Identification of carbamoylated thiol conjugates as metabolites of the antineoplastic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, in rats and humans

Abstract

The metabolic fate of 1-(2-chloroethyl)-3-cyclohexyl)-nitrosourea (CCNU) in rats and humans was investigated with a view to characterizing the nature of the carbamoylating species released upon in vivo transformation of the drug. CCNU undergoes oxidation in vivo to afford 4-hydroxy and 3-hydroxy CCNU which, along with the parent drug, decomposes to the corresponding isocyanates. Although the highly reactive nature of the isocyanate species precludes their identification in vivo, their existence as electrophilic intermediates was detected in the likeness of trapped glutathione (GSH) and N-acetylcysteine (NAC) conjugates. Conjugated thiol metabolites were purified by HPLC from the bile and urine of CCNU-dosed rats, and the urine of a patient on CCNU therapy. The metabolites were identified by atmospheric pressure chemical ionization LC/MS and LC/MS/MS. In addition, LC/MS and LC/MS/MS spectra of synthesized authentic standards corroborated the identity of the metabolites. In rats, 4-hydroxycyclohexyl, 3-hydroxycyclohexyl, and cyclohexyl isocyanate were identified as their GSH conjugates in bile and NAC conjugates in urine. In the case of the patient, the NAC conjugates of 4-hydroxycyclohexyl and 3-hydroxycyclohexyl isocyanate were identified as urinary metabolites. The identification of GSH and NAC conjugates reported herein marks a significant advance in the assessment of the in vivo carbamoylating activity of CCNU and its phase I metabolites.