title: 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.
authors.affiliation: Leiden Amsterdam Center for Drug Research, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: J P
authors.initials: JP
authors.lastname: Brakenhoff
authors.forename: J N
authors.initials: JN
authors.lastname: Commandeur
authors.forename: F J
authors.initials: FJ
authors.lastname: de Kanter
authors.forename: B L
authors.initials: BL
authors.lastname: van Baar
authors.forename: W C
authors.initials: WC
authors.lastname: Luijten
authors.forename: N P
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000378:metabolism*; D066298:In Vitro Techniques; D017953:Isocyanates / Q000737:chemistry / Q000493:pharmacokinetics*; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009607:Nitrosourea Compounds / Q000737:chemistry* / Q000493:pharmacokinetics*; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000737:chemistry* / Q000493:pharmacokinetics*; D051381:Rats; D017208:Rats, Wistar; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D017953:Isocyanates; D009607:Nitrosourea Compounds; D063065:Organophosphonates; D009943:Organophosphorus Compounds; C089229:diethyl (1-isocyanatoethyl)phosphonate; D005978:Glutathione; C054368:fotemustine