CYP2C19 genotype and phenotype determined by omeprazole in a Korean population

Omeprazole (20 mg orally) was given to 103 healthy Korean subjects and blood was taken 3 h after administration. The plasma concentration ratio of omeprazole and hydroxyomeprazole, used as an index of CYP2C19 activity, was bimodally distributed. Thirteen subjects (12.6%) were identified as poor metabolizers (PMs) with an omeprazole hydroxylation ratio of 6.95 or higher. Among the 206 CYP2C19 alleles, CYP2C19*2 and CYP2C19*3 were found in 43 alleles (21%) and 24 alleles (12%), respectively. Twelve subjects (12%) carried two defect alleles (*2/*2, *2/*3 or *3/*3), 43 subjects (42%) were heterozygous for a mutated (*2 or *3) and a wild type (*1) allele, and the remaining 48 subjects (47%) were homozygous for the wild type allele. The distributions of the metabolic ratio between these three genotype groups were significantly different (Kruskal-Wallis test: p < 0.0001). The genotypes of 19 additional Korean PMs has been identified in a previous mephenytoin study. From a total of 32 PMs, 31 were genotypically PMs by analysis of the CYP2C19*2 and *3 alleles and only one PM subject was found to be heterozygous for the *1 and *2 alleles. At present it cannot be judged whether this subject has a defective allele with a so-far unidentified mutation or a true wild type allele. We thus confirm a high incidence (12.6%) of PMs of omeprazole in Koreans and of the 32 Korean PMs 97% could be identified by the genotype analysis.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

Imipramine metabolism in relation to the sparteine and mephenytoin oxidation polymorphisms--a population study

1. Sparteine and mephenytoin phenotyping tests were carried out in 327 healthy Danish subjects. Two weeks later each subject took 25 mg imipramine followed by urine collection for 24 h. The urinary content of imipramine, desipramine, 2-hydroxy-imipramine and 2-hydroxy-desipramine was assayed by h.p.l.c. 2. The medians of the hydroxylation ratios (i.e. 2-hydroxy-metabolite over parent compound) were 6 to 14 times higher in 300 extensive metabolizers of sparteine (EMs) as compared with 27 poor metabolizers (PMs), but none of the ratios separated the two phenotypes completely. 3. There were 324 EM of mephenytoin (EMM) and three PM (PMM) in the sample. The demethylation ratios between desipramine, 2-hydroxy-desipramine and their corresponding tertiary amines showed statistically significant correlations with the mephenytoin S/R isomer ratio (Spearman's rs: -0.20 and -0.27, P < 0.05). 4. The demethylation ratios were higher in 80 smokers than in 245 non-smokers. This indicates that CYP1A2, which is induced by cigarette smoking, also catalyzes the N-demethylation of imipramine. 5. CYP2D6 genotyping was carried out by PCR in 325 of the subjects, and the D6-wt allele was amplified in 298 EMs, meaning that they were genotyped correctly. One PMs was D6-wt/D6-B, another PMs had the genotype D6-wt/ and hence both were misclassified as EMs. The remaining 25 PMs were D6-A/D6-B (n = 5), D6-B/ (n = 18) or D6-D/D6-D (no PCR amplification, n = 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of genetic polymorphisms of CYP2C9 and CYP2C19 on phenytoin metabolism in Japanese adult patients with epilepsy: studies in stereoselective hydroxylation and population pharmacokinetics

PURPOSE: The aim of this study was to clarify the effects of genetic polymorphisms of cytochrome P450 (CYP) 2C9 and 2C19 on the metabolism of phenytoin (PHT). In addition, a population pharmacokinetic analysis was performed. METHODS: The genotype of CYP2C9 (Arg144/Cys, Ile359/Leu) and CYP2C19(*1, *2 or *3) in 134 Japanese adult patients with epilepsy treated with PHT were determined, and their serum concentrations of 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, being major metabolites of PHT, were measured. A population pharmacokinetic analysis (NONMEM analysis) was performed to evaluate whether genetic polymorphism of CYP2C9/19 affects the clinical use of PHT by using the 336 dose-serum concentration data. RESULTS: The mean maximal elimination rate (Vmax) was 42% lower in the heterozygote for Leu359 allele in CYP2C9, and the mean Michaelis-Menten constants (Km) in the heterozygous extensive metabolizers and the poor metabolizers of CYP2C19 were 22 and 54%, respectively, higher than those without the mutations in CYP2C9/19 genes. (R)- and (S)-p-HPPH/PHT ratios were lower in patients with mutations in CYP2C9 or CYP2C19 gene than those in patients without mutations. CONCLUSIONS: Although the hydroxylation capacity of PHT was impaired with mutations of CYP2C9/19, the impairment was greater for CYP2C9. In view of the clinical use of PHT, two important conclusions were derived from this population study. First, the serum PHT concentration in patients with the Leu359 allele in CYP2C9 would increase dramatically even at lower daily doses. Second, the patients with CYP2C19 mutations should be treated carefully at higher daily doses of PHT.     

Association between CYP2C19 genotype and proguanil oxidative polymorphism

AIMS: To examine the relationship between proguanil metabolism and the number of mutations in CYP2C19 by comparing the CYP2C19 genotype and proguanil phenotype of 10 subjects. METHODS: Partial clearance and urinary metabolic ratio data were obtained from a previous study of 10 subjects [5]. Analysis of CYP2C19 genotypes was performed using PCR amplification followed by restriction endonuclease digestion of genomic DNA from a blood sample. RESULTS: The intrinsic partial clearance of PG to CG ranged from 0.41-10.11 h-1, and was related to the number of functional CYP2C19 alleles present. Genotypic PMs had metabolic ratios > 13, while genotypic heterozygote EMs had metabolic ratios < 9. CONCLUSIONS: Proguanil may be a suitable phenotyping probe for the CYP2C19 genetic polymorphism, however the exact antimode of the urinary metabolic ratio chosen to separate poor and extensive metabolisers needs further investigation.     

Genetic polymorphism of CYP2D6 in North Indian subjects

OBJECTIVES: CYP2D6 polymorphism of clinical relevance occurs with variable frequency in different ethnic groups. Since this polymorphism has not been studied in a North Indian population, the present study was undertaken. METHODS: One hundred healthy unrelated North Indian subjects received 30 mg dextromethorphan (DM) orally at bed-time. The amounts of DM and its metabolite, dextrorphan (DR), excreted in 8 h urine were estimated by high performance liquid chromatography. Metabolic ratio (DM/DR excreted in 8 h) was used as an index of the metabolic status of an individual. RESULTS: The analysis of the data by frequency distribution histogram, probit and NTV plots demonstrated bimodal distribution of the North Indian subjects with respect to hepatic CYP2D6. Out of 100 subjects, 97 were extensive metabolizers (EMs), whereas three were poor metabolizers (PMs). EMs and PMs excreted 29.82 and 2.67 micromol DR (mean value) and 2.59 and 8.82 micromol DM (mean value) in 8 h, respectively. MR and log MR was 197- and 2.2-fold higher in PMs versus EMs. The antimode value of zero was determined by visual observation in frequency distribution histogram and inflection point in probit plot. CONCLUSION: From this study, it can be concluded that the PM phenotype of CYP2D6 occurs with a frequency of 3% (95% confidence interval of 0.33%-6.33%) in North Indians.     

Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: application of the relative activity factor approach

The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4'-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CYP/mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblast-determined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6-17.9; n = 10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04-16.2; n = 10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immunoquantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.     

Reappraisal of human CYP isoforms involved in imipramine N-demethylation and 2-hydroxylation: a study using microsomes obtained from putative extensive and poor metabolizers of S-mephenytoin and eleven recombinant human CYPs

Cytochrome P450 (CYP) involved in the two major pathways of imipramine (IMI) was reappraised using human liver microsomes phenotyped for S-mephenytoin 4'-hydroxylation in vitro and 11 recombinant human CYP isoforms. Individual Eadie-Hoffstee plots for IMI N-demethylation and 2-hydroxylation showed a monophasic profile in microsomes obtained from three putative S-mephenytoin poor metabolizer (PM) livers, whereas the plots gave a biphasic relationship (except for one case in 2-hydroxylation) in those from the three extensive metabolizer (EM) livers. Effects of CYP-selective inhibitor/substrate probes on the two metabolic reactions were examined at the two IMI concentrations (2 and 400 microM) with microsomes obtained from the two PM and three EM livers. S-mephenytoin inhibited IMI N-demethylation by 50% at the low concentration in microsomes from the EM livers with no discernible effect on this pathway in those from the PM livers. Furafylline inhibited the N-demethylation by about 60% at the low and high substrate concentrations in microsomes from both the EM and PM livers. Quinidine abolished the 2-hydroxylation at the low and high concentrations in microsomes from both the EM and the PM livers. Among the recombinant human CYPs, CYP2C19, 2C18, 2D6, 1A2, 3A4 and 2B6 in rank order catalyzed the N-demethylation, whereas CYP2D6, 2C19, 1A2, 2C18 and 3A4 catalyzed the 2-hydroxylation. The Km values obtained from recombinant CYP2C19 and 1A2 approximated those of the high- and low-affinity components from human liver microsomes for IMI N-demethylation, respectively. For IMI 2-hydroxylation, the respective Km values obtained from recombinant CYP2D6 and 2C19 were close to those of the high- and low-affinity components from human liver microsomes. Our human liver microsomal study using the near-therapeutic IMI concentration (2 microM) suggests that 1) CYP2C19 and 1A2 are involved in the N-demethylation and the 2-hydroxylation is mediated exclusively by CYP2D6 and partially by CYP2C19 in the EM livers, and 2) CYP1A2 and 2D6 play a major role in IMI N-demethylation and 2-hydroxylation, respectively, in the PM livers. Our recombinant human CYP isoform study, in general, supports this conclusion.     

Significance of the plasminogen activator inhibitor of placental type (PAI-2) in pregnancy

OBJECTIVE: Genetic oxidation polymorphisms of debrisoquine (CYP2D6) and proguanil (CYP2C19) were studied in unrelated healthy South Pacific Polynesian volunteers recruited in the South Island of New Zealand. METHODS: Phenotyping for CYP2D6 and CYP2C19 activities was determined using debrisoquine and proguanil, respectively, as probe drugs by measuring the urinary metabolic ratio of parent drug and its metabolite. RESULTS: Of 100 Polynesian subjects phenotyped, the metabolic ratio of debrisoquine ranged from 0.01 to 9.94. Therefore, all South Pacific Polynesians were classified as extensive metabolizers of debrisoquine according to previously established criteria of the antimode. The prevalence of poor metabolizers of debrisoquine (CYP2D6) in this Polynesian population is 0% (95% confidence interval of 0-3.6%). Oxidation polymorphism of CYP2C19 using proguanil as a probe was also studied in 59 Polynesian volunteers. The frequency distribution of the proguanil/cycloguanil ratio was bimodal. The proguanil/cycloguanil ratios for these subjects ranged from 0.09 to 34.4. Using a recommended proguanil/cycloguanil ratio cut-off point of 10 established in Caucasian populations, eight Polynesian subjects were identified as poor metabolizers of proguanil (CYP2C19), which corresponds to a poor metabolizer phenotype frequency of 13.6% (a 95% confidence interval of 5.9-24.6%). CONCLUSION: The incidence of poor metabolizer phenotypes for debrisoquine (CYP2D6) in South Pacific Polynesians appears to lower than in Caucasian populations, while the prevalence of poor metabolizers for proguanil (CYP2C19) in this ethnic population is higher. The frequencies of the poor metabolizer phenotype for debrisoquine and also for proguanil in South Pacific Polynesians are similar to those reported in Asian populations.     

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (Km (substrate concentration producing maximum reaction velocity) 3-13 microM). Formation of dextrorphan from 25 microM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 microM); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, and -2D6 but not by those expressing CYP1A2, -2E1 or -3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 microM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean Km of 259 microM. For formation of 3-methoxymorphinan from 25 microM dextromethorphan the IC50 for ketoconazole was 1.15 microM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, -2D6, and -3A4, but not by those expressing CYP1A2 or -2E1. CYP2C19 had the highest affinity (Km = 49 microM) whereas CYP3A4 had the lowest (Km = 1155 microM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and -2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.     

Cortical reorganisation of sensory, motor and language functions due to early cortical damage

OBJECTIVES: To compare the pharmacokinetics and dynamics of omeprazole in white and Chinese subjects. METHODS: This double-blind two-stage study, performed in the clinical research center of a university hospital, evaluated 15 healthy nonsmoking men (eight white subjects and seven Chinese extensive metabolizers of mephenytoin). Blood samples were obtained over 24 hours after the eighth omeprazole dose (40 mg/day). Omeprazole, omeprazole sulfone, and hydroxyomeprazole pharmacokinetics were calculated from the respective plasma concentration-time curves. Twelve- and 24-hour integrated plasma gastriun (AUCgas12 and AUCgas24) were calculated from the respective plasma gastrin concentrations. A week before the initiation of omeprazole the activities of CYP2D6, CYP2C19, and CYP3A4 were determined by previously established methods. RESULTS: Omeprazole concentrations were significantly lower (mean area under the plasma concentration time curve extrapolated to infinity [AUCO-infinity] +/- SEM; 7.53 +/- 1.21 versus 12.80 +/- 2.13 mumol.hr.L-1, respectively; p < 0.05) and its oral clearance greater (319 +/- 60 versus 183 +/- 35 ml/min, respectively; p < 0.05) in the white subjects than in the Chinese subjects. Omeprazole and omeprazole sulfone AUCO-infinity values were well correlated with the S/R mephenytoin ratio (r = 0.82 and r = 0.84, respectively; p < 0.001) and with urinary 4'-hydroxymephenytoin (r = -0.58 [p < 0.03] and r = -0.52 [p < 0.02], respectively). Fasting gastrin, AUCgas12, and AUCgas24 were significantly greater in the Chinese subjects than in the white subjects (30.0 +/- 6.4 versus 14.4 +/- 1.2 pmol, respectively [p < 0.02]; 661 +/- 114 versus 334 +/- 38 pmol.hr.L-1, respectively [p < 0.002]; and 1414 +/- 228 versus 747 +/- 99 pmol.hr.L-1, respectively [p < 0.004]). In addition, the S/R mephenytoin ratio and omeprazole AUCO-infinity correlated with the extent of omeprazole induced hypergastrinemia. CONCLUSION: The metabolism of omeprazole and the rise in gastrin concentration after its administration is genetically determined and ethnically dependent.     

CYP2D6 phenotype-genotype relationships in African-Americans and Caucasians in Los Angeles

CYP2D6 genotyping (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*13, CYP2D6*16 alleles and gene duplications) was previously performed on 1053 Caucasian and African-American lung cancer cases and control individuals and no significant difference in allele frequencies between cases and control individuals detected. We have carried out additional genotyping (CYP2D6*6, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10, CYP2D6*17 alleles) and debrisoquine phenotyping on subgroups from this study to assess phenotype-genotype relationships. African-Americans showed significant differences from Caucasians with respect to frequency of defective CYP2D6 alleles, particularly CYP2D6*4 and CYP2D6*5. The CYP2D6*17 allele occurred at a frequency of 0.26 among 87 African-Americans and appeared to explain higher average metabolic ratios among African-Americans compared with Caucasians. CYP2D6*6, CYP2D6*8, CYP2D6*9 and CYP2D6*10 were rare in both ethnic groups but explained approximately 40% of higher than expected metabolic ratios among extensive metabolizers. Among individuals phenotyped with debrisoquine, 32 out of 359 were in the poor metabolizer range with 24 of these (75%) also showing two defective CYP2D6 alleles. Additional single strand conformational polymorphism analysis screening of samples showing large phenotype-genotype discrepancies resulted in the detection of three novel polymorphisms. If subjects taking potentially interfering drugs were excluded, this additional screening enabled the positive identification of 88% of phenotypic poor metabolizers by genotyping. This sensitivity was comparable with that of phenotyping, which identified 90% of those with two defective alleles as poor metabolizers.     

Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enzyme. Currently, two defective alleles are known--CYP2C19*2 and CYP2C19*3. The authors have developed an oligonucleotide ligation assay to detect these two alleles. This assay combines the hybridization of one common, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatched nucleotides. The probes are only ligated if they are base paired correctly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is removed in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured simultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese living in Denmark. All genotypes determined by the assay were consistent with the results from restriction enzyme cleavage. There were 12 poor metabolizers; 10 homozygous CYP2C19*2/CYP2C19*2, one heterozygous CYP2C19*2/CYP2C19*3, and one heterozygous CYP2C19*1/CYP2C19*2. The authors conclude that this assay is well-suited for a high throughput of samples in a routine laboratory. The finding of an apparently heterozygous CYP2C19*1/CYP2C19*2 poor metabolizer, confirms that there are still unknown mutations in CYP2C19.     

Fluvoxamine inhibits the CYP2C19-catalysed metabolism of proguanil in vitro

OBJECTIVE: The potent CYP1A2 inhibitor fluvoxamine has recently been shown also to be an effective inhibitor of the CYP2C19-mediated metabolism of the antimalarial drug proguanil in vivo. The purpose of the present study was to confirm this interaction in vitro. METHODS: A high-performance liquid chromatography (HPLC) method was developed to assay 4-chlorophenylbiguanide (4-CPBG) and cycloguanil formed from proguanil by microsomes prepared from human liver. The limit of detection was 0.08 nmol mg-'. h-I. RESULTS: The formation of 4-CPBG and cycloguanil could be described by one-enzyme kinetics, indicating that the formation of the two metabolites is almost exclusively catalysed by a single enzyme, i.e. CYP2C19 within the concentration range used, or that the contribution of an alternative low-affinity enzyme, probably CYP3A4, is very low. This notion was confirmed by the lack of potent inhibition by four CYP3A4 inhibitors: ketoconazole, bromocriptine, midazolam and dihydroergotamine. Fluvoxamine was a very effective inhibitor of the oxidation of proguanil, displaying Ki values of 0.69 micromol x l(-1) for the inhibition of cycloguanil formation and 4.7 micromol x l(-1) for the inhibition of 4-CPBG formation. As expected, the CYP2C19 substrate omeprazole inhibited the formation of both metabolites with an IC50 of 10 micromol x l(-1). Norfluoxetine and sulfaphenazole inhibited proguanil oxidation with Ki values of 7.3-16 micromol x l(-1), suggesting that the two compounds are moderate inhibitors of CYP2C19. CONCLUSIONS: Fluvoxamine is a fairly potent inhibitor of CYP2C19 and it has the potential for causing drug-drug interactions with substrates for CYP2C19 such as imipramine, clomipramine, amitriptyline and diazepam. The combination of fluvoxamine and proguanil can not be recommended.     

Detection of CYP2D6*3 and 2D6*4 allelic variants by PCR-restriction fragment length polymorphism

The mutant of CYP2D6*3 allele with A2637 deletion in exon 5 and the mutant of CYP2D6*4 allele G1934-->A, splice site defect are among the most common polymorphic alleles of CYP2D6 gene, resulting in a decreased or no activity of CYP isoenzyme. In this study, a reliable polymerase chain reaction-restriction fragment length polymorphism method for identification of CYP2D6*3 and CYP2D6*4 alleles was used to investigate the genotype and phenotype prevalence in the groups of normal controls, and of cirrhosis and cancer patients. The results showed none of 36 controls genotyped for 2D6*3 and 2D6*4 allele to have the 2D6*3 allele with frameshift mutation in exon 5, while 33% (n=12) were found to bear the 2D6*4 allele with G to A mutation at the intron 3-exon 4 junction. In breast cancer patients (n=35) genotyped for 2D6*3 and 2D6*4 alleles, none with 2D6*3 allele was found either, but 60% (n=18) were found to bear the 2D6*4 allele. In patients with head and neck squamous cell cancer, there was only one subject with 2D6*3 allele and he was heterozygous. Among them, as many as ten (40%) patients were found to bear 2D6*4 allele. In the cirrhosis group, none of the patients was found to have the 2D6*3 allele, while the CYP2D6*4 allele was found in 23% (n=6) patients. The phenotype predicted according to the genotype was as follows: in the control group, 3% of individuals were identified as poor metabolizers, 70% as extensive metabolizers, and 27% as heterozygote extensive metabolizers. In the group of breast cancer, 7% of the patients were identified as poor metabolizer, 57% as extensive metabolizer and 36% as phenotype. In squamous cell cancer and cirrhosis patients, the incidence of poor metabolizer was zero, and of heterozygotes extensive metabolizer 42% and 31%, respectively.     

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.     

A method for a selective local application of radioactive precursors of nucleic acids to the cow ovary

OBJECTIVE: To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 affects the in vivo metabolism of warfarin enantiomers. METHODS: Eighty-six Japanese patients heart disease who were given warfarin participated in the study. Plasma unbound concentrations of warfarin enantiomers and urinary (S)-7-hydroxywarfarin concentrations were measured by means of a chiral HPLC and ultrafiltration technique to calculate the unbound oral clearance (CLpo,u) for the enantiomers and the formation clearance (CLm) for (S)-warfarin 7-hydroxylation. Genotyping for CYP2C9 (the wild type [wt], Arg144/Cys, and I1e359/Leu) and for CYP2C19 (wt, ml, and m2) was performed with a polymerase chain reaction method. RESULTS: Three patients were heterozygous for the CYP2C9 Leu359 mutation but none were homozygous for the mutation (the allele frequency of 0.017). None had a CYP2C9 Cys144 allele. The medians for (S)-warfarin CLpo,u and its 7-hydroxylation CLm obtained from heterozygotes of CYP2C9 Leu359 were significantly less than those obtained from homozygotes of the wt allele, as follows: 234 ml/min (range, 156 to 269 ml/min) versus 632 ml/min (range, 180 to 2070 ml/min) (p < 0.001) and 0.20 ml/min (range, 0.05 to 0.77 ml/min) versus 0.80 ml/min (range, 0.05 to 14.9 ml/min) (p < 0.05), respectively. In contrast, no difference was observed in (R)-warfarin CLpo,u between the groups. The allele frequencies for CYP2C19 m1 and CYP2C19 m2 were 0.26 and 0.14, respectively, indicating 15% of patients were genotypically poor metabolizers of CYP2C19. No difference in CLpo,u for warfarin enantiomers was observed between the assumed CYP2C19 phenotypes. CONCLUSION: Heterozygotes for CYP2C9 I1e359/Leu allele have reduced in vivo metabolism of (S)-warfarin but not (R)-warfarin. Because (S)-warfarin has a greater anticoagulant potency than its (R)-congener, the genetic polymorphism of CYP2C9 may partly account for the large interpatient variability in therapeutic dosages of warfarin.     

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.     

Coexisting mental illness and alcohol and other drug dependencies in pregnant and parenting women

1. To determine whether dexfenfluramine is a substrate of cytochrome P450 2D6 (CYP2D6), its disposition has been studied in nine extensive (EM) and eight poor metabolizers (PM) of debrisoquine. 2. Following a 30 mg dose of dexfenfluramine hydrochloride, urine was collected in all subjects for 96 h post-dose and plasma samples were collected in 11 subjects (six EMs and five PMs). Dexfenfluramine and nordexfenfluramine were measured in urine by h.p.l.c. and in plasma by g.c. 3. Urinary recovery of dexfenfluramine was greater in PMs than EMs (4136 +/- 1509 micrograms vs 1986 +/- 792 micrograms; 95% CI of difference 926-3374; P < 0.05) whereas that of nordexfenfluramine was similar in both phenotypes (PM: 1753 +/- 411 micrograms vs 1626 +/- 444 micrograms). 4. Dexfenfluramine AUC was higher in PMs (677 +/- 348 micrograms l-1 h) than EMs 359 +/- 250 micrograms l-1 h). The apparent oral clearance of dexfenfluramine was greater in EMs than PMs (93.6 +/- 42.4 l h-1 vs 45.6 +/- 19.5 l h-1; 95% CI of difference 1.2-94.7; P < 0.05). The renal clearance was similar in both phenotypes (EMs: 5.88 +/- 2.83 l h-1; PMs 6.60 +/- 2.01 l h-1), indicating that the higher urinary recovery of dexfenfluramine in PMs reflects higher plasma concentrations, rather than phenotype differences in the renal handling, of dexfenfluramine. 5. The apparent nonrenal clearance of dexfenfluramine was substantially lower (P < 0.05; 95% CI of difference 3.0-94.1) in PMs (39.0 +/- 19.5 l h-1) than EMs (87.6 +/- 41.2 l h-1). 6. There was a significant inverse correlation (rs = 0.776 95% CI-0.31-0.94; n = 11; p = 0.005) between the debrisoquine metabolic ratio and the apparent nonrenal clearance of dexfenfluramine. 7. PMs had a higher incidence of adverse effects (nausea and vomiting) than EMs. 8. In conclusion, the metabolism of dexfenfluramine is impaired in PMs. Thus CYP2D6, the isoenzyme deficient in poor metabolizers of debrisoquine, must catalyse at least one pathway of dexfenfluramine biotransformation.     

The role of CYP2C in the in vitro bioactivation of the contraceptive steroid desogestrel

Desogestrel is a 3-deoxo progestogenic steroid that requires bioactivation to 3-ketodesogestrel. In these studies we have attempted to define the pathway of 3-ketodesogestrel formation and characterise the enzymes responsible for this biotransformation in vitro. Initial studies using deuterated desogestrel confirmed that desogestrel is metabolised by human liver microsomes via 3alpha-hydroxy and 3beta-hydroxydesogestrel to 3-ketodesogestrel. Metabolites were analysed by radiometric high-performance liquid chromatography and were identified by liquid chromatography-mass spectrometry and by cochromatography with authentic standards. Desogestrel was metabolised by microsomes from lymphoblasts containing cDNA-expressed CYP2C9 and CYP2C19 to 3alpha-hydroxydesogestrel with small amounts of 3beta-hydroxydesogestrel also being observed. The Km value for 3alpha-hydroxylation by CYP2C9 cell line microsomes was 6.5 microM and the corresponding Vmax value was 1269 pmole. mg-1. min-1. Sulfaphenazole potently inhibited 3alpha-hydroxydesogestrel formation by CYP2C9 microsomes with a Ki value of 0.91 microM. There was a significant negative correlation between 3-ketodesogestrel and CYP3A4 content/activity in a panel of human livers suggesting that the further metabolism of 3-ketodesogestrel is mediated by CYP3A4. Sulfaphenazole partially inhibited 3alpha-hydroxydesogestrel and 3-ketodesogestrel formation in human liver microsomes indicating a possible in vivo role for CYP2C9. In addition, when sulfaphenazole was combined with S-mephenytoin, further inhibition of 3alpha-hydroxydesogestrel formation was observed suggesting a possible role for CYP2C19. This was confirmed in incubations with inhibitory antibodies. Whereas an anti-CYP2C9/2C19 antibody completely abolished desogestrel metabolism, anti-CYP3A4 and anti-CYP2E1 were not inhibitory. We conclude that CYP2C9 and possibly CYP2C19 and important isoforms catalysing the initial hydroxylation of desogestrel.