Pharmacodynamic-Driven Sequence-Dependent Synergy Effects in Pemetrexed-Osimertinib Combination Against Non-Small Cell Lung Cancer (NSCLC): Optimizing Synergy Through Sequential Interval

Background: Combining pemetrexed (PEM) with Osimertinib (OSI) improves outcomes in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), but optimal scheduling remains undefined. Sequential PEM → OSI strategies may outperform concurrent administration; however, the critical dosing interval determining synergy has not been explored. Methods: PEM pharmacodynamics were divided into an OSI-antagonized early phase (S-phase arrest and DNA damage accumulation) and OSI-synergized late phase (DNA damage peak, apoptosis initiation, and feedback EGFR activation). Time-course profiling of cell cycle, DNA damage, apoptosis, and EGFR pathways was evaluated under monotherapy or sequential combination regimens to elucidate the mechanisms underlying synergistic/antagonistic effects. Results: OSI antagonizes PEM’s early phase via G1 arrest but potently enhances late-phase apoptosis through Rad51/thymidylate synthase suppression, Bim upregulation, and inhibition of EGFR signaling. The 48 h interval PEM → OSI uniquely enabled complete early-phase execution and aligned OSI exposure with late-phase initiation, yielding robust synergy across OSI-sensitive cell lines. In contrast, the 24 h interval PEM → OSI sequence demonstrated synergy only in PEM-sensitive PC9 cells. Both concurrent PEM + OSI and OSI → PEM sequence induced attenuated DNA damage and apoptotic signaling. Conclusions: The 48 h interval PEM → OSI sequence maximizes efficacy by temporally segregating antagonistic and synergistic interactions. This pharmacodynamically optimized regimen represents a promising strategy for clinical translation.