Synthesis, Cleavage Profile, and Antitumor Efficacy of an Albumin‐Binding Prodrug of Methotrexate that is Cleaved by Plasmin and Cathepsin B

Cathepsin B and plasmin are intra‐ or extracellular proteases that are overexpressed by several solid tumors. In order to exploit both proteases as molecular targets for tumor‐specific cleavage of prodrugs, an albumin‐binding formulation of methotrexate was developed that incorporated the peptide sequence D ‐Ala‐Phe‐Lys as the protease substrate. Albumin is a suitable carrier for cytostatic agents due to passive accumulation in solid tumors. Synthesis was performed by coupling the peptide linker EMC‐ D ‐Ala‐Phe‐Lys(Boc)‐Lys‐OH (EMC = ε‐maleimidocaproic acid) to the γ‐COOH group of α‐ tert ‐butyl protected methotrexate. After cleavage of the protective groups and purification on reverse phase HPLC, a highly water‐soluble methotrexate‐peptide derivative was obtained that binds rapidly and selectively to human serum albumin. The albumin‐bound form of the prodrug was shown to be efficiently cleaved by cathepsin B and plasmin as well as in an ovarian carcinoma homogenate (OVCAR‐3) liberating a methotrexate‐lysine derivative. In an OVCAR‐3 xenograft model, the prodrug at a dose of 4×15 mg/kg methotrexate equivalents demonstrated distinctly superior antitumor efficacy compared to free methotrexate at a dose of 4×100 mg/kg [T/C(%) for MTX = 69; T/C(%) for MTX prodrug = 29]. The data provide a further proof of concept for the development of albumin‐binding, enzymatically cleavable prodrugs of anticancer drugs.