中国健康人群P450 2C9基因多态性与甲苯磺丁脲代谢的关系

目的 研究甲苯磺丁脲人体代谢过程与中国健康志愿者P450 2C9基因多态性的关系。方法 不同P450 2C9＊1／＊1（n=53）、＊1／＊3（n=9）及＊3／＊3（n=1）基因型的健康志愿者单次口服甲苯磺丁脲500mg后，用HPLC方法测定血和尿中的甲苯磺丁脲及其代谢产物浓度，研究甲苯磺丁脲的药代过程。结果 P450 2C9＊1／＊3与P450 2C9＊3／＊3基因型的受试者与P450 2C9＊1／＊1基因型相比，AUC（0-∞）增加20％和116％，t1/2增加60％和813％，CL0为68％和11％，CLform仅为39％和3％。结论 P450 2C9＊1／＊3对甲苯磺丁脲的代谢活性较P4502C9＊1／＊1显著降低，P4502C9＊3／＊342谢活性更低，并呈现基因剂量关系。     

细胞色素P450 2C9酶活性测定探针的分析

P450 2C9是人体中重要的药物代谢酶.P450 2C9基因编码区的多态性造成氨基酸序列的变化,主要包括P450 2C9*2和P450 2C9*3两种突变体,变异纯合子对P450 2C9底物的代谢能力明显降低.目前常用的P450 2C9的探药包括甲苯磺丁脲、华法令和洛沙坦等,各种探药在检测方法、不良反应和灵敏度方面各有优缺点.     

用探药甲苯磺丁脲评价丹参片对健康人体内细胞色素P4502C9的影响

目的 用探药甲苯磺丁脲研究丹参片(抗心脑血管病中成药)对健康受试者体内细胞色素P4502C9的影响.方法 采用随机开放2周期试验设计,12例健康受试者口服丹参片14 d(1日3次,每次4片)的前后,单剂量口服甲苯磺丁脲0.5 g,以LC-MS测定给药后不同时间点甲苯磺丁脲和4-羟基甲苯磺丁脲的血浆浓度,计算口服丹参片前后的主要药代动力学参数,并进行配对t检验.结果 口服丹参片前后,甲苯磺丁脲Cmax、t1/2、CL/F和AUC0-36及4-羟基甲苯磺丁脲Cmax、t1/2和AUC0-36,经配对t检验差异均无显著意义.结论 口服丹参片14 d对人体内细胞色素P4502C9无诱导和抑制作用.     

药物代谢酶细胞色素P450 2C9研究进展

P450 2C9是人体中重要的药物代谢酶。P450 2C9基因编码区的多态性造成氨基酸序列的变化。主要包括P450 2C9*2和P450 2C9*3两种突变体，变异纯合子对P450 2C9底物的代谢能力明显降低。P450 2C9的底物包括甲苯磺丁脲、苯妥英、S-法华令、氟西汀、洛沙坦等。P450 2C9可被利福平诱导，被胺碘酮和氟康唑等多种药物抑制。     

CYP2C9基因多态性与磺酰脲类降糖药药代动力学药效学相关性的研究进展

磺酰脲类是目前临床上广泛使用的口服降糖药，在人体内通过具有基因多态性的CYP2C9酶代谢。不同基因型CYP2C9酶代谢活性的差异，是造成患者所需磺酰脲类口服降糖药剂量个体差异大的因素之一。本文综合国内外最新报道，阐述了CYP2C9基因多态性与磺酰脲类口服降糖药药动学、药效学相关性的研究进展，为制定磺酰脲类降糖药的个体化用药方案提供参者。     

RP-HPLC法测定人尿中甲苯磺丁脲及其代谢产物的浓度

目的:建立以反相高效液相色谱法测定人尿中甲苯磺丁脲及其代谢产物羧基甲苯磺丁脲和羟基甲苯磺丁脲浓度的方法。方法:以氯磺丁脲为内标,尿样经酸化后用异丙醚萃取,并进样分析;色谱柱为DiamonsilTM C18,流动相为乙腈-0.05 mol.L-1NaH2PO4(pH4.0,梯度洗脱),流速为1.0 mL.min-1,检测波长为230 nm,柱温为40℃。结果:甲苯磺丁脲、羧基甲苯磺丁脲、羟基甲苯磺丁脲尿药浓度均在0.3～10μg.mL-1范围内线性关系良好(r分别为0.999 9、1.000 0、0.999 9),最低检测浓度均为0.1μg.mL-1。各样本低、中、高浓度的方法回收率在95.00%～105.88%之间,日内、日间RSD均<6%。结论:本方法简便、准确、灵敏、重复性好,适用于细胞色素P4502C9表型分析及甲苯磺丁脲与其代谢产物的人体药动学研究。     

细胞色素P450(CYP450)遗传多态性研究进展

近年来对CYP450基因型和表型相关性的研究越来越受到重视,从临床合理用药方面来说,人们希望利用基因型分析来了解个体中药物代谢酶的活性,期望既能提高药物治疗水平同时又降低不良反应的发生;从新药研发角度来说,研究药物的代谢酶CYP450的功能能够指导新药的设计、筛选及优化。该文通过查阅国内外相关文献综述了近年来关于CYP450遗传多态性研究的进展,分别介绍了CYP2C19、CYP2C9、CYP3A4、CYP2D6、CYP1A2和CYP2E1这6种主要的药物代谢酶。研究CYP450对新药设计、筛选、评价及优化有重要意义。     

浙江沿海汉族人群CYP2C9基因多态性与甲苯磺丁脲代谢活性关系

目的探讨浙江沿海地区汉族人群CYP2C9基因多态性与甲苯磺丁脲代谢关系。方法采用寡核苷酸基因芯片,对浙江沿海地区汉族人群137名健康志愿者进行基因分型;选择含有突变型等位基因CYP2C9*3志愿者和部分野生型志愿者共20名参加甲苯磺丁脲药代动力学试验;统计分析不同CYP2C9基因型个体的代谢参数。结果在137名健康受试者DNA标本中发现CYP2C9*1/*3杂合突变型个体9名,CYP2C9*3/*3纯合突变个体1名,未发现CYP2C9*2、*4和*53种突变型等位基因,其中本研究受试人群中CYP2C9等位基因频率CYP2C9*1/*1占92.7%,CYP2C9*1/*3占6.6%,CYP2C9*3/*3占0.7%。药物代谢显示,CYP2C9*3纯合子个体的药物清除率显著低于CYP2C9*1纯合子个体。CYP2C9*1/*3杂合突变型个体组及1名CYP2C9*3/*3纯合型突变个体组,甲苯磺丁脲的代谢显著慢于野生型个体组。野生型(*1/*1)个体组、*1/*3杂合型突变个体组及1名*3/*3纯合型突变个体组,甲苯磺丁脲的药物代谢动力学参数差异与基因分型结果一致。结论CYP2C9多态性是甲苯磺丁脲药物代谢临床表现复杂性的主要因素,建立高效可靠的药代酶基因分型方法对CYP2C9代谢相关药物的临床个体化医疗具有一定的指导作用。     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

CYP2C9基因多态性对苯溴马隆药动学的影响

目的研究健康人体内不同细胞色素P450(CYP)2C9基因多态性对苯溴马隆药动学的影响。方法采用PCR-RFLP方法对120例健康志愿者进行了CYP2C9基因多态性筛查,其中20例参加苯溴马隆药动学试验。用高效液相色谱法检测受试者体内苯溴马隆的药动学参数,分析不同的CYP2C9基因分型药动学参数的差异。结果 120例志愿者中筛选出9例CYP2C9*1/*3杂合型突变体、1例CYP2C9*3/*3纯合型突变体和110例CYP2C9*1/*1野生型个体。9例CYP2C9*1/*3、1例CYP2C9*3/*3突变型(突变型组)和10例CYP2C9*1/*1(野生型组)志愿者参加了苯溴马隆的药动学试验。CYP2C9*3的等位基因频率为7.5%。突变型组的ρmax、t1/2、AUC0-24均高于野生型组,而CL/F低于野生型组,差异均具有统计学意义(P<0.05)。苯溴马隆给药24 h后,CYP2C9*1/*1野生型、CYP2C9*1/*3杂合突变型和CYP2C9*3/*3纯合突变型志愿者尿酸清除较服药前分别增加了130%、90%和75%,血尿酸浓度较服药前分别减少了60%、43%和41%;突变型组与野生型组相比,差异具有统计学意义(P<0.05)。结论 CYP2C9基因多态性对苯溴马隆的代谢具有显著的影响,苯溴马隆在CYP2C9*3突变个体的代谢显著低于CYP2C9*1野生型个体,检测突变型个体对指导临床个体化用药具有重要意义。     

用基因芯片技术测定健康人群P450 2C9基因多态性

目的 探讨基因芯片技术在P450 2C9基因分型中应用的可行性，研究中国人群P450 2C9基因多态性。方法 用基因芯片技术测定169例中国健康志愿者P4502C9基因分型，并用直接测序法对结果进行验证。结果 169例中国健康志愿者中，共发现9名P450 2C9*1／*3基因型，P450 2C9*3／*3基因型1名，无P4502C9*2，P450 2C9*4和P450 2C9*5变异。结论 基因芯片法适用于中国健康人群P450 2C9基因分型研究，健康人群：P450 2C9％3等位基因频率为0．03。     

Metabolism of steroids by cytochrome P450 2C9 variants

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild‐type CYP2C9 and ten mutants were co‐expressed with NADPH‐cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6β‐hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6β‐hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α‐hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α‐hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α‐hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α‐hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.     

Electrochemical characterisation of the human cytochrome P450 CYP2C9

The electrochemistry of human cytochrome P4502C9 (CYP2C9) was characterised using purified His-tagged enzyme. The His-tagged enzyme was shown to have similar functional characteristics to native CYP2C9 heterologously expressed in Escherichia coli and to the CYP2C9 activity of human liver microsomes. Evidence was observed for a reversible one-electron transfer between the P450 heme and the electrode. Both pH and ionic strength influenced the electrochemical behaviour of CYP2C9. A range of substrates was investigated to determine the effect of the heme-substrate interaction on CYP2C9 redox potential. In the absence of oxygen, tolbutamide, diclofenac, warfarin and sulfaphenazole did not alter the redox potential of the iron heme. In contrast, torsemide, carbon monoxide and oxygen led to an anodic shift in redox potential. These results suggest alternative mechanisms by which CYP2C9 (and by inference other P450 enzymes) may alter redox potential to facilitate electron delivery from physiological donors.     

Lornoxicam pharmacokinetics in relation to cytochrome P450 2C9 genotype

AIMS: To investigate the pharmacokinetics of lornoxicam and the relationship with CYP2C9 polymorphism in healthy Chinese subjects. METHODS: A single oral dose of 8 mg lornoxicam was administered to 18 healthy Chinese male subjects. Plasma was sampled for 24 h post dose, and plasma concentrations of lornoxicam were measured using a validated LC/MS/MS method. CYP2C9 genotype was determined by polymerase chain reaction-based restriction fragment length polymorphism or by direct sequencing of the coding region of the CYP2C9 gene. RESULTS: Of the 18 subjects, one subject was found to be a very poor metabolizer of lornoxicam with a long t(1/2) of 106 h, a low CL/F of 0.71 ml min(-1), and a high AUC(0-infinity) of 187.6 microg ml(-1) h. Genotyping studies revealed that this subject was heterozygous for CYP2C9*3 and a new variant CYP2C9 allele. Of the other 17 subjects, 13 were *1/*1 carriers, three were *1/*3 carriers, and one was a *1/*2 carrier. Mean AUC(0-infinity) values (95% confidence intervals) of lornoxicam were 9.25 (6.55, 11.95) vs. 4.75 (3.55, 5.95) microg ml(-1) h in *1 heterozygotes vs.*1 homozygotes, and mean CL/F values were 14.8 (10.2, 19.4) vs. 32.9 (24.5, 41.3) ml min(-1), respectively (P < 0.05 for both AUC and CL/F). CONCLUSIONS: The results show that the pharmacokinetics of lornoxicam are dependent on CYP2C9 polymorphism. In particular, the presence of the CYP2C9*3 allele impairs the oral clearance of lornoxicam.     

Frequency of cytochrome P450 CYP2C9 variants in a Turkish population and functional relevance for phenytoin

AIMS: The genetically polymorphic cytochrome P450 enzyme CYP2C9 metabolizes many important drugs. We studied the frequency of the amino acid variants cysteine144 (CYP2C9*2 ) and leucine359 (CYP2C9*3 ) in a Turkish population and the correlation between genotype and phenotype using phenytoin as probe drug. METHODS: CYP2C9 alleles *2 and *3 were measured in 499 unrelated Turkish subjects by PCR and restriction fragment length pattern analysis. Phenotyping was performed in a subgroup of 101 volunteers with a single oral dose of 300 mg phenytoin and concentration analysis in serum drawn 12 h after dosage. RESULTS: CYP2C9 allele frequencies in 499 unrelated Turkish subjects were 0.794 for CYP2C9*1, 0.106 for CYP2C9*2 and 0. 100 for CYP2C9*3. Mean phenytoin serum concentrations at 12 h after dosage were 4.16 mg l-1 (95% CI 3.86-4.46) in carriers of the genotype CYP2C9*1/1, 5.52 mg l-1 (4.66-6.39) in CYP2C9*1/2, and 5.65 mg l-1 (4.86-6.43) in CYP2C9*1/3. These differences were significant and accounted for 31% of total variability in phenytoin trough levels. Mean 12 h concentration ratios of 5-(para-hydroxyphenyl)-5-phenylhydantoin/phenytoin (p-HPPH/P) were 0. 43 (0.39-0.47) for CYP2C9*1/1 compared with 0.26 (0.21-0.31) for CYP2C9*1/2, 0.14 (0.13-0.14) for CYP2C9*2/2, 0.21 (0.18-0.24) for CYP2C9*1/3, and 0.02 for CYP2C9*3/3; all mutant genotypes were significantly different compared with CYP2C9*1/1. CONCLUSIONS: Frequency of the two CYP2C9 variants in Turkish subjects was in a similar range as in other Caucasian populations. A significant proportion of the interindividual variability in phenytoin trough levels is explained by the genotypes. The 12 h serum concentrations after a single phenytoin dose may be used for routine phenotyping of CYP2C9 mediated metabolic clearance and the p-HPPH/P ratios may be even more sensitive indicators of CYP2C9 activity.     

三维凝胶基因芯片法研究中国人群CYP2C9与CYP2C19基因多态性

目的：应用三维凝胶基因芯片方法研究中国人群CYP2C9与CYP2C19基因多态性,为临床合理用药提供新的技术手段与研究方法。方法：提取207名男性健康志愿者基因组DNA,PCR分别扩增包含CYP2C9＊3、＊13与CYP2C19＊2、＊3多态位点目标片段,制备三维凝胶基因芯片,荧光标记探针杂交,芯片扫描并基因分型,用直接测序法对结果进行验证。结果：207人中,11人为CYP2C9＊1/＊3基因型,1人为CYP2C9＊1/＊13基因型,1人为CYP2C9＊3/＊3基因型。2C9＊3、＊13等位基因发生率分别为2.90%、0.24%,慢代谢型发生率为0.48%。CYP2C19突变杂合子有97人,突变纯合子有26人,2C19＊2、＊3等位基因发生率分别为32.85%、3.62%,慢代谢型发生率为12.56%。四种等位基因型分布均符合Hardy-Weinberg平衡。结论：三维凝胶基因芯片方法可为临床快速基因分型提供高效平台。CYP2C9与CYP2C19基因在中国人群存在普遍多态性,检测其基因型可有效指导相关临床治疗药物应用,提高药效,降低不良反应。     

In vitro evaluation of valproic acid as an inhibitor of human cytochrome P450 isoforms: preferential inhibition of cytochrome P450 2C9 (CYP2C9)

AIMS: To evaluate the potency and specificity of valproic acid as an inhibitor of the activity of different human CYP isoforms in liver microsomes. METHODS: Using pooled human liver microsomes, the effects of valproic acid on seven CYP isoform specific marker reactions were measured: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), tolbutamide hydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1) and midazolam 1'-hydroxylase (CYP3A4). RESULTS: Valproic acid competitively inhibited CYP2C9 activity with a Ki value of 600 microM. In addition, valproic acid slightly inhibited CYP2C19 activity (Ki = 8553 microM, mixed inhibition) and CYP3A4 activity (Ki = 7975 microM, competitive inhibition). The inhibition of CYP2A6 activity by valproic acid was time-, concentration- and NADPH-dependent (KI = 9150 microM, Kinact=0.048 min(-1)), consistent with mechanism-based inhibition of CYP2A6. However, minimal inhibition of CYP1A2, CYP2D6 and CYP2E1 activities was observed. CONCLUSIONS: Valproic acid inhibits the activity of CYP2C9 at clinically relevant concentrations in human liver microsomes. Inhibition of CYP2C9 can explain some of the effects of valproic acid on the pharmacokinetics of other drugs, such as phenytoin. Co-administration of high doses of valproic acid with drugs that are primarily metabolized by CYP2C9 may result in significant drug interactions.