Evaluating the In Vitro Inhibition of UGT1A1, OATP1B1, OATP1B3

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Evaluating the In Vitro Inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in Predicting Drug-Induced Hyperbilirubinemia Jae H. Chang,* Emile Plise, Jonathan Cheong, Quynh Ho, and Molly Lin Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States S Supporting Information *

ABSTRACT: Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the body. Bilirubin elimination is a multifaceted process consisting of uptake of bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has been previously shown to be linked with hyperbilirubinemia. However, because drug transporters also contribute to bilirubin elimination, the purpose of this work was to investigate the in vitro inhibition of OATP1B1, OATP1B3, MRP2, and BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs investigated in this study were atazanavir and indinavir, which are associated with hyperbilirubinemia and elevations in serum transaminase; ritonavir and nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin, which are associated with severe idiosyncratic hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to limited solubility and poor ionization of bilirubin and its glucuronide, the formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1 activity, while the transport of pitavastatin, CDCF, and taurocholate were used as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2, and BSEP, respectively. It was assumed that any inhibition of the surrogate probe substrates by test drugs is indicative of the potential impact of test drugs to modulate the function of proteins involved in bilirubin disposition. In vitro inhibition was determined by calculating IC50. Moreover, Cmax and Cmax,free were integrated with IC50 values to calculate R and Rfree, respectively, which represents the ratio of probe drug glucuronidation/transport in the absence and presence of test drugs. Analysis of the data showed that Rfree demonstrated the best correlation to hyperbilirubinemia. Specifically, Rfree was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for atazanavir and indinavir. In contrast, Rfree was below this threshold for ritonavir and nelfinavir as well as for bromfenac, troglitazone, and trovafloxacin. For all test drugs examined, only minor inhibition against OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and BSEP may be suitable to assess bilirubin disposition. For protease inhibitors, inclusion of OATP1B1 and BSEP inhibition may improve the predictability of hyperbilirubinemia. KEYWORDS: UGT1A1, OATP1B1, OAT1B3, MRP2, BSEP, bilirubin, hypebilirubinemia

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the bile via MRP2 expressed on the bile caniculi,5 but in some cases, glucuronide metabolites may also be secreted into the blood by sinusoidally expressing MRP3.6,7 Hyperbilirubinemia is defined as the accumulation of bilirubin in the body resulting from increased formation of bilirubin and/or decreased capacity to eliminate bilirubin. Hyperbilirubinemia has been extensively studied in the context of neurotoxicity where it appeared that bilirubin may alter synaptic potentials and functions of neurotransmitters.8,9 Moreover, bilirubin may directly affect cellular processes as excess bilirubin can interfere with oxidative phosphorylation,

INTRODUCTION Blirubin is an endogenous molecule that is a byproduct of heme catabolism predominantly from red blood cells in the spleen. Heme oxygenase initially catalyzes the breakdown of heme to form biliverdin, carbon monoxide, and free iron. Biliverdin is then reduced to bilirubin by biliverdin reductase.1 The liver is the main organ involved in bilirubin elimination. As Figure 1 illustrates, bilirubin enters the liver through a carrier-mediated transport process facilitated by uptake transporters such as organic anion-transporting polypeptides OATP1B1 and OATP1B3.2,3 Once it enters the liver, bilirubin is extensively metabolized by UDP-glucuronosyltransferase 1A1 (UGT1A1) to form mono- and diglucuronide conjugates.4 Since the glucuronide metabolites are recognized by several multidrug resistance-associated proteins (MRPs), there are multiple pathways to exit the cells. The primary route is excretion into © 2013 American Chemical Society

Received: Revised: Accepted: Published: 3067

March 7, 2013 May 13, 2013 June 10, 2013 June 10, 2013 dx.doi.org/10.1021/mp4001348 | Mol. Pharmaceutics 2013, 10, 3067−3075

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Table 1. Summary of Select Clinical Information on Drugs Tested for UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP Inhibition

Figure 1. Proposed hypothesis where bilirubin elimination may be mediated by proteins (A) directly impacting bilirubin elimination such as UGT1A1, OATP1B1, OATP1B3, and MRP2 and (B) indirectly impacting bilirubin elimination such as BSEP which aids in maintaining the integrity of the liver by directing bile flow through secretion of monovalent bile salts.

drug

dose

Cmax (μM)

elevated bilirubin

f ua

atazanavir41,58 indinavir42 ritonavir43 nelfinavir44 bromfenac48,49 troglitazone54,55,59 trovafloxacin56,57,60

400 mg QD 800 mg TID 600 mg BID 1250 mg BID 50 mg QD 600 mg QD 200 mg QD

7.7 ± 1.9 13 ± 4 15 ± 5 3.3 ± 1.4 9.6 ± 3.5 6.4 ± 2.4 7.4 ± 2.4

yes yes no no yes yes yesb

0.066 0.228 0.007 0.001 0.127 0.008 0.211

a

Protein binding values measured using equilibrium dialysis. bElevated transaminase levels were not associated with concurrent elevation of bilirubin.

hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin, which are associated with severe idiosyncratic hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. The current aim was 2-fold as illustrated in Figure 1. The first was to study the inhibition of proteins directly involved in bilirubin elimination by not only examining UGT1A1, but also evaluating drug transporters OATP1B1, OATP1B3, and MRP2. The second was to evaluate the inhibition of BSEP which may not recognize bilirubin or its glucuronide as substrates but may have an indirect impact of bilirubin elimination through regulation of bile flow.

enhance DNA instability, interrupt protein synthesis, and block the activity of mitochondrial enzymes.10,11 Therefore, in addition to neurotoxicity, bilirubin may lead to non-neural organ dysfunctions, and hyperbilirubinemia can be considered as an early warning of possible adverse effects such as hepatotoxicity. There are several ways in which the disposition of bilirubin may be modulated. For example, certain disease states such as hemolytic anemia can heighten bilirubin formation,12,13 whereas liver diseases such as hepatitis, cholestasis, and cirrhosis can limit bilirubin elimination.14 Genetic modifications of proteins responsible for bilirubin metabolism and transport can also cause hyperbilirubinemia. Patients with Gilbert’s syndrome have reduced UGT1A1 transcription and those with CriglerNajjar disease are devoid of UGT1A1 expression.15 Bilirubin levels in these patients are high and can lead to phenotypes such as jaundice or even kernicterus if left untreated. DubinJohnson syndrome is an autosomal disease distinguished by mutations in the ATP-binding region of MRP2 characterized by high levels of conjugated bilirubin as efflux of the glucuronide metabolites into the bile is impaired.16 Similarly, patients with Rotor syndrome also display conjugated hyperbilirubinemia. However, Rotor syndrome is differentiated by deficiency in OATP1B1 and OATP1B3 function.17 Another mutation which affects the bile salt exporter pump (BSEP) is linked to progressive familial intrahepatic cholestasis.18 BSEP is the main transporter involved in the biliary secretion of monovalent bile salts and helps maintain the integrity of the liver by directing bile flow. However, unlike MRPs and OATPs, BSEP does not directly regulate bilirubin elimination. Instead, inhibiting BSEP results in reduction of bile flow and accumulation of bile salts leading to hepatocellular injury marked with chronic cirrhosis and jaundice.19 In addition to genetic factors, drugs can also trigger the onset of hyperbilirubinemia. NSAIDs, β-lactamase inhibitors, and sulfonamides have been shown to augment bilirubin production by stimulating hemolysis.20−22 Moreover, bilirubin levels may increase if drugs interfere with bilirubin elimination as demonstrated with in vitro inhibition of UGT1A1.23 However, because drug transporters also contribute to bilirubin elimination, the purpose of this work was evaluate the in vitro inhibition of drug transporters for select test drugs exhibiting hyperbilirubinemia. Test drugs investigated in this study as outlined in Table 1 were atazanavir and indinavir, which are associated with hyperbilirubinemia and elevations in serum transaminase; ritonavir and nelfinavir, which do not exhibit

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MATERIALS AND METHODS Materials. β-Estradiol, bromfenac, indinavir, nelfinavir, troglitazone, rosiglitazone, trovafloxacin, cyclosporine, cortisone, and potassium phosphate buffer were purchased from Sigma-Aldrich Company (St. Louis, MO). Pitavastatin, atazanavir, and ritonavir were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MK571 sodium salt was purchased from Calbiochem (Darmstadt, Germany). 3HTaurocholate and Microscint 20 liquid scintillation cocktail were purchased from PerkinElmer (Waltham, MA). Dulbecco’s modified Eagle’s medium was purchased from Invitrogen Inc. (Carlsbad, CA). All other chemicals and solvents were of analytical grade and were purchased from Sigma-Aldrich Co. The single-use RED plates for plasma protein binding were purchased from Thermo Scientific (Rockford, IL). MRP2 fluorescent PREDIVEZ vesicle transport kits and human BSEP vesicles were purchased from Solvo Biotechnology (Budapest, Hungary). Multiscreen HTS 96 well filter plates and HTS Vacuum Manifold were obtained from EMD Millipore (Darmstadt, Germany). Male human plasma containing K2EDTA was purchased from Bioreclamation, LLC (Westbury, NY). Human liver microsomes (HLM) were purchased from Xenotech, LLC (Lenexa, KS). Parent HEK cells and OATP1B1 and OATP1B3 transfected HEK cells were obtained from Dr. Y. Sugiyama at University of Tokyo, Japan. Microsomal Incubations. UGT1A1 inhibition experiments were conducted as described previously with slight modifications.24−26 Briefly, 0.5 mg/mL HLM was incubated at 4 °C along with 1 mM MgCl2 and 50 μg alamethicin/mg protein in 0.1 M potassium phosphate buffer, pH 7.4. After approximately 15 min, 20 μM β-estradiol (UGT1A1 probe substrate at Km) and a range of test compounds between 0.05 and 100 μM were spiked into the reaction mixture. Following preincubation for 15 min at 37 °C, reaction was initiated by the 3068

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point. The IC50 was estimated by a sigmoidal inhibition model and was fit to:

addition of uridine 5′-diphospho-glucuronic acid (final concentration 5 mM). The total reaction volume for all experiments was 200 μL. Reaction was terminated with the addition of equal volume of acetonitrile containing 100 nM cortisone as the internal standard. Each sample was centrifuged, and the supernatant was injected onto LC-MS/MS to monitor for the formation of estradiol 3-glucuronide (E3G). All experiments were done in triplicate. Plasma Protein Binding Incubations. Human plasma was thawed, and the pH adjusted to 7.4 with either sodium hydroxide or phosphoric acid. Test compound was spiked into the plasma to give a final concentration of 5 μM (0.1% v/v with DMSO). 300 μL of spiked human plasma was added to the donor chamber of the RED device, and 500 μL of phosphate buffer was added to the receiver chamber of the RED device. The plate was sealed and incubated on a Liconic Shaker at 37 °C for 4 h. Under a separate condition, 300 μL of spiked human plasma was transferred to a 96-well plate and was also incubated at 37 °C for 4 h along with the RED device. This served as the nondialyzed plasma used to calculate the recovery of the experiment. It was assumed that adsorption of the test compounds to the RED device was negligible. The RED device was removed from the incubator at 4 h as 40 and 4 μL were sampled from the receiver and donor chambers, respectively, and transferred to a 96-well plate for analysis. In addition to the RED device samples, a 4 μL aliquot of nondialyzed plasma was also taken. To the plasma samples, 36 μL of blank plasma was added to give a 1:10 dilution. To eliminate any matrix effect on LC-MS/MS quantitation, equal volumes of blank plasma and blank buffer were added to the buffer sample and plasma sample, respectively. The samples were then quenched with 150 μL of acetonitrile containing 2.5 nM cortisone as the internal standard. Each sample was centrifuged, and the supernatant was injected onto LC-MS/MS to monitor for the parent analyte. All experiments were done in triplicate. Fraction unbound ( f u) was calculated as: [buffer]/[plasma], where the square bracket indicates the concentration determined in each matrix. The f u value was accepted if the recovery of the experiment was within 75−125%. Recovery was calculated as: 100*([buffer] + [dialyzed plasma])/[non-dialyzed plasma]. OATP1B1 and OATP1B3 Inhibition Assay. The parental HEK cells and OATP1B1 and OATP1B3 transfected HEK cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 400 μg/mL geneticin at 37 °C with 5% CO2 and 95% humidity. The inhibition studies were carried out between 48 and 72 h after seeding the cells at a density of 5.0 × 105 cells/well in 24-well tissue culture plates coated with poly(D-lysine) and laminin. Cells were preincubated at 37 °C in Hank’s balanced salt solution (HBSS) for 10 min. The uptake inhibition studies were initiated with various concentrations of test compounds along with 0.1 μM pitavastatin. Following 5 min incubation at 37 °C, the reaction was terminated by rinsing the cells twice with 1 mL of ice-cold HBSS buffer. The cells were then sonicated in 200 μL of HBSS for 5 min and precipitated with an equal volume of acetonitrile. Each sample was centrifuged, and the supernatant was injected onto LC-MS/MS to monitor for pitavastatin. IC50 values are presented as mean ± standard deviation (SD) with a minimum of three wells was used to generate each data

V = V0/(1 + log(I /IC50)n )

where V is the uptake of pitavastatin in the presence of the test inhibitors, V0 is the uptake of pitavastatin in the absence of the inhibitor, I is the inhibitor concentration, and n is the hillslope. BSEP and MRP2 Inhibition Assay. Both assays were run according to the manufacturer’s instructions. Briefly, the assay and washing buffers for the BSEP inhibition assay were made according to Solvo’s instructions. The assay buffer consisted of 2 mM Hepes-Tris, 100 mM potassium nitrate, 10 mM magnesium nitrate, and 50 mM sucrose in distilled water. The wash buffer consisted of 10 mM Tris-HCl and 100 mM potassium nitrate, 50 mM sucrose, and 0.1 mM taurocholate in Milli-Q water. Both solutions were sterile filtered (0.22 μM) and stored at 4 °C during the assay. Membrane suspension with unlabeled- and 3H-taurocholate were added to a 96 well plate containing test compounds (0 and 0.137−100 μM) in triplicate. The plate was preincubated at 37 °C for 15 min before the addition of blank assay buffer in the presence or absence of ATP. The plate was incubated at 37 °C for an additional 5 min after ATP was added. Ice cold wash mix was added to stop the reaction, the suspension was transferred to the filter plate, and the samples were washed 3 times with ice cold wash mix. Liquid scintillation cocktail was added to the filter plate and read on a Topcount NXT (PerkinElmer, Waltham, MA) for 1 min per sample. For the MRP2 inhibition assay, MRP2 PREDIVEZ kit containing all required reagents was purchased from Solvo. Membrane suspension containing test compounds (0 and 0.137−100 μM in triplicate) and fluorescent probe substrate [5(6)-carboxy-2′,7′-dichlorofluorescein, CDCF] was incubated at 37 °C for approximately 10 min. Blank assay buffer or assay buffer with ATP was added, and the plate was incubated for 30 min at 37 °C before ice cold wash buffer was added to quench the reaction. Vesicles were transferred to the filter plate and washed three times with ice cold wash buffer, and the plate was dried. Detector solution provided by the kit was added, and the CDCF fluorescent signal (Ex: 485 nM Em: 538 nM) was obtained using a SpectraMax M5 multimode plate reader (Molecular Devices, Sunnyvale, CA). LC-MS/MS Analysis. All samples were analyzed using a Cohesive LX-2 Transcend Multiplexing system with Agilent 1100 series HPLC pumps from Agilent Technologies (Santa Clara, CA) and a HTS PAL autosampler from CTC Analytics (Carrboro, NC) connected to a Sciex API4000-QTrap mass spectrometer with a Turbo Ion Spray source (Foster City, CA). Multiple reaction monitoring was used to quantify compounds in the positive ion mode (for all test compounds) or negative ion mode (for E3G). The stationary phase for all the test compound chromatography evaluated for plasma protein binding was a Hypersil Gold C18 column (1.9 μm, 2.1 × 20 mm) from Thermo Electron Corporation (San Jose, CA). Chromatographic separation was achieved using a solvent system consisting of an aqueous mobile phase (Y, water containing 5 mM ammonium acetate with 0.1% acetic acid) and organic mobile phase (Z, acetonitrile containing 0.1% acetic acid). The flow rate through the system was 0.5 mL/min. The initial condition was set at 1% Z. After 12 s, the system was ramped to 98% Z over 48 s. After allowing the system to hold at 98% Z for 84 s, the gradient was changed 3069

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back to the initial condition of 1% Z and was allowed to equilibrate for 60 s before the next injection. The stationary phase for E3G chromatography was a HydroRP column (4 μm, 50 × 2 mm) from Phenomenex, Inc. (Torrance, CA). Chromatographic separation was achieved using a solvent system consisting of an aqueous mobile phase (A, water with 0.1% formic acid) and organic mobile phase (B, acetonitrile with 0.1% formic acid). The flow rate through the system was 0.7 mL/min. The initial condition was set at 5% B which was ramped to 40% B over 24 s. This was followed by a ramp to 90% B over 84 s. After allowing the system to hold at 90% B for 12 s, the gradient was changed back to the initial condition of 5% B and was allowed to equilibrate 54 s before the next injection. The stationary phase for pitavastatin chromatography was a LUNA C18 (4 μm, 50 × 2.1 mm) from Phenomenex, Inc. (Torrance, CA). Chromatographic separation was achieved using a solvent system consisting of an aqueous mobile phase (A, water with 0.1% formic acid) and organic mobile phase (B, acetonitrile with 0.1% formic acid). The flow rate through the system was 0.5 mL/min. The initial condition was set at 5% B which was ramped to 95% B over 40 s. After allowing the system to hold at 95% B for 20 s, the gradient was changed back to the initial condition of 5% B and was allowed to equilibrate 54 s before the next injection.

Table 2. Summary of UGT1A1 Inhibition drug

dose (mg)

Cmax (μM)

Cmax,free (μM)

UGT IC50a (μM)

atazanavir indinavir ritonavir nelfinavir bromfenac troglitazone trovafloxacin

400 2400 1200 2500 50 600 200

7.7 13 15 3.3 9.6 6.4 7.4

0.51 2.9 0.11 0.0033 1.2 0.051 1.6

0.31 ± 0.04 6.8 ± 1.0 3.1 ± 0.2 4.8 ± 0.1 77 ± 9 4.5 ± 0.2 >100

Rtotalb

Rfreec

26 2.9 5.8 1.7 1.1 2.4

2.6 1.4 1.0 1.0 1.0 1.0

a

Probe substrate was estradiol monitoring for the formation of E3G. Rtotal = 1 + [I]/IC50, where [I] is Cmax. cRfree = 1 + [I]free/IC50, where [I]free is Cmax,free.

b

the most potent blocker of UGT1A1 with IC50 = 0.31 ± 0.04 μM. IC50 values for indinavir, ritonavir, and nelfinavir ranged between 3 and 7 μM. In contrast, bromfenac was a poor blocker, whereas trovafloxacin did not alter UGT1A1 activity at the highest concentration examined at 100 μM. It was assumed that the interaction between the test drugs and UGT1A1 to reduce E3G formation was reversible and competitive. To assess the potential in vivo inhibition of UGT1A1, in vitro IC50 values were put into context of drug concentrations reached in the systemic circulation. Specifically, R values were calculated as outlined in Materials and Methods, where escalating values above 1.1 are correlated to higher likelihood for inhibition in vivo. Total (Cmax) and free (Cmax,free) concentrations were employed to represent systemic concentrations to evaluate Rtotal and Rfree, respectively. Cmax,free was determined by factoring the measured free fraction value in the plasma ( f u) summarized in Table 1 with Cmax. When considering total plasma concentrations, Rtotal exceeded 1.1 for atazanavir, indinavir, bromfenac, troglitazone, ritonavir, and nelfinavir, with the highest value observed for atazanavir at 26. When free concentration was utilized to calculate Rfree, only atazanavir and indinavir, which exhibit clinical hyperbilirubinemia, yielded values greater than 1.1 (Table 2). Inhibition of Efflux Transporters BSEP and MRP2. Membrane vesicles expressing BSEP and MRP2 were used to evaluate efflux transporter activity. Table 3 summarizes IC50 values of drugs examined against BSEP and MRP2 with 3[H]taurocholate and CDCF as surrogate probe substrates, respectively. IC50 of rosiglitazone, positive control for BSEP inhibition, was within range of published protocol given in the BSEP kit at 1.9 ± 0.5 μM. Trovafloxacin, atazanavir, indinavir, and ritonavir exhibited IC50 similar to rosiglitazone, while the values for nelfinavir and troglitazone were approximately 10fold higher. Bromfenac had no effect on BSEP. IC50 of MK0571, a positive control for MRP2 inhibition, was within range of published protocol given in the MRP2 kit at 2.7 ± 0.2 μM. Inhibition of MRP2 was only observed with troglitazone with IC50 of 17 ± 0.9 μM. IC50 values for these efflux transporters were also put into context of observed clinical concentrations to calculate Rtotal and Rfree. Rtotal was greater than 1.1 for all compounds for which the IC50 values were obtained in the BSEP and MRP2 assay. However, when accounting for the free concentration, Rfree exceeded 1.1 only for atazanavir, indinavir, and trovafloxacin in the BSEP assay. Rfree of all drugs examined was less than 1.1 for MRP2. Inhibition of Uptake Transporters OATP1B1 and OATP1B3. HEK cells expressing OATP1B1 and OATP1B3

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DATA ANALYSIS To determine the inhibition potency, IC50 values were generated by monitoring for the formation of E3G or flux of pitavastatin, CDCF and 3H-taurocholate, in the absence or presence of varying concentration of test drugs. Prism from Graphpad Software, Inc. (La Jolla, CA) was used fit the data to generate IC50 values. Absolute sum of squares and R2 generated by Graphpad were used to assess the goodness of the IC50 fit. IC50 plots for individual test compounds against UGT1A1, BSEP, MRP2, OATP1B1, and OATP1B3 are shown in the Supporting Information. To evaluate the likelihood that the in vitro inhibition values would translate into in vivo interaction, R value, which is the ratio of probe drug exposures in the absence or presence of an inhibitor, was calculated as follows: R = 1 + [I]/IC50 where [I] is the maximum inhibitor concentration that is achieved in vivo. [I] was approximated with Cmax since these values were available for the select test drugs. In addition, Cmax,free calculated from Cmax and experimental f u was used to estimate Rfree. The target threshold for R or Rfree was set at 1.1 as recommended by the FDA guidance, where values above 1.1 is indicative of potential in vivo liability.27,28 While the cutoff value of 1.1 does not predict the magnitude of in vivo interaction, it estimates the likelihood of drug interactions when the plasma concentration reaches 1/10th of the in vitro IC50 values.

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RESULTS Inhibition of UGT1A1. Biotransformation of estradiol to estradiol 3-β-glucuronide (E3G) is mediated by UGT1A1 with Km of approximately 20 μM.29 Inhibition of E3G formation has been previously demonstrated as a good surrogate to predict the modulation of bilirubin glucuronidation.30 Table 2 summarizes IC50 of E3G inhibition in HLM. Atazanavir was 3070

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Table 3. Summary of Efflux Transporter Inhibition BSEP and MRP2 BSEPa

MRP2b c

Rfree

d

drug

dose (mg)

Cmax (μM)

Cmax,free (μM)

IC50 (μM)

Rtotal

atazanavir indinavir ritonavir nelfinavir bromfenac troglitazone trovafloxacin

400 2400 1200 2500 50 600 200

7.7 13 15 3.3 9.6 6.4 7.4

0.51 2.9 0.11 0.0033 1.2 0.051 1.6

3.1 ± 0.7 3.1 ± 0.2 2.6 ± 0.1 24 ± 4 >100 18 ± 4 1.7 ± 0.5

3.5 5.1 6.9 1.1

1.2 1.9 1.0 1.0

1.4 5.4

1.0 1.9

IC50 (μM) >100 >100 >100 >100 >100 17 ± 0.9 >100

Rtotalc

Rfreed

1.4

1.0

Probe substrate was 3H-taurocholate; positive control was rosiglitazone with IC50 = 1.9 ± 0.5 μM. bProbe substrate was CDCF; positive control was MK-571 with IC50 = 2.7 ± 0.2 μM. cRtotal = 1 + [I]/IC50, where [I] is Cmax. dRfree = 1 + [I]free/IC50, where [I]free is Cmax,free. a

Table 4. Summary of Uptake Transporter Inhibition OATP1B1 and OATP1B3 OATP1B1a drug

dose (mg)

Cmax (μM)

Cmax,free (μM)

atazanavir indinavir ritonavir nelfinavir bromfenac troglitazone trovafloxacin

400 2400 1200 2500 50 600 200

7.7 13 15 3.3 9.6 6.4 7.4

0.51 2.9 0.11 0.0033 1.2 0.051 1.6

OATP1B3b

IC50 (μM)

Rtotalc

Rfreed

IC50 (μM)

Rtotalc

Rfreed

0.9 ± 4.1 ± 0.5 ± 2.0 ± >100 1.2 ± >100

0.2 1.8 0.4 0.9

9.6 4 32 3.8

1.6 1.7 1.2 1.0

3.1

1.1

0.3

6.3

1.0

3.7 ± 0.1 >100 >100 >100 >100 >100 >100

Probe substrate was pitavastatin; positive control was cyclosporine with IC50 = 0.23 ± 0.06 μM. bProbe substrate was pitavastatin; positive control was cyclosporine with IC50 = 0.42 ± 0.11 μM. cRtotal = 1 + [I]/IC50, where [I] is Cmax. dRfree = 1 + [I]free/IC50, where [I]free is Cmax,free.

a

the integrity of bile flow may play an indirect role in modulating bilirubin concentrations in the body. Therefore, the aim of this work was to evaluate the in vitro inhibitor potency of select test drugs associated with hyperbilirubinemia against not only UGT1A1 but also of drug transporters. Due to poor solubility33 and weak mass spectrometer response, bilirubin is not conducive for obtaining robust in vitro data. Although tritiated bilirubin has been used previously to elucidate the role of various OATPs, various experimental factors such as the need to supplement the incubation with serum albumin, high adsorption of bilirubin to plasticware, and uncertainty associated with actual incubation concentration may complicate data interpretation.2,34,35 In addition, there are many challenges when using poorly soluble compounds, especially in drug transporter experiments, which may compromise the integrity of the data such as compound precipitation, indeterminate integrity of the sink condition, and uncertain incubation concentrations.36 As a result, in the current study, typical probe substrates such as pitavastatin, estradiol (i.e., formation of E3G), CDCF, and taurocholate were employed to evaluate OATP1B1/OATP1B3, UGT1A1, MRP2, and BSEP, respectively. Although using surrogate probe substrates other than bilirubin or its glucuronide metabolite is not ideal and may limit data application, there is evidence supporting the utility of surrogates to yield reasonable estimation of inhibition liabilities related to bilirubin elimination. Indeed, a good correlation between the inhibition of E3G formation and bilirubin glucuronidation was observed.30 Therefore, it was assumed that any inhibition of the surrogate probe substrates by test drugs is indicative of the potential impact of test drugs to modulate the function of proteins involved in bilirubin disposition. To gauge the potential impact in vivo, IC50 values were put into context of concentrations observed in the clinic by calculating a R value which is the ratio of victim drug exposure

were used to examine uptake transporter activity. Pitavastatin was used as the surrogate probe substrate for both OATP1B1 and OATP1B3,31 and Table 4 summarizes IC50 values of the test drugs. Cyclosporine was run as a positive control for OATP1B1 and OATP1B3 inhibition yielding IC50 values of 0.23 ± 0.06 μM and 0.42 ± 0.11 μM, respectively, consistent with published values for OATP1B1.31,32 Ritonavir was a potent inhibitor of OATP1B1 with IC50 value comparable to cyclosporine. OATP1B1 activity was also attenuated in the presence of atazanavir, troglitazone, nelfinavir, and indinavir; whereas bromfenac and trovafloxacin did not have a significant effect on OATP1B1 with IC50 > 100 μM. Unlike OATP1B1, inhibition of OATP1B3 was observed only with atazanavir with IC50 3.7 ± 0.1 μM. Rtotal and Rfree were also calculated for OATP1B1 and OATP1B3. Rtotal was greater than 1.1 for all compounds for which the IC50 values were obtained in the uptake assays. However, when using the free concentration, Rfree exceeded 1.1 only for atazanavir, indinavir, and ritonavir with OATP1B1 and for atazanavir with OATP1B3.

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DISCUSSION Hyperbilirubinemia is best characterized among individuals with genetic diseases such as Gilbert’s syndrome, but druginduced hyperbilirubinemia are also observed and may need to be monitored for patients in various disease states and/or taking multiple therapeutic regimens. Particularly, drugs may limit bilirubin elimination by blocking different pathways associated with bilirubin disposition. As illustrated in Figure 1, once formed in the systemic circulation, bilirubin must first enter the hepatocytes via OATP1B1 and OATP1B3. Once in the hepatocytes, bilirubin is extensively metabolized by UGT1A1 to the diglucuronide metabolite which is then shunted into the bile by MRP2. In addition to proteins directly effecting bilirubin clearance, BSEP which aids in maintaining 3071

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Figure 2. Summary of UGT1A1, OATP1B1, OATP1B3, and BSEP inhibition by drugs evaluated in the current study (Rfree > 1.1). 1Represents protease inhibitors which exhibit clinical hyperbilirubinemia; 2Represents protease inhibitors which do not exhibit clinical hyperbilirubinemia; 3 Represents drugs which are associated with idiosyncratic toxicity and have been withdrawn from the market.

in the absence or presence of a perpetrator.28,37,38 The R value typically evaluates the likelihood of clinical drug interaction, and this assessment was applied to the current study where bilirubin and the test drugs were regarded as the victim and the perpetrator, respectively. In vivo liver concentrations may be best to represent [I], as it may be most relevant in translating in vitro parameters to hepatically expressed protein. Instead, Cmax was considered because, although it may not be the perfect surrogate as it has been shown to underestimate in vivo interactions,39 it was the most practical surrogate as liver concentrations are not available. Indeed, retrospective analysis showed that reasonable prediction of in vivo drug interaction was observed when using plasma.40 Therefore, R was defined as 1 + Cmax/IC50 by substituting [I] with Cmax. Furthermore, since the free drug may be critical for driving target engagement, both total and free Cmax were utilized to determine Rtotal and Rfree, respectively. While protease inhibitors atazanavir41 and indinavir42 are associated with clinical unconjugated hyperbilirubinemia, ritonavir43 and nelfinavir44 are not. Their inhibition profiles are summarized in Figure 2. In general, there was no effect on MRP2 and OATP1B3 (except for atazanavir which inhibited OATP1B3). These data suggest that the inhibition of these transporters may only play a minor role in correlating to hyperbilirubinemia. This is reasonable considering that differential regulation of MRP proteins is manifested in DubinJohnson patients, as loss of MRP2 activity can be compensated by MRP3, and since OATP1B1 may play a bigger role in bilirubin uptake than OATP1B3.34,45 In the current experiment, inhibition against OATP1B3 with atazanavir was consistent with previous literature reports.46 However, inhibition observed with indinavir and ritonavir were not reproducible with published literature IC50 values of 12.3 and 3.6 μM, respectively. It is not known if the disconnect are due to differences in probe substrate or the OATP1B3 transfected systems or both, and studies are presently ongoing to better understand the two systems. In contrast to MRP2 and OATP1B3, the protease inhibitors were all able to block UGT1A1, OATP1B1, and BSEP. However, the IC50 values were not able to correctly categorize

the drugs into appropriate clinical observations. For example, IC50 of indinavir against UGT1A1 was comparable to some drugs which do not exhibit hyperbilirubinemia (Table 2), whereas ritonavir was one of the most potent blocker of OATP1B1 and BSEP (Tables 3 and 4). The in vivo predictability was not improved when Rtotal was calculated using Cmax, as ritonavir demonstrated the highest liability across multiple proteins. Interestingly, hyperbilirubinemia became more predictable when free concentrations were used to calculate Rfree. Specifically, Rfree of UGT1A1, OATP1B1 and BSEP were above 1.1 for only atazanavir and indinavir which exhibit hyperbilirubinemia. Previous studies have shown the relevance of free concentration in predicting CYP-mediated drug interactions.40,47 However, these data suggest that free in vivo concentrations may also be relevant when trying to predict drug interactions mediated by drug transporters. Indeed, applying Cmax,free affords a better context to an in vitro IC50 value which already represents a free concentration. This observation is consistent with the free drug hypothesis which states that only free molecules are able to yield pharmacological/inhibitory effect. As such, the combination of high plasma concentration and potent in vitro inhibition of ritonavir, which should produce unfavorable consequences, may be negated by extensive plasma protein binding where just a small amount is actually able to inhibit proteins, thereby yielding insignificant in vivo effect. These data suggest that the mechanism underlying hyperbilirubinemia for protease inhibitors may be through the disruption of bilirubin elimination. As illustrated in Figure 2, blocking OTP1B1 and UGT1A1 would enhance the amount of unconjugated bilirubin in the body since the access to hepatocytes is reduced and the major enzymatic force involved in the breakdown of bilirubin is removed. In addition, inhibiting BSEP may disrupt the normal flow of bile needed to clear the hepatocyte system and to maintain the integrity of the hepatocytes. The link between UGT1A1 and hyperbilirubinemia with protease inhibitors has been previously reported,23 but the current study is the first to expand the investigation to include multiple drug transporters. Despite the limited data set, data infer that, in addition to UGT1A1, drug-induced 3072

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inhibition of not only UGT1A1 but also OATP1B1 and BSEP. While it is clear that inhibition of UGT1A1 is a necessary component, work is currently ongoing to assess if both transporters need to be inhibited or if inhibition of just one transporter is sufficient to enhance the predictability of hyperbilirubinemia. In contrast, bromfenac, troglitazone, and trovafloxacin had no effect on these proteins. Hence, hyperbilirubinemia for these test drugs may follow a separate mechanism and is not predicted with the tools utilized here, which is consistent with drugs associated with idiosyncratic toxicity.

hyperbilirubinemia may also require the inhibition of OATP1B1 and BSEP to yield a clinically meaningful effect. There is evidence to support that UGT1A1 itself is not sufficient to account for varying degrees of hyperbilirubinemia. For example, if UGT1A1 is the major protein responsible for bilirubin variations, its inhibition should have minimal impact in patients with Gilbert’s syndrome whose UGT1A1 activity is already attenuated. Yet, the severity of hyperbilirubinemia became exacerbated when these patients were administered atazanavir48 and indinavir,49 implicating the contribution by additional proteins other than UGT1A1 and supporting the hypothesis that the mechanism of hyperbilirubinemia may be multifaceted. The data also suggest that, while inhibition of UGT1A1 may be necessary, alteration of drug transporter activity alone may not be sufficient to trigger the onset of hyperbilirubinemia as exemplified by the nonhyperbilirubinemic ritonavir exhibiting Rfree of 1.2 against OATP1B1. In fact, studies with OATP1B1-expressing cells have indicated that OATP1B1 was insufficient to account for complete bilirubin transport,35 whereas polymorphism of OATP1B1 in Chinese patients did not correlate with bilirubin levels.50 In addition, although BSEP has been implicated in hepatotoxicity, BSEP inhibitor bosentan enhanced serum transaminase in the clinic without affecting bilirubin, implying that inhibition of BSEP activity alone was not sufficient to modulate bilirubin levels and that bosentan-induced hepatotoxicity was not mediated by increased bilirubin.51 Bromfenac,52,53 troglitazone,54,55 and trovafloxacin56,57 have been withdrawn from the market due to severe idiosyncratic hepatotoxicity characterized by pronounced increases in serum bilirubin and hepatic transaminase. Hepatotoxicity has been partly attributed to the formation of reactive metabolites that form covalent adducts to proteins and work is presently ongoing in this laboratory to assess their impact on UGT1A1, OATP1B1, and BSEP. The current scope was to investigate if the parent drug limited bilirubin clearance. As summarized in Figure 2, in contrast to the hyperbilirubinemic protease inhibitors, there was no significant effect on UGT1A1 and the various drug transporters that were examined with the exception of trovafloxacin against BSEP, suggesting that the mechanism of hyperbilirubinemia is not mediated through disrupting bilirubin elimination but may involve hepatocellular injury which fractures the integrity of the hepatocytes and the liver architecture. Consequently, hyperbilirubinemia may be a symptom of hypofunctional liver rather than a result of obstructed bilirubin disposition. In summary, these data demonstrate that the proposed surrogate probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and BSEP may be suitable to assess bilirubin disposition. In addition, the current work underpinned the importance of putting an in vitro IC50 value in context of in vivo concentrations to evaluate the likelihood of a drug interaction. Although some caveats associated with calculating the R have already been described, the value of 1.1 provided a reasonable cutoff for estimating the likelihood of unfavorable interaction with UGT1A1 and drug transporters contributing to bilirubin disposition. This is consistent with the current understanding of how to best predict in vivo drug interaction. As a result, the current work was able to describe and differentiate between two different mechanisms of hyperbilirubinemia. For atazanavir and indinavir, hyperbilirubinemia is mediated by direct and indirect disruption of bilirubin elimination which may be predictive through assessing the

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ASSOCIATED CONTENT

* Supporting Information S

IC50 plots for individual test compounds against UGT1A1, BSEP, MRP2, OATP1B1, and OATP1B3 are shown in S1, S2, S3, S4, and S5, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. Tel.: (650) 467-9708. Fax: (650) 467-3487. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors want to thank Ms. Gillian Smelick for her help in the early evaluation of UGT1A1. The authors would also like to thank Drs. Matthew Wright and Laurent Salphati for reviewing the manuscript and for their helpful comments.

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ABBREVIATIONS UGT, UDP-glucuronosyltransferase; OATP, organic-anion transporting polypeptide; MRP, multidrug resistance associated protein; BSEP, bile salt export pump; E3G, estradiol 3glucuronide; CDCF, 5(6)-carboxy-2′,7′-dichlorofluorescein; Cmax, therapeutic peak plasma concentration in humans; f u, fraction unbound in plasma; LC-MS/MS, liquid chromatography−mass spectrometry/mass spectrometry

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REFERENCES

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