Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance

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Relative Activity Factor (RAF)-based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes Saki Izumi, Yoshitane Nozaki, Hiroyuki Kusuhara, Koichiro Hotta, Toshiki Mochizuki, Takafumi Komori, Kazuya Maeda, and Yuichi Sugiyama Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00138 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Molecular Pharmaceutics

Relative Activity Factor (RAF)-based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes

Saki Izumi*, Yoshitane Nozaki, Hiroyuki Kusuhara, Koichiro Hotta, Toshiki Mochizuki, Takafumi Komori, Kazuya Maeda, and Yuichi Sugiyama

Drug Metabolism and Pharmacokinetics Tsukuba, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2635, Japan (S. I., Y. N., K. H., T. M., T. K.)

Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-003, Japan (H. K., K. M.)

Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, 1-6 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0045, Japan (Y. S.)

*Corresponding author

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Corresponding Author: Saki Izumi, PhD Drug Metabolism and Pharmacokinetics Tsukuba, Tsukuba Research Laboratories, Eisai Co., Ltd. 5-1-3, Tokodai, Tsukuba-shi, Ibaraki, 300-2635, Japan Telephone: +81-29-847-5656 Fax: +81-29-847-5672 E-mail: [email protected]

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Table of Contents/Abstract Graphic

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Abstract In vitro–in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CLuptake) to that in human hepatocyte suspensions (PSinf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin,

fexofenadine, fluvastatin, glibenclamide,

irbesartan, nateglinide, pitavastatin,

pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CLuptake was mainly accounted for by OATP1B1 (72.3% – 99.7%), and fell within the 3-fold of PSinf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PSinf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development.

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Keywords Organic anion transporting polypeptide (OATP), hepatic uptake, human hepatocytes, relative activity factor (RAF)

Abbreviations AUC, area under the plasma concentration-time curve; BSA, bovine serum albumin; BW, body weight; CCK-8, cholecystokinin octapeptide; CLh, hepatic clearance; CYP, cytochrome P450; PK, pharmacokinetic(s); HMG-CoA, hydroxymethylglutaryl-CoA; IVIVE, in vitro-in vivo extrapolation; KHB, Krebs Henseleit buffer; NTCP, Na+-taurocholate cotransporting polypeptide; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; RAF, relative activity factor; REF, relative expression factor.

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Introduction To identify drug candidates expected to have adequate pharmacokinetic (PK) profiles targeted therapeutic areas, the prediction of human PK profiles is routinely performed at nonclinical stages, wherein the full bottom-up approach is often adopted. Among the PK parameters, the prediction of hepatic clearance (CLh) of drug candidates is a key factor for estimating the impact of the first-pass effect after oral dose and hepatic elimination from systemic circulation on the extent of drug exposure. To avoid significant species differences in expression levels as well as substrate selectivity of drug metabolizing enzymes and transporters expressed in the liver

1-2

, in vitro–in vivo

extrapolation (IVIVE) using human liver tissue-derived in vitro systems, such as liver microsomes and hepatocytes, is widely used in pharmaceutical industries. In the liver, OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) play pivotal roles in the hepatic uptake of various anionic drugs, such as HMG-CoA reductase inhibitors (statins), angiotensin receptor blockers, antidiabetics, and direct-acting antivirals 3-4. Although many OATP1B substrate drugs are also subjected to hepatic metabolism, IVIVE based on uptake clearance determined in human hepatocytes offers better metrics for their overall hepatic intrinsic clearance in vivo (CLint,all,vivo) than that based on human microsomal metabolic clearance does

5-7

. Nevertheless,

this approach has some practical issues, such as low throughput (particularly in suspension systems), high assay cost, large inter-batch differences in the uptake activity of OATP1B substrates, and limited availability of human hepatocytes with adequate transporter-mediated uptake activities. To address these issues, in the present study, we used the relative activity factor (RAF) approach to extrapolate human hepatocyte uptake clearance from alternative in vitro systems of transporter-transfected cells. The RAF approach was originally developed for cytochrome P450 (CYP) enzymes

8

to

estimate the metabolic clearance and contribution of each CYP isoform in human liver microsomes from recombinant systems based on the metabolic activities of CYP-isoform selective substrates 9-10. This approach has also been applied to drug transporters, and many previous studies mainly focused on the estimating relative contribution of hepatic (OATP1B1 and OATP1B3) or renal (organic anion

6


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transporter (OAT) 1, OAT3, and organic cation transporter 2) uptake transporters to the total transporter-mediated tissue uptake clearance of the test substrates

11-18

. Hirano et al. (2004)

demonstrated that the uptake clearance of pitavastatin and estradiol-17β-glucuronide in human hepatocytes agreed well with that estimated by the RAF approach in three batches of human hepatocytes12. Kunze et al. (2014) also demonstrated that RAF and relative expression factor (REF, based on liquid chromatography with tandem mass spectrometry analysis) values were very similar to each other and that OATP1B1- and OATP1B3-mediated uptake clearance of some statins predicted by the RAF and REF approaches was within a two-fold error estimate of the uptake clearance observed in human hepatocytes17. These findings suggested that transporter-transfected cell systems can be a substitute for human hepatocytes to predict CLint,all,vivo of OATP1B substrates at nonclinical stages. Among the available methods, the RAF approach is considered useful for quantitative bridging of the different in vitro systems because this approach is practical and readily applicable to drug discovery and development settings due to the technical simplicity. In the present study, we comprehensively evaluated the capability of the RAF approach to estimate the active uptake clearance of 14 OATP1B substrate drugs across drug classes in human hepatocytes from transporter-transfected cell systems, focusing on OATP1B1 and OATP1B3. Hepatic uptake assay was conducted in two batches of human hepatocytes that had been fully characterized in terms of the uptake of OATP1B substrates and was validated by a good correlation with human CLint,all,vivo in our previous study 7.

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Materials and Methods Chemicals. [3H]Estrone-3-sulfate ([3H]E1S, 45.6 Ci/mmol) and [3H]cholecystokinin octapeptide ([3H]CCK-8, 98.7 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Pitavastatin calcium, pravastatin sodium, and glibenclamide (glyburide) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Atorvastatin calcium trihydrate, cerivastatin sodium, irbesartan, rosuvastatin calcium, and valsartan were from LKT Laboratories (St. Paul, MN), and fluvastatin sodium, fexofenadine hydrochloride, and bosentan were from Toronto Research Chemicals (Toronto, ON, Canada). Torasemide and nateglinide were obtained from Tokyo Chemical Industries (Tokyo, Japan) and Tocris Bioscience (Minneapolis, NM), respectively. Telmisartan, silicon oil (d = 1.05 g/mL), and mineral oil (d = 0.84 g/mL) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical or reagent grade and were from Wako Pure Chemical Industries.

Cell Culture. The human embryonic kidney (HEK) 293 cells stably expressing OATP1B1 (SLCO1B1, OATP1B1-HEK), OATP2B1-HEK)

OATP1B3 and

(SLCO1B,

corresponding

OATP1B3-HEK),

control

(control-HEK) were established previously

19-20

(empty

and

OATP2B1

vector-transfected)

(SLCO2B1,

HEK293

cells

. The cells were grown in Dulbecco’s modified

Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) of fetal bovine serum, penicillin (final concentration, 100 units/mL), streptomycin (100 µg/mL), and hygromycin B (80 µg/mL) at 37°C in a humidified incubator containing 5% CO2 gas. For cellular uptake study, the cells were seeded in the poly-D-lysine-coated 24-well plate (BD Biosciences, San Jose, CA) at a density of 2 × 105 cells/well and cultured for 48 hours in the CO2 incubator.

Cellular Uptake Study in Transporter-transfected HEK293 Cells. Cellular uptake study was performed as described previously

8


21

. Final concentrations of

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test compounds in Krebs Henseleit buffer (KHB, pH7.4) are given in Figure legends. For telmisartan, the uptake was also evaluated in KHB containing 0.25% and 1% bovine serum albumin (BSA). The amount of test compounds taken up into the cells were quantified by liquid scintillation counting (Tri-Carb 3100TR; PerkinElmer Life Sciences) for radiolabeled compounds or by liquid chromatography with tandem mass spectrometry (LC-MS/MS) for unlabeled compounds, as reported previously

21

. Protein content per well was quantified by using BCA Protein Assay Kit

(Thermo Fisher Scientific, Waltham, MA). The uptake of test compounds in the time-course experiments was expressed as an uptake volume (µL/mg protein), which was calculated by dividing the amount of the compound taken up into the cells (dpm/well or pmol/well) by the product of the buffer concentration (dpm/µL or pmol/µL) and protein content (mg protein/well).

Uptake Study in Cryopreserved Human Hepatocyte Suspensions. Two batches of cryopreserved human hepatocytes, lots VRR (pool of 50 donors) and OJE (single donor), purchased from Bioreclamation IVT (Baltimore, MD) were used in this study. These human hepatocyte batches had been fully characterized in terms of hepatic uptake of various OATP1B substrate drugs in our laboratories 7. The cryopreserved hepatocytes were recovered in Cryopreserved Hepatocyte Recovery Medium (Thermo Fisher Scientific) according to the manufacturer’s instructions (cell viability >85% in all preparations). The hepatic uptake assay was performed by using an oil-spin method according to our previous study

7

. Briefly, after

pre-incubating hepatocyte suspensions, an equal volume of KHB containing a radiolabeled or unlabeled test compound was added to the hepatocyte suspensions to yield a final compound concentration of 1 µmol/L or lower in 0.5 × 106 viable cells/mL. After incubation, an 80-µL aliquot of the reaction mixture was serially removed at designated time points and dispensed into a centrifuge tube containing 50 µL of 2 mol/L NaOH (for radiolabeled compounds) or 5 mol/L ammonium acetate (for unlabeled compounds) under a layer of 100 µL of silicon/mineral oil (silicon oil/mineral oil = 1/0.184, w/w), followed by centrifugation (Microfuge 16; Beckman Coulter, Fullerton, CA) to separate hepatocytes from KHB. The compound taken up into hepatocytes and

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remaining in KHB were quantified by liquid scintillation counting or LC-MS/MS. The time-course data were expressed as the uptake volume (Vd,uptake, µL/106 cells), which was obtained by dividing the amount of a compound taken up into hepatocytes (Xhep, pmol/106 cells) by the compound concentration in the incubation buffer (Cbuffer, µmol/L) at each time point.

LC-MS/MS Analysis. The unlabeled test compounds except for cerivastatin and irbesartan in the samples obtained from in vitro uptake studies were measured by LC-MS/MS under the conditions reported previously

7, 21

. LC-MS/MS analysis of cerivastatin and irbesartan was performed on Quattro

Premier mass spectrometer (Waters, Milford, MA) with Alliance HT 2795 separation module (Waters). Liquid chromatography was performed using an Atlantis T3 column (3.0 µm, 2.1 mm i.d., 50 mm, Waters) at a flow rate of 0.3 mL/min. Distilled water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) were used as the mobile phases, and the gradient program was as follows: 0% solvent B at 0 minute, linearly increasing to 80% over 3 minutes, further linearly increasing to 100% solvent B over the next 0.01 minutes, held at 100% solvent B for 1 minute. The column was equilibrated with initial mobile phase condition before each injection (injection volume, 10 µL). Analytes were ionized by electrospray ionization in a positive ion mode, and the selected ion monitoring transitions were 460.58>356.14 for cerivastatin and 429.81>179.78 for irbesartan.

Kinetic Analysis of Uptake into Transporter-transfected HEK293 Cells. In the uptake study in transporter-transfected HEK293 cells, transporter-mediated uptake of a test compound was calculated by subtracting the uptake in control-HEK from that in OATP1B1-, OATP1B3-, or OATP2B1-HEK. The following Michaelis-Menten equation was fitted to the concentration-dependent transporter-mediated uptake data to calculate kinetic parameters:

=

×

(1)

10


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where v, S, Vmax, and Km represent uptake rate of the substrate (pmol/min/mg protein), substrate concentration in the incubation buffer (µmol/L), maximum uptake rate (pmol/min/mg protein), and Michaelis constant (µmol/L), respectively. For the compounds which showed biphasic transporter-mediated uptake with low affinity (or apparently non-saturable) component at higher substrate concentration, the following equation was used for data fitting:

=

×

+ ×

(2)

where CLlow represents uptake clearance for the low affinity component of the transporter-mediated uptake. Data fitting was performed by a nonlinear least-squares regression method by using MULTI program 22. For the compounds the kinetic parameters (Km, Vmax, and CLlow) of which were available, the initial uptake clearance (CLuptake, µL/min/mg protein) via OATP1B1 (CLuptake,1B1) and/or OATP1B3 (CLuptake,1B3) in transporter-transfected HEK293 cells was calculated as follows:

=

(3)

or

=

+

(4)

For the compounds the uptake of which was not adequately greater in OATP1B3-HEK than that in control-HEK to calculate kinetic parameters, the CLuptake,1B3 was calculated from the initial uptake slope of transporter-mediated uptake obtained from the time-course data.

Kinetic Analysis of Uptake into Cryopreserved Human Hepatocytes. In the uptake study in human hepatocyte suspensions, the intrinsic uptake clearance (PSinf) of a test compound obtained at 37°C (PSinf,37°C) and on ice (PSinf,ice) was calculated as follows, according to a previous report 6: ()

!""# ()

= $%&' ×

()(*+,),

!""#

!""# ()

+ ./

(5)

where Xhep(t), Cbuffer(t), AUC(0-t),buffer, and V0 represent the amount of a test compound taken up into hepatocytes at time t, the compound concentration in the incubation buffer at time t, the area under the compound concentration-time curve from zero time to time t in the incubation buffer, and the 11



initial

distribution

volume,

respectively.

The

Xhep(t)/Cbuffer(t)

Page 12 of 38

was

plotted

against

AUC(0-t),buffer/Cbuffer(t), and PSinf was determined from the initial slope of the plots. Transporter-mediated active uptake clearance in human hepatocytes (PSinf,act) was defined by the following equation in the present study:

$%&',0 = $%&',12° − $%&',%0

(6)

RAF Method. According to a previous report 12, relative activity factor (RAF) values were calculated by using E1S and CCK-8 as reference substrates for OATP1B1 and OATP1B3, respectively.

567898 =

: ;C mutation in SLCO1B1 is associated with a higher AUC of OATP1B substrate drugs, such as atorvastatin, pitavastatin, and pravastatin

3

. Compared with

previously-reported RAF approach findings, the observed contribution of OATP1B1 for pitavastatin (90%–98%) and irbesartan (94%–99%) was in agreement with previously-reported values (88%– 95% and 71%–98%, respectively)

12, 18

. The present study yielded a slightly higher contribution of

OATP1B1 for rosuvastatin (95%–99%) and valsartan (77%–95%) than that obtained in previous studies (66%–84% and 22%–71%, respectively)

14-15

. This was mainly attributable to interbatch

differences in the relative contribution of OATP1B1 and OATP1B3 because the PSinf,act ratio of E1S to CCK-8 observed in the present study (20.6 for lot VRR, 50.4 for lot OJE) was equivalent to or greater than that observed in previous batches used for rosuvastatin (13.9–38.3) and valsartan (1.4–

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12.4) 14-15. Realizing the (i) strong correlation between human hepatocyte PSinf,act and CLint,all,vivo for OATP1B substrates and (ii) the major contribution of OATP1B1 in the hepatic uptake of the tested substrates prompted us to examine the performance of the RAF approach to directly correlate CLuptake in the OATP1B1-HEK cells with CLint,all,vivo for IVIVE purposes as follows: A;C) of SLCO1B1 on the clinical PK profile of fexofenadine the marked effect of rifampicin, simultaneously administered with fexofenadine

30

29

, and also by

. In addition, the

PSinf,act of telmisartan in human hepatocytes, which was as high as that of pitavastain, could not be predicted by the RAF approach due to the undetectable levels of OATP1B1- and OATP1B3-mediated transport (Figures 1 and 3). Since OATP1B3-mediated uptake was reportedly evident in the presence of albumin in the incubation buffer

31

, we performed an additional cellular

uptake study for telmisartan in the presence of BSA (Figure 6). Nevertheless, the OATP1B3- (or OATP1B1)-mediated uptake of telmisartan could not be detected in our expression systems. During this additional study, we clearly observed OATP2B1-mediated uptake of telmisartan in our overexpression systems (Figure 6), which was much greater than the previous report 31. Based on the REF method, we concluded that the contribution of OATP2B1 was marginal in that previous report. The discrepancy suggests the need to re-evaluate the contribution of OATP1B3 and OATP2B1 to the hepatic uptake of telmisartan in human hepatocytes. We also need to establish a method for distinguishing OATP2B1-mediated uptake from the net uptake in hepatocytes. The number of OATP1B substrates are increasing. In the most recent cases, direct-acting antivirals for treating the hepatitis C virus infection, such as asunaprevir and simeprevir, are OATP1B substrates 4, 32, whereas first generation antivirals such as telaprevir and boceprevir are not. Therefore, during the nonclinical drug development process, the pharmaceutical industry may have more opportunities to handle OATP1B substrate leads because of the broad substrate specificity of OATP1B. This study provides successful extrapolation of the low to high clearance of OATP1B substrates from the overexpression system to human hepatocytes, and also to in vivo. Further efforts are necessary to allow the RAF approach to avoid underestimation (by including other transporters

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in the analysis), and to increase the accuracy of CLint,all,vivo prediction. Since the RAF approach is a knowledge-intensive approach, inclusion of other transporters could improve future predictive performance. The RAF approach can be used to predict active uptake clearance of OATP1B substrates in human hepatocytes. This quantitative bridging of transporter-mediated uptake clearance between different in vitro systems will allow for the earlier estimation of human hepatocyte PSinf,act, a fundamental parameter for predicting the human CLh of OATP1B substrates.

20


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Supporting Information. PSinf,37°C and PSinf,ice values of OATP1B substrate drugs in cryopreserved human hepatocyte suspensions; Time profiles of the uptake of [3H]E1S and [3H]CCK-8 in cryopreserved human hepatocytes, lots VRR (pool of 50 donors) and OJE (a single donor); Time profiles of the uptake of OATP1B substrate drugs in cryopreserved human hepatocyte suspensions (lot OJE, single donor); Prediction of clinically-observed CLint,all,vivo of OATP1B substrate drugs from OATP1B1-HEK cells by the RAF approach.

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Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T., A pharmacokinetic analysis program

(multi) for microcomputer. Journal of Pharmacobio-dynamics 1981, 4 (11), 879-885. 23.

Ho, R. H.; Leake, B. F.; Roberts, R. L.; Lee, W.; Kim, R. B., Ethnicity-dependent

Polymorphism in Na+-taurocholate Cotransporting Polypeptide (SLC10A1) Reveals a Domain Critical for Bile Acid Substrate Recognition. Journal of Biological Chemistry 2004, 279 (8), 7213-7222. 24.

Roth, M.; Obaidat, A.; Hagenbuch, B., OATPs, OATs and OCTs: the organic anion and cation

transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology 2012, 165 (5), 1260-1287. 25.

Bi, Y.-a.; Scialis, R. J.; Lazzaro, S.; Mathialagan, S.; Kimoto, E.; Keefer, J.; Zhang, H.;

Vildhede, A. M.; Costales, C.; Rodrigues, A. D.; Tremaine, L. M.; Varma, M. V. S., Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes. The AAPS Journal 2017, 19 (3), 787-796. 26.

Williamson, B.; Soars, A. C.; Owen, A.; White, P.; Riley, R. J.; Soars, M. G., Dissecting the

relative contribution of OATP1B1-mediated uptake of xenobiotics into human hepatocytes using siRNA. Xenobiotica 2013, 43 (10), 920-931. 27.

Ismair, M. G.; Stieger, B.; Cattori, V.; Hagenbuch, B.; Fried, M.; Meier, P. J.; Kullak-Ublick, G.

A., Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver. Gastroenterology 2001, 121 (5), 1185-1190. 28.

Mathialagan, S.; Piotrowski, M. A.; Tess, D. A.; Feng, B.; Litchfield, J.; Varma, M. V.,

Quantitative prediction of human renal clearance and drug-drug interactions of organic anion transporter

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substrates using in vitro transport data: a relative activity factor approach. Drug Metabolism and Disposition 2017, 45 (4), 409-417. 29.

Niemi, M.; Kivistö, K. T.; Hofmann, U.; Schwab, M.; Eichelbaum, M.; Fromm, M. F.,

Fexofenadine pharmacokinetics are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1). British Journal of Clinical Pharmacology 2005, 59 (5), 602-604. 30.

Kusuhara, H.; Miura, M.; Yasui-Furukori, N.; Yoshida, K.; Akamine, Y.; Yokochi, M.;

Fukizawa, S.; Ikejiri, K.; Kanamitsu, K.; Uno, T.; Sugiyama, Y., Effect of coadministration of single and multiple doses of rifampicin on the pharmacokinetics of fexofenadine enantiomers in healthy subjects. Drug Metabolism and Disposition 2013, 41 (1), 206-213. 31.

Ishiguro, N.; Maeda, K.; Kishimoto, W.; Saito, A.; Harada, A.; Ebner, T.; Roth, W.; Igarashi, T.;

Sugiyama, Y., Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans. Drug Metabolism and Disposition 2006, 34 (7), 1109-1115. 32.

Eley, T.; Han, Y. H.; Huang, S. P.; He, B.; Li, W.; Bedford, W.; Stonier, M.; Gardiner, D.; Sims,

K.; Rodrigues, A. D.; Bertz, R. J., Organic anion transporting polypeptide-mediated transport of, and inhibition by, asunaprevir, an inhibitor of hepatitis C virus NS3 protease. Clinical pharmacology and therapeutics 2015, 97 (2), 159-66.

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Tables Table 1. RAF values for OATP1B1 and OATP1B3

Isoform

Reference substrate

Human hepatocytes

Transporter-transfected cell systems

Lot VRR

Km

Vmax

CLuptake,1B1,E1S

CLuptake,1B3,CCK-8

PSinf,act

(µmol/L)

(pmol/min/mg)

(µL/min/mg)

(µL/min/mg)

(µL/min/106 cells)

Lot OJE RAF

OATP1B1

E1 S

0.286a

36.4a

127

-

200 ± 57b

1.57

OATP1B3

CCK-8

5.77c (5.23, 6.30)

84.9c (75.0, 94.8)

-

14.6

9.72c (8.12, 11.3)

0.661

-, not applicable. a: Izumi et al. (2013) b: Mean ± SEM of 3 independent experiments. c: Mean of 2 independent experiments with individual values given in the parenthesis.

26


PSinf,act (µL/min/106 cells) 464c (463, 466) 9.21c (8.63, 9.77)

RAF

3.65 0.627

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Table 2. OATP1B1- and OATP1B3-mediated uptake of OATP1B substrate drugs in transporter-transfected HEK293 cells Test compounds

OATP1B1

OATP1B3

Km (µmol/L)

Vmax (pmol/min/mg)

CLlow (µL/min/mg)

CLuptake,1B1 (µL/min/mg)

0.761b

42.2b

-b

55.5c

4.27b 1.13 (0.857, 1.41)a

56.1b 87.7 (73.9, 101)a

-b

13.1c

-

77.6c

Fexofenadine

61.6b

41.0b

0.0199b

0.685c

Fluvastatin Glibenclamide

87.6b 38.1b 127 (116, 139)a 174b

-b 0.507b

Nateglinide

4.80b 1.24b 4.73 (3.84, 5.62)a 36.4b

Pitavastatin

2.48b

Pravastatin Rosuvastatin Telmisartan

Atorvastatin Bosentan Cerivastatin

Irbesartan

Vmax (pmol/min/mg) 16.8 (21.6, 12.0)a 7.07 (6.85, 7.29)a 9.00 (5.24, 12.8)a -

CLlow (µL/min/mg) 0.595 (1.05, 0.143)a 0.797 (0.958, 0.637)a

CLuptake,1B3 (µL/min/mg)

-

0.625c

18.3c 31.2c

Km (µmol/L) 0.923 (0.799, 1.05)a 1.43 (1.29, 1.57)a 14.4 (9.49, 19.2)a -

-

2.89 ± 0.42d 2.65 ± 0.82d

-

26.8c

-

-

-

4.28 ± 0.76d

-b

4.78c

0.632b

46.6c

27.0b 9.31b

187b 103b

-b 0.624b

6.93c 11.7c

27.6 (25.2, 30.0)a -

0.649 (0.972, 0.326)a -

1.97 ± 0.22d

114b

2.43 (1.77, 3.10)a -

0.859 ± 0.161d 1.33 ± 0.26d

-

-

-

ND

-

-

-

ND

13.6 (9.85, 17.3)a

54.9 (60.5, 49.3)a

-

0.344 ± 0.104d

-

4.04c

b

b

b

c

Torasemide

20.9

154

0.908

8.28

Valsartan

7.48b

42.3b

0.0545b

5.71c

18.8c 2.99 ± 0.15d 5.74c

12.0c

-, not applicable; ND, not detected. a: Kinetic parameters of test compounds for OATP1B1 and OATP1B3 obtained from transporter-transfected cells in the present study are expressed as mean ± SEM (n=3 or 4), or mean of 2 independent experiments with individual data given in a parenthesis. b: Izumi et al. (2015). c: CLuptake,1B1 or CLuptake,1B3 was calculated by using mean kinetic parameters according to Equation 3 or 4.

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

d: CLuptake,1B3 calculated from the initial slope of time course of OATP1B3-mediatd uptake is expressed as mean ± SEM of 3 to 4 independent experiments. The substrate concentrations (µmol/L) used are as follows: 0.1 for bosentan, glibenclamide, and irbesartan, 0.3 for fluvastatin, 1 for nateglinide, pravastatin, rosuvastatin, and torasemide.

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Table 3. Uptake clearance in cryopreserved human hepatocytes and RAF-based OATP1B1- and OATP1B3-mediated uptake clearance

Uptake clearance in human hepatocytes Lot VRR Lot OJE Test compounds

Atorvastatin

RAF1B1× CLuptake,1B1 (1B1 contribution)b

Compound concentration

PSinf,acta

(µmol/L) 0.1

(µL/min/106 cells) 73.7 173

87.1 (87.5%)

RAF-based OATP1B- and OATP1B3-mediated uptake clearance Lot VRR Lot OJE RAF1B1× RAF1B3× RAF1B3× CLuptake,1B3 CLuptake,1B1+1B3,RAFd (1B3 contribution)c (µL/min/106 cells) 12.4 (12.5%) 99.5

CLuptake,1B1 (1B1 contribution)b 318 (97.6%)

CLuptake,1B3 CLuptake,1B1+1B3,RAFd (1B3 contribution)c (µL/min/106 cells) 7.76 (2.4%) 326

Bosentan

0.1

30.6

56.4

20.6 (91.2%)

1.98 (8.8%)

22.6

75.2 (98.4%)

1.24 (1.6%)

Cerivastatin

0.1

81.7

166

122 (97.0%)

3.79 (3.0%)

126

445 (99.5%)

2.37 (0.5%)

447

Fexofenadine

1

10.9

11.2

1.08 (72.3%)

0.413 (27.7%)

1.49

3.94 (93.8%)

0.259 (6.2%)

4.20

Fluvastatin

0.3

91.2

225

28.7 (93.8%)

1.91 (6.2%)

30.6

105 (98.9%)

1.20 (1.1%)

106

Glibenclamide

0.1

83.1

175

49.0 (96.6%)

1.75 (3.4%)

50.8

179 (99.4%)

1.10 (0.6%)

180

Irbesartan

0.3

82.7

89.0

42.1 (93.7%)

2.83 (6.3%)

44.9

154 (98.9%)

1.77 (1.1%)

156

Nateglinide

1

9.92

37.4

7.50 (85.2%)

1.30 (14.8%)

8.80

27.4 (97.1%)

0.814 (2.9%)

28.2

Pitavastatin

0.3

143

307

73.2 (90.2%)

7.93 (9.8%)

81.1

267 (98.2%)

4.96 (1.8%)

272

76.4

Pravastatin

1

3.39

16.8

10.9 (95.0%)

0.568 (5.0%)

11.5

39.8 (99.1%)

0.356 (0.9%)

40.2

Rosuvastatin

1

12.0

38.3

18.4 (95.4%)

0.879 (4.6%)

19.3

67.2 (99.2%)

0.550 (0.8%)

67.8

Telmisartan

0.05

256

197

NA

NA

NA

NA

NA

NA

Torasemide

1

9.66

26.2

13.0 (98.3%)

0.227 (1.7%)

13.2

47.5 (99.7%)

0.142 (0.3%)

47.6

Valsartan

1

8.28

16.0

8.96 (77.0%)

2.67 (23.0%)

11.6

32.7 (95.1%)

1.67 (4.9%)

34.4

NA, not applicable because OATP1B1- and OATP1B3-mediated uptake of telmisartan was not observed in transporter-transfected HEK293 cells in this study. a: PSinf,act of test compounds in two batches of human hepatocytes (lots VRR and OJE) was calculated according to Equation 6 by using PSinf,37°C and PSinf,ice values (Table S1). b: 1B1 contribution (%) = RAF1B1 × CLuptake,1B1/(RAF1B1 × CLuptake,1B1 + RAF1B3 × CLuptake,1B3) × 100 c: 1B3 contribution (%) = RAF1B3 × CLuptake,1B3/(RAF1B1 × CLuptake,1B1 + RAF1B3 × CLuptake,1B3) × 100 d: CLuptake,1B1+1B3,RAF = RAF1B1 × CLuptake,1B1 + RAF1B3 × CLuptake,1B3

29



Figure Captions Figure 1. Time profiles of the uptake of [3H]CCK-8 and OATP1B substrate drugs in OATP1B3- and control-HEK. Uptake of [3H]CCK-8 (A, 0.001 µmol/L), atorvastatin (B, 0.1 µmol/L), bosentan (C, 0.1 µmol/L), cerivastatin (D, 0.1 µmol/L), fexofenadine (E, 1 µmol/L), fluvastatin (F, 0.3 µmol/L), glibenclamide (G, 0.1 µmol/L), irbesartan (H, 0.1 µmol/L), nateglinide (I, 1 µmol/L), pitavastatin (J, 0.1 µmol/L), pravastatin (K, 1 µmol/L), rosuvastatin (L, 1 µmol/L), telmisartan (M, 0.05 µmol/L), torasemide (N, 1 µmol/L), and valsartan (O, 1 µmol/L) in OATP1B3-HEK (closed circles) and control-HEK (open circles) cells was examined over a period of 10 (telmisartan) or 30 (other compounds) minutes at 37°C. Each point represents the mean ± SEM (n=3).

Figure 2. Concentration dependence of the OATP1B3-mediated uptake of [3H]CCK-8 (A, 0.001 – 100 µmol/L), atorvastatin (B, 0.03 – 30 µmol/L), cerivastatin (C, 0.03 – 30 µmol/L), fexofenadine (D, 0.1 – 100 µmol/L), pitavastatin (E, 0.1 – 100 µmol/L), and valsartan (F, 0.5 – 100 µmol/L). OATP1B3-mediated uptake was calculated by subtracting the uptake in control-HEK from that in OATP1B3-HEK, and the uptake was determined for 0.5 (atorvastatin and pitavastatin), 2 (valsartan and cerivastatin), or 5 (CCK-8 and fexofenadine) minutes at 37°C. Representative data from two independent experiments are shown as Eadie-Hofstee plot, in which OATP1B3-mediated uptake clearance (v/s, µL/min/mg protein, Y-axis) is plotted against the uptake rate (v, pmol/min/mg protein, X-axis). Each point represents mean ± SEM (n=3), and fitted lines (solid lines) were obtained by a nonlinear least-squares regression according to Equation 1 or 2 given under Materials and Methods.

Figure 3. Time profiles and concentration dependence of OATP1B1-mediated uptake of cerivastatin, irbesartan, and telmisartan. The uptake of cerivastatin (A, 1 µmol/L), irbesartan (B, 1 µmol/L), and telmisartan (C, 0.05 µmol/L) was examined in OATP1B1-HEK (closed circles) and control-HEK (open circles) cells for a period of 10 minutes at 37°C. Concentration dependence of OATP1B1-mediated uptake of cerivastatin (D, 0.01 – 10 µmol/L) and irbesartan (E, 0.03 – 30 µmol/L). OATP1B1-mediated uptake was calculated by subtracting the uptake in control-HEK from 30


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that in OATP1B1-HEK, and the uptake was determined for 0.5 minutes at 37°C. Representative data from two independent experiments are shown as Eadie-Hofstee plot, in which OATP1B1-mediated uptake clearance (v/s, µL/min/mg protein, Y-axis) is plotted against the uptake rate (v, pmol/min/mg protein, X-axis). The fitted lines (solid lines) were obtained by a nonlinear least-squares regression according to Equation 1 given under Materials and Methods. Each point represents mean ± SEM (n=3).

Figure 4.

Time profiles of the uptake of OATP1B substrate drugs in cryopreserved human

hepatocyte suspensions (lot VRR, pool of 50 donors). Uptake of atorvastatin (A, 0.1 µmol/L), bosentan (B, 0.1 µmol/L), cerivastatin (C, 0.1 µmol/L), fluvastatin (D, 0.3 µmol/L), glibenclamide (E, 0.1 µmol/L), irbesartan (F, 0.3 µmol/L), pitavastatin (G, 0.3 µmol/L), and telmisartan (H, 0.05 µmol/L) was determined at 37°C (closed circles) and on ice (open circles) up to 5 minutes by using an oil-spin method. The uptake value (µL/106 cells) was determined by dividing the amount of a compound taken up into human hepatocytes by the compound concentration in the incubation buffer at each time point. Representative 5-minute time course data from 2 to 7 independent experiments are presented. Each symbol represents mean of duplicate samples.

Figure 5. Comparison of RAF-based net uptake clearance mediated by OATP1B1 and OATP1B3 with human hepatocyte uptake clearance. The net uptake clearance mediated by OATP1B1 and OATP1B3 predicted from transfected cell systems by RAF approach (CLuptake,1B1+1B3,RAF = RAF1B1 × CLuptake,1B1 + RAF1B3 × CLuptake,1B3, X-axis) was compared with observed human hepatocyte active uptake clearance (PSinf,act = PSinf,37°C – PSinf,ice, Y-axis) in lots VRR (A) and OJE (B). Each symbol represents a mean value, and solid and dashed lines represent 1 to 1 and 1 to 3 (or 3 to 1) correlation lines, respectively. 1, atorvastatin; 2, bosentan; 3, cerivastatin; 4, fexofenadine; 5, fluvastatin; 6, glibenclamide; 7, irbesartan; 8, nateglinide; 9, pitavastatin; 10, pravastatin; 11, rosuvastatin; 12, torasemide; 13, valsartan. Telmisartan was excluded from the plots because the OATP1B1- and OATP1B3-mediate uptake was not observed in transporter-transfected HEK293 cell systems in the 31



present study.

Figure 6. Uptake of telmisartan in OATP1B1-, OATP1B3-, OATP2B1-HEK, and control-HEK cells in the presence of 0.25% (A) and 1% (B) BSA in the incubation buffer. The cells were incubated with KHB containing telmisartan (1 µmol/L) and BSA (0.25% or 1%) for 5 minutes at 37°C. The uptake was calculated by dividing the amount of telmisartan in the cells by total concentration of telmisartan (1 µmol/L) in the incubation buffer. Each bar represents the mean ± SEM (n=3). ***P < 0.001 between transporter-transfected HEK and control-HEK cells (one-way analysis of variance followed by Dunnett’s post hoc test). Time profiles of telmisartan uptake (0.05 µmol/L) in OATP2B1-HEK (closed circles) and control-HEK cells (open circles) for a period of 10 minutes at 37°C in KHB without BSA (C). Concentration dependence of the OATP2B1-mediated uptake of telmisartan (D). OATP2B1-mediated uptake of telmisartan (0.005 – 20 µmol/L) was calculated by subtracting the uptake in control-HEK from that in OATP2B1-HEK cells, and the uptake was determined for 1 minute at 37°C in KHB without BSA. Representative data from two independent experiments are shown as Eadie-Hofstee plot, in which OATP2B1-mediated uptake clearance (v/s, µL/min/mg protein, Y-axis) is plotted against the uptake rate (v, pmol/min/mg protein, X-axis). The fitted lines (solid lines) were obtained by a nonlinear least-squares regression according to Equation 1 given under Materials and Methods. Each point represents mean ± SEM (n=3).

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Illustrations: Figures Figure 1

33



Figure 2

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Figure 3

35



Figure 4

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Figure 5

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Figure 6

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Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance

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