Evaluation of Rosuvastatin as an Organic Anion Transporting...

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

Pharmaceutical Candidate Optimization (H.Shen., H.Su, T.L., M.Y., R.M.F., R.I., P.M., Y.L.,

A.D.R.), Genomic Technologies (G.M.), Bristol-Myers Squibb Research and Development,

Princeton, NJ; Department of Bioanalytical Service (L.L.), WuXi AppTec Co., Ltd, Shanghai,

China

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 4, 2015 as DOI: 10.1124/jpet.114.221804

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Running title: Use of Cynomolgus Monkey to Assess DDIs Involving OATPs

Corresponding author: Hong Shen, F1.3802, Route 206 & Province Line Road, Bristol-Myers

Squibb Company, Princeton, NJ 08543-4000. Telephone: (609) 252-4509; Facsimile: (609) 252-

6802; E-mail: [email protected]

Number of text pages: 31

Number of tables: 5

Number of figures: 6

Number of references: 32

Number of words:

Abstract: 250

Introduction: 986

Discussion: 1,886

Abbreviations: ADME, absorption, distribution, metabolism, and excretion; ATV, atorvastatin;

AUC, area under the concentration-time curve; BDC, bile duct cannulated; Cmax, maximum

plasma concentration; cNTCP, cynolmogus monkey sodium-taurocholate cotransporting

polypeptide; cOATP, cynomolgus organic anion transporting polypeptide; CsA, cyclosporin A;

CYP, cytochrome P450; DDI, drug-drug interaction; fu, fraction of unbound drug; HBSS, Hank’s

buffered saline solution; HEK, human embryonic kidney-293; HEPES, N-2-

hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HMG-CoA, 3-hydroxy-3-methylglutaryl-

coenzyme; hNTCP, human sodium-taurocholate cotransporting polypeptide; hOATP, human


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organic anion transporting polypeptide; HPLC, high-performance liquid chromatography; IC50,

concentration required to inhibit transport by 50%; IS, internal standard; ITC, International

Transporter Consortium; IVIVE, in vitro-in vivo extrapolation; Ki, inhibitory constant; Km,

Michaelis-Menten constant that corresponds to the substrate concentration at which the uptake

rate is half of Vmax; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LSC, liquid

scintillation counting; PCR, polymerase chain reaction; RIF, rifampin; RSV: rosuvastatin; Vmax,

maximum transport rate.

Recommended section: Metabolism, Transport and Pharmacogenomics


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Abstract

Organic anion transporting polypeptides (OATP) mediate hepatic drug uptake and serve as the

loci of drug-drug interactions (DDI). Consequently, there is a major need to develop animal

models and refine 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. Following a 3 mg/kg oral dose, mass balance was achieved following bile duct

cannulation (mean total recovery of radioactivity of 103.6%). Forty-two % 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)

towards cynomolgus monkey OATPs and sodium-taurocholate co-transporting polypeptide

(NTCP) was studied in vitro (primary hepatocytes and transfected transporters). 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 vs. human) and the increase (vs. vehicle control) in 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 cynomolgus monkey as a model to assess

interactions involving OATP inhibition.


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Introduction

Rosuvastatin (RSV) is a 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase

inhibitor (i.e., statin) used in the treatment of patients with hypercholesterolemia. DDIs involving

RSV that result in an increase in its systemic exposure might result in unwanted side effects

including myopathy and rhabdomyolysis. Such DDIs are clinically significant when one

considers that known transporter inhibitors like 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 (Martin et al., 2003b; Nezasa et al., 2002b) and is selectively transported and

distributed into the liver (Martin et al., 2003a; Martin et al., 2003b; Nezasa et al., 2002a; Nezasa

et al., 2002b), the overall disposition profile of oral RSV is thought to be highly dependent on

drug transporters such as OATP, NTCP, and breast cancer resistance protein (BCRP) (Bi et al.,

2013; Ho et al., 2006; Keskitalo et al., 2009; Kitamura et al., 2008; Pasanen et al., 2007). It has

been established that hepatic clearance and renal clearance are the primary pathways for the

elimination of RSV, account 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 to NTCP (Bi et al., 2013; Ho et al., 2006;

Kitamura et al., 2008). 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


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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

(Prueksaritanont et al., 2014; Schneck et al., 2004; Simonson et al., 2004), monkeys (Shen et al.,

2013), mice (Salphati et al., 2014), rats (Wen and Xiong, 2011), and pigs (Bergman et al., 2009).

When compared to rodents, however, the absorption, distribution, metabolism, and excretion

(ADME) profile of RSV in non-human primates has not been studied extensively (Martin et al.,

2003b; Nezasa et al., 2002b).

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 to those obtained in humans, reducing the utility

of such animals (Li et al., 2013; Shirasaka et al., 2010; Shitara et al., 2003). In fact, there is only

one member of the OATP1B family, Oatp1b2, which 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 dog, cow and horse suggested that there is only a single

Oatp in the 1B family, while cynomolgus monkey, a species much closer related to humans, both


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cOATP1B1 and cOATP1B3 orthologs have been cloned. (Shen et al., 2013). These results

indicate that, for many drugs, cynomolgus monkey may represent an acceptable in vitro and in

vivo model to investigate OATPs.

Several reports have described various rodent models, which can be used to support the

investigation of OATPs in vivo. For example, it is possible to delete the murine Oatp1b2 gene

(Chang et al., 2014; Lu et al., 2008; Zaher et al., 2008), introduce human OATP genes (van de

Steeg et al., 2009), and develop combinations of OATP knockout and knockin (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 (Iusuf et al., 2014;

Klaassen and Lu, 2008; Salphati et al., 2014). In addition, quantitative translation of data

obtained with knockout and/or knockin animals can be challenging. Indeed unexpected results

have been observed in mice and rats compared to 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 the humanized mice

did not significantly alter the liver or plasma concentration ratios of RSV and pitavastatin

compared to 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.

As described herein, the ADME of RSV was studied after administration of oral [3H]RSV

(3 mg/kg) to BDC cynomolgus monkeys. Because RSV has been shown to be a substrate of

human OATPs and sodium-taurocholate co-transporting polypeptide (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 human and


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cynomolgus monkey, and that the monkey can be used as a model to assess inhibition of OATP

both in vitro and in vivo.


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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 Inc. (Toronto, ON, Canada), CsA oral solution (Neoral, 100 mg/mL) was

purchased from Novartis Pharmaceuticals Corporation (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). 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, Inc (Manassas, VA).

Cryopreserved male human and cynomolgus hepatocytes were purchased from

BioreclamationIVT (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, Inc. (Wilmington, MA). All monkey excreta, bile, blood and

plasma were collected gravimetrically and stored at -20°C until analysis.

BDC Cynomolgus Monkey Study. All animal studies were performed under the

standards recommended by the Guide for the Care and Use of Laboratory Animals (Institute of

Laboratory Animal Resources, 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, Inc.) 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


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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 hr after dosing, animals were fed Certified

Primate Diet 5048 (PMI Nutrition International, Inc.). Bile was collected before dosing and from

0 to 8, 8 to 24, and 24 to 72 hr after dosing into containers that were surrounded by dry ice. Urine

and feces were collected before dosing and over 24 hr intervals through 168 hr postdose. Blood

samples were collected into tubes containing K2EDTA from the femoral artery before dosing and

at 1, 2, 6, 12, 24, and 48 hr 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 Biological Matrices from BDC Cynomolgus

Monkeys. Plasma, urine, bile and fecal samples were analyzed for radioactivity concentration

using Liquid Scintillation Counting (LSC). Portions of plasma (50-100 µL), urine (50 µL) and

bile (10 µL) were mixed with 5 mL Ecolite scintillation cocktail (PerkinElmer Life and

Analytical Sciences, Waltham, MA) 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 Soluene-350 and shaken slowly at room temperature overnight. The solubilized

homogenate mixtures were bleached with 1 mL 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 EcoliteTM 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, Inc., Fullerton, CA) for 5

min.


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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

14,000 g for 5 min. A portion of supernatant (25-50 µL) was injected into the HPLC for

biotransformation profiling and mass spectral analysis.

HPLC analysis was conducted on an Agilent 1290 Infinity LC system (Agilent

Technologies, Palo Alto, CA) interfaced to a Linear Ion Trap (LTQ) 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 min, 20% B; 40 min, 25% B; 47 min,

50% B; 50 min, 90% B; and 60 min, 20%. 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

LTQ mass spectrometer for profiling. The remaining 75% of the eluate was collected into

ScintiPlate™-96-well plates at 0.2-min intervals per well. EcoliteTM scintillation cocktail (200

µL) was then added to each well, and the radioactivity was counted for 20 min per well with a

PerkinElmer 1450 MicroBeta Wallac TRILUX Liquid Scintillation and Luminescence Counter

(PerkinElmer Life and Analytical Sciences, Waltham, MA). 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 in the positive ion

mode. Capillary temperature was 300°C and the electrospray ionizing voltage was maintained at

4.5 kV for all analyses. The collision energy was 15.0% for liquid chromatography-tandem mass


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spectrometry (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 HEK-293 cells with cynomolgus monkey NTCP (cNTCP)

were carried out as described previously (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 cynomolgus NTCP 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), 5’-AAG GGC TAG GCT GTG CAA GG-3’ (reverse primer,

reverse complementary to positions 1170-1189). PCR products were cloned into pJet1.2

(Fermentas) and several clones for each cDNA were sequenced. Sequences were deposited to

GenBank: cynomolgus monkey NTCP (ACC# KP453714). The coding sequence of the cDNA

was subcloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA) using

standard methods. The Gateway entry clones were recombined into a Gateway adapted version

of the expression vector pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA) using LR clonase II

(LifeTechnologies, 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 Dulbeccos’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 (Shen et al., 2013). After washing with serum-free

medium, the culture well was incubated at 37 °C for 4 h with 1 mL serum-free medium which


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contained 10 µL Lipofectamine 2000 (Invitrogen; Carlsbad, CA) 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; Carlsbad, CA). Expression of

cNTCP was verified by RT-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 (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 sub-cultured once a week. Cells were

seeded in 24-well poly-D-lysine coated plates at a density of 5 x 105 cells per well, and were

ready for experiment after 48 to 72 h. Before uptake experiments, cells were washed twice with

1.5 ml of pre-warmed Hank’s buffered saline solution (HBSS). The cells were then preincubated

with uptake buffer (HBSS with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

(HEPES), pH 7.4) containing CsA only (0.023 to 16.7 µM), at 37 °C for 15 min. Subsequently,

the cells were incubated with the test solution, containing CsA and the substrate RSV (0.1 µM) at

37 °C for 2 min. Over this time period, linearity of uptake has been confirmed (unpublished

data). Cells were washed three times with 1 ml of ice-cold HBSS buffer and lysed with 0.3 ml of

0.1% Triton X-100. Intracellular accumulation of RSV was determined using LC-MS/MS as

described later. Assessment of uptake of [3H]TCA, [3H]RSV, and [3H]atorvastatin (ATV) into

HEK-293 cells expressing cNTCP involved a 5 min 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 measured

using a bicinchoninic acid assay (BCA Protein Assay Kit; Pierce Chemical, Rockfold, IL).


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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 min

incubation at room temperature, the samples were transferred by a Tecan liquid handler 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, Rockfold, IL). The lysed samples (150 µL) were

mixed with 200 µL of acetonitrile containing d6-rosuvastatin, the internal standard (IS), 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, votexed and analyzed by LC-MS/MS. Cellular uptake was

normalized to the protein content.

The liquid chromatography system was the Shimadzu SCL 30AD Nexera comprised of

two LC-30AD pumps and a Shimadzu SIL-30AC autosampler. 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, Santa Clara, CA, USA). 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 min to 0.01 min,

keep B for 20%, from 0.01 min to 2.0 min, increase B linearly from 20% to 60%; from 2.0 min

to 2.1 min, increase B linearly from 60% to 95%, maintain 95% B for 0.4 min, from 2.5 to 2.6

min, decrease B from 95% to 20%. Then equilibrate column for 0.9 min for next injection. The

retention time for rosuvastatin and d6-rosuvastatin was 1.86 min. An AB Sciex Qtrap 6500

system (Applied Biosystems/MDS Analytical Technologies, Foster City, CA, USA) equipped

with electrospray ionization source was used for mass spectrometric detection. AnalystTM


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version 1.62 was used as the data acquisition software. The electrospray ionization 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 ms; source

temperature 400 ºC; 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 X100 introduced into the MS

source. At 0 to 0.8 min, the ultra performance liquid chromatography eluant was diverted to

waste and switched to MS source after one min. After 3.0 min, 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 (KHB) (BioreclamationIVT; Baltimore, MD). 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 2x106 viable cells/mL in

InVitroGRO KHB. 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 min. Uptake studies


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were initiated by an addition of 80 µL prewarmed buffer containing [3H]RSV (0.2 µM). Aliquots

(100 µL) of the mixture were removed at 15 and 90 sec, and then transferred to centrifuge tubes

and layered carefully on top of an oil layer. The tubes were centrifuged immediately at 13,000xg

for 15 sec. Following 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 h at an ambient temperature, and frozen in a -80°C freezer or on dry ice just prior to

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 150 µL 2N 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, China) using 3 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-h light/dark cycle. Animals were fed approximately 120 grams of Certified

Monkey Diet daily (Beijing Vital Keao Feed Corporation; Beijing, China). The monkeys were

fasted for a 12 h prior to dose administration, and water was made available ad lib. In the first

period, 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. Following a 1-week washout period, the

same animals were administered an equivalent oral dose of RSV one hour after CsA

administration (100 mg/kg, PO; 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 milliliter 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


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48 h 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 (3,000 x g for 10 min at 2 to 8°C) within one 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 hs post-dose into containers

cooled by wet ice. Total volume of urine samples for different periods were measured at the end

of collection and recorded. Aliquots (5mL) 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). Stock solution

(1 mg/mL) of rosuvastain-5S-lactone, a metabolite of RSV, was prepared in acetonitrile/0.1M

ammonium acetate pH 4.0 buffer (50:50 v/v) and stock solution (0.2 mg/mL) of atorvastatin (IS)

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)

0.1M ammonium acetate buffer (pH 4.5) followed by 20 μL of chilled 100 ng/mL atorvastatin


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solution in acetonitrile / 0.1M ammonium acetate pH 4.0 buffer (75:25 v/v). Following addition

of 300 μL 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 RT, and reconstituted with 200 μL chilled acetonitrile /0.1M 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) 0.1M ammonium acetate buffer (pH 4.5) followed by 20 μL of chilled 100

ng/mL atorvastatin solution in acetonitrile/0.1M ammonium acetate pH 4.0 buffer (75:25 v/v).

Following 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/0.1M 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, Santa Clara, CA) and a CTC PAL autosampler (CTC Analytics AG,

Zwingen, Switzerland) maintained at 4°C during analysis. The analytical column used was a

Shiseido CAPCELLPAK MG C18 (2.0 x 50 mm, 5.0 μm) (Shiseido Co., 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 min; ramp from 30 to 70% B from 0.5 to 1.5 min; hold at 70% B from 1.5

to 2.5 min; ramp to 30% B from 2.5 to 2.51 min; hold at 30% B until 3.7 min before the next


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injection. The flow rate was 0.4 mL/min. The retention times of RSV and atorvastatin were 2.15

and 2.50 min, respectively.

The HPLC was interfaced to a Sciex API 5000 mass spectrometer (MDS Sciex, Concord,

ON). The following positive electrospray source/gas conditions were used: curtain gas at 25; ion

spray voltage at 5500; temperature at 550°C; ion source gas 1 at 40; ion source gas 2 at 50. The

compound-dependent parameters used for RSV and atorvastatin were, respectively, as follows:

decluster-ing 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 atorvastatin.

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

rosuvastain-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 (0.2mg/mL) and of

Cyclosporine A-13C2,d4 (1 mg/mL, IS) 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,


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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 (IS) 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 analytical column used was a Venusil

XBP-C8, 5 µm (2.1 mm I.D. x 50 mm) (Agela) 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 min; hold at 100% B from 1.2 to 2.0 min; ramp to 40% B from 2.0 to 2.1 min; hold at 40%

B until 2.8 min 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 min, respectively.

The HPLC was interfaced to a Sciex API 5000 mass spectrometer (MDS 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


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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 Inc.; Mountain View, CA) using following equation:

where V is defined as the rate of RSV transport measured at given CsA concentration, γ is the

slope factor, C is the CsA concentration, V0 refers to the rate of RSV transport measured in the

absence of CsA, and Imax refers to the maximum inhibitory effect.

For the inhibition studies employing hepatocytes, the uptake rate of RSV (V(x-y);

pmol/min/106cells), determined from time x to time y (where y = 0.25 min when x = 1.5 min; or y

= 15 sec when x = 90 sec), was calculated using following equation:

IC50 values were then estimated by fitting V, the hepatic uptake at different CsA

concentrations, to the equation above using WinNonlin.

⎟⎟⎠

⎞⎜⎜⎝

⎛

+•

−=λγ

γ

CIC

CIVV

50

max0

( )yx

VVV yx

−−

=

y)-x(


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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):

where 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 hr is amount recovered in urine

over 48 hr. AUC(0-48 hr) was calculated using the log-linear-trapezoidal method. AUC(0-inf) was

calculated as the sum of AUC(0-48 hr) 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 (GMR) and its 90% confidence interval (CI) were

calculated by calculating the arithmetic difference of the log means and its associated 90% CIs

and then exponentiating these results back to the original (GMR) scale. The t-distribution was

assumed in the calculation of all.

hr) 48(0-

hr 480- e,R

inf)0(d

inf)0(

MRTDose /F

Dose /F

AUC

XCL

AUCV

AUCCL

=

•=

=

−

−


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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 hr vs. 24-72

hr: 34.3% vs. 4.7%). A majority of fecal excretion occurred by 72 hours. Although the urinary

excretion was small compared to biliary excretion, it continued up to the final collection period

(0-24 hr vs. 24-72 hr vs. 72-68 hr: 1.6% vs. 0.8% vs. 0.6%), indicating a slower rate of excretion

for a fraction of the dose (Table 1).

The radioactivity in the pooled bile and urine samples was analyzed by radio-HPLC. In

the pooled bile collected from 0 to 72 hr, unchanged RSV was the predominant component,

accounting for, on average, 62.8% of the sample radioactivity and 24.5% of the dose (Figure

1A). In addition to RSV, several metabolites were detected which accounted for 26% of the

sample radioactivity (Supplemental Figures 1 and 2). 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 (Figure 1B). Metabolites accounted for 37.2%

of the sample radioactivity and additional trace peaks were detected compared to biliary

chromatogram (Supplemental Figures 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


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containing individually expressed human and monkey OATP1B1 and OATP1B3 and monkey

NTCP. In these studies, cells were preincubated with CsA (0.02 to 16.7 µM) at 37°C for 15 min

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 (Figures 2 and 3A; 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 to the mock cells (Figure 3B).

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 (Figure 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.

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 co-administered with CsA (Figure 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/kg and 594.0 ± 216.7 L/kg, respectively. The mean CL/F and Vd/F for RSV when co-


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administered with CsA were 129.8 ± 54.8 mL/min/kg and 48.7 ± 29.1 L/kg, respectively. The t1/2

of RSV decreased with co-administration of CsA (9.1 ± 3.1 hr vs. 4.3 ± 1.3 hr; Table 3).

Urinary excretion of RSV was also increased in the presence of CsA (Figure 6; Table 4).

CsA increased total amount of RSV excreted in urine Xe, 0-48 hr by approximated 6-fold (214.1 ±

81.3 vs. 1234.8 ± 506.3 nmol). However, the renal clearance (CLR) of RSV did not change

significantly with co-administered CsA (Table 4).

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 hr (Figure 5B). The AUC over a 49-hr period was 10.0 ±

3.0 µM•hr, 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 (Noartis, 2005) and in monkeys at 50 and 100 mg/kg (Schuurman et al.,

2001).


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Discussion

OATP1B-mediated DDIs are a major concern in drug development and clinical practice

(International Transporter et al., 2010). OATP inhibition likely is not only dose-dependent but

also time-dependent, therefore, the extent and duration of inhibition is dynamic. As a result, a

mechanistic static model will often over-predict 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 the 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 h postdose. 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


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component in both pooled bile and urine from monkeys (62.8% and 45.7% of the radioactivity

were collected in bile and urine, respectively; Figure 1), indicating that RSV did not undergo

extensive metabolism before excretion in monkeys. The radioactivity in plasma was too low to

examine metabolite profile. Unfortunately, the metabolic profile of fecal samples was not

examined. If we assume no significant intestinal secretion and gastrointestinal (GI) 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) (US FDA, 2003; Martin et al., 2003b). The

extent of intestinal secretion of RSV is known to be low in dogs, with 3.3% of radioactive dose

was found in the feces collected from 0 to 72 hr after intravenous (IV) administration of 5 mg/kg

[14C]RSV to BDC dogs (US FDA, 2003). 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) (US FDA, 2003; (Martin

et al., 2003a). Putting these findings together, the similarities observed in absorption, metabolism

and excretion properties between cynomolgus monkey and human suggest that monkey is a

suitable surrogate animal model for further preclinical pharmacokinetic studies for RSV.

Previously, we have reported that 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 since CsA is commonly used as a


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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 (Figure 2A and 2C; Table 2). CsA also inhibited RSV

uptake mediated by hOATP1B3 and cOATP1B3, in a similar potent manner, with IC50 values of

0.13 ± 0.06 and 0.25 ± 0.09 μM, respectively (Figure 2B and 2D; Table 2). Moreover, CsA

decreased RSV uptake mediated by cNTCP in concentration-dependent manner with 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 (Figure 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 (Figure 4 and 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 ranged 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. have

shown that human OATP2B1 contributed minimally to the hepatic uptake of RSV by RIF

inhibition and DDI data (Prueksaritanont et al., 2014).

Using in vitro transporter inhibition studies as screening tools to evaluate 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


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transport studies using stably transfected HEK-293 cells, expressing individual monkey and

human OATP1B1, OAPT1B3, and OATP2B1, indicated that the RSV apparent Km (9.6 to 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 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).

Co-administration of CsA with RSV markedly increased AUC0-inf and Cmax of RSV in

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 hr after CsA administration (the first 24 hr after RSV administration) (Figure 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 vs. 1.0 µM). Interestingly, the impact of CsA on RSV in

monkeys appeared to be identical to that in heart transplant patients taking CsA (7.1-fold and

10.6-fold for AUC and Cmax, respectively) (Simonson et al., 2004). This finding, although not

unanticipated, suggests cynomolgus monkey is an appropriate model for the assessment of

OATP-mediated DDIs in a nonclinical setting. It is not clear however, if 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

and colleagues reported that site-directed mutagenesis of 3 amino acid residues in OATP1B1


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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 (DeGorter et al., 2012). Second, this IVIVE 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. The conclusions likely extend to pitavastatin. Takahashi and his

colleagues 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 (Takahashi et al., 2013).

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 (Bi et al., 2013; Ho et al., 2006),

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,


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because CsA did not impact the renal clearance of RSV (Table 4). In the present studies, 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 to the human counterparts. Consistent with human data

also is the marked effect of CsA on RSV exposure when compared to RIF (6.3- to 7.1-fold vs.

2.9- to 4.4-fold increase) (Shen et al., 2013; Simonson et al., 2004). 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. While RIF is also a potent inhibitor of OATPs (IC50:

0.42 to 1.69 µM), it is a weaker inhibitor of NTCP (IC50 of 277 uM vs 3.9 uM) and BCRP (IC50

of 14 µM vs. ~7 µM) (Nezasa et al., 2002b; Prueksaritanont et al., 2014; Shen et al., 2013) and

so it is hypothesized that the extent of inhibition of liver basolateral NTCP and OATP, and

intestinal apical BCRP, is greater for CsA (vs. 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 following 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.


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Authorship Contributions

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

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

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

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

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

Other: None.


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Lai (2013). Quantitative assessment of the contribution of sodium-dependent taurocholate

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fluvastatin. Biopharm Drug Dispos 34(8): 452-461.

Chang, J. H., J. Ly, E. Plise, X. Zhang, K. Messick, M. Wright and J. Cheong (2014).

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Footnotes

Reprint requests: Hong Shen, F1.3802, Route 206 & Province Line Road., Bristol-Myers Squibb

Company, Princeton, NJ 08543. Telephone: (609) 252-4509; Facsimile: (609) 252-6802

This study is supported by Bristol-Myers Squibb Company.


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Figure Legends

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 method. The retention time

for RSV was approximately 32 min. The % (of the radioactivity collected in bile or urine

samples) as parent RSV is indicated.

Fig. 2. Effect of CsA on the uptake of RSV (0.1 µM) in hOATP1B1-, hOATP1B3-,

cOATP1B1-, and cOATP1B3-HEK cells. The transporter-mediated uptake was obtained by

subtracting the transport rate during the first 2 min incubation at 37°C in vector control cells

from that in transporter-transfected cells. The data are expressed as a percentage of initial uptake

rate in the presence and absence of CsA. IC50 values were obtained by fitting the uptake data to

the equation (see Materials & Methods) and are summarized in Table 2. Values shown are mean

± S.D. for experiments performed in triplicate.

Fig. 3. Uptake of [3H]TCA, [3H]RSV and [3H]ATV in mock- and cNTCP-HEK cells was

determined after 5 minutes of incubation (A). Each value represents the mean ± SD (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 min 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 initial

uptake rate in the presence and absence of CsA. IC50 values were obtained by fitting the uptake


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data to either the equation (see Materials & Methods) and are summarized in Table 2. Values

shown are mean ± S.D. for experiments performed in triplicate.

Fig 4. Effect of CsA on the uptake of [3H]RSV (0.1 µM) by human and cynomolgus monkey

hepatocytes. The data are expressed as a percentage of initial uptake rate measured at 15 and 90

seconds at 37°C in the presence and absence of CsA. IC50 values were obtained by fitting the

uptake data to the equation (see Materials & Methods) and are summarized in Table 2. Values

shown are mean ± S.D. for experiments performed in triplicate.

Fig. 5. Mean plasma concentrations of RSV (A) and whole blood concentrations of CsA (B)

after single oral dose of RSV (3 mg/kg) alone (open circle) or with CsA (100 mg/kg, PO) (solid

circle or triangle) in cynomolgus monkeys. Inset in figure A depicts same data on semi-

logarithmic scale. CsA was dosed one hour ahead of RSV. Data are expressed as mean ± S.D. (n

= 3 animals).

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, PO) in cynomolgus monkeys. The amount of RSV excreted in urine in

monkeys was determined for the RSV alone (open bar) and CsA-treated (closed bar) groups.

CsA was dosed one hour ahead of RSV. Data are expressed as mean ± S.D. (n = 3 animals).


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Table 1

Cumulative excretion of radioactivity in the bile, urine and feces of bile-cannulated cynomolgus

monkeys (n = 3) after a single oral administration of [3H]RSV

Collection Interval (hr)

Excretion of Radioactivity (% of Dose)

Bile Urine Feces

0-8 20.8 ± 7.3 0.9 ± 0.3 NS** 8-24 13.5 ± 4.4 0.7 ± 0.2 30.1 ± 5.5 24-72 4.7 ± 2.9 0.8 ± 0.5 20.5 ± 17.0 72-168 NS* 0.6 ± 0.3 11.2 ± 12.4 0-168 39.0 ± 3.0 2.9 ± 1.3 61.7 ± 25.1 Total Recovery 103.6 ± 27.5


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Table 1 Legend:

*Only 0 to 72 hr bile samples were collected and analyzed

**Only 0 to 24 hr fecal samples were collected and analyzed

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

NS, no sample


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Table 2

In vitro evaluation of CsA as an inhibitor of RSV uptake into hepatocytes and

HEK-293 cells expressing individual transporter

IC50 (µM)*

Hepatocytes OATP1B1 OATP1B3 NTCP

Human 0.30 ± 0.08 0.21 ± 0.10 0.13 ± 0.06 2.1 ± 0.9**

Cynomolgus Monkey 0.29 ± 0.11 0.28 ± 0.11 0.25 ± 0.09 3.9 ± 2.0


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Table 2 Legend:

*IC50 values were generated at a low concentration of RSV (0.1 µM) and data are represented as

the mean ± SD (n = 3 determinations) (see Materials and Methods).

**IC50 was average of the values generated from 55 different test occasions using [3H]TCA (1

μM) as probe substrate.


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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

Parameter RSV Alone CsA + RSV* GMR (90% CI)

RSV

Cmax (nM) 14.3 ± 5.2 145.5 ± 53.1 10.2 (3.3-31.9)

tmax (hr) 2.7 ± 0.6 2.7 ± 0.6

t1/2 (hr) 9.1 ± 3.1 4.3 ± 1.3

AUC0-inf (nM•hr) 133.9 ± 34.5 897.5 ± 462.0 6.3 (3.1-12.9)

CsA*

Cmax (µM) 1.1 ± 0.3

tmax (hr) 4.3 ± 1.5

t1/2 (hr) 5.9 ± 0.4

AUC0-49 (µM•hr) 10.0 ± 3.0

C25 hr (µM) 0.05 ± 0.01


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Table 3 Legend:

*CsA was dosed one hr prior to RSV (see Materials and Methods).


GMR geometric mean ratio; CI confidence interval; Cmax maximum concentration; Tmax time of

Cmax; t1/2 terminal elimination half-life; AUC0-inf area under the concentration-time curve from

time zero to infinity, C25 hr concentration 25 hr post-dose.


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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

Parameter Animal 1 Animal 2 Animal 3 Average

RSV Alone

CsA + RSV RSV

Alone CsA + RSV RSV

Alone CsA + RSV RSV Alone CsA + RSV

Bodyweight (kg) 3.4 3.4 3.9 3.7 4.1 4.2 3.8 ± 0.4 3.8 ± 0.4

Xe, 0-48 hr (nmol) 120.6 925.1 253.9 1819.0 267.9 960.2* 214.1 ± 81.3 1234.8 ± 506.3

Xe, 0-48 hr (% dose) 0.6 4.5 1.1 8.1 1.1 3.8* 0.92 ± 0.29 5.5 ± 2.3

AUC0-inf (nM*hr) 95.8 706.3 162.9 1424.4 143.0 561.8 133.9 ± 34.5 897.5 ± 462.0

CLR (mL/min/kg) 6.1 6.3 6.7 5.7 7.6 6.8* 6.8 ± 0.7 6.3 ± 0.6

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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Legend to Table 4:

*The urine sample collected between 0 to 7 hr from animal 3 was contaminated, the amount of RSV excreted into urine during the

interval was not included into the cumulative excretion.


Xe, 0-48 hr is the amount or percentage of RSV recovered in urine over 48 hr; CLR renal clearance.




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Table 5

Percentage of dose recovery in monkeys and humans after oral administration of radio-labeled RSV

Species Oral Dose % Dose

Urine (parent) Feces (parent) Bile (parent) Total recovery Reference

BDC monkey 3 mg/kg 2.9 (1.3) 61.7 (ND) 39 (24.5) 103.6

Intact monkey 10 mg/kg 5.0 (1.0) 90.7 (75.0) 95.7 US FDA, 2003

Human 20 mg 10.6 (4.9) 90.0 (76.8) 100.6 Martin et al., 2003a




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Legend to Table 5:

ND not determined; BDC bile duct cannulated.




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1450

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