糖尿病状态下肝CYP3A等CYP450酶诱导对药物代谢影响及其在糖尿病病情发展中的作用

Based on both reports by literatures and our previous results, a new hypothesis was raised as follows. The induction of hepatic CYP3As etc CYP450s under diabetic condition not only alters drug metabolism, therapeutic effect and toxicological activity, but also leads to metabolism disturbance of some endogenous substrates including testosterone and retinol. In turn, the inducted hepatic CYP3As further worsens the diabetes. A series of experiments were designed to verify above hypothesis. II diabetic rats were included in the studies. Induction of CYP450s including CYP3As under diabetic rats and effects of the induced hepatic CYP450s on pharmacokinetics of nateglinide, repaglinide，testosterone and retinol were investigated. The pharmacokinetics and pharmacodynamics of therapeutic drugs were also measured during drug therapy. Mechanisms inducing CYP3As were also documented both in vivo and in vitro. Relationship between activity of hepatic CYP3A and diabetes by co-administration of CYP3A inhibitors as well as CYP3A inducer was studied to investigate whether the induced hepatic CYP3As worsens diabetes and its mechanisms. The above results were further verified using results from ob/ob mice and cyp3a11 knockout (cyp3a11-/-) mice. Hepatic cells charactering insulin residence induced by palmitic acid were also used to investigate relationship between hepatic CYP3A induction and insulin residence. The mechanisms leading to above results were also studied. The results may highlight roles of hepatic CYP3A induction in diabetic development, as well as give gudainces for drug and prediction of drug adverse effects in diabetic patients.

基于文献和前期成果提出"糖尿病状态下肝CYP3A等CYP450酶的诱导不但改变药物体内代谢，影响其疗效和毒性，也引起内源性底物代谢紊乱。同时这种肝CYP3A酶诱导反过来又加重糖尿病病情"推论，设计试验验证该推论。以II型糖尿病大鼠为模型研究肝CYP3A等CYP450酶诱导；这种诱导对瑞格列奈、那格列奈、睾丸酮和视黄醇等代谢影响；药物治疗过程中代谢动力学及疗效；糖尿病状态下引起肝CYP3A酶诱导机制。通过合用CYP3A诱导剂或抑制剂研究CYP3A酶活性与糖尿病关系，分析糖尿病状态下肝CYP3A酶诱导是否反过来加重糖尿病病情及机制。用ob/ob小鼠和基因敲除小鼠验证上述结果。用软脂酸诱导肝细胞胰岛素抵抗，研究CYP3A酶诱导与肝细胞胰岛素抵抗的关系及机制，用基因沉默和转染进一步验证上述结果。其成果对于阐明肝CYP3A等CYP450酶诱导在糖尿病病情发展中作用，合理用药和不良反应预测有重要意义。

用II型糖尿病大鼠研究糖尿病鼠肝CYp3a等Cyp450s 酶活性与表达及其对药物和内源性底物代谢动力学行为影响；分析Cyp3a活性改变是否影响糖尿病症状。结果显示糖尿病鼠灌胃和静注辛伐他汀后血浆中辛伐他汀和辛伐他汀酸浓度显著降低，清除率显著升高。体外结果显示糖尿病鼠肝摄取辛伐他汀能力显著增加，肝微粒体Cyp3a活性被诱导，辛伐他汀代谢加快。QT-PCR结果显示糖尿病鼠肝Cyp3a1和Oatp2的mRNA表达显著增加，提示糖尿病鼠血浆中辛伐他汀和辛伐他汀酸浓度降低可能与肝Cyp3a和Oatp2活性与表达上调有关。用HepG2和Fa2N-4细胞研究显示游离脂肪酸是糖尿病血清中诱导CYP3A4活性成分。软脂酸和油酸是通过不同通路诱导CYP3A4活性与表达。NF-KB抑制剂增加软脂酸诱导的CYP3A活性，而减弱油酸诱导CYP3A活性。PKC抑制剂抑制油酸诱导的CYP3A活性，不影响软脂酸介导的CYP3A诱导。进一步研究了Cyp3a等Cyp450活性诱导对糖尿病症状影响。结果显示阿伐他汀（20 mg/kg）治疗2周可以损伤亚糖尿病大鼠糖耐量，伴随肝CYP3A和CYP2C酶活性出现增加，肝和胰Cyp3a1/2的 mRNA表达被诱导。细胞试验显示低浓度阿伐他汀可以诱导HepG2细胞CYP3A4酶活性，并剂量依赖性地降低细胞的糖利用和增加ROS水平，这一作用可以被CYP3A抑制剂红霉素逆转；红霉素可以逆转阿伐他汀引起的INS-1细胞胰岛素释放抑制和阿伐他汀引起的ROS水平升高。结果提示阿伐他汀加重亚糖尿病的作用可能与其诱导P450酶和ROS过度生成有关。II糖尿病鼠灌胃后血浆中阿伐他汀浓度也显著降低，并呈病程依赖性。肝微粒体也证实阿伐他汀代谢加快，这可能与Cyp3a活性增加有关。阿伐他汀是Cyp3a和Oatp2底物，提示血浆中阿伐他汀浓度降低可能与Oatp2和Cyp3a表达增加有关。同时发现在阿伐他汀过程中伴随肝毒性增加，40mg/kg剂量组连续给药2周后，所有糖尿病动物因肝衰竭死亡。糖尿病人往往伴随着视黄醇水平紊乱。因此考察了糖尿病与视黄醇关系。结果显示糖尿病鼠血浆中视黄醇水平显著降低，但肝游离视黄醇水平增加。体外代谢研究显示糖尿病鼠肝微粒体和胞浆中视黄醛形成被抑制，相反视黄酸形成反应活性显著增强。其成果对于阐明肝CYP3A等CYP450酶诱导在糖尿病病情发展中作用有重要意义。