五环三萜酸类化合物的跨膜转运机制研究

Pentacyclic triterpenoids are a class of compounds derived from the linear hydrocarbon squalene, which are secondary plant metabolites widespread in numerous vegetal species. The lupane-, oleanane-, and ursane triterpenoids display plentiful pharmacological activities such as antitumor, anti-inflammatory, antibacterial, antiviral, antioxidant, hepatoprotective, and hypoglycemic properties without obvious adverse effects. Therefore, pentacyclic triterpenoids are promising leading compounds in the development of new multi-targeting bioactive agents for human health and disease. .However, due to the extreme low aqueous solubility, pentacyclic triterpenoids suffer from poor oral bioavailability usually less than 5%, which seriously hinders its application and development as a pharmaceutical preparation. In recently, acetyl-11-keto-beta-boswellic acid (AKBA) and rotundic acid have been found more than ten- to twenty-fold oral bioavailability compared with other pentacyclic triterpenoids. The remarkable differences in bioavailability are likely attributed to the involvement of transporter-mediated dispositions. Although intestinal drug transporters are a rate-determining barrier for the oral bioavailability, only a few have been investigated in the disposition of pentacyclic triterpenoids. .The present study is dedicated to explore the potential impact of uptake and efflux transporters on the bioavailability of the pentacyclic triterpenoids in depth. Typical triterpenoids, ursolic acid, oleanolic acid, AKBA, and rotundic acid, are chosen as model drugs with respect to pharmacological activities, structural features, pharmacokinetic profiles, and transport characteristics. Caco-2 and MDCK cell lines are used to assess the overall permeability and passive diffusion of pentacyclic triterpenoids, and transporter transfected cell lines expressing MDR1, BCRP, MRP2, OATPs, PEPTs and OCTs are established to characterize particular transporters involved in the absorption and disposition of pentacyclic triterpenoids. LC-MS and radiolabel analysis techniques are employed to determine the concentrations of model drugs and specific substrates in cell-based assay systems. The application of computational modeling algorithms combined with in vitro experimental results is conducted to gain insights into transporter–pentacyclic triterpenoids interaction domains. .Understanding transmembrane transport of pentacyclic triterpenoids will provide mechanistic explanations for the absorption and disposition, beneficial strategy for improvement of oral bioavailability, and reliable guidance for drug design and development.

五环三萜酸类化合物具有广泛的药理活性和诱人的开发前景，已成为天然产物研究和新药研发中最活跃的领域。文献报道，齐墩果酸、熊果酸等多数五环三萜酸的跨膜转运能力较弱，口服生物利用度普遍低于5%，极大限制了该类化合物的成药性研发；而本课题组近期发现，乙酰乳香酸、铁冬青酸等具有“反常”的跨膜能力，口服生物利用度分别达21.4%和55.4%，五环三萜酸类化合物跨膜转运的巨大差异现象值得深入探讨。本课题选取熊果酸、齐墩果酸、乙酰乳香酸、铁冬青酸等作为模型药物，综合应用多种人源化转运体基因转染细胞模型，联合液质联用、放射性同位素等分析方法，以及计算机模拟技术，探究影响其生物利用度悬殊差异的跨膜转运因素。研究结果将有助于揭示五环三萜酸类化合物的跨膜转运机制及其与结构的关系，从细胞及分子水平认识其在机体内的处置过程，为提高其生物利用度提供新的技术策略，为加快新药研发与临床应用提供可靠的理论依据及广阔的思路。

五环三萜酸类化合物是多种常见中药的有效成分，具有广泛的药理活性。但五环三萜酸类化合物由于结构的特殊性，跨膜转运能力较弱，口服生物利用度普遍较低（<5%），极大限制了该类化合物的成药性研发。本课题组近期发现，乙酰乳香酸、铁冬青酸等具有“反常”的跨膜能力，口服生物利用度分别达21.4%和55.4%，五环三萜酸类化合物跨膜转运能力的巨大差异现象值得深入探讨。.本课题首先对Caco-2细胞中药物转运蛋白的mRNA表达及活性进行了考察，并选取不同渗透性的工具药，应用Caco-2细胞模型评价其肠道渗透性，建立表观渗透系数与渗透性类别的相关性及与吸收程度的顺序关系，为准确评价五环三萜酸类化合物的总跨膜能力及渗透性水平提供可靠的模型体系。接着选择合适的宿主细胞特异性表达人源药物转运蛋白，构建了16种人源化药物转运体基因转染细胞模型，并进行了功能验证。选取熊果酸、齐墩果酸、乙酰乳香酸、铁冬青酸、甘草次酸、11-酮基-β-乳香酸(KBA)、3-乙酰基-β-乳香酸(ABA)七种五环三萜酸作为研究对象，考察其在Caco-2细胞模型中的渗透性水平、在ABC主动外排转运细胞模型中的外排转运机制、以及在SLC主动摄入转运细胞模型中的摄入机制，探究影响其生物利用度悬殊差异的跨膜转运因素。.铁冬青酸、甘草次酸的Papp(A→B)分别为3.56×10-6cm/s和4.15×10-6cm/s，具有中等偏上的渗透性，主要通过被动扩散方式跨膜吸收，均存在由MDR1介导的主动外排转运，但外排能力有限、易饱和；乙酰乳香酸、KBA的Papp(A→B)分别为0.816×10-6cm/s、2.28×10-6cm/s，跨膜能力中等偏下，均无外排型转运蛋白介导的主动外排转运，均存在由OATPs转运蛋白介导的主动摄入转运；齐墩果酸、熊果酸及ABA在Caco-2细胞的Papp(A→B)均小于0.5×10-6cm/s，跨膜能力较弱，为低或零渗透性化合物，均无外排或摄入型转运体介导的主动转运。.本研究揭示了七种五环三萜酸类化合物的跨膜转运机制，从细胞及分子水平认识其在机体内的处置过程，理解引起其生物利用度差异的跨膜转运机制因素。