慢性肾脏疾病状态下PTH下调CYP3A4活性的量效关系及机制研究

Chronic kidney disease (CKD) affects not only drugs’ renal elimination but also the activity of drug metabolizing enzymes. Studies have shown that the activity of cytochrome P450 3A4 are significantly decreased in CKD, but the dose-response relationship and specific mechanism remain unknown, which unable to be used for clinical transformational applications. Recent literature reports that the elevated serum level of parathyroid hormone (PTH) in patients with CKD can mainly explain for the down-regulation of activity of hepatic CYP3A4. Our pilot study also found that the expression of CYP3A1 and CYP3A2 ( Rat CYP3A1 and CYP3A4 correspond to human CYP3A4) in primary cultured rat hepatocytes were down-regulated by PTH possibility with a dose-response relationship. Therefore, we will investigate the dose-response relationship of PTH and CYP3A4 by in vivo and in vitro studies in our project; Then further explore the mechanism of PTH down-regulating the activity of CYP3A4 in CKD by techniques of Real-time PCR, Western Blot, Gene knockout technology and so on; Finally establish the population pharmacokinetic/pharmacodynamic model for individualizing dosage regimens based on the level of PTH, and verify the model and apply it to clinical practice. Our research will not only provide the theoretical basis and clinical guidelines for personalized medicine of CKD patients, but also provide the theoretical guidance for drug interaction studies and drug development in CKD special populations.

慢性肾脏疾病（CKD）不仅影响药物的经肾消除还影响药物代谢酶的活性。研究表明CKD时肝脏CYP3A4代谢酶活性显著下降，但量效关系及具体机制不明，难以临床转化应用。近期研究显示，CKD患者血清中甲状旁腺激素（PTH）水平的升高是引起肝脏CYP3A4下调的主要因素，本课题组前期研究也发现外源性PTH可下调原代大鼠肝细胞中CYP3A1和CYP3A2（人CYP3A4直系同源酶）的表达且其作用可能存在量效关系。因此，本项目将通过体内外实验研究PTH对CYP3A4影响的量效关系；采用Real-time PCR、Western Blot及基因敲除动物模型等研究CKD时PTH下调CYP3A4的分子机制；最后建立基于PTH水平的个体化给药群体药动/药效学模型，并在临床患者中验证与应用仿真模型。本研究不仅为CKD患者个体化用药提供理论依据和临床指导，还为CKD特殊人群药物相互作用和药物开发提供理论指导。

慢性肾脏疾病（CKD）患者血清中甲状旁腺激素（PTH）水平的升高是引起肝脏CYP3A4下调的主要因素，但其可能的量效关系不明。本项目通过体内外实验研究CKD时PTH对CYP3A4影响的量效关系及其分子机制，并建立基于PTH水平的个体化给药群体药动/药效学模型。我们通过在人原代肝细胞和肝癌细胞系（HepG2、Huh7和SMMC-7721）中给予浓度梯度（10-15～10-6 M）的PTH处理，探索体外PTH对CYP3A4表达及活性影响的量效关系；同时，在野生型及采用CRISPR/Cas9技术敲除PTH基因的小鼠模型中构建5/6肾切除模型，探讨CKD状态下，内源性PTH水平升高和外源性给予PTH对肝脏CYP3A11（人体CYP3A4的直系同源酶）表达和活性的量效关系，并进一步阐明CKD病理状态PTH升高下调肝脏CYP3A4表达的分子机制；接着，纳入169名使用硝苯地平控释片治疗的CKD患者进入前瞻性观察研究，收集临床数据，HPLC-MS/MS测定硝苯地平（CYP3A4底物）的血浆药物浓度，ELISA检测血清PTH水平，建立PPK/PD模型。我们发现，体外研究中，仅在HepG2细胞中，一定浓度的PTH可显著下调CYP3A4 mRNA和蛋白的表达及抑制其活性。 体内研究中，采用CRISPR/Cas9技术敲除PTH基因获得F0代后，扩繁过程发生敲除纯合子胚胎致死，因此仅获得PTH基因敲除杂合子小鼠模型（PTH+/-）。继而我们在野生型小鼠中建立5/6肾切除模型，所导致的内源性PTH水平升高程度较低，未能显著下调肝脏CYP3A11表达，进一步外源性腹腔注射PTH能够显著下调野生型小鼠肝脏CYP3A11表达，但在该浓度梯度内并未见明显量效关系。此外，PTH可能通过NF-κB/PXR/RXRα/CYP3A4的机制通路下调CYP3A4。CKD患者中，遗传因素CYP3A4 rs4646437及非遗传因素PTH、25(OH)D、血红蛋白、eGFR对硝苯地平控释片在CKD患者中的药动学有显著影响。CKD患者硝苯地平控释片的PopPK模型中，PTH、年龄、血红蛋白显著影响到硝苯地平的体内消除，ALT显著影响到硝苯地平的体内分布。本研究首次探讨了CKD病人升高的PTH水平和下调的肝药酶CYP3A4间可能的量效关系，为CKD患者的临床用药调整提供了新的标志物。