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 Mass Spectrometer 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 mass spectrometry (MS) intensity and identification accuracy. We then innovatively synthesized four 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 liquid chromatography tandem mass spectrometry (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, a 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 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.

SIGNIFICANCE STATEMENT The NanoLC Orbitrap Fusion Lumos Tribrid Mass Spectrometer was firstly applied to discover the components of CH-1, and one main peptide Pep72 (NYEPPTVVPGGDL) was innovatively synthesized and firstly found to have the strongest neuroprotective effect among 1347 peptides identified from CH-1. Our study is the first time to identify and verify the active ingredient of CH-1 from the perspective of pharmacokinetics and pharmacodynamics, and provides a systematic technical platforms and strategies for the active substance research of other protein hydrolysates.

This study was supported by the National Natural Science Foundation of China [Grant 82274194], the Jiangsu Natural Science Funds [Grant BK20211224], and the Natural Science Foundation of Hebei Province [Grants H2020208025, H2021208006, H2021302001, and C2021418001].

No author has an actual or perceived conflict of interest with the contents of this article.

↵1H.G. and H.Y. contributed equally to this work.

dx.doi.org/10.1124/dmd.123.001443.

↵This article has supplemental material available at dmd.aspetjournals.org.