Analysis of the CYP2D6 gene mutations and their consequences for enzyme function in a West African population

The data on differences in the metabolic handling of the CYP2D6 probe drugs sparteine and debrisoquine, and the relationship between phenotype and genotype and gene frequencies for several mutant CYP2D6 alleles in African populations are limited and sometimes controversial. Therefore, in a West African population (Ghana), we investigated (i) the phenotype for sparteine debrisoquine by phenotyping 201 individuals with both drugs and (iii) the genotype for CYP2D6 (n = 326) and debrisoquine (n = 201) oxidation, (ii) the coregulatory control of sparteine and alleles *3 and *4 in 133 individuals and for the alleles *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *14, *16, *17, *2b, *2xN, *2bxN in 193 individuals. Of the 326 individuals phenotyped with sparteine, eight had a metabolic ratio (MR)sp > 20 corresponding to a poor metabolizer frequency of 2.5% [95% (confidence interval) CI = 1.06-4.77]. The prevalence of the poor metabolizer phenotype for debrisoquine oxidation was 3% (95% CI = 1.1-6.39) with six of the 201 individuals having a MR greater than 12.6. The distribution of the MR of sparteine was trimodal whereas MR of debrisoquine was unimodally distributed with a pronounced kurtosis. In individuals phenotyped with both drugs, there was a significant correlation between the MRs (r(s) = 0.63, P < 0.001). The CYP2D6 alleles *1, *2 and *17 were the most common functional alleles occurring with frequencies of 43.7, 10.6 and 27.7%, respectively. The three other observed functional alleles *2xN, *10 and *20 had much lower frequencies (1.6%, 3.1% and 0.3%, respectively). Of the eight non-functional alleles, only *4 (6.3%) and *5 (6.0%) could be found. The allele *5 occurred with the same frequency as in Caucasian populations (4.1%) but the *4 allele had a much lower frequency (Caucasians 19.5%). One individual with *1/*1 was a poor metabolizer for sparteine and debrisoquine indicating the existence of as yet unknown non-functional alleles in this West African population. Although the prevalence of poor metabolizers and the number of heterozygotes for non-functional alleles was much lower in Ghanaians, the median MRsp of 0.7 was significantly higher in this population compared with a median MRsp of 0.4 in Caucasians, indicating a lower metabolic clearance for CYP2D6 substrates in the West Africans. The lower metabolic activity in Ghanaians could not be explained solely by the high frequency of the *17 allele, which is associated with an impairment of CYP2D6 enzyme function. In addition, a higher median MRsp of 0.5 corresponding to metabolic clearance of 346 ml/min was observed among extensive metabolizers with the genotype *1/*1. Thus, compared with the median of MRsp = 0.28 (CLmet 573 ml/min) in Caucasians homozygous for *1, the metabolic clearance of sparteine was 40% lower on average in respective Ghanaians.     

Phenotypes and genotypes for CYP2D6 and CYP2C19 in a black Tanzanian population

AIMS: CYP2D6 and CYP2C19 are polymorphically expressed enzymes that show marked interindividual and interethnic variation. The aim of this study was to determine the frequency of the defective alleles in CYP2D6 and CYP2C19 in Africans and to test whether the genotype for CYP2C19 is better correlated with the proguanil/cylcoguanil ratio than the mephenytoin S/R ratio. METHODS: Two hundred and sixteen black Tanzanians were phenotyped for CYP2D6 with the use of sparteine, and for CYP2C19 with the use of mephenytoin and proguanil. Of these 196 subjects were also genotyped for CYP2D6 (including the CYP2D6*1, CYP2D6*3 and CYP2D6*4 alleles) and 195 were genotyped for CYP2C19 (including the CYP2C19*1, CYP2C19*2 and the CYP2C19*3 alleles). Furthermore 100 subjects were examined for the allele duplication in CYP2D6, leading to ultrarapid metabolism, with long PCR. RESULTS: The sparteine metabolic ratio (MR) was statistically significantly higher in the Tanzanian group of homozygous, extensive metabolizers compared to a historical control group of white Danish extensive metabolizers. Only one poor metabolizer for CYP2D6 (MR=124 and genotype CYP2D6*1/CYP2D6*4 ) was found. The gene frequencies were 0.96 for the CYP2D6*1 allele and 0.04 for the CYP2D6*4 allele. No CYP2D6*3 alleles were found. Nine subjects had an allele duplication in CYP2D6 (9%). For CYP2C19 there were seven subjects (3. 6%) who were phenotyped as poor metabolizers, but only three subjects (1.5%) had a genotype (CYP2C19*2/CYP2C19*2 ) indicative of poor metabolism. The gene frequencies were 0.90 for the CYP2C19*1 allele and 0.10 for the CYP2C19*2 allele. No CYP2C19*3 alleles were found. The mephenytoin S/R ratios were not bimodally distributed. CONCLUSIONS: Both the genotyping and phenotyping results show that there is a substantial difference between an African black population and a Caucasian population in the capacity to metabolize drugs via CYP2D6 and CYP2C19.     

Allele and genotype frequencies of polymorphic cytochromes P450 (CYP2C9, CYP2C19, CYP2E1) and dihydropyrimidine dehydrogenase (DPYD) in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), c2 variant of CYP2E1 and DPYD alleles (*2 A-*6 ) was carried out in a total of 247 unrelated Egyptian subjects. An allele-specific fluorogenic 5' nuclease chain reaction assay was applied for detection of CYP2C9 and CYP2C19 variants. Other variants of the CYP2E1 and DPYD genes were determined using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR based assays. RESULTS: CYP2C9 allele frequencies in 247 Egyptian subjects were 0.820 for CYP2C9*1, 0.120 for CYP2C9*2 and 0.060 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.888, 0.110 and 0.002, respectively. CYP2C19*3, which is considered an Asian mutation, was detected in one subject (0.40%) who was heterozygous (*1/*3). Two subjects (0.80%) were homozygous for *2/*2, while no compound heterozygotes (*2/*3) or homozygotes for *3 were detected. For CYP2E1, only four subjects (1.70%) had the rare c2 variant, expressed heterozygously, giving an allele frequency of 0.009. Five variants of DPYD were analysed, with no splice sites (*2 A) or DeltaC1897 (*3) found in this population. The frequencies of other variants were 0.028, 0.115 and 0.090 for *4, *5 and *6, respectively. CONCLUSIONS: Comparing our data with that obtained in several Caucasian, African-American and Asian populations, we found that Egyptians resemble Caucasians with regard to allelic frequencies of the tested variants of CYP2C9, CYP2C19, CYP2E1 and DPYD. Our results may help in better understanding the molecular basis underlying ethnic differences in drug response, and contribute to improved individualization of drug therapy in the Egyptian population.     

Role of CYP2C9 and its variants (CYP2C9*3 and CYP2C9*13) in the metabolism of lornoxicam in humans.

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily with some 12 CYP2C9 alleles (*1-*12) being previously reported. Recently, we identified a new CYP2C9 allele with a Leu90Pro mutation in a Chinese poor metabolizer of lornoxicam [Si D, Guo Y, Zhang Y, Yang L, Zhou H, and Zhong D (2004) Pharmacogenetics 14:465-469]. The new allele, designated CYP2C9*13, was found to occur in approximately 2% of the Chinese population. To examine enzymatic activity of the CYP2C9*13 allele, kinetic parameters for lornoxicam 5'-hydroxylation were determined in COS-7 cells transiently transfected with pcDNA3.1 plasmids carrying wild-type CYP2C9*1, variant CYP2C9*3, and CYP2C9*13 cDNA. The protein levels of cDNA-expressed CYP2C9*3 and *13 in postmitochondrial supernatant (S9) from transfected cells were lower than those from wild-type CYP2C9*1. Mean values of Km and Vmax for CYP2C9*1, *3, and *13 were 1.24, 1.61, and 2.79 microM and 0.83, 0.28, and 0.22 pmol/min/pmol, respectively. Intrinsic clearance values (Vmax/Km) for variant CYP2C9*3 and CYP2C9*13 on the basis of CYP2C9 protein levels were separately decreased to 28% and 12% compared with wild type. In a subsequent clinical study, the AUC of lornoxicam was increased by 1.9-fold and its oral clearance (CL/F) decreased by 44% in three CYP2C9*1/*13 subjects, compared with CYP2C9*1/*1 individuals. This suggests that the CYP2C9*13 allele is associated with decreased enzymatic activity both in vitro and in vivo.     

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in aborigines from western Australia

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes. Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed. The wild-type allele *1 was the most frequent (85.81%) and the non-functional alleles (*4, * 5, * 16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5. For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype. For CYP2EI, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6* 10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively.     

Population differences in major functional polymorphisms of pharmacokinetics/pharmacodynamics-related genes in Eastern Asians and Europeans: implications in the clinical trials for novel drug development

Drug lag, recently discussed extensively in Japan, can be divided into two phases: clinical development time and application review time. The former factor is still an important problem that might be improved by promoting multi-regional clinical trials and considering the results from other similar populations with Japanese, such as Koreans and Chinese. In this review, we compare the allelic or genotype frequencies of 30 relatively common functional alleles mainly between Eastern Asians and Europeans as well as among 3 major populations in Eastern Asian countries, Japan, Korea, and China, in 12 pharmacokinetics (PK)/pharmacodynamics (PD)-related genes; CYP2C9 (*2 and *3), CYP2C19 (*2, *3 and *17), 13 CYP2D6 haplotypes including *4, *5 and *10, CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5, *6 and *7), GSTM1 and GSTT1 null genotypes, SLCO1B1 521T>C, ABCG2 421C>A, and HLA-A*31:01 and HLA-B*58:01. In this review, differences in allele frequencies (AFs) or genotype frequencies (GFs) less than 0.1 (in the cases of highest AF (GF) ≥0.1) or less than 0.05 (in the cases of lowest AF (GF) <0.1) were regarded as similar. Between Eastern Asians and Europeans, AFs (or GFs) are regarded as being different for many alleles such as CYP2C9 (*2), CYP2C19 (*2, *3 and *17), CYP2D6 (*4 and *10), CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5*7), GSTT1 null and ABCG2 421C>A. Among the 3 Eastern Asian populations, however, only AFs of CYP2C19*3, CYP2D6*10, HLA-A*31:01 and HLA-B*58:01 are regarded as dissimilar. For CYP2C19*3, the total functional impact on CYP2C19 could be small if the frequencies of the two null alleles CYP2C19*2 and *3 are combined. Regarding CYP2D6*10, frequency difference over 0.1 is observed only between Japanese and Chinese (0.147). Although environmental factors should be considered for PK/PD differences, we could propose that among Japan, Korea, and China, genetic differences are very small for the analyzed common PK-related gene polymorphisms. On the other hand, AFs of the two HLA alleles important for cutaneous adverse drug reactions are diverse even among Eastern Asians and thus should be taken into account.     

A new variant CYP2D6 allele (CYP2D6*21) with a single base insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype.

Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.     

Effects of the CYP 2D6 genotype and cigarette smoking on the steady-state plasma concentrations of fluvoxamine and its major metabolite fluvoxamino acid in Japanese depressed patients

The effects of the cytochrome P450 (CYP) 2D6 genotype and cigarette smoking on the steady-state plasma concentrations (C(ss)) of fluvoxamine (FLV) and its demethylated metabolite fluvoxamino acid (FLA) were studied in 49 Japanese depressed patients receiving FLV 200 mg/d. The C(ss) of FLV and FLA were measured by HPLC, and the wild-type allele (*1) and two mutated alleles causing absent (*5) or decreased (*10) CYP 2D6 activity were identified by PCR methods. The patients were divided into three genotype groups by the number of mutated alleles: 12 cases with no (*1/*1), 27 cases with one (*1/*5 and *1/*10), and 10 cases with two (*5/*10 and *10/*10) mutated alleles. The means +/- SD of the C(ss) of FLV and FLA and the FLA/FLV ratio of all patients were 169.1 +/- 147.5 ng/mL, 83.9 +/- 52.7 ng/mL, and 0.71 +/- 0.50, respectively. The C(ss) of FLV and FLA were not significantly different among the three genotype groups. However, the FLA/FLV ratio was significantly lower in the patients with one (P < 0.05) and two (P < 0.01) mutated alleles than in those with no mutated allele. There was no significant difference between nonsmokers (n = 34) and smokers (n = 15) in these values. In the stepwise multiple regression, the C(ss) of FLA (P < 0.05) and FLA/FLV ratio (P < 0.001) showed significant negative correlations with the number of mutated alleles, and the FLA/FLV ratio was significantly (P < 0.05) lower in women than in men. The present study suggests that the CYP 2D6 genotype and cigarette smoking have no major impact on the C(ss) of FLV and FLA, though CYP 2D6 is involved in the demethylation of FLV.     

The frequency of candidate alleles for CYP2D6 genotyping in the Japanese population with an additional respect to the -1584C to G substitution

The -1584C/G single nucleotide polymorphism (SNP) in the promoter region of CYP2D6 was suggested to have the potential to influence CYP2D6 activity. In this report, we demonstrated the frequencies of -1584C to G substitution-related alleles, such as CYP2D6*2, CYP2D6*21, CYP2D6*35 and CYP2D6*41, in the Japanese population. The frequencies of CYP2D6*2, *41 and *21 were 0.102, 0.026 and 0.005, respectively. We also showed a relationship between the SNP and other common alleles, CYP2D6*4, *5, *10, *14 and *18. Interestingly, the SNP was detected in all three subjects carrying CYP2D6*14. This finding suggests the -1584G is included in the CYP2D6*14 allele, which is a null-allele characteristic to the Japanese population. This report presents practical information on CYP2D6 alleles that should be considered in the pharmacokinetic study of CYP2D6 substrates in the Japanese population.     

Genetic variability of CYP2B6 in populations of African and Asian origin: allele frequencies, novel functional variants, and possible implications for anti-HIV therapy with efavirenz

The present study investigated CYP2B6 genetic variability by sequencing genomic DNA samples of African-American, Ghanaian, Taiwanese, Japanese and Korean subjects throughout all exons and exon-intron boundaries. The most common nonsynonymous single nucleotide polymorphisms (SNPs) were 15631G > T (Q172H) and 18053A > G (K262R, together defining allele 2B6*6), both of which had frequencies close to 50% in Ghanaians and 30% in African-Americans. These SNPs have recently been shown to affect efavirenz pharmacokinetics and response in HIV patients. Eight new missense mutations (76A > T [T26S], 83A > G [D28G], 85C > A, 86G > C [both R29T], 15618C>T [T168I], 18038G > A [D257N], 21034C > T [R336C], 21498C > A [P428T]), three new silent mutations and two new intronic SNPs defining six novel alleles (*17A and B, *18, *19, *20, *21) were identified. Heterologous expression in COS-1 cells revealed pronounced reduction in expression and/or bupropion hydroxylase activity for variants T168I, D257N, R336C and P428T, whereas the triple mutant 2B6.17 (T26S, D28G, R29T) appeared to be functionally normal. These data extend the CYP2B6 knowledge base and should be particularly relevant for anti-HIV-therapy with efavirenz.     

Effect of cyp2d6*10 allele on the pharmacokinetics of loratadine in chinese subjects.

Loratadine is known to be a substrate for both CYP3A4 and CYP2D6 based on a previous in vitro study. In view of the large interindividual variability in loratadine pharmacokinetics and the greater genetically determined variability of CYP2D6 activity than of CYP3A4 in vivo, we hypothesized that CYP2D6 polymorphisms may contribute to the pharmacokinetic variability of loratadine. The purpose of this study was to evaluate the effect of CYP2D6 genotype (specifically the CYP2D6*10 allele) on the pharmacokinetics of loratadine in Chinese subjects. Three groups of healthy male Chinese subjects were enrolled: group I, homozygous CYP2D6*1 (*1/*1, n=4); group II, heterozygous CYP2D6*10 (*1/*10 or *2/*10, n=6); and group III, homozygous CYP2D6*10 (*10/*10, n=7) carriers. Each subject received a single oral dose of 20 mg of loratadine under fasting conditions. Multiple blood samples were collected over 48 h, and the plasma concentrations of loratadine and its metabolite desloratadine were determined by high-performance liquid chromatography. In comparing homozygous CYP2D6*10 (group III) to heterozygous CYP2D6*10 (group II) to homozygous CYP2D6*1 (group I) subjects, loratadine oral clearance values were 7.17+/- 2.54 versus 11.06+/-1.70 versus 14.59+/-2.43 l/h/kg, respectively [one-way analysis of variance (ANOVA), p<0.01], and the corresponding metabolic ratios [area under the plasma concentration-time curve (AUC)(desloratadine)/AUC(loratadine)] were 1.55+/-0.73 versus 2.47+/- 0.46 versus 3.32+/- 0.49, respectively (one-way ANOVA, p<0.05), indicating a gene-dose effect. The results demonstrated that CYP2D6 polymorphism prevalent in the Chinese population significantly affected loratadine pharmacokinetics.     

Comprehensive analysis of the genetic factors determining expression and function of hepatic CYP2D6

Variable expression and function of the cytochrome P4502D6 (CYP2D6) leads to distinct phenotypes termed ultrarapid (UM), extensive (EM), intermediate (IM) and poor metabolizer (PM). Whereas the PM phenotype is known to be caused by two null-alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remained largely unexplained. In this study, we systematically investigated 76 liver biopsies from individuals with known sparteine metabolic ratios (MRS) for the relationships between CYP2D6 genotype, microsomal protein expression, bufuralol 1'-hydroxylase activity and in-vivo phenotype. Average CYP2D6 protein levels ranged from undetectable in PMs (MRS > 20) to 2.6 +/- 2.7 pmol/mg microsomal protein in IMs (1.2 < MRS< 20), 7.6 +/- 4.7 in EMs (0.2 < MRS < 1.2) and 23.8 +/- 7.7 in UMs (MRS < 0.2), respectively. Analysis with respect to genotype demonstrated gradually increased expression and function for individuals with no, one, two or three functional gene copies per genome. The recently discovered -1584 C/G promoter polymorphism was identified as another major factor for expression and function with the mutant [-1584G] promoter type being consistently associated with significantly higher expression than [-1584C]. To investigate functional differences between the detected variant protein forms CYP2D6.1, 2D6.2, 2D6.9 and 2D6.10, we expressed them recombinantly in insect cells. The most significant difference was a decrease in the relative P450 holoprotein content of all allelic forms, including the common functional variant 2D6.2, in comparison to 2D6.1, whereas only modest Km changes were observed. Taken together, these data provide further insight into the complex mechanisms that govern the highly variable expression and function of CYP2D6.     

G169R mutation diminishes the metabolic activity of CYP2D6 in Chinese.

The molecular basis of the reduced ability of a Chinese to metabolize debrisoquine was explored by sequencing all of the nine exons of the CYP2D6 gene. The subject has T188, A1846, T2938, and C4268 (CYP2D6*14) instead of C188, G1846, C2938, and G4268 as in wild-type subjects. XbaI restriction fragment length polymorphism indicated that the subject has a 29-kb allele and a gene deletion (11.5 kb) in another allele (CYP2D6*5). A CYP2D6*14 allele together with a CYP2D6*5 allele may cause the poor metabolism of the subject. T188, T2938, and C4268 are common haplotypes in Chinese-extensive metabolizers. The effect of G1846 to A mutation in CYP2D6 metabolism has not been reported. A polymerase chain reaction-based endonuclease digestion test was designed for the G/N1846 polymorphism and 124 Chinese subjects were screened. With DNA sequencing, two other subjects showed the heterozygous G/A1846 and have a relatively high metabolic ratio of debrisoquine hydroxylation. The site-directed mutagenesis was used to create recombinant CYP2D6 cDNA with T188, A1846, or C4268. The cDNA was then transfected into Rat-1 cells. The transfection was confirmed by Southern, Northern, and Western blots. Based on the same microsomal protein level, the bufuralol 1'-hydroxylation activity of CYP2D6(T188) or CYP2D6(A1846) was significantly lower than that of the wild-type CYP2D6. P34S mutation (C188 to T) significantly decreased CYP2D6 activity. G169R mutation (G1846 to A) also decreased CYP2D6 activity and may further reduce the metabolic activity of CYP2D6 protein with P34S, R296C, and S486T mutations.     

Effect of the CYP2D6 genotype on the pharmacokinetics of tropisetron in healthy Korean subjects

OBJECTIVE: To evaluate the effect of the CYP2D6 genotype on the pharmacokinetics of tropisetron in healthy Korean subjects. METHODS: A single 5-mg capsule of tropisetron was administered orally to 13 healthy subjects. Plasma concentrations were determined by validated HPLC procedures and data were analyzed by using noncompartmental linear PK methods. Four alleles, CYP2D6*1, CYP2D6*2 x2, CYP2D6*5, and CYP2D6*10, were identified by PCR. RESULTS: Thirteen subjects, consisting of two homozygous carriers of the wild type allele ( *1/*1), four heterozygous carriers of poor metabolizer (PM)-associated allele (* 1/*10), six homozygous carriers of PM-associated alleles (four with *10/*10 and two with *5/*10), and one carrier of a duplicated allele *1/*2 x2. All tested pharmacokinetic parameters (AUC(inf), AUC(inf)(NL70), Cmax, Cmax(NL70), T(1/2), and Tec) were significantly different among four different genotypic groups. The mean AUCs of carriers with the heterozygous PM-associated allele and the homozygous PM-associated allele were 1.9- and 6.8-higher than those of carriers with the wild type allele, respectively. In contrast, the mean AUC of carriers with a duplicated allele was 0.5-fold lower than that of those carriers with the wild type allele. CONCLUSION: The presence of CYP2D6*5, CYP2D6*10, and CYP2D6*2 x2 has an important impact on the pharmacokinetics of tropisetron, which may influence clinical response to tropisetron therapy.     

New cytochrome P450 2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes.

Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks.     

Duplications and defects in the CYP2A6 gene: identification, genotyping, and in vivo effects on smoking

In humans, 80% of nicotine is metabolized to the inactive metabolite cotinine by the enzyme CYP2A6, which can also activate tobacco smoke procarcinogens (e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). Previously, we demonstrated that individuals who are nicotine-dependent and have defective CYP2A6 alleles (*2, *3) smoked fewer cigarettes; however, we recognize that the genotyping method used for the CYP2A6*3 allele gave a high false-positive rate. In the current study we used improved genotyping methods to examine the effects of the defective CYP2A6*2 and CYP2A6*4 alleles on smoking behavior. We found that those with the defective alleles (N = 14) smoked fewer cigarettes per day than those homozygous (N = 277) for wild-type alleles (19 versus 28 cigarettes per day, P <.001). In addition, we identified a duplicated form of the CYP2A6 gene, corresponding to the gene deletion CYP2A6*4 allele, developed a genotyping assay, assessed the gene copy number, and examined its prevalence in Caucasian smokers (N = 296). We observed an ascending rank order for plasma cotinine and breath carbon monoxide levels (an index of smoke inhalation) in individuals with null (CYP2A6*2 and CYP2A6*4) alleles (N = 14), those homozygous for wild-type (CYP2A6*1/*1) alleles (N = 277), and those with our newly identified CYP2A6 gene duplication (N = 5). The phenotype, as determined by plasma nicotine/cotinine ratios, had a descending rank order for these three genotype groups that did not reach significance. Although further characterization is required for the duplication gene variant, these results extend our previous findings and suggest a substantial influence of CYP2A6 genotype and phenotype on smoking behavior.     

Cytochrome P450 2D6.1 and cytochrome P450 2D6.10 differ in catalytic activity for multiple substrates

CYP2D6 is involved in the metabolism of several classes of drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors and various amphetamines. CYP2D6*10 is an allelic variant, producing an enzyme with Pro34Ser and Ser486Thr amino acid substitutions. Approximately 75% of Asians possess the *10 allele. We sought to further characterize CYP2D6.10 catalytically in vitro in a baculovirus expression system using various substrates and inhibitors, in comparison to CYP2D6.1 (wild-type). Using dextromethorphan (DEX), P-methoxyamphetamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (+/-)3,4-methylenedioxymethamphetamine (MDMA), the ratios of intrinsic clearance (Vmax/Km) of *1 to *10 were 50, 34, 22 and 123, respectively. The CYP2D6 substrates amitriptyline, and (+) and (-) methamphetamine (MAMP) are both p-hydroxylated and N-demethylated (NDM). The intrinsic clearance *1/*10 ratios were 42, 30 and 67 for the p-hydroxylation; and 60, 120 and 157 for the NDM, respectively, illustrating chemical pathway and enantiomeric selectivity for MAMP. It was apparent that (+) and (-) MAMP NDM and MDMA demethylenation were most significantly different in CYP2D6.10. Using DEX as the substrate, the ratios of Ki(*10)/Ki(*1) for inhibitors were: budipine (1.3), sparteine (1.6), debrisoquine (8.1), fluoxetine (16), norfluoxetine (30), paroxetine (14), MDMA (21) and MMDA-2 (7.1), indicating that CYP2D6.10 shows drug-specific altered susceptibility to inhibition. Taken together, these data suggest that CYP2D6*10/*10 individuals may be expected to require different drug doses; and show altered susceptibility to toxicity, interaction risk and, in the case of the amphetamines, drug dependence and toxicity compared to CYP2D6*1/*1 individuals.     

Effect of CYP2D6 genotypes on the metabolism of haloperidol in a Japanese psychiatric population.

We investigated the effect of CYP2D6 genotypes on plasma levels of haloperidol (HAL) and reduced haloperidol (RHAL) in 88 Japanese schizophrenic inpatients being treated with HAL. Some subjects carrying CYP2D6*5 allele (CYP2D6*1/CYP2D6*5, CYP2D6*5/CYP2D6*10) showed extremely high concentrations of both HAL and RHAL, and the groups with CYP2D6*5 allele seemed to have higher plasma concentrations of HAL (1.14+/-0.69 ng/ml/mg) and RHAL (1.10+/-1.05 ng/ml/mg) than the other groups. Among those without CYP2D6*5 allele, there were no significant differences in plasma concentrations of HAL and RHAL between those without CYP2D6*10 allele (HAL=0.68+/-0.31 ng/ml/mg, RHAL=0.28+/-0.37 ng/ml/mg), those with one CYP2D6*10 (HAL=0.70+/-0.23 ng/ml/mg, RHAL=0.31+/-0.16 ng/ml/mg) and those with two CYP2D6*10 alleles (HAL=0.69+/-0.14 ng/ml/mg, RHAL=0.40+/-0.09 ng/ml/mg), although there was a tendency of higher plasma concentration of RHAL in those with two CYP2D6*10 alleles. At a lower daily dosage of HAL (<10 mg/day), the subjects with two or one CYP2D6*10 allele(s) showed significantly higher plasma concentrations of RHAL (0.43+/-0.23 ng/ml/mg, 0.34+/-0.16 ng/ml/mg) than those without CYP2D6*10 allele (0.18+/-0.16 ng/ml/mg). The results of this study indicate that CYP2D6*10 allele plays significant but modest role in HAL metabolism in Japanese; nevertheless, we should not lump CYP2D6*10 allele with CYP2D6*5 allele because these two mutated alleles seem to have different impacts in the metabolism of HAL.     

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

OBJECTIVES: CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status. METHODS: Wild-type and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine. RESULTS: CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N=560 alleles, 95% confidence interval, 0.8-3.1%) and was not detected in Caucasians (N=334 alleles), Chinese (N=288 alleles) or Japanese (N=104 alleles). In vitro, CYP2A6.23 had greatly reduced activity toward nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared with the wild-type enzyme. The trans-3'-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals (mean adjusted ratio of 0.60, n=5) compared with CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n=150) (P<0.04). CYP2A6*23 trended toward a higher allele frequency in nonsmokers (3.1%, N=9/286 alleles) compared with smokers (0.7%, N=2/274 alleles) (P=0.06). CONCLUSION: These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.