Analysis of human interindividual and racial differeness of drug response and metabolism : Approach by the method of diagnosis by gene analysis

对7名日本健康志愿者进行了甲苯妥英治疗，这些志愿者均为甲苯妥英4′-羟基化表型广泛（EM）和代谢不良（PM）。6名受试者被判定为EM， 1名受试者被判定为PM。12例日本人肝微粒体样本和5例高加索人肝微粒体样本也通过测定r -和s -甲苯托英4′-羟基化作用表型为EM和PM。9名日本人和5名高加索人被评为EM， 3名日本人被评为PM。从9个EM和4个PM中分离基因组DNA，分析DNA序列，并比较EM和PM中推测的甲苯托因4′-羟化酶（CYP2C形式）的序列。在CYP2C9/18序列上，EM和PM没有差异。在PM的CYP2C19外显子4序列中检测到无核苷酸替换，并在体内进行表型分析。在PM中CYP2C19外显子5前的内含子4序列检测到另一个突变，并在体外表型化。在这两种情况下，CYP2C19在PM肝脏中均不表达，我们利用EM和PM的肝微粒体体外研究了地西泮的代谢。在高底物浓度（0.2mM）下，地西泮的n -去甲基化速率约为3-羟化速率的三分之一，但随着底物浓度的降低（-0.02mM）， n -去甲基化速率增加。地西泮n -去甲基化似乎是由CYP2C形式介导的。另一方面，地西泮3-羟基化被CYP3A形式选择性催化。这些结果与在体内观察到的地西泮n -去甲基化和s -甲苯妥英4'-羟基化在人体中密切相关的结果一致。

Mephenytoin was administered to seven healthy volunteers of Japanese, and those were phenotyped extensive(EM)and poor metabolizers(PM)of mephenytoin 4'-hydroxylation.Six subjects were judged as EM,and one as PM.Twelve liver microsomal samples of Japanese and five samples of Caucasian were also phenotyped EM and PM by measuring R-and S-mephenytoin 4'-hydroxylation.Nine Japanese and five Caucasian subjects were judged as EM and three Japanese were PM.Genomic DNAs were isolated from nine EM and four PM,and DNA sequences were analyzed and the sequences of putative mephenytoin 4'-hydroxylases (CYP2C forms) were compared between EM and PM.No difference was observed on the sequences of CYP2C9/18 between EM and PM.Anucleotide substitution was detected in the sequence of exon 4 of CYP2C19 in PM,phenotyped in vivo.Another mutation was detected in the sequense of intron 4 just before exon 5 of CYP2C19 in PM,phenotyped in vitro.In either case, CYP2C19 does not express in PM livers, Metabolism of diazepam was studied in vitro by using liver microsomes obtained from EM and PM.The rate of diazepam N-demethylation was about one-third that of 3-hydoxylation at a high substrate concentration(0.2mM), however, the rate of N-demethylation increases with the decrease in the substrate concentration (-0.02mM).Diazepam N-demethylation seems to be mediated by CYP2C forms.On the other hand, diazepam 3-hydroxylation was selectively catalyzed by CYP3A forms.These results were consistent with the observation in vivo that diazepam N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans.