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

体内药物浓度调节系统；

• 批准号: 03557101
• 负责人: KAMATAKI Tetsuya
• 金额: $ 6.91万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557101/
• 关键词: Cytochrome P450; Poor metabolizer; Caffeine; N-acetyltransferase; Polymorphism; CYP1A2; CYP2D6; CYP2C; チトクローム P450; poor metabolizer; S-メフェニトイン; チトクロームP-450; P-450IID6; P-450IIC; 遺伝的多型性; P-450IA2; 薬物代謝酵素; eytensive metabolizer

(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

(1)DEBRIQUANE/SPARTEINE药物氧化多态性是由于CYP2D6基因突变或零等位基因导致的，导致大量具有治疗意义的药物和一些环境毒物不能代谢。在这项研究中，一组日本受试者通过测定尿液中司马豆碱的代谢率，对CYP2D6活性进行了表型分析。对具有较高代谢率(31.6)的PM的基因组DNA进行了序列分析。序列分析表明，PM的CYP2D6基因外显子和外显子-内含子连接在第9外显子上插入了9个碱基，这9个碱基的插入增强了蛋白质羧基末端的疏水性，经聚合酶链式反应-限制性片段长度多态性分析证实，这9个碱基的插入增强了PM的疏水性，并从母体遗传到PM。(2)CYP1A2参与多种致癌物的代谢活化。在人类中，CYP1A2活性的个体差异被认为是个体癌症易感性的决定因素。Probit Ana…非吸烟者(n=147)和吸烟者(n=58)的体内咖啡因试验结果表明，CYP1A2活性不呈正态分布，呈双峰分布。双峰概率图表明存在较差和广泛的表型。日本人不良表型个体的比例为14.1%。在吸烟者中观察到较高的摩尔比，证实了吸烟对细胞色素P1A2的诱导(p&lt；0.0001)。对8个家系的家系研究表明，CYP1A2的不良表型为常染色体隐性遗传。虽然不同表型间的基因外显子、外显子-内含子连接区和5-侧翼区(最大-2.6kb)的核苷酸序列没有差异。(3)N-乙酰转移酶(NAT)在咖啡因试验中用尿AFMU/1-甲基黄嘌呤的摩尔比进行表型鉴定。应用聚合酶链式反应-限制性片段长度多态性方法检测导致N-乙酰化缓慢的NAT突变。野生型的体内代谢率(AFMU/1x)为1.46，杂交型为0.972，p=0.007。同系变异型为0.131(p=0.001)。NAT2基因与表型完全相关，但与代谢比率无关。(4)测定了PM和EM的CYP2C18和CYP2C9基因的cDNA序列。PM组与EM组之间无明显差异。采用逆转录聚合酶链式反应(RT-PCR)方法检测了20例人肝组织中细胞色素P450 2C18基因的表达水平。肝脏细胞色素P450 2C18基因表达水平的个体差异超过10倍。肝脏细胞色素P4C18mRNA表达水平与甲苯妥因4-羟化的R/S比值之间存在良好的相关性(r=-0.758；p&lt；0.02)。较少

项目成果

25) Hisashi Hashimoto, Kenji Toide, Ryuji Kitamura, Masako Fujita, Sanae Tagawa, Susumu Itoh and Tetsuya Kamataki: "Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control." Eur.J.Biochem.218. 5

25) Hisashi Hashimoto、Kenji Toide、Ryuji Kitamura、Masako Fujita、Sanae Takawa、Susumu Itoh 和 Tetsuya Kamataki：“CYP3A4（人类肝脏中细胞色素 P450 的成人特异性形式）的基因结构及其转录控制。”

Kitamura,R.et al.: "Stable expression of cytochrome P-450lllA7 cDAN in human breast cancer cell line MCF-7 and its applicationto cytotoxicity testing.Archs." Archs,Biochem.Biophys.292. 136-140 (1992)

Kitamura, R.等人：“细胞色素 P-450lllA7 cDAN 在人乳腺癌细胞系 MCF-7 中的稳定表达及其在细胞毒性测试中的应用。Archs。”

T.Yokoi et al.: "Identification of protein disulfide isomerase and calreticulin as autoimmune antigens in LEC strain of rats" Biochim.Biophys.Acta. 1158. 339-344 (1993)

T.Yokoi 等人：“在大鼠 LEC 品系中鉴定蛋白质二硫键异构酶和钙网蛋白作为自身免疫抗原”Biochim.Biophys.Acta。

11) Toshiyuki Mori, Ryuji Kitamura, Susumu Imaoka, Yoshihiko Funae, Mitsukazu Kitada and Tetsuya Kamataki: "Examination for lipid peroxidation in liver microsomes of guinea pigs as a causal factor in the decrease in the content of cytochrome P-450 due to

11) Toshiyuki Mori、Ryuji Kitamura、Susumu Imaoka、Yoshihiko Funae、Mitsukazu Kitada 和 Tetsuya Kamataki：“豚鼠肝微粒体中的脂质过氧化检查是导致细胞色素 P-450 含量减少的原因之一

24) Toshiyuki Mori, Ryuji Kitamura, Susumu Imaoka, Yoshihiko Funae, Takashi Matsubara and Tetsuya Kamataki: "Increased CYP1A2 content and capacity to activate Glu-P-1 and Trp-P-2 in liver microsomes of scorbutic ODS rats." Carcinogen. 14. 2471-2475 (1993)

24) Toshiyuki Mori、Ryuji Kitamura、Susumu Imaoka、Yoshihiko Funae、Takashi Matsubara 和 Tetsuya Kamataki：“增加坏血病 ODS 大鼠肝微粒体中 CYP1A2 含量和激活 Glu-P-1 和 Trp-P-2 的能力。”