核受体及其靶基因CYPs的基因多态性对青蒿素类药物代谢调控的分子机制研究

As the first-line antimalrials in the clinic, artemisinin drugs have been found to show dramatic interindividual variations in their pharmacokinetic profiles. Genetic polymorphism exists for CYP2B6, CYP3A4 and their upstream regulators-nuclear receptors (NRs, CAR and PXR), which mainly contribute to the metabolism of artemisinin drugs. However, the relationship between the genetic polymorphism of NRs-CYPs and the metabolism of artemisinin drugs remains unclear. In our previous studies, we found that artemisinin drugs showed time-dependent pharmacokinetics, which was probably the result of auto-induction of CYPs. In addition, the mRNA expression and activity of CYP2B6 and CYP3A4 could be induced by artemisinin drugs. But the induction of CYP2B6 and CYP3A4 mRNA expression and enzyme activity showed significant interindividual difference, which could probably be caused by genetic polymorphism of these two CYP enzymes. In order to eludidate the molecular mechanism of genetic polymorphism in the metabolic regulation of artemisinin drugs, the present project will focus on: 1) the effect of the genotype of CYP2B6 and 3A4 in the regulation of the metabolism of artemisinin drugs; 2) the relationship between genetic polymorphism of CYPs (CYP2B6 and CYP3A4) and their enzyme activity with the treatment of artemisinin drugs; 3) the molecular mechanism of genetic polymorphism of the nuclear receptors PXR/CAR and their target genes CYPs in the metabolism of artemisinin drugs. The results will elucidate the effect of genetic polymorphism of two nuclear receptors (PXR and CAR) and their target CYPs (CYP3A4 and 2B6) on the auto-induction metabolism of artemisinin drugs, which will provide valuable information for the pharmacokinetic individual difference of this class of drugs.

临床一线抗疟药青蒿素类药物的临床药动学性质个体差异大，CYP2B6和3A4酶是其主要代谢酶，近年发现两酶及其调控核受体存在多种基因型，而基因多态性与青蒿素类药物代谢的相关性尚不清楚。在前期工作中，申请人发现青蒿素类药物存在时间依赖性药动学特征，这源于其自身诱导代谢；该类药物可上调靶基因CYP2B6和3A4的表达水平，并可诱导其活性，但调控程度却表现出显著的个体差异。为阐明基因多态性对青蒿素类药物代谢的调控机制，本项目拟(1)考察CYP2B6和3A4的基因型对该类药物的代谢调控；(2)研究在该类药物作用下CYP2B6和3A4的基因多态性与酶活性的相关性；(3)阐明核受体PXR/CAR的基因型在该药作用下对介导下游靶基因CYP2B6和3A4表达调控的分子机理。从而揭示PXR/CAR及其靶基因CYPs的基因多态性对青蒿素类药物代谢的调控机制，为最终阐明该类药物的代谢机理和临床个体差异提供新思路。

本课题组研究发现：（1）青蒿素类药物在健康人体内存在时间依赖性药动学现象。健康志愿者连续服用青蒿素（QHS）或双氢青蒿素（DHA）后，原形的曲线下面积AUC较单剂量给药后下降，而清除率CL/F升高。相应地，QHS的主要I相代谢产物和DHA的II相代谢物的生成率（AUC 代谢物/AUC原型）较单次给药升高。该结果验证了该类药物在人体内存在自身诱导代谢现象。（2） QHS对健康人CYP2B6/3A4酶活性的影响。QHS可诱导健康人CYP2B6（1.8-2.3）和CYP3A4（2.8-3.7）酶活性。（3）UGT基因型DHA在人体内药动学性质的影响。DHA及其代谢物在携带不同UGT1A9 (I399C>T)和UGT2B7 (802C>T)基因型健康人体内的血药浓度暴露水平无显著性差异。（4） CAR (C540T)基因多态性对QHS代谢动力学的影响。与野生型携带者（540CC）相比，突变杂合子（540CT）和突变纯合子（540TT）携带者中QHS及其代谢物去氧青蒿素的暴露水平降低，重复给药后显著（p<0.05）。（5） CAR (C540T)基因多态性对QHS自身诱导程度的影响。在CAR(540CC)携带者中，重复给予QHS后CL/F为单剂量的约1.3倍；突变杂合子和突变纯合子携带者无显著性差异（p>0.05）。（6） CAR (C540T)基因多态性对CYP2B6/3A4酶活性和被诱导程度的影响。CAR基因突变杂合子或纯合子携带者中CYP2B6/3A4的酶活性或被QHS诱导的程度无显著性差异（p>0.05）。（7） 青蒿素类药物对核受体PXR/3A4的诱导作用。采用体外转染和荧光素酶双报告基因技术，考察了QHS以及黄花蒿提取物对PXR/3 A4的诱导作用，结果显示青蒿素具有中度程度的诱导作用。.本课题揭示了青蒿素类药物在人体内的时间依赖性药动学特征与其自身诱导代谢密切相关，主要由I相代谢酶CYP2B6/3A4的诱导所导致，且受到核受体CAR和PXR的调控；核受体CAR (C540T)的基因多态性对青蒿素代谢动力学具有显著影响，而下游的CYP2B6/3A4或UGT1A9/2B7的基因多态性无影响。本课题达到了研究目标，受课题资助在Malar J和Drug Metab Pharmacokinet等SCI专业期刊中共发表8篇（2014-2017年）研究成果，共培养研究生5名。