PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) 30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus. The active endostatin fragment of PEP06 directly targets tumor cells and exerts an antitumoral effect. However, little is known about the kinetics and degradation products of PEP06 in vitro or in vivo. In this study, we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species; we further identified the degradation characteristics of its cleavage products. PEP06 underwent rapid enzymatic degradation in multiple types of living cells, and the liver, kidney, and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06. We identified metabolites of PEP06 using full-scan mass spectrometry (MS) and tandem MS (MS²), wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide, formed by successive losses of amino acids. The metabolites were C and N terminal truncated products of PEP06. The structures of 11 metabolites (M6, M7, M16, M17, M21, M25, M33, M34, M39, M40, and M42) were further confirmed by comparing the retention times of similar full MS spectrum and MS² spectrum information with reference standards for the synthesized metabolites. We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06, which can support further drug research.

PEP06 是一种新型内皮抑素-Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) 30-氨基酸多肽，在 N 末端具有末端融合的 RGDRGD 六肽。PEP06 的活性内皮抑素片段直接靶向肿瘤细胞并发挥抗肿瘤作用。然而，关于 PEP06 在体外或体内的动力学和降解产物知之甚少。在这项研究中，我们研究了 PEP06 与不同物种动物的活细胞一起孵育后的体外代谢稳定性；我们进一步确定了其裂解产物的降解特征。PEP06在多种活细胞中发生快速酶降解，肝脏、肾脏和血液在PEP06分子降解产生的肽的代谢和清除中起重要作用。² )，其中43种代谢物被表征并鉴定为来自母体肽的降解代谢物，由连续的氨基酸损失形成。代谢物是 PEP06 的 C 和 N 末端截短产物。11 种代谢物（M6、M7、M16、M17、M21、M25、M33、M34、M39、M40 和 M42）的结构通过将相似的全 MS 谱图和 MS ²谱图信息与参考标准的保留时间进行比较进一步确认对于合成的代谢物。我们已经在体外证明了 PEP06 的代谢稳定性，并鉴定了 PEP06 的一系列潜在生物活性下游代谢物，可以支持进一步的药物研究。