CHARACTERIZATION OF VERLUKAST METABOLITES ARISING FROM AN EPOXIDE INTERMEDIATE PRODUCED WITH HEPATIC MICROSOMES FROM BETA-NAPHTHOFLAVONE-TREATED RODENTS (P-4501A1)

Verlukast, methylamino-3-oxopropylthio)methyl)thio)-propionic acid (al so known as MK-0679 and L-668,019), is a potent leukotriene D4 antagon ist. Verlukast was incubated with hepatic microsomes from beta-naphtho flavone (betaNF) or isosafrole-treated rodents to evaluate whether P-4 501A1 or 1A2 mediated biotransformations could occur. With betaNF-indu ced mouse or rat microsomes, in which the induction of P-4501A1 had be en proven by Western blot analysis, incubations produced new metabolit es that were separated by reversed-phase HPLC and were initially chara cterized by UV (photodiode array). Metabolites were subsequently isola ted and characterized by NMR and MS, and were assigned as the 5'',6''- dihydrodiol and 6''-phenol (on the quinoline ring). The presumed 5'',' '-epoxide intermediate was also detected and was characterized by UV ( photodiode array) and MS. Microsomes from isosafrole-treated rodents p roduced the dihydrodiol to a much lesser extent and did not yield any other new metabolites. Alpha-naphthoflavone inhibited the dihydrodiol formation in incubations with microsomes from isosafrole- and betaNF-t reated rats. In incubations with microsomes from betaNF-treated rats, to which the epoxide hydrolase inhibitor 3,3,3-trichloropropene 1,2-ox ide had been added, the formation of dihydrodiol was inhibited, consis tent with a microsomal epoxide hydrolase hydrolysis of the epoxide int ermediate. When glutathione was added to incubations with microsomes f rom betaNF-treated rats, the dihydrodiol, phenol, and epoxide peaks we re reduced in size and a new material, the glutathione adduct, was for med. This material was isolated and characterized by NMR and capillary HPLC continuous-flow liquid secondary-ion MS. Glutathione S-transfera se was confirmed to be present in the microsomes by Western blot analy sis. A metabolic route for these biotransformation products is propose d.