Available online at www.sciencedirect.com Chemico-Biological Interactions 173 (2008) 59–67 Bioactivation of the tricyclic antidepressant amitriptyline and its metabolite nortriptyline to arene oxide intermediates in human liver microsomes and recombinant P450s Bo Wen a,∗,LiMab, Mingshe Zhu b a Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto, Palo Alto, CA 94304, United States b Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, NJ 08543, United States Received 8 January 2008; received in revised form 1 February 2008; accepted 4 February 2008 Available online 14 February 2008 Abstract Amitriptyline, the most widely used tricyclic antidepressant, has been associated with very rare but severe incidences of hepato- toxicity 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 spectrom- etry. 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. Published by Elsevier Ireland Ltd Keywords: Amitriptyline; Nortriptyline; Bioactivation; P450; Hepatotoxicity; Arene oxide 1. Introduction Amitriptyline (Scheme 1), along with other tricyclic antidepressants (TCAs), has been the cornerstone of antidepressive therapy for more than three decades. Cur- Abbreviations: P450, cytochrome P450; TCAs, tricyclic antide- rent treatment guidelines recommend the use of TCAs pressants; PI, precursor ion; EPI, enhanced product ion; HLM, human only in patients with psychotic features and treatment liver microsomes; GSH, glutathione. ∗ Corresponding author. Tel.: +1 650 855 5463; resistance [1]. Nevertheless, more than 1 million patients fax: +1 650 852 1070. received TCAs in the United States in 2000 [2] and E-mail address: [email protected] (B. Wen). amitriptyline is still used extensively in developing coun- 0009-2797/$ – see front matter. Published by Elsevier Ireland Ltd doi:10.1016/j.cbi.2008.02.001 60 B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 Scheme 1. Proposed bioactivation pathways of the tricyclic antidepressant amitriptyline and its metabolite nortriptyline. tries because of its favorable cost/benefit ratio. The nortriptyline, which is an N-dealkylated metabolite of clinical effects of amitriptyline are characterized by amitriptyline (Scheme 1) [11,12]. changes in mood, which is thought to be related to its While the mechanisms of drug-induced idiosyncratic ability to inhibit the neuronal uptake of the biogenic hepatotoxicity remain to be elucidated, there is a sub- amine, norepinephrine [3]. Despite its therapeutic ben- stantial amount of evidence that implicated chemically efits, treatment with amitriptyline has been associated reactive metabolites as toxicity mediators [13]. This prin- with very rare, but severe incidence of hepatic injury ciple could also apply to amitriptyline especially because [4–10], which is often described as idiosyncratic tox- its clearance pathway in humans is heavily dependent icity. Although the exact mechanism of hepatotoxicity on hepatic oxidative metabolism by cytochrome P450s caused by amitriptyline is currently unknown, a prob- [14–17]. Amitriptyline undergoes extensive metabolism able causal link between amitriptyline use and hepatic mainly by hydroxylation, N-dealkylation, N-oxidation injury has been established based on temporal relation- and glucuronidation [14]. Of significant interest in many ship between amitriptyline administration and the onset biotransformation pathways of amitriptyline in humans of hepatotoxicity [7,9]. Similar idiosyncratic hepatotox- is the detection and characterization of the dihydro- icity has also been observed with the antidepressant diol metabolite M2 (Scheme 1) of amitriptyline in urine B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 61 [16]. Formation of metabolite M2 can presumably occur by nucleophilic addition of water to the electrophilic epoxide intermediate as shown in Scheme 1. We pro- posed that the reactive epoxide intermediate, resulting from an initial P450-catalyzed bioactivation on an aro- matic ring of amitriptyline, could conjugate with water to yield the dihydrodiol M2, react with glutathione to form GSH adduct M1 or attack cellular proteins to trigger a toxicological response (Scheme 1). Considering that nor- Scheme 2. Detection and characterization of GSH adducts using the triptyline is a structurally close N-dealkylated metabolite PI-EPI approach with polarity switching between MS detection and of amitriptyline, formation of an epoxide intermediate MS/MS acquisition. from nortriptyline could contribute to the observed hep- atotoxicity caused by amitriptyline and nortriptyline, size, 5 ms pause between mass ranges and 2 s scan respectively (Scheme 1). In this context, it is noteworthy rate or 50 ms dwell. The TurboIonSpray® ion source to point out that formation of an epoxide intermediate has conditions were optimized and set as follows: curtain been implicated in the bioactivation of another tricyclic gas (CUR) = 35, collision gas (CAD) = medium, ion- antidepressant imipramine [18]. Although the structure spray voltage (IS) = −4500, temperature (TEM) = 500. has not been identified, the MS/MS spectra suggested Nitrogen was used as the nebulizer and auxiliary gas. a GSH conjugate resulting from nucleophilic attack Information dependent acquisition (IDA) was used to of the epoxide intermediate by glutathione, followed trigger acquisition of enhanced product ion (EPI) spec- by subsequent loss of water [18]. Thus, we exam- tra. The EPI scans were run in the positive ion mode ined the propensity of amitriptyline and nortriptyline to at a scan range for daughter ions from m/z 100 to undergo bioactivation in human liver microsomes and 1000. Polarity switching of this PI-EPI approach was recombinant P450s to reactive epoxide intermediates. applied between MS detection and MS/MS acquisition Contributions to GSH adduct formation from individual (Scheme 2). For metabolite profiling, full MS scans were P450 enzymes were also assessed. carried out using the enhanced MS (EMS)-EPI experi- ments. 2. Materials and methods 2.3. Microsomal incubations 2.1. Materials All incubations were performed at 37 ◦C in a water Reagents and solvents used in the current study were bath. Stock solutions of the test compounds were pre- of the highest grade commercially available. Amitripty- pared in methanol. The final concentration of methanol line, nortriptyline, NADPH and glutathione were in the incubation was 0.2% (v/v). There was no mea- purchased from Sigma–Aldrich (St. Louis, MO). Pooled surable effect on the P450 catalytic activities when the human liver microsomes and SupersomesTM con- 0.2% methanol was present [19]. Pooled HLMs and the taining cDNA-baculovirus-insect cell-expressed P450s human cDNA-expressed P450 isozymes were carefully (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, thawed on ice prior to the experiment. Amitriptyline CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) or nortriptyline was individually mixed with HLM pro- were obtained from BD Gentest (Woburn, MA). Formic teins (1 mg/ml) in 100 mM potassium phosphate buffer acid, methanol, and acetonitrile were purchased from (pH 7.4) supplemented with 1 mM GSH. The total incu- EM Scientific (Gibbstown, NJ). bation volume was 1 ml. After 3 min pre-incubation at 37 ◦C, the incubation reactions were initiated by the 2.2. Instrumentation addition of 1 mM NADPH. Reactions were terminated by the addition of 150 ␮l of trichloroacetic acid (10%) LC/MS/MS analyses were performed on an ABI after 60 min incubation. Incubations with the recombi- 4000 Q-TrapTM hybrid triple quadrupole linear ion trap nant cDNA-expressed P450 isozymes were performed mass spectrometer (Applied Biosystems, Foster City, similarly except that liver microsomes were substituted CA) interfaced online with a Shimadzu HPLC system by SupersomesTM (100 pmol P450/ml). Control samples (Columbia, MD). For complete profiling of reactive containing no NADPH or substrates were included. Each metabolites, the precursor ion (PI) scan of m/z 272 incubation