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Research ArticleMetabolism, Transport, and Pharmacogenomics

Evaluation of Rosuvastatin as an Organic Anion Transporting Polypeptide (OATP) Probe Substrate: In Vitro Transport and In Vivo Disposition in Cynomolgus Monkeys

Hong Shen, Hong Su, Tongtong Liu, Ming Yao, Gabe Mintier, Lun Li, R. Marcus Fancher, Ramaswamy Iyer, Punit Marathe, Yurong Lai and A. David Rodrigues
Journal of Pharmacology and Experimental Therapeutics May 2015, 353 (2) 380-391; DOI: https://doi.org/10.1124/jpet.114.221804

Abstract

Organic anion transporting polypeptides (OATPs) mediate hepatic drug uptake and serve as the loci of drug–drug interactions (DDIs). Consequently, there is a major need to develop animal models and refine in vitro–in vivo extrapolations. Therefore, the in vivo disposition of a model OATP substrate, [3H]rosuvastatin (RSV), was studied in the cynomolgus monkey and reported for the first time. After monkeys had received a 3-mg/kg oral dose, mass balance was achieved after bile duct cannulation (mean total recovery of radioactivity of 103.6%). Forty-two percent of the RSV dose was recovered in urine and bile, and the elimination pathways were similar to those reported for human subjects; 61.7%, 39.0%, and 2.9% of the dose was recovered in the feces, bile, and urine, respectively. The high levels of unchanged RSV recovered in urine and bile (26% of the dose) and the relatively low levels of metabolites observed indicated that RSV was eliminated largely by excretion. Also, for the first time, the in vitro inhibitory potential of cyclosporin A (CsA) toward cynomolgus monkey OATPs and sodium-taurocholate cotransporting polypeptide was studied in vitro (primary hepatocytes and transporter-transfected cells). It is concluded that one can study the CsA-RSV DDI in the cynomolgus monkey. For example, the in vitro IC50 values were within 2-fold (monkey versus human), and the increase (versus vehicle control) in the RSV AUC0–inf (6.3-fold) and Cmax (10.2-fold) with CsA (100 mg/kg) was similar to that reported for humans. The results further support the use of the cynomolgus monkey as a model to assess interactions involving OATP inhibition.

Introduction

Rosuvastatin (RSV) is a 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitor (i.e., statin) used in the treatment of patients with hypercholesterolemia. Drug–drug interactions (DDIs) involving RSV that result in an increase in its systemic exposure also might result in unwanted side effects including myopathy and rhabdomyolysis. Such DDIs are clinically significant when one considers that known transporter inhibitors such as cyclosporin A (CsA) increase RSV area under the plasma drug concentration–time curve (AUC) and maximum plasma concentration (Cmax) 7.1- and 10.6-fold, respectively (Simonson et al., 2004). Because RSV is not significantly metabolized across several species (Nezasa et al., 2002b; Martin et al., 2003b) and is selectively transported and distributed into the liver (Nezasa et al., 2002a,b; Martin et al., 2003a,b), the overall disposition profile of oral RSV is thought to be highly dependent on drug transporters such as organic anion transporting polypeptide (OATP), sodium-taurocholate cotransporting polypeptide (NTCP), and breast cancer resistance protein (BCRP) (Ho et al., 2006; Pasanen et al., 2007; Kitamura et al., 2008; Keskitalo et al., 2009; Bi et al., 2013). It has been established that hepatic clearance and renal clearance are the primary pathways for the elimination of RSV, accounting for 72% and 28% of total body clearance, respectively (Martin et al., 2003a). Active transport processes are responsible for approximately 90% of total hepatic uptake clearance of RSV. Indeed, RSV is a substrate of the hepatic OATP1B1 and OATP1B3 in vitro, the former contributing 77% and the latter 23% to sodium-independent active uptake in human hepatocytes. The contribution of both OATPs represents 70% of total hepatic active uptake; the remaining active uptake (30%) is attributed to NTCP (Ho et al., 2006; Kitamura et al., 2008; Bi et al., 2013). Consistent with the contribution of individual transporters to the overall hepatic uptake of RSV, OATPs and NTCP comprise two of the most abundant sinusoidal uptake transporters in human liver tissues, with relative abundance of quantifiable hepatic transporters of 29% and 13%, respectively, when quantified transporter protein expression in a human liver bank (n = 55) by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Wang et al., 2015). Furthermore, for most transporters, the expression in the liver tissues was comparable to that in the cryopreserved hepatocytes (Wang et al., 2015). In addition to in vitro assessment, RSV has often been used for kinetic studies in vivo in several species, including humans (Schneck et al., 2004; Simonson et al., 2004; Prueksaritanont et al., 2014), monkeys (Shen et al., 2013), mice (Salphati et al., 2014), rats (Wen and Xiong, 2011), and pigs (Bergman et al., 2009). When compared with rodents, however, the absorption, distribution, metabolism, and excretion (ADME) profile of RSV in nonhuman primates has not been studied extensively (Nezasa et al., 2002b; Martin et al., 2003b).

It has been reported that cynomolgus monkey OATPs (cOATPs) share a high degree of amino acid sequence identity and functional similarity to their human counterparts (Shen et al., 2013; Takahashi et al., 2013). Concomitant with these identities and similarities, there are investigations employing the cynomolgus monkey as an in vivo preclinical model to assess OATP DDIs (Shen et al., 2013; Takahashi et al., 2013). The DDIs have been investigated in other animal models; however, the sequences and transporting profiles obtained for xenobiotics in other animal species are frequently different from those obtained in humans, reducing the utility of such animals (Shitara et al., 2003; Shirasaka et al., 2010; Li et al., 2013). In fact, there is only one member of the OATP1B family, Oatp1b2, that is the closest ortholog of both human OATP1B1 and OATP1B3 (hOATP1B1 and hOATP1B3) and likely arises from a gene duplication after divergence of the rodent species (Hagenbuch and Meier, 2004). The comparison of predicted Oatps in the genomes of dogs, cows, and horses has suggested that there is only a single Oatp in the 1B family; in the cynomolgus monkey, a species much closer related to humans, both cOATP1B1 and cOATP1B3 orthologs have been cloned (Shen et al., 2013). These results indicate, for many drugs, that cynomolgus monkey may represent an acceptable in vitro and in vivo model to investigate OATPs.

Several reports have described various rodent models that can be used to support the investigation of OATPs in vivo. For example, it is possible to delete the murine Oatp1b2 gene (Lu et al., 2008; Zaher et al., 2008; Chang et al., 2014), introduce human OATP genes (van de Steeg et al., 2009), and develop combinations of OATP knockout and knock-in mice (Higgins et al., 2014; Salphati et al., 2014). However, the interpretation of data obtained with such models can be difficult in some cases when compensatory mechanisms are suspected (Klaassen and Lu, 2008; Iusuf et al., 2014; Salphati et al., 2014). In addition, quantitative translation of data obtained with knockout and/or knock-in animals can be challenging. Indeed, unexpected results have been observed in mice and rats compared with humans. For example, the blood and liver concentrations of atorvastatin in Oatp1a/b knockout mice were similar to wild-type animals (Chang et al., 2014). The expression of human OATP1B1 and OATP1B3 in humanized mice did not significantly alter the liver or plasma concentration ratios of RSV or pitavastatin compared with Oatp1a/1b knockout controls (Salphati et al., 2014). Therefore, there is a need for a better preclinical model to enable the prediction of OATP-mediated drug disposition and DDIs.

We studied the ADME of RSV after administration of oral [3H]RSV (3 mg/kg) to bile duct–annulated (BDC) cynomolgus monkeys. Because RSV has been shown to be a substrate of human OATPs and NTCP (Bi et al., 2013), we investigated CsA as an inhibitor of both monkey OATP- and NTCP-mediated RSV uptake in vitro. Finally, the CsA-RSV DDI was studied in vivo in cynomolgus monkeys. Our results indicate that there is a consistency in the elimination pathway of oral RSV between humans and cynomolgus monkeys, and that the monkey can be used as a model to assess inhibition of OATP both in vitro and in vivo.

Materials and Methods

Chemicals, Hepatocytes, and Cynomolgus Monkeys.

All chemicals and solvents of reagent or high-performance liquid chromatography (HPLC) grade were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Nonradiolabeled RSV was purchased from Toronto Research Chemicals (Toronto, ON, Canada), and CsA oral solution (Neoral, 100 mg/ml) was purchased from Novartis Pharmaceuticals (East Hanover, NJ). [3H]Taurocholic acid (TCA) (5.0 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). [3H]RSV calcium (10.0 mCi/mmol) and [3H]atorvastatin (ATV) sodium (20.0 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). The radiochemical purity of all compounds was determined to be greater than 98.2% by HPLC. Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA) or Mediatech (Manassas, VA). Cryopreserved male human and cynomolgus hepatocytes were purchased from Bioreclamation IVT (Baltimore, MD). The 24-well poly-d-lysine–coated plates were purchased from BD Biosciences (San Jose, CA). Intact and BDC male cynomolgus monkeys were procured from Charles River Laboratories (Wilmington, MA).

BDC Cynomolgus Monkey Study.

All animal studies were performed under the standards recommended by the Guide for the Care and Use of Laboratory Animals (NRC, 1996) and were approved by BMS Institutional Animal Care and Use Committee. The excretion of radioactivity into bile, feces, and urine and metabolism of RSV was investigated in BDC male cynomolgus monkeys (Charles River Laboratories) after administration of [3H]RSV. BDC monkeys (N = 3, weighing approximately 5.5 to 6.5 kg) were individually housed in metabolism cages and were freely mobile during the entire study with the exception of brief manual restraint for oral dosing. Each animal received a single oral dose of [3H]RSV administered by gavage at a dose level of 3 mg/kg (approximately 15 µCi/kg). Animals were fasted overnight before dosing. Approximately 4 hours after dosing, animals were fed Certified Primate Diet 5048 (PMI Nutrition International, Shoreview, MN). Bile was collected before dosing and from 0 to 8, 8 to 24, and 24 to 72 hours after dosing into containers that were surrounded by dry ice. Urine and feces were collected before dosing and over 24-hour intervals through 168 hours after dosing. Blood samples were collected into tubes containing K2EDTA from the femoral artery before dosing and at 1, 2, 6, 12, 24, and 48 hours after dosing. The blood samples were then centrifuged to obtain plasma, and the plasma samples were frozen at –70°C until analysis.

Determination of Radioactivity in Biologic Matrices from BDC Cynomolgus Monkeys.

Plasma, urine, bile, and fecal samples were analyzed for radioactivity concentration using liquid scintillation counting. Portions of plasma (50–100 µl), urine (50 µl), and bile (10 µl) were mixed with 5 ml of Ecolite scintillation cocktail (PerkinElmer Life and Analytical Sciences) into polystyrene tubes. For fecal samples, two portions (approximately 0.2 g each) of fecal homogenate were weighed individually in a scintillation vial, mixed with 1 ml of Soluene-350, and shaken slowly at room temperature overnight. The solubilized homogenate mixtures were bleached with 1 ml of 20% hydrogen peroxide, and then neutralized with 0.1 ml of a solution containing saturated sodium pyruvate in methanol, glacial acetic acid, and methanol (4:3:1, by volume). After addition of Ecolite cocktail, the samples were mixed and stored under refrigerated conditions in the dark overnight. Radioactivity was determined by LSC6000 or LS6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA) for 5 minutes.

Metabolite Profiling of Cynomolgus Monkey Bile and Urine.

Pooled bile and urine samples from BDC monkeys were prepared by combining a constant percentage of bile and urine volume across animals (3% and 2%, respectively). The pooled samples were then centrifuged at 14,000g for 5 minutes. A portion of supernatant (25–50 µl) was injected into the HPLC for biotransformation profiling and mass spectral analysis.

The HPLC analysis was conducted on an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA) interfaced to a linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Separation was achieved on a Agilent Zorbax SB-C18 column (4.6 mm × 250 mm, 5 µm) using a mobile phase consisting of 0.1% formic acid and 0.1% acetonitrile in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a constant flow rate of 0.8 ml/min. The gradient was as follows: 0–5 minutes, 20% B; 40 minutes, 25% B; 47 minutes, 50% B; 50 minutes, 90% B; and 60 minutes, 20% B.

The HPLC eluate was split via a Gilson Model FC 204 fraction collector (Gilson, Middleton, WI), with which 25% of the eluate was directed into the linear ion trap mass spectrometer for profiling. The remaining 75% of the eluate was collected into ScintiPlate 96-well plates at 0.2-minute intervals per well. Ecolite scintillation cocktail (200 µl) was then added to each well, and the radioactivity was counted for 20 minutes per well with a PerkinElmer 1450 MicroBeta Wallac TRILUX Liquid Scintillation and Luminescence Counter (PerkinElmer Life and Analytical Sciences).

Radiochromatographic profiles were prepared by plotting the net counts per minute values obtained from the counter versus time after injection. Mass spectral analyses were performed using electrospray ionization (ESI) in the positive ion mode. The capillary temperature was 300°C, and the ESI voltage was maintained at 4.5 kV for all analyses. The collision energy was 15.0% for LC-MS/MS analysis. Other instrument parameters were adjusted to give maximum sensitivity or fragmentation of drug-related components.

Generation of Stably Expressed Cynomolgus Monkey NTCP in HEK-293 Cells.

Cloning and stable transfection of human embryonic kidney 293 (HEK-293) cells with cynomolgus monkey NTCP (cNTCP) were performed as described elsewhere (Shen et al., 2013). In brief, cNTCP was cloned out of cDNA synthesized from Mauritian cynomolgus monkey liver total RNA. For polymerase chain reaction (PCR) on double-stranded cDNA from monkey liver, the following degenerate oligonucleotides, derived from the human and rhesus monkey NTCP gene sequences and predicted cNTCP transcript sequence, were used: 5′-CTT CCA CTG CCT CAC AGG AGG-3′ (forward primer, corresponding to the nucleotide positions 114–134 of human NTCP cDNA NM_003049.3), and 5′-AAG GGC TAG GCT GTG CAA GG-3′ (reverse primer, reverse complementary to positions 1170–1189).

The PCR products were cloned into pJet1.2 (Fermentas, Vilnius, Lithuania), and several clones for each cDNA were sequenced. Sequences were deposited to GenBank: cNTCP (KP453714). The coding sequence of the cDNA was subcloned into the Gateway entry vector pDONR221 (Invitrogen) using standard methods. The Gateway entry clones were recombined into a Gateway adapted version of the expression vector pcDNA5/FRT/TO (Invitrogen) using LR Clonase II (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Expression constructs were analyzed by agarose gel electrophoresis, and the sequence was confirmed.

Flp-In HEK-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM l-glutamine on a 6-well plate as described elsewhere (Shen et al., 2013). After washing with serum-free medium, the culture well was incubated at 37°C for 4 hours with 1 ml of serum-free medium that contained 10 µl Lipofectamine 2000 (Invitrogen) and 2.5 µg plasmid DNA. The cells stably transfected with pcDNA5/FRT/TO/cNTCP were then selected using hygromycin B (200 μg/ml) according to the protocol of the vendor (Invitrogen). Expression of cNTCP was verified by reverse-transcription PCR and functional characterization.

Uptake Studies Using Transporter-Expressing HEK-293 Cells.

The HEK-293 cells individually expressing human OATP1B1 (hOATP1B1), hOATP1B3, cynomolgus monkey OATP1B1 (cOATP1B1), cOATP1B3, or cNTCP were prepared and used as described previously elsewhere (Shen et al., 2013). In brief, all transporter- and vector-transfected cells were cultured at 37°C in an atmosphere of 95% air and 5% CO2 and subcultured once a week. Cells were seeded in 24-well poly-d-lysine–coated plates at a density of 5 × 105 cells per well, and were ready for experiment after 48 to 72 hours. Before uptake experiments, cells were washed twice with 1.5 ml of prewarmed Hanks’ buffered saline solution (HBSS). The cells were then preincubated with uptake buffer (HBSS with 10 mM HEPES, pH 7.4) containing CsA only (0.023–16.7 µM) at 37°C for 15 minutes. Subsequently, the cells were incubated with the test solution containing CsA and the substrate RSV (0.1 µM) at 37°C for 2 minutes. Over this time period, linearity of uptake was confirmed (Shen et al., 2013). Cells were washed 3 times with 1 ml of ice-cold HBSS buffer and lysed with 0.3 ml of 0.1% Triton X-100. The intracellular accumulation of RSV was determined using LC-MS/MS as described later.

Assessment of uptake of [3H]TCA, [3H]RSV, and [3H]ATV into HEK-293 cells expressing cNTCP involved a 5-minute incubation. The cells were lysed with 0.3 ml of 0.1% Triton X-100, and the radioactivity was determined by liquid scintillation counting. Accumulation was normalized to the protein content of the HEK-293 cells in each well, as measured using a bicinchoninic acid assay (BCA Protein Assay Kit; Pierce Chemical, Rockford, IL).

LC-MS/MS Analysis of RSV in Cell Lysates from In Vitro Transporter Studies.

Cells were solubilized and lysed in 300 µl of 0.1% Triton X-100 in water. After 60 minutes incubation at room temperature, the samples were transferred by a Tecan liquid handler (Tecan Group, Männedorf, Switzerland) from a 24-well plate to the first 96-well filter plate for LC-MS/MS analysis and the second 96-well plate for protein measurement. Protein concentrations in the cell lysates (20 µl) were measured using the BCA protein assay kit (Pierce Chemical). The lysed samples (150 µl) were mixed with 200 µl of acetonitrile containing d6-rosuvastatin (internal standard) in a 96-well hydrophilic filter plate (Millipore, Billerica, MA), stacked with a deep 96-well receiver plate, and centrifuged. The filtrates were dried under stream of nitrogen to half of the initial volume in the receiver plate, vortexed, and analyzed by LC-MS/MS. Cellular uptake was normalized to the protein content.

The liquid chromatography system was the Shimadzu SCL 30AD Nexera, composed of two LC-30AD pumps and a Shimadzu SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, MD). RSV and d6-rosuvastatin were separated under gradient elution on a Waters Acquity HSS T3 (50 mm × 2.1 mm internal diameter, 1.8 μm particle size) (Waters Corporation, Milford, MA). The column was maintained at room temperature. The mobile phase was a mixture of 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.60 ml/min. The gradient program was set as follows: from 0 to 0.01 minutes, keep B for 20%; from 0.01 to 2.0 minutes, increase B linearly from 20% to 60%; from 2.0 to 2.1 minutes, increase B linearly from 60% to 95%; maintain 95% B for 0.4 minutes; and from 2.5 to 2.6 minutes, decrease B from 95% to 20%. We then equilibrated the column for 0.9 minutes for the next injection. The retention time for rosuvastatin and d6-rosuvastatin was 1.86 minutes.

An AB Sciex Qtrap 6500 system (Applied Biosystems/MDS Analytical Technologies, Foster City, CA) equipped with ESI source was used for mass spectrometric detection. Analyst version 1.62 (AB Sciex, Framingham, MA) was used as the data acquisition software. The ESI source was used in the positive ion mode. The LC-MS/MS detector was operated at unit resolution in the multiple reaction monitoring mode using the transitions of the protonated forms of RSV at m/z 482.1 > 258.07 and d6-rosuvastatin at m/z 488.16 > 263.9. Optimized parameters were as follows: curtain gas, gas 1 and gas 2 (nitrogen) 60 and 75 units, respectively; dwell time 100 milliseconds; source temperature 400°C; and IonSpray voltage 5000 V. Declustering potential and collision energy were 116 V and 45 eV for both RSV and d6-rosuvastatin.

In addition, a divert valve was used to minimize the Triton X-100 introduced into the MS source. At 0 to 0.8 minutes, the ultraperformance liquid chromatography eluent was diverted to waste then switched to MS source after 1 minute. After 3.0 minutes, the eluent was diverted back to waste again. The range of standard curve was between 0.02 to 10.0 nM for RSV.

Uptake Studies in Cryopreserved Hepatocytes.

Cryopreserved human (Lots OJE, GST, LIO) and cynomolgus monkey hepatocytes (pool of four males; Lot VCE) were thawed according to the manufacturer’s instructions and resuspended in InVitroGRO Krebs-Henseleit buffer (Bioreclamation IVT). Human hepatocytes from three donors were then pooled together after thawing.

Cell number and viability were assessed using trypan blue exclusion. Cells with viability greater than 80% at the time of uptake assay were selected to reduce the background signal, and resuspended at a concentration of 2 × 106 viable cells/ml in InVitroGRO Krebs-Henseleit buffer. Centrifuge tubes (0.4 ml) were prepared by filling with 50 μl of 3N KOH solution and layering 100 μl of filtration oil on top of the KOH. The filtration oil was prepared as a mixture of 82 parts silicone oil to 18 parts mineral oil, resulting in an oil mixture with a density of 1.015 g/ml. Cell suspensions (100 µl) containing appropriate concentrations of CsA were added to glass tubes and preincubated in a water bath at 37°C for 5 minutes.

Uptake studies were initiated by an addition of 100 µl of prewarmed buffer containing [3H]RSV (0.2 µM). Aliquots (80 µl) of the mixture were removed at 15 and 90 seconds, and then transferred to centrifuge tubes and layered carefully on top of an oil layer. The tubes were centrifuged immediately at 13,000g for 15 seconds. After centrifugation of hepatocytes through the oil layer, the cell suspension buffer containing RSV was left in the supernatant.

The centrifuge tubes were incubated for at least 2 hours at an ambient temperature, then were frozen in a −80°C freezer or on dry ice just before cutting. The tubes were cut in the middle of the oil layer, allowing the bottom section to drop into a 20-ml scintillation vial. The cell pellets were resuspended in 120 µl 2 N HCl to neutralize the solution and read in a scintillation counter after addition of the scintillation cocktail.

In Vivo DDI Study.

The in vivo DDI pharmacokinetic studies were conducted by WuXi AppTec Corporation (Suzhou, P. R. China) using three young adult male cynomolgus monkeys weighing between 3.4 and 4.2 kg. The animals were housed in a temperature- and humidity-controlled room with a 12-hour light/dark cycle. Animals were fed approximately 120 g of Certified Monkey Diet daily (Beijing Vital Keao Feed Corporation, Beijing, P. R. China). The monkeys were fasted for 12 hours before dose administration, and water was made available ad libitum.

In the first period, the three monkeys received an oral dose of RSV at 3 mg/kg of body weight dissolved in sterile water by oral gavage followed by a water rinse. After a 1-week washout period, the same animals were administered an equivalent oral dose of RSV 1 hour after CsA administration (100 mg/kg p.o.; Neroral cyclosporine oral solution) in the second period. There was no substantial weight fluctuation of the animals between the times of RSV alone and coadministration.

Approximately 1 ml of blood was collected in tubes containing potassium (K2) EDTA by venipuncture via the cephalic or saphenous veins at 0.25, 0.5, 0.45, 1, 2, 3, 5, 7, 24, and 48 hours after RSV administration. In period 2 (i.e., coadministration treatment), 300 μl of whole blood was aliquoted and stored at ≤−15°C for CsA analysis. The remaining blood volume was centrifuged (3000g for 10 minutes at 2 to 8°C) within 1 hour of collection, transferred into polypropylene microcentrifuge tubes, and stored frozen at −70°C. The animals were placed in metabolic cages, and urine was collected at 0–7, 7–24, and 24–48 hours after dosing into containers cooled by wet ice. The total volume of urine samples for different periods was measured at the end of collection and recorded. Aliquots (5 ml) of urine samples collected from each period were frozen on dry ice and kept at −70°C until analysis.

LC-MS/MS Analysis of RSV in Plasma and Urine.

Stock solution (1 mg/ml) of RSV was prepared in acetonitrile/0.1 M ammonium acetate pH 4.0 buffer (80:20 v/v). A stock solution (1 mg/ml) of rosuvastatin-5S-lactone, a metabolite of RSV, was prepared in acetonitrile/0.1M ammonium acetate pH 4.0 buffer (50:50 v/v), and a stock solution (0.2 mg/ml) of ATV (internal standard) was prepared in 50% acetonitrile. For plasma assay, the following RSV calibration standards were prepared in monkey plasma: 0.1, 0.2, 1, 10, 50, 100, 200, and 250 ng/ml. Quality control (QC) samples were also prepared in monkey plasma at 0.3, 5, 90, and 190 ng/ml. For urine assay, the RSV calibration standards were prepared in monkey plasma at 0.3, 0.6, 3, 30, 150, 300, 600, and 750 ng/ml. QC samples were prepared in monkey urine with 0.75 mg/ml Triton X-100 at 4.5, 75, 1350, and 2850 ng/ml, and diluted with plasma (1:4, urine/plasma v/v), and quantified by plasma calibration curve.

Plasma sample extraction for RSV was conducted in 96-well plates using protein precipitation with acetonitrile containing 0.5% formic acid. In brief, 50 μl of calibration standards, QC samples, and study samples were first mixed with 50 μl of chilled (ice/water bath) 0.1 M ammonium acetate (pH 4.5) buffer followed by 20 μl of chilled 100 ng/ml atorvastatin solution in acetonitrile/0.1 M ammonium acetate (pH 4.0) buffer (75:25 v/v). After addition of 300 μl of chilled 0.5% formic acid in acetonitrile, vortex-mixing, and centrifugation at 4°C, an aliquot of 300 μl of each sample was transferred to a new 96-well plate, evaporated to dryness under N2 at room temperature, and reconstituted with 200 μl of chilled acetonitrile/0.1 M ammonium acetate (pH 4.0) buffer (50:50 v/v). A 10-μl aliquot was injected for analysis by LC-MS/MS.

Urine sample extraction for RSV was conducted in 96-well plates using protein precipitation with acetonitrile containing 0.5% formic acid. In brief, 50 μl of calibration standards, diluted QC samples, and diluted study samples were first mixed with 50 μl of chilled (ice/water bath) 0.1 M ammonium acetate (pH 4.5) buffer followed by 20 μl of chilled 100 ng/ml atorvastatin solution in acetonitrile/0.1 M ammonium acetate (pH 4.0) buffer (75:25 v/v). After addition of 300 μl chilled 0.5% formic acid in acetonitrile, vortex-mixing, and centrifugation at 4°C, an aliquot of 200 μl of each sample was transferred to a new 96-well plate. Chilled acetonitrile/0.1 M ammonium acetate (pH 4.0) buffer (200 µl) (50:50 v/v) was added and a 10-μl aliquot was injected for analysis by LC-MS/MS.

The HPLC system consisted of an Agilent 1200 pump, Agilent 1200 column oven (Agilent Technologies), and a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) maintained at 4°C during analysis. The analytic column used was a Shiseido CapCellPak MG C18 (2.0 × 50 mm, 5.0 μm) (Shiseido, Tokyo, Japan) and was maintained at 40°C. The mobile phase consisted of ammonium acetate pH 4.0 buffer (eluent A) and 100% acetonitrile (eluent B). The following gradient elution was used: start and maintain at 30% B from 0 to 0.5 minutes; ramp from 30 to 70% B from 0.5 to 1.5 minutes; hold at 70% B from 1.5 to 2.5 minutes; ramp to 30% B from 2.5 to 2.51 minutes; hold at 30% B until 3.7 minutes before the next injection. The flow rate was 0.4 ml/min. The retention times of RSV and ATV were 2.15 and 2.50 minutes, respectively.

The HPLC was interfaced to a Sciex API 5000 mass spectrometer (AB Sciex). The following positive ESI source/gas conditions were used: curtain gas at 25, ion spray voltage at 5500 V, temperature at 550°C, ion source gas 1 at 40, and ion source gas 2 at 50. The compound-dependent parameters used for RSV and ATV were, respectively, as follows: declustering potential of 80,80; collision energy of 48,30; collision cell exit potential of 14,14. The multiple reaction monitoring transitions used were as follows: m/z 482.2 → m/z 258.1 for RSV, and m/z 559.3 → m/z 440.2 for ATV.

To avoid the conversion of rosuvastatin-5S-lactone metabolite to RSV, plasma samples stored were thawed at 4°C (ice/water), and the aliquots for analysis were mixed with a pH 4.5 buffer. For the same reason, the acetonitrile used for protein precipitation and the reconstitution solution were also acidified. To avoid assay bias due to potential conversion of the metabolite in the mass spectrometer, the chromatographic conditions were optimized to achieve separation of the lactone metabolite from rosuvastatin. In addition, a special QC sample containing only rosuvastatin-5S-lactone (lactone-only QC) was included in every sample analysis run to gauge any conversion of the metabolite to RSV. The performance of this QC showed that the conversion was minimal (less than 5%) ensuring no contribution to the measured RSV concentrations.

LC-MS/MS Analysis of CsA in Blood.

Stock solutions of CsA (0.2 mg/ml) and of Cyclosporine A-13C2,d4 (1 mg/ml, internal standard) were prepared in methanol/acetone: (50:50 v/v). The concentrations of CsA calibration standards prepared in monkey whole blood were: 1, 2.5, 10, 25, 100, 400, and 1500 ng/ml. The QC samples were prepared in monkey whole blood at concentrations of 3, 40, 400, and 1200 ng/ml.

Whole blood sample extraction for CsA was conducted in polypropylene tubes using protein precipitation with methanol/ acetonitrile (50:50 v/v). In brief, 450 µl of methanol/ acetonitrile (50:50 v/v) was added into each polypropylene tube, mixed with 50 µl of CsA-13C2,d4 (internal standard) in methanol/ acetonitrile (50:50 v/v) followed by 50 µl sample. After vortex-mixing and centrifugation at room temperature, an aliquot of 200 μl supernatant of each sample was transferred to a 96-well plate, 200 μl of methanol/ acetonitrile (50:50 v/v) was added and mixed. A 20-μl aliquot was injected for analysis by LC-MS/MS.

The HPLC system consisted of Agilent 1200 HPLC pump, Agilent 1200 column oven and a CTC PAL autosampler maintained at 20°C. The analytic column used was a Venusil XBP-C8, 5 µm (2.1 mm I.D. × 50 mm) (Bonna-Agela Technologies, Wilmington, DE) and was maintained at 80°C. The mobile phase consisted of 0.2% acetic acid in water (eluent A) and 0.2% acetic acid in acetonitrile (eluent B). The following gradient elution was used: start at 40% B, ramp from 40% to 100% B from 0.0 to 1.2 minutes; hold at 100% B from 1.2 to 2.0 minutes; ramp to 40% B from 2.0 to 2.1 minutes; hold at 40% B until 2.8 minutes before the next injection. The flow rate was 0.6 ml/min. Retention times of CsA and CsA-13C2,d4 were 1.54 and 1.54 minutes, respectively.

The HPLC was interfaced to a Sciex API 5000 mass spectrometer (AB Sciex). The following positive ESI source/gas conditions were used: curtain gas at 20; ion spray voltage at 5500 V; temperature at 500°C; ion source gas 1 at 60; ion source gas 2 at 20. The compound-dependent parameters used for CsA and CsA-13C2,d4 were, respectively, as follows: declustering potential of 140,140; collision energy of 25,25; collision cell exit potential of 10,10. The multiple reaction monitoring transitions used were as follows: m/z 1219.45 → m/z 1202.70 for CsA, and m/z 1225.45 → m/z 1208.70 for CsA-13C2,d4.

Data Analysis.

Within the in vitro transporter studies, each assay was conducted in triplicate. The active transport of RSV by hOATP and cOATP was calculated after subtracting the uptake in mock-transfected cells from the total uptake in the transporter-expressing cells. IC50 values, the concentration of CsA required for 50% inhibition of transport of RSV, were calculated with WinNonlin (Pharsight, Mountain View, CA) using following equation:Embedded Imagewhere V is the rate of RSV transport measured at given CsA concentration, γ is the slope factor, C is the CsA concentration, V0 is the rate of RSV transport measured in the absence of CsA, and Imax is the maximum inhibitory effect.

For the inhibition studies employing hepatocytes, the uptake rate of RSV (Embedded Image pmol/min/106 cells), determined from time x to time y (where y = 0.25 minutes when x = 1.5 minutes; or y = 15 seconds when x = 90 seconds), was calculated using following equation:Embedded ImageThe IC50 values were then estimated by fitting V, the hepatic uptake at different CsA concentrations, to this equation using WinNonlin.

For the pharmacokinetic analysis, noncompartmental analysis of RSV and CsA concentration–time data was performed, and the following standard parameters were estimated using Kinetica (Thermo Fisher Scientific, Waltham, MA):Embedded Imagewhere CL/F and Vd/F are apparent clearance and volume of distribution after oral administration, MRT is mean residence time, CLR is renal clearance, and Xe, 0–48 h is the amount recovered in urine over 48 hours. The AUC from time 0 to 48 hours (AUC0–48 h) was calculated using the log-linear-trapezoidal method. The AUC from time 0 to infinity (AUC0–inf) was calculated as the sum of AUC0–48 h and Ct/λ, where λ is the apparent terminal rate constant and Ct is the predicted concentration at last time point. For each animal, λ was calculated by regression of the terminal log-linear portion of the plasma concentration-time profile, and the apparent terminal elimination half-life (t1/2) was calculated as the quotient of the natural log of 2 (ln [2]) and λ. The geometric mean ratio and its 90% confidence interval were calculated by calculating the arithmetic difference of the log mean and its associated 90% confidence intervals and then exponentiating these results back to the original (geometric mean ratio) scale. The t-distribution was assumed in the calculation of all.

Results

Excretion of Radioactivity in Bile, Urine, and Feces of Cynomolgus Monkeys.

Recovery of radioactivity in bile, urine and feces was complete (103.6% ± 27.5%) by 168 hours after administration of a single oral dose of [3H]RSV (3 mg/kg) to male BDC cynomolgus monkeys (Table 1). Fecal excretion was major and accounted for 61.7% ± 25.1% of the dose (Table 1). The balance was recovered in bile and in urine with 39.0% ± 3.0% and 2.9% ± 1.3% of the radioactive dose, respectively. Approximately 90% of the biliary excretion was complete between 0 and 24 hours with evidence of slower excretion beyond this time (0–24 hours versus 24–72 hours: 34.3% versus 4.7%). A majority of fecal excretion occurred by 72 hours. Although the urinary excretion was small compared with biliary excretion, it continued up to the final collection period (0–24 hours versus 24–72 hours versus 72–168 hours: 1.6% versus 0.8% versus 0.6%), indicating a slower rate of excretion for a fraction of the dose (Table 1).

View this table:
TABLE 1

Cumulative excretion of radioactivity in the bile, urine, and feces of bile duct–cannulated cynomolgus monkeys (n = 3) after a single oral administration of [3H]RSV

Data are reported as mean ± S.D., n = 3 different animals.

The radioactivity in the pooled bile and urine samples was analyzed by radio-HPLC. In the pooled bile collected from 0 to 72 hours, unchanged RSV was the predominant component, accounting for, on average, 62.8% of the sample radioactivity and 24.5% of the dose (Fig. 1A). In addition to RSV, several metabolites were detected which accounted for 26% of the sample radioactivity (Supplemental Figs. 1 and 2).

Fig. 1.
Fig. 1.

Representative radiochromatogram of pooled bile (A) and urine (B) from samples taken 72 and 168 hours after administration of a single oral dose of [3H]RSV at 3 mg/kg to bile duct–cannulated cynomolgus monkeys. Samples were profiled with HPLC. The retention time for RSV was approximately 32 minutes. The percentage (of the radioactivity collected in bile or urine samples) as parent RSV is indicated.

A similar chromatogram was obtained from the pooled urine samples, as unchanged RSV was the main radioactive compound accounting for 45.7% of the sample radioactivity (Fig. 1B). Metabolites accounted for 37.2% of the sample radioactivity, and additional trace peaks were detected compared with biliary chromatogram (Supplemental Figs. 1 and 3).

CsA as an Inhibitor of RSV Uptake Mediated by cOATP and cNTCP.

CsA was evaluated as an inhibitor of RSV (0.1 µM) uptake after incubation with HEK-293 cells containing individually expressed human and monkey OATP1B1 and OATP1B3 and monkey NTCP. In these studies, cells were preincubated with CsA (0.02–16.7 µM) at 37°C for 15 minutes before the addition of RSV. CsA inhibited uptake for hOATP1B1, hOATP1B3, cOATP1B1, cOATP1B3, and cNTCP with an estimated IC50 of 0.21 ± 0.10 µM, 0.13 ± 0.06 µM, 0.28 ± 0.11 µM, 0.25 ± 0.09 µM, and 3.9 ± 2.0 µM, respectively (Figs. 2 and 3B; Table 2). In addition, the expression of cNTCP was confirmed at a functional level by measuring the uptake of human NTCP model substrates (TCA, RSV, and ATV) into the HEK-293 cells transfected with cNTCP compared with the mock cells (Fig. 3A).

Fig. 2.
Fig. 2.

Effect of CsA on the uptake of RSV (0.1 µM) in hOATP1B1 (A), hOATP1B3 (B), cOATP1B1 (C), and cOATP1B3 (D). The transporter-mediated uptake was obtained by subtracting the transport rate during the first 2 minutes of incubation at 37°C in vector control cells from that in transporter-transfected cells. The data are expressed as a percentage of the initial uptake rate in the presence or absence of CsA. IC50 values were obtained by fitting the uptake data to the equation (see Materials and Methods) and are summarized in Table 2. Values shown are mean ± S.D. for experiments performed in triplicate.

Fig. 3.
Fig. 3.

(A) Uptake of [3H]TCA, [3H]RSV, and [3H]ATV in mock- and cNTCP‐HEK cells was determined after 5 minutes of incubation. Each value represents the mean ± S.D. (n = 3). ***P < 0.001, uptake was significantly different from mock HEK cells. (B) Effect of CsA on cNTCP-mediated uptake of RSV (0.1 µM) was determined by subtracting the transport rate during the first 2 minutes of incubation at 37°C in vector control cells from that in transporter-transfected cells in the presence of various concentrations of CsA. The data are expressed as a percentage of the initial uptake rate in the presence or absence of CsA. IC50 values were obtained by fitting the uptake data to either the equation (see Materials and Methods) and are summarized in Table 2. Values shown are mean ± S.D. for experiments performed in triplicate.

View this table:
TABLE 2

In vitro evaluation of CsA as an inhibitor of RSV uptake into hepatocytes and HEK-293 cells expressing individual transporter

Data are represented as mean ± S.D. (n = 3 determinations) (see Materials and Methods).

Parallel experiments were also conducted to evaluate the inhibitory effects of CsA on the uptake of 0.1 µM [3H]RSV in human and cynomolgus monkey hepatocytes (Fig. 4). Consistent with being a potent inhibitor of human and monkey OATPs using transporter-overexpressing cells, CsA demonstrated significant concentration-dependent inhibition of RSV uptake in human and monkey hepatocytes (maximal ∼80% inhibition) with IC50 values of 0.30 ± 0.08 µM and 0.29 ± 0.11 µM, respectively (Table 2). The IC50 values generated with hepatocyte suspensions were closer to the transfected OATP-derived values, which implies that RSV (0.1 µM) uptake is largely dominated by OATPs.

Fig. 4.
Fig. 4.

Effect of CsA on the uptake of [3H]RSV (0.1 µM) by human (A) and cynomolgus monkey hepatocytes (B). The data are expressed as a percentage of the initial uptake rate measured at 15 and 90 seconds at 37°C in the presence or absence of CsA. IC50 values were obtained by fitting the uptake data to the equation (see Materials and Methods) and are summarized in Table 2. Values shown are mean ± S.D. for experiments performed in triplicate.

Impact of CsA on RSV Pharmacokinetics in Cynomolgus Monkeys.

To assess the inhibitory effect of CsA on pharmacokinetics of RSV in vivo, a DDI study was conducted in male cynomolgus monkeys. The plasma concentrations of RSV were increased significantly when RSV was coadministered with CsA (Fig. 5A), and the pharmacokinetic results are summarized in Table 3. CsA increased the RSV AUC0–inf by 6.3-fold and Cmax by 10.2-fold relative to RSV administered alone. The mean CL/F and Vd/F for RSV alone are 784.6 ± 227.3 ml/min per kg and 594.0 ± 216.7 l/kg, respectively. The mean CL/F and Vd/F for RSV when coadministered with CsA were 129.8 ± 54.8 ml/min per kg and 48.7 ± 29.1 l/kg, respectively. The t1/2 of RSV decreased with coadministration of CsA (9.1 ± 3.1 hours versus 4.3 ± 1.3 hours; Table 3).

Fig. 5.
Fig. 5.

Mean plasma concentrations of RSV (A) and whole blood concentrations of CsA (B) after a single oral dose of RSV (3 mg/kg) alone (○) or with CsA (100 mg/kg p.o.) (● or ▴) in cynomolgus monkeys. Inset in A depicts same data on semilogarithmic scale. CsA was dosed 1 hour ahead of RSV. Data are expressed as mean ± S.D. (n = 3 animals).

View this table:
TABLE 3

Pharmacokinetics of RSV and CsA after a single oral dose of 3 mg/kg RSV with and without 100 mg/kg oral CsA in cynomolgus monkeys

Data reported as mean ± S.D., n = 3 different animals.

Urinary excretion of RSV was also increased in the presence of CsA (Fig. 6; Table 4). CsA increased the total amount of RSV excreted in urine Xe, 0–48 h by approximately 6-fold (214.1 ± 81.3 nmol versus 1234.8 ± 506.3 nmol). However, the renal clearance (CLR) of RSV did not change significantly with coadministered CsA (Table 4).

Fig. 6.
Fig. 6.

Effects of CsA on urinary exertion of RSV after single oral dose of RSV (3 mg/kg) alone or with CsA (100 mg/kg p.o.) in cynomolgus monkeys. The amount of RSV excreted in urine in the monkeys was determined for the RSV alone (open bar) and CsA-treated (closed bar) groups. CsA was dosed 1 hour ahead of RSV. Data are expressed as mean ± S.D. (n = 3 animals).

View this table:
TABLE 4

Urinary excretion of RSV after a single oral dose of 3 mg/kg RSV with and without 100 mg/kg oral CsA in cynomolgus monkeys

Data reported as mean ± S.D., n = 3 different animals.

At an oral dose of 100 mg/kg Neoral oral solution, CsA blood levels reached a peak of 1.1 ± 0.3 µM, with a tmax of 4.3 ± 0.4 hours (Fig. 5B). The AUC over a 49-hour period was 10.0 ± 3.0 µM⋅h, with an average concentration of 0.20 µM. The average concentration is comparable to the IC50 values of CsA obtained from monkey hepatocytes and cOATP-expressing HEK-293 cells. The CsA systemic exposures obtained in this study were comparable to those in patients at a therapeutic dose (Novartis, 2005) and in monkeys at 50 and 100 mg/kg (Schuurman et al., 2001).

Discussion

OATP1B-mediated DDIs are a major concern in drug development and clinical practice (International Transporter Consortium et al., 2010). OATP inhibition likely is not only dose dependent but also time dependent, so the extent and duration of inhibition is dynamic. As a result, a mechanistic static model will often overpredict a clinical DDI involving OATP inhibition, especially when assuming that the inhibitor concentration is represented by the maximum unbound concentration in the portal vein. The cynomolgus monkey model can be used to examine in vivo DDI, thus bridging the in vitro inhibitory potential to the extent of in vivo inhibition. However, the assumption is that the pharmacokinetics of probe substrate in monkeys is similar to that of human subjects. In recent studies, the monkey model has been successfully applied by investigators to quantitatively predict transport-based DDIs (Shen et al., 2013; Takahashi et al., 2013). The objective of our current study was to evaluate absorption, metabolism, and excretion of RSV in cynomolgus monkeys (BDC study), and in vitro and in vivo inhibitory effects of CsA on the transport of RSV, a probe substrate most commonly used in clinical DDI studies.

Given that the prediction of DDI of many OATP1B substrates, including RSV, is complex, requiring input parameters of fraction eliminated via each pathway, we evaluated the disposition profile of RSV in BDC monkeys using [3H]RSV. Recovery of radioactivity was complete after 168 hours after dosing. Excretory profiles of radioactivity in the urine, bile, and feces suggested that fecal excretion followed by biliary elimination was the major route of elimination of drug-derived radioactivity (61.7% and 39.0% of dose, respectively; Table 1). Metabolite profiles in urine and bile were qualitatively similar. Unchanged RSV was detected as the major component in both pooled bile and urine from monkeys (62.8% and 45.7% of the radioactivity were collected in bile and urine, respectively; Fig. 1), indicating that RSV did not undergo extensive metabolism before excretion in monkeys. The radioactivity in plasma was too low to examine the metabolite profile. Unfortunately, the metabolic profile of fecal samples was not examined.

If we assume no significant intestinal secretion and gastrointestinal metabolism, the total unchanged RSV recovered in bile and feces would be 86.2% of the dose administered in BDC monkeys. This is in agreement with what is observed in intact cynomolgus monkeys and humans (75.0% and 76.8%, respectively; Table 5) (Martin et al., 2003b; http://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/21-366_Crestor_Pharmr_P2.pdf). The extent of intestinal secretion of RSV is known to be low in dogs, with 3.3% of the radioactive dose found in the feces collected from 0 to 72 hours after intravenous administration of 5 mg/kg [14C]RSV to BDC dogs (http://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/21-366_Crestor_Pharmr_P2.pdf). The percentage of RSV absorbed in the BDC monkeys, estimated by adding the total radioactivity in bile and urine, was at least 41.9%, suggesting that the compound is reasonably absorbed in monkeys. This is comparable to the oral absorption fraction estimate of approximately 50% in humans (Table 5) (Martin et al., 2003a; http://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/21-366_Crestor_Pharmr_P2.pdf). Putting these findings together, the similarities observed in absorption, metabolism, and excretion properties between cynomolgus monkeys and humans suggest that the monkey is a suitable surrogate animal model for further preclinical pharmacokinetic studies for RSV.

View this table:
TABLE 5

Percentage of dose recovery in monkeys and humans after oral administration of radiolabeled RSV

Previously we reported that the cynomolgus monkey is an effective model to assess investigational drugs for OATP interaction in humans. Moreover, we provided evidence that RSV-rifampicin (RIF) is an OATP1B probe substrate/reference inhibitor combination applicable in several assay systems in vitro and in vivo (Shen et al., 2013). In the present study, we extended the application of the cynomolgus monkey model by validation of RSV-CsA as OATP1B probe substrate/reference inhibitor combination because CsA is commonly used as a clinical OATP1B inhibitor.

The potential hepatic transporter-mediated DDI between RSV with CsA was first investigated in monkey and human OATP1B and NTCP recombinant systems. CsA inhibited RSV uptake mediated by hOATP1B1 and cOATP1B1, with IC50 values of 0.21 ± 0.10 and 0.28 ± 0.11 μM, respectively (Fig. 2, A and C; Table 2). CsA also inhibited RSV uptake mediated by hOATP1B3 and cOATP1B3, in a similarly potent manner, with IC50 values of 0.13 ± 0.06 and 0.25 ± 0.09 μM, respectively (Fig. 2, B and D; Table 2). Moreover, CsA decreased the RSV uptake mediated by cNTCP in a concentration-dependent manner, with an IC50 value of 3.9 ± 2.0 μM, which is comparable to that of human NTCP generated from 55 different test occasions at Bristol-Myers Squibb (Fig. 3B; Table 2).

Furthermore, the potential hepatic transporter-mediated DDI between RSV with CsA was investigated in monkey and human hepatocyte inhibition assays. CsA significantly reduced uptake of RSV in both human and monkey hepatocytes in a concentration-dependent manner, with IC50 values of 0.30 ± 0.08 and 0.29 ± 0.11 μM, respectively (Fig. 4; Table 2). These data are in agreement with the recombinant data, which showed that CsA inhibited the hOATP1B- and cOATP1B-mediated transport of RSV, with IC50 ranging from 0.13 to 0.28 µM.

No attempt was made to study OATP2B1 inhibition by CsA because previous studies suggested that OATP2B1 is less likely to play a significant role in RSV disposition in both species. Moreover, CsA was a weak inhibitor of both monkey and human OATP2B1 (Shen et al., 2013). Similarly, Prueksaritanont et al. (2014) have shown via RIF inhibition and DDI data that human OATP2B1 contributed minimally to the hepatic uptake of RSV.

Using in vitro transporter inhibition studies as screening tools to evaluate the potential for DDI in vivo is based on the assumption that the victim drugs analyzed share the same transport kinetics between cynomolgus monkey and human OATP1B. Previous concentration-dependent transport studies using stably transfected HEK-293 cells, expressing individual monkey and human OATP1B1, OAPT1B3, and OATP2B1, indicated that the RSV apparent Michaelis–Menten constant Km (9.6–15.3 µM) is comparable across the three transporters (Shen et al., 2013). The transport kinetics of RSV in hepatocytes has been examined also. In this instance, the kinetics of RSV uptake were best described by a mixed model consisting of both a single saturable process and a passive component, yielding Km of 6.7 and 10.3 µM for monkey and human, respectively, which agreed well with the Km values obtained from the recombinant systems. These studies suggest no species difference in the transport kinetics of RSV in monkey and human OATP1B1-, OATP1B3-, and OATP2B1-overexpressing cells and hepatocytes (Shen et al., 2013).

Coadministration of CsA with RSV markedly increased the AUC0–inf and Cmax of RSV in the cynomolgus monkey by 6.3-fold and 10.2-fold, respectively (Table 3). After oral administration of 100 mg/kg CsA, blood concentrations of CsA in the range of 1.1 to 0.05 μM were achieved for the first 25 hours (the first 24 hours after RSV administration) (Fig. 5B). The Cmax of CsA after a single oral dose in monkeys was comparable to that at steady state after multiple dosing in patients (1.1 versus 1.0 µM) (Novartis, 2005).

Interestingly, the impact of CsA on RSV in monkeys appeared to be identical to that in heart transplant patients taking CsA (7.1- and 10.6-fold for AUC and Cmax, respectively) (Simonson et al., 2004). This finding, although not unanticipated, suggests the cynomolgus monkey is an appropriate model for the assessment of OATP-mediated DDIs in a nonclinical setting. It is not clear, however, whether this can be extended to other substrates of these transporters. First, although cynomolgus monkey OATPs share a high degree of amino acid sequence identity and functional similarity to their human counterparts, subtle amino acid differences can greatly impact substrate specificity. For example, DeGorter et al. (2012) reported that site-directed mutagenesis of three amino acid residues in OATP1B1 transmembrane domains 1 and 10, and extracellular loop 6 to the corresponding residues in OATP1B3 resulted in a gain of CCK-8 transport by OATP1B1, which is a high-affinity substrate for OATP1B3 but not OATP1B1. Second, this in vitro–in vivo extrapolation approach only makes a reasonable prediction if the relative contribution of OATP-mediated uptake clearance to the total body clearance and ADME profiles are well understood in both species. The BDC monkey study indicated that RSV is an appropriate in vivo probe for OATP-mediated DDI study in monkeys. These conclusions likely extend to pitavastatin. Takahashi et al. (2013) reported that the magnitude of hepatic OATP DDI was comparable between the monkey study and the clinical study by using pitavastatin as a substrate. They concluded that pharmacokinetic studies using pitavastatin as a probe in combination with drug candidates in cynomolgus monkeys are useful to support the assessment of potential clinical DDIs involving hepatic uptake transporters.

RSV has been shown in vitro to be a substrate of transporters other than OATP1B1, OATP1B3, and OATP2B1. For example, it has been reported that human NTCP plays an important role in hepatic uptake of RSV in human hepatocytes (Ho et al., 2006; Bi et al., 2013), although the relevance of this transporter in vivo has yet to be confirmed. In addition, BCRP and OAT3 (organic anion transporter 3) may play a role in RSV intestinal absorption and renal elimination, respectively (Yoshida et al., 2012). In the case of the former, patients expressing the ABCG2 variant 421C>A, a single-nucleotide polymorphism (SNP) associated with reduced efflux activity in vitro, showed a 140% increase in RSV exposure (Keskitalo et al., 2009). This implies that inhibition of intestinal BCRP can also bring about increased RSV exposure. Therefore, the impact on RSV pharmacokinetics will likely be determined by inhibition of OATPs, NTCP, BCRP, and OAT3. Consideration of OAT3 has been ruled out in this instance, because CsA did not impact the renal clearance of RSV (Table 4).

In our present study, we report for the first time that CsA is a potent inhibitor of cOATPs and cNTCP (IC50 values of ∼0.3 µM and 3.9 µM, respectively; Table 2). In addition, CsA inhibits these monkey hepatic transporters to a similar extent compared with the human counterparts. Consistent with human data also is the marked effect of CsA on RSV exposure when compared with RIF (6.3- to 7.1-fold versus 2.9- to 4.4-fold increase) (Simonson et al., 2004; Shen et al., 2013). Such results are consistent with the fact that DDI studies with CsA have been accepted by regulatory agencies as the worst-case scenario for substrates of transporters. Although RIF is also a potent inhibitor of OATPs (IC50: 0.42–1.69 µM), it is a weaker inhibitor of NTCP (IC50 of 277 μM versus 3.9 μM) and BCRP (IC50 of 14 µM versus ∼7 µM) (Nezasa et al., 2002b; Shen et al., 2013; Prueksaritanont et al., 2014), so it is hypothesized that the extent of inhibition of liver basolateral NTCP and OATP, and intestinal apical BCRP, is greater for CsA (versus RIF) in monkeys and humans. In addition, the decreased hepatic uptake could impact the extent of metabolism of RSV.

In summary, an in vitro–in vivo assay system previously used to evaluate the interaction between RIF with RSV in cynomolgus monkeys was extended to include CsA. Here we provided further evidence that the disposition of radiolabeled RSV in cynomolgus monkeys is comparable to that in humans after an oral dose. In addition, the magnitude of the DDI between CsA and RSV is similar to that reported clinically. Although additional transporter data are needed for monkey BCRP, the results described herein do suggest that RSV can be a useful substrate for probing OATP-mediated pharmacokinetics and DDIs in humans and monkeys.

Acknowledgments

The authors thank Dr. Lisa Elkin for providing IC50 values of CsA against hNTCP, which are generated from 55 different test occasions at Bristol-Myers Squibb.

Authorship Contributions

Participated in research design: Shen, Su, Mintier, Iyer, Marathe, Lai, Rodrigues.

Conducted experiments: Shen, Su, Liu, Yao, Mintier, Li.

Contributed new reagents or analytic tools: Shen, Liu, Mintier, Fancher.

Performed data analysis: Shen, Su, Liu, Mintier, Lai, Rodrigues.

Wrote or contributed to the writing of the manuscript: Shen, Marathe, Lai, Rodrigues.

Footnotes

Abbreviations

ADME
absorption, distribution, metabolism, and excretion
ATV
atorvastatin
AUC
area under the concentration-time curve
BDC
bile duct cannulated
CsA
cyclosporin A
DDI
drug–drug interaction
ESI
electrospray ionization
HBSS
Hanks’ buffered saline solution
HEK
human embryonic kidney
HPLC
high-performance liquid chromatography
LC-MS/MS
liquid chromatography–tandem mass spectrometry
NTCP
sodium-taurocholate cotransporting polypeptide
OATP
organic anion transporting polypeptide
PCR
polymerase chain reaction
QC
quality control
RIF
rifampin
RSV
rosuvastatin
TCA
taurocholic acid

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Research ArticleMetabolism, Transport, and Pharmacogenomics

Use of Cynomolgus Monkey to Assess DDIs Involving OATPs

Hong Shen, Hong Su, Tongtong Liu, Ming Yao, Gabe Mintier, Lun Li, R. Marcus Fancher, Ramaswamy Iyer, Punit Marathe, Yurong Lai and A. David Rodrigues
Journal of Pharmacology and Experimental Therapeutics May 1, 2015, 353 (2) 380-391; DOI: https://doi.org/10.1124/jpet.114.221804
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