title: Development of methotrexate alpha-peptides as prodrugs for activation by enzyme-monoclonal antibody conjugates.
abstract: Selective delivery of lethal concentrations of drugs to tumors, allowing the latter to be eradicated without damage to other tissues, continues to be a major goal in cancer chemotherapy. Prodrugs (i.e. drugs that have been derivatized to prevent uptake into cells or interaction with targets), activated by enzyme-monoclonal antibody conjugates positioned at tumor sites, offer promise for achieving this objective. Methotrexate alpha-peptides (derivatives in which an amino acid is linked to the alpha-carboxyl group of the glutamate moiety) are ideal prodrugs, since they are not transported into cells and can be converted to the parent drug by carboxypeptidases. The L,L-diastereomer of MTX-alpha-Phe, synthesized in good yield by treatment of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with Glu-alpha-Phe, was hydrolyzed readily by carboxypeptidase A (CP-A). Conjugate was prepared by derivatizing the enzyme and monoclonal antibody KS1/4 with linkers containing maleimide and sulfhydryl groups, respectively; interaction of these groups to form a stable thioether bond joined the proteins. When administered in vitro to UCLA-P3 human lung adenocarcinoma cells (ca. 5 x 10(4) antibody binding sites/cell) that had been pre-treated with the conjugate (whose antibody KS1/4 is targeted to these cells), and excess conjugate removed by extensive washing, MTX-Phe (ID50 = 6.3 x 10(-8) M) approached the toxicity of MTX (ID50 = 4.5 x 10(-8) M). In the absence of conjugate, MTX-Phe was much less toxic (ID50 = 2.2 x 10(-6) M).
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: F M
authors.initials: FM
authors.lastname: Huennekens
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D001711:Biotransformation; D002268:Carboxypeptidases / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D006801:Humans; D006868:Hydrolysis; D018796:Immunoconjugates; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D018796:Immunoconjugates; D011355:Prodrugs; C091716:methotrexate-alpha-phenylalanine; C066904:methotrexate-alpha-alanine; D010649:Phenylalanine; D002268:Carboxypeptidases; D008727:Methotrexate