多奈哌齐立体选择性代谢和转运机制及相关候选基因研究

Donepezil is the main drug in the treatment of Alzheimer's disease and administered as a racemate. There is a large variability in the clinical response to DNP which only be find after 6 month treatment. Pharmacogenomics and therapeutic drug monitoring become the focus to evaluate inter-individual differences in early usage of DNP. We found in our study that the plasma level of DNP was sereoselective and few patients the concentration ratio reversed. The mechanism of stereoselective plasma concentration and clinical respones remains poorly understood. We suppose that the metabolism and transport of "better enantiomers" maybe a key role for the pharmacogenomics activities.The "better enantiomers" maybe the more sensitive marker in therapeutic drug monitoring. In this study, we will use human liver microsomes, recombinant enzyme, MDCK-rMDR1,OCT-expressing HEK cells to investigate the mechanism of stereoselective in metabolism and transport and find the key P450 enzymes and transporters in drug stereoselective disposition. The impact of the key enzymes and transporters on the DNP disposition and clinical outcome will be evaluated in patients diagnosed with Alzheimer's disease and treated with donepezil. The objectives of the present study is to characterization of the DNP metabolic and transport of DNP enantiomers and to evaluate the correlate genetic variations in transporter and metabolism enzymes genes with functional alterations and plasma concentrations on the clinical relevance.

多奈哌齐（DNP）是治疗老年痴呆的一线药物，存在个体差异大，部分患者无效，给药6个月才能进行药效学评价等特点。为了在用药前或早期制定DNP的个体给药方案，药物基因组学和治疗药物监测成为研究重点但尚不成熟。.我们发现手性药物DNP体内浓度水平存在立体选择性，且少数患者对映体比例"倒置"，其机制及临床意义尚不明确。我们推测在药物基因组学研究中的候选基因确定与优势异构体的代谢和转运密切相关，同时优势异构体可能是与疗效相关的更加敏感的治疗药物监测的指标。本项目将利用微粒体、重组酶、rMDR1-MDCK、rOCT-HEK细胞模型从代谢及转运等环节阐明手性分子药动学的立体选择性及机制，确定关键酶及转运体的基因位点为候选基因，进行候选基因多态性、优势异构体血药浓度与疗效的关联性的临床研究。.本项目以代谢和转运的立体选择性机制为重点，探索建立候选基因筛查联合对映体单体血药浓度监测的个体化用药模式。

多奈哌齐（DNP）是治疗老年痴呆的一线药物，存在个体差异大，部分患者无效，给药6个月才能进行药效学评价等特点。为了在用药前或早期制定DNP 的个体给药方案，药物基因组学和治疗药物监测成为研究重点但尚不成熟。本研究在发现DNP血药浓度存在立体选择性的基础上，利用肝微粒体和重组酶体外实验证实了DNP代谢方面存在立体选择性；利用MDCK-MDR1细胞模型证实了DNP易于通过血脑屏障，对映异构体都不是P-gp的底物。临床研究方面测定了77例中国汉族AD患者的有效单体S-DNP稳态血药浓度及中国汉族人中变异频率大的相关CYP2D6基因型，探索了有效单体S-DNP在中国汉族AD患者体内的稳态血药浓度和其药效学的关联性。研究结果表明：基于CYP2D6基因多态性的中国汉族AD患者的S-DNP稳态血药浓度和DNP的临床治疗效果有显著关联。因此，S-DNP稳态血药浓度可能是DNP临床药效的潜在标记物。在DNP的临床治疗效果监测中，同时考虑患者CYP2D6基因型及测定有效单体S-DNP稳态血药浓度将是必要而有效的。但是S-DNP稳态血药浓度虽然与临床疗效相关，却不能准确确定范围，用来评价临床疗效。更灵敏的具备临床应用性的生物标记物有待探寻。.影响药物疗效的因素除了药代动力学还有药效学，本项目研究中发现APOE4显著影响药物疗效。但其机制不明确。该机制的明确也是下一步研究的方向之一。.为了探寻评价药物疗效的更为精准的生物标记物，发现药物无效组和有效组的产生机制，本项目进行了老年痴呆患者服用DNP之后的药物代谢物组学研究。研究结果表明药物有效组与药物无效组之间在PLS-DA上存在较为明显的代谢谱分离，说明药物无效组与药物有效组之间具有较为明显的代谢差异。与药物无效组相比，药物有效组中显著升高的代谢物是氨基酸如cysteine, tryptophan和homoserine等，而显著下降的是脂代谢相关代谢物（glycerol, glycerol-3-phosphate, glycerol-2-phosphate, 1-oleyolglycerol和methyl stearate）和甾醇类物质（dihydrocholesterol, campesterol和beta-sitosterol）。这些显著变化的物质可能作为区分药物有效还是无效的代谢标志物。