Novel insight into the mechanism underlying synergistic cytotoxicity from two components in 5-Fluorouracil-phenylalanine co-crystal based on cell metabolomics.

Co-crystallization of active pharmaceutical ingredients (API) with co-formers can induce synergistic effects on cytotoxicity; however, the underlying mechanism is unclear. Here, cell metabolomics was used to gain insight into the mechanisms of synergistic effect from API and co-former in co-crystal. The 5-Fluorouracil-phenylalanine co-crystal system was selected as the model owing to the apparent difference of cytotoxicity occurring between co-crystal and physical mixture of two components (PM). The cytotoxicity of 5-FU, PM and co-crystal on B16 cells were evaluated by MTT assay. Based on the IC values from MTT assays, the cytotoxicity mechanism of 5-FU, PM and co-crystal was evaluated using a comprehensive non-targeted metabolomics strategy based on multivariate data analysis and statistics using UHPLC-Q-TOF-MS/MS platform with IDA data acquisition. Co-crystal showed higher cytotoxicity than PM against B16 cells. In the cell metabolomics study, a total of 12 differential metabolites were found. Pathway analysis indicated that differences in purine and glycerophospholipid metabolism occurred between PM and co-crystal. The downregulated deoxyguanosine diphosphate and adenosine diphosphate in the purine metabolism and downregulated L-glycerophosphocholine and upregulated C16-dihydroceramide in the glycerophospholipid metabolism were associated with cellular antiproliferation and apoptosis, which directly influenced the cytotoxicity. Cell metabolomics was used to investigate the cytotoxicity mechanism of the pharmaceutical co-crystal, providing an effective and innovative method for clarifying the synergistic mechanism of API and CCF in co-crystal.