Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine™ and BCNU

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine™ (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine™ being equally potent inhibitors of purified human GR (IC50 values of 55.5 μM and 54.6 μM, respectively). Human erythrocytes, following exposure to 50 μM BCNU for 1 h at 37 °C, had an 84% decrease in GR activity, whereas 50 μM Cloretazine™ caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1 mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine™ compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.