Microsomal metabolism of the 5-lipoxygenase inhibitors L-746,530 and L-739,010 to reactive intermediates that covalently bind to protein: the role of the 6,8-dioxabicyclo[3.2.1]octanyl moiety.
Hepatic microsomes from different species were used to study the oxidative metabolism of L-746,530 and L-739,010, two potent and specific 5-lipoxygenase inhibitors. HPLC analysis of the incubates obtained from the microsomal incubations of L-739,010 and L-746,530 showed only traces of metabolites. However, recovery of the starting material in the supernatant was less than quantitative in all of the species studied (approximately 90% in rat, approximately 70% in the dexamethasone-induced rat, approximately 70-90% in humans, and approximately 60% in the rhesus monkey for both compounds). The recovery of the starting material was found to be time- and NADPH-dependent, suggesting that metabolite(s) were formed and reacting with the microsomal proteins. Evidence that the cytochrome P4503A (CYP3A) contributed to the formation of the reactive metabolite(s) was shown by the low recovery of material that was observed upon incubation with microsomes obtained from dexamethasone-treated rats (a CYP3A inducer), compared with microsomes obtained from untreated rats. Also, the recovery of material was improved when troleandomycin, a CYP3A inhibitor, was added to rhesus monkey microsomal incubations (25% more parent compound detected in the supernatant with 100 microM of troleandomycin). Using radiolabeled L-746,530 and gel electrophoresis analysis, it was confirmed that radiolabeled material was covalently bound to the microsomal protein. Incubations of L-739,010 and L-746,530 in the presence of semicarbazide resulted, in both cases, in the formation of two adducts. Using a combination of NMR, liquid secondary-ion MS, and UV techniques, these adducts were identified as isomers of an oxidized metabolite that had been trapped by semicarbazide. The site of oxidation was determined to be on the dioxabicyclo moiety. The importance of this moiety in the formation of reactive metabolite(s) was verified by incubating analogs of the 5-lipoxygenase inhibitors that contained blocking methyl groups at the proposed site of oxidation on the bicyclo moiety. Incubations of these gemdimethyl analogs of L-746,530 and L-739,010 with microsomes from different species resulted in significantly improved recovery of the starting material (approximately 94% in the rat, 85% in the dexamethasone-induced rat, 95% in humans, and 85% in the rhesus monkey for both compounds) and significantly less radioactive binding to the microsomal protein.
Chauret
N
N
Medicinal Chemistry Department, Merck Frosst Center for Therapeutic Research, Quebec, Canada.
Nicoll-Griffith
D
D
Friesen
R
R
Li
C
C
Trimble
L
L
Dubé
D
D
Fortin
R
R
Girard
Y
Y
Yergey
J
J
D000818:Animals; D015153:Blotting, Western; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D016859:Lipoxygenase Inhibitors / Q000378:metabolism*; D008253:Macaca mulatta; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011804:Quinolines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet
D001643:Bridged Bicyclo Compounds; C100577:L 739010; C100578:L 746530; D016859:Lipoxygenase Inhibitors; D011804:Quinolines