温脾止泻中药益智减轻伊立替康所致大鼠腹泻毒性的PK-PD作用机制研究

Clinically, chemotherapy-induced toxicity could be relieved partly through co-administration of chemotherapy drugs and traditional Chinese medicine. However, the mechanism(s) underlying these beneficial herb-durg interactions remain largely unknown. Irinotecan (also known as CPT-11)is approved for first-line therapy of advanced colorectal cancer in combination with 5-fluorouracil and/or leucovorin. The side effects of CPT-11 include vomiting, nausea and diarrhea, which can be serious. CPT-11 is converted into the active metabolite SN38 by hepatic and intestinal carboxyesterases (CES). SN-38 is metabolized by hepatic UDP glucuronosyltransferase (UGT) to form an inactive SN-38G that is excred into the bile. However, intestinal bacterial β-glucuronidase can transform SN-38G back into the active SN-38 form that can directly damage the intestine and result in intestinal inflammation. This application aims to investigate the mechanims of Yizhi extract and its components which reduce the gastrointestinal toxicity in murine models according to the toxic mechanisms caused by CPT-11.The concentration of CPT-11 and its metabolites （e.g., SN-38 and SN-38G), as well as Yizhi costituents, in various biomatrix samples (e.g., plasma, bile and feces) from the rats after intravenous administration will be analyzed using a ESI-interfaced LC-MS/MS method. The methods will be used to evaluate the influence of Yizhi extract and active components administered orally to rats on the exposure to CPT-11 and its metabolites after intravenous dosing. The present application will establish an in vitro system, using microsomal, cytosol preparations or others, as a model to determine the potential for Yizhi and its components to inhibit drug metabolic enzymes/transporter (e.g.,CES, CYP3A4, UGT1A1, β-glucuronidase, P-gp and Mrp2) might involved in the metabolic clearance and disposition of CPT-11. Furthermore, the current application also study the influence of Yizhi extracts and its components on COX-2, iNOS and PDE4B/CLYD, which are mediators of gut diarrhea and inflammation. This study investigates the roles of Yizhi from a new perspective and elucidates the underlying mechanisms. The investigation of such multicomponent herbal medicine, Yizhi, capable of reducing gastrointestinal toxicity could be useful for future clinical use for the treatment of cancers and the potential management of those complicated diseases.

临床上，化疗药物毒副作用常通过与中药联用得到不同程度缓解，阐明此种相互作用内在机制对于中医药创新发展具有重要意义。伊立替康（CPT-11）是治疗结直肠癌一线用药，但其存在严重腹泻毒性。课题组前期工作表明，温脾止泻中药益智可减轻CPT-11所致大鼠腹泻毒性，并基本确定其活性部位、组成成分及其初步药代动力学特征。针对CPT-11致毒机理，本项目拟从PK、PD两个角度研究活性部位及活性成分减轻CPT-11肠道毒性作用内在机制。通过体外和体内两种手段，借助LC-MS/MS技术，研究益智活性成分对CPT-11体内处置关键调控环节的抑制作用，评价药物体内暴露的变化；通过荧光PCR等分子生物学技术观察腹泻炎症信号通路中COX-2、iNOS mRNA、蛋白表达变化，结合血液炎症因子定量分析结果，探索益智抗炎作用分子生物学机理。本课题从全新角度研究益智功效，阐明部分科学内涵，为益智用于癌症治疗领域奠定基础。

常见恶性肿瘤确诊病例中，结直肠癌在男性占第三位，女性居第二位。伊立替康（CPT-11）是治疗转移性结直肠癌的一线药物，但具有剂量依赖性的迟发性腹泻毒性。将中药与化疗药物联合使用产生减毒增效作用成为我国临床上治疗癌症的有效手段，国际上也有使用源于古老中药方剂黄芩汤的标准化制剂PHY906减轻CPT-11所致模型动物的腹泻毒性的成功研究报道。在前期研究基础上，本课题提出从PK和PD两个角度研究温脾止泻中药益智减轻CPT-11所致大鼠腹泻毒性的内在机制。研究内容包括三个方面：i）益智提取物多成分的药代动力学研究；ii）益智提取物与CPT-11药代动力学相互作用研究；iii）益智提取物抗炎活性及分子生物学机制研究。研究发现：i）灌胃（p.o.）给药后，益智活性成分可快速被胃肠道吸收，Tmax小于0.5 h。在高剂量下（0.54 g干膏/Kg），除益智酮乙外，其余成分在大鼠血浆样品中均有显著暴露。二芳基庚烷类和黄酮类成分的药-时曲线呈现双峰现象。在所研究给药剂量范围内，除芹菜素-7,4'-二甲氧醚外，益智活性成分的系统暴露量AUC与给药剂量呈正相关。ii）益智活性成分苷元在胃肠道组织和肝脏中暴露水平最高，由高至低的顺序为胃>小肠>大肠>肝。各组织中诺卡酮暴露均最高。二芳基庚烷类和黄酮类成分在胃、小肠、大肠和肝组织中的药-时曲线也呈现双峰现象。iii）益智二芳基庚烷类成分和黄酮类成分主要以II相结合代谢产物形式从胆汁消除，苷元从尿消除仅占给药剂量的很小一部分。诺卡酮胆汁排泄和尿排泄相当。益智活性成分尿累积排泄量与给药剂量均呈正比关系。vi）益智二芳基庚烷类化合物对CYP450酶均有明显的抑制活性。v）同服益智提取物显著增加模型大鼠血浆中SN-38、SN-38G和APC的Cmax和AUC0-t。；vi）Western Blot法研究发现，与模型组相比，益智提取物、白杨素、诺卡酮、益智酮乙均显著降低结肠组织中iNOS、COX-2和MCP-1的蛋白表达；vii）Elisa法研究发现，与模型组比较，诺卡酮和益智酮乙能显著降低结肠组织中COX-2的含量，而白杨素和高剂量益智提取物组则显著降低结肠组织中iNOS的含量。本课题从PK和PD层面阐明温脾止泻中药益智减轻CPT-11 所致腹泻毒性的内在机理，为益智的创新发展和应用提供依据，为探索益智在临床上用于减轻化疗药物引起的腹泻毒性奠定基础。