体外重组人细胞色素P450 2C9 酿酒酵母表达体系的构建及其基因多态性对药物相互作用影响的初探

 目的 体外构建重组人细胞色素 P450 2C9（CYP2C9）酿酒酵母（Saccharomyces cerevisiae）表达体系,且利用该体系研究药物对 CYP2C9 基因多态性酶抑制程度的差异性。 方法 将 CYP2C9*1（野生型）和通过定点突变 PCR 法获得的等位基因突变克隆 CYP2C9*2（R144C 突变体）和 CYP2C9*3（I359L 突变体）电穿孔转化酿酒酵母后,差速离心法制备酿酒酵母微粒体,蛋白免疫印迹法检测 3 种 CYP2C9 微粒体蛋白的表达;HPLC 法检测 CYP2C9*1 与特异性底物双氯芬酸的反应,记录产物峰面积值,利用 PrismDemo 软件计算米氏常数（Km）值;利用荧光底物 BOMCC 对 3 个等位基因进行酶活性分析,分别计算 Km、最大酶促反应速度（Vmax）和内在清除率。采用荧光高通量方法测定 5 种药物（甲苯磺丁脲、磺胺苯比唑、酮康唑、氟西汀和泰洛平）对酶的抑制程度。 结果 蛋白免疫印迹结果表明,3 个等位基因均表达目的蛋白。CYP2C9*1 代谢双氯芬酸的 Km 值为 5.34 ± 0.96 μmol/L;以 BOMCC 为底物时,CYP2C9*1 和 CYP2C9*2 的 Km 值分别为 16.94 ± 4.78、34.73 ± 5.51 μmol/L,Vmax 分别为 0.21 ± 0.10、0.12 ± 0.01 pmol/（min&;#8226;pmol P450）,内在清除率分别为 0.012 ± 0.003、0.003 ± 0.0001 µl/（min&;#8226;pmol P450）;CYP2C9*3 无催化活性。5 种药物对 CYP2C9*1 的抑制程度：磺胺苯比唑 > 酮康唑 > 氟西汀 > 甲苯磺丁脲 > 泰洛平;对 CYP2C9*2 的抑制程度：磺胺苯比唑 > 甲苯磺丁脲 > 酮康唑 > 氟西汀 > 泰洛平。 结论 成功构建了重组人细胞色素 CYP2C9 及其多态性等位基因（CYP2C9*2、CYP2C9*3）酿酒酵母表达系统;初步建立了药物对 CYP2C9 基因多态性酶的抑制作用体外检测体系,为指导临床联合用药奠定了基础。     

细胞色素P450基因多态性与药物代谢

细胞色素P450(cytochrome P450,CYP)在众多外源性和内源性物质的代谢中具有重要作用.CYP家族1-3中编码P450的基因均存在多态性,特别是CYP2C9、CYP2C19、CYP2D6和CYP3A5.超过一半的临床药物是由多态性P450介导代谢,CYP基因的多态性是造成药物反应个体差异的主要原因.近几年,许多与P450酶活性和CYP基因表达相关的等位基因已被鉴定,因此通过分型CYP基因的功能性或标签(Tag)的遗传变异,就可以获得个体的代谢表型,有助于医生及时找到正确的用药方案,有效地提高药物疗效和降低毒副作用,特别是那些治疗指数窄的药物.显然,了解CYP基因的遗传变异对于临床药物治疗和药物开发是必不可少的.基因芯片技术具有高多重水平和高通量的特点,使同时分型大量CYP基因遗传变异成为可能,是实现个性化医疗的重要技术保障.然而,DNA制备制约了预测性CYP基因分型芯片的发展,其在临床上的广泛应用尚需时日.     

人细胞色素P4502 D64个单核苷酸突变株活性及其与药物相互作用的体外研究

目的探讨单核苷酸替换对人细胞色素P450 CYP2D6酶活性及其与药物相互作用的影响。方法体外构建CYP 2D6野生株（WT）和G169R、P34S、FA10K、V7M等4个单核苷酸突变株的表达载体并在酿酒酵母中诱导表达，裂解行提取锌酶的蛋白微粒体，用蛋白质印迹法验证其表达，冉分别测定各酶代谢底物丁夫洛尔和3-[2-（N，N二乙基-N-甲铵）乙基]-7-甲氧基-4-甲基香豆素（AMMC）的米氏常数（Km）和最大酶促反应速度（Vmax）。以AMMC为底物对CYP2D6wT和V7M、G169R进行高通量药物抑制实验，所选药物包括已知的2D6抑制剂（奎尼丁、舍曲林）、底物（氯丙嗪、氟西汀、阿米替林）和既非抑制剂又非底物的化合物（酮康唑、曲格列酮），求得不同药物对不同酶的半数抑制浓度（IC50）。结果蛋白质印迹法检测显示各酶均表达良好。CYP2D6WT代谢丁呋洛尔和AMMC的Km值分别为19．22、2．06μmol、L^-1，Vmax值分别为154．53、21．60pmol·min^-1·mg^-1，Vmax／Km值分别为8．04、11．49μmin·m^-1。与CYP2D6WT比较，V7M代谢2种底物的Vmax值均要高得多（P〈0．05），G169R和E410K均稍低，而P34S都要低很多（P〈0．05）：各突变株的Km值也发生了或多或少的改变。药物对CYP2D6WT和V7M和G169R的抑制程度排序均为奎尼丁〉氯丙嗪〉氟西汀〉阿米替林〉舍曲林〉酮康唑／曲格列酮；药物对V7M和G169R的IC50值与CYP2D6WT的IC50值比值，奎尼r分别为0．88和0．80，氯丙嗪0．54和0．80，氟西汀0．65和1．02，阿米替林0．85和0．71，舍曲林0．43和0．74。结论CYP2D6部分单核苛酸突变株的酶动力学特征与野生株有明显差异，单核苷酸替换可导致酶对药物抑制的敏感性发生变化。     

广州地区汉族人群细胞色素P450 1A1和细胞色素P450 2E1基因多态性

目的 探讨广州地区汉族人群细胞色素Ｐ４５０ １Ａ１（ＣＹＰ１Ａ１）和细胞色素Ｐ４５０ ２Ｅ１（ＣＹＰ２Ｅ１）基因的多态性分布规律。方法 用ＰＣＲ－ＲＦＬＰ和等位基因特异性扩增技术,对１５０名广州地区汉族正常人的ＣＹＰ１Ａ１和ＣＹＰ２Ｅ１基因多态性进行了检测,并与其他人群进行了比较。结果 ＣＹＰ１Ａ１基因３’端非翻译区的Ｍｓｐ１多态位点ｍ１（ＭＳＰⅠ－）、ｍ２（ＭｓｐⅠ＋）等位基因频率分别为６２．３     

细胞色素P450基因遗传多态性的研究进展

细胞色素P450（cytochrome P450，CYP）在众多外源性物质和内源性物质的代谢中具有重要作用。家族1-3中编码P450的基因均存在多态性，特别是CYP2C9、CYP2C19、CYP2D6和CYP3A5。超过一半的临床药物是由多态性P450介导代谢，CYP基因的多态性是造成药物反应个体差异的主要原因。近几年，许多与P450酶活性和CYP基因表达相关的等位基因已被鉴定，因此通过分型CYP基因的功能性遗传变异或标签（Tag）遗传变异，就可以获得个体的代谢表型，有助于医生及时找到正确的用药方案，有效地提高药物疗效和降低毒副作用，特别是那些治疗指数窄的药物。显然，了解CYP基因的遗传变异对于临床药物治疗和药物开发是必不可少。基因芯片技术具有高多重水平和高通量的特点，使同时分型大量CYP基因遗传变异成为可能，是实现个性化医疗的重要技术保障。然而，DNA制备制约了预测性CYP基因分型芯片的发展，其在临床上的广泛应用尚需时日。     

细胞色素P450 1A1的研究进展

细胞色素P450酶系参与生物体内许多内源性及外源性物质的生物转化。其中细胞色素 P4501A1(CYP1A1)广泛存在于多种肝外组织,参与了多种前致癌物和致突变物的活化代谢过程,并与 多种肿瘤发生相关。本文综述了CYP1A1在体内的分布、诱导和抑制,遗传多态性及癌症易感性,简要 介绍了该领域研究的进展情况。     

细胞色素P450 2A6的多态性研究

细胞色素P450 2A6(cytochrome P450 2A6，CYP2A6)是主要的尼古丁C-氧化酶和香豆素7-羟化酶。对已发现的CYP2A6的等位基因及其对CYP2A6活性的影响，CYP2A6的多态性和个体吸烟行为及肺癌、食管癌的易感性的关联进行综述。     

细胞色素P450基因多态性与肿瘤关系研究进展

毒物代谢酶基因多态是肿瘤易感性的一个重要方面。本就细胞色素Ｐ４５０同工酶中的ＣＹＰ１Ａ１、ＣＹＰ２Ｄ６和ＣＹＰ２Ｅ１的基因多态及其与肿瘤易感性的关系作一介绍。     

人类细胞色素P450的多态性

大量不同的细胞色素P450蛋白质在人体肝和其它组织中合成,这些酶氧化药物和致癌物以及内源性化学物质,如类固醇和花生酸。正如PetorGuengerich所阐述的那样,由于遗传和环境的影响,细胞色素P450酶的活性在不同个体存在差异,在有些情况下其机理已经阐明。这种变异在决定药物毒性、先天性类固醇代谢缺陷以及可能对发生癌症风险方面都具有较大的影响。细胞色素P450酶催化药物、致癌剂和其它共栖物以及身体内源物质氧化。对于实验动物及人体的研究提示,二十多种不同的     

The Inhibitory Activity of the Extracts of Popular Medicinal Herbs on CYP1A2, 2C9, 2C19 and 3A4 and the Implications for Herb-Drug Interaction

Background

Studies have suggested an increasing practice of concurrent herb-drug consumption. One of the major clinical risks of such concomitant herb-drug use is pharmacokinetic herb-drug interaction (HDI). This is brought about by the ability of phytochemicals to inhibit or induce the activity of metabolic enzymes. The aim of this study was to investigate the potential of the crude aqueous extracts of three popular medicinal herbs used in South Africa to inhibit major cytochrome P450 (CYP) enzymes.

Materials and Methods

The extracts of Bowiea volubilis, Spirostachys africana and Tulbaghia violacea were incubated with human liver microsomes (HLM) to monitor the phenacetin O-deethylation, diclofenac 4′-hydroxylation, S-mephenytoin 4′-hydroxylation and testosterone 6β-hydroxylation as respective probe reactions for CYP1A2, CYP2C9, CYP2C19 and CYP3A4. The inhibitory activity, where observed, was profiled against the extract concentration.

Results

Extracts of Bowiea volubilis inhibited the metabolic activity of CYP1A2 and CYP3A4 with IC₅₀ values of 92.3 ± 5.5 µg/mL and 8.1 ± 0.6 µg/mL respectively. Similar observation with Spirostachys africana showed inhibitory activity against CYP1A2 and CYP3A4 with respective IC₅₀ values of 14.3 ± 0.6 µg/mL and 47.4 ± 2.4 µg/mL. Tulbaghia violacea demonstrated relatively weak inhibitory activity against CYP1A2 (767.4 ± 10.8 µg/mL) and CYP2C9 (921 ± 15.3 µg/mL).

Conclusion

The results suggest the potential for HDI between the herbs and the substrates of the affected enzymes, if sufficient in vivo concentration is attained.     

Allele frequencies of single nucleotide polymorphisms (SNPs) in 40 candidate genes for gene-environment studies on cancer: data from population-based Japanese random samples

Knowledge of genetic polymorphisms in gene-environment studies may contribute to more accurate identification of avoidable risks and to developing tailor-made preventative measures. The aim of this study was to describe the allele frequencies of single nucleotide polymorphisms (SNPs) of select genes, which may be included in future gene-environment studies on cancer in Japan. SNP typing was performed on middle-aged Japanese men randomly selected from the general population in five areas of Japan. We genotyped and calculated allele frequencies of 153 SNPs located on 40 genes: CYP1A1, CYP1B1, CYP2C9, CYP2C19, CYP2E1, CYP17A1, CYP19A1, AHR, ESR1, ESR2, ERRRG, PGR, EPHX1, EPHX2, HSD17B2, HSD17B3, GSTM2, GSTM3, GSTT2, GSTP1, NAT1, NAT2, COMT, ADH1A, ADH1B, ADH1C, ALDH2, NOS2A, NOS3, IL1A, IL1B, OGG1, NUDT1 [MTH1], DRD2, DRD3, DRD4, SLC6A4, NR3C1 [GCCR], MTHFR, and NQO1. In the present study, the Japanese allele frequencies were verified by using nationwide population samples.     

Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population

The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.     

A common polymorphism G-50T in cytochrome P450 2J2 gene is associated with increased risk of essential hypertension in a Russian population

The present study was designed to test whether common polymorphism G-50T within the promoter of human CYP2J2 gene is associated with increased risk of essential hypertension in a Russian population. We studied 576 unrelated subjects, including 295 patients with hypertension and 281 healthy subjects. Genotyping for polymorphism G-50T of the CYP2J2 gene was performed by polymerase chain reaction and restriction fragment length polymorphism techniques. The frequency of a -50T variant allele of CYP2J2 gene was significantly higher in patients with hypertension versus healthy controls (OR 4.03 95%CI 1.80-9.04 p=0.0004). The association of a -50GT genotype with hypertension remained significant after adjustment for age, gender and family history of hypertension by multivariate logistic regression (OR 4.78 95%CI 1.87-12.27 p=0.001). It has been found that OR for -50GT genotype x gender interaction (OR 4.48 95%CI 1.93-10.39 p=0.00048) was slightly higher than OR for -50GT genotype (OR 4.43 95%CI 1.91-10.29 p=0.00052), suggesting a weak effect of gender on the risk of hypertension in the heterozygous carriers of -50GT genotype. A family history of hypertension has no effect on the association between a -50GT genotype and hypertension. In present study we demonstrate for the first time that a CYP2J2*7 allele of the CYP2J2 gene is clearly associated with an increased risk of essential hypertension. Furthermore, this study highlights the importance of P-450 epoxygenase pathway of arachidonic acid metabolism in the pathogenesis of hypertensive disease.     

Effect of inmecarb on alcohol consumption and state of the cytochrome P-450 system of the liver at different stages of experimental alcoholism in rats

Research Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. V. Val'dman). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 9, pp. 283–285, September, 1990.     

细胞代谢解毒酶CYP1A1、GSTM1基因多态性与吸烟对男性肺鳞癌发病的影响

 目的 探讨细胞代谢解毒酶细胞色素P450酶1A1（CYP1A1）、谷胱甘肽S-转硫酶M1（GSTM1）基因多态性和吸烟因素对男性肺鳞癌发病的影响。方法 采用基因芯片技术对125例男性肺鳞癌患者和125例男性健康对照者CYP1A1、GSTM1基因多态性进行检测。结果 CYP1A1 m2位点GG基因型和GSTM1缺失基因型在肺鳞癌组与健康对照者间存在显著性差异(P<0.05 )。吸烟者携带CYP1A1 m2位点至少一个变异G等位基因或携带GSTM1缺失型者患肺鳞癌的危险性进一步显著增加,OR值分别为4.50和3.81(P<0.01)。结论 吸烟与CYP1A1、GSTM1基因多态性与男性肺鳞癌的发生有关。     

TIM4基因多态性与湖北汉族变应性哮喘的相关性研究

目的分析湖北地区汉族人群T细胞免疫球蛋白域黏蛋白域蛋白4（T cells immunoglobulin domin and mucin domain protein 4，TIM4）基因8570G〉A、11515C〉A单核苷酸多态性，探讨其与变应性哮喘易感性之间的关系。方法采用聚合酶链反应和限制性片段长度多态性对145例变应性哮喘患者和130名健康对照T／M4基因8570G〉A和11515C〉A多态性进行分析，计算基因型和等位基因频率。结果湖北地区健康人群TIM48570G〉AGG、GA和AA基因型频率分别是0．985、0．015和0，而哮喘患者其频率分别为0．931、0．069、0，基因型和等位基因频率在病例组与对照组间差异有统计学意义（P=0．030，P=0．032）；未检测到T／M4基因11515C〉A的多态性。结论湖北汉族人群T／M4基因8570G〉A存在单核苷酸多态性变异，可能与湖北地区汉族变应性哮喘易感性有关。