基于药物转运蛋白-CYP450和SHR模型研究黔产杜仲在生理和病理状态下的药代动力学差异及其机制

Eucommia ulmoides is one of important and famous traditional Chinese medicine in Province Guizhou, which is commonly combined with other traditional Chinese medicine to treat hypertension. However, the study on the pharmacokinetics of Eucommia ulmoides are still carried out in the pathological states. Given the major role of drugs in the pathological state, the research of pharmacokinetics in pathological state will be more meaningful. More importantly, we have found that the pharmacokinetic behavior of Pinoresinol diglucoside and geniposidic acid with the hypotensive effect contained in Eucommia ulmoides were significantly different between normal rats and spontaneously hypertensive rats (SHR). However, the underlying mechanism for this difference is unknown and it is worthy of further exploration. It’s well known that drug transporters and cytochrome P450 (CYP450) play an important role in the absorption, distribution, metabolism and excretion of drugs. Therefore, in the present study, the pharmacokinetic difference of Five hypotensive active ingredients in Eucommia ulmoides between normal rats and SHR were investigated. Then, the expression of transport proteins in the small intestine and liver of rats and the activation of CYP450 in liver of rats were carried out to investigate the mechanism of pharmacokinetic difference. At last, this study will provide the theoretical basis for the further development of Eucommia ulmoides in Province Guizhou and its clinical application in the treatment of hypertension.

杜仲为贵州三宝之一，是中医临床上治疗高血压方剂中的常用中药。关于杜仲在病理状态下的药代动力学(Pharmacokinetic, PK) 研究尚属空白。鉴于药物主要是作用于病理状态的机体，因此开展病理状态下的PK研究更有意义。更为重要的是，我们发现杜仲的主要降压成分松脂醇二葡萄糖苷和京尼平苷酸在正常大鼠和自发性高血压大鼠（Spontaneously hypertensive rats, SHR）模型体内的PK行为存在显著差异。然而，这种差异的潜在机制不得而知，值得深入探讨。药物转运蛋白和细胞色素P450（CYP450）在药物的PK过程中起着至关重要的作用。有鉴于此，本项目旨在利用SHR模型，比较杜仲中的五个代表成分在正常大鼠和SHR模型体内的PK差异，并从药物转运蛋白以及CYP450的角度探讨杜仲代表成分PK差异的机制，以期为贵州杜仲药材的深度开发以及治疗高血压的临床应用推广奠定理论基础。

杜仲是中医临床上治疗高血压方剂中的常用中药。关于杜仲在正常大鼠和自发性高血压模型大鼠（SHR）体内的药代动力学(PK)的差异及其机制研究尚属空白。鉴于此，本研究首先运用UHPLC-Q-TOF/MS分析对比杜仲提取物、含药血清、空白血清指纹图谱，从含药血清中找到了26个入血成分，并明确了7个原型入血成分。结合文献和前期研究工作基础，选取京尼平苷酸（GA）、原儿茶酸（PCA）、松脂醇二葡萄糖苷（PDG）、松脂醇单葡萄糖苷（PG）、绿原酸（CA）、隐绿原酸（CCA）、新绿原酸（NCA）等7个成分作为杜仲提取物中活性成分研究的指标。应用大鼠离体外翻肠囊模型、在体循环肠灌流模型发现，杜仲提取物中7个成分在正常大鼠和SHR模型体内的肠吸收具有一定的差异。整体药代动力学结果表明，对于单次灌胃给予杜仲提取物后，与正常组相比，在SHR模型体内，GA的药动参数Tmax、AUC0-t、MRT0-t、Vd/F显著低于正常大鼠；PDG的Cmax、AUC0-t显著高于正常大鼠，而Tmax、清除率Cl/F显著低于正常大鼠；对于NCA和CA，除Tmax小于正常大鼠外，其余参数未见明显差异；CCA的 AUC0-t和MRT0-t显著高于正常大鼠；PCA的T1/2、AUC0-t、MRT0-t和Vd/F显著高于正常大鼠；PG的参数T1/2、AUC0-t和MRT0-t显著高于正常大鼠。对于多次给药，GA的T1/2、CL/F、Vd/F高于正常大鼠，Tmax、AUC0-t低于正常大鼠；PDG的T1/2、Vd/F、MRT0-∞低于正常大鼠。而且，7个成分在各时间点（10min、30 min、2 h、8h）SHR模型组与正常组中的主要组织脏器中的含量均有一定的差异。最后，采用Caco-2细胞模型和药物转运蛋白抑制剂发现转运蛋白P-gp、MRPs、BCRP、OATP2参与了PCA、PDG、GA、PG、NCA在Caco-2细胞的摄取转运。体内Cocktail探针药物法、Western blot、免疫组化和免疫荧光的结果表明，自发性的高血压病理状态会改变转运蛋白MRP1、MRP2、BCRP、OATP2的表达，同时会影响CYP2E1、CYP3A2、CYP2C11和CYP3A2的活性和表达，而这些改变可能是杜仲提取物中活性成分在生理病理状态下的PK差异的原因，本研究为杜仲深层次开发利用提供一定的理论基础。