A Correlation Between the in Vitro Drug Toxicity of Drugs to Cell Lines That Express Human P450s and Their Propensity to Cause Liver Injury in Humans

Home , Channel opener, Cinchophen, Ketotifen, Rimonabant, Troglitazone

toxicological sciences 137(1), 189–211 2014 doi:10.1093/toxsci/kft223 Advance Access publication October 1, 2013

A Correlation Between the In Vitro Drug Toxicity of Drugs to Cell Lines That Express Human P450s and Their Propensity to Cause Liver Injury in Humans

Frida Gustafsson,*,1 Alison J. Foster,* Sunil Sarda,† Matthew H. Bridgland-Taylor,† and J. Gerry Kenna2 *Global Safety Assessment and †Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom

1To whom correspondence should be addressed at 23S37-69, Molecular Toxicology, Safety Assessment, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom. Fax: +44(0)1625 513779. E-mail: [email protected]. 2Present address: Safety Science Consultant, Macclesfield, United Kingdom.

Received June 25, 2013; accepted September 12, 2013

drugs cause acute or chronic damage to the liver, which results Drug toxicity to T-antigen–immortalized human liver epi- in symptomatic liver injury but not liver failure, and some thelial (THLE) cells stably transfected with plasmid vectors drugs cause asymptomatic abnormalities in serum clinical that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, chemistry (most notably elevated concentrations of the enzyme or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% alanine aminotransferase (ALT), which is released from dam- dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the aged hepatocytes). DILI is an important cause of terminated culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3- development or failed registration of otherwise promising new carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduc- therapies, of withdrawal of licensed drugs, and of cautionary tion, was used to evaluate the cytotoxicity of 103 drugs after 24 h. and restrictive drug labeling (Kaplowitz and DeLeve, 2007). Twenty-two drugs caused cytotoxicity to THLE-Null cells, with For many drugs that cause DILI, the toxicity is not evident from

EC50 ≤ 200μM; 21 of these drugs (95%) have been reported to cause conventional preclinical safety studies undertaken in experi- human liver injury. Eleven drugs exhibited lower EC50 values in mental animals (Roth and Ganey, 2011) and occurs only infre- cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE- quently in humans, in certain susceptible drug-treated patients; Null cells; 10 of these drugs (91%) caused human liver injury. An this is termed “idiosyncratic DILI” (Kaplowitz and DeLeve, additional 8 drugs, all of which caused human liver injury, exhib- 2007). ited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not The mechanisms that underlie DILI are complex and include bovine serum. Fourteen of the drugs that exhibited potentiated both drug-related and patient-related processes. Drug-related THLE-3A4 cell toxicity are known to be metabolized by P450s to processes determine whether or not individual drugs have the reactive intermediates. These drugs included troglitazone, which propensity to cause DILI in vivo, in preclinical test species or in was shown to undergo metabolic bioactivation and covalent bind- humans. These arise via interaction of drugs and/or their metab- ing to proteins in THLE-3A4 cells. A single drug (rimonabant) olites with biochemical processes within liver cells that initi- exhibited marked THLE cell toxicity but did not cause human ate and propagate tissue injury, or trigger protective responses. liver injury; this drug had very low reported plasma exposure. Notable examples include drug metabolism within hepatocytes These results indicate that evaluation of toxicity to THLE-Null to chemically reactive intermediates, impairment of mitochon- and THLE-3A4 cell lines during drug discovery may aid selec- drial function, inhibition of hepatic transporters that mediate tion of drugs with reduced propensity to cause drug-induced liver transport of cytotoxic bile salts from hepatocytes into bile injury and that consideration of human exposure is required to enhance data interpretation. (especially the bile salt export pump [BSEP]), initiation of oxi- Key Words: drug hepatotoxicity; in vitro toxicity; THLE cell dative stress, and activation of inflammatory processes and cell lines; CYP3A4-dependent bioactivation. stress responses (Dawson et al., 2012; Dykens and Will, 2007; Greer et al., 2010; Thompson et al., 2011). Patient-related processes play a major role in influencing whether or not drugs cause DILI in individual patients. These include genotype, Many hundreds of licensed drugs may cause drug-induced underlying disease state, comedications that cause drug-drug liver injury (DILI) in humans, and the most concerning clinical interactions, adaptive immune responsiveness, gender, age, eth- outcome is life-threatening liver failure. However, numerous nicity, and other demographic factors (Pachkoria et al., 2007).

Many drug-related processes that initiate DILI can be eval- 8 and 40. Pooled cryopreserved human hepatocytes were obtained from Celsis/ uated using in vitro approaches. If used during drug discov- In Vitro Technologies (Brussels, Belgium). The following reagents were ery, when chemical choice is available, such methods could obtained from Invitrogen Ltd (Paisley, United Kingdom): Basal Pasadena Foundation for Medical Research Medium #4 (PMFR4); GlutaMAX; fotal aid design and selection of drugs with reduced propensity to bovine serum (FBS; nonheat inactivated); Hanks balance salt solution with- cause DILI (Greer et al., 2010; Thompson et al., 2011). Models out magnesium, calcium, and phenol red (HBSS−/−); Bis-Tris NuPAGE gels; that express cytochrome P450 (P450) activity are likely to be PVDF mini and regular iBlot stack gels; Novex sharp prestain molecular especially useful, due to the role played by chemically reactive weight markers; NuPAGE LDS sample buffer, sample reducing agent, and intermediates in DILI caused by numerous drugs and the impor- antioxidant. ECL plus, hyperfilm, and tracker tape were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). CellTiter 96 tance of P450 enzymes in drug bioactivation (Park et al., 2011). AQueous Non-Radioactive Cell Proliferation Assay reagent (3-(4,5-dimethyl- Because marked interspecies differences in P450-dependent thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium drug metabolism may arise, systems that express human P450s [MTS] reagent) was obtained from Promega UK Ltd (Southampton, United could be of greatest value. Freshly isolated human hepatocytes Kingdom). Pierce BCA protein assay was obtained from Thermo Fisher express high P450 activities but are not ideally suited to high- Scientific Inc (Rockford, Illinois). BioCoat collagen I flasks and BioCoat collagen I 96well and 384well plates were obtained from BD Bioscience (Bedford, volume screening due to their limited availability and high cost. United Kingdom). All pharmaceutical drugs were provided by AstraZeneca Furthermore, marked interdonor variability in P450 expression Compound Management, AstraZeneca R&D, Macclesfield, United Kingdom, may arise, and levels of expression of many P450s are down- apart from [14C]-troglitazone, which was obtained from Isotope Chemistry, regulated when hepatocytes are cultured using conditions used AstraZeneca R&D and had a purity > 98% and a specific activity of 1.42 in standard toxicity tests. kBq/nmol. SDS was obtained from VWR (Lutterworth, Leicester, United Kingdom). Ultima Gold liquid scintillation cocktail was obtained from Perkin An alternative approach is to utilize human liver–derived Elmer (Waltham, Massachusetts). HPLC-grade acetonitrile, methanol, and cell line constructs that express human P450s. These can be ammonium acetate were obtained from Fisher-Scientific (Loughborough, generated in several ways. An especially promising approach United Kingdom). The antisera used in Western blotting experiments were comprises transfection of SV40 large T-antigen–immortal- obtained from Abcam (Cambridge, United Kingdom) (product code numbers: ized human liver epithelial (THLE) cells (Pfeifer et al., 1993) mouse anti-P450 1A2, 56073; rabbit anti-P450 3A4, 22704; rabbit anti-P450 2C, 78100) or from Millipore (rabbit anti-P450 2D6, AB10081; mouse anti- with plasmid vectors that encode different human P450s (Bort glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CB1001). Horseradish et al., 1999; Macé et al., 1997), which has yielded cell lines that peroxidase (HRP)–conjugated secondary antisera (HRP-anti-mouse IgG and exhibit stable P450 expression and excellent cell culture prop- HRP-anti-rabbit IgG) were provided by Cell Signaling Technology Inc via New erties and have been used to explore the role played by P450s in England BioLabs (Ipswich, Massachusetts). All other reagents were obtained drug metabolism (eg, Coulet et al., 1998; Molden et al., 2000), from Sigma-Aldrich Company Ltd (Poole, Dorset, United Kingdom). metabolite-mediated genotoxicity (eg, Barceló et al., 1998; Pfeifer et al., 1993), and cell toxicity (Dambach et al., 2005; Analysis of P450 Expression in THLE Cell Lines by Western Blotting Foster et al., 2013). Recently, highly sensitive and specific dis- Collagen-coated 175-cm3 flasks were seeded with THLE-1A2, 2C9, 2C19, 2D6, 3A4, or Null cells at a density of 1 million cells per flask in 30 ml PMFR crimination between 27 drugs with marked or high propensity (+) medium (basal PMFR4 medium plus 2mM GlutaMAX, 10 μg/ml recombi- to cause idiosyncratic adverse reactions (primarily DILI) and 9 nant human insulin, 5.5 μg/ml human transferrin, 5 ng/ml sodium selenite, 1nM drugs with low propensity to cause such adverse reactions was hydrocortisone, 0.5 ng/ml epidermal growth factor, 35 μg/ml bovine pituitary observed when drug toxicity to THLE-3A4 cells or THLE-Null extract, 0.33nM retinoic acid, 3% FBS, 150 μg/ml G418). The flasks were incubated in a humidified chamber at 37°C under 5% CO for 3–4 days to enable (which do not express P450s) was assessed alongside several 2 the cells to adhere and grow to form confluent monolayers. The medium was other in vitro endpoints (Thompson et al., 2012). The additional removed, and the cells were washed with HBSS−/− and detached by addition of endpoints were mitochondrial toxicity to HepG2 cells, inhibi- 1.5 ml 0.025% trypsin per flask. The detached cells were resuspended in PMFR tion of the activities of BSEP and multidrug resistance protein (+) medium, sedimented by centrifugation at 300 × g for 3 min at room tem- type 2 (Mrp2), and dose-adjusted covalent binding (CVB) of perature, resuspended in radioimmunoprecipitation assay buffer at 7 million radiolabeled drugs to human hepatocyte proteins. cells/ml (as determined using a hemocytometer), and stored frozen at –20°C prior to Western blotting. Twenty-five micrograms protein per lane (determined To gain further insight into the value of THLE toxicity test- using the Pierce BCA protein assay, with bovine serum albumin as standard) ing for assessment of human DILI propensity, we have inves- was loaded onto Bis-Tris NuPAGE gels, which were run at 200 V for 1 h using tigated the toxicity of > 100 marketed drugs (83 of which NuPAGE equipment and reagents according to the NuPAGE Technical Guide. caused DILI in humans) to THLE cell lines that selectively Proteins were transferred onto nitrocellulose (THLE-1A2, 3A4, and 2D6) or express CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or PVDF (THLE-2C9 and 2C19) membranes using an iBlot (Invitrogen). The membranes were blocked by incubation for 1 h in 5% nonfat milk/Tris-buffered no P450s. saline (TBS) Tween 0.1% (TBST) and then incubated overnight at 4°C with primary antisera, which were diluted in 5% nonfat milk and TBS (anti-P450 1A2, 1:500; anti-P450 3A4, 1:2000; anti-P450 2D6, 1:2500; anti-P450 2C, Materials and Methods 1:750). To verify that equivalent amounts of cell protein had been loaded in each lane, the anti-P450 antisera were combined with anti-GAPDH antiserum Chemicals and Reagents (1:20 000). Membranes were then washed with TBST, were incubated for 1 h THLE cells expressing CYP1A2, 2C9, 2C19, 2D6, or 3A4, and THLE- at room temperature with appropriate HRP-conjugated secondary antisera Null cells, were obtained from Nestec Ltd (Lausanne, Switzerland) under an diluted in 5% nonfat milk and TBST (anti-mouse IgG diluted 1:2000; anti-rab- evaluation license. All experiments were conducted on cells between passages bit IgG diluted 1:2000), and after final washing were visualized by enhanced Drug Toxicity to THLE-CYP Cell Lines 191

chemiluminescence using ECL plus, as specified by the manufacturer. Pooled individual experiments were carried out in triplicate or duplicate. Because the cryopreserved human hepatocytes were used as controls. quantity of formazan product measured by the absorbance at 490 nm in the MTS assay is directly proportional to the number of living cells in culture, a reduced MTS signal can be due to a decrease in cell proliferation and cell Assessment of THLE Cell Cytotoxicity toxicity. Inspection of the dose-response curves, manual inspection of the cells In the majority of experiments, nonautomated manual methods were used by light microscopy, and evaluation of enzyme leakage using the lactate dehy- for compound dilutions, THLE cell culture, and assessment of cell toxicity. drogenase assay are therefore advised when testing compounds that affect the THLE-1A2, 2C9, 2C19, 2D6, 3A4, or Null cells were seeded into collagen- rate of cell proliferation. coated 96-well tissue culture plates at a density of 15 000 cells per well in An EC50 value of ≤ 200μM was used to identify drugs that caused toxicity 200 μl PMFR (+) medium, and the plates were incubated for 24 h at 37°C in in the assay, whereas an EC50 value of ≤ 100μM was used to indicate drugs that 5% CO2 and 95% humidity to allow the cells to adhere and form monolayers. exhibited marked toxicity. Potentiated toxicity to THLE-3A4 cells versus THLE- This cell seeding density was chosen because it resulted in a confluent cell Null cells was defined as a ratio of THLE-Null EC50/THLE-3A4 EC50 ≥ 1.4. monolayer, as assessed by light microscopy. The medium was then replaced with 200 μl of fresh PMFR (−) medium (basal PMFR (+) medium without Metabolism of P450 Probe Substrates by THLE Cells insulin, transferrin, selenium, or FBS), which contained the test compound and dimethyl sulfoxide (DMSO) vehicle. All except one of the test compounds THLE cells were seeded into collagen-coated 6-well plates at 450 000 were solubilized initially in 100% DMSO, at a concentration of 30 or 150mM, cells per well in 2.5 ml PMFR (+) medium and incubated for 24 h at 37°C in 5% CO and 95% humidity. Six-well plates were used in order to increase and compound solubility was verified by visual inspection. The appropriate 2 compound concentrations (between 0 and 500μM in a final concentration of the volume of culture media available for subsequent analysis. At the end of DMSO of 0.2 or 1% (vol/vol)) were achieved by serial dilution of stock solu- the incubation period, the medium was removed and each well was dosed tions into cell culture media, and cells were incubated with the test compounds with 2.5 ml PMFR (−) medium containing 20μM verapamil in 0.1% DMSO. After further incubation for 2 h at 37°C in 5% CO2 and 95% humidity, 200- for 24 or 48 h at 37°C in 5% CO2 and 95% humidity. One of the test compounds (streptomycin) was insoluble in 100% DMSO and so was dissolved from an µl aliquots were removed and were mixed with equal volumes of cold ace- aqueous stock solution into culture medium that contained 0.2% DMSO. To tonitrile and then stored at –80°C prior to analysis by HPLC. HPLC analyses assess toxicity, the media were then removed from each well and replaced with were undertaken on an Accela HPLC system consisting of a binary solvent 120 μl of fresh PMFR (−) medium containing MTS reagent at a dilution of manager, column oven, sample manager, and diode array detector (Waters, Millford, Massachusetts), using a Synergi Fusion 100 × 2 mm (2.5 μm) column 1:6. Following further incubation at 37°C under 5% CO2 for 1.5–2 h, absorbance at 492 nm was quantified using a Wallac EnVision spectrophotometer (Phenomenex, Macclesfield, United Kingdom). The mobile phases were 0.05% (Perkin Elmer). The CellTiter 96 AQueous Assay is composed of solutions of formic acid in water (A) and 0.05% formic acid in acetonitrile (B). The elution the tetrazolium compound MTS (inner salt) and the electron coupling reagent profile was as follows: linear gradient, 10%–90% B, 0.00–14.00 min; isocratic phenazine methosulfate. In metabolically active cells, MTS is reduced by mito- hold, 90% B, 14.01–18.00 min; reequilibration, 10% B, 18.01–20.00 min. The chondrial dehydrogenase enzymes to a formazan product that is soluble in tis- flow rate was 0.4 ml/min, and the column oven temperature was 30°C. The sue culture medium and absorbs light at 490 nm. The absorbance at 490 nm is sample injection volume was 20 µl. Ultraviolet spectra were acquired from directly proportional to the number of living cells; therefore, this assay enables 200 to 350 nm. assessment of cell toxicity. It is a sensitive assay that requires fewer steps than To investigate the effect of DMSO concentration on P450 probe substrate other procedures that use tetrazolium compounds, such as 3-[4,5-dimethylthia- clearance, THLE cells that had been seeded and incubated for 24 h postseed- zol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT). Toxicity studies undertaken ing in collagen-coated 96-well plates (as described for cell toxicity testing) with rimonabant in THLE-3A4 cells have shown a close correlation between were dosed with a probe substrate cocktail that contained 1μM phenacetin, decreased cellular MTS signal and reduced ATP content (Foster et al., 2013). dextromethorphan, and verapamil and 0.05 or 1% DMSO (vol/vol). The cells Toxicity of 103 drugs to THLE-Null and THLE-3A4 cell lines was inves- were incubated with the substrate cocktail with gentle mixing for 5 to 240 min at 37°C in 5% CO and 95% humidity. Aliquots (140 l) of the cell incubates tigated using a robotic platforms (Hamilton ML STAR [Hamilton Robotics]) 2 μ for compound dilution, sterile dosing, and toxicity evaluation, as described were then mixed with equal volumes of ice-cold acetonitrile and were stored elsewhere (Thompson et al., 2012). The most significant alterations from the frozen at –80°C prior to analysis by HPLC. manual method were the following: adoption of 384-well plates rather than 96-well plates (which necessitated seeding of cells at 4000 cells/well to ensure Metabolism of P450 Probe Substrates by Human Hepatocytes subsequent cell confluency); use of robotics for all compound dilutions and Loss of parent compound. Cryopreserved human hepatocytes were liquid-handling steps; use of 1% DMSO vehicle to enhance compound solubil- quickly thawed in a 37°C water bath (1.5 min) and then resuspended by careful ity; and dilution of test compounds directly into PMFR4 cell growth medium addition of 25 ml in Leibovitz buffer to 1 vial of cells. The cell suspension was (which contained insulin, transferrin, selenium, and FBS). equally divided into appropriate tubes, and centrifuged at 300 × g for 5 min, and The percentage of viable cells relative to the vehicle-treated control wells the cells were resuspended in prewarmed (37°C) Leibovitz buffer to a density was determined for each well, and EC values, which represent the concentra- 6 50 of 1 × 10 cells/ml. The cell suspension was transferred to 1-ml deep well plates, tions of the compounds that reduced MTS signal by 50%, were calculated with at 250 µl/well, and preincubated for 5 min at 37°C on a heated plate shaker. nonlinear regression for a sigmoidal dose response using the 4-parameter logis- Probe substrate cocktail (2.5 μl) that contained 100μM verapamil, phenacetin, tic equation in GraphPad Prism version 4.03 (GraphPad Software, San Diego, and dextromethorphan in 10% DMSO (final substrate concentration μ1 M, in California) or Origin version 7.5 (OriginLab, Northampton, Massachusetts): 0.1% DMSO) was added. After further incubation at 37°C on the heated plate shaker, 200-µl aliquots were removed after 0, 3, 6, 9, 12, and 15 min. These maxm− in were added immediately to 200 µl ice-cold methanol, to stop the reaction, and y = min+ -Hillslope centrifuged at 1800 × g for 15 min prior to analysis by HPLC. Protein determi-  x  (1) 1+  nations were undertaken using the Pierce BCA protein assay (Thermo Fisher EC or IC  50 50 Scientific Inc). The rate of loss of parent compound was calculated as the initial linear rate of clearance of the respective probe substrate over time (expressed where y is the MTS signal; min, the bottom of the curve (set to 0%); max, as μl/min/mg protein). the top of the curve of 100% (ie, the MTS signal of the vehicle control); x,

the compound concentration; and EC50, half-maximal effective concentration Appearance of metabolite. Cryopreserved human hepatocytes, characterizing the slope of the curve at its midpoint. All incubations within THLE-2C9, THLE-3A4, or THLE-Null cells, were incubated as above in 192 Gustafsson et al. suspension culture at 1 × 106 cells/ml, with a probe substrate cocktail contain- onto a fused-core C18 reversed-phase HALO column (150 × 4.6 mm, 2.7 μm, ing 2μM diclofenac and 3μM midazolam for 0, 5, 10, 15, 20, or 25 min, or Advanced Materials Technologies, Wilmington, Delaware) with a 2-mm with 10μM verapamil for 0, 3, 6, 12, or 15 min. At the indicated time points, (C18) precolumn guard filter (Hichrom Ltd, Reading, United Kingdom), all aliquots (60 or 200 μl) were removed from the incubations and were mixed maintained at 40°C. Elution was over 14 min, using Agilent 1200SL series with ice-cold methanol (20 or 200 μl) to stop the reactions. The samples were binary pumps (Agilent Technologies, Stockport, United Kingdom) at a flow centrifuged at 1800 × g for 15 min prior to analysis of the supernatants for rate of 0.5 ml/min. The mobile phases were 10mM ammonium acetate in H2O the respective isoform-specific metabolites via mass spectrometry LCMS. Data (unadjusted pH) (A) and 100% acetonitrile (B). The elution profile was as for the 1-OH-midazolam and 4-OH diclofenac metabolites were acquired on follows: 10% B, 0–2 min; linear gradient, 10%–95% B, 2.01–10 min; iso- an API4000 instrument (AB Sciex Instrument) with a HyPurity 50 × 2.1 mm, cratic hold, 95% B, 10.01–12 min; reequilibration, 10% B, 12.01–14 min. 5 μm column. The mobile phases were 0.1% formic acid, 5% acetonitrile in Retention times for troglitazone and troglitazone sulfate (which were available

H2O (A), and 0.1% formic acid in 95% acetonitrile (B). The elution profile as authentic standards) were typically 7.1 and 5.8 min, respectively, on this was as follows: linear gradient, 2%–3% B, 0.00–0.80 min; 3%–31% B, 0.80– chromatographic system. Mass spectrometric analyses were conducted using 3.10 min; 31%–98% B, 3.10–4.50 min; isocratic hold, 98% B, 4.50–5.00 min; a ThermoFinnigan LTQ linear ion trap instrument (Thermo Fisher Scientific, reequilibration, 2% B, 5.10–6.00 min. The flow rate was 0.75 ml/min, and the Bremen, Germany) fitted with an IonMax electrospray ionization source oper- column oven temperature was 40°C. The sample injection volume was 10 µl. ating in negative ion mode under, with parameters optimized for troglitazone The MS/MS ion transitions ranged from m/z 342.0 to 324 (positive ion mode) parent compound. The initial full MS scan ranged from m/z 400 to 600 in for 1′-hydroxymidazolam and from m/z 309.9 to 265.9 (negative ion mode) for negative ion mode. This was followed by an MS2 product ion scan of m/z 440.3 4′-hydroxydiclofenac. Data for the norverapamil metabolite were acquired on (troglitazone) and a further MS2 product ion scan of m/z 520.3 (troglitazone a Waters Platinum triple quadrapole with a Phenomenex max-rp 50 × 2.1 mm, sulfate). Collisionally induced fragmentation was conducted with an isolation 5 μm column. The mobile phases were 0.1% formic acid in water (A) and 0.1% width of 2 mass units (mu), a collision energy normalized to 35% for par- formic acid in methanol (B). The elution profile was as follows: linear gradient, ent compound and an activation time of 30 ms. All of the HPLC eluent was 5%–95% B, 0.00–3.00 min; isocratic hold, 95% B, 3.00–4.00 min; reequilibra- introduced to the mass spectrometer to increase detection sensitivity but was tion, 5% B, 4.01–5.00 min. The flow rate was 0.6 ml/min, and the column oven diverted away from the mass spectrometer to waste from 0 to 3 min and 12 to temperature was 60°C. The sample injection volume was 20 µl. The MS/MS 14 min, respectively. transition for detection of the norverapamil metabolite was m/z 441.3–165.4 Structural identification of troglitazone metabolites was assessed by quali- (positive ion mode). The standard curves were linear up to 500nM. Protein tative and quantitative analyses of the chromatographic profiles obtained via determinations were undertaken using the Pierce BCA protein assay. The rate [14C]-detection and consecutive fragmentation mass spectrometry (MSn). of specific metabolite formation was calculated as the initial linear rate of the Analysis of metabolites by HPLC-radioprofiling-MSn was undertaken using formation of the respective metabolite over time (expressed as pmol/min/mg the same LC-MS configuration as above, except that the injection volume was protein). 90 μl, the column temperature was set to 30°C, and elution was achieved over 60 min at a flow rate of 1 ml/min. The mobile phases were 10mM ammonium 14 acetate in H O containing 0.1% formic acid (A) and 0.1% (vol/vol) formic acid CVB to Proteins of [ C]-Troglitazone in THLE Cells 2 in acetonitrile (B). The elution profile was as follows: 5% B, 0–5 min; 5%–35% THLE-3A4 or THLE-Null cells were seeded into collagen-coated 25-cm2 B, 5–8 min; 35%–60% B, 8–50 min; 60%–95% B, 50–52 min; isocratic hold, tissue culture flasks at a density of 1.2 × 106 cells per flask in 5 ml PMFR (+) 95% B, 52–57 min; reequilibration, 5% B, 57–60 min. The postcolumn elu- medium. Flasks of size 25 cm2 were required to provide sufficient volumes of ent was split 1:5 vol/vol between the mass spectrometer and radiodetector, cells for subsequent detection of CVB. The flasks were incubated at 37°C for respectively, using a QuickSplit binary fixed-flow splitter (Analytical Scientific 24 h in 5% CO and 95% humidity, and the medium was removed and was 2 Instruments, El Sobrante, California), to enable simultaneous MS and radio- replaced with fresh PMFR (−) medium containing 10μM [14C]-troglitazone. detection. The HPLC eluent was diverted away from the mass spectrometer The cells were incubated for a further 24 h at 37°C in 5% CO and 95% humid- 2 to waste from 0 to 1 min and 51 to 60 min, respectively. Radiochemical detec- ity, washed with 2 × 5 ml HBSS−/−, scraped into 1 ml HBSS−/−, and then pel- tion was performed on-line using a LabLogic β-Ram 3 detector (LabLogic leted by centrifugation at 9000 × g for 1.5 min, at 4°C. To determine CVB, Systems Ltd, Sheffield, United Kingdom) with a 500-µl liquid flow cell the supernatants were discarded and 300 μl ice-cold acetonitrile was added and Ultima Flo-M scintillation cocktail (Packard Instruments, Pangbourne, to the pellets. The acetonitrile extracts were harvested onto filter paper using United Kingdom) running at 1.6 ml/min (ca. 2:1 vol/vol scintillant/eluent). a Brandel Cell Harvester ML-48TI (Brandel, Gaithersburg, Maryland) and Radiolabeled components were assumed to have the same specific activity as washed with methanol (80% (vol/vol) in water). The filter papers were punched the parent drug. Mass spectrometric acquisition data scans were performed as a out individually and transferred to 20-ml glass scintillation vials, to which 1 ml single segment throughout the analyses, with scan 1 being the full scan across of 5% SDS (wt/vol) in water was added. The vials were incubated overnight at a mass range of m/z 400–800, followed by multistage fragmentation studies 50°C. Aliquots (25 l) of the resulting solutions were analyzed for protein con- μ (MS2/3) conducted in collisional-induced dissociation (CID) mode in the linear tent using the Pierce BCA protein assay, with bovine serum albumin as stand- ion trap. Scan 2 was, therefore, typically conducted in data-dependent mode, ard. Liquid scintillation cocktail (10 ml) was added to the remaining solutions, with the most intense of the prelisted expected masses isolated using a 2.0-mu and total radioactivity was determined using a Beckman LS6500 MultiPurpose window, and collision energies were normalized to 25% (CID) for troglitazone Liquid Scintillation Counter. Each sample was counted for 5 min or until the (m/z 440). The subsequent scan 3 was typically a data-dependent scan derived variance was ≤ 1%, using a protocol with quench correction. The values were from the most intense ion afforded in scan 2, again isolated with a 2.0-mu win- corrected for background activity, and the levels of nonextractable radioactivity dow and collision energies normalized to 35%. were expressed as picomole equivalents per milligram protein (pmol eq/mg Other multistage fragmentation (MSn) experiments involved targeted protein), taking account of the protein concentration of each sample and the (defined) scans of selected precursor ions rather than data-dependent listed specific activity of the radiolabeled troglitazone. All incubations in individual ions, irrespective of the previous full scan data sets. These targeted MSn data experiments were performed in duplicate. sets used the same scan 2/3 settings described above. The radiometric data were collected and analyzed using LAURA version 14 Metabolism of [ C]-Troglitazone by THLE Cells 3.4.1.10e (Lablogic Systems Ltd, Sheffield, United Kingdom), whereas MSn For metabolite quantification by LC-MS/MS, 10-μl aliquots of culture data were collected using Xcalibur version 2.0.7 (Thermo Fisher Scientific, media obtained following incubation of THLE-3A4 and THLE-Null cells Waltham, Massachusetts). Components were identified as being derived from with 10μM [14C]-troglitazone for 24 h were injected using an autosampler [14C]-troglitazone if they displayed retention times corresponding with radi- (CTC Analytics HTC PAL, Presearch Ltd, Basingstoke, United Kingdom) olabeled peaks and subsequently demonstrated the characteristic deprotonated Drug Toxicity to THLE-CYP Cell Lines 193 molecular ion isotopic patterns and fragmentation components compatible with Data on the prescribed doses of the drugs, their extents of elements of the parent troglitazone molecule. plasma protein binding, their maximum reported plasma con- Statistical analysis. Statistical analyses were performed using GraphPad centration, and their estimated maximum unbound plasma Prism paired t test or ANOVA. concentrations (Cmax,u) were also extracted from the scientific literature and are summarized in Supplementary Table 1. Drugs in all 5 DILI categories exhibited similarly broad ranges of Results Cmax,u values (see Supplementary Figure 1). DILI Categorization of Tested Drugs P450 Enzyme Expression and Activity in THLE Cell Lines Currently, there is no agreed consensus approach for clas- Expression of P450 proteins in the THLE cell lines was sification of DILI in humans, which is suitable for use when investigated by Western blotting analysis, using specific anti- evaluating potential in vitro assays that might aid selection of sera. This revealed no detectable expression of P450 enzymes drugs with low propensity to cause clinically concerning DILI 1A2, 2C, 2D6, or 3A4 in the THLE Null cell line (Fig. 1). during drug discovery. We, therefore, used information on Selective expression of the P450 enzymes 1A2, 2D6, and DILI in humans caused by the 104 tested drugs, which was 3A4 was observed in the corresponding cell lines. The anti- extracted from the scientific literature and from U.S. FDA– P450 2C antiserum, which was unable to discriminate between approved product labels, to assign each drug to 1 of 5 DILI P450s 2C19 and 2C9, recognized proteins present in both the concern categories. These are summarized in Table 1, as are the THLE-2C19 and THLE-2C9 cell lines but not in the other cell pharmacology classes of the drugs and their patterns of human lines. Apparent levels of expression of CYP3A4 were similar in liver injury (hepatocellular, cholestatic, or mixed). Category 1 THLE-3A4 cell lysates and pooled human hepatocyte lysates, drugs (n = 24) caused cases of fatal liver failure, which had whereas higher apparent levels of expression of CYP1A2, resulted in either withdrawal or FDA Black Box Warnings and CYP2Cs, and CYP2D6 were observed in the corresponding were termed “Severe DILI.” Category 2 drugs (n = 27) caused THLE cell lysates than in human hepatocyte lysates (Fig. 1). cases of liver failure that resulted in cautionary labeling and P450 enzyme activities of the THLE cell lines were explored restricted drug usage, but not Black Box Warnings or drug using specific probe substrates Table 2( ). None of the probe sub- withdrawal, and were categorized as “High concern.” Category strates were metabolized in the THLE-Null cell line. Extensive 3 drugs (n = 32) caused isolated and extremely infrequent cases and specific clearance of phenacetin (CYP1A2 substrate), dex- of DILI, which also resulted in cautionary labeling and were tromethorphan (CYP2D6 substrate), and verapamil (CYP3A4 termed “DILI reports.” Category 4 drugs (n = 8) caused asymp- substrate) was observed in the appropriate transfected cell lines tomatic elevations in levels of serum ALT, but not liver injury, (THLE-1A2, THLE-2D6, and THLE-3A4, respectively). An illus- and were termed “ALT elevations.” Category 5 drugs (n = 13) trative LC profile for verapamil in the THLE-3A4 and THLE-2D6 have not been reported to result in any evidence of liver signals cells is shown in Supplementary Figure 2. Quantification of the in humans and were categorized as “No liver signals.” rates of disappearance of the parent compounds was used to Recently, 68 of the 103 drugs have been assigned to 1 of 3 estimate the corresponding enzyme activities. It was found that DILI concern classes (High, Low, or No Concern) by other the rates of clearance of phenacetin in THLE-1A2 cells and of investigators, who considered the clinical severity of DILI dextromethorphan in THLE-2D6 cells were similar to the rates reported in the clinic and labeling approved by U.S. FDA (Chen of clearance of these substrates in cryopreserved human hepato- et al., 2011). This information has been summarized in Table 1 cytes. However, the rate of clearance of verapamil in THLE-3A4 as “LTKB class.” All of the 24 category 1 drugs had been cells was about one-third of its rate of clearance in human hepato- assigned to LTKB classification “Most DILI concern,” as had cytes (Table 2). The impact of DMSO concentration on substrate 12 category 2 drugs. Six category 2 drugs had been described clearance in THLE cells was investigated because DMSO was as LTKB “Less DILI concern.” These were as follows: car- the vehicle used to facilitate drug solubility in cell toxicity stud- mustine, indomethacin, ranitidine, which have been reported to ies. The rate of dextromethorphan clearance in THLE-2D6 cells cause fatal hepatic injury; clozapine, which has been reported was similar when determined at 0.05% and 1% DMSO, whereas to cause fulminant hepatic failure; azathioprine, which has been the rates of phenacetin clearance in THLE-1A2 cells and of vera- associated with life-threatening hepatic veno-occlusive disease; pamil clearance in THLE-3A4 cells were reduced by about 40% and flurbiprofen, which may cause progressive liver injury that at the higher DMSO concentration (Table 2). results in fatality. Three category 3 drugs (acitretin, methima- The extents of clearance of the CYP2C9 substrate diclofenac zole, and zimelidine) have been classified as LTKB “Most DILI and the CYP3A4 substrate midazolam were insufficient to concern,” whereas 13 category 3 drugs were classified as LTKB enable robust quantification of the corresponding enzyme “Less DILI concern.” Two category 4 drugs (buspirone and dex- activities from the rates of loss of the parent compounds. amethasone) were assigned to LTKB class “Less DILI concern.” Therefore, for these substrates, metabolite formation was quan- Three category 5 drugs (betaine, isoproterenol, and streptomy- tified by LC-MS, using appropriate authentic reference stand- cin) were assigned to LTKB class “No DILI concern.” ards. Verapamil metabolite formation (catalyzed by CYP3A4) 194 Gustafsson et al. c LTKB Class LTKB Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern Most DILI concern , (2004) (1999), et al. et al. (2010), (2001) (1997) et al. References (1994), Zimmerman et al. et al. et al. Reuben package Manufacturer’s insert package insert Fung package insert (1999) Kaplowitz and DeLeve (2007) and DeLeve Kaplowitz Zimmerman (1999) Stricker (1992) Stricker Aranda-Michel Manufacturer’s package insert Manufacturer’s Stricker (1992) Stricker Kusnick (2007) Kusnick Sevilla-Mantilla Sevilla-Mantilla Stricker (1992) Stricker Stricker (1992) Stricker Stricker (1992) Stricker Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Kleiner Stricker (1992) Stricker Zimmerman (1999) Stricker (1992) Stricker Stricker (1992) Stricker Kaplowitz and DeLeve (2007) and DeLeve Kaplowitz Kaplowitz and DeLeve (2007) and DeLeve Kaplowitz Lee (2008) Stricker (1992) Stricker Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Baty b Consequence Most Severe Reported DILI Most Severe 1 to DILI 1998, relicensed 2004; DILI BBW doses; DILI BBW more frequent fibrosis progressing to cirrhosis; DILI BBW to DILI to DILI in clinical trials; development in clinical trials; development terminated development terminated development to DILI to DILI to DILI to DILI to DILI Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Fatal liver failure; withdrawn in EU withdrawn failure; liver Fatal Liver injury; withdrawn due to DILI injury; withdrawn Liver Acute severe liver injury; DILI BBW liver Acute severe Hepatocellular liver injury at high Hepatocellular liver Rare acute hepatocellular injury, Rare acute hepatocellular injury, Acute liver failure; withdrawn due withdrawn failure; Acute liver Acute liver failure; DILI BBW failure; Acute liver Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver and multi-organ failure failure and multi-organ liver Fatal Jaundice in clinical trials; Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Cholestatic liver injury; DILI BBW Cholestatic liver Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Fatal liver failure; withdrawn due withdrawn failure; liver Fatal Fatal liver failure; DILI BBW failure; liver Fatal Fatal liver failure; withdrawn due withdrawn failure; liver Fatal le b a T a DILI Pattern Categorization of the Pharmaceuticals Mixed Hepatocellular Mixed Mixed Hepatocellular Hepatocellular Hepatocellular Hepatocellular Mixed Hepatocellular Hepatocellular Mixed Mixed Mixed Hepatocellular Hepatocellular Hepatocellular Hepatocellular Cholestatic Hepatocellular Mixed Mixed Hepatocellular I L I D Pharmacology Class inhibitor dopamine reuptake inhibitor dopamine reuptake antidepressant Antidiabetic, thiazolidinedione Catechol-O-methyl transferase Catechol-O-methyl Antidepressant, norepinephrine- Antidepressant, 5HT antagonist Narcotic analgesic Antimetabolite and antifolate NSAID, Cox-2 inhibitor Antirheumatic Antifungal Antituberculosis and Monoamine oxidase inhibitor Nonsteroidal antiandrogen Nucleoside Nonsteroidal anti-inflammatory Anticonvulsant Antineoplastic alkylating agent Antineoplastic alkylating Analgesic Nonsteroidal anti-inflammatory Endothelin antagonist Antigout, uricosuric Nonsteroidal anti-inflammatory Antiarrhythmic Anxiolytic, imidazopyridine. Drug Name Troglitazone Tolcapone Nomifensine Nefazodone Naltrexone Methotrexate Lumiracoxib Leflunomide Ketoconazole Isoniazid Iproniazid Flutamide Fialuridine Fenclozic Acid Felbamate Dacarbazine Cinchophen Bromfenac Bosentan Benzbromarone Benoxaprofen Amiodarone Alpidem 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe DILI Category 1. Severe Drug Toxicity to THLE-CYP Cell Lines 195 c LTKB Class LTKB Less DILI concern Most DILI concern Most DILI concern Not assigned Less DILI concern Most DILI concern Less DILI concern Not assigned Most DILI concern Most DILI concern Not assigned Most DILI concern Not assigned Less DILI concern Not assigned Not assigned Less DILI concern Most DILI concern Less DILI concern Not assigned Most DILI concern

et al. et al. et al. et al. et al. et al. et al. (2010) et al. (2010) (2009), (2005), Figueira- (1994), Stricker (1994), Stricker References et al. et al. (1997), Manufacturer’s (1997), Manufacturer’s et al. et al. (2000) (2010), Manufacturer’s pack- (2010), Manufacturer’s age insert (2010) Coelho package insert (1992) (1992) package insert (2010) (2010), Manufacturer’s pack- (2010), Manufacturer’s age insert (2010) et al. package insert (2010), Reuben Manufacturer’s package Manufacturer’s insert (1999) package insert (2012), Manufacturer’s pack- (2012), Manufacturer’s age insert Stricker (1992), Ribeiro Stricker Stricker (1992), Reuben Stricker Stricker (1992), Reuben Stricker Coban Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Zimmerman (1999), Stricker Zimmerman (1999), Stricker Manufacturer’s package insert Manufacturer’s Koek Koek Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Stricker (1992), Reuben Stricker Stricker (1992) Stricker Stricker (1992), Reuben Stricker Stricker (1992), Reuben Stricker Zimmerman (1999), Macfarlane Zimmerman (1999), Macfarlane Stricker (1992), Robles Stricker El Hajj Stricker (1992), Zimmerman Stricker Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Stricker (1992) Stricker Stricker (1992), Martindale Stricker Stricker (1992) Stricker b Consequence Most Severe Reported DILI Most Severe bile duct syndrome combination with amoxicillin injury; cirrhosis Continued Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Cholestatic hepatitis and vanishing Cholestatic hepatitis and vanishing Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Fatal liver failure liver Fatal Acute liver failure Acute liver Fatal liver failure liver Fatal Cholestatic hepatitis, when used in Acute liver failure Acute liver Fatal liver failure liver Fatal Fatal liver failure liver Fatal Cholestatic or hepatocellular liver Cholestatic or hepatocellular liver Fatal liver failure liver Fatal Fatal liver injury; DILI BBW liver Fatal a LE 1— B TA DILI Pattern Cholestatic Mixed Hepatocellular Mixed Mixed Hepatocellular Hepatocellular Cholestatic Mixed Hepatocellular Hepatocellular Mixed Mixed Mixed Mixed Cholestatic Hepatocellular Mixed Mixed Mixed Mixed Pharmacology Class antagonist psychoactive antineoplastic mood-stabilizing antineoplastic mood-stabilizing Histamine H2-receptor Antibiotic Alpha-adrenergic agonist, Alpha-adrenergic Antibiotic Nonsteroidal anti-inflammatory Analgesic Nonsteroidal anti-inflammatory Antibiotic, beta-lactam Antibiotic Alcohol antagonist Antihypertensive Nonsteroidal anti-inflammatory Anti-infective Atypical antipsychotic Antibiotic Nonsteroidal anti-inflammatory Immunosuppressant and Anticonvulsant, Anticonvulsant, Immunosuppressant and Antimalarial Anticonvulsant, Anticonvulsant, Drug Name Ranitidine Nitrofurantoin Methyldopa Levofloxacin Indomethacin Glafenine Flurbiprofen Flucloxacillin Erythromycin Disulfiram Dihydralazine Diclofenac Dapsone Clozapine Clavulanate Celecoxib Carmustine Carbamazepine Azathioprine Amodiaquine Valproic acid Valproic 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern DILI Category 1. Severe 196 Gustafsson et al. c LTKB Class LTKB Less DILI concern Not assigned Not assigned Not assigned Less DILI concern Not assigned Less DILI concern Not assigned Not assigned Less DILI concern Less DILI concern Not assigned Less DILI concern Not assigned Less DILI concern Most DILI concern Not assigned Most DILI concern Not assigned Most DILI concern Most DILI concern Most DILI concern Most DILI concern Not assigned

et al. et al. et al. (2002), et al. , Zimmerman Kaplowitz and Kaplowitz (1986) (2009), Kreiss (2009), El-Naggar (2009), El-Naggar References (2011) et al. et al. (2002) (2010) (2008) et al. et al. Rodríguez-Gonzáles (2002) (1999) Manufacturer’s package Manufacturer’s insert Compendium (2013) Manufacturer’s package Manufacturer’s insert et al. (2010), Zimmerman (1999) FDA (2012) FDA Manufacturer’s package Manufacturer’s insert DeLeve (2007) DeLeve pack- (1999), Manufacturer’s age insert et al. (1998) (1999) et al. Stricker (1992), Javier (1992), Javier Stricker Kaplowitz and DeLeve (2007) and DeLeve Kaplowitz Zimmerman (1999) Stricker (1992) Stricker Stricker (1992), Zimmerman Stricker Stricker (1992) Stricker Zimmerman (1999), Konikoff Konikoff Electronic Medicines Stricker (1992) Stricker Stricker (1992) Stricker Stricker (1992) Stricker Kim Khemissa-Akouz Khemissa-Akouz Manufacturer’s package insert Manufacturer’s Leithead Stricker (1992), Reuben Stricker Kaplowitz and DeLeve (2007) and DeLeve Kaplowitz Zimmerman (1999), Stricker (1999), Stricker Zimmerman (1999), Reuben Stricker (1992), Blackard Stricker Stricker (1992), Zimmerman Stricker Floyd Floyd b Consequence Most Severe Reported DILI Most Severe liver injury liver liver injury liver Continued Acute liver failure, extremely rare extremely failure, Acute liver Hepatitis Cholestatic liver injury Cholestatic liver Cholestatic liver injury Cholestatic liver Hepatocellular liver injury Hepatocellular liver Cholestatic liver injury Cholestatic liver Cholestatic liver injury Cholestatic liver Cholestatic liver injury Cholestatic liver Cholestasis or cytolysis Hepatocellular and/or cholestatic Cholestatic hepatitis, fibrosis Mixed cholestatic/ hepatocellular Mixed Cholestatic hepatitis Hepatitis Hepatitis Fatal liver failure following overdose following failure liver Fatal Fatal liver failure following overdose following failure liver Fatal Hepatitis Acute liver failure Acute liver Acute severe liver injury liver Acute severe Acute liver failure Acute liver Acute hepatocellular injury Acute liver failure Acute liver Acute liver failure, extremely rare extremely failure, Acute liver a LE 1— B TA DILI Pattern Mixed Hepatocellular Cholestatic Cholestatic Hepatocellular Cholestatic Cholestatic Cholestatic Cholestatic Cholestatic Cholestatic Cholestatic Mixed Cholestatic Hepatocellular Mixed Hepatocellular Hepatocellular Hepatocellular Mixed Mixed Hepatocellular Mixed Mixed Pharmacology Class mood-stabilizing inhibitor phate receptor inhibitor Nonsteroidal anti-inflammatory Antidiabetic sulphonylurea Antidiabetic sulphonylurea Antibiotic Loop diuretic Antibiotic Immunosuppressant Antibiotic Fibrate, antihyperlipidemia Antidiabetic sulphonylurea Anticonvulsant, Anticonvulsant, Lipid lowering PPAR agonist PPAR Lipid lowering Antibiotic Calcium channel antagonist Antiviral, guanosine analogue Antiviral, Antipsoriatic retinoid Analgesic Antiasthmatic, 5-lipoxygenase Nonsteroidal anti-inflammatory Antiplatelet, adenosine diphos- Antifungal Cholinesterase inhibitor Nonsteroidal anti-inflammatory Antidiabetic, thiazolidinedione Drug Name Ibuprofen Gliclazide Glibenclamide Fusidic Acid Furosemide Dicloxacillin Cyclosporine A Cloxacillin Ciprofibrate Chlorpropamide Chlorpromazine Bezafibrate Amoxicillin Amlodipine Acyclovir Acitretin Acetaminophen Zileuton Tolmetin Ticlopidine Terbinafine Tacrine Sulindac Rosiglitazone Concern 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 2. High concern 2. High concern 2. High concern 2. High concern 2. High 2. High concern DILI Category 2. High concern Drug Toxicity to THLE-CYP Cell Lines 197 c LTKB Class LTKB Not assigned No DILI concern Not assigned Not assigned Not assigned Not assigned Less DILI concern Not assigned Less DILI concern Not assigned Most DILI concern Less DILI concern Not assigned Not assigned Not assigned Not assigned Less DILI concern Not assigned Less DILI concern Less DILI concern Not assigned Less DILI concern Less DILI concern Most DILI concern Not assigned (2012) (2003), (2012) (2000) (2009), References et al. et al. et al. et al. et al. package insert (1999) (1992) Manufacturer’s package Manufacturer’s insert Manufacturer’s package Manufacturer’s insert package insert No reports No reports Stricker (1992) Stricker Martindale (2012) Dawson Dawson Dawson Dawson Manufacturer’s package insert Manufacturer’s Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker Manufacturer’s package insert Manufacturer’s Rogers Zimmerman (1999) Stricker (1992) Stricker Zimmerman (1999) Stricker (1992), Zimmerman Stricker Stricker (1992) Stricker Stricker (1992) Stricker Zimmerman (1999), Stricker Zimmerman (1999), Stricker Stricker (1992) Stricker Floyd Floyd Zimmerman (1999) Jadallah Stricker (1992) Stricker Stricker (1992) Stricker Woeber (2002) Woeber Stricker (1992), Manufacturer’s (1992), Manufacturer’s Stricker b Consequence Most Severe Reported DILI Most Severe liver injury liver Continued No reported liver signals No reported liver No reported liver signals No reported liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Liver enzyme elevations Liver Jaundice Cholestatic Hepatocellular liver injury Hepatocellular liver Cholestatic hepatitis Hepatocellular injury Cholestatic liver injury Cholestatic liver Mild hepatic injury Hepatocellular Acute hepatocellular/ cholestatic Cholestatic hepatitis Hepatitis Acute hepatitis Acute hepatitis Cholestatic hepatitis Jaundice a LE 1— B TA DILI Pattern None None None None None None None None None None Mixed Cholestatic Hepatocellular Cholestatic Hepatocellular Cholestatic Hepatocellular Hepatocellular Mixed Mixed Mixed Mixed Mixed Mixed Hepatocellular Pharmacology Class tor antagonist and antiacne agent dihydropyridine calcium dihydropyridine channel blocker CNS stimulant Nutrient Vitamin B6 Vitamin Anthelmintic Monoamine oxidase B inhibitor Antihistamine Corticosteroid Antibiotic, Tuberculostatic Anxiolytic Cholinesterase inhibitor Antidepressant Antidiabetic sulphonylurea Nonsteroidal anti-inflammatory Antipsychotic, dopamine recep- Nonsteroidal anti-inflammatory Nonsteroidal anti-inflammatory Antibiotic Beta-blocker Uricosuric Antidiabetic, thiazolidinedione Chelating agent, antirheumatic Atypical antipsychotic Antianginal, antihypertensive, Antianginal, antihypertensive, Nonsteroidal anti-inflammatory Antithyroid Antihistamine Drug Name Caffeine Betaine Pyridoxine Praziquantel Pargyline Methapyrilene Dexamethasone Cycloserine Buspirone Aricept Zimelidine Tolbutamide Suprofen Sulpiride Salicylic Acid Salicylic Rifampicin Propranolol Probenecid Pioglitazone D-Penicillamine Olanzapine Nifedipine Naproxen Methimazole Ketotifen signals signals elevations elevations elevations elevations elevations elevations elevations elevations 5. No liver 5. No liver 5. No liver 5. No liver 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 4. ALT 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports DILI Category 3. DILI reports 198 Gustafsson et al. c LTKB Class LTKB Not assigned No DILI concern Not assigned Not assigned Not assigned Not assigned Not assigned No DILI concern Not assigned Not assigned Not assigned References Zimmerman (1999) No reports No reports No reports No reports No reports No reports No reports No reports No reports No reports b Consequence Most Severe Reported DILI Most Severe Continued No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver No reported liver signals No reported liver a LE 1— B TA DILI Pattern None None None None None None None None None None None Pharmacology Class inverse agonist inverse channel opener linesterase inhibitor antagonist http://www.fda.gov/ScienceResearch/BioinformaticsTools/LiverToxicityKnowledgeBase/ucm226811.htm Analgesic Antibiotic Cannabinoid type-1 receptor Beta adrenergic antagonist Beta adrenergic Antihypertensive, potassium Antihypertensive, Anticoagulant, antithrombotic Parasympathomimetic, cho- Parasympathomimetic, Beta adrenergic agonist Beta adrenergic benzodiazepine receptor Catecholamine Corticosteroid Drug Name Propionate Zomepirac Streptomycin Rimonabant Practolol Pinacidil Picotamide Physostigmine Isoproterenol Flumazenil Dopamine Clobetasol BBW = Black Box Warning. Mixed = mixed hepatocellular/cholestatic. Mixed = mixed Knowledgebase: Toxicity LTKB = Liver signals signals signals signals signals signals signals signals signals signals a b c signals 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver 5. No liver DILI Category 5. No liver Drug Toxicity to THLE-CYP Cell Lines 199 b c a a c 44 ND ND 263 60.9 Human 0% DMSO Hepatocytes a a b c 8 3.5 ND ND 0% DMSO THLE-3A4 Not metabolized a a a c 40 ND ND ND Metabolite Formation (pmol/min/mg Protein) Metabolite Formation 0% DMSO THLE-2C9

Fig. 1. P450 protein expression in pooled human hepatocytes: THLE-1A2, Not metabolized 2C9, 2C19, 2D6, 3A4, and Null cell lines. Protein extracts from the cell lines were probed using antisera, which recognized the indicated P450 enzymes, and/or

GAPDH. Abbreviation: THLE, T-antigen–immortalized human liver epithelial. a a 6.8 4.7 ND ND 11.5 Human

was also quantified using this approach in THLE-3A4 cells. 0% DMSO Hepatocytes Very similar rates of diclofenac metabolism were observed in THLE-2C9 cells and human hepatocytes. In contrast, the observed rate of midazolam and verapamil metabolite forma- a a 2.5 tion was approximately 30- and 17-fold lower, respectively, in ND ND 1% DMSO THLE-3A4 cells than in human hepatocytes (Table 2). THLE-3A4

Drug Toxicity to THLE Cells a a 2 Not metabolized Not metabolized 4.1 Initial THLE cell toxicity studies involved the use of manual ND ND le b

methods for compound dilution and liquid handling and were 0.05% DMSO a undertaken using cells cultured in 96-well plates in the pres- T l/min/mg Protein)

ence of 0.2% DMSO. Typical dose-response curves for 4 drugs µ a a

are shown in Figures 2 and 3. Marked toxicity (EC ≤ 100μM) 5.5 50 ND ND was evident when the cells were exposed to chlorpromazine, 1% DMSO troglitazone, and rosiglitazone for 24 h, which was more potent in THLE-3A4 cells than in THLE-Null cells or in THLE THLE-2D6 cells, which expressed other CYPs. However, no toxicity was a a Not metabolized Not metabolized 5.9 observed when the THLE cells were exposed to streptomycin. ND ND Metabolism of P450 Probe Substrates in the THLE Cell Lines Substrates in the Metabolism of P450 Probe

The EC values obtained with these drugs and an additional 0.05% DMSO 50 Compound ( Loss of Parent 4 drugs are summarized in Table 3. Clozapine and diclofenac exhibited both THLE-Null cell toxicity and potentiated toxicity a a

to THLE-3A4 cells (THLE-Null/THLE-3A4 EC ratio ≥ 1.4), 2.9 50 ND ND whereas ibuprofen and practolol did not. Both troglitazone and 1% DMSO rosiglitazone exhibited poor solubility in cell culture medium that contained 0.2% DMSO, whereas warming and repeated THLE-1A2 pipetting of the culture media were required in the case of clo- a a Not metabolized Not metabolized 5.3 zapine to achieve the higher drug concentrations required to ND ND

generate full dose-response curves for toxicity. 0.05% DMSO The solubility of rosiglitazone and clozapine in culture medium was improved markedly when the DMSO concentration was increased to 1%. Cell toxicity EC50 values obtained in the presence of 1% DMSO for the 8 drugs that had been evaluated at 0.2% DMSO are summarized in Table 3. The 3 drugs that did not cause cell toxicity in 0.2% DMSO (ibuprofen, Cells in suspension. practolol, and streptomycin) were also nontoxic in 1% DMSO. ND = not determined. 0.03% DMSO. Data were from single experiments performed in triplicate. Apart from the metabolite formation studies with verapamil in THLE-3A4 cells, THLE cell incubations were undertaken using plated cells. THLE cell incubations were undertaken THLE-3A4 cells, in Apart from the metabolite formation studies with verapamil performed in triplicate. Data were from single experiments a b c Probe Substrate (P450) (2D6) Dextromethorphan (3A4) Verapamil Diclofenac (2C9) Midazolam (3A4) were performed in suspension. Incubations with human hepatocytes Three drugs (chlorpromazine, clozapine, and rosiglitazone) Phenacetin (1A2) 200 Gustafsson et al.

Fig. 2. Toxicity of chlorpromazine, but not of streptomycin, to THLE-1A2, 2C9, 2C19, 2D6, 3A4, and Null cell lines following incubation for 24 h in 0.2% DMSO (vol/vol) vehicle (■ 1A2, ▽ 2C9, □ 2C19, ○ 2D6, △ 3A4, ● Null). Cell viability was determined using the MTS assay. (Panels A and C) Mean values ±

SD from 5 independent experiments, each involving triplicate determinations. (Panels B and D) The corresponding EC50 values; ***different from THLE-Null, p < 0.001. Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; THLE, T-antigen–immortalized human liver epithelial.

Fig. 3. Toxicity of troglitazone and rosiglitazone to THLE-2C19, 2D6, 3A4, and Null cell lines, following incubation for 24 h in 0.2% DMSO (vol/vol) vehicle (□ 2C19, ○ 2D6, △ 3A4, ● Null). Cell viability was determined using the MTS assay. (Panels A and C) Mean values ± SD from 5 independent experiments, each involving triplicate determinations. (Panels B and D) The corresponding EC50 values; **different from THLE-Null, p < 0.01. ***different from THLE-Null, p < .001. Abbreviations: DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; THLE, T-antigen–immortalized human liver epithelial. exhibited THLE-Null cell toxicity and potentiated toxicity to observed at 0.2% DMSO. For both troglitazone and diclofenac, THLE-3A4 cells at both DMSO concentrations, although the potentiated toxicity to THLE-3A4 cells was observed at 0.2%

EC50 values at 1% DMSO were up to 1.5-fold higher than those DMSO but not at 1% DMSO. Drug Toxicity to THLE-CYP Cell Lines 201 1 Ratio 1.6 1.0 1.0 1.0 1.0 1.0 0.7 1.0 1.0 1.0 1.0 1.9 1.0 1.0 0.9 2.2 1.1 2.1 1.0 1.0 1.9 1.9 1.0 1.0 1.9 1.2 1.5 1.0 > 50 > Null/3A4 EC 6

5 9 50 18 42 71 28 16 = 3) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 139 150 260 Null > n > > > > > > > > > > > > > > > > > ( 197 15** d

6 6 27 22 34 15 79 13 50 = 3) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 117 186 138 288 ± 3A4

n > > > > > > > > > > > > > > > ( 91 = 1) n 14*

5 7 ± 50

23 43 65 18 14 = 3) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 134 130 2D6 > n > > > > > > > > > > > > > > > > > > ( 1% DMSO ( 135 18

M) ±

7 9 μ 17 41 29 17 50 = 3) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 145 128 110 213 2C9 n > > > > > > > > > > > > > > > > > ( 221 ± SEM ( 50 18

7 8 50 14 32 93 38 96 10 = 3) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 150 193 1A2 > n > > > > > > > > > > > > > > > > > ( 162 sing the Manual Protocols U Ratio 2.4 1.0 1.5 2.1 2.1 1.0 1.8 50 > Null/3A4 EC 3 le 43 4 15 3

b ± ± ± ±

a THLE Cell Line Geomean EC = 5) = 8) = 8) = 5) = 4) = 3) 500 300 200 = 10) Null T n n n n n n 34 14 > > > n ( ( ( ( ( ( ( 179 131 d 25

1 d

2**

± 8*** 11**

= 5) = 8) ± = 8) = 5) = 4) 500 300

= 3) ± = 10) ± 3A4

7 n n n n n > > n n ( ( ( ( ( ( ( 119 16 62 84 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 2

51 4 3

16 0.2% DMSO

± ± ±

= 5) = 8) = 8) = 5) = 3) = 4) 500 300 = 10) 2D6 n n n n n n 35 15 > > n ( ( ( ( ( ( 98 198 ( 122 25 4 22

± ± ±

= 5) = 8) = 8) = 5) 500 300 ND ND = 10) 2C9 n n n n 36 > > n ( ( ( ( ( 235 147 THLE Cell Toxicity of 34 Pharmaceuticals When Tested Tested When of 34 Pharmaceuticals Toxicity THLE Cell 37 4 12

± ± ±

= 5) = 8) = 8) = 5) 500 300 ND ND = 10) 1A2 n n n n 33 > > n ( ( ( ( ( 173 122 DILI Category 5. No liver signals 5. No liver 3. DILI reports 5. No liver signals 5. No liver 2. High concern signals 5. No liver 3. DILI reports 2. High concern elevations 4. ALT 3. DILI reports 4. ALT elevations 4. ALT 2. High concern 3. DILI reports 4. ALT elevations 4. ALT 1. Severe 1. Severe 2. High concern 2. High concern 3. DILI reports 1. Severe 1. Severe 2. High concern 2. High concern 1. Severe 1. Severe 3. DILI reports 1. Severe 1. Severe 1. Severe 2. High concern b a a Flumazenil Ketotifen Rosiglitazone Dopamine Nitrofurantoin Betaine Ibuprofen Disulfiram Pyridoxine Furosemide Pargyline Ketoconazole Troglitazone Diclofenac Chlorpromazine Buspirone Felbamate Tolcapone Ticlopidine Clozapine Propranolol Dacarbazine Nomifensine Terbinafine Amodiaquine Bosentan Naltrexone Olanzapine Amiodarone Drug Name 202 Gustafsson et al.

In view of the improved compound solubility that could be achieved at 1% DMSO, this vehicle concentration was used Ratio 1.0 1.0 1.0 1.0 1.0 50 in subsequent cell toxicity studies. Data obtained for an addi- Null/3A4 EC tional 26 drugs using the manual protocol are shown in Table 3. Overall, 23 of the drugs evaluated under these conditions 300 300 300 300 300 caused DILI, and 13 drugs (57%) exhibited toxicity to one or Null > > > > > more THLE cell lines. Twelve drugs that were toxic to THLE cells (DILI category 1, 2, or 3) exhibited potencies of toxicity similar to that of THLE-Null, THLE-1A2, THLE-2C9, and d 300 300 300 300 300 3A4 > > > > > THLE 2D6 cell lines, whereas 8 drugs exhibited more potent = 1)

n toxicity to THLE-3A4 cells than to THLE-Null cells or the other cell lines. In addition, 1 drug that exhibited marked toxicity to THLE-Null cells (disulfiram) caused less potent toxicity 300 300 300 300 300 2D6 > > > > > to THLE-3A4 cells (see Table 3). None of the 11 drugs that did 1% DMSO ( not cause DILI (DILI category 4 or 5) were toxic to THLE cells. Investigation of the published scientific literature provided

M) evidence that 10 of the 11 drugs that exhibited potentiated tox- μ 300 300 300 300 300 2C9 > > > > > icity to THLE-3A4 cells when tested using the manual protocol (amiodarone, ketoconazole, troglitazone, amodiaquine, clo-

± SEM ( zapine, diclofenac, rosiglitazone, terbinafine, chlorpromazine, 50 and propranolol) are metabolized to reactive intermediates (see 300 300 300 300 300 1A2 > > > > > Table 4). No data could be found in the scientific literature, either positive or negative, on whether the remaining drug that exhibited potentiated THLE-3A4 cell toxicity (bosentan) might Ratio 1.0 1.0

50 be metabolized to reactive intermediates. Null/3A4 EC 14

Continued Metabolic Bioactivation of [ C]-Troglitazone in THLE-3A4 Cells THLE Cell Line Geomean EC = 5) 500 500 = 16) Null n > > n ( Radiochemical LC profiling of supernatants obtained follow- ( LE 3—

B ing incubation of radiolabeled troglitazone with THLE-Null

TA and THLE-3A4 cells revealed that numerous metabolites were d = 5) 500 500 produced in both cell lines (Fig. 4). Their chemical structures = 16) 3A4 n > > n ( ( were investigated by LC-MSn, which provided evidence of 2 sul- ND ND ND fated conjugates (M3 and M4), a hydroxylated metabolite (M5),

0.2% DMSO and a polar radiopeak that eluted early (M1) and whose chemi- = 5) 500 500 = 16) 2D6 n > >

n cal structure could not be assigned due to its close proximity to ( ( the solvent front (Table 5). In addition, a metabolite (M2) was detected in media from THLE-3A4 cells, but not in media from M. M. μ = 5) 500 500 μ

= 16) THLE-Null cells, which exhibited a molecular mass at m/z 745 2C9 n > > n ( ( that is consistent with direct conjugation of glutathione (a mass increment of 305 mu) with troglitazone (m/z 440). This metabolite afforded fragments corresponding to the loss of pyroglutamic = 5) 500 500 = 16) 1A2 n > > n (

( acid (m/z 618, m/z 747–129), as well as characteristic glutathione molecular (m/z 306; glutathione) and fragment ions (m/z 272;

glutathione-SH2 and m/z 254; glutathione-SH2-H2O), providing further confirmation that it was a glutathione conjugate. The < .001 statistically significant difference from THLE-Null. from < .001 statistically significant difference

p magnitude of irreversible binding of radiolabel derived from troglitazone to THLE-3A4 cell proteins was markedly greater than DILI Category 5. No liver signals 5. No liver 5. No liver signals 5. No liver 5. No liver signals 5. No liver 5. No liver signals 5. No liver 5. No liver signals 5. No liver its magnitude of binding to THLE-Null cell proteins (Table 6). < .01; *** p c Screening of 103 Drugs for Toxicity to THLE-Null and THLE-3A4 Cells < .05; ** p Maximum compound concentration achieved was 200 was Maximum compound concentration achieved ND = not determined. Maximum compound concentration achieved was 50 was Maximum compound concentration achieved and then added to culture medium containing the indicated concentration of DMSO. in water dissolved Streptomycin was In order to facilitate evaluation of THLE cell toxicity of a * a b c d Streptomycin Practolol Pinacidil Picotamide Isoproterenol Drug Name larger number of drugs, the manual protocol was modified to Drug Toxicity to THLE-CYP Cell Lines 203 Observed Potentiated Toxicity Was Was Toxicity THLE-3A4 Cell Protocol in Which Manual + automated Automated Manual Manual Automated Manual Manual Automated Manual + automated Manual Automated Manual Manual Automated Automated Manual + automated Manual Automated Automated

et al. (2009) (2011), (2005) (2001) (1994) (2008) (1992) (2013) (2000) (1995) et al. et al. et al. (1999), et al. et al. et al. et al. et al. et al. et al. Reference et al. (2003) Hargus Hargus (2006) (2001) Foster Foster Ohyama Ohyama Takakusa Takakusa Rodriguez and Buckholz Kalgutkar Kassahun Harrison Maggs Shen Wen and Moore (2011) Wen Alvarez-Sanchez Alvarez-Sanchez Iverson and Uetrecht Iverson Wen and Zhou (2009) Wen Nakayama Bergström Bergström in in in in and in vitro ; covalent ; covalent in vitro in human in vitro in vitro in vitro in vitro in vitro in vitro b in vivo in vitro in vivo Data Type in vivo in human microsomes by CYP3A4 ; time-dependent CYP3A4 inhibition 4 Other Data Demonstrating Reactive Metabolite Formation Other Data Demonstrating Reactive in vitro in vitro le in rat liver, via P450-mediated and UGT- in rat liver, b and in the rat ; irreversible binding to liver proteins in hepato- binding to liver ; irreversible a T binding to rat liver proteins binding to rat liver in liver microsomes and supersomes expressing microsomes and supersomes expressing in liver P4503A4 in vitro and membranes expressing recombinant P4503A4 and membranes expressing vitro ing to liver proteins in the rat ing to liver ing to liver microsome and neutrophil proteins ing to liver to a benzoquinone reactive intermediate, which bound to a benzoquinone reactive to proteins; also bioactivated covalently in vivo intermediates. to protein reactive mediated pathways, liver microsomes and by heterologously expressed microsomes and by heterologously expressed liver CYP3A4 and other CYPs vitro vitro microsomes and supersomes expressing CYP3A4 microsomes and supersomes expressing vitro THLE-3A4 cells; metabolism mediated and cytes THLE-3A4 cells toxicity to Mechanism-based CYP3A4 inhibition Liver microsome covalent binding microsome covalent Liver NA microsomes Metabolism in rat liver Quinone imine intermediate trapped with glutathione Glutathione conjugate formation in liver microsomes formation in liver Glutathione conjugate Glutathione conjugate formation and irreversible bind- formation and irreversible Glutathione conjugate NA Glutathione conjugate formation and irreversible bind- formation and irreversible Glutathione conjugate Bioactivated Bioactivated Glutathione conjugates formation Glutathione conjugates Glutathione conjugate formation in liver microsomes formation in liver Glutathione conjugate Glutathione conjugate formation in liver microsomes formation in liver Glutathione conjugate NA NA microsome glutathione trapping Liver Liver microsome covalent binding microsome covalent Liver NA Iminium ion intermediate trapped with cyanide in liver in liver Iminium ion intermediate trapped with cyanide (2012) (2012) (2012) (2012) (2012) (2012) (2012) (2012) (2012) (2009) (2012) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Reference In Vitro Thompson Thompson Thompson Thompson Thompson Thompson Thompson Thompson Thompson Nakayama Thompson Proteins a protein pmol/mg Covalent Binding to Human Hepatocyte Binding to Human Hepatocyte Covalent ND 99.7 ND ND 201.6 118.3 72.6 2.6 120.7 140.8 ND 32.4 ND ND 19.3 ND 9.4 ND 1114.8 Literature Evidence for Metabolic Activation of Drugs That Exhibited Potentiated Toxicity to the THLE-3A4 Cell Line to the Toxicity That Exhibited Potentiated of Drugs Activation Metabolic Evidence for Literature DILI Category 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 1. Severe 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 2. High concern 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports 3. DILI reports signals 5. No liver NA = no published data in the primary literature. NA = no ND = not determined. a b Drug Name Amiodarone Benzbromarone Bosentan Ketoconazole Nefazodone Troglitazone Amodiaquine Celecoxib Clozapine Diclofenac Glafenine Rosiglitazone Terbinafine Acitretin Amlodipine Chlorpromazine Propranolol Fusidic Acid Rimonabant 204 Gustafsson et al.

Fig. 4. Radiochromatograms of [14C]-troglitazone and its metabolites in media from THLE-Null (A) and THLE-3A4 cells (B) in 0.2% DMSO (vol/vol) vehicle for 24 h. The fragmentation scheme for troglitazone (C) derived from the data shown in Table 4 is also shown. Abbreviations: DMSO, dimethyl sulfoxide; GSH, glutathione; TGZ, troglitazone; THLE, T-antigen–immortalized human liver epithelial.

Table 5 Summary of LC-MSn Data for Troglitazone and Its Metabolites in Cell Culture Media From THLE-3A4 and THLE-Null Cell Lines

[M–H]− Relevant [M–H]− Fragment THLE-Null THLE-3A4 12 Peak ID tR (min) Assignment ( C Isotope) Ions (m/z) Cells Cells

TGZ 35.2 Troglitazone 440 412, 397, 369, 363, 276, 233, ✓ ✓ 222, 221, 199, 179, 178, 162, 145, 137, 119, 117 M1 — Unassigned (possible — — ✓ ✓ contaminant) M2 12.9 Glutathione conjugate 745 *729, 618, 440, 306, 272, 254 ✗ ✓ M3 19.2 Troglitazone sulfate 520 440, 412, 397, 369, 276, 162 ✓ ✓ M4 20.6 Troglitazone sulfate 520 440, 412, 397, 369, 276, 162 ✓ ✓ M5 26.2 Hydroxy troglitazone 456 413, 385, 379, 369, 278, 222, ✓ ✓ 179, 177, 162, 151, 145

Detection was undertaken by radiometric HPLC and structures were inferred from the mass ions detected by LC-MS/MS. *Fragments from m/z 747 (predominant 14C isotope) due to low levels of m/z 745. ✓Peak observed. ✗No peak observed. enable rapid and high-volume data generation. The most sig- No clear differences were evident between data obtained at nificant alterations were adoption of 384-well plates rather than these 2 time intervals; therefore, cells were exposed to test 96-well plates, use of robotics for all compound dilutions and compounds for 24 h in subsequent experiments. Typical cell liquid handling steps, evaluation only of THLE-3A4 and THLE- toxicity dose-response curves obtained with 3 drugs are shown Null cells, and dilution of the test compounds directly into in Figure 5. Toxicity data obtained with the 103 tested drugs in PMFR (+) growth medium that contained 2.7% bovine serum. THLE-Null and THLE-3A4 cells are summarized in Table 7 Compound dilution directly into the cell growth medium sim- and in Figure 6. plified the liquid handling protocol, and 1% DMSO was used Twenty-seven drugs exhibited evidence of toxicity when to facilitate compound solubility. Toxicity data obtained with evaluated using the automated protocol. For several drugs, 30 drugs using the automated protocol at 24 or 48 h after com- the effect was minimal and was only apparent at the high- pound administration are shown in Supplementary Figure 4. est tested drug concentration of 300μM. Visual inspection of Drug Toxicity to THLE-CYP Cell Lines 205

Table 6 dose-response curves indicated that an EC50 cutoff value of Covalent Binding of [14C]-Troglitazone to THLE Cell Proteins about 200μM enabled identification of toxicity, and this value was used in the subsequent data analysis. Twenty-two drugs Covalent Binding to Protein caused toxicity to THLE-Null cells, with EC50 values close (pmol drug eq/mg Protein) to or below this value (Fig. 6 and Table 7), whereas 11 drugs THLE Cell Line Geometric Mean Lower 95% CI Upper 95% CI exhibited potentiated toxicity to THLE-3A4 cells (Figs. 6 and 7 and Table 7) and 4 drugs exhibited greater toxicity to THLE- THLE-Null 80.5 29.3 221.2 Null cells than to THLE-3A4 cells (Table 7). THLE-3A4 209.3* 100 438.3 Seven of the 11 drugs that exhibited potentiated THLE-3A4 cell toxicity (THLE-Null/THLE-3A4 EC 14 50 Cells were incubated with 10μM [ C]-troglitazone for 24 h. Data are geo- ratio ≥ 1.4) when tested using the automated protocol metric means and 95% confidence intervals (95% CI) from 3 experiments, each performed in duplicate. have been reported to be metabolized to reactive interme- *p < .05 statistically significantly different from THLE-Null. diates (see Table 4). Three of these drugs (amiodarone,

Fig. 5. Toxicity of chlorpromazine, amiodarone, and practolol to THLE-3A4 and THLE-Null cell lines following incubation for 24 h in 1% DMSO (vol/vol) vehicle (▲ 3A4, ■ Null). (Panels A, C, and E) Mean values ± SD from 41 (chlorpromazine) or 7 (amiodarone and practolol) separate experiments, each performed

in duplicate. (Panels B, D, and F) The corresponding EC50 values; ***different from THLE-Null, p < .001. Abbreviations: DMSO, dimethyl sulfoxide; THLE, T-antigen–immortalized human liver epithelial. 206 Gustafsson et al.

Table 7 THLE Cell Toxicity of 103 Pharmaceuticals When Tested Using the Automated Protocol

Drug Name DILI Severity THLE-Null EC50 THLE-3A4 EC50 THLE-Null/3A4 EC50 Ratio

Amiodarone (n = 7) 1. Severe 24 ± 2 12 ± 1 2.0 Benzbromarone (n = 3) 1. Severe 89 ± 49 44 ± 9 2.0 Bosentan (n = 7) 1. Severe > 291 ± 4 ~260 ± 14 1.1 Cinchophen (n = 3) 1. Severe 210 ± 21 > 300 0.7 Ketoconazole (n = 7) 1. Severe 52 ± 2 46 ± 2 1.1 Lumiracoxib (n = 7) 1. Severe 102 ± 16 172 ± 23 0.6 Nefazodone (n = 7) 1. Severe 100 ± 22 66 ± 7 1.5 Tolcapone (n = 7) 1. Severe 22 ± 4 35 ± 8 0.6 Troglitazone (n = 7) 1. Severe 26 ± 1 24 ± 2 1.1 15 additional drugs 1. Severe > 290 > 290 1 Celecoxib (n = 3) 2. High concern 27 ± 2 12 ± 7 2.2 Clozapine (n = 8) 2. High concern 219 ± 19 150 ± 21 1.5 Diclofenac (n = 7) 2. High concern 130 ± 14 111 ± 7 1.2 Disulfiram (n = 7) 2. High concern 57 ± 11 48 ± 4 1.2 Glafenine (n = 3) 2. High concern > 300 > 192 1.6 Indomethacin (n = 3) 2. High concern 269 ± 16 284 ± 11 0.9 Rosiglitazone (n = 7) 2. High concern 183 ± 15 171 ± 6 1.1 Terbinafine n( = 7) 2. High concern 57 ± 3 54 ± 5 1 Ticlopidine (n = 7) 2. High concern 87 ± 19 92 ± 28 0.9 19 additional drugs 2. High concern > 290 > 289 1 Acitretin (n = 3) 3. DILI reports > 175 65 ± 15 2.7 Amlodipine (n = 3) 3. DILI reports 43 ± 4 18 ± 6 2.4 Chlorpromazine (n = 43) 3. DILI reports 43 ± 1 26 ± 1 1.7 Fusidic Acid (n = 3) 3. DILI reports 191 ± 22 124 ± 7 1.5 Glibenclamide (n = 3) 3. DILI reports 202 ± 42 290 ± 5 0.7 Ketotifen (n = 3) 3. DILI reports > 260 > 248 1 Nifedipine (n = 3) 3. DILI reports 104 ± 15 102 ± 14 1 25 additional drugs 3. DILI reports > 300 > 300 1 8 drugs 4. ALT elevations > 300 > 300 1 Picotamide (n = 3) 5. No liver signals > 269 > 300 0.9 Rimonabant (n = 2) 5. No liver signals 26 ± 2 11 ± 1 2.4 10 additional drugs 5. No liver signals > 300 > 300 1

Data are geomean EC50 ± SEM (μM). clozapine, and chlorpromazine) exhibited potentiated tox- reports (Table 8). There was no apparent association between icity to THLE-3A4 cells when tested using both protocols. the THLE cell toxicity data and whether the tested drugs were However, 8 drugs (bosentan, ketoconazole, troglitazone, reported to cause hepatocellular, cholestatic, or mixed patterns amodiaquine diclofenac, rosiglitazone, terbinafine, and of liver injury (Fig. 8). propranolol) exhibited potentiated THLE-3A4 cell toxicity One drug that exhibited marked THLE-Null cell toxicity when evaluated using the manual protocol, but not when and also markedly potentiated toxicity to THLE-3A4 cells did tested using the automated protocol. not cause DILI; this was rimonabant. The maximum unbound Twenty-one of the 22 drugs that exhibited THLE-Null cell plasma concentrations of this drug, and of the other 103 drugs, toxicity (95%) and 10 of the 11 drugs that exhibited potentiated were calculated from Cmax and plasma protein binding val- toxicity to THLE-3A4 cells (91%) have been reported to cause ues that were obtained from the published scientific literature DILI in humans. Therefore, the assay exhibited high specific- (see Supplementary Table 1). This value was much lower for ity/positive predictive value (PPV) for DILI. However, many rimonabant than for any of the other tested drugs (Fig. 9). drugs reported to cause DILI did not exhibit THLE-cell tox- Furthermore, the total uncorrected plasma Cmax of rimonabant icity; therefore, its DILI sensitivity/negative predictive value was much lower than the corresponding values for the other (NPV) was modest (Table 8). The drugs that exhibited THLE tested drugs (Supplementary Table 1). cell toxicity were distributed across all 3 DILI severity categories (Fig. 8 and Tables 7 and 8), although the frequencies of THLE-Null cell toxicity and of potentiated THLE-3A4 cell Discussion toxicity were higher among drugs categorized as Severe DILI The P450 expression and activities we observed in THLE than among drugs categorized as High DILI concern or DILI cell lines transfected with CYP1A2, CYP2C9, CYP 2C19, Drug Toxicity to THLE-CYP Cell Lines 207

CYP 2D6, and CYP 3A4, but not in cells transfected with which was not evident at 1% DMSO. Consequently, the higher an empty vector, are consistent with data obtained by previ- DMSO concentration was used to investigate THLE cell tox- ous investigators (Pfeifer et al., 1993; Soltanpour et al., 2012). icity of > 100 drugs, which utilized a fully automated assay Initial toxicity studies revealed limited solubility of several protocol. When evaluated using this protocol, 22 of 103 drugs drugs in the cell culture medium; therefore, concentrated stock exhibited toxicity to THLE-Null cells which can be attributed solutions were prepared in 100% DMSO and diluted into cul- to P450 independent mechanisms. A possible contributory fac- ture medium. Although numerous drugs could be dissolved tor is mitochondrial injury, which has been observed for the readily in medium that contained 0.2% DMSO, some com- majority of the drugs that exhibited such toxicity (Dykens and pounds exhibited relatively poor solubility in 0.2% DMSO, Will, 2007), although other mechanisms cannot be excluded. Four drugs (cinchophen, lumiracoxib, tolcapone, and glibenclamide) caused less potent toxicity to THLE-3A4 cells than to THLE-Null cells, which is suggestive of detoxification by CYP3A4, whereas 11 drugs exhibited more potent THLE-3A4 cell toxicity. These included rimonabant, which is metabolized by CYP3A4 to a chemically reactive and cytotoxic iminium ion intermediate (Foster et al., 2013), whereas 6 of the other 10 drugs (amiodarone, benzbromarone, nefazodone, clozapine, glafenine, and chlorpromazine) are also metabolized to reactive intermediates. No data could be identified on whether or not the remaining drugs (celecoxib, acitretin, amlodipine, and fusidic acid) are metabolized to reactive species. Eight drugs (bosentan, ketoconazole, troglitazone, amodiaquine, diclofenac, rosiglitazone, terbinafine, and propranolol) exhibited potentiated THLE-3A4 cell toxicity when tested using the manual protocol, which was not evident using the automated protocol. Three of these drugs (troglitazone, rosiglitazone, and diclofenac), all of which are metabolized to reactive intermediates, exhibited potentiated THLE-3A4 toxicity when tested using the manual protocol at 0.2% DMSO but not at 1% DMSO. Concentration-dependent suppression of several P450 Fig. 6. Association between THLE-Null cell toxicity and DILI. Drugs enzymes activities by DMSO has been observed (Easterbrook were incubated with the cells for 24 h in 1% DMSO (vol/vol) vehicle. Each data point represents the mean value obtained for a single drug in at least 3 inde- et al., 2001; Table 1), which implies a role for P450-dependent pendent experiments. Abbreviations: DILI, drug-induced liver injury; DMSO, metabolism in the toxicity as do the investigative studies dimethyl sulfoxide; THLE, T-antigen–immortalized human liver epithelial. undertaken with troglitazone. These identified a glutathione

Fig. 7. Association between potentiated drug toxicity to THLE-3A4 versus THLE-Null cells and DILI. Ratio = THLE-Null EC50/THLE-3A4 EC50. Drugs were incubated with the cells for 24 h in 1% DMSO (vol/vol) vehicle. Each data point represents the geomean value obtained for a single drug in at least 3 independent experiments. The symbols refer to the different DILI categories of the drugs (1. ● severe; 2. ○ high concern; 3. ■ DILI reports; 4. □ ALT elevations; 5. △ no liver signals). The dotted line at ratios of 1.4 and 0.7 denote the cutoff values for increased and decreased toxicity in the THLE-3A4 cells versus the THLE-Null cells. Abbreviations: ALT, alanine aminotransferase; DILI, drug-induced liver injury; DMSO, dimethyl sulfoxide; THLE, T-antigen–immortalized human liver epithelial. 208 Gustafsson et al.

Table 8 Correlation Between THLE Cell Toxicity and DILI

a DILI Category THLE-Null EC50 ≤ 200μM THLE-Null/THLE-3A4 EC50 Ratio ≥ 1.4

1. Severe (n = 24) n = 7 (29%) PPV 95% n = 3 (13%) PPV 91% 2. High concern (n = 27) n = 6 (22%) n = 3 (11%) 3. DILI reports (n = 32) n = 5 (16%) n = 4 (13%) 4. ALT elevations (n = 8) n = 0 (0%) NPV 23% n = 0 (0%) NPV 21% 5. No liver signals (n = 12) n = 1 (8%) n = 1 (8%)

NPV, negative predictive value; PPV, positive predictive value. aThe maximum soluble concentration of rimonabant was 30μM.

Fig. 8. Lack of correlation between toxicity to THLE cells and pattern of DILI reported in patients. Each data point represents an individual drug. A, Toxicity to THLE-Null cells. B, Ratio of toxicity to THLE-Null and THLE-3A4 cells. (Chol ● = cholestatic DILI; Hep □ = hepatocellular DILI; Mixed ∗ = mixed cholestatic/hepatocellular DILI.) The dotted line at ratios of 1.4 and 0.7 denote the cutoff values for increased and decreased toxicity in the THLE-3A4 cells versus the THLE-Null cells. Abbreviations: DILI, drug-induced liver injury; THLE, T-antigen–immortalized human liver epithelial. conjugate (implying formation of a reactive intermediate) in 4 of which have been reported to be metabolized by P450s to THLE-3A4 cells, but not THLE-Null cells, and demonstrated reactive intermediates) exhibited potentiated THLE-3A4 cell enhanced irreversible binding of radiolabel derived from trogl- toxicity in the presence of 1% DMSO when evaluated using itazone to THLE-3A4 cell proteins. A further 5 drugs (bosen- the manual protocol, but not using the automated protocol. tan, ketoconazole, amodiaquine, terbinafine, and propranolol; Because these drugs exhibit high plasma protein binding (> Drug Toxicity to THLE-CYP Cell Lines 209

in vivo at concentrations that are much higher than those in peripheral blood due to transport across the sinusoidal plasma membrane domain, which is mediated by solute carriers (Grime et al., 2008) not expressed in cultured THLE cells (Soltanpour et al., 2012) and first pass metabolism. Further work will be required to clarify the relationship between drug concentrations that cause THLE cell toxicity in vitro and within livers of patients in vivo. The marked THLE-3A4 cell toxicity of rimonabant, which was withdrawn due to severe behavioral adverse effects but did not cause DILI, highlights that care needs to be taken when extrapolating from in vitro toxicity signals observed with individual compounds to DILI hazard and risk in vivo. The esti-

Fig. 9. Comparison between Cmax,u, potency of THLE-3A4 cell toxicity mated plasma exposure of rimonabant was much lower than and DILI severity category. Each data point represents an individual drug (1. ● that of the other tested drugs due to its low PO daily dose of severe; 2. ○ high concern; 3. ■ DILI reports; 4. □ ALT elevations; 5. △ no liver 20 mg and its very high extent of plasma protein binding. This signals). Abbreviations: ALT, alanine aminotransferase; DILI, drug-induced liver injury; THLE, T-antigen–immortalized human liver epithelial. indicates that consideration of anticipated (or ideally measured) human exposure is required to aid interpretation of THLE cell toxicity data. 94%; see Supplementary Table 1) and 2.7% bovine serum was Our toxicity studies with 34 drugs did not reveal potentiated present when cells were incubated with test compounds in the THLE-1A2, THLE-2C9, or THLE-2D6 cell toxicity. However, automated protocol, but not in the manual protocol, binding to potentiated toxicity was observed when Huh7 cells transfected bovine serum proteins might help explain this finding. Other with CYP1A2 were exposed to furafylline, evoxine, and thi- differences between the protocols that could be relevant include merosal (Chu et al., 2011), whereas benzbromarone, losartan, the different cell seeding densities and ratios of cells to media and tienilic acid caused toxicity to HepG2 cells transfected with present in wells of 384well plates versus 96well plates. CYP2C9 when the Nrf2-dependent antioxidant response was Four drugs that exhibited potentiated THLE-3A4 cell tox- impaired by small inhibitory RNA knockdown (Iwamura et al., icity (amiodarone, clozapine, nefazodone, and troglitazone) 2011). Therefore, evaluation of additional drugs in the full have been reported to exhibit potentiated toxicity to other range of THLE-CYP cell lines may enable detection of drug human liver–derived and CYP3A4 transfected cell lines due to toxicity mediated by P450s other than CYP3A4. Undertaking CYP3A4-dependent metabolism (Hosomi et al., 2011; Vignati such studies using matched numbers of hepatotoxic and non- et al., 2005; Zahno et al., 2011). In addition, HepG2 cells hepatotoxic drugs should enhance understanding of the PPV transfected with multiple P450 enzymes (CYPA2, CYP2D6, and NPV of the data for human DILI. CYP2C9, CYP2C19, and CYP3A4) exhibited greater suscepti- Other investigators have observed a good correlation between bility to toxicity caused by chlorpromazine and diclofenac than toxicity to THLE-Null cells, and toxicity caused by test com- nontransfected HepG2 cells (Tolosa et al., 2013). Relatively pounds to various target organ systems in preclinical animal modest potentiation of drug toxicity to cells expressing P450s safety studies (Benbow et al., 2010). The relationship between was observed in these studies, which also is in line with our potentiated toxicity to THLE cells that express P450s and organ results. toxicity in animals was not explored in the present studies, but

The estimated EC50 values for THLE cell toxicity ranged merits investigation in the future. It will also be important to from low μM (eg, amiodarone, tolcapone) to > 100μM (eg, explore whether THLE cell toxicity data correlate with clini- clozapine, rosiglitazone). These concentrations are far higher cally important human adverse reactions to organs other than than the estimated Cmax,u concentrations of the majority of the the liver (eg, kidney or heart). drugs in human peripheral blood. However, our estimated EC50 A higher frequency of THLE cell toxicity was observed values assumed no binding of drugs to proteins or lipids; many among drugs categorized as Severe DILI than among drugs cat- of the drugs exhibit high plasma protein binding, and the auto- egorized as High DILI concern or DILI reports. However, the mated toxicity protocol included 2.7% FBS. Therefore, the true THLE cell toxicity data did not discriminate reliably between in vitro EC50 values could be much lower than the estimated drugs in the 3 DILI categories. They also did not differentiate values. It is also possible that our data have underestimated between drugs that caused hepatocellular, cholestatic, or mixed potencies of toxicity to liver cells in vivo, where DMSO is DILI patterns and provided only modest DILI sensitivity (ie, absent, due to suppression of P450 enzyme activity by DMSO many drugs that caused DILI did not exhibit THLE cell toxic- (Easterbrook et al., 2001) and because DMSO has antioxidant ity). THLE cells evaluate only some of the numerous adverse properties (Sanmartín-Suárez et al., 2011). Furthermore, many drug-related processes that may initiate DILI. Relevant addi- drugs and their metabolites accumulate within hepatocytes tional contributory mechanisms include mitochondrial injury, 210 Gustafsson et al. inhibition of the biliary transporters BSEP and Mrp2, and for- Benbow, J. W., Aubrecht, J., Banker, M. J., Nettleton, D., and Aleo, M. D. mation of reactive metabolites (Dawson et al., 2012; Greer (2010). Predicting safety toleration of pharmaceutical chemical leads: et al., 2010; Thompson et al., 2011). The value of multipara- Cytotoxicity correlations to exploratory toxicity studies. Toxicol. Lett. 197, 175–182. metric evaluation of DILI propensity has also been highlighted Bort, R., Castell, J. V., Pfeifer, A., Gómez-Lechón, M. J., and Macé, K. (1999). recently by other investigators (Sakatis et al., 2012). It has been High Expression of Human CYP2C in Immortalized Human Liver Epithelial proposed that THLE-Null and THLE-3A4 cell toxicity should Cells. Toxicol. In Vitro 13, 633–638. be evaluated alongside methods that assess these other mecha- Chen, M., Vijay, V., Shi, Q., Liu, Z., Fang, H., and Tong, W. (2011). FDA- nisms, when selecting drugs with reduced propensity to cause approved drug labeling for the study of drug-induced liver injury. Drug human adverse reactions (Thompson et al., 2012). The present Discov. Today 16, 697–703. results are consistent with this approach, which currently is Chu, C. C., Pan, K. L., Yao, H. T., and Hsu, J. T. (2011). Development of a being used within AstraZeneca to support compound selection whole-cell screening system for evaluation of the human CYP1A2-mediated metabolism. Biotechnol. Bioeng. 108, 2932–2940. during drug discovery. Coulet, M., Dacasto, M., Eeckhoutte, C., Larrieu, G., Sutra, J. F., Alvinerie, M., Although the automated protocol enabled convenient Macé, K., Pfeifer, A. M., and Galtier, P. (1998). 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