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May 30, 2013 - Department of Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, Califor...
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Bioactivation of Sitaxentan in Liver Microsomes, Hepatocytes, and Expressed Human P450S with Characterization of the Glutathione Conjugate by Liquid Chromatography Tandem Mass Spectrometry John C. L. Erve,*,†,§ Shawn Gauby,†,∥ John W. Maynard, Jr.,†,⊥ Mats A. Svensson,‡ George Tonn,†,⊥ and Kevin P. Quinn† †

Department of Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, California 94080, United States ‡ Schrödinger, Inc., 101 SW Main Street, Portland, Oregon 97204, United States ABSTRACT: Sitaxentan is a selective endothelin-A receptor antagonist that was marketed as Thelin in several European countries and Canada for pulmonary arterial hypertension. Sitaxentan was undergoing further clinical trials in the United States but due to four deaths and one case of liver transplantation from severe liver toxicity that appeared to be idiosyncratic in nature, it was withdrawn worldwide in December, 2010. Sitaxentan contains a 1,3-benzodioxole ring that undergoes enzymatic demethyleneation to an orthocatechol metabolite that can further oxidize to a reactive orthoquinone metabolite. Here, we report the detection and mass spectral characterization of a glutathione conjugate of this sitaxentan quinone reactive metabolite that was trapped in vitro using mouse, rat, dog, and human liver microsomes supplemented with NADPH and glutathione and that was also observed in rat and human hepatocytes. Using human liver microsomes, we also demonstrated that P450 3A4 undergoes time-dependent inhibition. Density functional calculations on the catechol metabolite of sitaxentan indicated that the reaction leading to the quinone was thermodynamically favorable with an enthalpy change of −6.3 kcal/mol. Using density functional methodology, we modeled the attack of glutathione on the quinone with an S-methyl thiolate anion which allowed us to predict, based on the difference in transition state energies, that the 2-position on the phenyl ring was more likely than the 5-position as the site of glutathione conjugation. Overall, our results demonstrated that sitaxentan is capable of facile formation of a reactive ortho-quinone metabolite capable of reacting with glutathione and may rationalize the idiosyncratic nature of the hepatotoxicity that led to its withdrawal.

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INTRODUCTION Sitaxentan, N-(4-chloro-3-methyl-isoxazol-5-yl)-2-[2-(6-methyl-1,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide, is a selective endothelin-A receptor antagonist that was marketed as Thelin in several European countries and Canada for pulmonary arterial hypertension.1 Sitaxentan demonstrated beneficial effects on exercise capacity as measured by the 6min walk distance test as well as improved hemodynamic parameters in pulmonary arterial hypertension patients and appeared to have a lower incidence of liver function test abnormalities than the FDA-approved endothelin-A receptor antagonist bosentan (Tracleer).2 Sitaxentan was undergoing further clinical trials in the United States but due to eight deaths from severe liver toxicity that appeared to be idiosyncratic in nature, it was withdrawn worldwide by Pfizer in December, 2010.3 Moreover, in contrast to bosentan, some of the reported cases of liver toxicity caused by sitaxentan were characterized by elevated bilirubin levels with histological evidence of lymphocytes and eosinophils, which are typical of idiosyncratic liver toxicity.4 Ambrisentan (Letaris), the newest endothelin-A receptor antagonist is a propanoic acid and has a © XXXX American Chemical Society

lower risk of liver toxicity than either bosentan or sitaxentan, which are sulfonamides.5 The structures of these endothelin-A receptor antagonists are shown in Scheme 1. Idiosyncratic drug toxicity, in particular, idiosyncratic liver toxicity remains a leading cause of drug related deaths in patients as well as a leading cause of drug withdrawal.6 Much circumstantial evidence implicates reactive metabolites as a common mechanistic link since many drugs associated with idiosyncratic drug toxicity that affect the liver, as well as other organs, are metabolized to reactive metabolites.7,8 Cytochrome P450 enzymes metabolize a large proportion of marketed drugs and may also bioactivate a drug to a reactive metabolite with electrophilic properties. An often invoked mechanism of idiosyncratic drug toxicity, the “Hapten Hypothesis”, posits that a reactive metabolite modifies susceptible nucleophilic sites on proteins, which initiates a damaging immune response.9,10 Because of their initial role in drug metabolism, P450s can often be targets of the reactive metabolites they generate, which may Received: March 18, 2013

A

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Scheme 1. Structures of the Endothelin-A Receptor Antagonists

result in the formation of autoantibodies,11 and/or cause mechanism based inhibition (MBI).12 Regarding MBI of P450s by drugs, many of the offending molecular features are recognized and include the 1,3-benzodioxazole moiety as found in sitaxentan.13 In light of these considerations, we wondered whether the idiosyncratic hepatotoxicity caused by sitaxentan could be explained, at least in part, by the formation of a reactive metabolite. Thus, the purpose of the present work was to investigate the in vitro metabolism of sitaxentan with particular focus on the generation of reactive metabolites. To this end, we used liver microsomes from mice, rats, dogs, and humans, as well as rat and human hepatocytes and human expressed P450s to generate a GSH conjugate which was characterized using LC/MS/MS. We also investigated the ability of sitaxentan to inhibit P450 enzymes and cause time-dependent inactivation (TDI) of P450s 3A4 and 2C9 using pooled human liver microsomes. In addition, we performed density functional calculations to support the observed facile conversion of the catechol metabolite to the quinone reactive metabolite and to propose the likely site of conjugation with GSH.

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mM) together with either mouse, rat, dog, or human liver microsomes (1.0 mg/mL), NADPH (1 mM), and GSH (13C15N-GSH:GSH ∼1:1, 5 mM), at 37 °C with gentle shaking for 30 min. The concentration of ACN in the incubation mixture was less than 0.5%. Incubations of Sitaxentan with Supersomes Containing cDNA-Expressed P450 Enzymes. Fifty picomole of cDNAexpressed P450 3A4 and 2C9 were incubated together with, sitaxentan (25 μM), NADPH (1 mM), and GSH (13C215N-GSH/GSH ∼1:1,5 mM) at 37 °C for 30 min. Microsomal Sample Preparation. At the end of the microsomal or Supersome incubations, 240 μL of ice-cold ACN/MeOH (1:1, v/v) was added to each incubation followed by vigorous mixing and centrifugation at 16,100g for 10 min. After centrifugation, the supernatant was transferred to autosampler vials with polypropylene inserts. Studies with Cryopreserved Rat and Human Hepatocytes. The cells were stored in liquid nitrogen until use. Immediately before use, vials of hepatocytes were rapidly thawed in a water bath (37 °C, < 2 min) and then quickly transferred to 50 mL of recovery media at 37 °C. The hepatocyte suspension was centrifuged at 100g for 10 min at room temperature, after which the supernatant was removed by aspiration. The cell pellet was resuspended in 2 mL of plating media containing 10% fetal bovine serum. Sitaxentan stock solution (40 μM in Krebs−Henseleit buffer for rats or Williams’ Eagle’s Media for humans) was added to the hepatocytes to give a final concentration of 13 or 20 μM, respectively, and placed in a 5% CO2 humidified incubator at 37 °C for 2 h with gentle shaking. A control prazosin stock solution (40 μM in Williams’ Eagle’s Media) was added to rat hepatocytes to give a final concentration of 20 μM. The cell number and viability were determined on a Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA). Incubations (400−600 μL) were performed in 1.5 mL Eppendorf tubes (for rats, approximately 3.1 million cells/mL, 84% viability; for humans, approximately 0.8 million cells/mL, 53% viability at time 0 h) for 2 h. Control rat hepatocyte incubations were assessed at 1 and 2 h and found to contain approximately 1.2 million cells/mL and 0.8 million cells/mL with viabilities of 78% and 50%, respectively. Control human hepatocyte incubations were assessed at 2 h and found to contain approximately 1.0 million cells/mL with viabilities of 31%. Hepatocyte Sample Preparation. At the end of the hepatocyte incubations, an equal volume of ice-cold 50:50 ACN/MeOH (v/v) was added, and the mixture was first vortexed and then centrifuged (1,640g for 10 min) to pellet the cells. The supernatant was transferred to a new Eppendorf tube, and the pellet was re-extracted (twice) with 500 μL of MeOH with sonication for 5 min. After each centrifugation, the supernatants were combined, and the volumes were reduced to approximately 100 μL under nitrogen gas at 20 °C prior to LC/MS analysis. In Vitro Competitive Inhibition. Sitaxentan and control inhibitors, terfenadine and sertraline, were incubated with pooled human liver microsomes at 37 °C based on a published method with slight modifications.14 The final protein concentration was 0.02 mg/ mL for all incubations in a buffer containing 0.1 mM potassium phosphate buffer (pH 7.4), 3.3 mM MgCl2, and 1.3 mM NADPH. Test compounds were incubated over an 11 point dose−response from 0.5 to 100 μM in the presence of either the probe substrates

MATERIALS AND METHODS

Sitaxentan sodium (purity 98.4%) was obtained from A Chemtek, Inc. (Worcester, MA). Terfenadine, sertraline, sulfaphenazole, GSH, NADPH, potassium phosphate monobasic, magnesium chloride, acetaminophen, dextrorphan, phenacetin, bupropion, pacitaxol, diclofenac, (S)-mephenytoin, dextromethorphan, Krebs-Henseleit buffer, and Williams Eagle Media were all purchased from SigmaAldrich Chemical Co. (St. Louis, MO). Liquid Chromatography−Mass Spectrometry (LC/MS) grade acetonitrile (ACN), methanol (MeOH), and Ultra Resi-analyzed water were obtained from J.T. Baker (Phillipsburg, NJ). Formic acid was obtained from Thermo (Pierce) (Rockford, IL). Stable isotope labeled GSH (glycine-13C2, 15 N, 65−70% purity) and deuterium oxide (99.8% purity) were purchased from Cambridge Isotope Laboratories (Andover, MA). Pooled liver microsomes from male CD-1 mice, male Sprague− Dawley rats, male beagle dogs, and mixed gender human liver microsomes (Ultra Pool HLM 150) were purchased from BD Biosciences (Woburn, MA). BD-Supersome (cDNA-expressed cytochrome P450 enzymes) 3A4 and 2C9 were purchased from BD Biosciences (Woburn, MA). Pooled rat cryopreserved hepatocytes (Lot # 2178746) and human cryopreserved hepatocytes from a male donor (Lot # 116, donor HH197) as well as CryoHepatocyte recovery media and CryoHepatocyte plating media were from BD Biosciences (Woburn, MA). Hydroxybupropion, [ 2H5] hydroxybupropion, [13C215N] acetaminophen, 6-α-hydroxypacitaxol, [2H5] 6-α-hydroxypacitaxol, 4′-hydroxydiclofenac, [13C6]-4′-hydroxydiclofenac, (S)-4′hydroxymephenytoin, [2H3] (S)-4′-hydroxymephenytoin, and [2H3] dextrorphan were purchased from BD Biosciences (Woburn, MA). All other reagents were of analytical grade. Incubations of Sitaxentan in Mouse, Rat, Dog, and Human Liver Microsomes with GSH. Sitaxentan (25 μM) was incubated in potassium phosphate buffer (50 mM, pH 7.4) containing MgCl2 (3 B

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Figure 1. Extracted ion chromatograms for sitaxentan and metabolites M1, M2, and M3 produced in liver microsomal incubations containing GSH from mice (A), rats (B), dogs (C), or humans (D). phenacetin (10 min incubation at 40 μM for P450 1A2 activity), bupropion (5 min at 80 μM for P450 2B6), pacitaxol (10 min at 10 μM for P450 2C8), diclofenac (5 min at 5 μM for P450 2C9), (S)mephenytoin (10 min at 40 μM for P450 2C19), dextromethorphan (5 min at 5 μM for P450 2D6), midazolam (5 min at 3 μM for P450 3A4), and testosterone (10 min at 50 μM for P450 3A4). Test compounds were prepared at 10 mM in ACN and then serially diluted into ACN to prepare a 100× stock for a final organic concentration of 1% in all incubations. All steps were performed on a Hamilton MicroLab STAR (Hamilton Company, Reno, NV) with CAT heater− shakers (CAT, Staufen, Germany). Each reaction was terminated with ACN and formic acid containing a stable-label isotope specific for each probe substrate. Samples were centrifuged at 1,900g on an Allegra 6R centrifuge (Beckman-Coulter, Fullerton CA) for 15 min at 4 °C, and the supernatants were analyzed for the formation of probe specific metabolites by LC/MS/MS. Peak areas were integrated with Analyst software, and the resulting metabolite to IS ratios were exported to XLFit Excel Add-in v4.3.1 (ID Business Solutions, LTD, Alameda, CA) for curve fitting and IC50 determinations by nonlinear regression with 4 Parameter Logistic Model. In Vitro Time Dependent Inactivation of P450 3A4 and P450 2C9. Sitaxentan was tested using the IC50 shift method for P450 3A4 and P450 2C9 as described previously.15 Briefly, compounds were spiked into 0.2 mg/mL pooled human microsomal protein at final concentrations of test compounds ranging from 0.5 to 100 μM. NADPH or incubation buffer was added and samples were preincubated for 30 min at 37 °C on a Hamilton MicroLab STAR. Following the preincubation step, microsomes were diluted 10-fold in buffer followed by the addition of NADPH and either midazolam or diclofenac as probe substrates. The second incubation was allowed to proceed for 5 min for both substrates before the reaction was

terminated. The reaction was stopped with ACN containing formic acid and stable-label isotopes as the internal standard for the particular probe substrate of interest and centrifuged as described above. The resulting supernatant was then analyzed for the formation of probe substrate specific metabolites by LC/MS/MS. The IC50 values were calculated as described above using 4 Parameter Logistic Model (XLFit Excel Add-in v4.3.1), and the shift was calculated from the ratio of the preincubation in the absence of the cofactor NADPH by preincubation in the presence of NADPH. Verapamil was used as the positive control for P450 3A4 with terfenadine as the negative control. Ticrynafen was used as the positive control for P450 2C9, and sulfaphenazole was the negative control. Liquid Chromatography Conditions. Metabolite characterization was performed on an Agilent 1290 Infinity binary pump (Agilent Technologies, Santa Clara, CA), and a PAL HTC-xt autosampler (LEAP Technologies, Carrboro, NC) was coupled to the mass spectrometer described below. Separations were accomplished on either a Poroshell 120 SB-C18 column (2.1 × 150 mm, 2.7 μm) (Agilent Technologies, Santa Clara, CA) or a Zorbax Eclipse XDB-Phenyl column (4.6 × 250 mm, 5 μm) (Agilent Technologies). The sample chamber in the autosampler was maintained at 4 °C, while the column was at ambient temperature of approximately 22 °C. For the Poroshell column, the mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B) and was delivered at 700 μL/min. The gradient started at 3% B and after 0.5 min was increased to 95% B over 15 min, maintained at this composition for 1.4 min before returning to initial conditions at 17 min. The total run time was 20 min. For the phenyl column, the mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in MeOH (B) and was delivered at 1 mL/min. The gradient started at 5% B and after 1.0 min was increased to 98% B over 20 min, maintained at this C

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1). LC/MS analysis indicated the presence of [M+H]+ ions at m/z 471 (M1, dog only), m/z 443 (M2), and m/z 748 (M3, GSH adduct), and m/z 455 (sitaxentan). Following incubation for 1 h, sitaxentan remained the largest component in all species based on mass intensity and UV response (λ = 260 nm). Metabolism of Sitaxentan with NADPH and GSHFortified Human P450 3A4 and P450 2C9 Supersomes. Sitaxentan incubated with supersomes fortified with NADPH and GSH revealed qualitatively similar metabolite profiles among the various P450s (data not shown). After 30 min of incubation with P450 2C9, sitaxentan remained the major component with both the phase 1 metabolite M2 and the GSH adduct M3 readily detected in the extracted ion chromatogram (XIC). After 30 min of incubation with P450 3A4, sitaxentan remained the major component, but only the GSH adduct, M3, was readily detected in the XIC. Regarding the GSH conjugate, the amount of GSH conjugate formed (based on peak height) was about the same with both P450 3A4 and P450 2C9 supersomes. In these incubations, high quality product ion spectra of M2 and the M3 were collected (data not shown) and revealed the same key product ions as obtained in the microsomal incubations as discussed below. Metabolism of Sitaxentan in Cryopreserved Rat and Human Hepatocytes. In rat or human cryopreserved hepatocytes, LC/MS analysis revealed sitaxentan and the GSH conjugate, M3, with trace amounts of M2 at the same retention times as observed in the microsomal incubations. The profiles produced by rat hepatocytes and human hepatocytes were very similar (data not shown), although the metabolism appeared to be less in humans than in rats. Compared to microsomes, the oxidative metabolite, M2, and the GSH conjugate, M3, were less abundant. There was no evidence of other conjugated metabolites derived from sitaxentan. Identification of Sitaxentan Metabolites. Sitaxentan had an HPLC retention time of about 20.1 min and showed a protonated molecular ion ([M+H]+) at m/z 455.0121, which is consistent with the elemental composition C18H16ClN2O6S2 (mass error 1.2 ppm). Loss of methylchloride from the molecular ion generated m/z 404. Cleavage at the sulfonamide bond with charge retention on the sulfonyl group generated m/z 323, representing the thiophene−benzodioxole system. Further loss of H2O or C2H2O2 from m/z 323 produced ions at m/z 304 and 277, respectively. Cleavage of the benzyl− carbonyl bond generated the product ion at m/z 149 representing the benzodioxole moiety. Cleavage of the sulfonamide bond with charge retention on the amino-group yielded the product ion at m/z 133, representing the chloromethyl isoxazole ring. Cleavage of the thiophene sulfone bond with loss of formaldehyde generated the product ion at m/z 229. LC/MS conducted with D2O revealed an [M+D]+ at m/z 457 indicating one exchangeable proton. The proposed fragmentation scheme and product ions of m/z 455 for sitaxentan are shown in Figure 2. M1. This metabolite was present in dog only and had a retention time of about 18.2 min on the HPLC system. The [M+H]+ was observed at m/z 471.0082, which is consistent with the elemental composition C18H16ClN2O7S2 (mass error 0.7 ppm), and is also16 Da larger than sitaxentan, indicating it had undergone mono-oxidation. Loss of methylchloride from the molecular ion generated m/z 420. The product ions at m/z 323, 304, and 277 were the same as sitexentan indicating an unchanged thiophene and benzodioxole rings. However, the

composition for 4.0 min before returning to initial conditions at 25.1 min. The run time was 26 min, with 5 min of postrun equilibration time. For hydrogen−deuterium exchange experiments, deuterium oxide was substituted for water in mobile phase A. CYP inhibition experiments were performed with the same chromatography hardware, but separations were conducted using an Eclipse Plus C18 column (2.1 × 30 mm and 3.5-um particle size, Agilent, Santa Clara, CA) with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B) and was delivered at 600 μL/min. The gradient started at 3% B, briefly held isocractically for 0.5 min, and then increased to 85% B over 2.2 min, maintained at this composition for 0.6 min before returning to initial conditions at 3.4 min. The total run time was 5 min. Mass Spectrometry Analysis. Metabolite characterization was conducted on an AB Sciex TripleTOF 5600 (triple quadrupole/timeof-flight) mass spectrometer (AB Sciex, Framingham, MA). It was equipped with a TurboV dual electrospray ionization/APCI source and operated in the electrospray positive ionization mode. Full scan data was collected from 50 to 1000 Da with a scan time of 0.25 s. Product ion spectra were collected for m/z 443, 455, 471, and 748, with a scan time of 0.3 s for each scan. A scan range of 50 to 500 Da was used for the 443 and 471 precursor ions, and a range of 50 to 800 Da was used for the 748 Da precursor ion. The IonSpray voltage was 5500 V, and the source temperature was 550 °C. The nebulization (GS1) and drying gas (GS2) flow were set to 40 and 50, respectively, and the curtain gas was set to 25. The declustering potential was set at 91, and the collision energy was set to 10 V for the TOF full scan, and for the product ion scans, 40 V, with a 10 V spread, was used for the 443 and 471 Da precursor ions, and 45 V, with a 25 V energy spread, was used for the 748 precursor ion. The TripleTOF was calibrated by the integrated calibration delivery system using the manufacturer’s positive calibration solution, with an injection flow rate of 300 μL/min, introduced via the APCI arm of the dual source. Calibration was performed at the start of every batch and repeated every 10 samples. Full-scan MS data was examined manually to look for possible metabolic transformations of the drug. Data acquisition was performed using Analyst TF software (version 1.5.1, AB Sciex), and data processing was performed using PeakView (version 1.1.1.2, AB Sciex). Inhibition experiment samples were analyzed on an AB Sciex Q-trap 5500 hybrid triple quadrupole/linear ion trap mass spectrometer with a Turbo V electrospray ion source (Applied Biosystems/MDS Sciex, Foster City, CA). All samples were detected in the positive ion mode and optimized with Analyst 1.5.2 software (Applied Biosystems/MDS Sciex). The ion spray was set to 5500 V, and the source temperature was 550 °C. Mass transitions for each probe substrate metabolite (acetaminophen, 152→110; hydroxybupropion, 256→238; 6-αhydroxy paclitaxol, 870→286; 4′-hydroxydiclofenac, 312→230; 4′hydroxymephenytoin, 312→230; and 4′-hydroxydiclofenac, 312→ 230) and their respective internal standard (IS) were monitored for 50 ms. Peak areas were integrated with Analyst software and then exported to Excel (Microsoft, Seattle WA). Density Functional Calculations. Geometries (bond lengths, bond angles, and torsional angles) of the structures were fully optimized in the gas phase and for some structures also in water. Relative electronic energies of the optimized structures were calculated as previously described for remoxipride16 using the gradient corrected hybrid density functional method B3LYP and the 6-31G** basis set, while solution phase transition energies were derived from single point calculations on the previously optimized structures, using the standard Poisson−Boltzmann continuum solvation model as implemented in the Jaguar program (version 8.0, Schrödinger LLC, New York, NY).

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RESULTS Metabolism of Sitaxentan in NADPH and GSHFortified Mouse, Rat, Dog, and Human Liver Microsomes. Incubation of sitaxentan with liver microsomes fortified with NADPH and GSH revealed qualitatively similar metabolite profiles among various species as judged by the extracted ion chromatogram of the incubation mixtures (Figure D

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Figure 2. Product ions of m/z 455 mass spectrum of sitaxentan and the proposed origin of key product ions.

Figure 3. Product ions of m/z 471 mass spectrum of M1 and the proposed origin of key product ions.

ion at m/z 133 was shifted 16 Da higher to m/z 149, which indicated hydroxylation had occurred on the chloromethyl isoxazole ring. LC/MS conducted with D2O produced an [M+D]+ at m/z 474 and indicated two exchangeable protons, one more than sitaxentan, consistent with hydroxylation (data not shown). Therefore, M1 was tentatively identified as hydroxy-sitaxentan, and the proposed fragmentation scheme and product ions are shown in Figure 3. M2. This metabolite had a retention time of about 17.1 min on the HPLC system. The [M+H]+ was observed at m/z 443.0131, which is consistent with the elemental composition C17H16ClN2O6S2 (mass error −0.5 ppm), and is also 12 Da less than that for sitaxentan, suggesting it was formed by demethylenation. Loss of methylchloride from the molecular ion generated m/z 392. The product ion at m/z 133, identical to sitaxentan, indicated an unchanged chloromethyl isoxazole ring. The ion at m/z 149 representing the benzodioxole moiety was absent, and a new peak 12 Da lower at m/z 137 was observed consistent with demethylenation. Product ions at m/z’s 323 and 305 in sitaxentan were also shifted 12 Da lower to m/z 311 and 292, respectively. LC/MS conducted with D2O produced an [M+D]+ at m/z 447 and indicated three exchangeable protons, two more than sitaxentan (data not shown). Therefore, M2 was tentatively identified as desmethylene sitaxentan, and the proposed fragmentation scheme and product ions are shown in Figure 4. M3, GSH Adduct. The GSH adduct had a retention time of about 5.4 min on the HPLC system. The [M+H]+ was observed at m/z 748.0818, which is 305 Da larger than M2 suggesting that it was a GSH adduct and consistent with the elemental composition C27H30ClN5O12S3 (mass error −0.48 ppm). In addition, the isotope pattern of the protonated molecule showed a profile consistent with the presence of

Figure 4. Product ions of the m/z 443 mass spectrum of M2 and the proposed origin of key product ions.

heavy labeled GSH (i.e., [13C2 15N]-GSH, a peak 3 Da larger than [M+H]+ with a relative intensity of approximately 50% that of [M+H]+). Loss of glycine or glutamic acid from E

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[M+H]+ generated product ions at m/z 673 and m/z 619, respectively. The product ion at m/z 133, identical to sitaxentan, indicated an unchanged chloromethyl isoxazole ring. Cleavage of the carbon−sulfur bond in GSH with sulfur retained on the catechol produced m/z 473, which subsequently lost H2O to generate ions at m/z 455 and 437, respectively. LC/MS conducted with D2O revealed an [M+D]+ at m/z 758, which indicated nine exchangeable protons, consistent with a GSH adduct. Therefore, M3 was tentatively identified as the GSH conjugate of M2, and the proposed fragmentation scheme and product ions are shown in Figure 5.

sitaxentan did not inhibit P450 1A2, P450 2B6, or P450 2D6 activity significantly at 100 μM and was a weak inhibitor of P450 3A4 with IC50 values of 28.1 μM and 33.1 μM for 1′-OH midazolam and 6β-OH testosterone formation, respectively. Sitaxentan was a modest inhibitor of P450 2C8 and P450 2C19 with IC50 values of 1.58 μM and 3.51 μM for the formation of 6-α-hydroxy pacitaxol and 4′-OH S-mephenytoin, respectively. However, sitaxentan, was a potent inhibitor of P450 2C9 activity with an IC50 suggested to be less than 0.5 μM, the lowest concentration tested. Time Dependent P450 Inhibition by Sitaxentan. Sitaxentan was also tested for time dependent inactivation (TDI) of P450 2C9 and P450 3A4 to identify mechanism based inhibition (MBI) using the IC50 shift method. Results for both isoforms are shown in Figure 6. The IC50 shift toward inhibiting

Figure 5. Product ions of the m/z 748 mass spectrum of M3, the GSH adduct, and the proposed origin of key product ions.

Figure 6. Inhibition (IC50) shift plots of P450 2C9 (A) and P450 3A4 (B) following a 30-min pretreatment in the presence (■) and absence (○) of NADPH with sitaxentan. A negligible IC50 shift (0.63) for P450 2C9 based on the formation of 4-hydroxy-diclofenac following pretreatment in the presence of NADPH (IC50 = 0.058 μM) compared to its absence (IC50 = 0.093 μM) suggest that sitaxentan is not a MBI of P450 2C9 but is a potent competitive inhibitor. In contrast, a robust IC50 shift (>5.6) for the P450 3A4 based formation of 1′-hydroxy-midazolam following pretreatment in the presence of NADPH (IC50 = 1.79 μM) compared to its absence (IC50 = >10 μM) suggests that sitaxentan is a strong MBI of P450 3A4 but a weak competitive inhibitor.

In Vitro Competitive Inhibition. Sitaxentan was tested for its ability to competitively inhibit P450 activity using isoform specific substrates in human liver microsomes in a dose− response fashion from 0.5 μM−100 μM of sitaxentan and two controls: terfenadine and sertraline (Table 1). In our system, Table 1. Competitive CYP Inhibition of Sitaxentan and Controls CYP isoform

substrate

sitaxentan IC50 (μM)

terfenadine IC50 (μM)

sertraline IC50 (μM)

CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4

phenacetin bupropion pacitaxol diclofenac (S)-mephenytoin dextromethorphan midazolam testosterone

>100 >100 1.6 100 28.1 33.1

>100 29.1 19.1 22.2 22.2 2.8 5.6 8.3

>100 6.3 29.7 >100 6.9 29.7 44.9 52.9

the formation of the P450 2C9 specific 4-hydroxy-diclofenac was 0.63, as shown in 6A, suggesting that sitaxentan is a negligible mechanism based inhibitor of P450 2C9. However, its potency with respect to its competitive inhibition was supported by the measured IC50 of 0.058 μM in the absence of NADPH in preincubation. In contrast, sitaxentan was a TDI of P450 3A4 where an IC50 shift of greater than 5.5-fold was observed. The IC50 for the formation of 1′-hydroxy-midazolam without NADPH in preincubation was greater than 10 μM, F

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Scheme 2. Proposed Transition States Formed by the Reaction of Quinone with -SCH3 and Their Relative Energy Difference

Scheme 3. Proposed Metabolic Pathways of Sitaxentan in Liver Microsomes Supplemented with Glutathione

whereas with NADPH in preincubation the IC50 was 1.79 μM as shown in Figure 6B. Density functional Calculations. The relative electronic energies (ΔE) defined by the oxidation reaction of the catechol

containing sitaxentan metabolites, M2 and M3, leading to the corresponding ortho-quinone and water (i.e., catechol +1/2 O2 + ΔE → quinone + H2O) were calculated using density functional theory. To identify the most likely site of GSH G

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hepatocyte studies, which may be due to experimental differences between our assay conditions or the expression of drug metabolizing enzymes in the specific batches of hepatocytes used. In addition to these potential factors, it has been suggested that the use of hepatocytes with greater than 75% viable cells is recommended, which may have contributed to the differences between the current work and Stavros’ observations of sitaxentan biotransformation.27 However, using prazosin as a positive control demonstrated that our rat hepatocytes did have glucuronidation activity as a known glucuronide metabolite of prazosin was observed. In light of these issues, our ongoing studies are employing newer hepatocyte models to determine a more complete metabolic profile of sitaxentan. The structure of the GSH conjugate, M3, was characterized by mass spectrometry, and the product ions produced by fragmentation indicated that the phenyl ring, rather than the thiophene or isoxazole ring, was the site of conjugation consistent with a 1,6 Michael addition reaction. However, as the observed mass spectrometric fragmentation did not allow discrimination between the 2- or 5-position as the site of conjugation, we used density functional methods, which led us to propose the 2-position in the phenyl ring as the most likely site of GSH conjugation. We modeled the nucleophilic attack of GSH on the quinone using an S-methyl anion, i.e., an unprotonated methyl sulfhydryl, and determined the relative energy difference between the corresponding transition states (see TS “A” and “B” in Scheme 2). Using this approach, the relative energy difference between the two possible TS following S-methyl conjugation was 2.8 kcal/mol with the 2position (leading to TS “A”) on the phenyl ring more favorable than the 5-position (leading to TS “B”) suggesting that the structure of the GSH conjugate (M3) that we indentified was at the 2-position (see Figure 5 and Scheme 2). The calculated energy difference between TS “A” and TS “B” implies an approximate selectivity of 100-fold.28 Recently, a computational study calculated the activation energy, ΔG⧧, for the reaction between GSH and bisphenol A-3,4-quinone,29 although they did not address the selectivity, which was our objective. We did attempt to obtain an NMR spectrum of the GSH conjugate that would allow experimental determination of the site of conjugation by conducting an HPLC purification of M3 from a large batch human liver microsomal incubation. Although we succeeded with the initial purification of M3, subsequent efforts proved fruitless, ostensibly due to the stability of M3, which prevented a quality NMR spectrum from being collected. The instability of the isolated adduct may be a reflection of our calculations that indicated that further oxidation of M3 was thermodynamically favorable (−6.9 kcal/mol), so it is highly possible that M3 underwent further intramolecular chemistry to form a cyclic structure analogous to the 1,4-benzothiazine formation observed with the GSH conjugates of bromo-3(glutahion-s-yl)hydroquinone.30 Indeed, it has been reported that thiol conjugates are often more prone to oxidation than the original nonconjugated species because the electron-deficient quinone can be stabilized by the sulfanyl ring substituent,31 which is consistent with our calculations for the reoxidation of M3. Our modeling that we performed in our work illustrates the value of computational methods to allow a rational choice for the likely site of conjugation to be made in the absence of NMR data. In addition to sitaxentan, bosentan (Tracleer) and ambrisentan (Letaris) are endothelin-A receptor antagonists

conjugation on the phenyl ring, the nucleophilic attack of GSH was modeled using an S-methyl anion and comparing the difference in energies between the transition states (TS) after the S-methyl anion attack on either the 2- or 5-position, i.e., before protonation and collapse to the product (see Scheme 2). The relative electronic energies of the reactions of M2 leading to the ortho-quinone was −6.3 kcal/mol. These results indicate that oxidation of M2 to the ortho-quinone is thermodynamically possible. Reoxidation of the GSH conjugate, M3, to the ortho-quinone was also thermodynamically favorable as the relative electronic energy of the reaction of M3 to the corresponding ortho-quinone was calculated to be −6.9 kcal/mol. Regarding the site of GSH conjugation, TS “A” resulting from S-methyl anion attack on the 2-position of the phenyl ring was found to be favored by 2.8 kcal/mol over TS “B” formed by attack at the 5-position of the phenyl ring as depicted in Scheme 2.

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DISCUSSION Sitaxentan was withdrawn voluntarily from the worldwide market in 2010 by Pfizer due to increasing evidence that it could cause serious hepatotoxicity in susceptible individuals.3 Of the approximately 2,000 patients treated with sitaxentan, at least four patients had died due to liver failure, and one required liver transplantation.17 On the basis of the clinical findings in these case reports, heptotoxicity had the hallmarks of an idiosyncratic nature, such as increased alanine amino transferases together with markedly elevated bilirubin levels (>300 μM/L), eosinophil and lymphocyte infiltration and a positive response to glucocorticoid treatment suggesting immune system involvement.4 Idiosyncratic hepatotoxicity remains a concern for both pharmaceutical firms producing new chemical entities and doctors prescribing these agents as it can cause serious harm to patients and may lead to the significant labeling changes or market withdrawal. A mechanistic explanation for indiosyncratic hepatotoxicity often invokes the metabolism of a drug to a reactive metabolite capable of combining chemically with critical biological macromolecules.18 Many drugs that are associated with idiosyncratic toxicity have been shown to generate reactive metabolites.19 Sitaxentan contains a 1,3-benzodioxole moiety that has the potential to undergo metabolism to an orthocatechol metabolite that can subsequently oxidize to a reactive ortho-quinone. Other drugs that contain this toxicophore include the antidepressant drug, paroxetine, which is associated with idiosyncratic hepatotoxicity,20,21 the nootropic drug, fipexide,22 which was withdrawn due to hepatotoxicity,23 and the designer drug Ecstasy, which can cause hepatotoxicity in recreational drug abusers. Paroxetine,24 fipexide,22 and Ecstasy25 all form thiol GSH conjugates of ortho-quinone metabolites. In this study, we demonstrated that the orthocatechol metabolite of sitaxentan formed by mouse, rat, dog, and human liver microsomes as well as by rat and human hepatocytes can oxidize to the corresponding ortho-quinone metabolite and form an adduct with GSH (Scheme 3). Regarding the metabolism of sitaxentan in human and rat hepatocytes, our results indicated the presence of both M2 and M3, albeit at low abundance. A poster abstract by Stavros et al. described the observation of the catechol, i.e., M2, which was subsequently methylated, glucuronidated, or sulfated but not trapped with GSH, in mouse, rat, dog, and human hepatocytes, although experimental details were not provided.26 We did not observe these conjugated sitaxentan metabolites of M2 in our H

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spectrometry as has been done for raloxifene, a mechanismbased inhibitor of P450 3A4.43 Despite the TDI of P450 3A4 and potent competitive inhibition of P450 2C9, no clinically relevant drug−drug interactions (DDIs) requiring dose adjustment were noted in the product insert for the P450 3A4 substrates cyclosporine, ketoconazole, and sildenafil or the P450 2C9 substrates omeprazole and warfarin.44,45 The lack of clinically relevant DDIs may be due to concurrent induction of P450 3A4 by sitaxentan.46 The investigational endothelin-A receptor antagonist CI-1034, which also contained a benzodioxole system, both inhibited P450 3A4 and induced P450 3A4 mRNA levels 15-fold, and it was suggested that these effects could cancel so that a clinical result would not be observed, although this was not verified by an actual clinical trial.47 Empirical evidence suggests that drugs given at doses of greater than 10 mg per day have an increased risk of causing idiosyncratic liver toxicity.48 Sitaxentan was approved at a daily dose of 100 mg/day, and during clinical studies, a dose of 300 mg/day was also used. Both of these doses caused idiosyncratic hepatotoxicity in humans including several cases in which patients died. For comparison, bosentan dosing typically starts at 62.5 mg twice a day for four weeks before switching to a maintenance dose of 125 mg/day. Although higher than 10 mg/day, no evidence of reactive metabolite formation has been reported nor is the hepatotoxicity idiosyncratic in nature. In contrast to sitaxentan or bosentan, the newest endothelin receptor antagonist, ambrisentan, is more potent, and a 5 mg/ day dose is effective. Although no reactive metabolites of ambrisentan have been reported, their formation would not be expected to be of clinical significance, and indeed, no evidence of idiosyncratic hepatotoxicity is known. Thus, the dose of sitaxentan that was given to patients and was associated with idiosyncratic hepatotoxicity are consistent with the notion that high dose drugs pose a greater risk of idiosyncratic toxicity especially when they can also be metabolized to a reactive metabolite. It should be noted that there are marketed drugs that contain a 1,3-benzodioxole ring and are not associated with heptotoxicity, which includes tadalafil (Cialis), which is dosed at up to 20 mg/day.49

approved for pulmonary arterial hypertension for which clinical safety data is available. Bosentan, the first marketed endothelinA receptor antagonist, also causes liver damage in approximately 11% of patients, although unlike sitaxentan, severe liver damage is rare (three case reports describe serious liver damage) and is generally reversible when the drug is stopped.32 In contrast to sitaxentan and bosentan, which are sulphonamides, the newest endothelin receptor antagonist, ambrisentan, is a propanoic acid. To date, ambrisentan appears to have a better hepatic safety profile.33 Early studies to understand the mechanisms of bosentan hepatotoxicity focused on its ability to inhibit hepatic transporter proteins, such as, the sodiumtaurocholate cotransporting polypeptide (NTCP) and the bile salt export pump (BSEP).34,35 A subsequent study compared the ability of these three endothelin-A receptor antagonists to inhibit transport proteins for influx (NTCP and OATP) and efflux (BSEP). In that study, both bosentan and sitaxentan had the ability to inhibit these transporters, while ambrisentan was devoid of these properties36 suggesting that a possible contributing factor to the mechanism for hepatotoxicity involves inhibition of bile salt excretion and/or uncoupling lipid−bile salt secretion with subsequent accumulation of cytotoxic bile salts. Our work reveals an additional unique liability for sitaxentan, namely, the potential to form a reactive metabolite that might help explain the idiosyncratic nature of the hepatotoxicity compared to bosentan. We have demonstrated the ability of the reactive metabolite to react with GSH and possibly lead to protein modification, although subsequent investigations would be necessary to determine if the latter can occur. On the basis of our IC50 shift-assay results, sitaxentan caused TDI of P450 3A4 but not P450 2C9, indicating that the bioactivation of sitaxentan generates a reactive metabolite that is capable of inactivating P450 3A4. Inactivation can either take place through a covalent modification of the P450 apoprotein, alkylation or arylation of the prosthetic heme moiety, or destruction of the prosthetic heme groups via the metabolic intermediate complex37,38 and are indicative of a reactive metabolite that can interact with a macromolecule. Our work has revealed evidence for a reactive ortho-quinone metabolite as a possible mechanism for inactivation by covalently modifying P450 3A4. Covalent modification of P450 enzymes can also lead to the generation of autoantibodies that recognized the modification and subsequently lead to an adverse immune response of toxicological significance.39 Thus, it is plausible that the metabolism of sitaxentan to the ortho-quinone reactive metabolite, which we trapped with GSH, is responsible for TDI and is mechanistically relevant to the idiosyncratic nature of the hepatotoxicity. Autoantibodies to the P450s that metabolize a drug to a reactive metabolite have been detected in some patients that have experienced idiosyncratic liver toxicity to these drugs, such as, tienilic acid, 40 disulfiram, 41 and halothane.42 However, it is not known if those patients taking sitaxentan that experienced idiosyncratic liver toxicity had autoantibodies to P450 3A4. The TDI that we observed here may also be unrelated to the quinone reactive metabolite that we trapped with GSH as it is well-known that 1,3-benzodioxole systems can be metabolized to reactive carbene species that react with the P450 heme group to form a metabolite− intermediate complex that is catalytically inactive.12 Thus, further investigations are required to determine if sitaxentan is modifying P450 3A4 via protein modification, which could include characterizing the protein modification(s) by mass

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AUTHOR INFORMATION

Corresponding Author

*849 W. Orange Avenue, South San Francisco, CA 94080. Tel: +267-334-3572. E-mail: [email protected]. Present Addresses §

J.C.L.E.: Jerve Scientific Consulting, 849 W. Orange Avenue, South San Francisco, CA 94080. ∥ S.G.: Crescendo Bioscience, South San Francisco, CA 94080. ⊥ G.T. and J.W.M.: Novartis Institutes for Biomedical Research, MAP, Emeryville, CA 94608. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the efforts of Dr. Smriti Khera of Agilent Technologies (Santa Rosa, CA) to characterize the GSH conjugate by NMR spectroscopy.

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ABBREVIATIONS LC/MS, liquid chromatography/mass spectrometry; ACN, acetonitrile; GSH, glutathione; ESI, electrospray ionization; I

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metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States. Chem. Res. Toxicol. 24, 1345−1410. (20) Azaz-Livshits, T., Hershko, A., and Ben-Chetrit, E. (2002) Paroxetine associated hepatotoxicity: a report of 3 cases and a review of the literature. Pharmacopsychiatry 35, 112−115. (21) Akin, M., Isler, M., Senol, A., Aksakal, G., Kockar, C., and Songur, Y. (2012) Paroxetine induced toxic hepatitis: case report. Turk. Klin. Tip Bilimleri Derg. 32, 264−266. (22) Sleno, L., Varesio, E., and Hopfgartner, G. (2007) Determining protein adducts of fipexide: mass spectrometry based assay for confirming the involvement of its reactive metabolite in covalent binding. Rapid Commun. Mass Spectrom. 21, 4149−4157. (23) Sleno, L., Staack, R. F., Varesio, E., and Hopfgartner, G. (2007) Investigating the in vitro metabolism of fipexide: characterization of reactive metabolites using liquid chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 21, 2301−2311. (24) Zhao, S. X., Dalvie, D. K., Kelly, J. M., Soglia, J. R., Frederick, K. S., Smith, E. B., Obach, R. S., and Kalgutkar, A. S. (2007) NADPHdependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: identification of an electrophilic quinone metabolite of paroxetine. Chem. Res. Toxicol. 20, 1649−1657. (25) Hiramatsu, M., Kumagai, Y., Unger, S. E., and Cho, A. K. (1990) Metabolism of methylenedioxymethamphetamine: formation of dihydroxymethamphetamine and a quinone identified as its glutathione adduct. J. Pharmacol. Exp. Ther. 254, 521−527. (26) Stavros, F. L., Kramer, W. G., Ogilvie, B. W., and Parkinson, A. (2008) The victim potential of sitaxentan: metabolism by human CYPP450 enzymes, in European Respiratory Society Abstract 1062, European Respiratory Society, Berlin, Germany. (27) McGinnity, D. F., Soars, M. G., Urbanowicz, R. A., and Riley, R. J. (2004) Evaluation of fresh and cryopreserved hepatocytes as in vitro drug metabolism tools for the prediction of metabolic clearance. Drug Metab. Dispos. 32, 1247−1253. (28) Liljenberg, M., Brinck, T., Herschend, B., Rein, T., Rockwell, G., and Svensson, M. (2010) Validation of a computational model for predicting the site for electrophilic substitution in aromatic systems. J. Org. Chem. 75, 4696−4705. (29) Kolsek, K., Sollner, D. M., and Mavri, J. (2013) Computational study of the reactivity of bisphenol A-3,4-quinone with deoxyadenosine and glutathione. Chem. Res. Toxicol. 26, 106−111. (30) Monks, T. J., Highet, R. J., and Lau, S. S. (1990) Oxidative cyclization, 1,4-benzothiazine formation and dimerization of 2-bromo3-(glutathion-S-yl)hydroquinone. Mol. Pharmacol. 38, 121−127. (31) Butterworth, M., Lau, S. S., and Monks, T. J. (1997) Formation of catechol estrogen glutathione conjugates and γ-glutamyl transpeptidase-dependent nephrotoxicity of 17β-estradiol in the golden Syrian hamster. Carcinogenesis 18, 561−567. (32) Eriksson, C., Gustavsson, A., Kronvall, T., and Tysk, C. (2011) Hepatotoxicity by bosentan in a patient with portopulmonary hypertension: a case-report and review of the literature. J. Gastrointest. Liver Dis. 20, 77−80. (33) McGoon, M. D., Frost, A. E., Oudiz, R. J., Badesch, D. B., Galie, N., Olschewski, H., McLaughlin, V. V., Gerber, M. J., Dufton, C., Despain, D. J., and Rubin, L. J. (2009) Ambrisentan therapy in patients with pulmonary arterial hypertension who discontinued bosentan or sitaxsentan due to liver function test abnormalities. Chest 135, 122− 129. (34) Fattinger, K., Funk, C., Pantze, M., Weber, C., Reichen, J., Stieger, B., and Meier, P. J. (2001) The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 69, 223−231. (35) Leslie, E. M., Watkins, P. B., Kim, R. B., and Brouwer, K. L. R. (2007) Differential inhibition of rat and human Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) by bosentan: a mechanism for species differences in hepatotoxicity. J. Pharmacol. Exp. Ther. 321, 1170−1178.

FBS, fetal bovine serum; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MBI, mechanism based inhibitor; MS, mass spectrometry; MS/MS, tandem mass spectrometry; Q-TOF, quadrupole time-of-flight; TOF, timeof-flight; TDI, time-dependent inhibition; XIC, extracted ion chromatogram

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K

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