Galactosylated Camptothecin (Gal-CPT) as a New Anticancer Prodrug Sequentially Activated by Dual Tumor-Specific Enzymes

Abstract

Camptothecin (CPT) and its clinically used derivatives are still plagued by severe off-target toxicities. In this study, we designed a galactosylated CPT (Gal-CPT) prodrug incorporating an enzymatically cleavable diethylene glycol linker. In vivo experimental results verified that Gal-CPT exhibited markedly better antitumor efficacy (59.98 ± 4.75% vs 20.52 ± 3.10%) and stronger tumor-specific accumulation than free CPT. This prodrug maintained excellent stability against hydrolysis and remained stable in acidic/neutral solutions, liver microsomes, plasma, and CYP450 metabolic systems. Notably, it could achieve the targeted release of active CPT through sequential cleavage of glycosidic and ester bonds. This activation process was triggered by tumor-enriched CYP1B1 and CYP3A5 at both the enzymatic and cellular levels. Molecular docking and molecular dynamics (MD) simulations further validated this activation mechanism. Additionally, Gal-CPT possessed favorable water solubility and prominent tumor-targeting capability, accompanied by a prolonged half-life (T1/2 = 2.6 h vs 0.75 h). Overall, this dual-enzyme-responsive prodrug offers a promising strategy for precise chemotherapy.