A novel, glutathione-activated prodrug of pimasertib loaded in liposomes for targeted melanoma therapy

Pimasertib is a reversible, non-allosteric MEK inhibitor targeting the MAPK signalling pathway, which is frequently dysregulated in cancers such as melanoma in 90% of cases [1]. Although promising in clinical trials, particularly for BRAF/NRAS-mutant melanoma, Pimasertib has not gained regulatory approval due to poor selectivity and a short half-life [2]. To overcome these limitations, we developed a prodrug strategy using a redox-sensitive design responsive to the elevated glutathione (GSH) levels characteristic of the tumour microenvironment [3]. We synthesized PROPIMA, a novel Pimasertib prodrug featuring a disulfide linker that enables selective drug release in GSH-rich cancer cells. To further enhance its therapeutic profile, PROPIMA was encapsulated in liposomes using a scalable ethanol injection method. Liposomal delivery offers several advantages, including improved drug loading, stability, and targeted release [4]. Our study was conducted in two phases. In the first phase, we compared Pimasertib and PROPIMA (free and liposome-encapsulated) in the A375 melanoma model. We assessed cytotoxicity, proliferation, membrane integrity, MAPK pathway inhibition (via pERK/ERK ratio), and 2D cell migration. Specificity was also evaluated in U87 glioblastoma (MAPK-independent) and hCMEC (normal, low oxidative stress) cell lines, as demonstrated by our observation of lower glutathione peroxidase activity compared to A375. PROPIMA showed similar cytotoxicity to Pimasertib but with a more controlled release, greater inhibition of melanoma migration, higher tumour specificity, and significantly improved liposomal incorporation. In the second phase, we expanded the evaluation to both A375 and its metastatic derivative A375-MA2. PROPIMA (free and liposomal) was tested for effects on cell viability, proliferation and MAPK inhibition. We further assessed its impact on key processes in melanoma progression: 1) ECM adhesion using gelatine substrates and β1-integrin expression one of the proteins which mediate attachment to gelatine [5]; 2) 2D migration via wound healing assay; 3) Invasion of ECM through analysis of MMP-2 activation intracellularly and in culture medium [6]. PROPIMA significantly reduced cell viability, proliferation, MAPK signalling, ECM adhesion, β1-integrin levels (in A375-MA2) and 2D migration. While total MMP-2 levels remained unchanged, PROPIMA effectively reduced MMP-2 activation both intracellularly and in culture medium, showing promise in reducing also ECM invasion. In conclusion, PROPIMA offers a more selective and effective alternative to Pimasertib, employing redox-responsive release and greater liposomal incorporation for targeted action in melanoma. Its design makes it well-suited for tumours with MAPK alterations and oxidative stress, and it holds promise in reducing also tumour metastasis formation.