Identification and validation of active ingredient in cerebrotein hydrolysate-I based on pharmacokinetic and pharmacodynamic studies

Cerebrotein hydrolysate-1 (CH-1), a mixture of small peptides, polypeptides and various amino acids derived from porcine brain, has been widely used in the treatment of cerebral injury. However, the bioactive composition and pharmacokinetics of CH-1 are still unexplored because of their complicated composition and relatively tiny amounts in vivo. Herein, NanoLC-Orbitrap-Fusion-Lumos-Tribrid-MS/MS was firstly used to qualitatively analyze the components of CH-1. A total of 1347 peptides were identified, of which 43 peptides were characterized by high MS intensity and identification accuracy. We then innovatively synthesized 4 main peptides for activity verification, and the results suggested that Pep72 (NYEPPTVVPGGDL) had the strongest neuroprotective effect on both in vivo and in vitro models. Next, a quantitative method for Pep72 was established based on LC-MS/MS with the aid of Skyline software, and then used in its pharmacokinetic studies. The results revealed that Pep72 had a high elimination rate and low exposure in rats. In addition, hCMEC/D3 based in vitro model was built and firstly used to investigate the transport of Pep72. We found that Pep72 had extremely low blood-brain barrier (BBB) permeability, and was not a substrate of efflux transporters. The biotransformation of Pep72 in rat fresh plasma and tissues was investigated to explore the contradiction between pharmacokinetics and efficacy. A total of 11 main metabolites were structurally identified, with PGGDL and EPPTVPGGDL being the main metabolites of Pep72. Notably, metalloproteinase and cysteine protease were confirmed to be the main enzymes mediating Pep72 metabolism in rat tissues.