicate that more than 10% of the dogs studied were CYP1A2-null. Because CYP1A2-null phenotype in dogs affects the results of pharmacokinetic, toxicological and pharmacological studies of drug candidates, these findings are important in the pharmaceutical and the veterinary fields.

Drug development and use in the elderly: search for the right dose and dosing regimen

Across the globe over the last few decades, there has been a remarkable increase in the proportion of the population which is elderly, and this trend is set to continue. Data from 10 countries that accounted for about 55% of the world population of 6079 million in the year 2000 provide an overview of this significant shift in demography. Table 1 summarizes the projections for the next 25 years. Hitherto, increases in life expectancy have been most obvious in the affluent countries; however, improvements in public health and disease control are also leading to increases in life expectancy in the less affluent countries. As a result of relatively larger increases in life expectancy at birth, significant shifts in demography are also expected in countries that are at present economically less affluent. The number of elderly people in the world is therefore expected to continue to increase for some considerable time. Interestingly, the most dramatic increases in the proportion of the population who are elderly are anticipated in countries where total populations are projected to decrease (such as Japan, Germany and Italy). Table 1 Age-related demography in 2000 and projected changes in 10 major countries The 2001 population census of the UK revealed that since the census of 1951, the proportion of elderly population above 65 years and 85 years has increased from 16% and 0.4% respectively to 21% and 1.9%, respectively. These changes in demography will have significant effects on the economics generally as well as on the provisions for healthcare. Cardiovascular and neurological diseases and cancers are the most prevalent in the elderly. These three groups of diseases account for about 54% of the total burden of disease in Europe in terms of disability adjusted life years. Of the total gross Hospital and Community Health Services expenditure of £31.9 billion in the year 2001–2002 in the UK, 13% was expended on people aged 65–74 years, 16% on those aged 75–84 years and 10% for people aged 85 years or more. Furthermore, about 55% of the community prescriptions during 2001 in the UK were dispensed for the elderly. Cardiovascular and psychoactive drugs accounted for more than 40% of these prescriptions. In view of many age-related changes in the pharmacokinetics and pharmacodynamics of a drug, the safe and effective prescribing of medicines in the elderly will continue to present a major challenge. This review provides an overview of the issues relevant to development and clinical use of drugs in the elderly population, with particular reference to determining the right dosage and dose regimen and the regulatory requirements that facilitate this process. It also examines whether, as a result of advancing age per se, the dosing regimens in the elderly and the frail elderly might be different from those in the non-elderly.

Toward individualized pharmaceutical care of East Asians: the value of genetic testing for polymorphisms in drug-metabolizing genes.

Research into the relationship between genetics and drug response has focused on polymorphisms in genes that encode drug-metabolizing enzymes, particularly the genes of cytochrome P450 superfamily 2, which affect the clearance of the anticoagulant warfarin, proton pump inhibitors, tricyclic antidepressants, and many other clinically relevant drugs. Much of this work has targeted East Asians, a genetically distinguishable and populous group. Researchers have identified polymorphisms that inactivate gene function, compared polymorphism frequencies in East-Asian and Caucasian populations, and determined the effects on the pharmacokinetic parameters of drugs. Detection in an individual of polymorphisms known to inactivate a drug-metabolizing enzyme is predictive of poor metabolism of drugs processed by that pathway, which itself may be predictive of an atypical drug response. Genetic tests can be used to screen for individuals with poor metabolizer phenotypes, with the ultimate goal of predicting the clinical effects of drugs.

Human cytochrome p450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1'-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1'-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1'-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.

Fenproporex N-dealkylation to amphetamine—enantioselective in vitro studies in human liver microsomes as well as enantioselective in vivo studies in Wistar and Dark Agouti rats

Fenproporex (FP) is known to be N-dealkylated to R(−)-amphetamine (AM) and S(+)-amphetamine. Involvement of the polymorphic cytochrome P450 (CYP) isoform CYP2D6 in metabolism of such amphetamine precursors is discussed controversially in literature. In this study, the human hepatic CYPs involved in FP dealkylation were identified using recombinant CYPs and human liver microsomes (HLM). These studies revealed that not only CYP2D6 but also CYP1A2, CYP2B6 and CYP3A4 catalyzed this metabolic reaction for both enantiomers with slight preference for the S(+)-enantiomer. Formation of amphetamine was not significantly changed by quinidine and was not different in poor metabolizer HLM compared to pooled HLM. As in vivo experiments, blood levels of R(−)-amphetamine and S(+)-amphetamine formed after administration of FP were determined in female Dark Agouti rats (fDA), a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats (mDA), an intermediate model, and in male Wistar rats (WI), a model of the human CYP2D6 extensive metabolizer phenotype. Analysis of the plasma samples showed that fDA exhibited significantly higher plasma levels of both amphetamine enantiomers compared to those of WI. Corresponding plasma levels in mDA were between those in fDA and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher amphetamine plasma levels, which did not significantly differ from those in fDA. The in vivo studies suggested that CYP2D6 is not crucial to the N-dealkylation but to another metabolic step, most probably to the ring hydroxylation. Further studies are necessary for elucidating the role of CYP2D6 in FP hydroxylation.

Identification of a novel variant CYP2C9 allele in Chinese.

Cytochrome P450 (CYP) 2C9 metabolizes about 16% of drugs in current clinical use, including lornoxicam and tolbutamide. SNPs in the CYP2C9 gene have increasingly been recognized as determinants of the metabolic phenotype that underlies interindividual and ethnic differences. The present study focused on a Chinese poor metabolizer (PM) whose apparent genotype (CYP2C9*1/CYP2C9*3) did not agree with his PM phenotype for both lornoxicam and tolbutamide. By sequencing his CYP2C9 gene, we identified a new variant CYP2C9 allele involving a T269C transversion in exon 2 that leads to a Leu90Pro substitution in the encoded protein. The CYP2C9 genotype analysis in the family of the poor metabolizer showed the new exon 2 change and CYP2C9*3 occurred on different alleles. Thus, the PM status of this subject could be attributed to his being heterozygous for the CYP2C9 T269C allele together with the CYP2C9*3. Frequency analysis in 147 unrelated Chinese males indicated approximately 2% of the Chinese population carry the allele. This study suggests that this novel CYP2C9 allele was correlated with reduced plasma clearance of drugs that are substrates for CYP2C9.

A CYP2D6 phenotype-genotype mismatch in Japanese psychiatric patients.

The aim of the present study was to seek a CYP2D6 genotypic-phenotypic discordance possibility in Japanese patients under psychotropic drug treatment where the CYP2D6 status and pharmacodynamic responses differ from those in Caucasian psychiatric patients. Ninety drug-free, healthy volunteers and 14 patients undergoing psychotropic drug treatment were phenotyped for their individual CYP2D6 activity using dextromethorphan as a probe, and then the metabolic ratio (MR) was calculated. For the genotyping, eight mutant alleles of the CYP2D6 genes were identified. Serum concentrations of two frequently co-medicated psychotropic drugs, biperiden and levomepromazine, were determined by GC/MS. Genotyping revealed no poor metabolizers (PMs) enrolled in our study. Healthy volunteers exhibited an identical phenotype-genotype concordance, whereas 7 of the 14 patients had significantly high (p < 0.05) MRs compared with genotype-matched volunteers. Three of the patients who had the extensive metabolizer (EM) genotype had extremely high MRs and were classified as phenotypic PMs. Five patients plus all of the seven high MR patients were treated with levomepromazine and/or biperiden, respectively. Their mean serum steady-state concentrations were 27.4 and 7.6 ng/ml, respectively. A CYP2D6 phenotype-genotype mismatch (phenocopying) can occur in Japanese psychiatric patients receiving clinical doses of some psychotropic drugs where the prevalence of PMs is low and the pharmacodynamic responses to those drugs are enhanced compared to Caucasian patients.

Human pharmacology of MDMA: pharmacokinetics, metabolism, and disposition.

MDMA (3,4-methylenedioxymethamphetamine, ecstasy) is a widely misused psychostimulant drug abused among large segments of the young population. Pharmacologically it displays effects related to amphetamine-type drugs and a set of distinctive effects (closeness to others, facilitation to interpersonal relationship, and empathy) that have been named by some authors "entactogen" properties. MDMA is a potent releaser and/or reuptake inhibitor of presynaptic serotonin (5-HT), dopamine (DA), and norepinephrine (NE). These actions result from the interaction of MDMA with the membrane transporters involved in neurotransmitter reuptake and vesicular storage systems. The most frequent effects after MDMA/ecstasy administration are euphoria, well-being, happiness, stimulation, increased energy, extroversion, feeling close to others, increased empathy, increased sociability, enhanced mood, mild perceptual disturbances, changed perception of colors and sounds, somatic symptoms related to its cardiovascular and autonomic effects (blood pressure and heart rate increase, mydriasis), and moderate derealization but not hallucinations. Acute toxic effects are related to its pharmacologic actions. The serotonin syndrome (increased muscle rigidity, hyperreflexia, and hyperthermia), among others, is characteristic of acute toxicity episodes. MDMA metabolism is rather complex and includes 2 main metabolic pathways: (1) O-demethylenation followed by catechol-O-methyltransferase (COMT)-catalyzed methylation and/or glucuronide/sulfate conjugation; and (2) N-dealkylation, deamination, and oxidation to the corresponding benzoic acid derivatives conjugated with glycine. The fact that the polymorphic enzyme CYP2D6 partially regulates the O-demethylenation pathway prompted some expectations that subjects displaying the poor metabolizer phenotype may be at higher risk of acute toxicity episodes. In this metabolic pathway a mechanism-based inhibition of the enzyme operates because the formation of an enzyme-metabolite complex that renders all subjects, independently of genotype, phenotypically poor metabolizers after the administration of 2 consecutive doses. Therefore, the impact of CYP2D6 pharmacogenetics on acute toxicity is limited. One of the interesting features of MDMA metabolism is its potential involvement in the development of mid- to long-term neurotoxic effects as a result of progressive neurodegeneration of the serotonergic neurotransmission system.