Investigation of Fluorescein Derivatives as Substrates of Organic

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Investigation of fluorescein derivatives as substrates of organic anion transporting polypeptide (OATP) 1B1 to develop sensitive fluorescence-based OATP1B1 inhibition assays Saki Izumi, Yoshitane Nozaki, Takafumi Komori, Osamu Takenaka, Kazuya Maeda, Hiroyuki Kusuhara, and Yuichi Sugiyama Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00664 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on January 7, 2016

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Molecular Pharmaceutics

1

Investigation of fluorescein derivatives as substrates of organic anion transporting

2

polypeptide (OATP) 1B1 to develop sensitive fluorescence-based OATP1B1 inhibition

3

assays

4 5

Saki Izumi*, Yoshitane Nozaki, Takafumi Komori, Osamu Takenaka, Kazuya Maeda, Hiroyuki

6

Kusuhara, and Yuichi Sugiyama

7 8 9 10

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

11 12

Modeling & Simulation, Clinical Pharmacology, Eisai Co., Ltd., 4-6-10 Koishikawa, Bunkyo-

13

ku, Tokyo 112-8088, Japan (O. T.)

14 15

Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The

16

University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (K. M., H. K.)

17 18

Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, 1-6

19

Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0045, Japan (Y. S.)

20

1 ACS Paragon Plus Environment


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Corresponding Author:

2

Saki Izumi

3

Drug Metabolism and Pharmacokinetics Japan, Tsukuba Research Laboratories, Eisai Co. Ltd.,

4

5-1-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2635, Japan

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Telephone: +81-29-847-5656

6

FAX: +81-29-847-5672

7

e-mail: [email protected]

8 9


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1

Table of Contents/Abstract Graphic

Comparison of the Ki values obtained with 2',7'-dichlorofluorescein (DCF) and [3H]estradiol-17 -glucuronide (E2G) 1000

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

100

Ki ( M), DCF

13

7

1

8

12 10

6

4

0.1

11

9

10

5

2 3 1

estrone-3-sulfate cyclosporine A sulfobromophthalein ritonavir rifampin tacrolimus erythromycin estradiol-17 -glucuronide ketoconazole taurocholate verapamil gemfibrozil probenecid

10 00

10 0

10

1

0. 1

0.01 0. 01

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 48 49 50 51 52 53 54 55 56 57 58 59 60


Ki ( M), [3H]E2G

2 3 4



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1

Abstract

2

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic

3

uptake of various drugs.

4

evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug

5

development. For establishing highly sensitive, high-throughput fluorescence-based OATP1B1

6

inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and

7

evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-

8

expressing human embryonic kidney 293 cells. We identified 2′,7′-dichlorofluorescein (DCF),

9

4′,5′-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with Km

10

values of 5.29, 4.16, and 54.1 µM and Vmax values of 87.9, 48.1, and 187 pmol/min/mg protein,

11

respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG and DBF were identified

12

as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates.

13

Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The Ki

14

values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good

15

agreement with those obtained using [3H]estradiol-17β-glucuronide (E2G) as a substrate,

16

whereas [3H]estrone-3-sulfate and [3H]sulfobromophthalein yielded higher Ki values for all

17

inhibitors than DCF. Mutual competitive inhibition observed between DCF and E2G suggested

18

that they share the same binding site on OATP1B1. Therefore, DCF as well as E2G can be used

19

as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of

20

false-negative DDI predictions potentially caused by substrate-dependent Ki variations.

Because OATP1B1 is a site of drug–drug interactions (DDIs),

21


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1

Keywords

2

Organic anion transporting polypeptide (OATP), fluorescent substrate, inhibition assay, drug-

3

drug interaction (DDI)

4 5

Abbreviations

6

BCECF, 2’,7’-bis(2-carboxyethyl)-5-(or 6)-carboxyfluorescein; BSP, sulfobromophthalein;

7

cAMP, cyclic adenosine monophosphate; CCK-8, cholecystokinin octapeptide; CDCA-NBD,

8

chenodeoxycholyl-(Nε-nitrobenz-2-oxa-1,3-diazole)-lysine; CDCF, 5-(and-6)-carboxy-2’,7’-

9

dichlorofluorescein; 5-CF, 5-carboxyfluorescein; 6-CF, 6-carboxyfluorescein; CGamF, cholyl-

10

glycylamide-fluorescein; CLF, cholyl-L-lysyl-fluorescein; CsA, cyclosporine A; DBF, 4’,5’-

11

dibromofluorescein; DCF, 2’,7’-dichrolofluorescein; DDI, drug-drug interaction; E2G, estradiol-

12

17β-glucuronide; E1S, estrone-3-sulfate; 8-FcA, 8-fluorescein-cAMP; FL, fluorescein; FMTX,

13

fluorescein-methotrexate; KH, Krebs Henseleit; OATP, organic anion transporting polypeptide;

14

OG, Oregon green; statin, 3-hydroxymethylglutaryl-CoA reductase inhibitor.

15 16



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1

Introduction

2

Membrane transporters play key roles in the pharmacokinetics of a wide range of drugs,

3

and transporter-mediated drug–drug interactions (DDIs) have been reported in clinic.1-3 After

4

publication of the so-called “transporter white paper” by the International Transporter

5

Consortium,4 DDI (draft) guidelines/guidance was released by regulatory agencies in the EU,

6

US, and Japan, wherein systematic approaches to assessing the risk of DDIs involving

7

transporters as well as drug metabolizing enzymes are given.5-7 Currently, the nonclinical and

8

clinical evaluation of transporter-mediated DDIs is widely recognized as an integral element of

9

drug development.

10

Organic anion transporting polypeptide (OATP) 1B1 (encoded by SLCO1B1), OATP1B3

11

(SLCO1B3), and OATP2B1 (SLCO2B1) are multispecific organic anion transporters mediating

12

the hepatic uptake of anionic drugs,8,9 and inhibition of OATPs could result in clinically relevant

13

DDIs.

14

hydroxymethylglutaryl-CoA reductase inhibitors (statins),10 increased in the clinic with the

15

concomitant administration of cyclosporine A or rifampin, which is believed to be due primarily

16

to the inhibition of the hepatic uptake process via OATP1B1 and OATP1B3.11-13 Therefore,

17

from the viewpoint of clinical safety, understanding of DDI potentials of drug candidates as

18

OATP inhibitors is crucial.

Indeed, the systemic exposures to various OATP substrates, such as 3-

19

To evaluate the inhibitory potencies of drug candidates against OATP1B1, cell-based

20

inhibition assays with representative probe substrates are widely employed. Because of their

21

high detection sensitivity and quantitative nature, radiolabeled compounds, such as

22

[3H]estradiol-17β-glucuronide (E2G), have been preferably used as in vitro probe substrates of

23

OATP1B1, particularly for regulatory submission in the pharmaceutical industries. On the other

24

hand, fluorescent-based methods could also provide highly sensitive, quantitative, and high-

25

throughput measures for prioritizing synthesized compounds based on the OATP1B1 inhibition

26

liabilities in the early stage of drug development. The following fluorescent substrates of


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1

OATPs have been employed in inhibition assays: chenodeoxycholyl-(Nε-nitrobenz-2-oxa-1,3-

2

diazole)-lysine (CDCA-NBD), cholyl-glycylamido-fluorescein (CGamF), cholyl-L-lysyl-

3

fluorescein (CLF), fluorescein-methotrexate (FMTX), 8-fluorescein-cAMP (8-FcA), fluo-3, and

4

fluorescein (FL) (Table 1).

5

Although these fluorescent substrates are useful tools for in vitro inhibition assays, they

6

do not adequately fulfill the requirement of drug discovery. Bile salt derivatives chemically

7

conjugated with NBD or FL as the fluorophore, namely CDCA-NBD, CGamF, and CLF, are

8

substrates of both OATP1B1 and OATP1B3;14-16 however, they are not commercially available,

9

and they need to be synthesized before use. FMTX, 8-FcA, fluo-3, and FL are readily available Fluo-3, a typical fluorescent Ca2+ indicator, is a substrate of

10

from commercial sources.

11

OATP1B3 as well as OATP1B1,17,18 but the fluorescence quantum yield (Φf) in the presence of

12

Ca2+ (0.18) is lower than that of FL (Φf = 0.91).19 FMTX and FL are substrates of both

13

OATP1B1 and OATP1B3,20,21 and the uptake of FL was equivalent to or slightly lower than that

14

of FMTX in OATP1B1- and OATP1B3-expressing cells. For applying FMTX to OATP1B1

15

inhibition assays, the authors noted the need for further optimization.20 8-FcA, a fluorescent

16

analog of cAMP, is one of the most successfully used fluorescent probe substrates for OATP1B1

17

and OATP1B3.22 8-FcA can provide a robust screening measure to identify OATP1B1 or

18

OATP1B3 inhibitors; however its reagent cost is higher than FL.

19

To identify better fluorescent substrates for in vitro OATP1B1 inhibition assays, the

20

present study focused on FL and its derivatives as potential substrates, and a comprehensive

21

analysis was conducted using OATP1B1-, OATP1B3-, and OATP2B1-expressing cells. The

22

fluorescent compounds examined include FL, Oregon green (OG), 2′,7′-dichlorofluorescein

23

(DCF), 4′,5′-dibromofluorescein (DBF), 5-carboxyfluorescein (5-CF), 6-carboxyfluroescein (6-

24

CF), 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDCF), 2′,7′-bis(2-carboxyethyl)-5-(or 6)-

25

carboxyfluorescein (BCECF), calcein, fluo-3, and 8-FcA (Figure 1). We newly identified DCF

26

as a good fluorescent substrate of OATP1B1 in the present study, and the inhibitory effects of 14



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1

test compounds on OATP1B1 using DCF as a probe were compared with those determined

2

using the prototypical radiolabeled substrates.

3 4


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1

Experimental Section

2

Chemicals and Reagents

3

[3H]E2G

(48.9

Ci/mmol),

[3H]estrone-3-sulfate

(E1S)

(45.6

Ci/mmol),

and

4

[3H]cholecystokinin octapeptide (CCK-8) (80.0 Ci/mmol) were purchased from PerkinElmer

5

Life Sciences (Boston, MA). Unlabeled E2G sodium salt, E1S sodium salt, sulfobromophthalein

6

(BSP),

7

hydrochloride, and probenecid were purchased from Sigma-Aldrich (St. Louis, MO).

8

Cyclosporine A (CsA), erythromycin, and cimetidine were from Wako Pure Chemical Industries

9

(Osaka, Japan). BCECF, calcein, and fluo-3 were from Dojindo (Kumamoto, Japan). FL and

10

DCF were purchased from Tokyo Chemical Industry (Tokyo, Japan). CDCF mixed isomer and

11

OG were obtained from Invitrogen (Carlsbad, CA).

12

ritonavir obtained from Calbiochem (Darmstads, Germany), AnaSpec (Fremont, CA), Biolog

13

Life Science Institute (Bremen, Germany), LKT Laboratories (St. Paul, MN), and Abbott

14

Laboratories (Abbott Park, IL), respectively, were used. All other chemicals were of analytical

15

grade and available from commercial sources.

DBF,

rifampin,

sodium

taurocholate,

ketoconazole,

gemfibrozil,

verapamil

5-CF, 6-CF, 8-FcA, tacrolimus, and

16 17

Transporter-expressing Cells.

18

The OATP1B1-expressing human embryonic kidney (HEK) 293 cells and the control

19

(empty-vector transfected) HEK293 cells that we established previously23 were used.

20

OATP1B3- and OATP2B1-expressing HEK293 cells were established in the present study.

21

Briefly, the open reading frame of OATP1B3/SLCO1B3 or OATP2B1/SLCO2B1 was subcloned

22

into a mammalian expression vector pcDNA3.1/Hygro (+) (Invitrogen).

23

transfected into HEK293 cells using Lipofectamine 2000 according to manufacturer’s protocol

24

(Invitrogen).

25

hygromycin B selection.

26

(Invitrogen) supplemented with 10% (v/v) of fetal bovine serum, penicillin (final concentration,

The vector was

HEK293 cells stably expressing OATP1B3 or OATP2B1 were obtained by The cells were grown in Dulbecco’s Modified Eagle Medium



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1

100 units/mL), streptomycin (100 µg/mL), and hygromycin B (80 µg/mL) in a humidified

2

incubator with 5% CO2 at 37°C.

3 4

Cellular Uptake and Inhibition Assays

5

The cellular uptake of radiolabeled or fluorescent substrates, and inhibition assays with a

6

fluorescent substrate (DCF) were conducted as described previously.23 To quantify the amount

7

of fluorescent compounds taken up by transporter-expressing cells or control cells, the cells

8

were lysed overnight with 0.1 N NaOH (500 µL/well) at room temperature, and 200 µL each of

9

the cell lysate samples was directly used for measuring the fluorescence intensity with a

10

microplate spectrofluorometer (SpectraMax M2; Molecular Devices, Sunnyvale, CA).

11

Calibration curves were prepared by spiking each fluorescent compound solution to 0.1 N

12

NaOH (0.1 to 1000 nmol/L). For calcein or fluo-3, 4 µL of 50 mM CaCl2 was added to 200 µL

13

of the cell lysate and samples for calibration curves before measuring the fluorescence intensity

14

because they require Ca2+ to emit fluorescence under the alkaline condition.19,24

15

The inhibitor solutions were prepared as reported earlier.23 Briefly, CsA, BSP, ritonavir,

16

rifampin, tacrolimus, E2G, and gemfibrozil were dissolved in DMSO. Their DMSO original

17

solutions were serially diluted with DMSO, and the prepared DMSO working solutions were

18

diluted 1,000 fold with KH buffer. E1S, erythromycin, TCA, probenecid, and cimetidine were

19

directly dissolved in KH buffer, followed by serial dilution with KH buffer. Ketoconazole and

20

verapamil were dissolved in KH buffer under the acidic pH conditions, followed by serial

21

dilution with KH buffer to prepare the working solutions. The pH of the working solutions was

22

adjusted to 7.4 before use. Then, DCF DMSO stock solution (3 mM) with or without additional

23

blank DMSO was spiked to the inhibitor-containing KH buffer (final DMSO concentration,

24

0.2%). We confirmed that DMSO did not affect the uptake of DCF in OATP1B1-expressing

25

cells and control cells up to a concentration of 0.3% (Figure S1).

26


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1

Estimation of Kinetic Parameters for Transporter-Mediated Uptake of Fluorescent

2

Substrates or Radiolabeled Substrates

3

Uptake of a fluorescent or radiolabeled substrate in transporter-expressing cells and

4

control cells was expressed as the uptake volume (µL/mg protein).23 Transporter-mediated

5

uptake was calculated by subtracting the uptake into control cells from that into transporter-

6

expressing cells. Concentration dependence of the transporter-mediated uptake of selected

7

fluorescent substrates (FL, OG, DCF, DBF, and 8-FcA) or [3H]E2G was examined, and the data

8

were fitted to the Michaelis-Menten equation to calculate the Michaelis constant (Km, µM ) and

9

maximum uptake rate (Vmax, pmol/min/mg protein) values:

10

v =

Vmax × S Km + S

(1)

11

where v is the uptake rate (pmol/min/mg protein), and S is the substrate concentration (µM).

12

Data fitting was performed by the iterative nonlinear least-squares regression analysis with

13

MULTI program.25

14

The effect of an inhibitor on OATP1B1-mediated uptake of a substrate (% of control) was

15

examined at various inhibitor concentrations, and the half maximal inhibitory concentration

16

(IC50) was estimated from the following equation as reported previously:23

17

uptake (% of control ) =

CL i × 100 = CL

P [ I] 1+ IC 50

(2)

18

where CL and CLi represent the uptake clearance in the absence and presence of an inhibitor,

19

respectively, and [I] is the inhibitor concentration. P (initial value = 100) was set as a free

20

parameter to achieve the best fit in the nonlinear iterative least squares regression analysis. For

21

the inhibition of OATP1B1-mediated uptake of DCF by E1S, the data were fitted to the

22

following equation to estimate the IC50 value:



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1

uptake (% of control ) =

CL i × 100 = CL

P + P0 [ I] 1+ IC 50

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(3)

2

where P0 represents the non-inhibitable component that was observed even at the maximum

3

tested concentration of E1S. P and P0 (initial value of P + P0 = 100) were set as free parameters.

4

The MULTI program was also used for estimating IC50 values of inhibitors.

5 6 7

The inhibition constant Ki was estimated from the following equation, with the assumption of competitive inhibition:26 Ki =

IC50 S 1+ Km

(4)

8 9

Statistical Analysis

10

The data are given as mean ± S.D. The Student’s two-tailed nonpaired t test or a one-way

11

analysis of variance followed by Dunnett’s post hoc test were performed to identify significant

12

differences between groups where appropriate (Prism 6, GraphPad Software, La Jolla, CA).

13

The differences were considered significant when P < 0.05.

14


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1

Results

2

Uptake of FL and Its Derivatives by OATP1B1-, OATP1B3-, and OATP2B1-expressing

3

Cells and Control Cells

4

To investigate FL and its derivatives (Figure 1) as potential OATP substrates, their uptake

5

was evaluated using OATP1B1-, OATP1B3-, and OATP2B1-expressing cells and control cells

6

for 5 min of exposure at 1 (for FL, OG, DCF, DBF, and 8-FcA) or 10 µM (for 5-CF, 6-CF,

7

CDCF, BCECF, calcein, and fluo-3 because of the limit of detection) (Figure 2).

8

OATP1B1-, OATP1B3-, and OATP2B1-expressing cells used in the present study displayed

9

greater uptake of the typical substrates (184 µL/5 min/mg protein for 0.004 µM [3H]E2G in

10

OATP1B1; 94.5 µL/5 min/mg protein for 0.001 µM [3H]CCK-8 in OATP1B3; 126 µL/5 min/mg

11

protein for 0.005 µM [3H]E1S in OATP2B1) than the control cells (3.02, 4.76, and 5.86 µL/5

12

min/mg protein, respectively), indicating that the transporter-expressing cell systems had

13

adequate uptake activities.

The

14

Under such conditions, DCF, OG, and DBF were newly identified as good substrates of

15

OATP1B1 (Figure 2). Among the fluorescent compounds examined, DCF exhibited the greatest

16

uptake in OATP1B1-expressing cells, which was 41-fold higher than that in control cells. DCF

17

uptake in OATP2B1-expressing cells was also up to 4.8-fold higher than that in control cells.

18

No significant uptake was observed for DCF in OATP1B3-expressing cells. OG was identified

19

as a substrate of all OATPs, and the uptake in OATP1B1-, OATP1B3-, and OATP2B1-

20

expressing cells were 31-, 6.3-, and 5.8-fold higher than that in control cells, respectively. DBF

21

was also a substrate of all OATPs examined, with the highest uptake recorded in OATP2B1-

22

expressing (20-fold higher than that in control cells), followed by OATP1B1- (8.0-fold) and

23

OATP1B3-expressing cells (2.5-fold). As previously reported, the present study confirmed that

24

FL and 8-FcA were substrates of both OATP1B1 and OATP1B3, and fluo-3 was a substrate of

25

OATP1B3.17,21,22 The uptake of FL and 8-FcA in OATP1B1-expressing cells was 6.8- and 3.3-

26

fold higher than that in control cells, respectively. In addition, FL and CDCF displayed slightly



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1

but significantly higher uptake in OATP2B1- and OATP1B1-expressing cells, respectively, than

2

in control cells. No significant uptake was observed for 5-CF, 6-CF, BCECF, and calcein in any

3

transporter-expressing cells tested.

4

For FL, OG, DCF, DBF, and 8-FcA, the time profiles of the uptake in OATP1B1-

5

expressing cells and control cells were evaluated (Figure 3). Their uptake was linear over 2

6

(DBF) or 10 min (FL, OG, DCF, and 8-FcA) in OATP1B1-expressing cells; thus, an incubation

7

time of 30 s (DBF) or 5 min (FL, OG, DCF, and 8-FcA) was used in the subsequent experiments.

8 9 10

Kinetic Analysis of OATP1B1-mediated Uptake of FL, OG, DCF, DBF, and 8-FcA and OATP2B1-mediated Uptake of DBF

11

The concentration dependence of the OATP1B1-mediated uptake of FL (0.3–100 µM),

12

OG (1-500 µM), DCF (0.1-100 µM), DBF (0.3-100 µM), and 8-FcA (1-100 µM) was

13

investigated using OATP1B1-expressing cells and control cells. Representative Eadie–Hofstee

14

plots of the uptake are shown in Figure 4, and the obtained Km, Vmax, and Vmax/Km values are

15

summarized in Table 2. The OATP1B1-mediated uptake of the five fluorescent compounds

16

were composed of one saturable component, and DBF (Km, 4.19 µM) and DCF (5.29 µM)

17

displayed smaller Km values for OATP1B1 than 8-FcA (9.05 µM), FL (19.1 µM), and OG (54.1

18

µM). On the basis of the Vmax/Km values, DCF exhibited the greatest OATP1B1-mediated

19

uptake clearance (16.5 µL/min/mg protein), followed by DBF (13.9), OG (3.53), 8-FcA (2.97),

20

and FL (1.37).

21

As DBF was also identified as a good OATP2B1 substrate in the single-time point assay

22

(Figure 2), the time profiles of DBF uptake in OATP2B1-expressing cells and control cells and

23

the concentration dependence of its OATP2B1-mediated uptake were evaluated (Figure S2).

24

The uptake of DBF was linear at least up to 1 min in OATP2B1-expressing cells, and the Km,

25

Vmax, and Vmax/Km values for OATP2B1 were 10.0 µM, 236 pmol/min/mg protein, and 27.1

26

µL/min/mg protein, respectively.


Page 14 of 48

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1 2

OATP1B1 Inhibition Assay with DCF as a Fluorescent Probe Substrate

3

Because the OATP1B1-mediated uptake activity and signal-to-noise ratio of DCF were

4

the highest among the FL derivatives tested, we focused on DCF for further evaluation, and the

5

in vitro inhibitory effects of 14 compounds (E1S, CsA, BSP, ritonavir, rifampin, tacrolimus,

6

erythromycin, E2G, taurocholate, ketoconazole, gemfibrozil, verapamil, probenecid, and

7

cimetidine) on OATP1B1 were investigated using DCF as the fluorescent probe substrate

8

(Figure 5). All tested compounds, except for cimetidine, inhibited the OATP1B1-mediated

9

uptake of DCF in a concentration-dependent manner, and the Ki values of the inhibitors ranged

10

from 0.0449 µM (E1S) to 39.8 µM (probenecid) (Table 3). Cimetidine slightly inhibited the

11

OATP1B1-mediated uptake of DCF only at the highest concentration examined (81% of control

12

at 1mM).

13

Ki values determined with DCF as a probe substrate were compared with those we

14

previously determined under the same experimental conditions using prototypical radiolabeled

15

substrates [3H]E2G, [3H]E1S, and [3H]BSP (Figure 6).23 This comparison revealed that the Ki

16

values obtained using DCF exhibited good agreement with those obtained using [3H]E2G, with

17

the differences in the Ki values falling within 2.1-fold.

18

inhibitors examined for [3H]E1S and [3H]BSP uptake were higher than those for DCF uptake.

19

Compared with DCF, [3H]E1S yielded more than 10-fold higher Ki values for ritonavir (150-

20

fold), rifampin (20-fold), and gemfibrozil (21-fold). Similarly, [3H]BSP yielded more than 10-

21

fold higher Ki values for ritonavir (11-fold), erythromycin (12-fold), taurocholate (18-fold), and

22

probenecid (19-fold) compared with DCF.

Conversely, the Ki values of all

23 24

Mutual Inhibition of the OATP1B1-mediated Uptake of DCF and E2G

25

To evaluate the mode of inhibition between DCF and E2G, the concentration dependence

26

of the OATP1B1-mediated uptake of DCF and [3H]E2G was investigated in the presence or



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 48 49 50 51 52 53 54 55 56 57 58 59 60

1

absence of unlabeled E2G and DCF, respectively. The representative data are given in Figure 7,

2

and the obtained kinetic parameters (Km and Vmax) are summarized in Table 4. In the presence

3

of 3 or 10 µM DCF, the Km value of [3H]E2G (7.96 µM) increased to 10.7 and 16.6 µM,

4

respectively, with a minimal effect on Vmax value. Similarly, in the presence of 3 or 10 µM

5

unlabeled E2G, the Km value of DCF (4.82 µM) increased to 6.38 and 12.6 µM, respectively,

6

with minimal effect on Vmax value. This mutual inhibition study suggested that DCF and E2G

7

competitively inhibited each other on OATP1B1.

8


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1

Discussion

2

To develop drugs with low or no OATP1B1-mediated DDI risk as perpetrators, it is

3

important to evaluate the inhibitory potencies of synthesized compounds against OATP1B1

4

from the early stage of drug development, wherein fluorescence-based in vitro assays are

5

preferably used for the purpose of high-throughput screening. This study was designed to

6

identify fluorescent OATP1B1 probe substrates which are readily available from commercial

7

sources and applicable to developing fluorescence-based OATP1B1 inhibition assays.

8

To understand the substrate specificities of hepatic OATPs for FL derivatives, their uptake

9

was evaluated in a single time-point assay using OATP1B1-, OATP1B3-, and OATP2B1-

10

expressing cells and control cells (Figure 2). Consistent with previous studies, the present study

11

confirmed that FL and 8-FcA are substrates of both OATP1B1 and OATP1B3,21,22 and fluo-3 is

12

a substrate of OATP1B3.17 Although fluo-3 was also reported to be a substrate of OATP1B1

13

with much lower transport activity than that via OATP1B3,18 the uptake in OATP1B1-

14

expressing cells was similar to that in control cells in the present study. CDCF, which was

15

suggested to be a substrate of hepatic Oatps in rats,27 was identified as an OATP1B1 substrate in

16

this study; however, its uptake in OATP1B1-expressing cells was only 2.5-fold higher than that

17

in control cells.

18

In the present study, DCF, DBF, and OG were newly identified as good fluorescent

19

OATP1B1 substrates with higher uptake activities and signal-to-noise ratios than the known

20

fluorescent substrates FL and 8-FcA (Figure 2). Among them, DCF displayed the highest

21

uptake in OATP1B1-expressing cells (41-fold higher than that in control cells). OG and DBF

22

were also newly identified as OATP1B3 substrates. Although the uptake of OG in OATP1B3-

23

expressing cells (3.5 µL/mg protein for 5 min) was lower than that of 8-FcA (16.68 µL/mg

24

protein for 5 min), the ratio of uptake in OATP1B3-expressing cells to that in control cells was

25

slightly greater for OG (6.1 fold) than that for 8-FcA (4.8 fold). FL, OG, DCF, and DBF were

26

identified as OATP2B1 substrates, of which DBF displayed the highest uptake in OATP2B1-



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 48 49 50 51 52 53 54 55 56 57 58 59 60

1

expressing cells (27-fold higher than that in control cells). Conversely, no significant uptake of

2

5-CF, 6-CF, BCECF, and calcein was observed in any transporter-expressing cells.

3

The substrate selectivity and transport activities of the hepatic OATPs against FL

4

derivatives were largely affected by the position and/or species of substituents introduced into

5

FL. The majority of fluorescent OATP1B1 substrates (FL, OG, DCF, and DBF) have one

6

carboxylic acid group at the 3-position of the benzene ring (Figure 1 and 2). The introduction of

7

another carboxylic acid group on the benzene ring in addition to the 3-position dramatically

8

reduced OATP1B1-mediated uptake activity, and 5-CF and 6-CF were nonsubstrates of

9

OATP1B1. The same tendency was observed in CDCF, the uptake of which via OATP1B1 was

10

lower than that of DCF. Among FL, OG, and DCF, DCF exhibited the highest OATP1B1-

11

mediated uptake, followed by OG and FL. This suggested that the substitution of the 2′ and 7′

12

positions of xanten with halogen atoms could selectively enhance OATP1B1-mediated uptake

13

activity with minimal effects on OATP1B3 and OATP2B1. Although substitution of the 4′ and

14

5′ positions of xanten with bromine atoms also improved OATP1B1-mediated uptake activity,

15

the effect was more evident for OATP2B1, as demonstrated using DBF. Therefore, the positions

16

of halogen substitution on xanten could affect the recognition by OATP1B1 and OATP2B1.

17

Comparing FL and 8-FcA, their uptake via OATP1B1 was similar; however, the OATP1B3-

18

mediated uptake of 8-FcA was greater than that of FL. Substitution of the 5-position of the

19

benzene ring with a large moiety may affect the recognition by OATP1B3.

20

For further characterization of the OATP1B1-mediated uptake of DCF, DBF, and OG,

21

their time- and concentration-dependent uptake was examined in comparison with FL and 8-

22

FcA (Figure 3 and 4). Their uptake in OATP1B1-expressing cells was higher than that in

23

control cells over the periods examined (Figure. 3). Among them, OATP1B1-mediated uptake

24

clearance defined by Vmax/Km was greatest for DCF (16.5 µL/min/mg protein), followed by DBF

25

(13.9), OG (3.53), 8-FcA (2.97), and FL (1.37) (Table 2). Because the uptake clearances of DCF,

26

DBF, and OG were greater than those of FL and 8-FcA which have been used as the probes in


Page 18 of 48

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1

the fluorescence-based OATP1B1 inhibition assays,21,22 DCF, DBF, and OG also serve as

2

fluorescent probes. Considering the superiority in the detection sensitivity resulting from the

3

higher OATP1B1-mediated transport activity and signal-to-noise ratio, this study focused on

4

DCF for further evaluation. We also identified DBF as a good substrate of OATP2B1 (Figure

5

S2). Further investigation with various inhibitors will be needed to support the idea that in vitro

6

OATP2B1 inhibition assays with DBF as a probe can evaluate DDI risk involving OATP2B1 in

7

the intestines and other tissues.28

8

When evaluating the inhibitory potency of a compound on OATP1B1 in vitro, care is

9

required in probe substrate selection because substrate-dependent inhibition was reported for

10

OATP1B1.23,26,29 We previously investigated the inhibitory potencies of 14 test compounds on

11

OATP1B1 using three prototypical probe substrates, [3H]E2G, [3H]E1S, and [3H]BSP, of which

12

[3H]E2G provided the lowest Ki value for all inhibitors tested, whereas [3H]E1S or [3H]BSP

13

yielded 10-fold or higher Ki values for several inhibitors.23 Moreover, the Ki values of CsA,

14

rifampin, and gemfibrozil for OATP1B1 determined with clinically used substrate drugs (e.g.,

15

statins) were generally comparable to those obtained using [3H]E2G as a substrate, rather than

16

[3H]E1S and [3H]BSP.26

17

substrates or E2G as a surrogate should be used as the OATP1B1 probe substrate in the in vitro

18

inhibition assays.26 To clarify whether DCF could be classified as an E2G-type substrate, we

19

examined the inhibitory effects of the 14 compounds on the OATP1B1-mediated uptake of DCF

20

(Figure 5), and the obtained Ki values were compared with those previously determined using

21

the prototypical probe substrates (Table 3 and Figure 6). There was an excellent agreement of

22

the Ki values between DCF and [3H]E2G, whereas [3H]E1S and [3H]BSP yielded higher Ki

23

values for all inhibitors tested than DCF. Therefore, DCF behaved similarly as [3H]E2G in

24

terms of the evaluation of the susceptibility to OATP1B1 inhibition, suggesting that DCF as

25

well as [3H]E2G can be used as surrogate probes for clinically used OATP1B1 substrate drugs in

26

its inhibition assays. Moreover, as with [3H]E2G, DCF can also provide more conservative Ki

On the basis of these findings, we proposed that clinically used



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Page 20 of 48

1

values for OATP1B1 than [3H]E1S and [3H]BSP, which will be helpful to avoid false-negative

2

DDI predictions potentially caused by substrate-dependent variations in Ki values.

3

Multiple binding sites were suggested on OATP1B1.23,30

To evaluate the mode of

4

inhibition between DCF and E2G, mutual inhibition studies were conducted (Figure 7).

5

OATP1B1-mediated uptake of DCF and E2G inhibited each other in a competitive manner,

6

suggesting that both substrates share the same binging site on OATP1B1. This may be an

7

underlying mechanism of similar susceptibility to OATP1B1 inhibition between DCF and E2G.

8

Although the present study focused on single transporter-transfected cell systems,

9

fluorescent probe substrates can also be used for other in vitro assay systems. De Bruyn et al.

10

demonstrated saturable uptake of FL in rat and human hepatocytes.21 Considering that DCF was

11

more selectively taken up by OATP1B1 among the hepatic OATPs than FL and 8-FcA under our

12

assay conditions (Figure 2), DCF may also be used as an OATP1B1-selective fluorescent probe

13

substrate in human hepatocytes, which would be useful for rapidly evaluating the inter-batch

14

differences in OATP1B1-mediated transport functions.31

15

inhibitors32 will clarify the quantitative contribution of OATP1B1 to the hepatic uptake of DCF.

16

In addition, fluorescent probe substrates can also be used for evaluating the DDI potential of

17

drug candidates via hepatobiliary transport processes using in vitro systems such as sandwich-

18

cultured human hepatocytes and double-transfected cells.33,34. To achieve this, an understanding

19

of the fluorescent probes as substrates of biliary efflux transporters is crucial. Therefore, further

20

investigation of DCF as a potential substrate of MRP2, P-glycoprotein, and breast cancer

21

resistance protein will be required to extend its utility. The metabolic stability of DCF also

22

needs to be evaluated when applied to more physiologically relevant in vitro systems such as

23

hepatocytes.

Further studies using selective

24

In conclusion, DCF, DBF, and OG were newly identified as good fluorescent substrates of

25

OATP1B1, and DCF displayed the highest OATP1B1-mediated uptake among the fluorescent

26

compounds examined.

DCF behaved similarly as [3H]E2G in terms of susceptibility to


Page 21 of 48

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1

OATP1B1 inhibition, indicating that DCF as well as [3H]E2G can be used as surrogate probes

2

for clinically used OATP1B1 substrate drugs and can provide more conservative Ki values than

3

[3H]E1S and [3H]BSP in vitro. Therefore, in vitro fluorescence-based OATP1B1 inhibition

4

assays with DCF as a probe can both offer high-throughput, highly sensitive measures for

5

evaluating the inhibitory potential of newly synthesized compounds and help mitigate the risk of

6

false-negative DDI prediction potentially caused by substrate-dependent Ki variations. Our

7

findings will contribute for creating drugs with low or no risk of OATP1B1-mediated DDIs.

8 9



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 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Acknowledgements

2

We sincerely thank Hisae Noguchi (Eisai Co., Ltd) for technical support.

3 4

Supporting Information

5

IC50 values of 14 compounds for OATP1B1-mediated uptake of DCF (Table S1); effect of

6

DMSO on the uptake of DCF by OATP1B1-expressing cells and control cells (Figure S1);

7

uptake of DBF in OATP2B1-expressing cells and control cells (Figure S2).

8


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1

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Ohtsuki, S.; Schaefer, O.; Kawakami, H.; Inoue, T.; Liehner, S.; Saito, A.; Ishiguro, N.;

Hirano, M.; Maeda, K.; Shitara, Y.; Sugiyama, Y. Drug-drug interaction between

Swift, B.; Pfeifer, ND.; Brouwer, KL. Sandwich-cultured hepatocytes: an in vitro model

Sasaki, M.; Suzuki, H.; Ito, K.; Abe, T.; Sugiyama, Y. Transcellular transport of organic

Maglova, LM.; Jackson, AM.; Meng, XJ.; Carruth, MW.; Schteingart, CD.; Ton-Nu,

33


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Tables Table 1 Reported fluorescent substrates of Hepatic OATP/Oatp family Fluorescent substrates CDCA-NBD CA-NBD DCA-NBD LCA-NBD UDCA-NBD CGamF CLF FMTX Flutax-2 FL

Substances labeled with fluorophores Bile acid (Chenodeoxycholic acid) Bile acid (Cholic acid) Bile acid (Deoxycholic acid) Bile acid (Lithocholic acid) Bile acid (Urosodeoxycholic acid) Bile acid (Cholic acid) Bile acid (Cholic acid) Methotrexate Paclitaxel -

Fluorophores

Transporters

References

NBD

OATP1B1, OATP1B3, Oatp(s)

14, 35

NBD

OATP1B1, OATP1B3, Otap(s)

14, 35

NBD

OATP1B1, OATP1B3

14

NBD

OATP1B1, OATP1B3

14

NBD

OATP1B1, OATP1B3

14

FL

OATP1B1, OATP1B3, Oatp(s)

15, 35

FL

OATP1B1, OATP1B3

16

FL OG FL

OATP1B1, OATP1B3 OATP1B3 OATP1B1, OATP1B3, Oatp(s)

20 20 20, 21

8-FcA

cAMP

FL

OATP1B1, OATP1B3

22

Fluo-3

-

Fluo-3

OATP1B1, OATP1B3

17, 18

-

CDCF

Oatp(s)

27

CDCF -: Not applicable.

Oatp: rat organic anion transporting polypeptide.

27

ACS Paragon Plus Environment


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Table 2 Kinetic parameters of OATP1B1-mediated uptake of FL, OG, DCF, DBF, and 8FcAa Substrates FL OG DCF DBF a

19.1 54.1 5.29 4.16

Km

Vmax

Vmax/Km

µM

pmol/min/mg protein

µL/min/mg protein

± ± ± ±

6.5 9.6 1.51 2.53

26.5 187 87.9 48.1

± ± ± ±

10.9 11 35.9 8.0

1.37 3.53 16.5 13.9

± ± ± ±

0.33 0.63 3.7 5.8

8-FcA 9.05 ± 3.44 26.0 ± 8.1 2.97 ± 0.69 Kinetic parameters were estimated by nonlinear least-squares regression analysis based on Eq.

1 as described under Experimental Section and are shown as mean ± S.D. of 3 (OG), 4 (8-FcA), 5 (FL and DBF), or 6 (DCF) independent experiments.


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Table 3. Ki values of test compounds for OATP1B1-mediatd uptake of DCFa Inhibitors Ki (µM) E1 S 0.0449 ± 0.0039 CsA 0.116 ± 0.015 BSP 0.102 ± 0.012 Ritonavir 0.320 ± 0.024 Rifampin 0.355 ± 0.061 Tacrolimus 0.533 ± 0.046 Erythromycin 5.27 ± 0.63 E2 G 6.63 ± 0.91 Ketoconazole 11.5 ± 0.9 Taurocholate 8.97 ± 1.97 Verapamil 15.3 ± 2.3 Gemfibrozil 18.1 ± 3.9 Probenecid 39.8 ± 4.4 a OATP1B1-mediated uptake of DCF (3 µM) was determined for 5 min in the presence and absence of inhibitors (Figure 5), and the IC50 values were obtained by a nonlinear least-squares regression analysis based on Eq. 2 or Eq. 3. Using the IC50 values (Table S1), the Km value of DCF for OATP1B1 (5.29 µM), and substrate (DCF) concentration (3 µM), the Ki values were calculated according to Eq. 4. Each value represents parameter estimate ± computer-calculated S.D.



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Table 4. Kinetic parameters of OATP1B1-mediated uptake of [3H]E2G and DCF in the presence and absence of DCF and unlabeled E2Ga Substrates 3

Inhibitors

DCFc

[ H]E2G

No inhibitor DCF (3 µM)

Km µM 7.96 ± 1.60 10.7 ± 1.7

DCF (10 µM)

16.6

E2G (3 µM) E2G (10 µM) NA, not applicable. a

b

± NA NA

2.5***

Vmax pmol/min/mg protein 259 ± 76 254 ± 66 213

±

Km µM 4.82 ± 1.23 NA

Vmax pmol/min/mg protein 68.3 ± 13.1 NA

NA

NA

44

NA NA

6.38 12.6

± ±

1.30 1.2

***

59.3

±

10.2

63.3

±

12.3

Kinetic parameters were estimated by nonlinear least-squares regression analysis based on Eq. 1 under Experimental Section and are presented as

mean ± S.D. of 3 independent experiments. b

c

Concentration-dependent uptake of [3H]E2G (0.002-100 µM) for 5 min was examined in the absence and presence of DCF at 3 or 10 µM.

Concentration-dependent uptake of DCF (0.1-100 µM) for 5 min was examined in the absence and presence of unlabeled E2G at 3 or 10 µM.

***

P < 0.001 compared with parameters determined without any inhibitors.


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Figure Captions Figure 1. Chemical structures of FL and its derivatives used in this study. (A) FL, (B) OG, (C) DCF, (D) DBF, (E) 5-CF, (F) 6-CF, (G) CDCF, (H) BCECF, (I) calcein, (J) fluo-3, and (K) 8-FcA.

Figure 2. Uptake of FL and its derivatives in OATP1B1-, OATP1B3-, and OATP2B1expressing cells and control cells.

Transporter-expressing cells and control cells were

incubated with KH buffer containing FL (A), OG (B), DCF (C), DBF (D), 5-CF (E), 6-CF (F), CDCF (G), BCFCF (H), calcein (I), fluo-3 (J), or 8-FcA (K) for 5 minutes at 37°C. Final substrate concentration in the KH buffer was 1 µM (for FL, OG, DCF, DBF, and 8-FcA) or 10 µM (for CDCF, BCECF, 5-CF, 6-CF, calcein, and fluo-3, due to the limit of detection). Each point represents the mean ± S.D. (n = 3). *P < 0.05 and ***P < 0.001 between transporterexpressing cells and control cells.

Figure 3. Time profiles of the uptake of FL, OG, DCF, DBF, and 8-FcA in OATP1B1expressing cells and control cells. OATP1B1-expressing cells (closed circles) and control cells (open circles) were incubated with FL (A), OG (B), DCF (C), DBF (D), or 8-FcA (E) at 1 µM over a period of 30 minutes at 37°C. Each point represents the mean ± S.D. (n = 3).

Figure 4. Concentration dependence of OATP1B1-mediated uptake of FL, OG, DCF, DBF, and 8-FcA. OATP1B1-expressing cells and control cells were incubated with FL (A, 0.3-100 µM), OG (B, 1-500 µM), DCF (C, 0.1-100 µM), DBF (D, 0.3-100 µM), or 8-FcA (E, 1-100 µM) for 5 minutes (FL, OG, DCF, and 8-FcA) or 30 seconds (DBF) at 37°C. Their OATP1B1mediated uptake were obtained by subtracting the uptake in control cells from that in OATP1B1-expressing cells. Representative data from 3 (OG), 4 (8-FcA), 5 (FL and DBF), or 6 (DCF) independent experiments are shown as Eadie-Hofstee plots, and each point represents the



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mean ± S.D. (n = 3). Fitted lines that were obtained by a nonlinear least-squares regression analysis based on Eq. 1 are presented as solid lines.

Figure 5. Inhibitory effects of 14 compounds on OATP1B1-mediated uptake of DCF. Uptake of DCF (3 µM) mediated by OATP1B1 was examined for 5 minutes at 37°C in the presence and absence of (A) E1S (0.01-10 µM), (B) CsA (0.01-10 µM), (C) BSP (0.01-10 µM), (D) ritonavir (0.01-10 µM), (E) rifampin (0.01-10 µM), (F) tacrolimus (0.01-10 µM), (G) erythromycin (1-1000 µM), (H) E2G (0.01-100 µM), (I) taurocholate (1-1000 µM), (J) ketoconazole (1-100 µM), (K) gemfibrozil (1-500 µM), (L) verapamil (1-300 µM), (M) probenecid (1-1000 µM), and (N) cimetidine (1-1000 µM).

The data are taken from 2

independent experiments with 6 samples per concentration and are shown as % of control as described under Experimental Section. Solid lines represent fitted lines for the inhibition of DCF uptake by the inhibitors that were obtained by a nonlinear least-squares regression analysis based on Eq. 2 or Eq. 3. Each point represents the mean ± S.D. (n = 6).

Figure 6. Comparison of Ki values for OATP1B1 between DCF and [3H]E2G (A), [3H]E1S (B), or [3H]BSP (C). Ki values determined with DCF are summarized in Table 3. Ki values determined with radiolabeled substrates were obtained from our previous study.23 Each point represents parameter estimate ± computer-calculated S.D. The solid line and dashed lines represent the line of unity and the lines of 1:10 and 10:1 correlations, respectively. 1, E1S; 2, CsA; 3, BSP; 4, ritonavir; 5, rifampin; 6, tacrolimus; 7, erythromycin; 8, E2G; 9, ketoconazole; 10, taurocholate; 11, verapamil; 12, gemfibrozil; 13, probenecid. The Ki value of E1S for [3H]BSP uptake was eliminated from the panel (C) because E1S showed biphasic inhibition for [3H]BSP uptake.23 The linear regression equations and correlation coefficients (R) were Y = 0.506 × X + 1.577 and 0.978 (A), Y = 0.0701 × X + 3.9030 and 0.699 (B), and Y = 0.0524 × X + 3.0958 and 0.973 (C), respectively.


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Figure 7. Mutual inhibition of OATP1B1-mediated uptake of DCF and [3H]E2G. (A) Concentration-dependent uptake of [3H]E2G (0.002 to 100 µM) for 5 minutes in the absence (closed circles) or presence of DCF at 3 (open squares) or 10 µM (open diamonds). (B) Concentration-dependent uptake of DCF (0.1 to 100 µM) for 5 minutes in the absence (closed circles) or presence of unlabeled E2G at 3 (open squares) or 10 µM (open diamonds). Representative data from 3 independent experiments are shown as Eadie-Hofstee plots. Each point represent the mean ± S.D. (n = 3). Fitted lines were obtained by a nonlinear least-squares regression analysis based on the Eq. 1 under Materials and Methods.



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Illustrations: Figures Figure 1.


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Figure 2.



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Figure 3.


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Figure 4.



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Figure 5.


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Figure 6.



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Figure 7.


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Figure 1 233x162mm (300 x 300 DPI)



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Figure 2 272x182mm (300 x 300 DPI)


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Figure 3 274x185mm (300 x 300 DPI)



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Figure 4 268x182mm (300 x 300 DPI)


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Figure 5 184x281mm (300 x 300 DPI)



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Figure 6 276x106mm (300 x 300 DPI)


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Figure 7 261x139mm (300 x 300 DPI)



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TOC 172x106mm (300 x 300 DPI)


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Investigation of Fluorescein Derivatives as Substrates of Organic

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