反相HPLC法同时测定人尿中茶碱及其两种代谢产物

目的:建立一种同时测定人尿中茶碱及其1,3-二甲基尿酸(1,3-DMU)和3-甲基噻嗪(3-MX)代谢产物的HPLC方法.方法:尿样用异丙醇/二氯甲烷(2/8)混合液提取,有机相在空气吹干,用流动相复溶后进行HPLC分析.色谱柱为Diamonsil ODS C_(18)5 μm,150 mm×4.6 mm I.D),流动相由0.1%甲酸液和乙腈(95:5)组成,流速1.0 mL/min,测定波长280 nm.测定12名受试者单剂量和多剂量口服茶碱后24 h内尿中茶碱及其代谢物累计排泄量.结果:尿中茶碱及其代谢物1,3 DMU和3-MX的线性范围分别为0.312～40.0、0.156～20.0、0.078～10.μg/mL,最低可定量浓度分别为0.312、0.156、0.078μg/mL.批间和批内的变异小于15%,回收率大于70%.结论:该方法的特异性、灵敏度能够满足临床上对人尿中茶碱及其代谢产物同时测定的要求.     

Characterization of human liver cytochromes P-450 involved in theophylline metabolism.

Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of decursinol angelate on the pharmacokinetics of theophylline and its metabolites in rats

Herb-drug interactions represent a serious problem as herbal medicine is used extensively in the modern world. This study investigated the effects of decursinol angelate on the pharmacokinetics of theophylline, a typical substrate of the cytochrome P450 1A2 enzyme, in rats. After 3 days of decursinol angelate pretreatment, on the fourth day, rats were administered decursinol angelate and theophylline concomitantly. Blood theophylline and its major metabolite [1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 1-methyluric acid (1-MU), and 1,3-dimethyluric acid (1,3-DMU)] levels were monitored by liquid chromatography-tandem mass spectroscopy. The results indicated that theophylline clearance significantly decreased and the area under the concentration–time curve (AUC) increased in decursinol angelate (25 mg/kg)-pretreated rats administered theophylline (10 mg/kg). The elimination half-life (t1/2) of theophylline was increased by 20%. In the presence of decursinol angelate (25 mg/kg), the pharmacokinetic parameters of three metabolites (1-MX, 1,3-DMU, and 1-MU) were significantly altered (half-life for 1-MU, and AUC24 h for 1-MX, 1,3-DMU, and 1-MU). Our results suggest that patients receiving CYP1A2-metabolized drugs, such as caffeine and theophylline, should be advised of the potential herb-drug interaction to reduce the risk of therapeutic failure or increased toxicity of conventional drug therapy.     

Pharmacokinetics of theobromine and its metabolites in rabbits

The pharmacokinetics of theobromine (3, 7-DMX) and its metabolites was investigated in detail in four male rabbits after bolus intravenous injection (4 mg/kg) of the compound. Apparent first-order rate constants for the metabolic processes involved in the formation of 3,7-DMX metabolites and their excretion in urine were calculated. Theobromine, 7-methylxanthine (7-MX) and 3-methylxanthine (3-MX) were measured in blood and urine, and the other metabolites were determined only in urine. An appropriate model of 14 compartments is formulated to describe the disposition of 3,7-DMX and its metabolites.     

Theophylline metabolism by human, rabbit and rat liver microsomes and by purified forms of cytochrome P450

The capacity of human, rabbit and rat liver microsomes and purified isozymes of cytochrome P450 to metabolize theophylline has been assessed. In all three species the 8-hydroxylation of theophylline to 1,3-dimethyluric acid (1,3-DMU) was the major pathway. In human, control rabbit and rat liver microsomes this metabolite accounted for 59, 77 and 94%, respectively, of the total metabolites formed. In both human and control rabbit liver microsomes the N-demethylation of theophylline to 1-methylxanthine (1-MX) accounted for 20% of the total metabolites formed. N-demethylation of theophylline to 3-methylxanthine (3-MX) accounted for 21% of theophylline metabolism in human microsomes but was a minor pathway in control rabbit and rat microsomes. Acetone and phenobarbitone pretreatment markedly increased the formation of 1,3-DMU by rabbit liver microsomes. Rifampicin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration caused a slight but significant increase in this pathway. In general the N-demethylation pathways in rabbit liver microsomes were refractory to induction. In the rat, the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU were all significantly increased in Aroclor 1254, dexamethasone, phenobarbitone and 3-methylcholanthrene-treated microsomes. In reconstitution experiments the polycyclic hydrocarbon inducible rabbit cytochrome P450 Forms 4 and 6 and the constitutive Form 3b all metabolized theophylline to its three metabolites. In human liver microsomes from four subjects anti-rabbit cytochrome P450 Form 4 IgG inhibited the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU by approximately 30%. These data indicate that theophylline is metabolized by multiple forms of cytochrome P450 in human, rabbit and rat liver microsomes.     

Pharmacokinetics of theobromine and its metabolites in rabbits

The pharmacokinetics of theobromine (3, 7-DMX) and its metabolites was investigated in detail in four male rabbits after bolus intravenous injection (4 mg/kg) of the compound. Apparent first-order rate constants for the metabolic processes involved in the formation of 3,7-DMX metabolites and their excretion in urine were calculated. Theobromine, 7-methylxanthine (7-MX) and 3-methylxanthine (3-MX) were measured in blood and urine, and the other metabolites were determined only in urine. An appropriate model of 14 compartments is formulated to describe the disposition of 3,7-DMX and its metabolites.     

Associations between CYP2E1 promoter polymorphisms and plasma 1,3-dimethyluric acid/theophylline ratios

Objective Theophylline is metabolized to 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine, and 1-methylxanthine by CYP1A2 and partly by CYP2E1. Because 1,3-DMU is the major metabolite of theophylline, the 1,3-DMU/theophylline ratio is viewed as a good indicator of theophylline metabolic clearance. Here, we investigated the associations between 1,3-DMU/theophylline ratios and genetic polymorphisms of CYP2E1 and CYP1A2.Methods Polymerase chain reaction (PCR) and direct sequencing or PCR-restriction fragment length polymorphism (RFLP) were performed to analyze CYP2E1 and CYP1A2 promoter polymorphisms in 62 Korean asthma patients. Plasma theophylline and 1,3-DMU levels were measured by liquid chromatography-tandem mass spectrometry.Results Eleven polymorphisms including Ins₉₆, −1566 T>A, −1515 T>G, −1414 C>T, −1295 G>C, −1055 C>T, −1027 T>C, −930 A>G, −807 T>C, −352 A>G, and −333 T>A were detected in the 5′ flanking region of the CYP2E1 gene (numbering according to GenBank Accession number NT_017795). Of these, five single nucleotide polymorphisms (SNPs) (−1566 T>A, −1295 G>C, −1055 C>T, −1027 T>C, and −807 T>C) were closely linked. Another three polymorphisms (Ins_96, −930 A>G, and −352 A>G) and two polymorphisms (−1515 T>G and −333 T>A) were also closely linked. The five closely linked polymorphisms were associated with significantly different 1,3-DMU/theophylline ratios between heterozygotes plus homozygotes of a rare allele (n=23, 0.0368±0.0171) and common allelic homozygotes (n=39, 0.0533±0.0343) (p=0.024 by Mann-Whitney U test). In the CYP1A2 gene, the −2964G>A polymorphisms exhibited a significant difference in 1,3-DMU/theophylline levels between heterozygotes plus homozygotes of a rare allele (n=30, 0.0406±0.0272) and homozygotes of a common allele (n=32, 0.0534±0.0316) (p=0.032).Conclusion We confirm that hydroxylation at the 8 position of theophylline (1,3-DMU) is significantly affected by genetic polymorphism in CYP2E1 in addition to CYP1A2.     

Renal clearance of theophylline and its major metabolites: Age and urine flow dependency in paediatric patients

Summary The renal clearance of theophylline (TH) and its metabolites 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), and 1-methyluric acid (1-MU) has been studied in 10 children aged 8 months to 14 years. Individual renal clearances were calculated from serum levels and amounts excreted in urine after i.v. administration of the parent drug. The clearance of 1,3-DMU was found to depend both upon urine flow rate and age, which are interrelated. An effect of urinary pH was expected, but was not studied. Consistent age-dependent changes in the relative quantities of metabolites excreted were not observed.     

Effects of water deprivation on the pharmacokinetics of theophylline and one of its metabolites, 1,3-dimethyluric acid, after intravenous and oral administration of aminophylline to rats

It has been reported that the expressions of hepatic microsomal cytochrome P450 (CYP) 1A1/2, 2B1/2 and 3A1/2 were not changed in rats with water deprivation for 72 h (rat model of dehydration) compared with the controls. It has been also reported that 1,3-dimethyluric acid (1,3-DMU) was formed from theophylline via CYP1A1/2 in rats. Hence, it could be expected that the formation of 1,3-DMU could be comparable between the two groups of rats. As expected, after both intravenous and oral administration of theophylline at a dose of 5 mg/kg to the rat model of dehydration, the AUC of 1,3-DMU was comparable to the controls. After both intravenous and oral administration of theophylline to the rat model of dehydration, the Cl(r) of both theophylline and 1,3-DMU was significantly slower than the controls. This could be due to significantly smaller urinary excretions of both theophylline and 1,3-DMU since the AUC of both theophylline and 1,3-DMU were comparable between the two groups of rats. The smaller urinary excretion of both theophylline and 1,3-DMU could be due to urine flow rate-dependent timed-interval renal clearance of both theophylline and 1,3-DMU in rats.     

大豆苷元在人肝微粒体中的单羟化代谢机制大豆苷元在人肝微粒体中的单羟化代谢机制

目的探讨大豆苷元在人肝微粒中羟基化代谢所涉及的肝细胞色素P450(CYP)同工酶，为研究其在人体内的代谢提供基础。方法通过分析大豆苷元在肝微粒体中和重组CYP酶中形成的单羟化代谢物的酶促动力学,分析其酶学模型，然后用不同CYP同工酶选择性抑制剂或底物进行抑制实验，初步筛选出介导大豆苷元单羟化代谢所涉及的CYP同工酶。结果代谢物的形成动力学符合米氏方程单酶模型。CYP1A2选择性抑制剂呋喃茶碱和CYP1A2单克隆抗体均能明显抑制3种单羟化代谢物的形成。而其他CYP选择性的抑制剂对3种代谢物的形成没有或较小产生抑制作用。用重组酶实验得出相同结果。结论体外肝微粒体研究表明，大豆苷元的单羟基代谢主要由CYP1A2所介导。     

Mechanism of formation of 6-amino-5-(N-methylformylamino)-1-methyluracil and 3,7-dimethyluric acid from theobromine in the rat in vitro: involvement of cytochrome P-450 and a cellular thiol

1. The involvement of glutathione (GSH) and cytochrome P-450 in the conversion of theobromine to 6-amino-5-(N-methylformylamino)-1-methyluracil (3,7-DAU) and 3,7-dimethyluric acid (3,7-DMU) has been investigated in rat liver microsomal incubations. 2. The ratio of formation of 3,7-DAU to 3,7-DMU increased with increasing GSH concentration, reaching a maximum (approximately 12:1) at 2 mM. For any given added GSH concentration the formation of 3,7-DAU plus 3,7-DMU remained constant. 3. 3,7-DAU and 3,7-DMU formation were increased approx. 12- and 1.6-fold in liver microsomes from rats treated with 3-methylcholanthrene and phenobarbitone, respectively. Cimetidine, metyrapone and SKF-525A each inhibited the conversion of theobromine to 3,7-DAU and 3,7-DMU. 4. Apparent Km and Vmax values for the combined formation of 3,7-DAU and 3,7-DMU were the same in the absence and presence of GSH, 2 mM. 5. L-Cysteine and N-acetyl-L-cysteine were as effective as GSH in causing a shift from 3,7-DMU to 3,7-DAU formation, but the non-thiol reducing agents ascorbic acid and alpha-tocopherol were ineffective. 6. Data are consistent with the hypothesis that 3,7-DAU and 3,7-DMU are derived from a common oxidized intermediate of theobromine, the formation of which is rate-limiting. The putative intermediate normally serves as a precursor to 3,7-DMU but in the presence of GSH, or some other cellular thiol, it may be reduced to give 3,7-DAU.     

Diet-induced alterations in theobromine disposition and toxicity in the rat

To assess the potential influence of diet on theobromine (TBR) disposition and the development of TBR-induced thymic and testicular toxicity, male Sprague-Dawley rats were given the following diets ad libitum for 28 days: (1) semipurified (S); (2) commercial chow (Ch); (3) semipurified + 0.6% TBR (S + TBR); or (4) commercial chow + 0.6% TBR (Ch + TBR). Toxicity endpoints determined in each TBR group indicated that Ch + TBR-treated animals did not exhibit the marked reduction in body weight or testicular atrophy induced by the S + TBR diet, although thymic weight was lower regardless of diet. Metabolic studies performed after the 28-day feeding period using 5 mg/kg TBR + 10 microCi [8-14C]TBR revealed an overall inductive effect of Ch on TBR metabolism as shown by increased urinary excretion (0-24 hr) of the major TBR metabolite, 6-amino-5[N-methylformylamino]-1-methyluracil (6-AMMU), as well as 7-methylxanthine + 3-methylxanthine (7-MX + 3-MX) and 3,7-dimethyluric acid (3,7-DMU). Consumption of 0.6% TBR for 28 days in either S or Ch diets also induced its own metabolism, as shown by decreased urinary excretion of unchanged TBR and increased conversion primarily to 3,7-DMU. Fecal 14C elimination (0-24 hr) was similar between animals fed S and Ch diets, indicating no effect of control diet on TBR bioavailability. Since serum TBR concentrations and overall toxicity were lower in Ch + TBR-treated animals than in S + TBR treated animals, yet TBR bioavailability was similar, this effect was attributed to the inducing potential of the Ch diet on TBR metabolism and clearance. Investigators are cautioned to consider the potential effect of diet on metabolism when performing and evaluating toxicological studies.     

Biotransformation of caffeine by cDNA-expressed human cytochromes P-450

Objectives: The biotransformation of caffeine has been studied in vitro using human cytochrome P-450 isoenzymes (CYPs) expressed in human B-lymphoblastoid cell lines, namely CYP1A1, 1A2, 2A6, 2B6, 2D6-Val, 2E1 and 3A4, and microsomal epoxide hydroxylase (EH). In addition, CYP 2D6-Met was also studied, in which a valine in the wild type (CYP2D6-Val) has been replaced by a methionine due to a G to A mutation in position 112. Results: At caffeine 3 mmol·l^-1, five CYPs (1A1, 1A2, 2D6-Met, 2E1 and 3A4) catalysed the biotransformation of caffeine. Among the enzymes studied, CYP1A2, which predominantly catalysed paraxanthine formation, had the highest intrinsic clearance (160 l h^-1·mmol^-1 CYP). Together with its high abundance in liver, it should be considered, therefore, to be the most important isoenzyme in caffeine metabolism. The affinity of caffeine for CYP1A1 was comparable to that of its homologue 1A2. CYP2D6-Met, which catalysed caffeine metabolism by demethylation and 8-hydroxylation, also had a relatively high intrinsic clearance (3.0 l·h^-1mmol^-1 CYP), in particular for theophylline and paraxanthine formation, with k_M values between 9–16 mmol·l^-1. In contrast, the wild type, CYP2D6-Val, had no detectable activity. In comparison, CYP2E1 played a less important role in in vitro caffeine metabolism. CYP3A4 predominantly catalysed 8-hydroxylation with a k_M value of 46 mmol·l^-1 and an intrinsic clearance of 0.60 l·h^-1·mmol^-1 CYP. Due to its high abundance in human liver, the latter CYP may contribute significantly to the in vivo formation of TMU. Conclusion: The findings of this study indicate that i) microsomes from transfected human B-lymphoblastoid cell lines give results close to those obtained with microsomes isolated from human liver, ii) at least four CYP isoforms are involved in caffeine metabolism, iii) at a substrate concentration <0.1 mmol·l^-1, CYP1A2 and 1A1 are the most important isoenzymes, iv) at higher concentrations the participation of other isoenzymes, in particular CYP3A4, 2E1 and possibly also CYP2D6-Met, are important in caffeine metabolism, and v) the nucleotide composition at position 1120 of CYP2D6 determines the activity of this isoenzyme in caffeine metabolism.Abbreviations AFMU 5-acetylamino-6-formylamino-3-methyluracil - CYP human cytochrome P-450 - PAH polycyclic aromatic hydrocarbon - 17X paraxanthine - 37X theobromine - 13X theophylline - 137U trimethyluric acid.     

Expression and activity of CYP2E1 in circulating lymphocytes are not altered in diabetic individuals

Cytochrome P4502E1 (CYP2E1) plays an important role in ROS production thus favouring accelerated membrane lipid peroxidation. This isoform is strongly expressed in the liver but it can be also found in lymphocytes. As such, lymphocyte may provide a non-invasive accessible pool for screening CYP2E1 expression in man. We have, therefore, analysed CYP2E1 expression and activity in lymphocyte microsomes from 12 healthy controls, 11 type 1 and 12 type 2 diabetic subjects by using Western blot and enzymatic activities. Immunoblotting did not show difference among CYP2E1 protein bands in controls, type 1 and type 2 diabetics. To assess CYP2E1 activity we used the 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), as a fluorescent substrate. The rate of deethylation of 7-EFC from controls did not differ from type 1 and type 2 diabetic subjects. The lack of any difference in CYP2E1 activity also was confirmed by the NADPH-dependent microsomal lipid peroxidation CCL4-induced assay showing similar peroxidation rates among controls and diabetic subjects. The results show that CYP2E1 expression/activity in lymphocytes is not enhanced in diabetes.     

慢性间断性低氧对大鼠肝脏CYP3A_2和CYP2E_1的影响研究

目的研究慢性间断性低氧对大鼠肝脏CYP3A2和CYP2E1的影响。方法将大鼠随机分为对照组、低氧3d组、低氧7d组、低氧14d组、低氧28d组,低氧处理结束24h后,常规腹腔注射麻醉,摘取眼球血液2ml制备血清,并测定丙氨酸氨基转移酶(ALT)、天门冬酸氨基转移酶(AST)、红霉素N-脱甲基酶(ERD)、苯胺羟化酶(ANH)活性;取新鲜肝组织以制备微粒体和提取核糖核酸(RNA),并以RT-PCR进行基因片段扩增以检测大鼠肝脏细胞色素CYP3A2、CYP2E1的mRNA表达水平。结果慢性间断性低氧对血清ALT、AST活性无明显影响;低氧7d后,大鼠肝脏ERD、ANH活性明显升高,28d时诱导率分别为155.5%、42.2%;同时,CYP3A2、CYP2E1mRNA的表达水平也分别增加了220.5%、102.8%。结论慢性间断性低氧能显著增加大鼠肝脏ERD、ANH活性,其机制可能与其在转录水平上提高肝脏CYP3A2和CYP2E1的基因表达水平有关。     

咖啡因探针法测定正常人肝脏药物代谢酶CYP1A2活性

目的:建立咖啡因4种主要代谢物含量的测定方法,探讨咖啡因代谢物在药物代谢酶CYP1A2活性评价中的意义。方法:采用反相高效液相梯度洗脱法测定尿液内咖啡因代谢产物5—乙酰氨基—6—甲酰氨基—3—甲基尿酸(AFMU)、1—甲基尿酸(1U)、1—甲基黄嘌呤(1X)和1,7—二甲基尿酸(17U)的相对含量,计算代谢物比率(AFMU+1X+1U)/17U,绘制频数分布直方图,评价CYP1A2活性。结果:受试者代谢物比率平均值为4.27,呈正态分布。结论:本方法简便、准确、快速,适合于尿液中咖啡因代谢物的测定及CYP1A2活性的研究。     

The human hepatic metabolism of simvastatin hydroxy acid is mediated primarily by CYP3A,and not CYP2D6

AIMS: To identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of simvastatin hydroxy acid (SVA), the most potent metabolite of simvastatin (SV). METHODS: The metabolism of SVA was characterized in vitro using human liver microsomes and recombinant CYPs. The effects of selective chemical inhibitors and CYP antibodies on SVA metabolism were assessed in human liver microsomes. RESULTS: In human liver microsomes, SVA underwent oxidative metabolism to three major oxidative products, with values for Km and Vmax ranging from about 50 to 80 microM and 0.6 to 1.9 nmol x min(-1) x mg(-1) protein, respectively. Recombinant CYP3A4, CYP3A5 and CYP2C8 all catalysed the formation of the three SVA metabolites, but CYP3A4 was the most active. CYP2D6 as well as CYP2C19, CYP2C9, CYP2A6, CYP1A2 did not metabolize SVA. Whereas inhibitors that are selective for CYP2D6, CYP2C9 or CYP1A2 did not significantly inhibit the oxidative metabolism of SVA, the CYP3A4/5 inhibitor troleandomycin markedly (about 90%) inhibited SVA metabolism. Quercetin, a known inhibitor of CYP2C8, inhibited the microsomal formation of SVA metabolites by about 25-30%. Immunoinhibition studies revealed 80-95% inhibition by anti-CYP3A antibody, less than 20% inhibition by anti-CYP2C19 antibody, which cross-reacted with CYP2C8 and CYP2C9, and no inhibition by anti-CYP2D6 antibody. CONCLUSIONS: The metabolism of SVA in human liver microsomes is catalysed primarily (> or = 80%) by CYP3A4/5, with a minor contribution (< or = 20%) from CYP2C8. CYP2D6 and other major CYP isoforms are not involved in the hepatic metabolism of SVA.     

Identification of the human cytochromes P450 catalysing the rate-limiting pathways of gliclazide elimination

AIMS: To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6beta-hydroxy (6beta-OHGz), 7beta-hydroxy (7beta-OHGz) and hydroxymethyl (MeOH-Gz) metabolites of gliclizide (Gz). METHODS: 6beta-OHGz, 7beta-OHGz and MeOH-Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high-performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. RESULTS: Microsomes from six human livers converted Gz to its 6beta-OHGz, 7beta-OHGz, and MeOH-Gz metabolites, with respective mean (+/- SD) K(m) values of 461 +/- 139, 404 +/- 143 and 334 +/- 75 microm and mean V(max) values of 130 +/- 55, 82 +/- 31 and 268 +/- 115 pmol min(-1) mg(-1), respectively. V(max)/K(m) ratios for the microsomal reactions parallelled relative metabolite formation in vivo. Sulfaphenazole inhibited microsomal 6beta-OHGz, 7beta-OHGz and MeOH-Gz formation by 87, 83 and 64%, respectively, whereas S-mephenytoin caused significant inhibition (48%) of only MeOH-Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6beta-OHGz and 7beta-OHGz. CONCLUSION: Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH-Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics.     

CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响(英文)

目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶。方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学。鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶。结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征。当底物浓度为氟西汀25μmol/L和100μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r_1=0.821,P_1=0.001;r_2=0.668,P_2=0.013),当底物浓度为氟西汀100μmol/L时,N-FLU的生成率与S-美芬妥因4’-羟化酶活性显著相关(r=0.717,P=0.006)。PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73％vs 45％,P<0.01)。结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主。