Binding affinity and binding sites to albumin reshape efficacy of gemcitabine prodrugs

Covalent albumin conjugation revolutionizes drug circulation prolongation, where binding affinity and binding sites critically govern therapeutic efficacy, but mechanisms remain unclear. Here, we report four gemcitabine (GEM) prodrugs whose pharmacodynamics and safety profiles strictly depend on the above parameters. Specifically, two prodrugs, MG and BG, employ albumin-binding modification modules derived from N-ethylmaleimide and acryloyl chloride, while others containing succinimide and propionyl chloride serve as non-albumin-binding controls. Regarding affinity, MG exhibits the highest albumin affinity, with 45-fold higher area under the curve (AUC) and 9-fold longer half-life compared to GEM, and the strongest antitumor activity. However, its reduced safety contrasts with the moderate-affinity BG, which achieves optimal efficacy-to-safety balance. Binding sites further modulate outcomes: pre-conjugation of BG to albumin remarkably increases bioavailability (23-fold higher AUC than GEM), while pre-binding of MG diminishes efficacy. These results highlight the need to optimize both binding affinity and binding sites in albumin-conjugated prodrugs.