小肠靶向释放NO前药作为P-gp抑制剂促进P-gp底物药物口服吸收的研究

Intestinal transporter P-glycoprotein limits the intestinal absorption of drugs by flushing out the substrate drugs out from a cell. The use of P-gp inhibitors could be an effective way to improve the bioavailability of drugs. Although many P-gp inhibitors could enhance drug absorption though P-gp regulation, most of them showed nonspecific action and nonselective distribution. Therefore, development of more specific P-gp inhibiting agents may be of great interest. Recently, nitric oxide donors have been reported to increase the drug accumulation in multi-drug resistant cell by inhibition of P-gp. These studies support a novel hypothesis that nitric oxide donors may be a new strategy to bypass the efflux of small-intestinal P-gp and enhance P-gp substrate drugs oral absorption. In our preliminary work, α-glucosidase activatable small-molecule NO prodrugs were synthesized, which could release NO gas in the presence of intestinal homogenate and significantly increased the intestinal absorption of P-gp substrate drug puerarin in vitro and in vivo. However, small-molecule NO prodrugs also could be absorbed by small intestine and lead to nonselective distribution to nontarget organs. To achieve small intestine-selective distribution and enhance the attachment of NO donors to small intestinal mucosa, we plan to further synthesize small intestinal-targeting release nitric oxide donors by covalently linking α-glucosidase activatable small-molecule NO prodrugs to the mucoadhesive polymer chitosan, and validate the effect of chitosan-based NO donors on P-gp efflux function. We will also evaluate the enhancement effect of chitosan-based NO donors on the oral absorption of P-gp substrate in vitro and in vivo.

研发具有低毒、高选择性的高分子P-gp抑制剂是提高P-gp底物药物口服吸收的有效途径之一。NO供体是一种新型P-gp抑制剂，可以抑制P-gp表达和外排功能，促进耐药细胞对药物的摄取。在前期工作中，基于前药原理我们合成了α-葡萄糖苷酶活化的小分子NO前药，能够在小肠中选择性活化释放NO，并且可以促进P-gp底物药物葛根素的口服吸收。为了避免小分子NO前药被小肠吸收所带来的药物相互作用，同时增加NO前药在小肠中的滞留时间，本项目拟将α-葡萄糖苷酶控制释放小分子NO前药共价连接到壳聚糖上，以制备消化道系统特异性分布的小肠靶向释放高分子NO供体，评价其对P-gp功能和P-gp底物药物吸收的影响，以期得到安全、高选择性P-gp抑制剂作为口服吸收促进剂解决P-gp底物药物口服吸收差的问题。

如何实现一氧化氮（NO）的按需释放是NO供体领域面临的挑战之一，也是限制NO临床应用的关键因素之一。本项目通过自分解链将葡萄糖片段与自发释放型NO供体（偶氮二醇烯鎓，NONOate）相连，首次合成alpha-葡萄糖苷酶诱导释放的NO供体，通过在自分解链上引入不同基团调节NO释放速度；并利用“click”反应将其共价连接到高分子量的壳聚糖上，得到无法穿过肠道屏障的高分子NO供体。电子顺磁共振(EPR)自旋捕获方法和NO荧光探针活体成像实验表明NO在肠道中alpha-葡萄糖苷酶诱导下，能够靶向肠道释放NO； 药代实验表明该NO供体可以改变P-糖蛋白(P-gp)底物药物的药代动力学性质，增加阿霉素的口服利用度；另外该NO供体还可以显著减轻非甾体抗炎药引起的肠道损伤，为药物研发提供新型药物辅料。此外，我们还通过化学生物学手段，基于“凸凹互补”原理对半乳糖苷酶进行“凹”的定点突变，并对其底物β-半乳糖基化NO供体进行“凸”的修饰，实现NO的定点释放，并将此突变酶/底物应用于治疗下肢缺血和急性肾缺血模型，结果显示能够促进血管的新生，缓解急性肾损伤。