摘要:

The CYP3A4 enzyme contributes to the disposition of more than 60 therapeutically important drugs and displays marked person-to-person variability of the catalytic function. However, the extent of genetic contribution to variability in CYP3A4 activity remains elusive. Recently, we showed that a comparison of between- (SDb2) and within-person (SDw2) variances provides an estimate of the genetic component of variability in drug disposition. The aim of the present analysis was to assess the genetic control of CYP3A4 activityin vivo. A computerized literature search was conducted covering 1966 to September 1999 to identify studies reporting repeated administration of CYP3A4 substrates. The genetic contribution (rGC) to disposition of each CYP3A4 substrate was obtained by the formula (SDb2−SDw2)/SDb2. TherGCvalues approaching 1.0, point to overwhelming genetic control, whereas those close to zero suggest that environmental factors dominate. A total of 16 studies with 10 different CYP3A4 substrates were identified (n= 161 subjects). TherGCfor hepatic CYP3A4 activity as measured by midazolam plasma clearance or the erythromycin breath test was 0.96 (0.92–0.98) (95% CI) and 0.89 (0.65–0.98), respectively (P<0.05). The point estimates ofrGCfor composite (hepatic + intestinal) CYP3A4 activity measured after oral administration of cyclosporine, ethinylestradiol, ethylmorphine, nifedipine and nitrendipine, ranged from 0.66–0.98 (median: 0.83) (P<0.05). Cyclosporine data suggested a higher genetic control of CYP3A4 at night than during the day. These data indicate that further molecular genetic investigations are warranted to identify genetic variants atCYP3A4or elsewhere in the genome which contribute to regulation of CYP3A4 activity.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Tryptases are serine proteases involved in mast cell-mediated inflammatory responses which represent potential targets of drugs against diseases such as asthma, arthritis and inflammatory bowel disease. In order to interpret pharmacodynamic data on the tryptase inhibitors undergoing clinical trials, we defined the genetic variability of the tryptase 1 (TPS1) and tryptase 2 (TPS2) loci by screening a reference population of 32 individuals representing three major ethnic groups (Caucasian, African American, Asian). Using overlapping PCR products, we resequenced the entire tryptase genes with the only exclusion ofTPS2intron 1 and 20 bp ofTPS25′ untranslated region included in exon 1 and we identified 21 novel single nucleotide polymorphisms inTPS1and 17 single nucleotide polymorphisms plus a large polymorphic deletion in theTPS2gene. We also compared the type, frequency and distribution of single nucleotide polymorphisms inTPS1andTPS2and we observed that the polymorphism frequency within these two loci is unexpectedly high (approximately 1 SNP every 90 bp) and that some of the allele frequencies differ significantly among the three ethnic groups. Based on differences observed in preclinical studies using a cynomolgus monkey (Macaca fascicularis) asthma model system, we investigated the difference between monkey and human tryptase genes in order to better understand the mechanism of action of our tryptase inhibitors.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The human norepinephrine transporter (hNET) gene has five sequence polymorphisms that predict amino acid substitutions in the transporter protein: Val69Ile, Thr99Ile, Val245Ile, Val449Ile, and Gly478Ser. In order to functionally characterize the naturally occurring transporter variants, we used site-directed mutagenesis to establish the hNET variants and compared some basic pharmacological properties (uptake of norepinephrine and its inhibition by the tricyclic antidepressant desipramine) in COS-7 cells transiently expressing variant hNETs and wild-type hNET. None of the hNET variants displayed changes in the potency (Ki) of desipramine for inhibition of norepinephrine uptake. Furthermore, variants Val69Ile, Thr99Ile, Val245Ile, and Val449Ile did not affect kinetic constants (Km,Vmax) of norepinephrine uptake. However, COS-7 cells expressing the hNET variant Gly478Ser displayed an approximately four-fold increase in theKmfor norepinephrine, while theVmaxwas unaffected. The increase in theKm, which is equivalent to a four-fold reduction in the affinity of the variant hNET for its natural substrate norepinephrine, indicates that the glycine in position 478 is part of a substrate recognition domain. The reduced clearance of released norepinephrine by reuptake through the Gly478Ser variant might cause an increase in the synaptic and the circulating concentration of norepinephrine. Elevated norepinephrine concentrations have been associated with human diseases and it will be interesting to explore a possible contribution by the Gly478Ser variant to certain desease states.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Human prostatic steroid 5α-reductase, encoded by theSRD5A2gene on chromosome band 2p23, catalyses the irreversible conversion of testosterone to dihydrotestosterone (DHT), the most active androgen in the prostate, with NADPH as its cofactor. This enzyme has never been purified but a number of competitive inhibitors have been developed for this enzyme since increased steroid 5α-reductase activity may cause benign prostatic hypertrophy and prostate cancer. We report here the detailed biochemical and pharmacogenetic dissection of the human enzyme by analysing 10 missense substitutions and three double mutants which are all naturally found in humans. Nine of these 13 mutants reduce activity (measured asVmax) by 20% or more, three increase steroid 5α-reductase by more than 15% and one results in essentially unaltered kinetic properties suggesting that it is a truly neutral (`polymorphic') amino acid substitution. Substantial pharmacogenetic variation among the mutants was also observed when three competitive inhibitors, finasteride, GG745 (dutasteride) and PNU157706, were investigated. Our studies not only define the substrate and cofactor binding sites of human steroid 5α-reductase, but also have significant consequences for the pharmacological usage of steroid 5α-reductase inhibitors in human patients treated for prostatic conditions.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Cytochrome P450 3A subfamily members (CYP3A) are the most abundant liver cytochrome P450 forms, responsible for the biotransformation of over 50% of all drugs. The expression and activity of isoforms CYP3A4 and CYP3A5 show wide inter-individual variation, influencing both drug response and disease susceptibility. The molecular basis for this variation has never been defined. In this study, we used midazolam to characterize CYP3A5 phenotype in a panel of liver samples. A clear bimodality in metabolism was observed. Analysis of the 5′ flanking region of theCYP3A5gene identified two linked polymorphisms, T−369G and A−45G, located in transcriptional regulatory elements which are associated with increased expression and activity of the gene. A polymerase chain reaction based detection assay is described facilitating future studies into both the metabolic consequences of this variation and disease association studies relating to CYP3A5.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The effects of gender, time variables, menstrual cycle phases, plasma sex hormone concentrations and physiologic urinary pH on CYP2D6 phenotyping were studied using two widely employed CYP2D6 probe drugs, namely dextromethorphan and metoprolol. Phenotyping on a single occasion of 150 young, healthy, drug-free women and men revealed that the dextromethorphan : dextrorphan metabolic ratio (MR) was significantly lower (P<0.0001) in 56 female extensive metabolizers (0.008±0.021) compared to 86 male extensive metabolizers (0.020±0.040). Urinary pH was a significant predictor of dextromethorphan : dextrorphan MRs in men and women (P<0.001). Once-a-month phenotyping with dextromethorphan of 12 healthy young men (eight extensive metabolizers and four poor metabolizers) over a 1-year period, as well as every-other-day phenotyping with dextromethorphan of healthy, pre-menopausal women (10 extensive metabolizers and 2 poor metabolizers) during a complete menstrual cycle, did not follow a particular pattern and showed similar intrasubject variability ranging from 24.1% to 74.5% (mean 50.9%) in men and from 20.5% to 96.2% (mean 52.0%) in women, independent of the CYP2D6 phenotype (P=0.342). Using metoprolol as a probe drug, considerable intrasubject variability (38.6±12.0%) but no correlation between metoprolol : α-hydroxymetoprolol MRs and pre-ovulatory, ovulatory and luteal phases (mean±SD metoprolol : α-hydroxymetoprolol MRs: 1.086±1.137 pre-ovulatory; 1.159±1.158 ovulatory and 1.002±1.405 luteal phase;P>0.9) or 17β-oestradiol, progesterone or testosterone plasma concentrations was observed. There was a significant inverse relationship between physiologic urinary pH and sequential dextromethorphan : dextrorphan MRs as well as metoprolol : α-hydroxymetoprolol MRs in men and women, with metabolic ratios varying up to six-fold with metoprolol and up to 20-fold with dextromethorphan (ANCOVAP<0.001). We conclude that apparent CYP2D6 activity is highly variable, independent of menstrual cycle phases, sex hormones, time variables or phenotype. Up to 80% of the observed variability can be explained by variations of urinary pH within the physiological range. An apparent phenotype shift as a result of variations in urinary pH may be observed in individuals who have metabolic ratios close to the population antimode.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysedN-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of theFMO3gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for theFMO3Thr82allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus–insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse theN-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMAN-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype–phenotype relationships in trimethylaminuria.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Paraoxonase (PON1) is tightly associated with high-density lipoprotein particles and is believed to contribute to the prevention of atherosclerosis by metabolizing oxidized lipids. PON1 also hydrolyses the bioactive oxon forms of organophosphorus pesticides such as parathion, diazinon and chlorpyrifos. Two common polymorphisms have been identified in the coding sequence of human PON1: L55M and R192Q. Several previous studies have found that the presence of thePON1R192allele raises the risk of cardiovascular disease while others found no correlation. The studies, however, have focused on the genotype of PON1 and not the expression level of the protein. We found that the PON1 expression level in plasma, as determined by the rates of paraoxon and diazoxon hydrolysis, varies widely among individuals and within a genotype. Previous studies found that individuals having Met at PON155have lower levels of both PON1 mRNA and activity. In this study, we determined the plasma activity levels of PON1 and examined the relationships betweenPON155genotype and PON1 level. As withPON1192, we found considerable overlap in activity among thePON155genotypes. Of the 317 individuals whose PON1 status was determined in this study, 48.9% werePON1Q192homozygotes. Analysis of the PON1QQ192population showed that while the average PON1 activity (diazoxon hydrolysis) was 12266 U/L for PON1LL55and 7777 U/L for PON1MM55, a given PON1MM55individual could have more than twice the activity of a PON1LL55individual. PON1 status, which includes PON1 level as well asPON1192genotype, may be a better predictor for cardiovascular disease or organophosphate susceptibility thanPON1genotype alone.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The relationship of breast cancer to cigarette smoking is inconsistent in the literature, possibly due in part to heterogeneity in carcinogen metabolism.N-acetyltransferase 2 (NAT2) enzyme activity is believed to play a role in the activation of tobacco smoke carcinogens. We examined the effect ofNAT2genetic polymorphisms on risk of breast cancer from active and passive smoking. Women were recruited from those who had suspicious breast masses detected clinically and/or mammographically. Questionnaire data were collected prior to biopsy diagnosis to blind subjects and interviewers. Histopathology showed 113 cases with mammary carcinoma (30 carcinomain situ) and 278 controls with benign breast disease.NAT2genotype was determined using allele-specific polymerase chain reaction amplification to detect slow acetylator mutations. Effects of passive and active tobacco smoke and ofNAT2genotype on breast cancer risk were examined with logistic regression controlling for known risk factors. Models first included all controls, and subsequently 107 with no or low risk (normal breast or no hyperplasia), and finally 148 with high risk (hyperplasia, atypical hyperplasia, complex fibroadenomas). Referents had no active or passive smoke exposure. We found no association between breast cancer risk andNAT2, smoking status (never, former, current), smoking duration, or cigarettes per day. There were no effects of passive exposure among never-smokers. Models were unchanged across control groups. There were no statistical interactions between tobacco smoke exposure andNAT2. The results were similar when restricting the analysis to invasive cancers. These findings do not support the hypothesis thatNAT2is a risk factor for breast cancer or that it alters susceptibility to tobacco smoke.

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0960-314X    

出版商:OVID    

年代:2000

数据来源: OVID