Eleven methotrexale-alpha-peptides containing arginine (MTX-Arg), asparagine (MTX-Asn), cysteine (MTX-Cys), glutamine (MTX-Gln), histidine (MTX-His), lysine (MTX-Lys), methionine (MTX-Met), phenylalanine (MTX-Phe), proline (MTX-Pro), tryptophan (MTX-Trp) and tyrosine (MTX-Tyr) were produced by solid phase synthesis using the Fmoc method. Purity of the MTX derivatives was assessed by capillary zone electrophoresis (CZE). An enzymatic assay was then developed using CZE for monitoring the hydrolysis of the MTX-alpha-peptides by bovine pancreatic carboxypeptidase A (CP-A) leading to the release of free MTX. MTX-Phe appeared to be the most suitable substrate for CP-A out of the eleven tested with a hydrolysis rate comparable to that of hippuryl-L-phenylalanine, a natural substrate for CP-A. In vitro assays on a human ovarian teratocarcinoma cell line (CRL-1572), showed that MTX-Phe was non toxic, but when combined with 1 mU of CP-A, MTX-Phe cytotoxicity was enhanced considerably showing only two times less pharmacological activity than MTX. These results suggest that MTX-Phe is a potent prodrug and could be used in a drug targeting model combining monoclonal antibodies coupled with CP-A for a more specific approach in cancer therapy.

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