摘要:

There is a marked variation between people in the activity of CYP3A4 in liver and intestine. We reasoned that if CYP3A4 was expressed in peripheral blood cells, a simple blood based test of CYP3A4 phenotype might be feasible. We prepared peripheral blood smears from healthy volunteers and performed immunostaining with a rabbit polyclonal antibody that selectively reacts with enzymes within the CYP3A subfamily. Staining was observed only within the cytoplasm of neutrophils (PMNs). cDNA prepared from isolated PMNs and mononuclear cells was subjected the polymerase chain reaction using as primers synthetic oligonucleotides that selectively amplify fragments of each known CYP3A cDNAs (CYP3A3, CYP3A4, CYP3A5, and CYP3A7). Amplification was only obtained with the CYP3A5 specific oligonucleotides, predominantly in PMNs, and the identity of the amplified fragment was confirmed by sequencing. Next, whole white cell homogenate prepared from human blood was reacted on immunoblots with a monoclonal antibody that recognizes all CYP3A proteins or an absorbed polyclonal antibody that recognizes only CYP3A5. Both antibodies recognized a protein in the white cells that comigrated with purified CYP3A5. However, metabolism of midazolam, a substrate of CYP3A, could not be detected in homogenates of isolated granulocytes, in homogenates of the whole WBC fractions, or in incubations with unlysed WBC preparations. We conclude that CYP3A4 is not expressed in peripheral blood and hence a blood phenotyping test for this enzyme will not be feasible. Our discovery that CYP3A5 is present may be important since this enzyme is also present in the liver, intestine and kidney of many people.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Carisoprodol is a muscle relaxant analgesic, which has an active metabolite i.e. meprobamate. We conducted an open three-panel single-dose administration study with 15 healthy volunteers: five poor metabolizers of mephenytoin, five poor metabolizers of debrisoquine and five extensive metabolizers of both substrates. The aim was to investigate if the elimination of carisoprodol and meprobamate is dependent on the two metabolic polymorphisms of mephenytoin and debrisoquine. The subjects were given single oral doses of 700 mg carisoprodol and 400 mg meprobamate on separate occasions. The disposition of carisoprodol was clearly correlated to the mephenytoin hydroxylation phenotype. The mean serum clearance of carisoprodol was four times lower in poor metabolizers of mephenytoin than in extensive metabolizers, which confirms the hypothesis from our previous study that N-dealkylation of carisoprodol cosegregates with the mephenytoin hydroxylation polymorphism. However, mean serum clearance of meprobamate did not differ between the two groups. Also, polymorphic debrisoquine hydroxylation did not influence the elimination of carisoprodol or meprobamate. Poor metabolizers of mephenytoin thus have a lower capacity to metabolize carisoprodol and may therefore have an increased risk of developing concentration dependent side-effects such as drowsiness and hypotension, if treated with ordinary doses of carisoprodol.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The CYP2D6 gene of a Japanese sparteine poor metabolizer (PM, proband) showing a urinary sparteine metabolic ratio of 31.6 was analysed, and a heterozygous CYP2D6(D), a deletional type, was found by restriction fragment length polymorphism analysis with Xba I enzyme. The PM did not have any other previously described mutations in the CYP2D6 gene causing the loss of catalytic activity of the CYP2D6 enzyme. Thus, a possible new allele(s) responsible for the PM phenotype was analysed. The results indicated that the PM possessed a new 9-base insertion in exon 9, designated CYP2D6(/9). The CFP2D6(/9) and CYP2D6(D) alleles were clarified to be inherited from the mother [2D6(W)/2D6(J9)] and the father [2D6(W)/2D6(D)], respectively. The 9-base insertion caused a large increase in the apparent Km value for bufuralol l'-hydroxylation as examined by expression of the enzyme protein in yeast. Four of 300 Japanese carried a heterozygous CYP2D6(J9) allele (0.7%, 4/600 chromosomes) as determined by a polymerase chain reaction analysis.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Human α 1-acid glycoprotein (AAG), a plasma drug transport protein, has three main genetic variants, the A variant and the Fl and S variants, which are encoded by two different genes. The binding of disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone to the two main gene products of AAG - the A variant and a mixture of the Fl and S variants (60% Fl and 40% S) - separated by chromatography from native commercial AAG, a mixture of almost equal proportions of the Fl, S and A variants, was studied by equilibrium dialysis. A selective binding of disopyramide and methadone to the A variant and a preferential binding of dipyridamole to the F1S variant mixture were found. Lignocaine and chlorpromazine had a slight preference for binding to the A variant and to the F1S mixture, respectively, but progesterone showed no selectivity with regard to any of the variants of AAG. The differences in drug-binding demonstrated between the A variant and the F1S mixture confirmed those of a previous study, in which a selective binding of imipramine to the A variant and of warfarin and mifepristone to the F1S mixture have been found. These results indicate specific drug transport roles for each AAG variant, according to its separate genetic origin. The results of control binding experiments performed with (unfractionated) commercial AAG and the series of tested Iigands concurred with that for the separate AAG variants, with respect to the proportion of the A variant (27%) and that of the Fl and S variants (73%) in the commercial protein. In addition, disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone were used in equilibrium dialysis displacement experiments to study interactions on binding sites labelled with imipramine for the A variant and with warfarin for the F1S variant mixture. The four latter Iigands were found to competitively inhibit the binding of warfarin to the F1S variant mixture and all of them that of imipramine to the A variant. The Iigands association constants to each AAG variant obtained from such inhibitory experiments were comparable to those determined in the direct binding studies. As the stochiometry of the interactions of the A variant and the F1S variants, respectively, with their specific Iigands was approximately one (1), it was concluded that these Iigands bind to each of these variants via a single common binding site. These results indicate that the AAG molecule would have for its Iigands at least two separate binding sites, showing different specificity and localization, and not one site, as it is generally assumed. The possible pharmacological and clinical consequences of the binding results with the separate AAG variants are discussed.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The CYP2D6 gene on human chromosome 22 encodes a cytochrome P450 responsible for oxidative metabolism of over 30 clinically used drugs. The CYP2D6 gene is highly polymorphic with more than 20 alleles described to date. Some of these harbour loss-of-function mutations which lead to the poor metabolizer phenotype in 5–10% of Caucasians. These individuals are at increased risk of suffering from adverse side effects or to experience therapeutic failure following drug treatment. Phenotype determination requires ingestion of a probe drug and has other inherent problems. Due to the increasing number of alleles known, comprehensive CYP2D6 genotyping using the conventional assays has become cumbersome and time consuming. We have therefore developed a streamlined and more rapid CYP2D6 genotyping procedure. Use of long distance PCR allowed the amplification of a 4666 bp fragment which contains the entire CYP2D6 gene. The 4.7 kb fragment serves as a template for a multiplex allele-specific PCR assay to simultaneously identify the five PM-associated alleles, CYP2D6*3 (A), *4 (B), *6 (T), *7 (E), and *8 (G). Together with the CYP2D6 deletion allele CYP2D6*5 (D), which can be detected in a separate PCR assay, these alleles are responsible for the PM phenotype in approximately 99% of Caucasian individuals. We tested the reliability of the procedure by analysing DNA from more than 80 individuals with known CYP2D6 genotypes. Twelve different genotypes were present among these samples and all of them were correctly identified.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The prevalence and distribution of seven point mutations at the coding region of the highly polymorphic NAT2 gene were studied in 1008 chromosomes from healthy Spanish subjects. Most of the genes studied (78.4%) had one or more mutations, distributed in seventeen allelic variants of the NAT2 gene. Three alleles were present at high frequencies, namely NAT2*5B (41.6%), NAT2*6A (23.6%) and NAT2*4 (21.6%). The frequencies for the rest of alleles were: NAT2*12A (2.5%), NAT2*6B (2.0%), NAT2*13 (1.9%), NAT2*5A (1.5%), NAT2*7B (1.2%), NAT2*12C (1.0%), NAT2*5C (0.8%), NAT2*14C (0.8%), NAT2*14A (0.6%), NAT2*5D (0.3%), NAT2*12B (0.2%), and NAT2*14D (0.1%). In addition, we identified two new allelic variants with mutations at 191A +341C +803G (0.1%) and 282T + 59OA +803G (0.3%) which to our knowledge are described here for the first time. No other combination of mutations was identified, including the previously described allelic variants NAT2*14B, NAT2*14E, NAT2*5E and NAT2*7A. The phenotype predictive capacity of simplified PCR tests including analyses for mutations at 341C and 590A, and more sophisticated tests analysing seven mutations revealed that, in the population studied, the analysis of these two mutations is enough to predict as rapid acetylators over 99.5% subjects with two rapid genes, and about 94% subjects with one rapid gene. Given a prevalence of poor acetylators of about 55% subjects, the simplified analysis would predict the phenotype in about 97.5% subjects.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Cytochrome P450 CYP2C9 metabolizes a wide variety of clinically important drugs, including phenytoin, tolbutamide, warfarin and a large number of non-steroidal anti-inflammatory drugs. Previous studies have shown that even relatively conservative changes in the amino acid composition of this enzyme can affect both its activity and substrate specificity. To date six different human CYP2C9 cDNA sequences, as well as the highly homologous CYP2C10 sequence have been reported suggesting that the CYP2C9 gene is polymorphic. Only nine single base substitutions in the coding region of CYP2C9 account for the differences seen between the CYP2C9 proteins. In this report we have developed polymerase chain reaction (PCR)-based assays to distinguish all seven sequences, and have determined their allele frequencies in the Caucasian population. Of the seven sequences studied in one hundred individuals only three appeared to be CYP2C9 alleles. These alleles termed CYP2C9*1, CYP2C9*2 and CYP2C9*3 had allele frequencies of 0.79, 0.125 and 0.085 respectively. The CYP2C10 gene could not be found in any of the samples studied. The assays developed here will allow the prediction of CYP2C9 phenotype, thus identifying those individuals who may exhibit different drug pharmacokinetics for CYP2C9 substrates.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

One hundred and fifty-two healthy Korean volunteers were phenotyped with debrisoquine and mephenytoin and genotyped with respect to CYP2D6. The debrisoquine metabolic ratio (MR) varied between 0.09 and 6.3, and all subjects were thus classified as extensive metabolizers of debrisoquine. Polymerase chain reaction (PCR)-based amplification of genomic DNA with primers specific for the C188U2192T mutation present in exon 1 of the CYP2D6*10B allele was performed and revealed an allele frequency of 0.51 in this Korean population. Fortythree subjects (28%) were homozygous for CYP2D6*10B, 69 subjects (45%) were heterozygous for this allele, while in 40 subjects (26%) no exon 1 mutation could be found. AH subjects except one homozygous for the wild type allele had MRs below 0.75 whereas the MR was higher than 0.99 in all subjects homozygous for the CYP2D6*10B allele. The MRs in the three genotype groups were significantly different (p<0.0001; Kruskal-Wallis test). Eco RI RFLP analysis of DNA from six subjects with debrisoquine MRs≤0.11 revealed that only one (MR 0.09) carried a duplicated CYP2D6*2-gene (CYP2D6*2X2) as indicated by the Eco RI 12.1 kb haplotype. It is concluded that, as shown earlier for Chinese and Japanese populations, the CYP2D6*20B-alleIe containing the C188U2192 T mutation is the major cause of diminished CYP2D6 activity in Koreans. In this Korean population, the MR of debrisoquine was shifted towards higher values (lower CYP2D6 activity) compared with Caucasian populations but the shift appeared to be less pronounced than earlier shown for Chinese. Twenty-four subjects (16%) were poor metabolizers of S-mephenytoin as indicated by the S/R mephenytoin ratio of about 1. Twenty-three of these were genotyped with respect to the defect CYP2C19- alleles CYP2C19*2 and CYP2C19*3. Of the 46 poor metabolizer alleles, 32 (70%) were CYP2C19*2 and the remaining 14 (30%) were CYP2C19*3. Thus, the defect CYP2C19*2 and CYP2C19*3-alIeles explained 100% of the 23 Korean poor metabolizers of S-mephenytoin.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

To determine which cytochrome P450 form is involved in the promethazine [10-(2-dimethyIaminopropyl) phenothiazine] metabolism,in vitroanalysis using human liver microsomes were performed. Promethazine was mainly biotransformed to ring-hydroxylated, S-oxidized and N-demethylated metabolites. The promethazine hydroxylase in human liver microsomes was inhibited by SKF-525A, propranolol, sparteine, quinidine and anti-CYP2D6 serum suggesting involvement of a P450 related to CYP2D6. Lineweaver-Burk plots for the hydroxylation, S-oxidation and N-demethylation indicated that the hydroxylation occurred with a low Kmvalue in human liver microsomes. Microsomes from genetically-engineered human B-lymphoblastoid cells expressing CYP2D6 hydroxylated promethazine most efficiently as compared to other P450 forms, indicating that it was the principal P450 responsible for the metabolism of promethazine in human liver microsomes. The inhibition of CYP2D6- catalysed bufuralol 1-hydroxylase by various histamine H1antagonists including promethazine suggested that promethazine and some other histamine H1antagonists could be inhibitors of this P450 in human liver microsomes.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Bacterial vaginosis (BV) is the most common cause of vaginal discharge in women of childbearing age. A common symptom of this condition is a fishy-smelling vaginal discharge. Trimethylamine (TMA) is the substance which is primarily responsible for this distinctive odour. The ability to metabolize TMA is polymorphically distributed such that the majority of individuals metabolize a large part of the body burden of TMA to the odourless trimethylamine N-oxide (TMAO) which is excreted in urine with a small amount of TMA. However, in certain individuals, N-oxidation of TMA is impaired which results in the excretion of large amounts of TMA in the urine, breath, sweat and vaginal secretions. In the present study the metabolism of TMA to TMAO was determined in women with clinically diagnosed BV, women with fishy-smelling vaginal discharge but with no other evidence of BV and control women with no evidence of fishy-smelling vaginal discharge. An index of TMA N-oxidation was established for all subjects after analysis of a 24 h urine sample for free TMA and total TMA after reduction of TMAO. We show that, irrespective of diagnosis of BV or not, women with fishy-smelling vaginal discharge excrete significantly more free TMA and have a similarly significantly reduced capacity to N-oxidize TMA when compared to healthy control women. Thus, the results of this study suggest that a woman's ability to metabolize TMA to TMAO is an important factor which predisposes to a fishy-smelling vaginal discharge.

ISSN:0960-314X    

出版商:OVID    

年代:1996

数据来源: OVID