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Arch ToxicolDOI 10.1007/s00204-013-1056-y

In vITrO sysTems

In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid

Alison V. M. Rodrigues · Helen E. Rollison · Scott Martin · Sunil Sarda · Timothy Schulz‑Utermoehl · Simone Stahl · Frida Gustafsson · Julie Eakins · J. Gerry Kenna · Ian D. Wilson

received: 18 December 2012 / Accepted: 3 April 2013 © springer-verlag Berlin Heidelberg 2013

rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. no effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxic-ity to THLe cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BseP and mrP2 or mitochon-drial toxicity to THLe or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxic-ity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

Keywords Biotransformation · Fenclozic acid · Hepatotoxicity · Covalent binding · reactive intermediate

Introduction

Fenclozic acid (ICI 54,450, 2-(p-chlorophenyl)thiazol-4-yl acetic acid, myalex) (structure 1) emerged in the late 1960s as a promising carboxylic acid non-steroidal anti-inflammatory drug candidate that demonstrated potent anti-inflammatory, anti-pyretic and analgesic properties (Hepworth et al. 1969; newbould 1969). safety testing revealed a good safety profile, and the only concerning tox-icity evident in experimental animals (rats, dogs and mice) was intestinal ulceration at high doses (Alcock 1970). Fen-clozic acid was administered to healthy male volunteers and rheumatoid arthritis patients without adverse effects, leading to further clinical trials comparing fenclozic acid with aspirin (Chalmers et al. 1969a, 1969b). Clinically, doses of 100 mg fenclozic acid twice daily resulted in no adverse effects. However, numerous cases of jaundice and

Abstract The carboxylic acid nsAID fenclozic acid exhibited an excellent preclinical safety profile and prom-ising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [14C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in nADPH-supplemented liver microsomes, although no metabolites were detected in these incuba-tions or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharac-terizable metabolite was also observed. In addition, cova-lent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabo-lite was detected when liver microsomes from human,

Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1056-y) contains supplementary material, which is available to authorized users.

A. v. m. rodrigues molecular and Clinical Pharmacology, mrC Centre for Drug safety science, sherrington Building, University of Liverpool, Ashton street, Liverpool L69 3Ge, UK

H. e. rollison (*) · s. martin · s. sarda · T. schulz-Utermoehl · I. D. Wilson Drug metabolism and Pharmacokinetics Im, AstraZeneca, Alderley Park, macclesfield, Cheshire sK10 4TG, UKe-mail: helen.rollison@astrazeneca.com

s. stahl · F. Gustafsson · J. eakins · J. G. Kenna molecular Toxicology, Global safety Assessment, AstraZeneca, Alderley Park, macclesfield, Cheshire sK10 4TG, UK

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abnormal hepatic function emerged after administration of 400 mg/day for more than 14 days (Alcock 1970). A detailed review of eight of the patients with abnormal liver parameters revealed that the clinical symptoms included raised body temperature and pruritus, that two patients had enlarged livers and one patient had a rash; in addition, raised alkaline phosphatase, serum AsT and ALT activities were observed in all patients (Hart et al. 1970). Due to the evident hepatotoxicity in humans, fenclozic acid was with-drawn from clinical development, all of the preclinical data were reassessed and additional experimentation in a wide range of preclinical species was undertaken in an attempt to elucidate the underlying cause (Alcock 1970).

The hepatotoxicity observed in humans could not be replicated in any of the animal species tested. The drug therefore represents a classic example of an apparently human-specific hepatotoxicant. The pattern of liver injury exhibited a clear dose–response relationship and was evi-dent in numerous patients during clinical trials; in one trial, 2 of 12 patients who received fenclozic acid at 400 mg/day became jaundiced after 4 weeks. Therefore, the pattern of liver injury cannot be considered idiosyncratic. The cru-cial role played by metabolism in many instances of drug hepatotoxicity only became clear after fenclozic acid had been discontinued (mitchell et al. 1973), although it was recognized that drugs could affect the hepatic uptake of bilirubin (Levi et al. 1969), and that nsAIDs could inhibit bilirubin metabolism (Hargreaves 1965). nevertheless, several Phase 1 and Phase 2 metabolites of fenclozic acid were identified in the preclinical in vivo drug metabolism studies conducted during its development. In rat and dog it was reported that [14C]-fenclozic acid was hydroxylated at position 4 on the phenyl ring resulting in the loss or migration, via an apparent ‘nIH shift’, of the ring chlorine substituent (Foulkes 1970). Fenclozic acid and its metabo-lites were excreted in the urine of these species as glucuro-nide conjugates. In the monkey, Phase 1 metabolism was not observed and only an acyl glucuronide conjugate was detected (Foulkes 1970). Glucuronide and taurine conju-gates were found in the bile and urine of rats, although the balance of conjugation appeared to be dose dependent with the percentage of the taurine conjugate increasing from 16 to 32 % in the urine as the dose was increased from 2 to 100 mg/kg (Bradbury et al. 1981). The production of

hydroxy- and glucuronide metabolites remained unchanged with increasing dose, suggesting that metabolic pathways such as hydroxylation and glucuronidation became satu-rated at high doses, forcing metabolism down alternative metabolic routes such as amino acid conjugation.

very recently, it has been proposed that the use of a multi-parametric in vitro approach may provide novel insights into mechanisms by which a variety of drugs cause hepatotoxicity in humans (Thompson et al. 2012). This approach assesses five toxicity-related endpoints using an in vitro assay panel and also by quantifying covalent bind-ing of radiolabelled drug to human hepatocyte proteins. When evaluated using this approach, fenclozic acid caused no detectable activity in the in vitro assay panel but exhib-ited high levels of covalent binding to human liver proteins in vitro, suggesting that metabolic bioactivation might play a key role in its human-specific liver toxicity (Thompson et al. 2012). In the present study, this preliminary panel of experiments has been extended via a detailed investiga-tion of the in vitro toxicity, biotransformation and covalent binding to liver protein of fenclozic acid. This involved the assessment of: cytotoxicity to an immortalized human liver-derived (THLe) cell line expressing a range of dif-ferent human P450 enzymes; mitochondrial impairment in HepG2 cells and in THLe cells; effects on the activities of rat and human orthologues of the bile salt efflux pump (BseP) and multi-drug resistance protein type 2 (mrP2); in vitro metabolism by and covalent binding to human, rat and dog liver microsomal and hepatocytes; and pos-sible time-dependent inhibition of multiple human P450 enzymes.

Experimental procedures

materials

[3H]-Taurocholic acid (1 mCi/ml) was purchased from Per-kin elmer Life and Analytical sciences (Waltham, mA, UsA). Fenclozic acid was obtained from the AstraZeneca compound collection (Alderley Park, UK) and was dis-solved in purified water by addition of a minimum amount of potassium hydroxide (0.1 m). [14C]-Fenclozic acid (the site of radiolabelling is shown in structure 1) was synthe-sized and purified (>99 %) by Isotope Chemistry, Astra-Zeneca, Alderley Park, UK, and dissolved in ethanol to a final concentration of 2 mm, with a specific activity of 60.6 μCi/μmol. [14C]-ethoxycoumarin (specific activ-ity 14.1 μCi/μmol) (Ge Healthcare UK Ltd, Chalfont st. Giles, UK) was used as a positive control to verify the viability of the liver microsomes and was prepared at a concentration of 6 mm in methanol. [14C]-Diclofenac (specific activity 35.1 μCi/μmol) (American radiolabeled

Structure 1 The structure of fenclozic acid showing the site of 14C-labelling

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Chemicals, st Louis mO, UsA) was used as a positive control for the covalent binding experiments and was pre-pared at a concentration of 2 mm in methanol. Analyti-cal grade acetonitrile, formic acid and trifluoroacetic acid were obtained from Fisher scientific (Loughborough, UK). Ultima Flo-m™ and Ultima Gold™ scintillants were obtained from Packard Instruments (seer Green, UK). The Pierce® BCA Protein Assay Kit was obtained from Perbio science (Cramlington, UK). HepG2 cells were supplied by the AstraZeneca cell bank and were characterized by sTr fingerprinting. THLe cell lines were obtained under an evaluation licence from nestec. Ltd. (Lausanne, switzer-land). THLe cell line enzyme and transporter gene expres-sion studies were completed previously (soltanpour et al. 2012). The CellTiter 96® AQueous non-radioactive mTs Cell Proliferation Assay and the CellTiter-Glo® Lumines-cent Cell viability Assay were obtained from Promega (southampton, UK). PmFr P-004 medium was obtained from Pasadena Foundation for medical research (CA, UsA). GlutamAX™, heat inactivated foetal bovine serum, Dmem and the Bac-to-Bac® baculovirus expression sys-tem were obtained from Invitrogen (Paisley, UK). Bacu-lovirus expressing human BseP (ABCB11) was provided by Prof. Dr. Bruno stieger (University of Zurich, switzer-land). cDnAs encoding sprague–Dawley rat liver mrp2 (Abcc2) and human liver mrP2 (ABCC2) were from Ori-Gene Technologies (rockville, mD, UsA). Protease inhibi-tor tablets were from roche (Basel, switzerland). All other chemicals were obtained from the sigma-Aldrich Company Ltd (Poole, UK).

In vitro metabolism of [14C]-fenclozic acid

Human (pool of 28 donors) and dog (Beagle) liver micro-somes (20 mg/ml protein) were obtained from BD Gen-test™ (Franklin Lakes nJ, UsA). rat liver microsomes were prepared from 12 male rats (Wistar-derived, Alderley Park) and pooled (22.7 mg/ml protein). All liver micro-somes were stored at −80 °C and thawed immediately prior to use. Human (pooled donors), dog (male Beagle) and rat (male Wistar) cryopreserved hepatocytes were obtained from CellzDirect (Durham, nC, UsA). Hepatocyte viabil-ity was determined by trypan blue exclusion prior to and during experiments.

Microsomes

[14C]-Fenclozic acid (10 μm) or [14C]-7-ethoxycoumarin (30 μm) was incubated with human, rat or dog liver micro-somes (final protein concentration of 2 mg/ml) and 2 mm nADPH or 4 mm UDPGA dissolved in phosphate buffer (0.1 m with 10 mm mgCl2, pH 7.0), or without cofactor, at 37 °C for 120 min. In UDPGA-supplemented incubations

the microsomes were preincubated with alamethicin (25 μg/mg protein) for 15 min at 37 °C before addition of other components. samples were taken in duplicate at 0 and 120 min, and metabolic reactions terminated in chilled 3 % formic acid in acetonitrile in order to stabilize any acyl glucuronide metabolites (shipkova et al. 2003). samples were stored at −20 °C, then analysed by high performance liquid chromatography with radiochemical and mass spec-trometric detection (HPLC-rAD-ms). Details of sample preparation and analysis methods are provided in Online resource 1.

Hepatocytes

Hepatocytes at 106 cells/ml in Leibovitz’s-L15 buffer were incubated with 10 μm aqueous [14C]-fenclozic acid or [14C]-diclofenac with or without 1 mm 1-aminoben-zotriazole (ABT). samples were taken at 0, 60, 120 and 180 min into chilled acetone and stored at −20 °C. Details of sample preparation and analysis methods are provided in Online resource 1.

In vitro covalent binding of [14C]-fenclozic acid

Microsomes and hepatocytes

Liver microsomes or hepatocytes were incubated under the conditions stated above, but at 1 mg/ml microsomal pro-tein, with 10 μm [14C]-fenclozic acid or [14C]-diclofenac. samples were taken in triplicate at 0, 30, 60, 120 min and in hepatocytes also at 180 min and were mixed with chilled acetone, vortexed and stored at −20 °C to aid protein pel-let formation until processed further as described below. Additional covalent binding experiments were carried out in human liver microsomes with nADPH plus the trap-ping agents glutathione, cyanide, semicarbazide, methoxy-lamine, lysine or cysteine, each at 5 mm, with sampling at 0 and 120 min. separate incubations of trapping agents with [14C]-fenclozic acid in phosphate buffer were per-formed to determine any intrinsic chemical reactivity.

Sample analysis

Analysis was based on a published method (evans et al. 2003). Briefly, the proteins present in the hepatocyte and microsome samples were washed with 80 % methanol and collected on Whatman GF/B fired filter paper (Chal-font st.Giles, UK) using a Brandel 96-sample cell har-vester (Gaithersburg mD, UsA). Proteins were solubilized with 5 % sDs overnight at 50 °C and quantified using the Pierce BCA Protein Assay Kit® with bovine serum albumin as a standard and the BioTek® synergy HT multidetec-tion microplate reader with KC4 software (Potton, UK).

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Protein-bound radioactivity was measured by liquid scin-tillation counting using Ultima Gold™ scintillant (Perkin elmer) on a Packard 1900Tr analyser (Berkshire, UK).

Data analysis

Covalent binding was calculated as follows:

equation 1: Covalent binding (pmol equivalent)

equation 2: Covalent binding (pmol equivalents per mg protein)

Covalent binding data were compared pairwise at each individual time-point by one-way AnOvA using Tukey’s method to adjust for multiple comparisons. A value of P < 0.05 was considered significant. Covalent binding data from different incubation conditions were analysed by an unpaired student’s t test with two-tailed distribution. Data are presented as mean covalent binding in pmol equivalents per milligram of protein ± standard deviation.

Reactive metabolite trapping experiments

samples were analysed using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher scientific, Bremen, Germany) connected to a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corp., milford, mA, UsA) as described in Online resource 1.

Time-dependent P450 inhibition

Human liver microsomes (1 mg/ml) were preincubated with fenclozic acid (10 μm) in phosphate buffer (0.1 m with 10 mm mgCl2, pH 7.0) containing 1 % DmsO for 30 min in the presence or absence of 1.05 mm nADPH and were diluted fivefold into a secondary incubation medium which contained a cocktail of probe substrates and fur-ther nADPH (1 mm). The probe substrate cocktail con-sisted of 75 μm phenacetin (CyP1A2), 10 μm diclofenac (CyP2C9), 100 μm s-mephenytoin (CyP2C19), 20 μm bufuralol (CyP2D6) and 10 μm midazolam (CyP3A4). 10 μm furafylline, 10 μm tienilic acid, 5 μm ticlopidine, 5 μm mDmA (3,4-methylenedioxy-n-methylampheta-mine), 1.5 μm troleandomycin and 5 μm mifepristone were incubated in parallel as positive control time-depend-ent inhibitors for CyP1A2, CyP2C9, CyP2C19, CyP2D6,

pmol equiv.sample=Radioactivity/sample (DPM)

÷Specific activity of compound (DPM/pmol)

pmol equiv./mg protein = [covalent binding(pmolequiv.)

÷protein concentration(µg/mL)]

× 1000

CyP3A4 and CyP3A4, respectively. 1 % DmsO vehi-cle was also incubated in parallel as a negative control. The secondary incubation was terminated after 15 min by the removal of 200 μL incubate into 200 μL acetoni-trile containing a proprietary internal standard. An aliquot of 200 μL of water was added to every sample, samples were centrifuged at 1,479g and the formation of paraceta-mol, 4′OH diclofenac, 4′OH mephenytoin, 1′OH bufuralol and 1′OH midazolam was quantified by HPLC–ms2. The percentage of time-dependent and reversible inhibition was calculated relative to the positive control using the peak area in the presence and absence of nADPH, respectively.

THLe cell cytotoxicity

THLe cell lines transfected with vectors encoding individual human P450 isoforms (1A2, 2C9, 2C19, 2D6, 2e1, 3A4), or with an empty vector (THLe-null), were seeded in col-lagen I coated 96-well plates at 15,000 cells/well and incu-bated at 37 °C in 95 % air, 5 % CO2 for 24 h. medium was then replaced with serum-free culture medium containing fenclozic acid (0.3–500 μm) in 1 % DmsO vehicle, or vehi-cle alone. Incubations were performed in triplicate in a sin-gle experiment. After incubation for a further 24 h at 37 °C in 95 % air, 5 % CO2, cell viability was measured using the Promega CellTiter 96® AQueous non-radioactive mTs Cell Proliferation Assay according to the manufacturer’s instruc-tions. Absorbance was measured at 492 nm on a Perkin elmer envision™ 2100 spectrophotometer (seer Green, UK).

mitochondrial toxicity determination

HepG2 cells were seeded at 5,000 cells/well in 96-well black collagen-coated plates, in either Dmem supple-mented with 25 mm glucose, 4 mm glutamine, 1 mm pyru-vate, 5 mm HePes and 10 % FBs or Dmem supplemented with 10 mm galactose, 4 mm glutamine, 1 mm pyruvate, 5 mm HePes and 10 % FBs and left overnight to attach at 37 °C in 95 % air: 5 % CO2. Fenclozic acid (0.25–250 μm) in 0.5 % DmsO vehicle was added to the cells, whereas control cells were incubated with 0.5 % DmsO vehicle alone, and incubated for a further 24 h. Cytotoxicity was assessed using the CellTiter-Glo® Luminescent Cell via-bility Assay according to the manufacturer’s instructions. eC50 values were determined using Origin® 7.5 (Origin-Lab, northampton, mA, UsA). studies in THLe-null cells involved culture in PmFr P-004 media supplemented with either 25 mm glucose or 10 mm galactose.

Inhibition of BseP and mrP2 transporter activity in vitro

membrane vesicles were prepared from Spodoptera fru-giperda Sf21 insect cells transfected with pFastBac1

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vectors containing cDnAs encoding rat mrp2 or Bsep, or human mrP2 or BseP, using the Bac-to-Bac® baculovirus expression system as described previously (Dawson et al. 2012) with the modification that, at the end of the isolation procedure, vesicles were resuspended in buffer containing 50 mm sucrose, 10 mm HePes/Tris, pH 7.4 and protease inhibitor tablets (roche, Basel, switzerland). ATP-depend-ent transport of probe substrates into inside-out membrane vesicles was measured by a rapid filtration method (Daw-son et al. 2012). Details of the buffer composition and incu-bation conditions are provided in Online resource 1.

Results

microsomal metabolism of [14C]-fenclozic acid

When [14C]-fenclozic acid (10 μm) was incubated in the presence of human, rat and dog liver microsomes (2 mg/mL protein) for 120 min, either with or without nADPH, no metabolites were detected by radiochemical HPLC. A [14C]-labelled metabolite was detected in incubations sup-plemented with UDPGA with a relative retention time (rrT) of 0.85–0.86, accounting for 4.5, 0.9 and 4.9 % of the total radioactivity in human, rat and dog microsome incubations, respectively. HPLC–ms Data-dependent analysis in positive esI of this UDPGA-dependent metabo-lite gave an ion at m/z 432. This result is consistent with the addition of a glucuronic acid moiety (+176) to [14C]-fenclozic acid (m/z 256) and indicates that a glucuronide metabolite was formed. Given the carboxylic acid struc-ture within [14C]-fenclozic acid, it is likely that this was an acyl glucuronide as reported in the original in vivo studies (Bradbury et al. 1981; Foulkes 1970).

metabolism of fenclozic acid in hepatocyte incubations

In rat and dog hepatocyte incubations, a metabolite was detected at rrT of 0.69–0.73. HPLC–ms data-dependent analysis in negative ion mode afforded an ion at m/z 361, subsequently generating a characteristic fragment ion at m/z 124, indicating that this metabolite was a taurine con-jugate of [14C]-fenclozic acid. The amount of this taurine conjugate increased with time and was most abundant in rat hepatocyte incubations, reaching 9.6 % of the total radioactivity after 180 min (in the presence of ABT, which was included to inhibit P450 activity). The production of this [14C]-fenclozic acid–taurine conjugate was similar in incubations performed with rat hepatocytes (6.7 % without ABT and 9.6 % with ABT, after 180 min) and dog hepato-cytes (1.4 % without ABT and 1.8 % with ABT, after 180 min). In human hepatocyte incubations, in the absence of ABT, an unidentified metabolite (rrT 0.87) was also

detected by radiochemical HPLC from 120 min onwards, but full characterization could not be undertaken due to its low abundance (<1 % of the radioactivity). HPLC–ms analysis of this unknown human metabolite, which was not detected when human hepatocytes were incubated with ABT, did however confirm that it was neither an acyl glu-curonide nor a taurine conjugate.

Covalent binding to liver microsomes

To investigate covalent binding to proteins, [14C]-fenclozic acid (10 μm) was incubated with human, rat and dog liver microsomes (1 mg/mL protein) for 120 min in the pres-ence and absence of either nADPH (Fig. 1a) or UDPGA (Fig. 1b). In the absence of nADPH, the quantities of non-extractable radiolabel observed were low (11.9 ± 6.2, 11.7 ± 4.7 and 12.5 ± 5.9 pmol equivalents per milligram

Fig. 1 Covalent binding to human-, rat- or dog-derived liver micro-somal protein in the presence of a nADPH or without cofactor. b UDPGA or without cofactor. Data are presented as mean val-ues ± standard deviation of three technical replicates

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of protein for human, rat and dog liver microsomes, respec-tively). However, in the presence of nADPH markedly higher levels of non-extractable binding to liver microsomal protein in rat (P = 0.001) were evident after 30 min, with significant effects also seen in human (P = 0.0005) and dog (P < 0.05) after 60 min (one-way AnOvA, adjusted by Tukey’s method). The greatest amount of non-extract-able binding was observed with human microsomal incu-bations, followed by dog and then rat liver microsomes. The observed values were 125.8 ± 20.1, 70.2 ± 19.8 and 38.5 ± 6.8 pmol equivalents per milligram of protein for human, dog and rat liver microsomes, respectively (sum-marized in Online resource 2). The metabolic viability of the microsomes was confirmed by investigation of the metabolism of [14C]-7-ethoxycoumarin, and their capac-ity to exhibit covalent binding was established in sepa-rate incubations in which [14C]-diclofenac was used as a positive control. In this experiment, the equivalent results for [14C]-diclofenac were 108.6 ± 3.6, 145.8 ± 20.5 and 126.9 ± 18.0 pmol equivalents per milligram of protein for human, rat and dog, respectively. When [14C]-fenclo-zic acid incubations were undertaken in the presence of UDPGA, the extent of non-extractable binding to protein after 120-min incubation was not statistically different to the binding in the absence of cofactor. This indicates that metabolism of fenclozic acid to an acyl glucuronide metabolite does not result in significant levels of covalent binding under these conditions. In contrast, covalent bind-ing of [14C]-diclofenac to liver microsomes in the pres-ence of UDPGA reached 155.1 ± 17.7, 113 ± 21.4 and 131.4 ± 21.2 pmol equivalents per milligram of protein for human, rat and dog, respectively.

The effect of a variety of trapping agents (glutathione (GsH), cyanide, methoxylamine, semicarbazide, cysteine and lysine) on the amount of nADPH-dependent non-extractable binding of fenclozic acid to human liver micro-somal protein is shown in Fig. 2 (and summarized in Online resource 3). While the extent of nADPH-depend-ent non-extractable binding after 120-min incubation was unaffected by semicarbazide or lysine, it was significantly reduced in the presence of GsH (P = 0.0002), cyanide (P = 0.0002), cysteine (P < 0.0001) and methoxylamine (P < 0.005) (T test; unpaired, two-tailed distribution). It is notable that higher levels of non-extractable binding were evident at 0 min in the presence of GsH plus nADPH than when incubations were undertaken with nADPH but not GsH (P < 0.005; t test; unpaired, two-tailed distribu-tion). In contrast, elevated levels of non-extractable bind-ing were not observed when incubations were undertaken in the presence of nADPH and GsH for 120 min. The rea-son for this unexpected observation, which was replicated in a second experiment, is not known. However, it may be significant that the ‘0 min’ time-point involved initiation of

the reaction, mixing and termination of a sample in chilled acetone, which would have been sufficient to enable metab-olism for a brief time interval of several seconds.

Despite the clear reduction in non-extractable binding that was evident with some of the trapping agents, analyses undertaken by radiochemical HPLC analysis or by UPLC-Uv or UPLC–ms (using common fragment analysis) were unable to detect any compound-related peaks or identify any reaction products with the various trapping agents that could be attributed to the formation of reactive metabolites in these incubations.

Covalent binding to hepatocytes

To further investigate the role of metabolism in covalent binding, [14C]-fenclozic acid was incubated with cryopre-served human, rat and dog hepatocytes for 180 min with, and without, the P450 inhibitor ABT. The greatest amount of non-extractable binding occurred for rat, then dog and finally human hepatocytes, amounting to 539.0 ± 69.6, 269.7 ± 143.5 and 74.9 ± 13.6 pmol equivalents per mil-ligram of protein, respectively (see Fig. 3, summarized in Online resource 4). This binding was time-dependent in

Fig. 2 Covalent binding in human liver microsomes in the pres-ence of nADPH and various trapping agents. Data are presented as mean ± standard deviation of three technical replicates (except for cysteine 0 min, n = 2; GsH, +nADPH and w/o cofactor, data are mean ± standard deviation of a minimum of 5 replicates from two independent experiments). significance is relative to +nADPH time-point **P < 0.005, ***P < 0.0005, ****P < 0.0001

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hepatocytes derived from all three species. In the rat hepat-ocyte incubations, there was a greater extent of binding at the 180 min time-point in the presence than in the absence of ABT (P < 0.05; t test; unpaired; two-tailed) (Fig. 3). Conversely, in human hepatocyte incubations, there was a significantly greater extent of binding at the 180 min time-point when ABT was absent (P < 0.05; t test; unpaired; two-tailed). At all other time-points, comparisons between incubations undertaken with and without ABT revealed no statistically significant differences. Incubation of hepato-cytes with [14C]-diclofenac for 3 h resulted in amounts of non-extractable binding of 302.7 ± 5.4, 204.1 ± 26.7 and 224.8 ± 0.6 pmol equivalents per milligram of protein for human, rat and dog, respectively.

Time-dependent inhibition of CyPs

The liver microsome non-extractable binding data indi-cate an nADPH-dependent mechanism of bioactivation and covalent binding of fenclozic acid. experiments were therefore performed to determine whether fenclozic acid affected the activities of five major hepatic P450 enzymes. Incubations undertaken using positive control compounds revealed time-dependent inhibition of all enzymes, thus validating the experimental procedure (data not shown). However, no time-dependent inhibition of the enzymes by fenclozic acid was observed. Although 24 % reduction in P450 1A2-mediated phenacetin O-deethylase activity was observed in the presence of fenclozic acid, this was also evident in the absence of nADPH in the preincubation,

suggesting that the enzyme inhibition had occurred via a reversible mechanism. reversible inhibition of other P450 enzyme activities by fenclozic acid was not observed.

THLe cell cytotoxicity

Toxicity of fenclozic acid to a panel of immortalized human liver-derived THLe cell lines which expressed either indi-vidual human P450s (THLe-1A2, 2C9, 2C19, 2D6, 2e1 or 3A4 cells), or no detectable CyP activity (THLe-null cells), was assessed using the mTs assay. Following expo-sure of the cell lines to fenclozic acid for 24 h at concen-trations up to 500 μm, no evidence of cell toxicity was observed.

mitochondrial toxicity

The THLe cell cytotoxicity experiments summarized above were undertaken using cells cultured in media con-taining high glucose concentrations. It has been observed that cells cultured in media that contain galactose in place of glucose exhibit much greater sensitivity to mitochondrial dysfunction, since galactose supports generation of rela-tively little ATP from glycolysis (marroquin et al. 2007). Further experiments were therefore undertaken to deter-mine the cytotoxicity of fenclozic acid to THLe-null cells cultured in galactose media. In addition, equivalent experi-ments were performed using human hepatocyte-derived HepG2 cells, which have been utilized by previous investi-gators to assess mitochondrial impairment by various other drugs (marroquin et al. 2007). no evidence of toxicity was observed following exposure of the two cells lines to fenclozic acid for 24 h at concentrations up to 250 μm, in either glucose or galactose media.

Inhibition of BseP and mrP2 transporter activity

Fenclozic acid did not affect the ATP-dependent transport of probe substrates of either the human or rat orthologues of BseP/Bsep and mrP2/mrp2 at concentrations up to and including 62.5 μm, when assessed using recombinant membrane vesicle assays (Fig. 4). At test compound con-centrations which exceeded 125 μm, evidence of dose-dependent transporter inhibition was evident and the maxi-mum observed reduction in uptake of [3H]-taurocholate (at 1,000 μΜ fenclozic acid) was 54 ± 12 and 78 ± 10 % of control values for rat Bsep and human BseP, respec-tively. This is in line with recently published data on other pharmaceuticals in which IC50 values of >1,000 μm were reported for inhibition of rat Bsep and human BseP by fenclozic acid (Dawson et al. 2012). In the presence of 1,000 μm fenclozic acid, 5(6)-carboxy 2′,7′-dichlorofluo-rescein (CDF) uptake into vesicles expressing rat mrp2 or

Fig. 3 Covalent binding to human rat or dog cryopreserved hepato-cytes in the presence and absence of 1-aminobenzotriazole. Data are presented as mean values ± standard deviation of three technical rep-licates (except for dog hepatocytes + ABT at 180 min, n = 2)

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human mrP2 was reduced to 77 ± 14 and 60 ± 13 % of the activities of control incubations, respectively (summa-rized in Online resource 5).

Discussion

The mechanism(s) behind the human-specific hepatotoxic-ity of the potent nsAID fenclozic acid have been investi-gated through a panel of modern in vitro techniques which were not available at the time of development, see Fig. 5. Our inability to detect cell cytotoxicity in any of the THLe cell lines suggests that the toxicity cannot be attributed to cytotoxic effects of the parent compound itself or of metabolites formed by the major human P450 enzymes that were investigated (1A2, 2C9, 2C19, 2D6, 2e1 or 3A4). In addition, the studies in which HepG2 cells were exposed to fenclozic acid in media containing galactose in place of glucose, which has been shown by other investigators to enable detection of mitochondrial injury caused by many other drugs (marroquin et al. 2007), did not provide evi-dence that fenclozic acid impaired mitochondrial func-tion. Although partial inhibition of the activities of the biliary efflux transporters BseP and mrp2 activities was observed, this was evident only at very high concentrations

of fenclozic acid (apparent IC50 ≥ 1,000 μm) and so is of questionable clinical relevance. Biliary transporter inhibi-tion and cell cytotoxicity studies were not undertaken using the acyl glucuronide metabolite of fenclozic acid since the relative instability of a synthetic fenclozic acid acyl glu-curonide (t1/2 in pH 7.4 buffer ca. 30 min, Karlson et al. in preparation) made such in vitro experiments impracti-cal. However, it may be noteworthy that no hepatotoxicity was observed when in vivo safety studies were undertaken in the monkey, where the acyl glucuronide was the major metabolite (Foulkes 1970).

The most compelling mechanistic data were provided by the covalent binding studies undertaken using [14C]-fenclozic acid in isolated hepatocytes and liver microsomes from various species. In human, rat and dog liver micro-somes, the amount of irreversible binding increased over time when nADPH was present. Furthermore, the cova-lent binding that was evident at the last time-point in the human hepatocyte incubations was reduced in the presence of the P450 inhibitor ABT. There was also evidence of a Phase 1 metabolite in the later time-points of the human hepatocyte incubation, which was absent when ABT was present. These data strongly indicate that oxidative metab-olism was responsible for the observed covalent binding and that nADPH-dependent bioactivation of fenclozic

Fig. 4 a Inhibition of rat Bsep (rBsep) and human BseP (hBseP) mediated uptake of [3H]-taurocholate in membrane vesicles by fenclozic acid. b Inhibition of rat mrp2 (rmrp2) and human mrP2 (hmrP2) mediated uptake of CDF by fenclozic acid. All data are pre-sented as mean values ± stand-ard deviation of three independ-ent experiments, with duplicate incubations per experiment

Fig. 5 The carboxylic acid nsAID fenclozic acid caused hepatotox-icity in clinical trials. Although modern in vitro toxicity approaches did not provide useful insight into the underlying mechanism, cova-

lent binding studies showed significant levels of time-dependent irre-versible binding to protein in the presence of nADPH, indicative of Phase 1 mediated bioactivation

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acid had resulted in the formation of a chemically reac-tive, transient metabolite(s) which rapidly bound to micro-somal proteins. The nADPH-dependent covalent binding in human liver microsomes was twofold greater than the value observed in microsomes from the dog and 3.8-fold greater than that evident in microsomes from the rat. In addition, the levels of covalent binding of fenclozic seen in nADPH-supplemented microsomes and hepatocytes were comparable to values obtained with the positive control drug diclofenac, which could be an indication of potential to cause hepatotoxicity in vivo. The covalent binding val-ues were not corrected for possible differences in metabolic turnover between species, as has been proposed previously (Thompson et al. 2012), since the in vitro metabolism stud-ies undertaken with fenclozic acid in microsomes supple-mented with CyP cofactors provided no evidence of turno-ver other than to reactive intermediates.

If the degree of covalent binding is taken as an indication of a drug’s propensity to initiate hepatotoxicity, as has been proposed by numerous investigators (Bauman et al. 2009; Lu et al. 2006; nakayama et al. 2009; Obach et al. 2008; Thompson et al. 2011, 2012; Usui et al. 2009), then these data raise the possibility that that there could be quantitative differences between the potential of fenclozic acid to elicit hepatotoxicity in humans and in non-clinical species via reactive metabolite mediated processes. If such differences were to occur in vivo, they might perhaps help explain why liver toxicity was seen in humans but not when fenclozic acid was evaluated in the numerous in vivo safety studies that were undertaken in animals. However, it is important to note that while the level of covalent binding of fenclo-zic acid that was observed in human hepatocytes was two-fold lower than that observed in human liver microsomes, the levels of covalent binding in rat and dog hepatocytes were 14-fold and threefold greater than was seen in the cor-responding liver microsomes, respectively. Consequently, the extent of covalent binding of fenclozic acid to human hepatocyte proteins was lower than its extent of covalent binding to dog or rat hepatocyte proteins. Inconsistencies between microsome and hepatocyte data are not uncom-mon and have been observed by many previous investiga-tors. In particular, nakayama et al. found that the covalent binding data for 42 drugs in human liver microsomes and hepatocytes had a weak correlation (r = 0.25) (nakayama et al. 2009). Indeed, it is often the case that apparent intrin-sic clearance of a drug by hepatocytes is lower than that seen for liver microsomes (Lu et al. 2006), due to intact hepatocytes having a plasma membrane barrier between the drug and metabolizing enzymes. Therefore, the metabolic fate of fenclozic acid in hepatocytes could be dependent on its uptake or diffusion into the cells, which limits the rate of bioactivation and covalent binding. It is also possible that differential uptake rates of the drug into the hepatocyte

preparations we have used could account for the inter-spe-cies differences in covalent binding that were observed.

It is also notable that fenclozic acid (see structure 1 for its chemical structure) contains a number of structural motifs that might provide clues as to the likely nature of the toxicophore. Thus, the aromatic chlorine substituent pro-vides an obvious potential for metabolic activation, while the thiazole structure represents a known structural alert for reactive metabolites. A standard, and well tried, approach to trying to understand the mechanism(s) underlying covalent modification of proteins by chemically reactive metabolites is the use of so-called trapping experiments (Bauman et al. 2009; Obach et al. 2008). Our attempts to characterize the putative reactive intermediate(s) formed by the metabolism of fenclozic acid by chemical trapping were unsuccessful, although we observed reductions in levels of covalent binding when microsomal incubations were undertaken in the presence of various ‘trapping’ mol-ecules. The most marked decrease in covalent binding to microsomal protein was evident in the presence of either GsH or cysteine, both of which are thiol-containing soft nucleophiles. This suggests that the reactive intermediate is likely to be a soft electrophile and raises the possibility that sulphur atoms could play an important role in the mecha-nism of the nADPH-dependent covalent binding. It is also noteworthy that neither semicarbazide nor lysine, which are hard nucleophile trapping agents, inhibited nADPH-dependent covalent binding. These results indicate that the reactive intermediate is unlikely to be a hard electrophile, such as an iminium ion or reactive aldehyde (Argoti et al. 2005; Goldszer et al. 1981). Partial inhibition of nADPH-dependent covalent binding was achieved in the presence of the hard nucleophile trapping agents methoxylamine and cyanide. However, in view of the lack of inhibitory effect of semicarbazide and lysine, it appears most likely that the reduction in covalent binding observed in the presence of methoxylamine and cyanide is attributable to inhibition of the metabolism of fenclozic acid, rather than inhibition of binding of a reactive intermediate to protein.

Although we were unable to detect any evidence of in vitro formation of stable oxidative metabolites of fenclozic acid [despite prior evidence from in vivo studies of exten-sive metabolism by such pathways (Foulkes 1970)], two Phase 2 metabolites were detected. Co-incubation of liver microsomes with UDPGA demonstrated conjugation of [14C]-fenclozic acid with glucuronic acid to form an acyl glucuronide metabolite. The fenclozic acid acyl glucuronide was previously identified by Foulkes and by Bradbury in rats, dogs and monkeys in vivo (Bradbury et al. 1981; Foulkes 1970). It has been proposed that acyl glucuron-ide metabolites of other drugs are potential toxicophores (Boelsterli 2002) and this possibility has been highlighted in the recent FDA Guidance on metabolites in safety

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Testing (FDA 2008). However, since we observed no detectable covalent binding when radiolabelled fenclozic acid was incubated with liver microsomes in the presence of UDPGA, it is reasonable to infer that acyl glucuronide formation is not a route of bioactivation of fenclozic acid but rather is a detoxification pathway. In addition, a taurine conjugate of [14C]-fenclozic acid was formed in the hepato-cyte incubations. The production of amino acid conjugates is a common metabolic pathway for xenobiotic carboxylic acids (Knights et al. 2007), and a glutamine conjugate of fenclozic acid was previously identified in vivo as a major metabolic pathway in man (Platt 1971). moreover, dose-dependent formation of fenclozic acid–taurine conjugates was noted in the rat (Bradbury et al. 1981). The identifica-tion of the fenclozic acid–taurine conjugate in vitro implies that an acyl-CoA intermediate is formed in the mitochon-dria, since this is a necessary step in amino acid conjuga-tion. Acyl-CoA thioesters can covalently modify proteins in a similar manner to acyl glucuronides and so may be of toxicological relevance (Boelsterli 2002). However, it remains unclear whether such metabolites play a causative role in human adverse drug reactions and, as mentioned above, we were unable to obtain evidence of mitochondrial toxicity caused by fenclozic acid.

Overall, our in vitro investigations indicate biotransfor-mation of fenclozic acid to a reactive metabolite as a poten-tial cause for the liver injury observed in humans treated with relatively high doses of the drug. The investigations also highlight the valuable insights that can be obtained when studies of biotransformation are combined with evaluation of toxicity and of covalent binding to proteins. There are several additional lines of research which would complement the present study. It will be important to iden-tify the enzyme (or enzymes) that catalyse the bioactivation of fenclozic acid, to characterize the reactive intermediate and also to explore the nature of the target proteins. since detectable metabolite adducts could not be trapped and characterized using conventional LC–ms/ms technology, it is possible that the adducted site(s) on target proteins are chemically fragile. The uncharacterized metabolite that was observed in human hepatocyte incubations also requires consideration; its absence in the presence of ABT suggests that its formation is mediated by Phase 1 enzymes and raises the possibility that it might be involved in the mechanism of covalent binding. It will also be important to search for evidence of subtle biological perturbations caused by fenclozic acid and arising from reactive metabo-lite formation in cell systems in vitro and in animals in vivo that could have toxicological relevance in humans, such as induction of cell stress response pathways. such studies are warranted since fenclozic acid caused relatively frequent liver toxicity in humans when administered at a dose of 400 mg/day, but not when administered at 200 mg/day, and

exhibited no evidence of overt hepatotoxicity in numerous preclinical test species. Improved insights into its mecha-nism of human liver toxicity may therefore help advance our ability to assess the safety of new investigational drugs prior to their introduction into humans.

Acknowledgments We would like to thank Prof. Dr. Bruno stieger for provision of hBseP expressing baculovirus stocks; Johan e. Palm, DmPK imeD, AstraZeneca r&D mölndal, for provision of the hmrP2 encoding plasmid; and the reagents and Assay Develop-ment team, Discovery sciences, AstraZeneca r&D Alderley Park, for preparation of membrane vesicles. Pavandeep rai is acknowledged for help with the assessment of mitochondrial toxicity. Gillian smith is acknowledged for completion of the CyP time-dependent inhibi-tion assay.

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