基于PEPT1和PSA双靶向原理的帕拉米韦拟肽类前药的研究

Oral administration was the dominant route for the patient. But, at present, more emphasis has been placed on the affinity of new chemical entities (NCE) to the target receptor during the process of drug development, which lead to the unsuitability for some drugs to oral administration because of the undesirable pharmacokinetic characteristics. The barrier to oral administration could be conquered by a certain drug delivery system. But this strategy was not effective in some circumstance. The intestinal membrane transporters and some aminopeptidases widely expressed in the organism have become promising targets for oral drug delivery. In the present study, some peptidomimetics of peramivir doubly targeted to peptide transporter 1 (PEPT1) and puromycin-sensitive aminopeptidase (PSA) were synthesized by attaching amino acid to the carboxyl group. The peptidomimetics were evaluated by the affinity to PEPT1 and the hydrolysis rate by PSA. To help the rational design of successful prodrugs, the quantitative structure-affinity/hydrolysis rate relationships were constructed by analyzing the physiochemical and structural parameter of the prodrug. The pharmacokinetics of the candidate prodrug was studied in rat and dog to delineate the hydrolysis mechanism, the species difference and the pharmacokinetic behavior. The preliminary result showed the present study was feasible.

口服给药是最主要的给药途径，但是由于目前药物体外筛选注重与靶受体的亲和性，而导致有的药物性质并不适合口服给药。虽然有时通过给药系统可以克服，但并不都能行得通。肠道中广泛分布的药物转运蛋白以及生物体内广泛分布的氨基肽酶，利用它们为靶点来提高药物的口服吸收是非常有前途的，而且适合我国目前新药创制的条件。本课题以肠寡肽转运蛋白（PEPT1）和嘌呤霉素敏感的氨基肽酶（PSA）为靶点，选择口服吸收差的帕拉米韦（Peramivir）为模型药物，在帕拉米韦的羧基位引入氨基酸，从与PEPT1亲和性和PSA对前药的降解速率两方面对帕拉米韦拟肽类前药进行考察和筛选；结合药物理化和结构参数，建立定量结构-亲和性/降解速率关系，指导前药的合理设计。选择适宜的前药进行大鼠和犬体内药动学研究，考察体内的活化机制、种属差异和药动学行为，并筛选出最佳候选前药。前期工作结果表明本项目可行性良好。

帕拉米韦是一个高效的神经氨酸酶抑制剂，用于流感病毒A和B的防治。然后由于极性大和较低的膜渗透性，帕拉米韦的口服生物利用很低。本课题以PEPT1和嘌呤霉素敏感的氨基肽酶（PSA）为靶点，合成了7个氨基酸酯类前药和7个氨基酸酰胺前药。Caco-2细胞实验表明，14个氨基酸前体药物的膜渗透率比帕拉米韦都要高，其中Peramivir-(CH2)2-L-Val膜渗透率是10.5×10-6 cm/s，是帕拉米韦的10.9倍，其次是L-异亮氨酸酰胺前药Peramivir-L-Ile，它的膜渗透率是帕拉米韦的9.13倍，因此选择Peramivir-(CH2)2-L-Val和Peramivir-L-Ile做下一步的研究。建立了膜渗透性LogP和和结构LogD之间的关系。随着Peramivir-(CH2)2-L-Val和Peramivir-L-Ile浓度的增加，Gly-Sar被Caco-2摄取的量逐渐减少，两个前药对Gly-Sar摄取的半数抑制浓度(IC50)分别是1.34 ± 0.31 mM和1.78 ± 0.48 mM。Peramivir-(CH2)2-L-Val在MDCK-PEPT1细胞上摄取量是空白MDCK细胞的5.5倍，Peramivir-L-Ile在MDCK-PEPT1细胞上摄取量是空白MDCK细胞的4.6倍，并且能够被Gly-Sar抑制。在MDCK-PEPT细胞上Peramivir-(CH2)2-L-Val和Peramivir-L-Ile在0.01-50 mM范围内摄取量符合米氏方程。Peramivir-(CH2)2-L-Val和Peramivir-L-Ile的最大转运速度分别是14.89 ± 0.65 and 14.07 ± 0.54 mM·mg-1protein·10 min-1, 米氏常数被分别是4.23±0.11 and 5.92±0.14 mM。稳定性研究表明Peramivir-L-Ile是PSA的底物。口服帕拉米韦、Peramivir-(CH2)2-L-Val和Peramivir-L-Ile后， 帕拉米韦的生物利用度分别是4.1%，65.3%，37.3%; 还考察了Peramivir-(CH2)2-L-Val犬体内药动学。因此口服Peramivir-(CH2)2-L-Val和Peramivir-L-Ile能提高Peramivir的口服生物利用度。

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