Bioactivation of the Tricyclic Antidepressant Amitriptyline and Its

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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 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. 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 proposed that the reactive epoxide intermediate, resulting from an initial P450-catalyzed bioactivation on an aromatic 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 hepatotoxicity 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 experiments. 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 was performed in duplicate. The difference was run in the negative ion mode with 0.2 Da step between two measurements was generally less than 10%, 62 B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 the ratio of the difference over the smaller measurement. m/z 601 → 472 and 601 → 276; for GSH adduct 7: m/z Samples were centrifuged at 10,000 × g for 15 min at 587 → 458 and 587 → 262. Data were analyzed using 4 ◦C to pellet the precipitated proteins, and supernatants Analyst 4.1 version software (Applied Biosystems, Fos- were concentrated by solid phase extraction as described ter City, CA). below, prior to LC/MS/MS analyses. 3. Results 2.4. Solid-phase extraction 3.1. Formation of dihydrodiol metabolites Samples resulting from incubations were desalted and concentrated by solid-phase extraction (SPE), prior In order to simulate hepatic metabolism, it is neces- to the negative precursor ion scan MS/MS analyses. sary to estimate drug concentrations in hepatic tissue. At SPE was performed using Oasis® solid-phase extraction ordinary therapeutic dosing of amitriptyline, the serum cartridges packed with 60 mg of sorbent C18 (Waters, levels of amitriptyline are up to 500 nmol/l in humans Milford, MA). Cartridges were first washed with 2 ml [20]. Assuming hepatic tissue concentrations of 10–20 methanol and then conditioned with 2 ml of water. Super- times the serum levels [21,22],10␮M of amitriptyline natants resulting from centrifugation were loaded onto was used in order to be clinically relevant in all incuba- the cartridges, and cartridges were washed with 2 ml tions performed in this study. of water and then eluted with 2 ml of methanol. The For the LC/MS/MS analysis of amitriptyline metabo- methanol fractions were dried under a gentle stream lites, samples generated from incubations with human of nitrogen gas and reconstituted with 100 ␮lofa liver microsomes were desalted and concentrated by water–methanol (70:30) mixture. Aliquots (20 ␮l) of the solid-phase extractions (SPE), and resulting samples reconstituted solutions were subjected to LC/MS/MS were subjected to the EMS-EPI experiments. Compar- analysis. ison between samples with or without SPE treatments revealed that SPE recovered all metabolites including 2.5. LC/MS/MS analysis the hydroxylated and N-dealkylated metabolites identified previously (data not shown). Approximately 60% For complete profiling of reactive metabolites, sam- of amitriptyline was converted to metabolites in the ples were first subjected to chromatographic separations 60 min incubation with human liver microsomes based with a Shimadzu HPLC system coupled with an Agilent on UV detection at 254 nm. Of interest from the current Eclipse XDB-Phenyl C18 column (3.0 mm × 150 mm, perspective was the detection of two metabolites with 3.5 ␮m, Agilent Technologies, Palo Alto, CA). The molecular weights (MH+) at 312 and 298 that eluted at HPLC mobile phase A was 10 mM ammonium acetate retention time (Rt) = 11.4 and 10.6 min, respectively. The in water with 0.1% formic acid, and mobile phase B MS/MS spectrum of the component at Rt = 11.4 (Fig. 1A) was acetonitrile with 0.1% formic acid. A Shimadzu revealed product ions at m/z = 294, 267, 249, 239, 221, LC-20AD solvent delivery module (Shimadzu Scien- 177 and 133. The fragment ions at m/z 177, 239, and 267 tific Instruments, Columbia, MD) was used to produce were consistent with the addition of two hydroxyl groups the following gradient elution profile: 5% solvent B for to the aromatic benzene nucleus in amitriptyline. The 2 min, followed by 5–70% B in 10 min and 70–90% B in fragment ions at m/z 221 and 249 corresponded to loss 2 min. The HPLC flow rate was 0.3 ml/min. At 24 min, of water from the product ions m/z 239 and 267, respec- the column was flushed with 90% acetonitrile for 3 min tively. N-Oxidation of the tertiary amine nitrogen was before re-equilibration at initial conditions. LC/MS/MS ruled out as a site of oxidation because of the fragmenta- analyses were performed on a 20 ␮l aliquot of sam- tion patterns and corresponding product ions. Therefore, ple obtained from the solid phase extraction columns. the proposed structure of this metabolite, which would When a positive peak was detected in the negative pre- be consistent with the observed MS2 spectra is shown cursor ion scanning over the range m/z 270–1000, a in Fig. 1A and was subsequently identified as the dihy- collision-induced dissociation (CID) MS/MS spectrum drodiol metabolite M2 of amitriptyline. This metabolite was simultaneously obtained to further elucidate the was previously identified in both human and dog urine structure of the GSH adduct. [16,23]. For relative comparison of GSH adduct levels, the In addition to the dihydrodiol metabolite of mass spectrometer was operated in the multiple reac- amitriptyline, a second diol metabolite (MH+ = 298, tion monitoring (MRM) mode. MRM transitions were Rt = 10.6 min) was also detected in the microsomal incu- simultaneously monitored for detecting GSH adduct 5: bations of amitriptyline. The MS/MS spectrum of this B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 63

Fig. 1. Formation of the dihydrodiol metabolites from incubations of amitriptyline in human liver microsomes. (A) CID MS/MS spectrum of the diol metabolite M2 at m/z 312; (B) CID MS/MS spectrum of the diol metabolite M3 at m/z 298. metabolite (Fig. 1B) afforded product ions at m/z 280, dependent data acquisition (Scheme 2). As shown in 267, 249, 239, 221, 177 and 159. Similarly, the fragment Fig. 2, a total of two major components were detected ions at m/z 177, 239, and 267 were consistent with the by the negative precursor ion scanning of m/z 272 and addition of two hydroxyl groups to the aromatic benzene they were arbitrarily designated as M1 (9.2 min) and nucleus, suggesting an unaltered tricyclic ring system M4 (6.7 min), respectively. Neither of these two peaks and an N-demethylation reaction at the side chain. The was detected when either amitriptyline or NADPH was fragment ions at m/z 159, 221 and 249 corresponded to absent from the incubations. These data suggested that loss of water from the product ions m/z 177, 239 and GSH adducts were formed from reactive metabolites of 267, respectively. Thus, the proposed structure is shown amitriptyline via oxidative metabolism. in Fig. 1B as the dihydrodiol metabolite M3 of nortripty- Structures of these detected components were simul- line. These dihydrodiol metabolites of amitriptyline are taneously veriﬁed based on MS/MS spectra in positive presumably formed by nucleophilic addition of water to ion mode. The PI-directed positive MS2 spectrum of an arene oxide reactive precursor. [M+H]+ ion at m/z 601 of the most abundant adduct M1 (Fig. 3B) provided characteristic product ions at m/z 3.2. Characterization of GSH conjugates of 472 and 454, resulting from neutral loss of pyrogluta- amitriptyline mate (129 Da) and subsequent loss of water, respectively (Fig. 3B). This conﬁrmed that M1 was a GSH adduct For the LC/MS/MS analysis of GSH conjugates, formed in the incubation of amitriptyline. The molecular resulting samples from solid-phase extractions were ion [M+H]+ at m/z 601 was consistent with the addi- subjected to the PI-EPI experiments using polarity tion of a glutathionyl moiety to the hydrated metabolite switching of a hybrid triple quadrupole linear ion trap of amitriptyline. Double cleavage at the thioether and mass spectrometer (Scheme 2). MS detection was car- ether motifs formed the product ions at m/z 278 and 276. ried out using the negative precursor ion scanning of Cleavages at the side chain afforded product ions at m/z m/z 272, corresponding to deprotonated ␥-glutamyl- 543 and 516. A proposed structure for M1, which is dehydroalanyl-glycine originated from the glutathionyl consistent with the CID cleavage, is shown in Fig. 3A. moiety [24]. MS/MS spectra were simultaneously Another component, namely M4, was also detected acquired in positive ion mode using information- in the microsomal incubations of amitriptyline. The 64 B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67

Fig. 2. LC/MS/MS detection of GSH adducts using the precursor ion scanning of m/z 272 in negative ion mode. Two components M1 and M4 were detected in microsomal incubations of amitriptyline.

MS/MS spectrum of [M+H]+ ion at m/z 587 of M4 essentially identical MS/MS spectrum (data not shown). afforded characteristic product ions at m/z 458 and 440, A proposed structure for M4, which is consistent with the resulting from neutral loss of pyroglutamate (129 Da) CID cleavage, is shown in Fig. 4A. The overall substrate and subsequent loss of water, respectively (Fig. 4B). turnover for M1 and M4 were estimated to be ∼6% and The mass shift of 14 Da between components M1 ∼1%, respectively based on UV detection at 254 nm. and M4 suggested that M4 was derived from an N-demethylated metabolite of amitriptyline. The molec- 3.3. GSH adduct formation with recombinant P450s ular ion [M+H]+ at m/z 587 was consistent with the addition of a glutathionyl moiety to the hydrated metabo- To investigate the roles of individual human P450 lite of nortriptyline, an N-demethylated metabolite of isozymes in the bioactivation of amitriptyline and nor- amitriptyline. Direct evidence of bioactivation of nortriptyline, the formation of GSH adducts M1 and M4 triptyline comes from incubations of nortriptyline with was examined in the incubations of amitriptyline and human liver microsomes with supplemental glutathione. nortriptyline with insect cell-expressed recombinant LC/MS/MS analysis of samples from incubations of nor- P450s, respectively. As shown in Fig. 5A, at the same triptyline revealed the same GSH conjugate M4 with enzyme concentration (100 pmol P450/ml), CYP2D6 is

Fig. 3. LC/MS/MS analysis of component M1. (A) Extracted ion chromatogram of [M−H]− ion at m/z 599 in negative ion mode; (B) CID MS/MS spectrum of M1 at m/z 601 in positive ion mode. B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 65

Fig. 4. LC/MS/MS analysis of component M4. (A) Extracted ion chromatogram of [M−H]− ion at m/z 585 in negative ion mode; (B) CID MS/MS spectrum of M4 at m/z 587 in positive ion mode. the major enzyme for the formation of M1 in the incu- well with a previous report that at low substrate con- bations of amitriptyline (10 ␮M). CYP1A2, CYP3A4 centrations, CYP2C19, but not CYP2D6, was the most and CYP3A5 also catalyzed M1 formation, and lev- important enzyme with regard to the demethylation of els of M1 formation range from approximately 30% to amitriptyline [26]. 60% of that formed by CYP2D6. Only trace amounts or no M1 formation were detected in the incubations with other P450 enzymes including CYP2A6, CYP2B6, 4. Discussion CYP2C8, CYP2C9, CYP2C19 and CYP2E1. In contrast, CYP3A4 and CYP3A5 are the major enzymes for the for- The results from the current investigation consti- mation of M4 in the incubations of amitriptyline, while tute the first report on the cytochrome P450-catalyzed the level of M4 formation by CYP2D6 was less than bioactivation of the antidepressants amitriptyline and 20% of that formed by CYP3A4. This is probably due nortriptyline. Apart from the literature reports on the to the high catalytic efficiency of CYP3A enzymes to involvement of CYP2D6 and CYP3A4 in the hydrox- N-demethylate amitriptyline to form nortriptyline [25], ylation of amitriptyline and nortriptyline, our studies which were subsequently oxidized to the reactive epox- demonstrated major roles for these enzymes in the ide intermediate and trapped by glutathione to yield M4. metabolic activation of the two antidepressants. Forma- Except CYP3A4, CYP3A5 and CYP2D6, no other P450 tion of the glutathione conjugate M1 is consistent with a enzymes were found to catalyze formation of M4. bioactivation sequence involving initial P450-catalyzed Interestingly, CYP2D6 is the major enzyme for the oxidation of the benzene nucleus in amitriptyline to formation of M4 in the incubations of nortriptyline an electrophilic epoxide, which reacts with water and (10 ␮M) (Fig. 5B). Similarly to M1 formation from glutathione generating the dihydrodiol metabolite M2 amitriptyline, CYP1A2, CYP3A4 and CYP3A5 cat- and the sulfydryl conjugate M1, respectively. Forma- alyzed the formation of M4 from nortriptyline, but levels tion of the glutathione conjugate M4 is mediated by of M4 were between approximately 20% and 60% of a similar oxidative pathway after P450-mediated N- that formed from CYP2D6. Taken together, these results demethylation of amitriptyline to afford nortriptyline. suggested that CYP2D6 is a major enzyme for bioactiva- Bioactivation of nortriptyline was further confirmed by tion of amitriptyline and nortriptyline, but a poor enzyme incubations of nortriptyline with human liver micro- for N-demethylation of amitriptyline. These data agree somes and recombinant P450s. 66 B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67

tion, other enzymes which may not be present in our model system such as dihydrodiol dehydrogenase and soluble epoxide hydrolase may also contribute to the bioactivation of amitriptyline. For example, dihydrodiol dehydrogenase may oxidize the dihydrodiol metabolites M2 and M3 to reactive ortho-quinone intermediates which can contribute to the toxicity of this drug via redox-cycling. However, the roles of these individual non-microsomal enzymes in the bioactivation of amitriptyline and nortriptyline remain to be established. Clinical experience to date indicates that amitriptyline therapy is associated with very rare idiosyncratic hepatotoxicity. Hepatic injury has also been observed with the closely related antidepressant nortriptyline. While the mechanism of hepatotoxicity associated with amitriptyline and nortriptyline use remains to be elucidated, it is proposed that reactive metabolites of both amitriptyline and nortriptyline may play a causative role in susceptible patients. The current investigation provides support for this hypothesis. A further link between hepatotoxicity and the microsomal bioactivation of amitriptyline and nortriptyline is the in vivo detection of potential downstream stable metabolites derived from further processing of reactive metabolites. In the case of amitriptyline, dihydrodiol M2 was detected in human urine [16]. Likewise, both dihydrodiols M2 and M3 were detected in dog urine. By trapping these putative reactive intermediates with glutathione, the current study provides Fig. 5. Formation of GSH adducts in incubations with cDNA- direct evidence for the bioactivation of amitriptyline expressed recombinant P450 isozymes. (A) Formation of M1 and and nortriptyline at clinically relevant concentrations. M4 in incubations of amitriptyline; (B) formation of M4 in incuba- These ﬁndings are of signiﬁcance in understanding tions of nortriptyline. The enzyme activities were an average of two to biochemical mechanisms of idiosyncratic toxicity measurements. of tricyclic antidepressants amitriptyline and nortriptyline. Formation of M1 from amitriptyline and formation of M4 from nortriptyline are mainly catalyzed by CYP2D6. In contrast, CYP3A4 and CYP3A5 are References the major enzymes involved in formation of M4 from [1] M.L. Crismon, M. Trivedi, T.A. Pigott, A.J. Rush, R.M. incubations of amitriptyline. These data are consistent Hirschfeld, D.A. Kahn, C. DeBattista, J.C. Nelson, A.A. with the previous reports that CYP3A4 and CYP2C19 Nierenberg, H.A. Sackeim, M.E. Thase, The Texas Medication are the major enzymes catalyzing N-demethylation of Algorithm Project: report of the Texas Consensus Conference amitriptyline to nortriptyline, while CYP2D6 has poor Panel on Medication Treatment of Major Depressive Disorder, J. N-demethylation activity towards amitriptyline [15,25]. Clin. Psychiatry 60 (1999) 142–156. [2] G.T. Tucker, Advances in understanding drug metabolism and its However, it is noteworthy to point out that contributions contribution to variability in patient response, Ther. Drug Monit. from individual P450s may change at different sub- 22 (2000) 110–113. strate concentrations. This is supported by different roles [3] A.G. Gilman, L.S. Goodman, T.W. Rall, F. Murad, Tricyclic of P450 isozymes in the metabolism of amitriptyline antidepressants, in: Goodman and Gilman’s The Pharmacologi- and nortriptyline at different substrate concentrations cal Basis of Therapeutics, Macmillan Publishing Company, New York, 1985, pp. 413–423. [15,26]. At high substrate concentrations or toxic doses, [4] M.L. Cunningham, Acute hepatic necrosis following treatment CYP2D6 may be saturated and CYP3A4 may play a with amitriptyline and diazepam, Br. J. Psychiatry 111 (1965) dominant in the bioactivation of both drugs. In addi- 1107–1109. B. Wen et al. / Chemico-Biological Interactions 173 (2008) 59–67 67

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