Cytochrome P450 3A4-Mediated Bioactivation of Raloxifene: Irreversible Enzyme Inhibition and Thiol Adduct Formation
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- 作者：Qing Chen ; Jason S. Ngui ; George A. Doss ; Regina W. Wang ; Xiaoxin Cai ; Frank P. DiNinno ; Timothy A. Blizzard ; Milton L. Hammond ; Ralph A. Stearns ; David C. Evans ; Thomas A. Baillie ; and Wei Tang
- 刊名：Chemical Research in Toxicology
- 出版年：2002
- 出版时间：July 2002
- 年：2002
- 卷：15
- 期：7
- 页码：907 - 914
- 全文大小：103K
- 年卷期：v.15,no.7(July 2002)
- ISSN：1520-5010
文摘
Raloxifene is a selective estrogen receptor modulator which is effective in the treatment ofosteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 isinhibited by raloxifene in human liver microsomal incubations. The nature of the inhibitionwas irreversible and was NADPH- and preincubation time-dependent, with KI and kinact valuesestimated at 9.9 
M and 0.16 min-1, respectively. The observed loss of P450 3A4 activity wasattenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified inincubations with human liver microsomes; substitution with GSH occurred at the 5- or7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. Theseadducts are postulated to derive from addition of GSH to raloxifene arene oxides followed bydehydration and aromatization. Alternatively, raloxifene may be oxidized to an extendedquinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifenemost likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolishedwhen liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4IgG. The GSH adducts also were detected in incubations of raloxifene with rat or humanhepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile andurine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicatethat P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzymeactivity. The significance of these findings with respect to the clinical use of raloxifene remainsto be determined.
M and 0.16 min-1, respectively. The observed loss of P450 3A4 activity wasattenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified inincubations with human liver microsomes; substitution with GSH occurred at the 5- or7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. Theseadducts are postulated to derive from addition of GSH to raloxifene arene oxides followed bydehydration and aromatization. Alternatively, raloxifene may be oxidized to an extendedquinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifenemost likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolishedwhen liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4IgG. The GSH adducts also were detected in incubations of raloxifene with rat or humanhepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile andurine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicatethat P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzymeactivity. The significance of these findings with respect to the clinical use of raloxifene remainsto be determined.