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Methotrexate--Phenylalanine: Optimization of Methotrexate Prodrug for Activation by Carboxypeptidase A-Monoclonal Antibody Conjugate¹

Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

Methotrexate--phenylalanine (MTX-Phe), a second-generation prodrug in the MTX -peptide series designed for activation to MTX by carboxypeptidase-mAb conjugates, was synthesized by reaction of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with L-glutamyl--L-phenylalanine. Production of MTX from MTX-Phe, catalyzed by bovine pancreas carboxypeptidase A (CPA), was 250-fold faster than the corresponding reaction involving methotrexate--alanine, previously the best MTX peptide substrate for the enzyme. The amount of CPA required to make MTX-Phe equitoxic with MTX, when tested against UCLA-P3 human lung adenocarcinoma cells in vitro, was more than 10-fold lower than that required to achieve the same result with MTX--alanine. When the lung tumor cells were treated with CPA conjugated to KS1/4 (a mAb targeted to these cells) and excess conjugate removed by extensive washing, the ID₅₀ for MTX-Phe improved from 2.2 x 10^-6 M (no enzyme present) to 6.3 x 10^-8 M; the latter value was comparable to that of the parent drug MTX (4.5 x 10^-8 M). [³H]MTX-Phe was synthesized and used to investigate the mechanism by which the prodrug exerts its cytotoxic effect in the presence and absence of CPA. The present results demonstrate that, for use in conjunction with CPA-mAb conjugates, the -phenylalanine derivative is the optimal prodrug form of MTX (and probably other antifols that contain the glutamate moiety).

¹ This work was supported by Outstanding Investigator Grant CA 39836 from the National Cancer Institute and by Grant CH-31 from the American Cancer Society.

² Present address: Byk Gulden Pharmazeutika, Konstanz, Federal Republic of Germany.

³ To whom requests for reprints should be addressed.

Received 11/ 8/94. Accepted 12/19/94.

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