CHEMICAL AND GLUTATHIONE CONJUGATION-RELATED DEGRADATION OF FOTEMUSTINE - FORMATION AND CHARACTERIZATION OF A GLUTATHIONE CONJUGATE OF DIETHYL (1-ISOCYANATOETHYL) PHOSPHONATE, A REACTIVE METABOLITE OF FOTEMUSTINE

Fotemustine is a chemotherapeutic drug for the treatment of melanoma. In this study, we investigated the metabolic and chemical stability of fotemustine with P-31-NMR and FAB-MS. In the absence of GSH, 95% of f otemustine decomposed rapidly into a reactive diethyl ethylphosphonate (DEP) isocyanate, both in rat liver S-9 fraction and in HEPES buffer (pH = 7.4). DEP-isocyanate in turn hydrolyzed rapidly into diethyl (1- aminoethyl)phosphonate, which reacted subsequently with the parent DEP -isocyanate. The remaining 5% of fotemustine was shown to decompose vi a dechlorination into diethyl nitroso-2-oxoimidazolidin-1-yl)ethyl]-ph osphonate. In the presence of GSH, hydrolysis of DEP-isocyanate was bl ocked, and a glutathione conjugate (DEP-SG) was formed instead. DEP-SG was relatively stable at 37 degrees C in HEPES buffer. Only two minor and as yet unidentified decomposition products were formed. Addition of N-acetyl-L-cysteine (NAC) to DEP-SG in HEPES buffer converted DEP-S G rapidly into the corresponding NAC conjugate of DEP-isocyanate (DEP- NAC). The formation of DEP-SG from DEP-isocyanate and GSH appeared to be spontaneous. The extent of formation of DEP-SG from fotemustine and GSH was equal in both enzymatically active and inactive rat liver S-9 fractions. In the presence and in the absence of GSH, the half-lives of decomposition (t(1/2)) of fotemustine were 33 +/- 6 and 27 +/- 3 mi n, respectively. The formation of the DEP-isocyanate and 2-chloroethan ediazohydroxide intermediates from fotemustine appeared to be rate lim iting, and not the hydrolysis of the DEP-isocyanate nor its conjugatio n to GSH. Active or inactive rat liver Ss fractions accelerated the de composition of fotemustine slightly; i.e., the t(1/2) of fotemustine d ecreased from 39 +/- 3 to 29 +/- 1 min. Further knowledge of the metab olic and chemical stability of fotemustine and DEP-isocyanate will con tribute to a better understanding of fotemustine-related cytostatic ef fects and toxic side effects and to the design of chemoprotection agai nst undesired toxic side effects.