Bioactivation of the tricyclic antidepressant amitriptyline and its metabolite nortriptyline to arene oxide intermediates in human liver microsomes and recombinant P450s.
Amitriptyline, the most widely used tricyclic antidepressant, has been associated with very rare but severe incidences of hepatotoxicity in patients. While the mechanism of idiosyncratic hepatotoxicity remains unknown, it is proposed that metabolic activation of amitriptyline and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether amitriptyline undergoes cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing amitriptyline and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed the formation of GSH conjugates derived from the addition of the sulfydryl nucleophile to hydrated metabolites of amitriptyline and nortriptyline, the major N-dealkylated metabolite of amitriptyline. Formation of GSH conjugates was primarily catalyzed by heterologously expressed recombinant CYP2D6, CYP3A4, CYP3A5, and to a less extent, CYP1A2. Corresponding dihydrodiol metabolites of amitriptyline and nortriptyline were also detected by tandem mass spectrometry. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed oxidation of the aromatic nucleus in amitriptyline to an electrophilic arene oxide intermediate, which is subsequently attacked by glutathione and water yielding the sulfydryl conjugate and the dihydrodiol metabolite, respectively. The results from the current investigation constitute the first report on the cytochrome P450-catalyzed bioactivation of the antidepressants amitriptyline and nortriptyline. It is proposed that the arene oxide intermediate(s) may represent a rate-limiting step in the initiation of amitriptyline and nortriptyline-mediated hepatotoxicity.
Wen
Bo
B
Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto, Palo Alto, CA 94304, United States. bo.wen@roche.com
Ma
Li
L
Zhu
Mingshe
M
D000639:Amitriptyline / Q000494:pharmacology*; D000929:Antidepressive Agents, Tricyclic / Q000494:pharmacology*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009661:Nortriptyline / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D000929:Antidepressive Agents, Tricyclic; D011994:Recombinant Proteins; D000639:Amitriptyline; D003577:Cytochrome P-450 Enzyme System; D009661:Nortriptyline; D005978:Glutathione