【作者】 司大勇；

【导师】 周慧；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 细胞色素P450(CYP)2C亚家族包括CYP2C9、CYP2C19、CYP2C8等,都是重要的药物代谢酶。据估计,CYP2C9负责代谢清除15%的在人体内的经过一相代谢的药物。而CYP2C19也代谢很多临床重要药物。他们的基因的多态性和在体内受诱导或抑制的程度被认为决定了相关药物代谢的个体和种族差异。在本文的第一部分研究中,一个新的CYP2C9等位基因CYP2C9~*13(Leu90Pro单氨基酸取代突变)被首次识别,约2.0%的中国汉族人是本突变的杂合携带者。CYP2C9~*13酶活力较野生型显著下降。计算机结构模拟研究表明这主要由于底物口袋体积的缩小和形状的改变。本文第二部分研究了CYP2C9和CYP2C19基因多态性与受试者格列奇特代谢能力减弱的相关性,首次发现是CYP2C19基因多态性决定了格列奇特代谢速度个体差异。本文也研究了CYP2C9和CYP2C19基因多态性对人体中卡利普多和格列本尿代谢速度的影响。第三部分研究了CYP2C9受抑制的特性,发现大多数黄酮类和黄酮醇类化合物都能在体外强烈抑制CYP2C9,计算机结构模拟研究核定点突变证明抑制位点有两处,分别在底物结合位置和别构位点上;另外本文也研究了CYP2C9多态性对其受抑制能力的影响。更多还原

【Abstract】 Cytochrome P450s（CYPs） are the most important superfamily of biotransformation enzymes that are involved in oxidative metabolism of a wide variety of endogenous and exogenous compounds.The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism and plays a key role in the drugs pharmacological and toxicological effects.CYP2C9 is the most abundant 2C subfamily isozyme in human liver which is responsible for the metabolic clearance of a wide variety of the therapeutic agents,estimated up to 16%of drugs in current clinical used.CYP2C19 ranks amongst the most important drug metabolizing enzymes in human,too.In the first part of the article,CYP2C9*13 was found in a person with lornoxicam half-life of about 105 hours was markedly longer than that of other subjects.This new variant CYP2C9 allele involving a T269C transversion in exon 2 that leads to a Leu90Pro substitution in the encoded protein（CYP2C9*13）.Frequency analysis in 147 unrelated Chinese males indicated approximately 2%of the Chinese population carry the allele.Then, the pharmacokinetics of tolbutamide in the PM subjects was investigated, and he was found to be a phenotypically poor metabolizer of tolbutamide as well.Since tolbutamide is widely accepted as a probe substrate for the assessment of hepatic CYP2C9 activity in vivo,this study confirms that the study subject has severely impaired CYP2C9 catalytic efficiency.This novel CYP2C9 allele was correlated with reduced plasma clearance of both lornoxicam and tolbutamide.In order to explore the structure-activity relationship of CYP2C9*13,the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1（PDB code 1R9O） by molecular dynamics simulations.The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam.The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified.The backbone of residues 106-108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type,which is believed to be the dominant mechanism of the catalytic activity reduction.Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al.identifies the most important residues for enzyme-substrate complexes.In the second part of the article,we investigated the influence of CYP2C9 and CYP2C19 genetic polymorphisms on the pharmacokinetics of gliclazide modified release（MR） in healthy Chinese subjects.In a single-dose pharmacokinetic study,24 healthy male subjects with various CYP2C9 and CYP2C19 genotypes received an oral dose of 30 mg gliclazide MR and plasma was sampled for 72 h postdose.In a multiple-dose pharmacokinetic study,17 other CYP2C9*1 homozygotes with various CYP2C19 genotypes received 30 mg gliclazide MR once daily for 6 days and plasma was sampled after the last dose.The plasma concentrations of gliclazide were measured using a validated LC/MS/MS method.CYP2C9 and CYP2C19 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis.In the single-dose study,no significant difference in any pharmacokinetic parameters was found in CYP2C9*1/*1,*1/*3 and *1/*13 subjects.In contrast,the AUC_0-∞ of gliclazide was significantly increased by 3.4-fold [95%confidence interval（CI） 2.5,4.7;P＜0.01]in CYP2C19 poor metabolizer（PM） subjects compared with CYP2C19*1 homozygotes.The half-life(t_1/2) was prolonged from 15.1 to 44.5 h（P＜0.01）.Similar differences were found in the multiple-dose study.The parameters of gliclazide AUCss,AUC_0-∞ and C_max were 3.4-fold（95%CI 2.9,4.0）, 4.5-fold（95%CI 3.8,5.4） and 2.9-fold（95%CI 2.4,3.4） increased（P＜0.01） in CYP2C19 PM subjects,respectively,compared with CYP2C19*1 homozygotes,and t_1/2 was increased from 13.5 to 24.6 h（P＜0.01）.The pharmacokinetics of gliclazide MR are affected mainly by CYP2C19 genetic polymorphism instead of CYP2C9 genetic polymorphism.We also investigate the pharmacokinetics of carisoprodol at dosage of 350 mg and 700 mg and the influence of CYP2C19 polymorphism on its pharmacokinetics in healthy Chinese volunteers.In a randomized, two-period crossover study,12 healthy Chinese male volunteers were administered carisoprodol 350 mg or 700 mg,and plasma was sampled for 36 h post dose.The plasma concentrations of carisoprodol and it main metabolite meprobamate were measured using a validated LC/MS/MS method.CYP2C19 genotyping was conducted on these volunteers by PCR-RFLP analysis.The study indicates that saturation of metabolism may occur at higher dose of carisoprodol.CYP2C19 polymorphism influences the pharmacokinetics of carisoprodol markedly in Chinese volunteers.We investigate the impact of CYP2C9*3 on the pharmacokinetics of glibenclamide.CYP2C9*3 greatly affects both the pharmacokinetic profiles of glibenclamide.The elimination of glibenclamide significantly decreased in subjectswith CYP2C9*1/*3 genotype.In the third part of the article,an in vitro investigation of the inhibition of cytochrome p450 2C9（CYP2C9） by a series of flavonoids was described. In recent years,flavonoid intake in the form of dietary supplements and plant extracts has been steadily increasing.Some clinical studies have demonstrated that flavonoids may alter drug metabolism.Moreover,in China,some flavonoids are administered intravenously as drugs.In the current investigation,using CYP2C9 substrate diclofenac,one of the most commonly employed probes for determining CYP2C9 catalytic activity,a number of flavones（flavone,6-hydroxyflavone,7-hydroxyflavone,chrysin, baicalein,apigenin,luteolin,scutellarein,wogonin） and flavonols（galangin, fisetin,kaempferol,morin,quercetin） were shown to substantially inhibit CYP2C9-mediated diclofenac 4’-hydroxylation in CYP2C9 RECO^? system （K_i≤2.2μM） with galangin being the most potent inhibitor（K_i=0.15μM）. These findings raise concerns about possible drug-drug interactions between supplements,remedies and drugs containing flavonoids and the some 100 therapeutic drugs metabolized by CYP2C9.Cytochrome P450 noncompetitive inhibitors have been reported widely, particularly to CYP1A2 and CYP2C9,with molecular basis unknown.In the current investigation,most flavones and flavonols are found to be competitive inhibitor,indicating they bind to the substrate-binding site of CYP2C9.On the basis of docking simulation studies using the 1R9O crystal structure,this binding site is shown to be close to the heme and is the same site as flurbiprofen in the 1R9O crystal structure.On the other hand, noncompetitive inhibitor 6-hydroxyflavone binds to a site further from the heme and oriented away from the other flavones and flavonols,but consistent with the reported warfarin allosteric binding site revealed by the crystal structure of 1OG5,and also consistent with the reported dapsone allosteric site that can activate CYP2C9-mediated flurbiprofen 4’-hydroxylation.To confirm the role of Phe 100 in the noncompetitive binding of 6-hydroxyflavone to CYP2C9,site-directed mutagenesis was constructed generating CYP2C9 variants Phe100Asp and Phe100Trp.These mutants showed diclofenac 4’-hydroxylase activity similar to that of CYP2C9.1 and were both competitively inhibited by 6-hydroxyflavone.This demonstrates the presence of a direct interaction between 6-hydroxyflavone and Phe 100 in the CYP2C9 noncompetitive inhibition.The binding sites of flavones and flavonols to CYP2C9 determine their inhibiting mechanism.更多还原