A novel anti-tumor candidate drug, CHA for injection, was used for the treatment of malignant glioma in clinical trial (phase I) in China. The isolation and identification of the metabolites of CHA injection in humans were investigated in the present study. Urine and feces samples obtained after intramuscular administration of CHA injection to healthy adults have been analyzed by high-performance liquid chromatography coupled with high-resolution mass and multiple-stage mass spectrometry (HPLC-HRMS/MSn). No metabolite was detected in human feces; however, in human urine, a total of six metabolites were identified including isomerized CHA (P1 and P2), hydrolyzed CHA (M1and M2), and methylated CHA (M3 and M4). Among them, M3 and M4 were the main metabolites and target analytes for human mass balance study. Additionally, the structure of M3 and M4 was characterized by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPENMR), and the results demonstrated that the methoxy group of M3 and M4 was exclusively attributed to C-3′ and C-4′, respectively. Due to the unavailability of commercial reference, the pure products of M3 and M4 were synthesized by CHA methylation and followed by isolation and purification. Moreover, the potential activity of M3 and M4 on malignant glioma was predicted using a reverse molecular docking analysis on eight malignant glioma-related pathways.

The results showed that M3 and M4 had various interactions against malignant glioma-related targets.

Our study provides an insight into the metabolism of CHA injection in humans and supports the clinical human mass balance study.

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