双峰驼CYP450酶系及对探针药物动力学特征的影响

The Bactrian camel is a unique species of livestock living in the desert and semi-desert Gobi steppe, and they are exceptionally well-adapted to drought and heat, and is able to survive and reproduce in conditions not tolerated by other domestic animals. This project is a basic applied research project which was estabolished on the basis of systemic study of Bactrian camel genome and unique biological activities of camel milk and poll gland secretions, as well as, according to the physiological characteristics of Bactrian camel feed some of the poisonous plants without poisoning and the particularities of Bactrian camel CYP gene copy numbers. Firstly, the influence of enzyme inhibitors and enzyme inducers on Bactrian camel CYP enzymes were studied by using liver microsomes incubation system and primary hepatocytes culture system in vitro, respectively. And then the interaction between enzyme inhibitors/ inducers and the Bactrian camel CYP enzyme subfamilies were studied deeply by using Coctail probe drug techniques. The achievement of the project will reveal the interaction between the Bactrian camel CYP enzymes and drug metabolizing enzyme inducers and inhibitors, as well as, will clarify the correlation between the Bactrian camel CYP enzymes and probe drug pharmacokinetics in vivo. The research results will also provide a scientific basis for reasonable use of medicine in clinic while will fill the research gaps in this area on the world, as well as will lay scientific foundation for further studies of Bactrian camel CYP genes and its subfamilies, CYP gene expression and enzyme activity.

双峰驼是生活在荒漠、半荒漠戈壁草原的一种特有畜种，对干旱地区严酷的生存条件有着独特的适应性。本项目是在系统研究双峰驼全基因组以及驼乳和公驼枕腺分泌物独特的生物学活性的基础上，根据双峰驼对部分有毒植物的耐受性生理特性以及双峰驼CYP基因拷贝数的特殊性而提出来应用基础研究项目。项目首先采用肝脏微粒体体外孵育法和肝脏原代细胞体外培养法分别研究药酶抑制剂和诱导剂对双峰驼CYP酶系活性的影响，然后采用Coctail探针药物法研究双峰驼体内CYP酶亚型及其活性以及探针药物与CYP酶活性的相关性。项目成果将揭示双峰驼CYP酶系与药酶诱导剂和抑制剂的相互作用关系，阐明双峰驼CYP酶系与探针药物代谢动力学之间的相关性，为临床合理用药提供科学依据，同时还能填补该领域的国内外空白，并为进一步深入研究双峰驼CYP基因及其亚型、CYP基因的表达与酶活性的深入研究奠定基础。

细胞色素P450（CYP）酶系在动物内源性化合物和外源性化学物质代谢以及某些外来毒性物质和致癌物质的代谢中发挥着至关重要的作用。目前已发现60多种CYP酶系，其中CYP1A、CYP2C、CYP2D和CYP3A等参与90%以上的药物和外来化学物质代谢，且这些酶主要表达在肝脏中，在小肠、肺、胎盘和肾脏中部分表达。为探讨双峰驼代谢途径的特殊性和处置外来物质的能力是否与其CYP酶系的多样性和活性有关，按照课题计划任务对双峰驼CYP1-3三个亚型酶体内外活性进行系统研究。.（一）双峰驼肝微粒体的制备和CYP总酶活性的检测.首先采用钙离子沉淀法和差速离心法成功制备了双峰驼肝微粒体，然后分别采用CO还原差示光谱法和还原差示光谱法测定了CYP450总酶含量和细胞色素b5（Cytb5）的含量，并证明了后者更适合于制备高质量的肝微粒体。.（二）双峰驼CYP酶体外活性研究.首先分别建立双峰驼CYP1A、CYP2C、CYP2D、CYP3A亚酶的体外孵育体系，并找出最适宜的反应条件。然后向孵育体系分别加入各亚酶的特异性探针底物，共同孵育，并通过HPLC - UV和UPLC - UV检测其中各探针药物的动态浓度及其主要代谢产物的动态浓度，从而间接检测双峰驼CYP1A、CYP2C、CYP2D、CYP3A亚酶的体外活性，计算出各亚酶的酶动力学参数。其次研究了各亚酶特异性抑制剂对双峰驼肝微粒体CYP1A、CYP2C、CYP2D、CYP3A酶活性的影响。.（三）CYP酶系活性的体内研究.双峰驼CYP酶系中CYP1A和CYP3A参与70%以上内源性和外源性物质代谢。本项目分别以对乙酰氨基酚和咪达唑仑作为双峰驼CYP1A酶和CYP3A酶的特异性探针药物研究了双峰驼CYP1A酶和CYP3A酶的体内活性。首先肌肉注射CYP1A和CYP3A酶的特异性探针药物，按照事先设定的时间点采血并进行血样处理，通过HPLC - UV法检测特异性探针药物的动态血药浓度，并借助phoenix 7.0药动学软件找出两种底物的主要动力学参数。还研究了洛美沙星对双峰驼CYP1A酶体内活性的影响和伊曲康唑对CYP3A酶体内活性的影响。.（四）双峰驼CYP2J酶在各组织中相对基因表达量研究.根据双峰驼CYP基因家族中CYP2J亚族基因拷贝数明显多于其他家畜的特殊性，我们增加了《CYP2J亚族在双峰驼各组织中相对表达量比较研究》内容。