Inhibition of MDR3 Activity in Human Hepatocytes by Drugs

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INHIBITION OF MDR3 ACTIVITY BY DRUGS ASSOCIATED WITH LIVER INJURY IN HUMAN HEPATOCYTES Kan He, Lining Cai, Qin Shi, Hao Liu, and Thomas Woolf Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00201 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 10, 2015

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Chemical Research in Toxicology

INHIBITION OF MDR3 ACTIVITY IN HUMAN HEPATOCYTES BY DRUGS ASSOCIATED WITH LIVER INJURY Kan He*, Lining Cai, Qin Shi, Hao Liu, Thomas F. Woolf Biotranex LLC, Monmouth Junction, NJ 08852 KEYWORDS: MDR3, ABCB4, hepatocyte, DILI, hepatotoxicity, cholestasis, VBDS

ABSTRACT: MDR3 dysfunction is associated with liver diseases. We report here a novel MDR3 activity assay involving in situ biosynthesis in primary hepatocytes of deuterium (d9)-labeled PC and LC-MS/MS determination of transported extracellular d9-PC. Several drugs associated with DILI such as chlorpromazine, imipramine, itraconazole, haloperidol, ketoconazole, sequinavir, clotrimazole, ritonavir and troglitazone inhibit MDR3 activity. MDR3 inhibition may play an important role in drug-induced cholestasis and vanishing bile duct syndrome. Several lines of evidences demonstrate that this reported assay is physiologically relevant and can be used to assess the potential of chemical entities and their metabolites to modulate MDR3 activity and/or PC biosynthesis in hepatocytes.

INTRODUCTION

EXPERIMENTAL PROCEDURES

Multidrug resistance protein 3 (MDR3, ABCB4) is a 1279 amino acid P-glycoprotein primarily expressed in the canalicular membrane of hepatocyte, and is responsible for 1,2 biliary secretion of phosphatidylcholine (PC). PC combined with bile salts form mixed micelles in bile secretions, which solubilizes cholesterol and prevents highly concentrated bile salts from damaging biliary canaliculi epithelium cells. Mutations in the human MDR3 gene are associated with a wide spectrum of liver diseases, such as progressive familial intrahepatic cholestasis type 3 (PFIC3), intrahepatic cholestasis of pregnancy, lowphospholipid-associated cholelithiasis, primary biliary cirrhosis, cholangiocarcinoma and drug-induced liver 1,3,4 injury (DILI).

Chemicals and Reagents. Deuterium (d9)-Choline was purchased from Cambridge Isotope Laboratories Inc (Tewksbury, MA). Human hepatocytes and InVitroGRO media were obtained from BiorecalamationIVT (Baltimore MD). Human hepatocytes were pooled from 10 individual donors. Williams Medium E was purchased from Life Technologies (Grand Island, NY). Other reagents were purchased from Sigma-Aldrich (St Louis, MO) unless stated otherwise in the text.

Several MDR3 assays have been reported, including MDR3-transfected LLC-PK and HEK293 cells, Mdr2expressed yeast secretory vesicles, and MDR3-over 1,5,6 expressed Sf9 insect cell membrane vesicles. Many of these assays use as an MDR3 probe substrate the PC analog containing a fluorescence group of 7-nitro-2,1,3benzoxadiazol (NBD). These assays have several disadvantages, including a lack of physiological relevance to hepatocytes, artificial substrates, low signal-to-noise ratio and inability to assess a test agent’s metabolism on MDR3 1 transport . Because of difficulties in establishing a robust and accurate MDR3 assay in physiologically relevant systems, there is much less known about drugs interacting with MDR3 than other transporter proteins. Here we report a novel MDR3 activity assay using primary hepatocytes and use of the assay in screening drugs associated with DILI for their potential to inhibit MDR3 activity.

Preparation of Hepatocytes. Fifty mL InVitroGRO HT medium was pre-warmed in a 37oC water bath. A vial of hepatocyte was removed from a liquid N2 tank and quickly warmed up in a 37oC water bath by holding in hand with slow rotation. As soon as the edge of the frozen cells was separated from the wall of the vial, the frozen cells were poured into the pre-warmed InVitroGRO HT medium and the remaining cells in the vial were collected using pipette. The tube was centrifuged at 50 g, 25oC for 5 minutes, the supernatant was removed and the cell pellet was re-suspended in 8 mL of pre-warmed Williams E buffer, the cell numbers were counted in a hemocytometer. Cell viability was determined using Trypan blue method. The hepatocyte concentration was adjusted with Williams E buffer to meet the objectives of various experiments. Incubations with Hepatocytes in Suspension. Human hepatocytes (0.25 million/mL) were incubated in Williams E buffer in the presence of 100 µg/mL d9choline and 2.5 or 5 mM taurocholic acid (TCA) at 37 oC under 5% CO2 and saturated humidity for 2 hours. In some experiments the incubation times varied from 0-2 hours, or the hepatocyte concentrations varied from 0-1 million/mL. Test drugs were incubated at various concen-

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trations ranging from 0-100 µM in hepatocyte incubations to assess their effect on MDR3 activity. The effect of TCA on MDR3 activity and PC synthesis was evaluated at concentrations ranging from 0 to 5 mM. Following incubation for designated time periods, extracellular media were isolated by differential centrifugation at 3310 g for 15 min or by filtration with a 96-well membrane filter (pore size 0.2 µm). The resulting supernatants or eluates from the filtration were mixed with 2-3X volumes of isopropanol containing the analytical internal standard (see below section) for LC-MS/MS analysis. Hepatocyte pellets after centrifugation were sonicated in the presence of 100 µL water for 5 min, following with addition of 2-3 X volumes of an isopropanol:hexane (9:1) solution, mixing at 2000 rpm for 1 min, and centrifugation at 3310 g for 10 min. Organic phases were dried under nitrogen flow. The residues were reconstituted with isopropanol containing the analytical internal standard for LC-MS/MS analysis. LC/MS/MS Assays. Liquid chromatography was carried out using a Shimadzu (Columbia, MD) HPLC system consisting of two LC-10ADvp pumps, a SIL-HTC autosampler, and an automated switching valve. The switching valve was used to divert the column effluent to either waste or to the MS instrument. The Shimadzu HPLC system was used for sample injection and analyte separation. Each sample was loaded onto a reverse phase column, Phenomenex (Torrance, CA) Luna C8 (5 µm, 2 mm x 50 mm). The column chamber’s temperature was ambient. The HPLC mobile phases are 50% acetonitrile in water containing 0.1% formic acid (A) and isopropanol:acetonitrile (9:1)(B). The flow rate was 0.5 mL/min. The amount of B in the mobile phase was ramped linearly from 0% up to 62% over a 0.5-minute period followed by a slow increase to 63% B in 4 minutes, then a rapid increase to 95% B in 0.1 min. After holding at 95% B for 2.3 minutes, the mobile phase was reset to the initial conditions in 0.1 minute. The analytical column was equilibrated with the starting mobile phase for 3 minutes. The total run time for each sample analysis was approximately 10 minutes. The HPLC elute was injected into an AB Sciex API4000 LC/MS/MS system (Framingham, MA) equipped with a Turbo IonSpray source set with a desolvation temperature of 550°C. Data for PC was acquired in the positive ion mode using multiple reaction monitoring methods (MRM). The ion transitions of the MRM method for specific detection of PC species were developed in standard fashion. Ionspray voltage was set at 5000 V and the collision gas (CAD) set at 4. Declustering potential and collision energy was set at 50, 42 respectively for PC. The PC species were monitored using the following transitions: 768→193, 770→193, 796→193, 798→193, 792→193, 794→193, 816→193, respectively. (S)-(+)-Methyl 2-(4,5,6,7tetrahydrothieno[3,2-c]pyridin-5-yl)-2-(2-chlorophenyl) acetate hydrogen sulfate was used as an internal standard with the following ion transition: 322.2→152.1. Propane-1sulfonic acid {3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3b]pyridine-3-carbonyl]-2,4difluoro-phenyl}-amide was also used as an internal standard with the following ion transition: 490.1→383.1.

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Data Analysis. IC50 values were calculated using medi7 an-effect plots.

RESULTS AND DISCUSSION The assay we report here involves in situ biosynthesis in primary hepatocytes of d9-labeled PC species from the precursor d9-choline, isolation of extracellular medium, and determining the amount of d9-labeled PC

Figure 1. Selected ion chromatograms of d9-PC species from extracellular medium of human hepatocyte incubations by LC-MS/MS. Pooled hepatocytes were incubated in the

presence of 100 µg/mL d9-choline and 2.5 mM TCA at 37oC for 2 hours. species transported into extracellular medium with a sensitive and specific LC-MS/MS method. The PC species consist of a d9-choline head group, a glycerophosphoric acid backbone and two C16-C22 fatty acids. Several PC species are formed by human hepatocytes and transported into extracellular medium when incubated at 37oC for 2 hr in Williams E buffer in the presence of 100 µg/mL d9choline and 2.5 mM taurocholic acid (TCA) (Figure 1). The major PC species detected in the extracellular medium, in order of abundance, have the following ratios of fatty acid carbon numbers to double-bond numbers: 34:2, 34:1, 36:4, 36:3, 36:2, 38:6 and 36:1. The two most abundant PC species, 34:2 and 34:1, correspond to 1-palmitoyl 2-linoleyl and 1-palmitoyl 2-oleol, respectively. The PC species profile observed in extracellular medium is similar to that 8 reported in human bile. In comparison, the PC species profile obtained from the lipid extracts of the culture medium of HEK/ABCB4 cells displays a PC species profile with the most abundant PC species being 34:1, 32:1 and 36:2, which differs from those obtained in our hepatocytes 9 assay and reported in human biliary secretions. In our assay, the transport of the PC species increases with an increase in incubation time (0-2 hours) and hepatocyte concentrations (0-1 million cells/mL) (data not shown). These results demonstrate that MDR3 activity can be determined using primary hepatocytes, and that the PC species profile in the extracellular medium is similar to that reported in human bile.


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Table 1. Inhibition of MDR3 activity by drugs associated with or without drug-induced liver injury (DILI) in human hepatocyte incubations

140 PC amount (fold change)

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Extracellular medium Cellular and extracellular

120 100 80

Drug

IC50 (µM)*

DILI Type**

Biotin

>100

None

60

Chlorpromazine 9.7

CHL/VBDS

40

Clotrimazole

CHL, Mixed

20

Cyclosporine

~100^

CHL

Furosemide

>100

None

0 0

2

4

6

Taurocholic acid concentration (mM)

Figure 2. Effects of taurocholic acid on MDR3 activity (extracellular PC level) and PC synthesis (cellular and extracellular PC level) in human hepatocyte incubations. PC (34:2) was analyzed by LC-MS/MS for concentrations in the extracellular medium and cellular fractions. Pooled human hepatocytes were incubated in the presence of 100 µg/mL d9choline and of various concentrations of TCA from 0-5 mM o at 37 C for 2 hours. PC amounts were determined in extracellular medium and cell extract using LC-MS/MS.

Bile salts are major components in bile and present at 10 high concentrations up to 40 mM. Previous reports showed that bile salts could stimulate MDR3 activity in 5,9 various non-hepatocyte experimental systems. A discrepancy among these reports involves whether the stimulating effect is due to TCA monomers or TCA micelles. As shown in Figure 2, the amount of PC transported into extracellular medium is dramatically increased in human hepatocytes by TCA at concentrations greater than 1.25 mM. The total amount of each PC species formed in hepatocytes, combining extracellular and cellular amounts, is not affected by the TCA concentration, suggesting that the TCA effect is on MDR3 transport or release of PC from the plasma membrane of hepatocytes. The potential effect of bile salts including TCA on lipid composition, and in turn, the membrane integrity of hepatocyte plasma membrane is subject for further investigation. Previous reports suggest that TCA does not have a clear critical micellar concentration (CMC), but continuously self-aggregates over a wide range of concentra11 tions. Morita et al reported that TCA had a CMC of 2.5 mM in DMEM medium using light scattering measure9 ments. It is reasonable to speculate that TCA at concentrations greater than 2.5 mM exists primarily in micellar or aggregated forms. Therefore, the stimulation effect on MDR3 by TCA in micelles might be more substantial than that by TCA monomers. Our results demonstrate that bile salts at concentrations that form micelles have dramatic stimulating effect on MDR3 activity in primary hepatocytes. Verapamil and itraconazole are known MDR3 inhibi1,12 tors. In the present study, these drugs

4.6

Haloperidol

10.7

CHL/VBDS

Imipramine

14.2

CHL/VBDS, mixed

Itraconazole

2.1

CHL

Ketoconazole

5.6

HC, CHL

Penicillamine

>100

None

Ritonavir

9.6

HC, CHL

Saquinavir

12.9

HC, CHL

Troglitazone

10.3

Mixed, CHL

Verapamil

6.3

Mixed, CHL

*Experiments were carried out in triplicate using pooled human hepatocytes. The coefficients of variation were less than 20%. The mean was used for IC50 calculation 13

**Based on the LiverTox database ; CHL: cholestasis; VBDS: vanishing bile duct syndrome; HC: hepatocellular injury; Mixed: mixed types of injury ^30-50% inhibition at concentrations ranging from 1-100 µM with no clear dose response

show potent MDR3 inhibition with estimated IC50 values of 6.3 and 2.1 µM, respectively (Table 1), which is consistent with previous findings. Furthermore, haloperidol, ketoconazole, saquinavir, clotrimazole, ritonavir and troglitazone, all associated with DILI and potent inhibi14 tion of the bile salt export pump (BSEP), are also potent inhibitors of MDR3 transport. The liver injuries, particularly cholestatic liver injury, associated with these drugs could involve the inhibition of BSEP and/or MDR3. Interestingly, chlorpromazine, imipramine, and itraconazole, all associated with DILI and weak or poor BSEP inhibition, show potent inhibition of MDR3 (Table 1), suggesting an important role MDR3 inhibition may play in the cholestatic liver injuries caused by these drugs. It is noteworthy that inhibition of PC biosynthesis may also result in reduced MDR3 activity as determined based on the extracellular level of PC. Chlorpromazine and imipramine are well known drugs associated with acute and chronic cholestatic liver injury and liver test abnormalities in patients on long term ther13 apy. In most cases the liver abnormalities are usually self-limited and reversible. In some cases, the liver injury may result in prolonged jaundice and vanishing bile duct syndrome. Both chlorpromazine and imipramine, as mentioned above, are not potent BSEP transporter inhibitors, suggesting that bile salt transport abnormalities may not 14 be involved in their ability to cause DILI. Contrastingly,



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both chlorpromazine and imipramine are potent inhibitors of MDR3 activity as shown in Table 1. Since these drugs undergo extensive hepatic metabolism, the MDR3 inhibition observed may be reflective of inhibition by parent and/or formed metabolites in our metabolically competent assay. Itraconazole is associated with typical cholestatic liver injury, and in rare cases with vanishing bile 12,13 duct syndrome. As shown in Table 1, itraconazole caused potent inhibition of MDR3. Previous studies showed that itraconazole markedly reduced biliary PC levels due to inhibition of MDR3 activity while biliary bile 12 salt levels remained unchanged. Combined, these results support the hypothesis that this class of drugs/chemical entities may interfere with biliary secretion of PC species, resulting in disruption of bile salts-PC mixed micelles formation and deleterious effects of highly concentrated free bile salts on canaliculi and bile ducts, eventually leading to vanishing bile duct syndrome. MDR3 has a 76% amino acid sequence identity with MDR1 (ABCB1, p-glycoprotein), yet these two transporters exhibit distinct substrate specificities. Several MDR1 inhibitors/substrates, including verapamil, intraconazole, cyclosporine, valspodar, vinblastine and paclitaxel are reported to inhibit NBD-PC transport activity in Mdr21,15 expressed yeast secretory vesicles. But concerns have been raised that NBD-PC may involve transport by both MDR1 and Mdr2. In the present study, we tested several MDR1 inhibitors for their potential to inhibit MDR3 activities in human hepatocytes (Table 1). Our results showed that several potent MDR1 inhibitors, such as verapamil, ketoconazole and ritonavir, inhibit MDR3 activity. However, cyclosporine, a potent inhibitor of MDR1, BSEP and other transporters, does not inhibit MDR3 activity. Additionally, MDR3 activity is inhibited by the drugs imipramine, chlorpromazine and clotrimazole which are not potent MDR1 inhibitors. Collectively, these results suggest that MDR3 has not only distinct substrates, but also different inhibitor specificity from MDR1. In conclusion, we report here a novel assay that measures MDR3 activity using primary human hepatocytes. Several drugs associated with DILI demonstrate potent inhibition of MDR3 activity. Our assay provides the ability to rapidly and accurately test the potential of chemical entities and/or their metabolites to inhibit MDR3 transport activity and/or PC biosynthesis in a physiologically relevant in vitro model.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * Kan He, PhD, Biotranex LLC, Monmouth Junction, NJ 08852; Phone: 609-240-8333; Fax: 732-230-3062; Email: [email protected]

ABBREVIATIONS ABC: ATP-binding cassette; BSEP: bile salt export pump; CMC: critical micellar concentration; DILI: drug-induced

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liver injury; d9: nine deuterium atoms; MDR1: multidrug resistance protein 1; MDR3: multidrug resistance protein 3; NBD: 7-nitro-2,1,3-benzoxadiazol; PC: phosphatidylcholine; PFIC3: progressive familial intrahepatic cholestasis type 3; TCA: taurocholic acid.

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Inhibition of MDR3 Activity in Human Hepatocytes by Drugs

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