System for the regulation of in vivo drug concentration ; Analyzes of genetic polymorphisms of drug metabolizing enzymes

(1)碎片喹/sparteine药物氧化多态性是由于CYP2D6等位基因突变或缺失导致大量重要治疗药物和一些环境毒物无法代谢。在这项研究中，一组日本受试者通过测定尿中sparteine的代谢比率，对CYP2D6活性进行表型分析。对代谢率高（31.6）的PM基因组DNA进行序列分析。对CYP2D6基因外显子和外显子-内含子连接的序列分析显示，第9外显子有9个碱基插入。9个碱基的插入增强了该蛋白羧基末端的疏水性，并通过PCR（聚合酶链反应）-RFLP验证了该蛋白从PM母亲遗传给PM。(2) CYP1A2负责多种致癌物质的代谢激活。在人类中，CYP1A2活性的个体间差异被认为是个体癌症易感性的决定因素。利用日本人群体内咖啡因试验的数据，对非吸烟者（n=147）和吸烟者（n=58）进行了更多的分析，结果表明CYP1A2活性不是正态分布的，呈现双峰型。双峰概率图表明存在不良和广泛的表型。日本人的不良表型个体比例为14.1%。吸烟对CYP1A2的诱导作用通过在吸烟者中观察到更高的摩尔比得到证实（p<0.0001）。8个家系的家庭研究表明CYP1A2的不良表型是一种常染色体隐性遗传性状。尽管在CYP1A2基因的外显子、外显子-内含子连接和5-侧翼区域（高达2.6kb）的核苷酸序列在各表型之间没有差异。这是第一次对CYP1A2基因的表型和遗传多态性进行比较研究。(3)通过尿中AFMU/1-甲基黄嘌呤的摩尔比对n -乙酰转移酶（NAT）进行表型分析。采用PCR-RFLP法测定导致n -乙酰化缓慢的NAT突变。野生型的体内代谢比（AFMU/1X）为1.46，而杂种型为0.972,p=0.007。同种突变型为0.131 （p=0.001）。NAT2基因型与表型完全相关，但与CYP1A2代谢率无关。(4)分别从pm和EMs中测序CYP2C18和CYP2C9 cdna。PM和EM之间没有差异。采用逆转录- pcr法检测20例人肝脏组织中CYP2C18 mRNA的表达水平。肝脏CYP2C18 mRNA表达水平的个体差异超过10倍。肝脏CYP2C18 mRNA水平与甲苯妥英4，-羟基化的r /S比呈良好的相关性（r=-0.758; p<0.02）。少

(1) The debrisoquine/sparteine drug oxidation polymorphism is due to mutant or null alleles of CYP2D6 causing an incapability to metabolize a large number of therapeutically-important drugs and some environmental toxicants. In this study, a group of Japanese subjects were phenotyped for CYP2D6 activity by determination of urine metabolic ratios of sparteine. Genomic DNA from a PM,having a high metabolic ratio(31.6), was subjected to sequence analysis. Sequence analysis of exon and exon-intron junction in CYP2D6 from the PM revealed nine base-insertion in exon 9. The nine base-insertion enhances the hydrophobicity at the carboxy-terminal region of the protein, and was demonstrated to inherit from mother of PM to PM as examined by PCR (polymerase chain reaction)-RFLP.(2) CYP1A2 is responsible for the metabolic activation of various carcinogens. In human, interindividual differences of CYP1A2 activities have been regarded as determinants of individual cancer susceptibility. The probit ana … More lyzes of non-smokers (n=147) and smokers (n=58) , using data from in vivo caffeine test in Japanese population, suggested that the CYP1A2 activity was not normally distributed and appeared bimodal. The bimodal probit plot suggested the existence of poor and extensive phenotypes. The percentage of individuals with the poor phenotype in Japanese was 14.1%. Induction of CYP1A2 by cigarette smoking was confirmed by the higher molar ratio observed in smokers (p<0.0001) . Family study in eight pedigrees suggested that the poor phenotype of CYP1A2 inherited as an autosomal recessive trait. Although no differences of nucleotide sequence were observed in exons, exon-intron junctions and 5-flanking regions (up to-2.6kb) of CYP1A2 gene between each phenotypes. This is the first report in which the CYP1A2 phenotype and a genetic polymorphism in the CYP1A2 gene were comparably investigated.(3) N-Acetyltransferase (NAT) was phenotyped by the urinary molar ratio of AFMU/1-methylxanthine in the caffeine test. PCR-RFLP method was applied to determine the NAT mutations causing slow N-acetylation. In vivo metabolic ratio (AFMU/1X) of wild-type was 1.46, whereas hetero-type showed 0.972, p=0.007. Homo-mutated type showed the value of 0.131 (p=0.001). The genotype of NAT2 completely correlated well with the phenotype but did not correlate with metabolic ratio due to CYP1A2.(4) We sequenced CYP2C18 and CYP2C9 cDNAs from PMs and EMs. No difference between PM and EM was observed. The hepatic expression level of CYP2C18 mRNA was examined in 20 human liver subjects by reverse transcriptase-PCR method. Individual variations in hepatic expression levels of CYP2C18 mRNA were over 10-fold. A good correlation (r=-0.758 ; p<0.02) was observed between the hepatic levels of CYP2C18 mRNA and R/S ratios of mephenytoin 4, -hydroxylation. Less