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

Abstract

Fotemustine is a chemotherapeutic drug for the treatment of melanoma. In this study, we investigated the metabolic and chemical stability of fotemustine with 31P-NMR and FAB-MS. In the absence of GSH, 95% of fotemustine decomposed rapidly into a reactive diethyl ethylphosphonate (DEP) isocyanate, both in rat liver S9 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 via dechlorination into diethyl [1-(3-nitroso-2-oxoimidazolidin-1-yl)ethyl]-phosphonate. In the presence of GSH, hydrolysis of DEP-isocyanate was blocked, 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-SG 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 S9 fractions. In the presence and in the absence of GSH, the half-lives of decomposition (t1/2) of fotemustine were 33 +/- 6 and 27 +/- 3 min, respectively. The formation of the DEP-isocyanate and 2-chloroethanediazohydroxide intermediates from fotemustine appeared to be rate limiting, and not the hydrolysis of the DEP-isocyanate nor its conjugation to GSH. Active or inactive rat liver S9 fractions accelerated the decomposition of fotemustine slightly; i.e., the t1/2 of fotemustine decreased from 39 +/- 3 to 29 +/- 1 min. Further knowledge of the metabolic and chemical stability of fotemustine and DEP-isocyanate will contribute to a better understanding of fotemustine-related cytostatic effects and toxic side effects and to the design of chemoprotection against undesired toxic side effects.

Identification of novel reaction products of methylene-bis - phenylisocyanate (“MDI”) with oxidized glutathione in aqueous solution and also during incubation of MDI with a murine hepatic S9 fraction

“…The novelty of the study is one strength; while S9-mediated transformation of mono-isocyanates (e.g. decomposition products of fotemustine, carmustine, and other anti-cancer drugs) has been shown to involve GSH (12, 13), to the best of our knowledge, similar studies with diisocyanates have yet to be reported. Additional strengths of the present investigation include the use of LC-MS and LC-MS/MS to characterize MDI reaction products with glutathione under well controlled conditions in vitro.…”

“…While GSH conjugation and metabolism of mono-isocyanates via the mercapturic acid pathway has been established as a major route by which these chemicals are excreted in vivo, the role of glutathione in metabolism of MDI (and other diisocyanates) remains unclear (12–15). Many studies to date have inferred diisocyanate excretion in urine based on the levels of the corresponding diamine released following acid or base hydrolysis (16–18).…”

Mechanism of Action of Fotemustine, a New Chloroethylnitrosourea Anticancer Agent: Evidence for the Formation of Two DNA-Reactive Intermediates Contributing to Cytotoxicity

“…The likely candidates for the reaction intermediates revealed by this work are not obvious. Briscoe et al (1990) suggested pathways for breakdown of the chloroethyl moiety of CENUs based on BCNU; Brakenhoff et al (1993Brakenhoff et al ( , 1994 also provide a scheme for the breakdown of FM. These mechanisms involve the formation of 2-chloroethyldiazohydroxide, which is thought to be unstable and hence a reasonable candidate for the fast reaction component.…”

“…Estimates of the half-life of the intact drug in plasma have ranged from 12 min in cynomolus monkeys (drug administered as a bolus) to 23 min in a human (drug administered as an intravenous infusion over a 1 h period (Gordon et al, 1989). In HEPES buffer, the halflife was estimated to be 24-40 min (Brakenhoff et al, 1994). Our studies showed a biphasic effect with a substantial loss in cytotoxic activity, N 7 -guanine alkylation and DNA interstrand cross-linking occurring in the first 5 min of addition of drug to aqueous medium.…”

“Commandeuring” Xenobiotic Metabolism: Advances in Understanding Xenobiotic Metabolism

“…In order to elucidate the mechanism of toxicity of fotemustine, various metabolites were synthesized. 32 Also, the metabolic and chemical stability of fotemustine was investigated with 31 P NMR and FAB-MS. 33 In the absence of glutathione (GSH), 95% of fotemustine decomposed rapidly into a reactive diethyl ethylphosphonate (DEP)-isocyanate, both in rat liver S9 fraction and in buffer. DEP-isocyanate, in turn, hydrolyzed rapidly into diethyl (1-aminoethyl)phosphonate, which reacted subsequently with the parent DEP-isocyanate.…”