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Research ArticleArticle

Specific Inhibition of the Distribution of Lobeglitazone to the Liver by Atorvastatin in Rats: Evidence for a Rat Organic Anion Transporting Polypeptide 1B2–Mediated Interaction in Hepatic Transport

Chang-Soon Yim, Yoo-Seong Jeong, Song-Yi Lee, Wonji Pyeon, Heon-Min Ryu, Jong-Hwa Lee, Kyeong-Ryoon Lee, Han-Joo Maeng and Suk-Jae Chung
Drug Metabolism and Disposition March 2017, 45 (3) 246-259; DOI: https://doi.org/10.1124/dmd.116.074120

Abstract

Cytochrome P450 enzymes and human organic anion transporting polypeptide (OATP) 1B1 are reported to be involved in the pharmacokinetics of lobeglitazone (LB), a new peroxisome proliferator–activated receptor γ agonist. Atorvastatin (ATV), a substrate for CYP3A and human OATP1B1, is likely to be coadministered with LB in patients with the metabolic syndrome. We report herein on a study of potential interactions between LB and ATV in rats. When LB was administered intravenously with ATV, the systemic clearance and volume of distribution at steady state for LB remained unchanged (2.67 ± 0.63 ml/min per kg and 289 ± 20 ml/kg, respectively), compared with that of LB without ATV (2.34 ± 0.37 ml/min per kg and 271 ± 20 ml/kg, respectively). Although the tissue-to-plasma partition coefficient (Kp) of LB was not affected by ATV in most major tissues, the liver Kp for LB was decreased by ATV coadministration. Steady-state liver Kp values for three levels of LB were significantly decreased as a result of ATV coadministration. LB uptake was inhibited by ATV in rat OATP1B2-overexpressing Madin–Darby canine kidney cells and in isolated rat hepatocytes in vitro. After incorporating the kinetic parameters for the in vitro studies into a physiologically based pharmacokinetics model, the characteristics of LB distribution to the liver were consistent with the findings of the in vivo study. It thus appears that the distribution of LB to the liver is mediated by the hepatic uptake of transporters such as rat OATP1B2, and carrier-mediated transport is involved in the liver-specific drug–drug interaction between LB and ATV in vivo.

Introduction

Lobeglitazone (LB), an agonist for peroxisome proliferator–activated receptor γ (PPARγ), is approved in Korea for the treatment of type 2 diabetes mellitus (T2DM). Previous studies (Sauerberg et al., 2003; Kim et al., 2004; Lee et al., 2005) indicate that LB has a therapeutic advantage over rosiglitazone and pioglitazone (i.e., clinically approved/used PPARγ agonists with similar structural motifs) in terms of potency for the PPARγ receptor. As a result, an improvement in insulin sensitivity is expected even at a lower dosage (e.g., 0.5 mg/d): the lower dosage of LB is likely to reduce the incidence of adverse effects (Kim et al., 2014).

We previously demonstrated that LB was primarily metabolized by major cytochrome P450 enzymes (namely, CYP1A2, CYP2Cs, and CYP3A4) (Lee et al., 2015a,b) and interacts with human organic anion transporting polypeptide (hOATP) 1B1, with an IC50 value of 2.44 μM (Lee et al., 2015b). This suggests that drug–drug interactions (DDIs) are possible for LB in drug metabolism as well as in its distribution to tissues. Clinical pharmacokinetics studies of LB with other prescription drugs such as warfarin or amlodipine were recently conducted in Korea, and no significant differences in the systemic pharmacokinetics of LB were reported after coadministration (Jung et al., 2015; Kim et al., 2015). These studies were designed to investigate potential changes in the systemic pharmacokinetics of LB when these drugs were coadministered, considering the fact that they are known to share some of the same metabolic pathways of LB (i.e., CYP1A2, CYP2C9, and CYP3A4 with warfarin; CYP3A4 with amlodipine) (Guengerich et al., 1991; Kaminsky and Zhang, 1997). The lack of appreciable differences in pharmacokinetics indicates that a DDI mediated by drug metabolism is less likely for LB.

In addition to DDIs caused by complications in drug metabolism, based on the literature, it is clear that DDIs are possible as a result of interactions at the drug transporter level. For example, it was reported that cyclosporine inhibits the transport of rosuvastatin to the liver, probably mediated by hOATP1B1, thereby increasing the plasma level of rosuvastatin by 7.1-fold (Simonson et al., 2004). Interestingly, however, evidence for interactions caused by drug transporters may be less obvious and would be difficult to detect in some cases. For example, prior studies reported that concentrations of metformin in the liver and kidney are elevated after coadministration of cimetidine or pyrimethamine, which is probably caused by the inhibition of mouse multidrug and toxin extrusion 1 protein expressed in the liver and kidney of mice (Ito et al., 2012; Shingaki et al., 2015) without any apparent indication of DDI in the systemic pharmacokinetics. It was also noted that the concentration of ciprofloxacin in the liver was decreased after coadministration of 24-nor-ursodeoxycholic acid by inducing the expression of mouse multidrug resistance-associated protein 4 in the basolateral side of the mouse liver (Wanek et al., 2016), whereas the systemic pharmacokinetics remained unchanged. These results indicate that conventional methods for detecting DDIs are not adequate for some interactions involving transporters, and the possibility of DDI needs to be examined in the target tissue. For LB, however, the possibility of transporter-mediated DDIs is not known.

Atorvastatin (ATV), one of the most frequently prescribed statins (Hsyu et al., 2001; Laufs et al., 2016), is reported to be metabolized by CYP3A4 (Park et al., 2008) and is transported by hOATP1B1 and hOATP1B3 (Knauer et al., 2010). It is now well established that hyperlipidemia can occur simultaneously with T2DM (Iglay et al., 2016); thus, a combined treatment involving statins and hypoglycemic agents (e.g., LB) may become necessary clinically (Gu et al., 2010). It is noteworthy that ATV and LB share similar pathways for drug metabolism (CYP3A4) and distribution to tissues (hOATP1B1). Unfortunately, however, the possibility of DDIs between ATV and LB does not appear to have been examined to date.

The objective of this study, therefore, was to determine whether DDIs between LB and ATV occur in rats. We were particularly interested in the possibility of DDI at the level of metabolism by CYP3A and the carrier-mediated transport via rat organic anion transporting polypeptide (rOATP) 1B2 (i.e., ortholog of hOATP1B1 and hOATP1B3) (Hagenbuch and Meier, 2004) in the pharmacokinetics of LB in the absence or presence of ATV coadministration. The findings indicate that although no apparent interaction was found between LB and ATV in their systemic pharmacokinetics, there was a significant interaction for the hepatic distribution of LB, most likely at the level of the hepatic distribution via rOap1b2, after coadministration of ATV.

Materials and Methods

Chemicals and Reagents

LB (98.5% purity) was kindly provided by Chong Kun Dang Pharmaceuticals (Seoul, Korea). ATV (98% purity) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Glipizide (96% purity), bicinchoninic acid (BCA), bovine serum albumin (BSA), Hanks’ balanced salt solution, sodium bicarbonate, HEPES, and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). [3H]-Inulin (194 mCi/g) and [3H]-estradiol-17β-d-glucuronide (specific activity, 41.4 Ci/mmol) (both from American Radiolabeled Chemicals, St. Louis, MO) were also used in this study. SDS and polyethylene glycol 400 were purchased from Georgia Chemical (Norcross, GA) and Duksan Pure Chemicals (Ansan, Korea), respectively. Dulbecco’s modified Eagle’s medium, nonessential amino acid solution, Dulbecco’s phosphate-buffered saline (DPBS), penicillin/streptomycin, and fetal bovine serum were obtained from Welgene (Daegu, Korea). High-performance liquid chromatography (LC)– grade acetonitrile (ACN) and formic acid were purchased from Fisher Scientific (Pittsburgh, PA) and Fluka (Cambridge, MA), respectively. An ammonium formate solution (5 M; Agilent Technologies, Santa Clara, CA) was also used. Pooled male rat liver microsomes and an NADPH-regenerating system solution were purchased from Corning Gentest (Woburn, MA). Zoletil 50 (tiletamine-HCl/zolazepam-HCl) was purchased from Virbac Laboratories (Carros, France) and Rompun (xylazine-HCl) was from Bayer Corp. (Shawnee Mission, KS). All other chemicals were of reagent grade or greater and were used without further purification.

Animals

Male Sprague-Dawley (SD) rats (body weight, 240–270 g; Orient Bio Inc., Seongnam, Korea) were used in all in vivo/hepatocyte isolation studies. Experimental protocols involving the animals used in this study were reviewed and approved by the Seoul National University Institutional Animal Care and Use Committee, according to the National Institutes of Health Principles of Laboratory Animal Care (publication number 85-23, revised in 1985).

Intravenous Bolus Administration Study

Identification of Systemic DDIs in Rats.

Overnight fasted male SD rats were anesthetized by an intramuscular administration of 50 mg/kg tiletamine-HCl/zolazepam-HCl (Zoletil 50) and 10 mg/kg xylazine-HCl (Rompun). After we confirmed the induction of anesthesia, the femoral artery (for collecting blood samples) and vein (for administering and supplementing body fluids) were catheterized with polyethylene tubing (PE 50; Clay Adams, Parsippany, NJ) filled with heparinized saline (20 U/ml; for arterial cannulae) and normal saline (for venous cannulae), respectively. Upon recovery from the anesthesia, four types of dosing solutions (i.e., 0.5 mg/ml LB, 0.5 mg/ml LB with 2.5 mg/ml ATV, 2.5 mg/ml ATV, or 2.5 mg/ml ATV with 0.5 mg/ml LB) were prepared immediately prior to administration and given intravenously to rats by a bolus injection. The vehicle for the dosing solutions consisted of DMSO/polyethylene glycol 400/saline [0.5:4:5.5 (v/v/v)] and the injection volume was 2 ml/kg. Blood samples (150 μl for each sample) were collected from the arterial catheter at 0, 5, 15, 30, 60, 120, 240, 360, and 480 minutes (for the study of LB pharmacokinetics) or at 0, 5, 15, 30, 60, and 120 minutes (for the study of ATV pharmacokinetics) after administration. Immediately after blood collection, a volume of saline identical to the volume of the blood sample was given to the animal to compensate for fluid loss. The plasma fraction was separated from blood samples by centrifugation (16,100g for 5 minutes at 4°C) and stored at –80°C until it was used in a LC–tandem mass spectrometry (MS/MS) assay for LB and ATV (see below).

Intravenous Bolus Administration Study

Identification of DDIs in Rat Tissues.

Overnight fasted male SD rats were anesthetized and catheterized as described above. Upon recovery from anesthesia, four types of dosing solutions (i.e., 0.5 mg/ml LB, 0.5 mg/ml LB with 2.5 mg/ml ATV, 2.5 mg/ml ATV, or 2.5 mg/ml ATV with 0.5 mg/ml LB) were similarly prepared and administered intravenously to rats by a bolus injection. The animals were euthanized at 30, 60, 120, or 240 minutes after administration, and tissue (i.e., lungs, adipose, brain, heart, kidneys, muscle, liver, spleen, and gut) samples were collected. In this study, samples of trunk blood were also collected. After tissue isolation, the samples were washed four times with ice-cold DPBS and weighed. Twice the volume of the tissue weight of DPBS was then added for homogenization (Ultra Turrax homogenizer; IKA, Staufen, Germany). The mixture was then stored at −80°C until used in determining the concentration of LB and ATV.

When it was necessary to determine the volume of trapped blood in tissue samples, rats were injected intravenously with a solution of inulin containing a trace amount of [3H]-inulin in normal saline (injection volume, 1 ml/kg). Blood samples were collected at 0.5 minutes, and the animals were immediately euthanized by cervical dislocation and tissue samples were collected. Blood samples were then centrifuged at 10,770g for 10 minutes and the supernatant was collected. For tissue samples, approximately 100 mg of the tissue sample was weighed and digested with 2 ml Soluene-350 (PerkinElmer Life and Analytical Sciences, Waltham, MA). Hydrogen peroxide (200 μl; 35%; Junsei Chemical, Tokyo, Japan) was then added and the resulting sample was incubated overnight at room temperature to prevent quenching (Saito et al., 2001). Aliquots of plasma (50 μl) or tissue sample were mixed with Ultima Gold (PerkinElmer), and the radioactivity of the sample was determined by liquid scintillation counting (Tri-Carb 3110TR Liquid Scintillation Analyzer; PerkinElmer). Considering the possibility that inulin might not be distributed to erythrocytes (Gaudino and Levitt, 1949), the estimated trapped plasma volume was subsequently divided by 0.55 (i.e., plasma to blood volume ratio). In all subsequent studies in which a tissue concentration of drugs was reported, the concentration found in the sample was corrected by subtracting the portion in the trapped blood volume.

Intravenous Infusion study of LB with and without ATV in Rats

When it was necessary to further determine the distribution of LB to the liver, a constant LB concentration in the plasma was achieved by intravenous infusion and the liver concentration was determined in the absence and presence of ATV coadministration. Thus, overnight fasted male SD rats, weighing 240–270 g, were anesthetized and catheterized, as described above. After the rats recovered from anesthesia, LB was intravenously infused at a rate of 50, 250, or 1000 ng/min with or without ATV solution (10 μg/10 μl per min of DMSO/polyethylene glycol 400/saline [0.5:2:7.5 (v/v/v)]) using an infusion pump (Harvard Apparatus, Holliston, MA). Blood samples (150 μl) were collected from the catheter connected to the artery at 30, 60, 120, 240, 300, and 360 minutes. Upon collecting the last blood sample, the animals were euthanized and a liver sample was collected. The plasma and liver samples were processed for analysis as described above.

Determination of Blood-to-Plasma Concentration Ratio and Plasma Protein Binding of LB

LB was added to fresh blank blood at final concentrations of 0.1, 1, or 10 μM, and the mixture was incubated at 37°C in a water bath for 60 minutes. After incubation, the mixture was centrifuged (16,100g) and the plasma was collected as the supernatant: the concentration of LB was then determined by LC-MS/MS analysis to calculate the blood-to-plasma concentration ratio (B/P).

In this study, the extent of plasma protein binding was also determined for LB by the rapid equilibrium dialysis method, according to the manufacturer’s recommended protocol (Thermo Fisher Scientific, Waltham, MA). Briefly, the plate of the dialysis device was rinsed with 20% ethanol for 10 minutes. LB [0.1, 0.3, 1, 3, or 10 μM as the final concentration for the rat plasma; 0.5, 5, or 50 μM for rat liver microsomes (see below); 0.5, 1, 2, 5, 10, or 20 μM for 5 μM BSA (see below) in the transport medium (9.7 g/l Hanks’ balanced salt solution, 2.38 g/l HEPES, and 0.35 g/l sodium bicarbonate, pH adjusted to 7.4)] and ATV [final concentration: 0.1, 1, or 10 μM for 5 μM BSA in the transport medium] were added to the medium [i.e., the plasma, the microsomal incubation medium, or the transport medium]. Aliquots of the mixture (200 μl) and protein-free solution (350 μl) were placed into the sample chamber and the buffer chamber, respectively, and the device was sealed/incubated on a shaker (150 rpm) at 37°C for 4 hours. After the incubation was complete, an aliquot (50 μl) was collected from each side of the chamber. To ensure that the matrix of the samples matched, a 50-μl aliquot of blank medium was then added to the protein-free solution sample, and an equal volume of protein-free solution was also added to the medium. The resulting sample was then analyzed for LB and ATV to determine the unbound fraction (fu). In this study, the recovery was between 70% and 100% in all experiments.

Stability of LB in Rat Liver Microsomes

The metabolic stability of LB with or without ATV in rat liver microsomes was determined in this study. The reaction mixture (total volume of 1 ml) consisted of an NADPH-regenerating solution containing 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 3.3 mM MgCl2, as recommended by the manufacturer’s protocol. Depending on the study, an aqueous solution (100 mM potassium phosphate buffer, pH 7.4) containing LB (final concentration in the reaction mixture: 1, 2, 5, 10, 20, or 50 μM) without ATV, or LB (the final concentration: 5 μM) with ATV (the final concentration: 1, 5, 10, 20, 50, 100, or 200 μM) was also added. The concentration of organic solvent (i.e., DMSO) in the incubation mixture was maintained at less than 1% to limit its influence on microsomal enzymes. After preincubation (37°C for 10 minutes), the reaction was initiated by the addition of rat liver microsomes (50 μl; the final microsomal protein concentration was 0.5 mg/ml). Preliminary experiments were carried out to determine the incubation time for the given LB concentrations for a linear rate of LB disappearance (data not shown). Samples were collected (50 µl) at various predetermined times and then an ice-cold ACN solution (i.e., for the termination of the reaction) containing glipizide (100 ng/ml, internal standard) was added. The LB concentration in the mixture was determined by the LC-MS/MS assay.

rOATP1B2 Cloning

The protein coding region of rOATP1B2 (GenBank accession number NM_031650.3) was cloned from a rat total liver mRNA library (Takara Shuzo, Kyoto, Japan). The RNA (1 μg) was reverse transcribed using the PrimeScript first-strand cDNA synthesis kit (Takara) by incubating the mixture at 65°C for 5 minutes, cooling to 4°C followed by adding RNase inhibitor and RTase into the reaction mixture, and then the following reaction was performed by incubating at, 42°C for 60 minutes, heating to 95°C for 5 minutes, and cooling to 4°C. Specific primers for cloning of the rOATP1B2 coding region were 5′-GCTAGCAGTGATTGCAGACGTTCCCA-3′ (sense strand; the NheI site is underlined) and 5′-AAGCTTGTCCATCCTTGCCCCATTCT-3′ (antisense strand; the HindIII site is underlined). The polymerase chain reaction was performed using ExTaq DNA polymerase (Takara) using the following settings: 30 cycles of denaturation at 94°C for 30 seconds, a primer-annealing step at 63°C for 30 seconds, and extension at 72°C for 3 minutes. The amplicon was cloned into pcDNA5/FRT (flippase recognition target) vector (Invitrogen, Carlsbad, CA), and the identity of the insert confirmed by sequencing. The plasmid containing the wild type form of rOATP1B2 was selected and transfected into Madin–Darby canine kidney (MDCK) II/FRT cells using the FuGENE transfection reagent. The transfected cells were incubated in the culture medium containing 0.1 mg/ml hygromycin B (Invitrogen) for several weeks for selection. Expression of rOATP1B2 was confirmed by the reverse transcription polymerase chain reaction in the transfected cells. Functional expression was determined by comparing the uptake of radiolabeled estradiol-17β-glucuronide (a standard substrate of the transporter; Cattori et al., 2000) in the transfected cells with that in mock-transfected MDCKII/FRT cells (Supplemental Fig. 1). In this study, at least a 60-fold higher uptake was found in rOATP1B2 transfected cells with estradiol-17β-glucuronide. Since the measurements in this study involved uptake across the apical membrane, this observation indicates that the transporter is expressed ubiquitously in the plasma membrane, rather than localized in the basolateral membrane (i.e., endogenous site of expression), in the transfected cells. Protein concentrations were determined by a BCA assay (Smith et al., 1985) and used for the correction of the transport function.

Uptake of LB in rOATP1B2-Transfected MDCKII/FRT Cells

To determine the kinetic characteristics of rOATP1B2 transport, MDCKII/FRT cells expressing the anion transporter were used. Thus, rOATP1B2 cells were grown in Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acid solution, 100 U/ml penicillin/streptomycin, and 10 mM HEPES. All cells were kept at 37°C with 5% CO2 and 95% relative humidity. Cells were seeded in poly(l)-ornithine (Sigma-Aldrich)–coated 24-well (2.0 cm2) cell-culture plates (Corning, Corning, NY) in supplemented Dulbecco’s modified Eagle’s medium at a density of 0.5 × 106 cells/well and were then grown in a humidified atmosphere of 5% CO2 at 37°C for 2 days. Preliminary studies were carried out with LB to determine the appropriate reaction time in rOATP1B2 cells as well as mock cells. In this study, the uptake of LB was proportionally increased with the incubation time up to 12 minutes (Supplemental Fig. 2); thus, a 10-minute incubation time was used in subsequent studies involving LB and rOATP1B2-expressing cells.

For measurement of LB transport in rOATP1B2 cells, the cells were first washed twice with prewarmed DPBS and preincubated with transport medium containing 2% (final concentration) DMSO (i.e., solubilizing agent for LB). From a pilot experiment, it was found that DMSO up to 2% in the transport medium had no appreciable effect on the function of the transporter (Supplemental Fig. 3), consistent with other literature findings (Da Violante et al., 2002; Taub et al., 2002). LB was prepared in DMSO and diluted with 5 μM BSA in transport medium (final DMSO concentration of 2%). We found that the addition of BSA was necessary to suppress the nonspecific adsorption of the drugs on the experimental apparatus and/or cell surface (Takeuchi et al., 2011). The uptake of LB was measured in six different concentrations ranging from 0.5 to 20 μM (i.e., 0.5, 1, 2, 5, 10, or 20 μM) in rOATP1B2 and in mock cells. After preincubation for 10 minutes at 37°C, the transport medium was removed and replaced with prewarmed medium containing LB in the presence of and absence of ATV (200 μl). Upon completion of the incubation, the aqueous medium was first aspirated and the cells were washed twice with ice-cold DPBS containing 0.1% BSA (400 μl) and once with ice-cold DPBS (400 μl). The final rinsing solution was then aspirated and 0.1% (w/v) SDS added to the cell suspension for lysis. A preliminary experiment indicated that the addition of BSA up to 3% in the rinsing solution had no effect on the protein concentration in the cell lysate. In addition, the effect of limiting the nonspecific binding on the experimental apparatus and/or on cell surface was not statistically different for BSA concentrations ranging from 0.1% to 10% in the rinsing solution (Supplemental Fig. 4). The resulting mixture was agitated for 30 minutes, and an ACN solution containing glipizide (100 ng/ml, internal standard) was added. After vortexing for 5 minutes and centrifugation for 5 minutes (16,100g), an aliquot (50 µl) of the supernatant was collected and the concentration of LB in the supernatant was determined by LC-MS/MS assay (see below). The transport rate was normalized by the protein concentration in the sample as determined by a BCA assay. When necessary, the number of cells was estimated using the predetermined relationship [y (mg protein/ml) = 0.304 × 106 ⋅ x (cells/ml) + 0.0335, r2 = 0.994] between the protein concentration (in mg protein/ml) and the number of cells per milliliter (in 106 cells/ml).

When the inhibitory effect of ATV on the transport function of LB in rOATP1B2 cells was studied, the uptake of LB (5 µM) was determined in the cells in the presence of various concentrations of ATV (i.e., 0, 0.01, 0.1, 1, 10, or 30 μM). The transport medium containing DMSO (2%, final concentration) was prepared and BSA (5 μM, final concentration for minimizing nonspecific binding) was added. After a 10-minute incubation at 37°C, the uptake was terminated by aspiration of the transport medium. The cells were then rinsed twice with ice-cold DPBS containing 0.1% BSA (400 μl) and once with ice-cold DPBS (400 μl). Cells were lysed for 30 minutes in 0.1% (w/v) SDS and the mixture was agitated. An ACN solution containing glipizide (100 ng/ml, internal standard) was then added to the mixture. After vortexing for 5 minutes and centrifugation for 5 minutes (16,100g), an aliquot (50 µl) of the supernatant was collected and the concentration of LB was determined by an LC-MS/MS assay. The data were normalized by the protein concentration in the sample as determined by BCA assay. Preliminary studies were carried out in an attempt to determine whether the opening of the tight junction is required for transport in the case of rOATP1B2-expressing cells. Therefore, the cells were pretreated with EDTA according to a literature report (Wang et al., 2016) with minor modifications. Briefly, a Ca2+/Mg2+-free transport medium containing 500 μM EDTA was prepared and rOATP1B2-expressing cells were pretreated for 2 hours. The transport experiment was then performed as described above. Although the uptake of LB was slightly increased in both mock and rOATP1B2-expressing cells with comparable Km and Ki values, this finding suggests that the kinetic properties of carrier-mediated transport are not significantly altered for LB by EDTA pretreatment. Therefore, we chose to exclude the EDTA pretreatment step before the transport experiment, to prevent the formation of any possible artifacts resulting from the EDTA pretreatment.

Uptake of LB in Isolated Rat Hepatocytes

Rat hepatocytes were isolated using the two-step collagenase perfusion method with minor modifications (Kotani et al., 2011). Briefly, under anesthesia, a cannula was inserted into the portal vein followed by perfusing with a Ca2+/Mg2+-free buffer at the flow rate of 20 ml/min for 5 minutes. Upon the initiation of the perfusion, the inferior vena cava was dissected to allow the perfusate to exit. When necessary, the perfusion medium was switched to a buffer containing 50 mM CaCl2 and 0.5 mg/ml collagenase (Sigma-Aldrich) at a flow rate of 20 ml/min for 10 minutes. Hepatocytes from the digested liver were dispersed in fresh perfusion buffer and separated from the connective tissue by filtration through a sterile 50-mesh (the size of the sieve opening of 280 μm). Rat hepatocytes were separated from nonparenchymal cells in the crude hepatocyte fraction as a pellet by centrifugation at 50g for 5 minutes. The cells were then resuspended in Krebs-Henseleit buffer (KHB) and centrifuged again at 50g for 5 minutes. In this study, hepatocytes having a viability of greater than 80%, as determined by a trypan blue exclusion assay, were used in subsequent experiments. The resulting cell pellet was resuspended in Williams’ media E containing 10% (v/v) fetal bovine serum, 1% (v/v) penicillin/streptomycin, and 0.01% (v/v) insulin/transferrin/selenium (i.e., 10 mg/ml insulin, 5.5 mg/ml transferrin, and 5 μg/ml selenium) premix (Sigma-Aldrich) at pH 7.4. The hepatocytes were plated in 24-well collagen I (Sigma-Aldrich)–coated plates at a density of 5 × 105 viable cells per well in 0.5 ml supplemented Williams’ media E. The plate was equilibrated from 5 to 6 hours at 37°C in an atmosphere containing 5% CO2 to allow the cells to adhere to the collagen-coated surface.

In this study, hepatocyte uptake was measured for six concentrations of LB (i.e., 0.5, 1, 2, 5, 10, or 20 μM). After approximately 5 to 6 hours of cell plating, the medium was aspirated and the attached cells were rinsed twice with prewarmed DPBS. LB was dissolved in KHB containing DMSO (5%, final concentration) and BSA (5 μM, final concentration). In a preliminary study, the addition of DMSO up to a final concentration of 10% did not appear to have an appreciable impact on transport function (Supplemental Fig. 5), consistent with previous observations (Da Violante et al., 2002). The transport reaction was initiated by the addition of a KHB solution containing LB (200 μl) on the top of the plate. According to our study (Supplemental Fig. 6), the accumulation of LB in the hepatocytes was proportional to the incubation time up to 2 minutes and this incubation time was used in subsequent studies. Cellular uptake was terminated by aspiration of the transport medium; cells were rinsed twice with ice-cold DPBS containing 0.1% BSA (400 μl), followed by an additional washing with ice-cold DPBS (400 μl). The hepatocytes were lysed for 30 minutes by the addition of 0.1% (w/v) SDS and agitation. An ACN solution containing glipizide (100 ng/ml, internal standard) was then added to the lysate and the concentration of LB determined by a LC-MS/MS assay. When necessary, the transport rate was normalized by the amount of protein in the sample as determined by a BCA assay. The number of hepatocytes was also estimated using the predetermined relationship [y (mg protein/ml) = 0.600 × 106 ⋅ x (hepatocytes/ml) − 0.007, r2 = 0.998] between the protein concentration (in mg protein/ml) and the number of hepatocytes per milliliter (in 106 hepatocytes/ml).

To determine the inhibitory effect of ATV on the transport of LB in rat isolated hepatocytes, LB (5 µM) uptake was measured in the presence of various concentrations of ATV (i.e., 0, 0.001, 0.01, 0.1, 1, 10, 30, or 100 μM). Thus, the medium was aspirated, followed by rinsing twice with prewarmed DPBS. The DPBS was then aspirated, and the cells were preincubated for 60 minutes at 37°C with KHB containing various concentrations of ATV (i.e., 0, 0.001, 0.01, 0.1, 1, 10, 30, or 100 μM) and BSA (5 μM, final concentration). After preincubation for 60 minutes (Amundsen et al., 2010; Shitara et al., 2013), the medium was switched to KHB containing BSA (5 μM, final concentration) and 5 μM LB along with various concentrations (i.e., 0, 0.001, 0.01, 0.1, 1, 10, 30, or 100 μM) of ATV. After incubation at 37°C for 2 minutes, uptake was terminated by aspirating the medium, followed by rinsing twice with ice-cold DPBS containing 0.1% BSA (400 μl) and once with ice-cold DPBS (400 μl). The hepatocytes were lysed for 30 minutes in 0.1% (w/v) SDS and the mixture was agitated. An ACN solution containing glipizide (100 ng/ml, internal standard) was then added to the lysate. The concentration of LB was determined by an LC-MS/MS assay. When necessary, the transport rate was normalized by amount of protein in the sample as determined by a BCA assay.

LC-MS/MS Assay for LB and ATV

The concentration of LB and ATV in rat plasma samples, tissue homogenates, or cell lysates was determined using an LC-MS/MS assay method as previously described (Lee et al., 2009; Kim et al., 2012) with a minor modification. Briefly, an aliquot (50 μl) of the sample was vortex mixed with an ACN solution containing glipizide (100 ng/ml, internal standard) followed by centrifugation (16,100g for 5 minutes at 4°C). An aliquot (5 µl) of the supernatant was directly injected onto the LC-MS/MS system. In this study, the LC-MS/MS system was equipped with a Waters e2695 high-performance LC system (Milford, MA) and API 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA). The mobile phase, consisting of (A) 0.1% formic acid in ACN and (B) 10 mM ammonium formate in purified water, was delivered at the flow rate of 0.3 ml/min using a gradient elution involving 30% of A (0 minutes), from 30% to 80% of A (0–0.5 minutes), 80% of A (0.5–1.5 minutes), from 80% to 30% of A (1.5–2 minutes), and 30% of A (2–5 minutes). Chromatographic separation was carried out on a reversed-phase high-performance LC column (Eclipse XDB-C18, 3.5 μm, 2.1 × 100 mm; Agilent Technologies) at 25°C while the temperature in the autosampler was maintained at 4°C during the analysis. Samples were ionized using a turbo ion spray interface in the positive ionization mode and monitored at the following Q1/Q3 transitions (mass/charge ratio): 481.3/258.2 for LB, 559.3/440.4 for ATV, and 445.8/320.9 for glipizide. The common source/gas conditions for LB, ATV, and glipizide were as follows. The pressure of the curtain gas, ion spray voltage, source temperature, ion source gas 1, and ion source gas 2 was 25 psi, 4500 V, 550°C, 60 psi, and 60 psi, respectively. The declustering potentials for LB, ATV, and glipizide were 77.9, 45.0, and 47.5 V, respectively. The entrance potentials were 7.4, 4.0, and 4.0 V, collision energies were 47, 27, and 17 V, and the collision cell exit potentials were 4.0, 6.0, and 8.0 V, respectively. The detector response was linear in the concentration range examined (i.e., 5–5000 ng/ml) for both LB and ATV in the rat plasma samples with interday/intraday precisions of less than 10% and an accuracy within 8% of the theoretical value, indicative of a valid assay for LB and ATV. In addition to plasma samples, the calibration curves for LB and ATV in the rat tissue homogenates were linear (i.e., 0.992 ≤ r2 ≤ 0.999 for LB and 0.995 ≤ r2 ≤ 0.999 for ATV, respectively) in the concentration range studied. The concentration of LB in cell lysates was also determined with the linear calibration curves (i.e., 0.998 ≤ r2 ≤ 0.999 in cell lysates from mock and rOATP1B2 cells, and 0.998 ≤ r2 ≤ 0.999 in cell lysates from isolated rat hepatocytes).

Data Analysis

In Vitro Kinetic Analysis.

When necessary, the in vitro metabolic kinetic parameters (e.g., Km, Vmax, and Ki) were determined by fitting the data to eqs. 1 and 2 (Segel, 1975):Embedded Image(1)Embedded Image(2)where V was the rate of metabolic reaction, Vmax is the maximal rate of metabolism, Km is the Michaelis–Menten constant, Ki is the inhibition constant, [S] is the LB concentration as a substrate, and [I] is the ATV concentration as an inhibitor, respectively.

In addition, the cellular transport rate (J) was estimated from the amount of LB uptake in cell lysates, divided by the incubation time, and expressed in eqs. 3 and 4.

In the case where only passive transport was present,

Embedded Image(3)

In the case of parallel passive and carrier-mediated transports,Embedded Image(4)where PS is the unbound clearance via passive diffusion and [S], Km, and Jmax are the unbound concentration of LB, the unbound Michaelis-Menten constant, and the maximal rate of the carrier-mediated transport, respectively. Kinetic parameters (e.g., PS, Km, and Jmax) were estimated by the simultaneous fitting of the in vitro data to eqs. 3 and 4 using nonlinear regression analysis. The measured value for uptake in mock cells may contain some statistical variability and the correction may not be adequate in some cases (e.g., resulting in a negative transport value after the correction). As indicated in the literature (DeLean et al., 1978), a simultaneous fitting approach may result in more reliable values in estimating vitro kinetic parameters. Therefore, we chose to use this method of kinetic analysis.

When it was necessary to analyze the kinetics of the concentration-dependent inhibition of LB transport by ATV, eq. 5 was fitted to the in vitro data to estimate the half maximal inhibitory concentration (IC50). In addition, the Ki value was estimated using eq. 6 (Cheng and Prusoff, 1973):Embedded Image(5)Embedded Image(6)where R, Rmax, R0, [I], [S], and Km are the ratio of the amount of LB transport to the value for the control group treated without ATV, the maximum value of R, the minimum value of R, the ATV concentration, the unbound concentration of LB as a substrate, and the unbound Michaelis–Menten constant, respectively. It has been reported that the adoption of Hill’s slope factor in the Cheng–Prusoff equation may introduce additional errors (Lazareno and Birdsall, 1993). Therefore, the IC50 was estimated without using Hill’s slope to minimize additional errors (eq. 5). WinNonlin Professional 5.0.1 software (Pharsight Corporation, Mountain View, CA) was used for the nonlinear regression analysis using the equations described above.

Noncompartmental Pharmacokinetic Analysis.

Standard noncompartmental analyses were carried out using WinNonlin to calculate the pharmacokinetic parameters (Gibaldi and Perrier, 1982), including the area under the plasma concentration–time curve (AUC) from time zero to infinity (AUCinf), elimination clearance (CL), terminal half-life, mean residence time, and apparent volume of distribution at steady state (Vss).

The tissue-to-plasma concentration ratio of LB (i.e., Kp) was calculated using eq. 7 (Sawada et al., 1985) after the administration of an intravenous bolus of LB and/or ATV to rats:Embedded Image(7)where AUCinf,tissue represents the area under the drug concentration–time curve in tissue and AUCinf,plasma represents the area under the drug concentration–time curve in plasma after the intravenous bolus administration. For the calculation of the tissue-to-plasma partition coefficient (Kp), the contribution of trapped blood in the collected tissue was corrected using the volume of blood trapped in tissue samples.

Physiologically Based Pharmacokinetic Modeling and Simulation.

In this study, a physiologically based pharmacokinetic (PBPK) model (see the Appendix) was constructed assuming the simultaneous intravenous bolus administration for LB and ATV. The PBPK model consisted of nine tissues corresponding to the different tissues of the body (lung, adipose, brain, heart, kidneys, muscle, liver, spleen, and gut) and the tissues were assumed to be connected by the circulatory system (arterial and venous side). Physiologic and anatomic variables, required in the PBPK calculation, were obtained from the literature (Brown et al., 1997) [i.e., essentially the default values found in Simcyp software (Jamei et al., 2009), version 15, release 1; Simcyp Limited, Sheffield, UK]. In this calculation, the rate of LB/ATV distribution to tissues, including the liver, was assumed to be perfusion rate limited and the standard mass balance differential equations were used with minor modifications (Lee et al., 2011) (see the Appendix).

After the adequacy of the constructed PBPK model (for single intravenous bolus administration of LB and ATV, respectively) (Supplemental Fig. 7) was confirmed with the Simcyp animal simulator (version 15, release 1), a simulation was carried out with Berkeley Madonna software (version 8.3.18; University of California, Berkeley, CA) since a DDI module for rats was not available in the Simcyp software. In this study, the fourth order of the Runge–Kutta method was used for the numerical integration.

Statistical Analysis.

When it was necessary to compare the mean values between/among groups, the unpaired t test or one-way analysis of variance (ANOVA), followed by the Tukey post hoc test, was used. In this study, data are expressed as means ± S.D. P < 0.05 was denoted as statistically significant.

Results

DDI between LB and ATV in Systemic and Tissue Pharmacokinetics in Rats

The mean plasma concentration-time profiles are shown in Fig. 1A for an intravenous bolus administration of 1 mg/kg LB to rats with or without simultaneous administration of 5 mg/kg ATV. The LB concentration profile in the case of ATV coadministration was comparable to that without ATV (Fig. 1A). The pharmacokinetic parameters, as estimated from a standard moment analysis, are summarized in Table 1. The CL of LB with or without ATV was 2.67 ± 0.63 and 2.34 ± 0.37 ml/min per kg, respectively, and these values were not significantly different from each other. The Vss for LB with and without ATV was 289 ± 20 and 271 ± 20 ml/kg, respectively. Furthermore, the secondary pharmacokinetic parameters for LB (e.g., AUCinf, terminal half-life, and mean residence time) were not affected by the coadministration of ATV. Similar to the case of LB, ATV concentrations in rat plasma with or without LB co-administration were not changed, as shown in Fig. 1B and the summary of pharmacokinetic parameters for ATV (Table 1). Collectively, these observations suggest that there is no appreciable interaction in systemic pharmacokinetics when LB and ATV are simultaneously given in rats.

Fig. 1.
Fig. 1.

(A) Temporal profiles for the plasma concentration of LB after an intravenous bolus administration of 1 mg/kg LB alone (filled circles) and 1 mg/kg LB with 5 mg/kg ATV (open circles) to rats. (B) Temporal profiles for the plasma concentration of ATV after an intravenous bolus administration of 5 mg/kg ATV alone (filled circles) and 5 mg/kg ATV with 1 mg/kg LB (open circles) to rats. Data are expressed as means ± S.D. of quadruplicate runs in (A) and pentaplicate runs in (B).

View this table:
TABLE 1

Pharmacokinetic parameters for LB and ATV after an intravenous bolus administration of LB (1 mg/kg dose) with and without coadministration of 5 mg/kg ATV to rats, and after intravenous bolus administration of 5 mg/kg ATV with and without 1 mg/kg LB to rats, respectively

Data are expressed as means ± S.D. of quadruplicate runs for LB and pentaplicate runs for ATV.

The mean tissue concentration-time profiles for LB and ATV are depicted in Fig. 2 for an intravenous bolus administration to rats at the LB dose (i.e., 1 mg/kg) with or without ATV coadministration (i.e., 5 mg/kg). The tissue-to-plasma partition coefficient (Kp) found in this study, along with the volume of blood trapped in tissues for LB and ATV, is listed in Table 2. When LB was administered intravenously without ATV, the liver appeared to be the primary organ for distribution, consistent with our previous findings (Lee et al., 2015b). The estimated Kp,liver value for LB was 2.34 in the absence of ATV coadministration, whereas the value was reduced to 1.55 in the presence of ATV coadministration. For the case of the distribution of LB to tissues other than the liver, ATV coadministration did not appear to significantly affect the distribution of LB. For the case of ATV without LB coadministration, the liver was one of the primary organs for this distribution (i.e., Kp,liver = 97.8 and Kp,gut = 125). However, the coadministration of LB reduced the Kp,liver and Kp,gut values for ATV to 65.4 and 64.1, respectively. These observations suggest that DDIs might be a possibility at the level of drug distribution when LB and ATV are simultaneously administered.

Fig. 2.
Fig. 2.

(A) Temporal profiles for plasma and tissue concentration of LB after an intravenous bolus administration of 1 mg/kg LB alone (open circles) and 1 mg/kg LB with 5 mg/kg ATV (closed circles) to rats. (B) Temporal profiles for the plasma and tissue concentrations of ATV after intravenous bolus administration of 5 mg/kg ATV alone (filled triangles) and 5 mg/kg ATV with 1 mg/kg LB (open triangles) to rats. Data are expressed as means ± S.D. of triplicate runs.

View this table:
TABLE 2

Tissue-to-plasma partition coefficient (Kp) for LB and ATV in rats and the volume of blood trapped in rat tissues

Kp values were obtained from triplicate runs. Volumes of trapped blood in the tissue are expressed as means ± S.D. of triplicate runs.

Dose Dependence of the Liver-to-Plasma Concentration Ratio of LB in Rats

In this study, different levels of LB in steady state were attained, and the concentrations of LB in the plasma/liver were determined, in an attempt to examine the dependence of Kp,liver on LB dosage and ATV coadministration. The mean plasma concentration–time profiles are shown in Fig. 3A for intravenous infusions of LB to rats at rates of 50, 250, or 1000 ng/min with and without simultaneous infusion of ATV at a rate of 10 μg/min. For all infusion rates, the concentrations of LB in the plasma for 4, 5, and 6 hours of infusion were not statistically different among the three time points for a given rate, confirming that a steady state had been reached by 4 hours after the infusion. It was also noted that ATV coadministration did not result in a change in LB concentration in plasma at a 6-hour infusion.

Fig. 3.
Fig. 3.

(A) Temporal profiles for the plasma concentration of LB after an intravenous infusion at dosing rates of 50 ng/min, 250 ng/min, and 1000 ng/min (shown as filled circles, triangles, and diamonds for LB alone and as open circles, triangles, and diamonds for LB with 10 μg/min ATV, respectively). (B) Liver-to-plasma concentration ratio of LB at 6 hours after an intravenous infusion at the dosing rates of 50, 250, and 1000 ng/min to rats. Asterisks in the black circles indicate statistical differences from the lowest LB dose (i.e., by one-way ANOVA, followed by the Tukey post hoc test), and asterisks in the white circles indicate statistical differences from the control (i.e., the absence of ATV) by the unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001). Data are expressed as means ± S.D. of quadruplicate runs.

The LB concentration was determined in the liver at 6 hours: the liver-to-plasma concentration ratio for LB without ATV coadministration was 3.14 ± 0.46 (at 50 ng/min), 1.55 ± 0.52 (at 250 ng/min), and 0.959 ± 0.259 (at 1000 ng/min) (i.e., P < 0.001 by one-way ANOVA). In addition, the liver-to-plasma concentration ratio for LB with ATV coadministration was 1.38 ± 0.39 (at 50 ng/min), 0.701 ± 0.013 (at 250 ng/min), and 0.714 ± 0.184 (at 1000 ng/min), indicating that the coadministration of ATV significantly decreases the liver-to-plasma concentration ratio for LB (Fig. 3B). For the 1000-ng/min LB infusion condition, the liver-to-plasma concentration ratio for LB was not further decreased by ATV, probably because the transport of LB would be fully saturated by the high concentration of LB. Collectively, these observations indicate that the distribution of LB to the liver is mediated by saturable transport process(es) and that transport to the liver is inhibited by ATV in rats.

B/P and Plasma Protein Binding

The B/P ratios of LB were 0.631 ± 0.023, 0.611 ± 0.022, and 0.667 ± 0.077 (i.e., no statistical difference, by one-way ANOVA) at concentrations of 0.1, 1, and 10 μM, respectively. Considering the lack of concentration dependence in the distribution to blood cells, the average B/P for LB (i.e., 0.636) for the three concentrations was assumed to be representative and the value was used in subsequent calculations.

The fraction of unbound LB in the rat plasma (fu,plasma) was also independent in the LB concentration range from 0.3 to 10 μM. The representative (i.e., averaged) fu,plasma value of LB was then calculated as 0.00267 ± 0.00036. The fraction of unbound LB in the microsomal incubation medium (fu,MIC) was independent in the concentration range from 0.5 to 50 μM, and the representative fu,MIC value of LB was estimated to be 0.479 ± 0.063. However, the unbound fraction of LB at 5 μM BSA in the transport medium (fu,BSA) increased with increasing LB concentration in the range from 0.5 to 20 μM (i.e., approximately 0.111–0.447). For the case of fu,BSA of ATV, the value was independent in the concentration range from 1 to 100 μM, and the representative value for fu,BSA of ATV was estimated to be 0.588 ± 0.045. These values were used in subsequent calculations.

Stability of LB in the Presence and Absence of ATV in Rat Liver Microsomes

The concentration-metabolic reaction rate profile for LB in rat liver microsomes is shown in Fig. 4A. The Eadie–Hofstee transformation of the data is also presented as an inset in Fig. 4A. In general, the metabolic rate decreased proportionally with the increasing V/[LB] value, suggesting that a saturable process is involved in the metabolic reaction. As a result, assuming that simple Michaelis–Menten kinetics is involved as shown in eq. 1, a nonlinear regression analysis indicates that the Km,MIC and Vmax,MIC values are 4.37 ± 0.90 μM and 996 ± 110 nmol/min per milligram of protein, respectively. The inhibitory effect of ATV on the metabolic reaction rate of LB in rat liver microsomes was also determined (Fig. 4B) in this study. Based on a nonlinear regression analysis using eq. 2, the Ki,MIC value was estimated to be 27.0 ± 5.7 μM. Using the kinetic characteristics obtained in this study, the estimated extraction ratio was calculated to be 0.03 for LB. Therefore, we assumed that a correction of Kp,liver for LB was not necessary and this value is reported without correction.

Fig. 4.
Fig. 4.

(A) The concentration-metabolic reaction rate curve for LB in an incubation with rat liver microsomes. The solid line was generated by the best-fit parameters obtained from the nonlinear regression analysis based on Michaelis–Menten kinetics and indicates the linearly regressed line of the data (eq. 2). The inset represents an Eadie–Hofstee plot of the concentration-metabolic reaction rate curve. (B) The inhibitory effect of ATV on the metabolic reaction rate of LB (5 μM) for an incubation with rat liver microsomes. The solid line was generated by the best-fit parameters obtained from the nonlinear regression analysis based on eq. 3. Each symbol represents the mean ± S.D. of triplicate runs.

Uptake of LB in rOATP1B2-Transfected MDCK Cells and Isolated Rat Hepatocytes

The unbound concentration-uptake rate profile for LB in mock cells and rOATP1B2 cells is shown in Fig. 5A. Since both LB and ATV are drugs that bind highly to plasma proteins (i.e., fu,plasma values of 0.00267 and 0.0567, respectively), it is possible that they might also bind nonspecifically to the surfaces of the experimental apparatus, thereby causing experimental artifacts. In this study, we chose to include BSA in the incubation medium and washing buffer to prevent/minimize nonspecific binding to the experimental apparatus and/or cell surface (Takeuchi et al., 2011). As a result, the fu,BSA values of LB were multiplied by the total LB concentration to estimate the concentration of unbound LB in the transport medium. Using this experimental design and a nonlinear regression analysis, Km,OATP and Jmax,OATP values were 2.06 ± 0.45 μM and 2.42 ± 1.27 pmol/min per 106 cells, respectively (eqs. 3 and 4).

Fig. 5.
Fig. 5.

(A) The unbound concentration-uptake rate curve for LB in mock-MDCK cells (filled circles) and rOATP1b2-expressing MDCK cells (open circles). (B) Unbound concentration-uptake rate curve for LB in rat hepatocytes at 4°C (filled circles) and 37°C (open circles). The solid lines were generated by the best-fit parameters obtained from the simultaneous nonlinear regression analysis based on eqs. 4 and 5, respectively. (C and D) The inhibitory effect of ATV on the uptake of LB in rOATP1b2-expressing MDCK cells (C) and rat hepatocytes (D). The solid line was generated by the best-fit parameters obtained from the nonlinear regression analysis based on eq. 6. Asterisks indicate statistical differences (**P < 0.01; ***P < 0.001) from the control group (i.e., without ATV) by one-way ANOVA, followed by the Dunnett post hoc test. Each symbol represents the mean ± S.D. of triplicate runs in (A) to (C) and octuplicate runs in (D).

The profile for the unbound concentration-uptake rate for LB in rat hepatocytes is shown in Fig. 5B. Similar to the case of transfected cells, the PS, Km,hepa, and Jmax,hepa values were estimated to be 21.0 ± 6.0 µl/min per 106 cells, 16.3 ± 10.0 μM, and 637 ± 384 pmol/min per 106 cells, respectively (eqs. 3 and 4), based on a nonlinear regression analysis. Based on the inhibitory effect of ATV on the transport rate of LB in rOATP1B2 cells (Fig. 5C) and rat hepatocytes (Fig. 5D), a nonlinear regression analysis indicated that the IC50,OATP and IC50,hepa values were 0.473 ± 0.278 and 0.984 ± 0.812 μM, respectively. Based on the relationship given by Cheng and Prusoff (1973), the Ki,OATP and Ki,hepa values were estimated to be 0.296 ± 0.174 and 0.875 ± 0.739 μM by eq. 6. Based on nonlinear regression analysis of virtual data (Supplemental Table 1) and eqs. 5 and 6, we found that the assigned and calculated Ki (in μM) were 0.875 and 0.833 ± 0.520 (Supplemental Table 2). These observations suggest that the variability found in the current study does not significantly affect the outcome of the estimation of Ki values.

Simulation of LB Pharmacokinetics after a Single Intravenous Dose and DDIs Using a PBPK Model

In this study, we attempted to use a PBPK model to predict the plasma and tissue kinetics of LB at a dose of 1 mg/kg (Fig. 2A) using primarily in vitro experimental results (Table 3). Our calculations indicate that the profiles for the plasma and liver concentration could be reasonably predicted when LB was given to rats intravenously without ATV coadministration (Fig. 6). Accordingly, the ratios of AUCplasma to the AUCliver (i.e., Kp,liver) were calculated to be 2.18 ± 0.09 (Table 4) from in vitro data (i.e., Km,MIC, Vmax,MIC, PS, Km,hepa, and Jmax,hepa), which is reasonably close to the experimentally determined value (i.e., 2.34; Table 4).

Fig. 6.
Fig. 6.

(A) Observed and simulated plasma concentration–time profiles for LB after intravenous bolus administration of 1 mg/kg LB with or without 5 mg/kg ATV to rats. Black circles, solid line, white circles, and the dashed line represent the observed (LB alone), simulated (LB alone), observed (LB with ATV), and simulated (LB with ATV, with the Ki,OATP value) plasma concentrations of LB, respectively. (B) Observed and simulated liver concentration–time profiles for LB after intravenous bolus administration of 1 mg/kg LB with or without 5 mg/kg ATV to rats. Black circles, solid line, white circles, and the dashed line represent the observed (LB alone), simulated (LB alone), observed (LB with ATV), and simulated (LB with ATV, with the Ki,OATP value) liver concentration of LB, respectively. Symbols represent the mean ± S.D. of triplicate runs. The input parameters for LB and ATV are summarized in Table 3.

View this table:
TABLE 3

Summary of kinetic parameters for LB and ATV used in PBPK calculation

View this table:
TABLE 4

Observed and simulated pharmacokinetic parameters for LB for an intravenous bolus administration of 1 mg/kg LB alone and 1 mg/kg LB with 5 mg/kg ATV to rats

Simulated data are expressed as means ± S.D. of three virtual rats from in vitro experiments in triplicate runs.

Assuming that the current PBPK model for LB is predictive, the PBPK model for LB may also be used to mimic in vivo DDIs between LB and ATV by using the metabolic and transport kinetic parameters for LB obtained from in vitro studies. The observed and simulated LB concentration profiles for the plasma and liver are presented in Fig. 6, and their pharmacokinetic parameters are summarized in Table 4. For the case of the Kp,liver value of LB with ATV, the predicted value (1.71) was similar to that based on an experimental determination (1.55) when the Ki,OATP value was used in the calculation.

Discussion

The possibility of a DDI occurring between LB and ATV was examined, considering the likelihood of the concurrent development of hyperlipidemia/T2DM (Ervin, 2009) and overlapping metabolic (CYP3A)/transport (rOATP1B2) pathways for the two drugs. No apparent DDI was found (Fig. 1; Table 1) between the two drugs in their respective systemic pharmacokinetics, even though interactions between LB and ATV were both detected in vitro at the metabolic level (Fig. 4B) and at transport (Fig. 5, C and D). Despite its high lipophilicity, the volume of distribution was limited for LB (i.e., 271 ± 20 ml/kg), likely because it extensively binds to plasma proteins. In addition, the reduction in Kp,liver for LB (i.e., from 2.34 to 1.55) by ATV did not appear to be significant in the total Vss, considering the fact that the contribution of the reduced distribution of LB to the liver would be approximately 27 ml/kg, which is relatively small for the Vss of 271 ml/kg. In other clinical DDI studies involving LB with metformin (i.e., T2DM agent), LB with warfarin (i.e., an anticoagulant with a narrow therapeutic index), or LB with amlodipine (i.e., an antihypertensive agent) (Shin et al., 2012; Jung et al., 2015; Kim et al., 2015), no apparent changes in the systemic pharmacokinetics was found. In contrast, however, the liver concentration of LB was significantly decreased by the coadministration of ATV (Fig. 2; Table 2). A concentration-dependent decrease was readily apparent for the Kp,liver of LB for different levels of steady-state LB concentrations (Fig. 3) and the extent of liver distribution was determined. These findings are the first indication of this “liver-specific” DDI between LB and ATV in rats. Other tissue-specific DDIs have also been noted. A decrease in the concentration of metformin in the liver and kidney was reported, without any apparent change in the systemic pharmacokinetics, for the coadministration of cimetidine or pyrimethamine: this tissue-specific change was mediated by the inhibition of mouse multidrug and toxin extrusion 1 protein (Ito et al., 2012; Shingaki et al., 2015). In addition, an increase in the donepezil concentration in the heart and brain was reported, without any appreciable change in systemic pharmacokinetics, after cilostazol coadministration, probably by inhibition of the breast cancer resistance protein (Takeuchi et al., 2016)

The metabolic rate of LB was also decreased by ATV (Fig. 4), although the Ki,MIC values for ATV (i.e., 15.0 μM, unbound concentration) as estimated from in vitro data were relatively high. Since the estimated unbound liver concentration of ATV in vivo would be close to 71.8 nM (calculated from the observed Cmax,liver of ATV when ATV was administered with LB in the tissue study by multiplying fu,plasma,ATV/Kp,liver,ATV), the maximum concentration of free ATV in the liver (i.e., 1.27 μM) would still be approximately 12-fold lower than the calculated Ki,MIC value, even if all ATV in the plasma is present in the unbound form. Taken together, the hepatic metabolism of LB is not likely to be significantly inhibited by the ATV dose administered in this study. In addition, the reduced metabolic activity by ATV from in vitro studies did not appear to be consistent with in vivo observations in which a lower hepatic concentration of LB by ATV coadministration was found. Therefore, we did not pursue the mechanism responsible for the metabolic interaction between LB and ATV in vitro any further.

It is now well established that rOATP1B2 is primarily expressed in the liver in rats and ATV is one of its representative substrates (Knauer et al., 2010). These findings indicate that the transport of LB in rOATP1B2-expressing cells had a Km,OATP of 2.06 ± 0.45 μM with a Ki,OATP by ATV of 0.296 ± 0.174 μM (Fig. 5, A and C). The uptake of LB was saturable and inhibited by ATV, with Ki,hepa values of 0.875 ± 0.739 μM in the study involving isolated rat hepatocytes. Collectively, our observations suggest that the distribution of LB to the liver is mediated by hepatic uptake transporter(s), such as rOATP1B2, and the liver distribution of LB is inhibited by the coadministration of ATV, a rOATP1B2 substrate. As corroborative evidence for the involvement of rOATP1B2 in liver-specific DDIs, we found that the Kp,liver of ATV declined from 97.8 to 65.4 by the coadministration of LB (Table 2).

Based on in vitro studies, LB appeared to be transported to the liver via passive diffusion and carrier-mediated transport(s). Assuming that a similar process occurs in vivo, evaluating the Kp,liver,pass of LB, representing the liver-to-plasma concentration ratio for LB by passive diffusion only, was necessary. We used the method of Rodgers and Rowland (2006) for this (i.e., 0.794 for LB). Interestingly, the current value was comparable to the Kp,liver value measured from the intravenous infusion study at the highest dose of LB with ATV (i.e., 0.714 ± 0.184; Fig. 3B). The ATV plasma concentration was approximately 4.8 µM and the estimated Ki,OATP value by ATV was approximately 0.3 μM in rOATP1B2-expressing cells. Therefore, carrier-mediated transport by rOATP1B2 appears to be fully inhibited under the above conditions. In subsequent calculations, the computational estimate was assumed to be adequate and was used.

It has been reported that “albumin-mediated uptake mechanism(s)” in hepatocytes may occur for highly bound drugs such as LB and ATV (Poulin et al., 2016). The calculated fu,plasma,surf value for ATV was approximately 5-fold higher than fu,plasma for ATV (i.e., fu,plasma,surf and fu,plasma for ATV were 0.280 and 0.0567, respectively), indicating that the potency of ATV for inhibiting the hepatic uptake of LB might be underestimated by as much as 5-fold if the fu,plasma of ATV is used instead of the fu,plasma,surf of ATV (Poulin and Haddad, 2015). In this study, the simulation using fu,plasma,surf, instead of fu,plasma, yielded improved predictions (Supplemental Fig. 8).

The theoretical Kp,liver assuming LB without the coadministration of ATV was calculated to be 2.18 ± 0.09, comparable to the observed Kp,liver value (i.e., 2.34). In addition, the estimated Kp,liver assuming that LB was coadministered with ATV was 1.71 ± 0.13, close to 1.55 under a similar in vivo situation. Taken together, the simulated Kp,liver values for LB with or without ATV were in reasonable agreement with the observed data. It is noteworthy that the kinetic calculation was primarily based on parameters obtained from in vitro studies using rOATP1B2-expressing cells. Therefore, the liver-specific DDI between LB and ATV may be due to the interaction of rOATP1B2 between the two drugs. Since rOATP1B2 is known to be functionally similar to hOATP1B1 (Hagenbuch and Meier, 2003), a similar interaction (e.g., the lack of a DDI in systemic exposure while the liver concentration for LB reduced by ATV) may occur between LB and ATV in humans. This aspect of a DDI involving LB and ATV warrants further investigation.

Although LB was reported to be a PPARγ agonist, it possessed a strong affinity for PPARα (i.e., EC50 = 0.02 μM) (Lee et al., 2007). It was reported that fenofibrate, a PPARα agonist, may improve insulin sensitivity, probably by interactions between the drug and its receptor, as shown in an animal model of diabetes and obesity (Koh et al., 2003). Interestingly, the effective dose of LB was significantly lower than that for rosiglitazone and pioglitazone [i.e., (in effective dose) LB, 0.5 mg/d; rosiglitazone, 2–8 mg/d; pioglitazone, 15–45 mg/d] (Balfour and Plosker, 1999; Hauner, 2002; Park et al., 2014). Therefore, it is possible that the higher potency of LB may be partly mediated by the interaction of LB with PPARα. Assuming this, the liver may be regarded as an additional site of action for LB, since the receptor is highly expressed in the liver (Braissant et al., 1996). Considering that the coadministration of LB and ATV led to a liver-specific decrease in the distribution of the two drugs, efficacy could be affected for the two drugs. In particular, since the liver is likely to be closely linked to the pharmacological activity of the drugs (i.e., the primary site of action for ATV and potentially the secondary site for LB), additional studies may be necessary to confirm the impact of the liver-specific DDI on the pharmacological activity.

In conclusion, ATV coadministration led to a significant decrease in the liver concentration of LB with no appreciable change in its plasma concentration in rats. Metabolic interactions between LB and ATV appeared to cause only minor kinetic changes and are not likely to account for the liver-specific decrease in LB levels in vivo. LB was a substrate for rOATP1B2 and carrier-mediated transport was inhibited by ATV, even at low concentrations. The transport variables obtained from in vitro studies and the PBPK model assuming a carrier-mediated transport process in distribution to the liver were adequate for mimicking the in vivo pharmacokinetics of LB with or without ATV coadministration. Considering that LB and ATV might be used in combination, the liver-specific DDI caused by the combination found in this study may be therapeutically relevant.

Authorship Contributions

Participated in research design: Yim, Chung.

Conducted experiments: Yim, Jeong, S.-Y. Lee, Pyeon, Ryu.

Contributed new reagents or analytic tools: J.-H. Lee.

Performed data analysis: Yim, Jeong, Maeng, Chung.

Wrote or contributed to the writing of the manuscript: Yim, Jeong, J.-H. Lee, K.-R. Lee, Maeng, Chung.

Footnotes

    • Received November 1, 2016.
    • Accepted January 5, 2017.

  • This research was supported by the National Research Foundation of Korea, funded by the Korean government [Grant 2009-0083533].

  • A portion of this work was previously presented as a poster Interaction between Lobeglitazone and Atorvastatin in Rats: Potential Involvement of Transporter-Mediated Interaction in the Hepatic Uptake. Yim CS, Jeong YS, Lee SY, Ryu HM, Lee W, Dae-Duk K, Chung SJ: (2016) 11th International Society for the Study of Xenobiotics Meeting; 2016 Jun 12–16; Busan, Republic of Korea.

  • dx.doi.org/10.1124/dmd.116.074120.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

ACN
acetonitrile
ANOVA
analysis of variance
ATV
atorvastatin
AUC
area under the concentration-time curve
B/P
blood-to-plasma concentration ratio
BCA
bicinchoninic acid
BSA
bovine serum albumin
CL
systemic clearance
DDI
drug–drug interaction
DMSO
dimethylsulfoxide
DPBS
Dulbecco’s phosphate-buffered saline
FRT
flippase recognition target
fu
fraction unbound
hOATP
human organic anion transporting polypeptide
J
rate of cellular uptake
KHB
Krebs-Henseleit buffer
LB
lobeglitazone
LC
liquid chromatography
MDCK
Madin–Darby canine kidney
MRT
mean residence time
MS/MS
tandem mass spectrometry
OATP
organic anion transporting polypeptide
PBPK
physiologically based pharmacokinetic
PPARγ
peroxisome proliferator–activated receptor γ
PS
unbound passive diffusional clearance
rOATP
rat organic anion transporting polypeptide
SD
Sprague-Dawley
T2DM
type 2 diabetes mellitus

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DDIs between Lobeglitazone and Atorvastatin in Rats

Chang-Soon Yim, Yoo-Seong Jeong, Song-Yi Lee, Wonji Pyeon, Heon-Min Ryu, Jong-Hwa Lee, Kyeong-Ryoon Lee, Han-Joo Maeng and Suk-Jae Chung
Drug Metabolism and Disposition March 1, 2017, 45 (3) 246-259; DOI: https://doi.org/10.1124/dmd.116.074120
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