Comprehensive pharmacokinetics and metabolite profiling of the novel anti-tumor polypeptide CPP-4-2 in rats using...

CPP-4-2, a novel anti-tumor peptide, has been shown to disrupt tumor growth by competitively binding to Staphylococcal nuclease domain-containing protein 1 (SND1), inducing its degradation and inhibiting the SND1-Metadherin (MTDH) interaction. Despite the established efficacy of this approach, comprehensive in vivo analytical methodologies remain limited. Therefore, based on the physicochemical characteristics of CPP-4-2, its anti-hepatocellular carcinoma activity was confirmed using CCK-8, colony formation, and cell migration assays. A comprehensive quality standard for CPP-4-2, encompassing description, identification, tests, assay, and related substances, was established, alongside a validated HPLC method for in vitro quantification. Additionally, a highly sensitive, specific, reproducible, and rapid UHPLC-QTRAP-MS/MS method for in vivo CPP-4-2 quantification was developed and fully validated, meeting the requirements for biological sample analysis in plasma, heart, liver, spleen, lung, and kidney tissues. Application of this method to SD rats following intravenous injection (10 mg/kg) revealed a rapid peak plasma concentration, followed by a swift decline, indicating a primary efficacy window of 0-2 h. 16 metabolites, including the parent compound, were identified using UHPLC-Q-TOF-MS/MS. This study highlights the potential of CPP-4-2, establishes quality control, provides key in vivo data, and lays the foundation for further structural modifications, development of novel dosage forms, and investigations into safe medication practices.