Inhibition of Hepatobiliary Transport Activity by the Antibacterial Agent

Article pubs.acs.org/crt

Inhibition of Hepatobiliary Transport Activity by the Antibacterial Agent Fusidic Acid: Insights into Factors Contributing to Conjugated Hyperbilirubinemia/Cholestasis Kimberly Lapham,† Jonathan Novak,† Lisa D. Marroquin,† Rachel Swiss,† Shuzhen Qin,∥ Christopher J. Strock,∥ Renato Scialis,†,⊥ Michael D. Aleo,‡ Thomas Schroeter,† Heather Eng,† A. David Rodrigues,† and Amit S. Kalgutkar*,§ †

Pharmacokinetics, Dynamics, and Metabolism Department, ‡Investigative Toxicology, Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States § Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, United States ∥ Biological Screening and Assay Development, Cyprotex, Watertown, Massachusetts 02472, United States ABSTRACT: Conjugated hyperbilirubinemia accompanied by cholestasis is a frequent side effect during chronic treatment with the antimicrobial agent fusidic acid. Previous studies from our laboratory, addressing mechanisms of musculoskeletal toxicity arising from coadministration of fusidic acid with statins, demonstrated the ability of fusidic acid to potently inhibit human organic anion transporting polypeptides OATP1B1 (IC50 = 1.6 μM) and OATP1B3 (IC50 = 2.5 μM), which are responsible for the uptake-limited clearance of statins as well as bilirubin glucuronide conjugates. In the present work, inhibitory effects of fusidic acid were characterized against additional human hepatobiliary transporters [Na+/ taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance-associated proteins MRP2 and MRP3] as well as uridine glucuronosyl transferase (UGT1A1), which mediate the disposition of bile acids and bilirubin (and its conjugated metabolites). Fusidic acid demonstrated concentration-dependent inhibition of human NTCP- and BSEP-mediated taurocholic acid transport with IC50 values of 44 and 3.8 μM, respectively. Inhibition of BSEP activity by fusidic acid was also consistent with the potent disruption of cellular biliary flux (AC50 = 11 μM) in the hepatocyte imaging assay technology assay, with minimal impact on other toxicity end points (e.g., cytotoxicity, mitochondrial membrane potential, reactive oxygen species generation, glutathione depletion, etc.). Fusidic acid also inhibited UGT1A1-catalyzed β-estradiol glucuronidation activity in human liver microsomes with an IC50 value of 16 μM. Fusidic acid did not demonstrate any significant inhibition of ATPdependent LTC4 transport (IC50’s > 300 μM) in human MRP2 or MRP3 vesicles. R values, which reflect maximal in vivo inhibition, were estimated from a static mathematical model by taking into consideration the IC50 values generated in the various in vitro assays and clinically efficacious unbound fusidic acid concentrations. The magnitudes of in vivo interaction (R values) resulting from the inhibition of OATP1B1, UGT1A1, NTCP, and BSEP transport were ∼1.9−2.6, 1.1−1.2, 1.0−1.1, and 1.4− 1.7, respectively, which are indicative of some degree of inherent toxicity risk, particularly via inhibition of OATP and BSEP. Collectively, these observations indicate that inhibition of human BSEP by fusidic acid could affect bile acid homeostasis, resulting in cholestatic hepatotoxicity in the clinic. Lack of direct inhibitory effects on MRP2 transport by fusidic acid suggests that conjugated hyperbilirubinemia does not arise via interference in MRP2-mediated biliary disposition of bilirubin glucuronides. Instead, it is possible that elevation in the level of bilirubin conjugates in blood is mediated through inhibition of hepatic OATPs, which are responsible for their reuptake and/or downregulation of MRP2 transporter as a consequence of cholestatic injury.

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INTRODUCTION Fusidic acid (Figure 1) is a steroidal bacteriostatic agent with anti-staphylococcal activity against methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant S. aureus strains.1,2 Fusidic acid lacks significant cross-resistance to other antibacterial classes and is under consideration to treat both healthcare-acquired and community-associated MRSA in the United States.3 A novel oral dosing regimen of fusidic acid has been recently studied in comparison to linezolid in a phase 2 © 2016 American Chemical Society

clinical trial for the treatment of acute bacterial skin and structure infections with comparable clinical success.4 Phase 2 clinical studies evaluating the potential of oral fusidic acid in the treatment of prosthetic joint infections are also in progress in the United States.5 Although not approved in the United States, oral and intravenous forms of fusidic acid have been used in Received: July 29, 2016 Published: September 27, 2016 1778

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Chemical Research in Toxicology

In conjunction with previous in vitro studies addressing mechanisms of musculoskeletal toxicity arising from coadministration of fusidic acid with statins, we demonstrated44 the ability of fusidic acid to potently inhibit human OATP1B1 (IC50 = 1.6 μM) and OATP1B3 (IC50 = 2.5 μM) isoforms, which are responsible for the uptake-limited clearance of statins.45−47 Inhibition of OATP-mediated hepatic uptake by fusidic acid can cause elevated blood and tissue concentrations of statins, potentially manifesting in myopathy including rhabdomyolysis. In the present work, we have extended the in vitro studies with fusidic acid by also examining inhibitory effects on human NTCP and BSEP and in the hepatocyte imaging assay technology (HIAT) assay48,49 in an attempt to rationalize the cholestasis findings in humans. In addition, the propensity of fusidic acid to inhibit UGT1A1 activity was explored in human liver microsomes, and MRP2/3 transport was examined in membrane vesicles. To gauge the potential impact in vivo, the IC50 values generated in the various in vitro assays were placed in perspective with clinically efficacious unbound fusidic acid concentrations using a static mathematical model.

Figure 1. Chemical structure of fusidic acid.

Europe and Australia for more than 40 years to treat chronic airway, skin, and bone and joint infections.6−8 From a toxicological standpoint, the widespread clinical use of fusidic acid in suppressive antibiotic therapy has been associated with several cases of life-threatening (and sometimes fatal) rhabdomyolysis upon coadministration with the 3hydroxy-3-methylglutaryl coenzyme A reductase inhibitors atorvastatin, simvastatin, and rosuvastatin.9−14 In addition, a frequent (and major) side effect of fusidic acid use is hyperbilirubinemia/jaundice, which can occur as early as 2 days after starting treatment,15−23 with a higher incidence in patients given fusidic acid intravenously (17−48%) rather than via oral administration (6−13%).20 Hyperbilirubinemia is accompanied by very high serum levels of bilirubin (predominantly in the conjugated form) and elevations in serum transaminases, alkaline phosphatase activity, and bile acids, suggestive of mixed cholestatic hepatocellular druginduced liver injury (DILI).15,24 As such, a temporal relationship linking elevated fusidic acid systemic exposure to DILI has been established;18−20 in virtually all cases, cholestatic jaundice is reversible and serum bilirubin levels return to normal within 4−6 days after fusidic acid treatment is stopped.15,18 Bile acids are predominantly taken up from the sinusoidal blood into hepatocytes by human Na+/taurocholate cotransporting polypeptide (NTCP) in a Na+-dependent fashion, followed by biliary elimination by the ATP-binding cassette transporter bile salt export pump (BSEP), which is expressed at the canalicular domain of hepatocytes.25 Inhibition of the hepatobiliary transport of bile acids (e.g., NTCP and/or BSEP) can lead to a reduction of bile flow and accumulation of cytotoxic bile salts, leading to hepatocellular injury marked with jaundice.26−28 Genetic studies have also shown that polymorphism(s) in the gene coding for BSEP and/or inherited mutations lead to progressive familial intrahepatic cholestasis and severe liver disease.29 Bilirubin, the principal product of heme catabolism, is mainly eliminated through a multifaceted process in the liver. Unconjugated bilirubin is transported into the liver through a carrier-mediated transport process facilitated by human OATP1B1 and OATP1B3 isoforms30,31 followed by glucuronidation by uridine glucuronosyl transferase (UGT) 1A1 to form mono- and diglucuronide conjugates,32,33 which are excreted into bile via multidrug resistance-associated protein (MRP) 2 expressed on the bile caniculi.34,35 In cases where biliary excretion is impaired, conjugated bilirubin can also be secreted into blood by sinusoidally expressing MRP3,36,37 which enables sinusoidal reuptake of bilirubin glucuronides by more downstream hepatocytes via OATP1B1 and 1B3.30,38,39 Inhibitory effects on the elimination processes of bilirubin (and its corresponding glucuronide conjugates) have been recognized as critical elements of drug-induced unconjugated and/or conjugated hyperbilirubinemia.39−43

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MATERIALS AND METHODS

Materials. Fusidic acid sodium salt (purity ≥98%), β-estradiol, βestradiol-3-glucuronide sodium salt, alamethicin (from Trichoderma virid), uridine 5′-diphosphoglucuronic acid (UDPGA) triammonium salt, nicardipine, and taurocholic acid were purchased from SigmaAldrich (St. Louis, MO). 1-Naphthyl-glucuronide sodium salt was purchased from Sequoia Research Products (Pangbourne, UK). Pooled male and female human liver microsomes (n = 50 donors) were purchased from BD Gentest (Woburn, MA). All other commercially obtained chemicals and solvents were of high-performance liquid chromatography or analytical grade. UGT1A1 Inhibition Assay. The potential for fusidic acid to inhibit UGT 1A1 enzyme activity in human liver microsomes was investigated in duplicate using established protocols.50 An incubation mixture containing human liver microsomes (0.025 mg/mL), 100 mM TrisHCl buffer (pH 7.5 at 37 °C), 5 mM magnesium chloride, 10 μM βestradiol, and 10 μg/mL alamethicin was preincubated on ice for 15 min to allow for microsome pore formation. Aliquots (178 μL) of this mixture were placed into a 96-well polypropylene plate and warmed to 37 °C prior to the addition of fusidic acid, nicardipine (nonspecific UGT inhibitor as a control51), or solvent control. The final concentrations for fusidic acid and nicardipine were 0.3, 1, 3, 10, 30, and 100 μM and 0.03, 0.1, 0.3, 1, 3, 10, and 30 μM, respectively. Reactions were initiated with the addition of 5 mM UDPGA and terminated at 60 min by transferring 100 μL aliquots to 200 μL of acetonitrile containing 10 μM 1-naphthyl-glucuronide as internal standard. After centrifugation, samples (200 μL) were evaporated with nitrogen gas and then reconstituted in 100 μL of 10:90 acetonitrile/ water containing 0.1% formic acid. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and quantified against a standard curve of β-estradiol-3-glucuronide. Standard curve concentrations ranged from 0.01 to 5 μM βestradiol-3-glucuronide. β-Estradiol-3-glucuronide concentrations in microsomal incubations were measured by LC-MS/MS relative to a standard curve in the presence of an internal standard (1-naphthyl-glucuronide). The LCMS/MS system consisted of an AB Sciex 4000 Q-Trap mass spectrometer equipped with an electrospray source (AB Sciex, Framingham, MA), Agilent Infinity 1290 LC pumps (Agilent Technologies, Santa Clara, CA), and a Leap CTC HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland). The mass spectrometer source was maintained at 550 °C, and the ion spray capillary voltage was set to −4.2 kV with the declustering potential, entrance potential, and collision exit potential set at −55, −10, and −15 V, respectively. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode for quantitative analysis. The 1779

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Chemical Research in Toxicology collision energy was kept at −30 eV. The mass transitions for βestradiol-3-glucuronide and the internal standard were as follows: 447.2 → 113.2 (β-estradiol-3-glucuronide) and 319.1 → 112.9 (1naphthyl-glucuronide). Estradiol and β-estradiol-3-glucuronide were separated on a Phenomenex Luna 5 μm, C18(2) 100 × 30 mm column (Phenomenex Inc., Torrance, CA) using a binary gradient composed of water/0.1% formic acid (mobile phase A) and acetonitrile/0.1% formic acid (mobile phase B). The mobile phase composition of 25% B was held over 2.0 min, ramped to 90% B over 0.1 min, held at 90% B for 0.5 min, returned to 25% B for 0.1 min, and held for an additional 1.3 min. The flow rate was 0.7 mL/min. Standard curve fitting was accomplished with Analyst software (version 1.6.2; AB Sciex, Framingham, MA). Data was fit linearly using 1/x2 weighting. IC50 estimates for inhibition of glucuronidation were determined by nonlinear fitting with GraphPad Prism (version 6.03, GraphPad Software, Inc., La Jolla, CA) and defined as the concentration of inhibitor required to inhibit control glucuronidation reactions by 50%. Since activity remaining was normalized to the solvent controls, the model forced the curve to run from 100 to 0%. The model also assumed that the dose−response curve had a standard slope, equal to a Hill slope of −1.0. Human NTCP Inhibition Assay. Human NTCP-expressing embryonic kidney (HEK) 293 cells were generated at Pfizer Inc. (Sandwich, UK). HEK-NTCP cells were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum. Cells were seeded at a density of 5.0 × 104 cells per well on BioCoat 96-well poly-D-lysine coated plates (Corning Inc., Corning NY). Cells were washed with transport buffer (118 mM NaCl, 24 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl2 adjusted to pH 7.4) prior to transport assays. For inhibition studies, the uptake of 5 μM taurocholic acid-d5 sodium salt (Medical Isotopes, Pelham, NH) was investigated in the absence and presence of fusidic acid (0.3−300 μM) in HEK-NTCP cells for 3 min in triplicate at 37 °C [n = 3]. Cellular uptake was terminated by quickly washing the cells four times with 0.2 mL of ice-cold uptake buffer. The cells were then lysed with 100 μL of ice-cold methanol containing indomethacin (0.1 μg/mL) as the internal standard. The lysate was mixed with 100 μL of water and vigorously vortex-mixed, and a 10 μL aliquot was injected onto an LCMS/MS system. Mass spectrometric analysis was accomplished on an AB Sciex API-4000 electrospray mass spectrometer equipped with an electrospray source, Agilent Infinity 1290 LC pumps, and a Leap CTC HTS PAL autosampler. The mass spectrometer source was maintained at 550 °C, and the ion spray capillary voltage was set to −4.25 kV with the declustering potential set at −70. The collision energy was kept at -80 eV. The mass spectrometer was operated in negative ion mode and MRM mode for quantitative analysis. The MRM transitions for taurocholic acid-d5 and indomethacin were 519.3 → 80.1 and 356.0 → 311.8, respectively. Analytes were separated on a Phenomenex Kinetex XB-C18 30 × 2.1 mm, 2.6 μm particle size column using a binary gradient composed of water/0.1% formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B). The mobile phase composition of 10% B was held over 0.5 min, ramped to 90% B over 1.25 min, held at 90% B for 1.5 min, returned to 10% B for 1.5 min, and held for an additional 1.5 min. The flow rate was 0.3 mL/ min. Standard curve fitting was accomplished with Analyst software (version 1.6.2). Data was fit linearly using 1/x2 weighting. GraphPad Prism (La Jolla, CA) was used to fit the data to generate IC50 values. Results are expressed as percent of activity of taurocholate transport in the presence of fusidic acid relative to untreated control. Human BSEP Inhibition Assay. Human BSEP vesicles were purchased from Solvo Biotechnology (Budapest, Hungary). The membrane vesicles were harvested from recombinant baculovirusinfected Hi5 insect cells transfected with human BSEP and processed for inside-out vesicles.28 BSEP vesicles (16 μg) were incubated with fusidic acid (0.2−200 μM) [n = 5] or solvent (DMSO) and taurocholic acid (2 μM) for 40 min at 25 °C in buffer containing 4 mM ATP, 50 mM HEPES pH 7. 4, 100 mM KNO3, 10 mM Mg(NO3)2, and 50 mM sucrose. The final DMSO concentration was 1% (v/v). The transport reaction was stopped by addition of ice-cold

stop buffer (5 M EDTA, 10 mM Tris pH 7.4, 100 mM KNO3, 10 mM Mg(NO3)2, and 50 mM sucrose). The vesicles were washed three times with ice-cold stop buffer, and taurocholic acid was extracted from the membrane with a mixture of 80:20 methanol/water. Taurocholic acid concentrations were measured by LC-MS/MS. Mass spectrometric analysis was accomplished on a Applied Biosystems Sciex Triple Quad 6500 system with an electrospray IonDrive Turbo V Ion Source, Agilent Infinity 1290 LC pumps, and a Apricot/Sound Analytics ADDA autosampler (Sound Analytics, Niantic, CT). The ion spray capillary voltage was set to 5.0 kV, with the declustering potential set at 105. The collision energy was kept at 25 eV. The mass spectrometer was operated in positive ion mode and MRM mode for quantitative analysis. The main MRM transitions for taurocholic acid was m/z 516.3 → 462.2. Analytes were separated on an ACE C18 20 × 2.1 mm, 5 μm particle size column (Advanced Chromatography Technologies, Aberdeen, Scotland) using a binary gradient comprised of water/0.1% formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B). The mobile phase composition started at 5% solvent B and was held for 0.3 min, ramped to 90% solvent B over 0.5 min, held at 90% B for 0.3 min, and then switched back to starting conditions in 0.02 min. The column was re-equilibrated at 5% solvent B for 0.38 min, for a total run time of 1.5 min. The flow rate was set to 0.8 mL/min. Data was analyzed using Sciex MultiQuant software, version 3.0.2, and results are expressed as percent of activity of taurocholate transport in the presence of fusidic acid relative to untreated control. IC50 value for BSEP inhibition by fusidic acid was estimated using a nonlinear regression for a sigmoidal dose−response using a four-parameter logistic equation. Human MRP2 and MRP3 Inhibition Assay. Human MRP2 and MRP3 (BD Gentest) inside-out vesicles were prepared from Sf9 insect cells using a baculovirus system. The vesicle uptake of leukotriene C4 (LTC4) was carried out in 96-well filter plate using a rapid filtration system. A reaction mixture containing 50 μg of vesicles, 2.5 mM glutathione, and 1 μM LTC4 in uptake buffer (47 mM 3-(Nmorpholino)propanesulfonic acid, 65 mM potassium chloride, and 7 mM magnesium chloride, pH 7.4) was preincubated at 37 °C for 5 min with either vehicle (DMSO), fusidic acid (0.3−300 μM) [n = 3], or MK-571 (0.3−100 μM) [n = 3] (a cysteinyl leukotriene receptor antagonist and MRP2/MRP3 inhibitor)52 in DMSO. The uptake was initiated by addition of either ATP or AMP (final concentration of 5 mM), followed by incubation at 37 °C for 5 min. The assay was terminated by addition of cold wash buffer (40 mM 3-(Nmorpholino)propanesulfonic acid and 70 mM potassium chloride, pH 7.4) and transferred to a 96-well filter plate. The filter plate was washed three times with cold washing buffer using a 96-well vacuum filtration manifold. The intact membrane vesicles were trapped on the filter bed, whereas unbound LTC4 was washed away. After drying the filter plate, the residue was reconstituted in water (100 μL) and 100 μL of indomethacin (100 ng/mL) as internal standard in methanol was added into the sample wells to release the trapped LTC4. After a 10 min incubation at room temperature, the released LTC4 was eluted into an analytical 96-well plate and analyzed by LC-MS/MS. Mass spectrometric analysis was accomplished on a Applied Biosystems MDS Sciex API-4000 electrospray mass spectrometer equipped with an electrospray source, Agilent Infinity 1290 LC pumps, and a Leap CTC HTS PAL autosampler. The mass spectrometer source was maintained at 550 °C, and the ion spray capillary voltage was set to 4.75 kV, with the declustering potential set at 64. The collision energy was kept at 29 eV. The mass spectrometer was operated in MRM mode for quantitative analysis. The MRM transitions for LTC4 and indomethacin were m/z 626.4 → 189.5 and m/z 358.0 → 139.0, respectively. Analytes were separated on a Phenomenex Kinetex XBC18 30 × 2.1 mm, 2.6 μm particle size column using a binary gradient composed of water/0.1% formic acid (solvent A) and acetonitrile/ 0.1% formic acid (solvent B). The mobile phase composition of 10% B was held over 0.5 min, ramped to 90% B over 1.25 min, held at 90% B for 1.5 min, returned to 10% B for 1.5 min, and held for an additional 1.5 min. The flow rate was 0.3 mL/min. Quantification analysis was performed using Sciex Analyst 1.6 software. The ATP-dependent uptake activity was reported as uptake activity in the presence of ATP 1780

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Cellular Biliary Flux. Cells were seeded at a density of 12 000 cells/ well in a 384-well plate format. After 24 h, medium was removed and 25 μL of 0.25 mg/mL matrigel was added to each well. Medium was then added after allowing the overlay to form, and the cells were put back in the incubator for 3 days for canaliculi to form. After 3 days, the cells were treated with fusidic acid for 30 min followed by addition of cholyllysyl fluoroscein (Corning). The cells were incubated for 20 min at 37 °C, for 10 min at room temperature, and finally for 5 min at 4 °C. The cells were then washed 2× with phosphate-buffered saline. Cells were imaged on the ArrayScan VTI with the FITC channel. Canaliculi were determined using total spot area in the Spot Detector bioapplication (Thermo Fisher). The AC50 value for this end point represents the media concentration of test compound that results in a 50% reduction in the visualized bile ducts with cholyllysyl fluoroscein dye as calculated by area. Measurement of Direct Mitochondrial Uncoupling and Inhibition. Rat liver mitochondria were isolated, and oxygen consumption was monitored in a 96-well plate format using a phosphorescent oxygen-sensitive probe as previously described54,55 with minor modifications. Briefly, A65N-1 oxygen probe was reconstituted in 10.5 mL of mitochondrial incubation buffer54 to a concentration of approximately 100 nM, and 100 μL aliquots were transferred into a 96-well plate (10 pmol of probe/well). Fusidic acid in DMSO (final DMSO content 2-fold above background (indicative of a cell with increased permeability). Glutathione Depletion. The AC50 value for this end point (cells stained with monochlorobimane) represents the media concentration of a test compound that causes a loss of greater than 50% of total cellular endogenous glutathione. Reactive Oxygen Species. The AC50 value for this end point (cells stained with CM-H2DCFDA) represents the media concentration of a test compound that causes >2-fold increase in reactive oxygen species in hepatocytes. Cytokine-Mediated Cytotoxicity. Cells were treated for 48 h (single treatment) with fusidic acid in the presence and absence of human cytokines as described in Cosgrove et al.53 The cytokines (R&D Systems, Minneapolis, MN) were added at the following final concentrations: 20 ng/mL interleukin 6, 100 ng/mL tumor necrosis factor alpha, interleukin 1 alpha 20 ng/mL, and 20 μg/mL lipopolysaccharide. After 48 h, the cells were stained with 4 μg/mL Hoechst 33342 and 2 μg/mL propidium iodide, and cytotoxicity was determined using the Arrayscan VTI. 2-fold or greater shifts in extracellular AC50 values in the presence over absence of cytokines are considered positive for cytokine synergy, as calculated by total cells subtracted by the percent of high responders in the PI channel. Lipid Content. Cells treated for 48 h without cytokine were fixed by the direct addition of formaldehyde in Hanks balanced salt solution with phenol red to a final concentration of 3.7%. After incubation in the fixation medium for 30 min at room temperature, cells were rinsed twice with phosphate-buffered saline before labeling with the fluorescent reporter dye, LipidTox Deep Red (Bioreclamation), for neutral lipid accumulation, to evaluate lipid content. The AC50 value for this end point represents the media concentration of test compound that causes induction (>2.5-fold of baseline) of steatosis.

R=1+

Iin,max,u IC50

where Iin,max,u represents the estimated maximum unbound concentration of fusidic acid at the inlet to the liver and is defined as follows

⎛ ka × Fa × Fg × dose ⎞ Iin,max,u = fu,b × ⎜⎜Cmax + ⎟⎟ Qh ⎠ ⎝

ka =

0.693 absorption t1/2

where f u,b is the unbound fraction of fusidic acid in human blood and was assumed to be equal to f u in plasma (i.e., the blood-to-plasma ratio was assumed to be unity), Cmax represents maximal systemic exposure of fusidic acid after oral dosing in humans, Fa is fraction of the oral fusidic acid dose absorbed from the gut to the portal vein, Fg is the fraction of the absorbed fusidic acid dose escaping gut wall extraction, 1781

DOI: 10.1021/acs.chemrestox.6b00262 Chem. Res. Toxicol. 2016, 29, 1778−1788

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Chemical Research in Toxicology ka is the oral absorption rate constant, and Qh is the human hepatic blood flow of 97 l/h/70 kg.58

NTCP- and BSEP-mediated taurocholic acid transport, with IC50 values of 44 ± 1.0 and 3.8 ± 1.2 μM, respectively. In human MRP2 or human MRP3 vesicles, fusidic acid stimulated MRP2- and MRP3-mediated LTC4 transport (∼125−140% of control) at the concentration range used to assess inhibition (Figure 4) without any meaningful inhibitory effects. Some inhibitory effect (∼49% inhibition of LTC4 transport) against MRP3 was noted at the top fusidic acid concentration of 300 μM. Under these experimental conditions, the MRP inhibitor and cysteinyl leukotriene receptor antagonist MK-57152 also stimulated MRP2- and MRP3-mediated LTC4 transport at the lower concentrations followed by inhibition at the higher concentrations, resulting in IC50 values of 19.9 and 7.48 μM, respectively. The IC50 value for MRP2 inhibition by MK-571 in our study is comparable to the one (IC50 = 21.2 μM) previously determined by Csandl et al.60 for inhibition of MRP2-mediated transport of the structurally diverse ligand 17β-estradiol glucuronide. Furthermore, stimulation and inhibitory effects of MK-571 and related cysteinyl leukotriene receptor antagonists on MRP2-mediated transport were also noted.60 R values, which reflect maximal in vivo inhibition of drug metabolizing enzymes or hepatobiliary transport, were estimated using a static mathematical model depicted in the Materials and Methods section. At its clinically efficacious oral dose (500 mg, three times daily), the total fusidic acid Cmax in plasma ranges from 50 to 100 μg/mL (unbound Cmax = 600− 1200 ng/mL or 1.2−2.3 μM, plasma f u value of 0.012 (Pfizer data on file), molecular weight of fusidic acid = 516.7) and occurs at 2 h (Tmax), which results in a ka value of 1.7 h−1.61 For estimation of unbound liver inlet Iin,max,u concentrations of fusidic acid, Fa and Fg were set to unity considering that fusidic acid is a low clearance agent (plasma clearance = 0.3 mL/min/ kg) with a high oral bioavailability (>90%).62 Under these conditions, the Iin,max,u values for fusidic acid ranged from ∼1.4 to 2.5 μM. In the case of BSEP (IC50 = 3.8 μM), NTCP (IC50 = 44 μM), and UGT1A1 (IC50 = 16 μM), the R values were 1.4− 1.7, 1.0−1.1, and 1.1−1.2, respectively. Utilizing the previously determined44 OATP1B1 and OATP1B3 IC50 values of 1.6 and 2.5 μM yielded R values ranging from 1.9 to 2.6 and 1.6 to 2.0, respectively. HIAT Results. As shown in Table 1, fusidic acid did not demonstrate cytokine synergy since the ratio of cell loss at 48 h in the absence of exogenous cytokines over cell loss in the presence of cytokine stimulation was 1.25.56,57 To evaluate the utility of this methodology in predicting the toxicity risk with fusidic acid, the IC50 values for inhibition of OATP, NTCP, and BSEP and projected unbound portal vein concentrations (1.4−2.5 μM) of fusidic acid (at its clinically efficacious dose of 500 mg, three times daily) were used to estimate R values from a static mathematical model.79 The R values obtained using unbound portal vein concentrations as a surrogate of fusidic acid liver concentrations exceeded the proposed cutoff value of 1.25, indicating some degree of inherent toxicity risk with fusidic acid, particularly via inhibition of OATP (R value = 1.9−2.6 (OATP1B1) and R value = 1.6−2.0 (OATP1B3)) and BSEP (R value = 1.4−1.7). The R value approach utilizing in vitro transporter inhibition and human pharmacokinetic data has been previously used to predict clinical drug-induced hyperbilirubinemia potentially arising from OATP inhibition.41 Strong OATP inhibitors and drugs associated with clinical hyperbilirubinemia such as indinavir (OATP1B1 IC50 ∼ 8.3 μM, OATP1B3 IC50 ∼ 16 μM), cyclosporine (OATP1B1 IC50 ∼ 0.057 μM, OATP1B3 IC50 ∼ 0.13 μM), and rifamycin SV (OATP1B1 and 1B3 IC50 ∼ 0.05 μM) demonstrate relatively high R values (1.5−2.0 (indinavir), 4.9−9.6 (cyclosporine), 101−126 (rifamycin)) in comparison to weak OATP inhibitors and drugs that do not cause hyperbilirubinemia in the clinic (e.g., saquinavir (OATP1B1 IC50 ∼ 0.41 μM, OATP1B3 IC50 ∼ 0.47 μM, R value ∼ 1.1). As a combined BSEP inhibitor and mitochondrial uncoupler, the risk for significant DILI increases as reductions in cellular ATP levels further decrease hepatic transporter function.80 As such, it is interesting to note that unusually high fusidic acid plasma concentrations (195 μg/mL, 16 h post the last dose of fusidic acid) have been measured in a patient with cholestatic jaundice,24 which translates into an unbound portal inlet concentration of ∼4.7 μM, further increasing the R values to 4.7 (OATP1B1), 2.9 (OATP1B3), 1.1 (NTCP), and 2.2

Figure 6. Concentration-dependent effects on cellular biliary flux in human hepatocytes in the hepatocyte imaging assay technology (HIAT) after exposure to fusidic acid.

where biliary excretion is impaired, conjugated bilirubin can be secreted into blood by MRP3, which enables reuptake of bilirubin glucuronides by more downstream hepatocytes in the sinusoid by OATP1B1 and 1B3. Additional support for this hypothesis stems from a recent finding on the OATP/BSEP (but not MRP2) inhibition by a tyrosine kinase inhibitor CP724,714, which caused elevations in total (conjugated/nonconjugated) bilirubin and hepatobiliary cholestasis in humans.70,71 The synergistic contribution of fusidic acid metabolites toward inhibition of hepatobiliary transport cannot be excluded at this point. Metabolism can potentially increase the inhibitory activity of the parent molecule, as has been shown for troglitazone.72 In vitro membrane transporter assays are not metabolically competent; therefore, metabolite activity against drug transporters cannot be assessed in these assays. Fusidic acid is extensively metabolized in humans,73−76 followed by exclusive biliary excretion of the metabolites. The taurine and glycine conjugates of fusidic acid (e.g., taurodihydrofusidate and glycodihydrofusidate) demonstrate detergent-like properties similar to native bile salts and have also been shown to inhibit bile salt secretion in rats, monkeys, and hamsters.23 Of the metabolites eliminated in human bile, the major ones include an acyl glucuronide, a dicarboxylic acid (derived from oxidation of one of the terminal methyl group), and a hydroxyl derivative 1785

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Chemical Research in Toxicology

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(BSEP). The significance of these observations and values from in vitro assays are not without physiologic meaning. In humans, there is a dose- and time-dependent rise in serum bile acids after oral administration of fusidic acid (1 to 2 g), showing disruption in hepatic uptake and excretion of bile acids after a meal challenge.19 This in vivo effect demonstrates the relevance of the in vitro transporter IC50 values in relation to portal vein exposure estimates at clinical doses. With a daily dose of ∼1.5 g, fusidic acid reaffirms the empirical concept that high daily dose drugs are frequently associated with DILI arising from a single or multifactorial pathway(s) (e.g., reactive metabolite formation, mitochondrial toxicity, etc.). The case study with fusidic acid also reiterates the need to examine inhibition of hepatobiliary transporters as a causative factor for DILI in preclinical drug discovery, particularly for compounds projected to be high daily dose agents and/or ones with physicochemical and structural properties close to endogenous transport substrates such as bile acids.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: amit.kalgutkar@pfizer.com. Phone: (617) 551-3336. Present Address

⊥ (R.S.) Bristol-Myers Squibb, Princeton, New Jersey, United States.

Notes

The authors declare the following competing financial interest(s): At the time of data generation, all authors were employees and stockholders of Pfizer Inc.

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ABBREVIATIONS MRSA, methicillin-resistant Staphylococcus aureus; DILI, druginduced liver injury; NTCP, sodium/taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; BSEP, bile salt export pump; UGT, uridine 5′-diphosphoglucuronosyltransferase; MRP, multidrug resistance-associated protein; HIAT, hepatocyte imaging assay technology; UDPGA, uridine 5′-diphosphoglucuronic acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring; HEK, human embryonic kidney; LTC4, leukotriene C4; AC50, cell proliferation inhibition constant

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REFERENCES

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