title: Synthesis, cleavage profile, and antitumor efficacy of an albumin-binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B.
abstract: 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 = epsilon-maleimidocaproic acid) to the gamma-COOH group of alpha-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 4x15 mg/kg methotrexate equivalents demonstrated distinctly superior antitumor efficacy compared to free methotrexate at a dose of 4x100 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.
authors.affiliation: Tumor Biology Center, Freiburg, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Warnecke
authors.forename: Iduna
authors.initials: I
authors.lastname: Fichtner
authors.forename: Gretel
authors.initials: G
authors.lastname: Sass
authors.forename: Felix
authors.initials: F
authors.lastname: Kratz
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000378:metabolism; D002401:Cathepsin B / Q000494:pharmacology*; D005260:Female; D005341:Fibrinolysin / Q000494:pharmacology*; D006801:Humans; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D011355:Prodrugs / Q000138:chemical synthesis* / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D011355:Prodrugs; D012709:Serum Albumin; D005341:Fibrinolysin; D002401:Cathepsin B; D008727:Methotrexate