Combination of MDM2 inhibitor and lipophilic gemcitabine: Strong synergism in lung cancer, while antagonism in brain cancer

Cancer remains the leading cause of mortality worldwide, even in the post-COVID-19 era. Lung and brain cancers account for a substantial proportion of these deaths. Although multiple anticancer agents are available, single-agent therapies often fail to achieve durable responses due to limited efficacy or the development of drug resistance. Combination therapy is therefore recognized as a powerful strategy to enhance therapeutic efficacy and delay resistance.In this study, we investigated the synergistic potential of Idasanutlin (Ida), a second-generation MDM2 inhibitor, and Gemcitabine Elaidate (Gem Eli), a lipophilic prodrug of Gemcitabine, for the treatment of non-small cell lung cancer (NSCLC) and glioblastoma (GBM). Both molecules exhibit hydrophobic characteristics, prompting their incorporation into the lipid bilayer of liposomal carriers. Using a modified hydration method, we developed a liposomal nanoformulation co-loaded with Ida and Gem Eli (IG). A major challenge was the rapid insoluble precipitation of Ida, a brick-dust molecule. To address this, we introduced the cationic ionizable lipid DLin-DMA to enable the formation of a charge-tunable complex between anionic Ida and cationic DLin-DMA. Ionizable lipids remain neutral at physiological pH but acquire a positive charge in acidic conditions (e.g., the tumor microenvironment, pH ~5.5), which facilitates tumor-targeted delivery while minimizing systemic toxicity. The IG formulation was evaluated in NSCLC cell lines (A549 and H460) and a GBM cell line (U-87), all harboring wild-type p53. Strong synergism was observed in NSCLC, with combination index values of 0.07 (A549) and 0.3 (H460). In contrast, U-87 cells demonstrated no synergism; instead, Gem Eli displayed antagonistic behavior, with a ~7-10-fold increase in IC₅₀ when combined with Ida across ratios ranging from 1:1 to 1:20. A 3D spheroid assay further confirmed antagonism in GBM, after 10 days of alternate-day treatment, IG, Gem Eli, and Ida reduced spheroid area by 81%, 61.9%, and 81%, respectively, compared to controls. Synergism in NSCLC was further validated using Combenefit analysis, which demonstrated robust synergy in 3D synergy maps. The optimized IG liposomes exhibited a particle size of 131.1 ± 0.68 nm, a PDI of 0.172 ± 0.13, and pH-responsive zeta potentials of -2.06 ± 0.06 (pH 7.4) and +8.88 ± 0.31 (pH 5.5), supporting their suitability for tumor-targeted delivery. Hemocompatibility testing in mouse red blood cells showed <1% hemolysis at concentrations up to 100 µM. Cryo-electron microscopy confirmed uniform particle morphology with no evidence of aggregation. Ongoing studies are focused on elucidating the cellular mechanisms underlying the observed antagonism in GBM.