detection of novel reactive metabolites of...

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DETECTION OF NOVEL REACTIVE METABOLITES OF

TRAZODONE: EVIDENCE FOR CYP2D6-MEDIATED

BIOACTIVATION OF M-CHLOROPHENYLPIPERAZINE

Bo Wen§, Li Ma, A. David Rodrigues, and Mingshe Zhu

Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton,

NJ 08543

DMD Fast Forward. Published on January 31, 2008 as doi:10.1124/dmd.107.019471

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on January 31, 2008 as DOI: 10.1124/dmd.107.019471

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Running title: Cytochrome P450 2D6-Mediated Bioactivation of m-CPP

Number of text pages: 20

Number of tables: 1

Number of figures: 8

Number of References: 39

Number of words in Abstract: 234

Number of words in Introduction: 641

Number of words in Discussion: 1172

Address correspondence to:

Bo Wen, Ph.D.

Drug Metabolism and Pharmacokinetics

M/S S3-2-E 218A

Roche Palo Alto

Palo Alto, CA 94304

Telephone: (650)-855-5463

Fax: (650)-852-1070

Email: [email protected]

ABBREVIATIONS: P450, cytochrome P450; GSH, glutathione; HLM, human liver

microsomes; MS/MS, tandem mass spectrometry; CID, collision-induced dissociation; m-

CPP, 1-(3'-chlorophenyl)piperazine; p-CPP, 1-(4'-chlorophenyl)piperazine.


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Abstract

Several new glutathione adducts (M3-M7) of trazodone were tentatively identified in

human liver microsomal incubations using liquid chromatography-tandem mass

spectrometry (LC/MS/MS). Following incubations with trazodone in the presence of

glutathione, 1-(3'-chlorophenyl)piperazine (m-CPP), a major circulating and

pharmacologically active metabolite of several antidepressants including trazodone,

nefazodone, and etoperidone, was trapped with glutathione to afford the corresponding

quinone imine-sulfydryl adducts M4 and M5. Two novel glutathione adducts of

deschloro-m-CPP and deschloro-trazodone, M3 and M6, were also detected by tandem

mass spectrometry. The identities of these m-CPP derived glutathione adducts were

further confirmed by LC/MS/MS analyses of microsomal incubations of m-CPP. To

investigate the bioactivation mechanism, a regioisomer of m-CPP, 1-(4'-

chlorophenyl)piperazine (p-CPP), was incubated in human liver microsomes. Blockage

of bioactivation by 4'-chloro-substititution at least partially suggested that formation of

m-CPP derived glutathione adducts M3, M4 and M5 is mediated by a common quinone

imine intermediate. A tentative pathway states that upon formation of the trazodone- and

m-CPP-1',4'-quinone imine intermediates through initial 4'-hydroxylation, GSH attacks at

the chlorine position by an ipso substitution, resulting in 4'-hydroxy-3'-glutathion-

deschloro-trazodone (M6) and 4'-hydroxy-3'-glutathion-deschloro-m-CPP (M3),

respectively. In contrast to CYP3A4-dependent bioactivation of trazodone itself,

formation of M4 was mediated specifically by CYP2D6, as evidenced by cDNA-

expressed CYP2D6-catalyzing formation of M4 from m-CPP, strong inhibition of

formation of M4 by quinidine, a specific CYP2D6 inhibitor, in both incubations of


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trazodone and m-CPP with human liver microsomes, and concentration-dependent

inhibition of M4 formation by quinidine.


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Introduction

Trazodone is a second-generation triazolopyridinone antidepressant drug

(Scheme 1), which is structurally distinct from selective serotonin reuptake inhibitors, tri-

and tetracyclics, and monoamine oxidase inhibitors. It is thought to act through combined

5-HT2 antagonism and 5-HT reuptake blockage (Haria et al., 1994). Trazodone is often

co-prescribed with other antidepressants as a sleep-inducing agent because of its more

sedating and less anticholinergic side effects. Despite its therapeutic benefits, treatment

with trazodone has been associated with rare, but severe incidence of hepatic injury (Chu

et al., 1983; Longstreth et al., 1985, Beck et al., 1993; Hull et al., 1994), which is often

described as idiosyncratic toxicity. Although the exact mechanism of trazodone

hepatotoxicity is not clearly understood, a probable causal link between trazodone use

and the onset of hepatic injury has been established (Fernandes et al., 2000; Rettman et

al., 2001).

As shown in Scheme 1, trazodone contains a triazolopyridinone moiety and a 3-

chlorophenylpiperazine ring system. In humans, trazodone undergoes extensive hepatic

metabolism mainly by hydroxylation, N-dealkylation and N-oxidation (Baiocchi et al.,

1974; Yamato et al., 1974; Jauch et al., 1976). Of particular interest in the

biotransformation pathways of trazodone in humans is the detection and characterization

of a dihydrodiol metabolite and 4'-hydroxytrazodone as major metabolites in urine

(Baiocchi et al., 1974; Jauch et al., 1976). Formation of the dihydrodiol metabolite can

presumably occur by nucleophilic addition of water to an electrophilic epoxide

intermediate. On the other hand, 4'-hydroxytrazodone can undergo a two-electron

oxidation leading to formation of an electrophilic quinone imine intermediate, which is


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capable of reacting with cellular proteins and other nucleophiles such as glutathione

(Scheme 1). In human liver microsomal incubations, two glutathione adducts have been

previously identified (Kalgutkar et al., 2005a) arising from quinone imine and epoxide

intermediates (M1 and M2 depicted in Scheme 1). The epoxidation of triazolopyridinone,

para-hydroxylation of 3-chlorophenylpiperazine and subsequent oxidation to quinone

imine were shown to be mediated by cytochrome P450 (CYP) 3A4 (Kalgutkar et al.,

2005a). These findings are significant as the first line of evidence to suggest that

cytochromes P450-mediated reactive metabolites may play an important role in toxicity

of the drug.

It is noteworthy that 1-(3'-chlorophenyl)piperazine (m-CPP, Scheme 1), resulting

from N-dealkylation of trazodone, is a major circulating metabolite in humans common

to several antidepressants including trazodone, nefazodone and etoperidone (Otani et al.,

1997; von Moltke et al., 1999; Melzalka et al., 1980; Fong et al., 1982). The metabolite

m-CPP is of significant interest because it has 5-HT2C agonistic and 5-HT2A antagonistic

properties (Conn and Sanders-Bush, 1987; Fiorella et al., 1995). It has also been

suggested that m-CPP may contribute to the antidepressant efficacy of trazodone (Maes et

al., 1997). While oxidative metabolism of trazodone, nefazodone and etoperidone are

controlled by CYP3A4 (Zalma, et al., 2000; Rotzinger et al., 1998a; von Moltke et al.,

1999; Yan et al., 2002), 4'-hydroxylation of m-CPP is specifically mediated by CYP2D6

(Rotzinger et al., 1998b). Consistently, a clinical study showed that haloperidol, an

inhibitor of CYP2D6, significantly increased plasma concentration of m-CPP, but not of

trazodone (Mihara et al., 1997). Because 4'-hydroxy-m-CPP may undergo further two-

electron oxidations to form an electrophilic quinone imine intermediate, characterization


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of reactive metabolites and responsible CYP enzymes would be very helpful for complete

understanding of the biochemical mechanisms of idiosyncratic toxicity associated with

m-CPP containing drugs, such as trazodone. In this study, we report a total of five

products of reactive metabolite formation in human liver microsomal incubations of

trazodone, three of which were generated from bioactivation of the active metabolite m-

CPP. Two novel deschloro glutathione adducts of m-CPP and trazodone presumably

derived from an ipso substitution of chlorine by GSH were tentatively identified. In

addition, it was found that formation of reactive metabolites of m-CPP was specifically

mediated by CYP2D6. These data are important for further understanding the relationship

between metabolic activation and hepatotoxicity of m-CPP containing antidepressant

drugs.

Materials and Methods

Materials. Reagents and solvents used in the current study were of the highest

grade commercially available. The following chemicals were purchased from Sigma-

Aldrich (St. Louis, MO): acetaminophen, dextromethorphan, dextrophan, glutathione

(GSH), ketoconazole, α-naphthoflavone, trazodone, nefazodone, phenacetin, testosterone,

6β-hydroxy-testosterone, tolbutamide, sulfaphenazole, quinidine, trichloroacetic acid,

NADPH, m-CPP hydrochloride and 1-(4'-chlorophenyl)piperazine (p-CPP).

Tranylcypromine was obtained from BIOMOL international, L.P. (Plymouth Meeting,

PA). (S)-mephenytoin, 4'-hydroxy-(S)-mephenytoin, and 4-hydroxy-tobutamide were

purchased from Ultrafine Chemicals (Manchester, UK). Pooled human liver microsomes

and SupersomesTM containing cDNA-baculovirus-insect cell-expressed P450s (CYP1A2,


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CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and

CYP3A5) were obtained from BD Gentest (Woburn, MA). Formic acid, methanol, and

acetonitrile were purchased from EM Scientific (Gibbstown, NJ).

Instrumentation. LC/MS/MS analyses were performed on an API 4000 Q-

TrapTM hybrid triple quadrupole linear ion trap mass spectrometer (Applied Biosystems,

Foster City, CA) interfaced online with a Shimadzu HPLC system (Columbia, MD). For

complete profiling of reactive metabolites, the precursor ion (PI) scan of m/z 272 was run

in the negative mode with 0.2 Da step size, 5 ms pause between mass ranges and 2 s scan

rate or 50 ms dwell. The TurboIonSpray® ion source conditions were optimized and set

as follows: curtain gas (CUR) = 35, collision gas (CAD) = medium, ionspray voltage (IS)

= –4500, temperature (TEM) = 500. Nitrogen was used as the nebulizer and auxiliary gas.

Information dependent acquisition (IDA) was used to trigger acquisition of enhanced

product ion (EPI) spectra. The EPI scans were run in the positive mode at a scan range

for daughter ions from m/z 100 to 1000. For NL-EPI analysis, the ion source conditions

were set as follows: CUR = 35, CAD = 6, IS = 4500, TEM = 500. Data were processed

using Analyst 4.1 software (Applied Biosystems, Foster City, CA).

A Shimadzu HPLC system was coupled with an Agilent Eclipse XDB-Phenyl

C18 column (3.0 × 150 mm, 3.5 µm, Agilent Technologies, Palo Alto, CA). The HPLC

mobile phase A was 10 mM ammonium acetate in water with 0.1% formic acid, and

mobile phase B was acetonitrile with 0.1% formic acid. A Shimadzu LC-20AD solvent

delivery module (Shimadzu Scientific Instruments, Columbia, MD) was used to produce

the following gradient elution profile: 5% solvent B for 2 min, followed by 5-70% B in

20 min and 70-90% B in 2 min. The HPLC flow rate was 0.3 ml/min.


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Microsomal Incubations. All incubations were performed at 37°C in a water

bath. Stock solutions of the test compounds were prepared in methanol. The final

concentration of methanol in the incubation was 0.2% (v/v). Pooled HLMs and the

human cDNA-expressed P450 isozymes were carefully thawed on ice prior to the

experiment. Trazodone, nefazodone, m-CPP or p-CPP (10 µM) was individually mixed

with HLM proteins (1 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4)

supplemented with 1 mM GSH. The total incubation volume was 1 mL. After 3 min pre-

incubation at 37°C, the incubation reactions were initiated by the addition of 1 mM

NADPH. Reactions were terminated by the addition of 150 µL of trichloroacetic acid

(10%) after 60 min incubation. Incubations with the recombinant cDNA-expressed P450

isozymes were performed similarly except that liver microsomes were substituted by

SupersomesTM (100 pmol/ml). Control samples containing no NADPH or substrates were

included. Samples were centrifuged at 10,000 g for 15 min at 4°C to pellet the

precipitated proteins, and supernatants were subjected to LC/MS/MS analysis of GSH

adducts. Each incubation was performed in triplicate. For the negative precursor ion

scanning of GSH adducts, supernatants were concentrated by solid phase extraction as

described below, prior to LC/MS/MS analyses.

P450 Inhibition by Chemical Inhibitors. The effect of specific inhibitors of

individual P450 enzymes on the formation of reactive metabolites was examined using

pooled human liver microsomes. Incubation mixtures consisted of trazodone (10 µM) or

m-CPP (10 µM), individual chemical inhibitors, GSH (1 mM) and HLMs (1 mg/ml). The

P450 specific inhibitors α-naphthoflavone (1 µM), sulfaphenazole (5 µM),

tranylcypromine (15 µM), quinidine (2 µM) and ketoconazole (1 µM) were used to


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investigate the involvement of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4,

respectively. Incubations containing trazodone or m-CPP were started with the addition

of 1 mM NADPH, and reactions were terminated by trichloroacetic acid. Controls

containing no chemical inhibitors were included. Each incubation was performed in

duplicate. The effectiveness of individual P450 inhibitors was also evaluated using P450

marker substrates 50 µM phenacetin (CYP1A2), 150 µM tolbutamide (CYP2C9), 100 µM

(S)-mephenytoin (CYP2C19), 10 µM dextromethorphan (CYP2D6), and 100 µM

testosterone (CYP3A4) in HLMs. Individual marker substrates were pre-incubated for 5

min at 37°C in the presence and absence of P450 specific inhibitors. Reactions were

started with the addition of 1 mM NADPH, and terminated after 20 min. Formation of

metabolites from individual P450 marker substrate were analyzed by LC/MS/MS as

previously described (Walsky and Obach, 2004) with minor modifications. A comparison

was made relative to the controls without inhibitors, and P450 activity was expressed as

the percentage of control activity.

For concentration-dependent inhibition, the specific CYP2D6 inhibitor quinidine

was used to further access the role of CYP2D6 for the reactive metabolite formation from

incubations of m-CPP in human liver microsomes. Under similar incubation conditions

described above, quinidine at various concentrations (0, 0.1, 0.3, 0.7, 1.2, 2, 4 or 6 µM)

was added to the incubation mixture containing m-CPP (50 µM).

Solid-Phase Extraction. Samples resulting from incubations were desalted and

concentrated by solid-phase extraction (SPE), prior to the negative precursor ion scan

MS/MS analyses. SPE was performed using Oasis® solid-phase extraction cartridges

packed with 60 mg of sorbent C18 (Waters, Milford, MA). Cartridges were first washed


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with 2 ml methanol and then conditioned with 2 ml of water. Supernatants resulting from

centrifugation were loaded onto the cartridges, and cartridges were washed with 2 ml of

water and then eluted with 2 ml of methanol. The methanol fractions were dried by

nitrogen gas and reconstituted with 100 µl of a water-methanol (70:30) mixture. Aliquots

(20 µl) of the reconstituted solutions were subjected to LC/MS/MS analysis.

LC/MS/MS Analysis. For complete profiling of reactive metabolites, samples

were first subjected to chromatographic separations with a Shimadzu HPLC system

coupled with an Agilent Eclipse XDB-Phenyl C18 column (3.0 × 150 mm, 3.5 µm,

Agilent Technologies, Palo Alto, CA). The HPLC mobile phase A was 10 mM

ammonium acetate in water with 0.1% formic acid, and mobile phase B was acetonitrile

with 0.1% formic acid. A Shimadzu LC-20AD solvent delivery module (Shimadzu

Scientific Instruments, Columbia, MD) was used to produce the following gradient

elution profile: 5% solvent B for 2 min, followed by 5-70% B in 20 min and 70-90% B in

2 min. The HPLC flow rate was 0.3 ml/min. At 24 min, the column was flushed with

90% acetonitrile for 3 min before re-equilibration at initial conditions. LC/MS/MS

analyses were performed on 20 µl aliquots of cleaned samples. A positive peak detected

in the negative precursor ion scan over the range m/z 270 to 1000 was used to trigger the

acquisition of a collision-induced dissociation (CID) MS/MS spectrum to further

elucidate the structure of the GSH adduct.

For relative comparison of GSH adduct levels, the mass spectrometer was

operated in the multiple reaction monitoring (MRM) mode. MRM transitions were

simultaneously monitored for detecting M1: m/z 693→564 and 693→420; for M2: m/z


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695→566 and 695→422; for M4: m/z 518→389 and 518→243. Data were analyzed

using Analyst 4.1 version software (Applied Biosystems, Foster City, CA).

Molecular Modeling. The structure of m-CPP was drawn and the geometry was

optimized using the NDDO (neglect of diatomic differential overlap) semiempirical

method of PM3 by steepest decent (Stewart, 2004; Pople and Segal, 1965) with

ArgusLab 4.0 (Planaria Software, Seattle, WA). This was aligned and edited into the

active site of the X-ray crystal structure of CYP2D6 (Protein Data Bank code: 2F9Q)

(Rowland et al., 2006). The structure was manipulated using WebLab Viewer Lite 4.0

(Accelrys, San Diego, CA) and ArgusLab 4.0, so that it was orientated in a geometrically

reasonable position with no significant Van der Waals overlap with the protein, providing

us a working model of the m-CPP bound CYP2D6.

Results

Characterization of GSH Adducts of Trazodone. For the LC/MS/MS analysis

of GSH adducts, samples generated from incubations with human liver microsomes were

desalted and concentrated by solid-phase extractions, and resulting samples were

subjected to the PI-EPI experiments. MS detection was carried out using the negative

precursor ion scanning of m/z 272, corresponding to deprotonated γ-glutamyl-

dehydroalanyl-glycine originating from the glutathionyl moiety (Dieckhaus et al., 2005).

MS/MS spectra were acquired in positive ion mode using information-dependent data

acquisition (Hopfgartner et al., 2003). As shown in Fig. 1A, a total of seven major

components were detected by the negative precursor ion scanning of m/z 272 and they

were arbitrarily designated as M1 (10.9 min), M2 (12.7 min), M3 (6.0 min), M4 (8.0


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min), M5 (8.5 min), M6 (9.3 min) and M7 (9.8 min), respectively. None of these peaks

was detected when either trazodone or NADPH was absent from the incubations. These

data suggested that GSH adducts were formed from reactive metabolites of trazodone via

oxidative metabolism.

Structures of these detected components were simultaneously identified based on

MS/MS spectra in the positive ion mode. The PI-directed positive MS/MS spectrum of

the most abundant adduct, M1, showed an [M + H]+ ion at m/z 693, suggesting that this

component was one of the two GSH adducts previously identified (Kalgutkar et al.,

2005a). This was supported by the MS/MS spectrum of [M + H]+ ion at m/z 693 that

showed product ions at m/z 564, 547, 489, 444, 420, 283, 176 and 148. This component

was subsequently identified as the corresponding quinone imine-sulfydryl adduct (M1,

Scheme 1). The MS/MS spectrum of M2 showed an [M + H]+ ion of m/z 695, with

product ions at m/z 566, 548, 422, 404, 372, 352, 237 and 208. This component was

subsequently detected as the corresponding epoxide-sulfydryl adduct previously

identified (Kalgutkar et al., 2005a).

There are five other components, M3-M7, detected in the microsomal incubations

of trazodone (Fig. 1A). Among these five components, M4 was the most abundant peak.

The deprotonated molecular ion of component M4 was m/z 516 in the negative ion mode

(Fig. 2A). A chlorine isotope peak was observed at m/z 518 (~35% of the [M – H]– ion),

which indicated that formation of a new GSH adduct that contained a chlorine atom.

Under the positive ion mode, the MS/MS spectrum of [M + H]+ ion at m/z 518 provided

characteristic product ions at m/z 443 and 389, resulting from neutral losses of glycine

(75 Da) and pyroglutamate (129 Da), respectively (Fig. 2B). This confirmed that M4 was


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a new GSH adduct formed in the incubation of trazodone. The molecular ion [M + H]+ at

m/z 518 was consistent with the addition of one molecule of glutathione to a mono-

hydroxylated m-CPP metabolite of trazodone after P450-mediated N-dealkylation.

Double cleavage at the piperazine ring with neutral losses of glycine and pyroglutamate

formed the product ion at m/z 269. A loss of NH3 from the product ion at m/z 389 resulted

in the fragment ion at m/z 372. The occurrence of the product ion at m/z 243 was

consistent with the presence of an aromatic thioether motif in this GSH adduct (Baillie,

1993). A proposed structure for M4, which is consistent with the chlorine isotope cluster

and the CID cleavage, is shown in Fig. 2B. The parent ion of component M5 was also

m/z 516 in negative ion mode with a chlorine isotope pattern. The MS/MS analysis

showed that components M4 and M5 had essentially identical spectra (Fig. 2B),

suggesting that they are likely positional isomers.

The parent ion of component M3 was m/z 482 in negative ion mode with no

chlorine isotope peak (Fig. 3A). Under the positive ion mode, the MS/MS spectrum of

[M + H]+ ion at m/z 484 provided characteristic product ions at m/z 409 and 355,

resulting from neutral losses of glycine and pyroglutamate, respectively (Fig. 3B). The

molecular ion [M + H]+ of m/z 484 had a mass difference of 34 Da from that of M4 or

M5, suggesting loss of a chlorine atom from m-CPP. Similar to the CID patterns in the

MS/MS spectra of M4 and M5, the MS/MS spectrum of M3 provided the product ion at

m/z 235, presumably resulting from double cleavage at the piperazine ring together with

neutral losses of glycine and pyroglutamate. The occurrence of the product ion at m/z 209

was also consistent with the presence of an aromatic thioether motif in this GSH adduct

(Baillie, 1993). Taken together, these data suggest that M3 is a GSH adduct of a


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deschloro-mono-hydroxylated m-CPP. A proposed structure for M3, which is consistent

with the CID cleavage and loss of chlorine from m-CPP, is shown in Fig. 3B.

Components M3, M4 and M5 were also identified in the incubations of nefazodone

which contains the same m-CPP moiety (data not shown).

Another component displaying no chlorine isotope cluster in MS detection was

M6 (Fig. 4A). The [M + H]+ ion of m/z 659 was 34 Da less than that of M1, suggesting

loss of a chlorine atom from trazodone. The MS/MS spectrum of [M + H]+ ion at m/z 659

afforded the diagnostic product ion at m/z 530, resulting from neutral loss of

pyroglutamate (Fig. 4B). The presence of product ions at m/z 148 and 176 suggested that

the N-propyl-triazolopyridinone moiety is unaltered. The product ions at m/z 249, 386,

410, 455 and 530 all had a mass shift of 34 Da from the corresponding product ions of

M1 at m/z 283, 420, 444, 489 and 564, respectively. These data clearly suggested that the

aromatic chlorine atom is lost in this GSH adduct, which is consistent with the absence of

a chlorine isotope cluster. The occurrence of the product ion at m/z 386 also suggested

that M6 contains an aromatic thioether motif. A proposed structure of the deschloro-

mono-hydroxylated trazodone for M6 is shown in Fig. 4A.

Component M7 had a molecular ion [M + H]+ at m/z 689, which was 4 Da less

than that of M1 (Fig. 5B). The deprotonated molecular ion of component M7 was m/z

687 in the negative ion mode (Fig. 5A). A chlorine isotope peak was observed at m/z 689

(~35% of the [M – H]– ion), indicating retention of the chlorine atom. The MS/MS

spectrum of M7 afforded the characteristic product ion at m/z 560, resulting from neutral

loss of the pyroglutamate moiety (Fig. 5B). The presence of product ions at m/z 148 and

176 suggested that the N-propyl-triazolopyridinone moiety was unchanged. The product


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ions at m/z 279, 416, 440, 485 and 560 all had a mass decrease of 4 Da from the

corresponding product ions of M1 at m/z 283, 420, 444, 489 and 564, respectively (Fig.

5B). These data suggested that the piperazine ring of trazodone may have undergone a

P450-mediated dehydrogenation reaction. Such dehydrogenation reactions are often seen

in drug metabolism (Ortiz de Montellano, 1989; Obach, 2001). The occurrence of the

product ion at m/z 416 also suggested that M7 contains an aromatic thioether motif

(Baillie, 1993). A proposed structure of M7, which is consistent with the CID cleavage, is

shown in Fig. 5B.

Bioactivation of m-CPP. Characterization of GSH adducts formed from

incubations of trazodone showed that three components, namely M3, M4, M5, were

generated from the metabolite m-CPP which was presumably released from trazodone

after N-dealkylation. To investigate the mechanisms of m-CPP bioactivation and further

confirm the identities of m-CPP derived GSH adducts generated from incubations of

trazodone, m-CPP and a regioisomer 1-(4'-chlorophenyl)piperazine (p-CPP) were

incubated with human liver microsomes or recombinant CYP2D6. The regioisomer p-

CPP was used to investigate if a quinone imine was involved in the m-CPP bioactivation,

by blocking 4'-hydroxylation of m-CPP. As shown in Fig. 1B, three components, M3,

M4 and M5, were detected in the incubation of m-CPP and those components had the

same HPLC retention times as the corresponding components from the incubation of

trazodone (Fig. 1A). Also the MS/MS spectra of these components were essentially

identical as those of the corresponding GSH adducts from the trazodone incubation with

HLMs or recombinant CYP2D6 (Fig. 2, Fig. 3 and Fig 1C). These data confirmed the

structural identities of m-CPP derived GSH adducts formed in the incubations of


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trazodone. Under the same incubation conditions, the regioisomer p-CPP showed no

bioactivation in human liver microsomes (Fig. 1D). The blockage of bioactivation of p-

CPP was also confirmed by NL scanning of m/z 129 and 75, respectively (data not

shown).

GSH Adduct Formation with Recombinant P450s. To investigate the roles of

individual human P450 isozymes in the bioactivation of m-CPP and trazodone, the

formation of GSH adducts M1, M2 and M4 was examined in incubations of m-CPP and

trazodone with insect cell-expressed recombinant P450s. As shown in Fig. 6A, at the

same enzyme concentration (100 pmol/mL), CYP2D6 was the predominant enzyme for

the formation of M4 in the incubations of m-CPP. CYP3A4 also catalyzed M4 formation,

but the level of M4 was less than 2% of that formed by CYP2D6. Only trace amounts or

no M4 were detected in incubations with other P450 enzymes including CYP1A2,

CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A5.

In the incubations of trazodone, formation of M1, M2 and M4 was examined.

Consistent with observations by others (Kalgutkar et al., 2005a), it was found that

formation of M1 and M2 was primarily mediated by CYP3A4, and to a less extent by

CYP3A5 (Fig. 6B). CYP2D6 also catalyzed formation of M1 and M2, but both levels

were less than 10% of those formed by CYP3A4. No single P450 isozyme was capable of

catalyzing formation of M4 from the incubations of trazodone (Fig. 6B). This result

agrees well with a previous report that the N-dealkylation of trazodone to form m-CPP

was primarily mediated by CYP3A4 (Rotzinger, 1998a). In marked contrast, the

formation of M4 was dramatically increased in the presence of both CYP2D6 and

CYP3A4 (Fig. 6B). These data clearly suggested that while bioactivation and N-


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dealkylation of trazodone is mediated by CYP3A4/CYP3A5, bioactivation of the

metabolite m-CPP is specifically mediated by CYP2D6.

Chemical Inhibition of Trazodone and m-CPP Bioactivation. The inhibitory

effects of P450 isozyme specific inhibitors on the formation of M1, M2 and M4 were

examined using pooled human liver microsomes. Inhibitory activity was confirmed using

P450 marker substrates (Walsky and Obach, 2004). In the incubations of trazodone,

formation of M1 and M2 was greatly inhibited by CYP3A4/3A5 selective inhibitor

ketoconazole. Ketoconazole also strongly inhibited the formation of M4 (>85%)

compared to the control value. This can be explained by the inhibition of CYP3A4-

dependent N-dealkylation of trazodone (Rotzinger, 1998a). Quinidine, a specific

CYP2D6 inhibitor, strongly inhibited M4 formation by 83%, but did not inhibit the

formation of M1 and M2 (Table 1).

In the incubations of m-CPP, formation of M4 was only inhibited by quinidine,

which is consistent with the predominant role of CYP2D6 for M4 formation in the

incubations with recombinant P450s. In both incubations of trazodone and m-CPP, the

inhibitory effects on the formation of M1, M2 and M4 were minimal (<10%) for other

P450 specific inhibitors including α-naphthoflavone (CYP1A2), sulfaphenazole

(CYP2C9) and tranylcypromine (CYP2C19) (Table 1). It is noteworthy to point out that

ketoconazole did not inhibit the formation of M4 from m-CPP (<10% inhibition).

Concentration-dependent Inhibition with Quinidine. The formation of M4 was

further examined using the specific CYP2D6 inhibitor quinidine in human liver

microsomal incubations of m-CPP. As shown in Fig. 7, the CYP2D6 inhibitor quinidine

resulted in a concentration-dependent inhibition of M4 formation from incubations of m-


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CPP. These results further support that CYP2D6 is the P450 enzyme involved in the

formation of M4 from m-CPP.

Structural Modeling of CYP2D6-m-CPP Complex. Although a crystal structure

of CYP2D6-substrate complex has not been reported yet, the recently published CYP2D6

structure (Rowland et al., 2006) provides an excellent template for modeling CYP2D6-m-

CPP complex. It is well known that substrates of CYP2D6 typically contain a basic

nitrogen atom and a planar aromatic ring, which is the structural feature of m-CPP.

Figure 8 depicts the binding mode of m-CPP in the CYP2D6 active site. The

terminal nitrogen atom of the piperazine ring of m-CPP formed an ionic hydrogen bond

with the negatively charged carboxylate group of Glu-216, which lies on the underside of

the F-helix. This is in agreement with mutagenesis studies in which Glu-216 was

identified as a key determinant in the binding of basic substrates (Paine et al., 2003). The

chlorophenyl ring is proximate to the heme group, consistent with the fact that the

oxidation of m-CPP by CYP2D6 occurs in this part of the molecule. Most noticeably, the

binding orientation of m-CPP is precisely controlled by the hydrophobic π-π interaction

between the chlorophenyl ring and Phe-120 positioned on the B'-C loop, and

concomitantly by the hydrogen bond with Glu-216 on the top (Fig. 8). The distance

between C-4' and the heme iron atom is 4.6 Å, suggesting that the active high-valent

iron-oxo attacks the C-4' during the oxidation reaction. From this CYP2D6-m-CPP

binding mode, it was expected that docking of trazodone would encounter significant

steric hindrance from residues on the F-G helices loop, situated on the top of the active

site cavity. This result agrees well with the observation that even sharing a common


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structural feature of CYP2D6 substrates, trazodone is a poor substrate of this P450

isoform.

Discussion

In the present study, several new GSH adducts (M3-M7) were detected in the

incubations of trazodone with human liver microsomes using LC/MS/MS. The results

constitute the first report on bioactivation of m-CPP, a major circulating metabolite for

several antidepressant drugs including trazodone, nefazodone and etoperidone. It was

found that formation of GSH adducts M3, M4 and M5 from m-CPP was mediated

specifically by CYP2D6, in contrast to the CYP3A4-catalyzed bioactivation of trazodone

and nefazodone (Kalgutkar et al., 2005a and 2005b). In addition, two novel deschloro-

GSH adducts, M3 and M6, derived from m-CPP and trazodone were tentatively

identified by tandem mass spectrometry. These findings are important to fully understand

the bioactivation pathways of trazodone and potential links to the mechanism of toxicity.

Direct evidence of bioactivation of m-CPP comes from incubations of m-CPP in

human liver microsomes and recombinant P450 enzymes. Bioactivation of m-CPP is of

particular interest because not only it is a major circulating metabolite of several

antidepressants but also it is a pharmacologically active serotonin receptor 5-HT2C

agonist. Very recently, m-CPP was found used as an ecstasy-like substance, becoming a

potential target for drug abuse (Lecompte et al., 2006; Bossong et al., 2005). The same

set of m-CPP derived GSH adducts was generated in incubations of trazodone and m-CPP

respectively (Fig. 1), suggesting that M3, M4 and M5 were formed via bioactivation of

m-CPP. A two-step oxidation mechanism has previously been proposed for the


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bioactivation of the 3-chlorophenylpiperazine ring of trazodone; direct oxidation at the C-

4' position resulting in 4'-hydroxytrazodone, followed by further oxidation to form a

quinone imine (Kalgutkar et al., 2005a). Sharing the identical 3-chlorophenylpiperazine

ring structure, M4 and M5 are likely formed by the same bioactivation pathways

(Scheme 2). Upon generation of m-CPP after CYP3A4-mediated N-dealkylation of

trazodone followed by a two-step oxidation pathway, an m-CPP quinone imine was

trapped by GSH to form M4 and M5.

Unlike M4 and M5, M3 was identified as a GSH adduct of m-CPP containing no

chlorine atom. Recently, a similar deschloro GSH adduct of diclofenac was identified by

LC/MS/MS and NMR and proposed to be derived from an ipso substitution of chlorine

by GSH from a quinone imine intermediate (Yu et al., 2005). Because the proposed ipso

substitution pathway is a two-step oxidation pathway and requires a initial oxidation on

the C-4' position, we further investigated the metabolic mechanisms using a regioisomer

of m-CPP, p-CPP, to determine if 4'-hydroxylation is required for formation of M3.

There was no M3 detected in the incubations of p-CPP with human liver microsomes and

recombinant P450 enzymes, suggesting formation of M3 requires the oxidation at the C-

4' position. Moreover, a total blockage of all three m-CPP derived GSH adducts M3-M5

in incubations of p-CPP suggested that they are likely formed via a common reactive

quinone imine intermediate by two-electron oxidations, after the initial 4'-hydroxylation

on the chlorophenyl ring (Scheme 2). Other evidence to support this bioactivation

pathway is that 4'-hydroxy-m-CPP is the major metabolite in incubations of m-CPP (data

not shown). Given these observations, we speculated that M3 detected in this study is 4'-

hydroxy-3'-(glutathione-S-yl)-deschloro-m-CPP formed via a common quinone imine


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which is shared by M4 and M5, followed by the ipso substitution by GSH (Scheme 2).

Similarly, the structure of M6 is proposed to be 4'-hydroxy-3'-(glutathione-S-yl)-

deschloro-trazodone. Both proposed structures would have expected fragmentation

patterns consistent with the CID MS/MS spectra (Fig. 3B and 4B, respectively). An

alternate pathway for deschloro adduct formation is P450-mediated epoxidation between

the C-3' and C-4' or the C-3' and C-2' positions on the chlorophenyl ring. Loss of chlorine

would result from an attack of GSH on the epoxide. This mechanism has been proposed

for the formation of the deschloro GSH adduct of diclofenac in human liver microsomal

incubations (Yan et al., 2005). While the current study cannot rule out this bioactivation

pathway, one would expect that such potential epoxides could be formed in incubations

of the regioisomer of m-CPP, p-CPP, for example, an epoxide between the C-4' and C-3'

positions. The formed epoxides can readily be trapped by GSH to afford corresponding

conjugates. However, both PI and NL scans failed to detect any GSH adducts in the

incubations of p-CPP.

Our structural modeling of the CYP2D6-m-CPP complex revealed a well defined

binding mode of m-CPP, with its orientation precisely controlled by a hydrophobic π-π

interaction with Phe-120 at the bottom and an ionic hydrogen bond with Glu-216 from

the top (Fig. 8). The binding orientation of m-CPP in the CYP2D6 active site clearly

suggested that the C-4' is the most susceptible site for an attack from the active high-

valent iron-oxo, which is consistent with the fact that 4'-hydroxy-m-CPP is the major

metabolite in incubations of m-CPP with both human liver microsomes and recombinant

P450s. The abundance of 4'-hydroxy-m-CPP makes the ipso substitution pathway a more

favorable mechanism for formation of M3 and M6. It is highly desirable to further


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confirm the absolute structures of these newly detected GSH adducts using synthetic

standards. However, efforts to synthesize M3 and M6 have currently been hampered by

the lack of a feasible chemistry strategy. Attempts were made to isolate M3 and M6

directly from incubations of trazodone with HLMs using preparative LC, but failed to

obtain enough of the adducts in the desired purity for NMR analysis, due to the low

abundance of GSH adducts formed in the incubations. Regardless, formation of M3 and

M6 represents unique reactive metabolites formed by trazodone.

Apart from CYP3A-mediated M1 and M2 formation, the formation of M4 from

m-CPP was found to be mediated specifically by CYP2D6. This conclusion is supported

by the following observations: 1) recombinant CYP2D6 catalyzed-formation of M4 in

incubations of m-CPP; 2) formation of M4 was strongly inhibited by quinidine, a specific

CYP2D6 inhibitor, in both incubations of m-CPP and trazodone; 3) concentration-

dependent inhibition of formation of M4 by quinidine. CYP3A4 also catalyzed M4

formation, but the conversion rate was much lower than that of CYP2D6. Therefore,

CYP3A4 is unlikely to play a significant role in formation of M4 from m-CPP. This is

supported by the observation that no single P450 isozyme was able to catalyze formation

of M4 from the incubations of trazodone. Lack of M4 formation by CYP3A4 in

incubations of trazodone can be explained by lower binding affinity of the formed m-CPP

compared to trazodone, while the reason that CYP2D6 did not form M4 is that the N-

dealkylation of trazodone to form m-CPP was primarily mediated by CYP3A4. This

conclusion is further supported by the formation of M4 when trazodone was incubated

with a mixture of CYP2D6 and CYP3A4 enzymes.


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In conclusion, several novel reactive intermediates were detected in incubations

of trazodone and m-CPP with human liver microsomes. In contrast to CYP3A-mediated

metabolism and bioactivation of trazodone, formation of GSH adducts of m-CPP was

found to be specifically mediated by CYP2D6, suggesting a possible relevance of

CYP2D6 polymorphism and/or drug interactions to m-CPP toxicokinetics (Staack et al.,

2007). Further studies are currently underway to study the balance of reactive metabolite

formation from m-CPP and CYP2D6 phenotypes. It is our hypothesis that formation of

GSH adducts from the 3-chlorophenylpiperazine ring moiety is mediated by a common

quinone imine species. These findings are of significance in understanding biochemical

mechanisms of idiosyncratic toxicity of several m-CPP containing antidepressant drugs.

Acknowledgment

We thank Sid Nelson and Griff Humphreys for their insightful suggestions and

comments during preparation of this manuscript.


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Footnotes § Current Affiliation: Department of Drug Metabolism and Pharmacokinetics, Roche Palo

Alto, Palo Alto, CA 94304


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Figure Legends FIG 1. LC/MS/MS detection of GSH adducts using the negative precursor ion scanning

of m/z 272 in (A) HLM incubations of trazodone; (B) HLM incubations of m-CPP; (C)

incubations of m-CPP with recombinant CYP2D6; (D) HLM incubations of 1-(4'-

chlorophenyl)piperazine (p-CPP).

FIG 2. LC/MS/MS analysis of component M4. (A) MS detection in negative ion mode;

(B) CID MS/MS spectrum of M4 at m/z 518 in positive ion mode.







FIG 6. Formation of GSH adducts in incubations with cDNA-expressed recombinant

P450 isozymes. (A) formation of M4 in incubations of m-CPP; (B) formation of M1, M2

and M4 in incubations of trazodone. The enzyme activities were an average of three

measurements.

FIG 7. Formation of M4 in the presence of the specific CYP2D6 inhibitor quinidine in

incubations of m-CPP with human liver microsomes.

FIG 8. A partial structure of the CYP2D6-m-CPP complex. m-CPP is shown with respect

to a stick model of heme (bottom) and two residues Phe-120 and Glu-216 in the CYP

2D6 active site.


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SCHEME 1. Cytochrome P450 3A4-mediated metabolism and proposed bioactivation pathways of trazodone.

N N

N

O

N

N

Cl

Trazodone

CYP3A4

N N

N

O

N

N

Cl

N N

N

O

N

N

Cl

O OH

GSHGSH

N N

N

O

N

N

Cl

SGHO

N N

N

O

N

N

Cl

OH

SG

M1M2

Triazolopyridinone epoxide4'-hydroxytrazodone

CYP3A4

N N

N

O HN

N

Cl

m-CPP

O

H+

Triazolopropionic aldehyde

N N

N

O

N

N

OH

M6

SG

N N

N

O

N

N

Cl

OH

SG

M7


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SCHEME 2. Proposed bioactivation pathways leading to the formation of M3, M4 and M5.

m-CPP

NHNCYP2D6

NHN O

4'-OH m-CPP

NHN OH

Cl Cl ClGSH

NHN O

SGCl

NHN O

SG

[H]NHN OH

SG

NHN O

Cl

GSH

OR

CYP2D6

M3

M4, M5

1'

2' 3'

4'

5'6'

NHN OH

Cl

SG

NHN1'

2' 3'

4'5'6'

Cl

Xp-CPP


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TABLE 1

Effects of P450 isoform-specific inhibitors on the formation of M1, M2 and M4 in HLM incubations of trazodonea

P450 Inhibitor

M1

Formation

M2

Formation

M4

Formation

M4

Formationb

P450

Activityc

α-Naphthoflavone (CYP1A2) 95.2 91.8 96.8 97.2 12.7

Sulfaphenazole (CYP2C9) 96.6 92.4 93.2 95.3 33.2

Tranylcypromine (CYP2C19) 91.8 95.4 98.9 97.4 27.6

Quinidine (CYP2D6) 99.2 102.1 17.2 22.4 8.9

Ketoconazole (CYP3A4/3A5) 35.8 24.8 11.7 94.2 22.4

a Values are an average of duplicated measurements and expressed as percentage relative to the

control without inhibitors. b M4 formation from the incubations of m-CPP. c P450 activities were

determined using known P450 substrates.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (min)

(A)100

0

Rel

ativ

e A

bun

danc

e%

M1

M2

M3

M4

M5

M6

M7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (min)

(B)100

0

Rel

ativ

e A

bun

danc

e

%

M3

M4

M5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22Time (min)

(C)100

0

%

Rel

ativ

e A

bun

danc

e

M3

M4

M5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (min)

100

0

Rel

ativ

e A

bun

danc

e

%

(D)

Figure 1

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

m/z

516.1

518.2

100

%

(A)

0

Rel

ativ

e A

bund

ance

0

m/z

100

%

Rel

ativ

e A

bund

ance

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

389.0

243.0 518.2

269.0372.2

443.1500.2

(B)

179.0

S

HN

O NH

COOH

OHOOC

NH2

HN

N

Cl

OH

389372- NH3

443

269

243

Figure 2




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400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

m/z

482.1100

%

0

Rel

ativ

e A

bund

ance

(A)

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

355.0

484.2209.1

235.0 338.1 409.1466.2

177.0

m/z

100

%

0

Rel

ativ

e A

bund

ance

(B)

S

HN

O NH

COOH

OHOOC

NH2

HN

N

355338- NH3

409

235

209OH

Figure 3




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100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

176.0530.4 659.3

148.1

386.5

249.1

642.2

100

0

%

Rel

ativ

e A

bund

ance

(B)

410.4 455.2

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

657.2

m/z

100

%

0

Rel

ativ

e A

bund

ance

(A)N

NN

O

N

N

S

HN

O NH

COOH

OHOOC

NH2

530 455

386

- NH3 410

176148

249

OH

Figure 4




DM

D Fast Forw


I: 10.1124/dmd.107.019471



500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

m/z

687.1

689.2

100

%

(A)

0

Rel

ativ

e A

bund

ance

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

689.2

560.2

176.0 416.2

148.1

279.1 368.2 440.2 671.3

100

0

%

Rel

ativ

e A

bund

ance

485.0 543.3

N

NN

O

N

N

Cl

OHS

HN

O NH

COOH

OHOOC

NH2

560

485416

- NH3 440

176148

279

- NH3

543

(B)

Figure 5




DM

D Fast Forw


I: 10.1124/dmd.107.019471



0

20

40

60

80

100

120

CYP1A2

CYP2A6

CYP2B6

CYP2C8

CYP2C9

CYP2C19

CYP2D6

CYP2E1

CYP3A4

CYP3A5

For

mat

ion

of M

4 fr

om m

-CP

P (

%) (A)

0

20

40

60

80

100

CYP1A2

CYP2A6

CYP2B6

CYP2C8

CYP2C9

CYP2C19

CYP2D6

CYP2E1

CYP3A4

CYP3A5

CYP2D6/

CYP3A4

For

mat

ion

of G

SH

add

ucts

fr

om tr

azod

one

(%)

M1M2M4

(B)

Figure 6




DM

D Fast Forw


I: 10.1124/dmd.107.019471



0

20

40

60

80

100

0 1 2 3 4 5 6

Quinidine concentration (µM)

Fo

rmat

ion

of

M4

fro

m m

-CP

P (

%)

Figure 7




DM

D Fast Forw


I: 10.1124/dmd.107.019471



Figure 8




DM

D Fast Forw


I: 10.1124/dmd.107.019471



detection of novel reactive metabolites of...

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