Many
tumors contain elevated levels of
plasminogen activator and thus produce elevated levels of the
protease plasmin in the milieu of the
tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl
prodrug derivatives of anticancer drugs that would be locally activated by
tumor-associated
plasmin. As an initial test of this hypothesis, we synthesized the peptidyl
prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic
acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (
phenylenediamine mustard) by mixed
anhydride coupling of the parent
drug with the protected tripeptide, Boc-D-
Val-Leu-Lys(Boc)-
OH, followed by deprotection with
trifluoroacetic acid. The
prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of
plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of
plasminogen activator). In the presence of the
plasmin inhibitor,
p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the
phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free
drug. Furthermore, the
prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the
prodrug derivative of
acivicin did not display selective toxicity for transformed cells when the cells were cultured in
plasminogen-free medium. These results suggest that
plasmin hydrolysis is necessary for the activation of the
prodrugs. The
prodrugs were tested in vivo for antitumor activity. The
prodrug of
acivicin, like
acivicin itself, was inactive against the
B16 melanoma, a murine
tumor that produces high levels of
plasminogen activator. This
prodrug was active against the M5076
carcinoma, a
tumor that displays only moderate levels of
plasminogen activator; however, despite the fact that the
prodrug was 2- to 3-fold less toxic on a molar basis than
acivicin, there was no evidence of an increased therapeutic index. The
prodrug of
phenylenediamine mustard was also slightly less toxic than the parent
drug, but again there was no evidence for an improved therapeutic index against the B16
tumor.