Evaluation of OATP1B-Mediated Drug-Drug Interaction Risks Of

Home , Paclitaxel

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Title

Alteration in the plasma concentrations of endogenous OATP1B-biomarkers in non-small cell lung cancer patients treated with paclitaxel

Authors Downloaded from

Daiki Mori1, Hiroo Ishida2, Tadahaya Mizuno1, Sojiro Kusumoto3, Yusuke Kondo1, Saki Izumi4, Genki

1 4 1 2 5 1

Nakata , Yoshitane Nozaki , Kazuya Maeda , Yasutsuna Sasaki , Ken-ichi Fujita , Hiroyuki Kusuhara dmd.aspetjournals.org

Affiliations at ASPET Journals on September 26, 2021

1 Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the

University of Tokyo, Tokyo, Japan

2 Division of Medical Oncology, Department of Medicine, Showa University School of Medicine,

Tokyo, Japan

3 Division of Respiratory Medicine and Allergology, Department of Medicine, Showa University

School of Medicine, Tokyo, Japan

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

Ibaraki, Japan.

5 Division of Cancer Genome and Pharmacotherapy, Department of Clinical Pharmacy, Showa

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University School of Pharmacy, Tokyo, Japan

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021

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Running Title

Paclitaxel effects on the endogenous OATP1B biomarkers.

Corresponding Author

Hiroyuki Kusuhara, Ph.D. Downloaded from

Affiliation: Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences,

The University of Tokyo dmd.aspetjournals.org

Address: 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Tel: +81-3-5841-4770; Fax: +81-3-5841-4766; Email: [email protected] at ASPET Journals on September 26, 2021

Numbers for manuscript elements

Text pages:37

Number of tables: 2

Number of figures:5

Number of references:44

Number of Supplemental Tables: 1

Number of Supplemental Figures:11

Number of references in Supplemental Materials:1

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Number of words in the Abstract: 226/250

Number of words in the Introduction: 642/750

Number of words in the Discussion: 1421/1,500

Abbreviations Downloaded from

AUC, area under the plasma concentration–time curve; C4, 7-hydroxy-4-cholesten-3-one; CA,

cholate; CCK-8, cholecystokinin octapeptide; CPI, coproporphyrin I; CPIII, coproporphyrin III; DDI, dmd.aspetjournals.org drug–drug interaction; E217G, estradiol 17 glucuronide; GCA, glycocholate; GCDCA, glycochenodeoxycholate; GCDCA-G, glycochenodeoxycholate glucuronide; GCDCA-S, at ASPET Journals on September 26, 2021 glycochenodeoxycholate sulfate; GDCA, glycodeoxycholate; GDCA-G, glycodeoxycholate glucuronide; GDCA-S, glycodeoxycholate sulfate; GLCA, glycolithocholate; HEK293, human embryonic kidney cells 293; LCA, lithocholate; LC-MS/MS, liquid chromatography-tandem mass spectrometry; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; SEM, standard error of the mean; TCA, taurocholate; TLCA, taurolithocholate

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Abstract

Paclitaxel has been considered to cause OATP1B-mediated drug–drug interactions at therapeutic doses; however, its clinical relevance has not been demonstrated. This study aimed to elucidate in vivo inhibition potency of paclitaxel against OATP1B1 and OATP1B3 using endogenous OATP1B

biomarkers. Paclitaxel is an inhibitor of OATP1B1 and OATP1B3 with Ki of 0.579 ± 0.107 M and Downloaded from

5.29 ± 3.87 M, respectively. Preincubation potentiated its inhibitory effect on both OATP1B1 and

OATP1B3 with Ki of 0.154 ± 0.031 M and 0.624 ± 0.183 M, respectively. Ten non-small cell lung dmd.aspetjournals.org cancer patients who received 200 mg/m2 of paclitaxel by a 3-h infusion were recruited. Plasma concentrations of 10 endogenous OATP1B biomarkers, namely, coproporphyrin I, coproporphyrin III, at ASPET Journals on September 26, 2021 glycochenodeoxycholate-3-sulfate, glycochenodeoxycholate-3-glucuronide, glycodeoxycholate-3- sulfate, glycodeoxycholate-3-glucuronide, lithocholate-3-sulfate, glycolithocholate-3-sulfate, taurolithocholate-3-sulfate, and chenodeoxycholate-24-glucuronide, were determined in the non-small cell lung cancer patients on the day before paclitaxel administration, and after the end of paclitaxel infusion for 7 hours. Paclitaxel increased the area under the plasma concentration–time curve (AUC) of the endogenous biomarkers 2–4-fold although a few patients did not show any increment in the

AUC ratios of lithocholate-3-sulfate, glycolithocholate-3-sulfate, and taurolithocholate-3-sulfate.

Therapeutic doses of paclitaxel for the treatment of non-small cell lung cancer (200 mg/m2) will cause significant OATP1B1 inhibition during and at the end of the infusion. This is the first demonstration

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that endogenous OATP1B biomarkers could serve as surrogate biomarkers in patients.

Significance Statement

Endogenous biomarkers can address practical and ethical issues in elucidating transporter-mediated drug–drug interaction (DDI) risks of anticancer drugs clinically. We could elucidate a significant Downloaded from increment of the plasma concentrations of endogenous OATP1B biomarkers following a 3-h infusion

(200 mg/m ) of paclitaxel, a time-dependent inhibitor of OATP1B, in non-small cell lung cancer dmd.aspetjournals.org patients. The endogenous OATP1B biomarkers are useful to assess the possibility of OATP1B- mediated DDIs in patients, and help in appropriate designing dosing schedule to avoid the DDIs. at ASPET Journals on September 26, 2021

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Introduction

Organic anion transporting polypeptides 1B1/SLCO1B1 (OATP1B1) and 1B3/SLCO1B3

(OATP1B3) are specifically expressed on the sinusoidal membrane of hepatocytes to mediate the uptake of their substrates from the blood circulation into hepatocytes for further elimination to the bile and metabolism (Maeda, 2015; Patel et al., 2016; Hannah H. Lee and Ho, 2017; McFeely et al., 2019). Downloaded from

OATP1B1 and OATP1B3 are multispecific transporters that recognize various anionic drugs and play

key roles in the clearance of such drugs. Therefore, OATP1B-mediated drug–drug interactions (DDIs dmd.aspetjournals.org via induction or inhibition) could modulate the pharmacokinetics of drugs, and thereby, their pharmacological effect or toxicity (Maeda, 2015; Patel et al., 2016; Hannah H. Lee and Ho, 2017; at ASPET Journals on September 26, 2021

Asaumi et al., 2019; McFeely et al., 2019). Regulatory agencies in Japan, the US, and EU strongly recommend pharmaceutical industries in their guidelines to assess the possibility of OATP1B- mediated DDIs with their new chemical entities routinely in vitro, and when the DDI risk predicted is above the regulatory thresholds, clinically.

The principle of DDI prediction is to compare the unbound concentration at the site of DDI and

inhibition constants (Ki) (Giacomini et al., 2010). However, there is a limitation in the current DDI risk prediction for improvement in preclinical stages to avoid false-positive and -negative predictions

(Vaidyanathan et al., 2016). Endogenous biomarkers for drug transporters have emerged as surrogate

DDI probes to strengthen the in vitro DDI prediction (Chu et al., 2018; Müller et al., 2018; Rodrigues

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et al., 2018). Using the typical drug transporter inhibitors, the magnitude of changes of endogenous biomarkers in the area under the plasma concentration–time curves (AUC), and/or renal clearance have been reported for MATEs, OAT1 and OAT3, and OATP1B1 and OATP1B3. The most important advantage of endogenous biomarkers is that researchers are freed from administration of probe drugs, which could increase the opportunity to assess the DDI risk in humans. Downloaded from

An antimicrotubule agent, paclitaxel, has been used in chemotherapy of various tumors, such as

non-small cell lung cancer, breast cancer, and ovarian cancer. The major elimination pathway of dmd.aspetjournals.org paclitaxel from the blood circulation is CYP2C8-mediated metabolism to form 6-hydroxy paclitaxel

(Sonnichsen et al., 1995). DDI with paclitaxel as victim drug has been studied in patients who received at ASPET Journals on September 26, 2021 clopidogrel, which caused time-dependent inhibition of CYP2C8 (Floyd et al., 2012); patients who received both clopidogrel and paclitaxel exhibited higher incidence of severe paclitaxel neuropathy than those treated with paclitaxel and low-dose aspirin, although the impact on paclitaxel pharmacokinetic parameters remains to be determined (Agergaard et al., 2017). In contrast, paclitaxel

was also suggested to inhibit OATP1B based on comparison of in vitro Ki values for OATP1B1 and

OATP1B3 with its clinically relevant unbound concentrations (Marada et al., 2015; Murata et al.,

2019). However, the clinical relevance of OATP1B inhibition by paclitaxel has not been confirmed because of ethical and practical issues in conducting clinical DDI studies in healthy subjects or patients.

We and other groups identified glycochenodeoxycholate-3-sulfate (GCDCA-S) (Takehara et al., 2017,

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2018), other sulfated bile acids (Thakare et al., 2017; Mori, Kashihara, et al., 2019; Takehara et al.,

2019), coproporphyrin I (CPI) and CPIII (Lai et al., 2016; Barnett et al., 2019), tetradecanedioate and hexadecanedioate (Yee et al., 2016, 2019; Barnett et al., 2019) as endogenous biomarkers for

OATP1B1/1B3. Accumulation of clinical data in healthy subjects to support the suitability of these

OATP1B-endogenous biomarkers could allow us to evaluate the OATP1B-mediated DDI risks of Downloaded from anticancer drugs in patients.

This study was a clinical study designed to assess the changes in plasma concentrations of such dmd.aspetjournals.org endogenous OATP1B biomarkers in patients with non-small cell lung cancer who received paclitaxel chemotherapy (paclitaxel 200 mg/m2 given intravenously by a 3-h infusion, plus dexamethasone, at ASPET Journals on September 26, 2021 diphenhydramine and famotidine given 30 min prior to paclitaxel administration). In addition, the patients also received drug therapy for treatment of their complications (Table 1), but there was no drug that was administered to all patients.

Materials and Methods

Chemicals

[3H]Estradiol 17 -D-Glucuronide (E217G) and [3H]CCK-8 were purchased from PerkinElmer

Life and Analytical Sciences (Boston, MA). Authentic compounds including stable isotope-labeled compounds used in this study were all commercially available (Supplemental Materials). Other

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reagents and organic solvents were of a commercially available analytical grade.

In vitro transport study using transporter-expressing cells

HEK293 cells, which were stably transfected with OATP1B1, OATP1B3, OAT1, OAT3, or empty vector, were established previously (Deguchi et al., 2004; Hirano et al., 2004). The transport study Downloaded from was performed according to the previous reports (Deguchi et al., 2004; Hirano et al., 2004). Briefly,

5 HEK293 cells were seeded at 2  10 cells/well in 24-well plate coated with poly-L-lysine (50 mg/L) dmd.aspetjournals.org and poly-L-ornithine (50 mg/L). After 48 h, the medium was substituted by DMEM containing 5 mM sodium butyrate. After 24 h, the transport study was conducted. Cells were washed twice and at ASPET Journals on September 26, 2021 preincubated with Krebs–Henseleit (KH) buffer at 37°C for 5 min. Then, substrates were added to initiate the uptake. At the designated times, incubation buffer was removed and ice-cold KH buffer was added to terminate the uptake.

To evaluate the inhibition potency of paclitaxel and 6-hydroxy paclitaxel, either paclitaxel or

6-hydroxy paclitaxel was added simultaneously with the test substrate. When the time dependence of the inhibitory effect was assessed (shown in Figure 1 and S8), cells were preincubated in the presence of paclitaxel or 6-hydroxy paclitaxel for 15 min. In a separate experiment (shown in Figure

S6A), cells were preincubated in KH buffer containing paclitaxel for the designated periods (5, 10, 30,

60 min), then washed. After washing, uptake was initiated in the absence of inhibitor. To assess the

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duration of the inhibitory effect (shown in Figure S6B), cells were preincubated with KH buffer containing paclitaxel for 30 min, incubated with paclitaxel-free KH buffer for designated periods (0,

10, 30, 60 min), then uptake was initiated in the absence of paclitaxel.

For quantification of nonradiolabeled compounds, the cells were collected in Milli-Q water using a scraper and disrupted using a bioruptor (Sonic-bio, Kanagawa, Japan). A 20 L aliquot was mixed Downloaded from with 60 L of acetonitrile containing the corresponding internal standard solution. After vortex mixing,

the mixtures were centrifuged (20,000g for 5 min at 4°C) and the supernatants were used for the dmd.aspetjournals.org following LC-MS/MS analyses. For quantification of the radioactivity, the cells were solubilized with

200 L 0.2 N NaOH overnight at 4°C and then neutralized with 100 L 0.4 N HCl. Aliquots of the at ASPET Journals on September 26, 2021 lysates (200 L) were transferred to scintillation vials containing a scintillation cocktail (Clear-sol I;

Nacalai Tesque, Kyoto, Japan), and the radioactivity was measured in a liquid scintillation counter

(LS6000SE, Beckman Instruments, Inc., Fullerton, CA). The protein concentration was determined using the protein assay method of Lowry (Lowry et al., 1951) using bovine serum albumin as the protein standard.

Western blotting to detect OATP1B1 and OATP1B3 in whole cells and cell surface

Cells were harvested by cell scraper and collected by centrifugation (12,000g, 5 min, 4°C). They were lysed by cell lysis buffer (Cell Signaling, Danvers, MA). The samples were centrifuged at

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12,000g for 10 min at 4°C, and then, the supernatants were used in the analysis. 4SDS sample buffer

(12% SDS, 25% glycerol, 150 mM Tris-HCl pH 7.0, 0.05% bromophenol blue, 6% - mercaptoethanol) and 2-mercaptoethanol were added one-fourth and one-thirtieth volume of the sample, respectively, and heated at 60°C for 5 min. Then, the whole cell samples were subjected to western blot analysis. Downloaded from

For cell surface analysis of OATP1B1 and OATP1B3 proteins, cell surface biotinylation was

conducted as previously described (Mizuno et al., 2015). Briefly, cells were incubated with NHS-SS- dmd.aspetjournals.org

Biotin solution (0.5 mg/mL; Pierce Biotechnology, Inc., Rockford, IL) for 20 min at 4°C and washed using phosphate-buffered saline supplemented with Ca2+, Mg2+, and 100 mM glycine before at ASPET Journals on September 26, 2021 preparation of cell lysate with cell lysis buffer (Cell signaling). The cell lysate was mixed with 50 L streptavidin–agarose (Thermo Fisher Scientific, Waltham, MA) and kept under end-over-end rotation for 2 h at 4°C. After centrifugation at 5,900g for 1 min at 4°C, the supernatant was removed. The beads were washed with lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton-X 100, pH 7.5) twice, high salt buffer (50 mM Tris, 500 mM NaCl, 5 mM EDTA, 0.1% Triton-X 100, pH 7.5) twice, low salt buffer (50 mM Tris, pH 7.5) once. Biotinylated proteins were eluted with 40 L loading buffer

(Milli-Q water/4 SDS loading buffer/2-mercaptoethanol, 35/4/1) for 1 h at 60°C and obtained specimens were subjected to western blotting analysis.

The specimens were loaded into wells of a 10% SDS–PAGE plate with a 3.75% stacking gel and

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subjected to immunoblotting as previously described (Hirano et al., 2004). Immunoreactivity was detected with an ECL Prime Western Blotting Detection Kit (Amersham Biosciences, Piscataway, NJ).

Clinical study design

This was a prospective study performed with Japanese non-small cell lung cancer patients Downloaded from who received the first cycle of paclitaxel and carboplatin regimen. To evaluate the difference in

plasma concentration profiles of the endogenous substrates with or without the paclitaxel infusion, dmd.aspetjournals.org the blood samples were collected on two consecutive days at the same time points. On day 1, plasma samples were collected before and after the paclitaxel infusion as shown below, while on at ASPET Journals on September 26, 2021 day 0, plasma samples were collected at the same time points as day 1. The study was conducted in accordance with the principles of the Declaration of Helsinki. This study protocol was approved by the Institutional Review Board of Showa University and Graduate School of Pharmaceutical

Sciences, The University of Tokyo. All patients were asked for written informed consent for their peripheral blood samples and medical information to be used for research purposes. The study was registered at the University Hospital Medical Information Network-Clinical Trials Registry

Japan (UMIN000032370).

Eligibility

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All patients 20 years or older with metastatic or recurrent and histologically confirmed non- small cell lung cancer who received paclitaxel, had an Eastern Cooperative Oncology Group performance status of 0–1, and no history of chemotherapy within 4 weeks were eligible. Each patient was confirmed to have adequate bone marrow function (absolute neutrophil count, at least

1.5  109/L; platelet count, at least 150  109/L), liver function (serum bilirubin level, less than Downloaded from

1.5 mg/dL; transaminases, less than 2.5 times the upper limit of normal), and renal function

(serum creatinine level, less than 1.5 mg/dL) (Table 1). dmd.aspetjournals.org

Treatment at ASPET Journals on September 26, 2021

The therapeutic dose of paclitaxel (200 mg/m2) and carboplatin (area under the blood concentration–time curve of 6 mg/mL×min) was administered to patients with non-small cell lung cancer. Combination chemotherapy with bevacizumab (15 mg/kg) was permitted. Paclitaxel was administered as an intravenous drip infusion over the course of 3 h after the patient received premedication with oral 50 mg diphenhydramine followed by intravenously given 3 mg granisetron, 50 mg ranitidine, and 20 mg dexamethasone over the course of 10 min (Figure S1).

Thereafter, carboplatin was given as an intravenous drip infusion over the course of 1 h. The patients were given a dietary supplement containing an equivalent amount of total daily lipids, to minimize the influence of these dietary ingredients on the synthesis or disposition of the

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endogenous substrates of OATP1B1 and 1B3 such as bile acids. Note that regular meals were served to patients at 8:00 AM and 12:00 PM with the same menu on day 0 and day 1, and paclitaxel infusion was performed from 11:00 AM to 2:00 PM.

Blood sampling Downloaded from

Blood samples were taken from the arm opposite the infusion site at the beginning of

paclitaxel infusion and 0, 2, 4, and 7 h after the end of the 3-h infusion of paclitaxel on day 1. dmd.aspetjournals.org

Blood samples were also collected at the same time points on day 0 to assess the difference in plasma concentration profiles of the endogenous substrates in the presence or absence of at ASPET Journals on September 26, 2021 paclitaxel infusion. The blood samples were immediately centrifuged, and the plasma was stored at –80°C until analysis.

Genotyping of SLCO1B1 in patients

This study focused on two SLCO1B1 single nucleotide polymorphisms, 388G>A and 521T>C, that form haplotypes, *1a, *1b, and *15. The genotypes were determined as described previously

(Akiyama et al., 2012) using genomes prepared from peripheral blood cells of the patients.

Determination of the unbound fraction of paclitaxel in the plasma

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The unbound fraction of paclitaxel in the plasma (fp) was determined at 0 h and 7 h using a High

Throughput Dialysis Model HTD96b and Dialysis Membrane Strips MWCO 12–14 kDa obtained from HTDialysis, LLC (Gales Ferry, CT).

Quantification of paclitaxel, 6α-hydroxy paclitaxel in the plasma specimens

Plasma aliquot (20 L) was added to acetonitrile (180 L), vortexed, and centrifuged at 20,000g Downloaded from for 5 min for protein precipitation. The supernatant (10 L) was mixed with 0.1% formic dmd.aspetjournals.org acid/acetonitrile = 1/1 solvents (150 L) and analyzed using LC-MS/MS. Chromatography was performed on a Prominence UFLC system (Shimadzu, Kyoto, Japan) and separation was achieved as

described in Supplemental Methods. Data were collected on an AB Sciex API5500 (QTRAP) mass at ASPET Journals on September 26, 2021 spectrometer (Foster City, CA, USA) using positive Turbo IonSpray™ electrospray ionization (ESI) and MRM mode (Supplemental Methods). Data acquisition and processing were carried out with

Analyst software version 1.6.2. (Applied Biosystems/MDS Sciex, Foster City, Canada).

Quantification of endogenous OATP1B biomarkers and bile acids in the plasma specimens

A QTRAP5500 mass spectrometer (AB Sciex, Foster City, Canada) equipped with Prominence

UFLC system (Shimadzu, Kyoto, Japan), or Nexera UHPLC (Shimadzu, Kyoto, Japan) was used for quantification of endogenous compounds. Quantification of C4, bile acids, and bile acid sulfates

(LCA-S, TLCA-S, and GLCA-S) was performed as described previously (Mori, Kashihara, et al.,

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2019; Mori, Kimoto, et al., 2019). The measurement method is summarized in Supplemental

Methods. For separation of stereoisomers, such as CPI and CPIII, GCDCA-S and GDCA-S, and

GCDCA-G and GDCA-G, the chromatographic separation was modified as described in the

Supplemental Methods. The chromatographic separation and analytical condition are described in the Supplemental Methods. Downloaded from

Calculation of AUC of the endogenous compounds dmd.aspetjournals.org

The area under the plasma concentration–time curve (AUC) was calculated from –3 to 10 h using the linear trapezoidal method using Excel. at ASPET Journals on September 26, 2021

Determination of the inhibition constants (Ki) of paclitaxel and 6α-hydroxy paclitaxel for

OATP1B1 and OATP1B3

Iterative nonlinear least-squares method using GraphPad Prism8 (GraphPad Software, San

Diego, CA) was conducted to determine the Ki values of paclitaxel or 6α-hydroxy paclitaxel. The

uptake of test compound determined in the presence and absence of inhibitor (CLuptake (+inhibitor) and

CLuptake (control), respective) was fitted to the following equation:

퐶퐿푢푝푡푎푘푒(푐표푛푡푟표푙) 퐶퐿푢푝푡푎푘푒(+𝑖푛ℎ𝑖푏𝑖푡표푟) = 1 + 퐼⁄퐾𝑖 where I represents the inhibitor concentration.

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Statistics

Paired t-test was used to examine statistical significance in the effect of paclitaxel administration on the plasma concentrations of endogenous OATP1B biomarkers using GraphPad Prism8 (GraphPad

Software, San Diego, CA). Geometric means and 95% confidence of intervals of AUC ratio were calculated using GraphPad Prism8. Downloaded from

Results dmd.aspetjournals.org

Effect of paclitaxel on OATP1B1- and OATP1B3-mediated uptake in HEK293 cells

Paclitaxel inhibited the OATP1B1-mediated uptake of [3H]E217G in a concentration-dependent at ASPET Journals on September 26, 2021

manner (Figure 1) with Ki of 0.579 ± 0.107 M with a competitive manner (Figure S2). A 15-min

preincubation with paclitaxel potentiated its inhibitory effect, reduced the Ki to 0.154 ± 0.031 M

(Figure 1). Paclitaxel showed lower inhibition potency to OATP1B3 than OATP1B1 (Figure 1) with

the estimated Ki of 0.624 ± 0.183 and 5.29 ± 3.87 M with or without preincubation, respectively

(Figure 1). OATP1B1 and OATP1B3 were not inhibited by drugs administered prior to paclitaxel administration (Figure S3). Paclitaxel did not inhibit either OAT1 or OAT3 at 10 M (Figure S4).

Plasma concentration–time profile of paclitaxel in non-small cell lung cancer patients

Plasma concentrations of paclitaxel and its hydroxy metabolite, 6-hydroxy paclitaxel, were

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determined in the patients (Figure 2). Plasma concentrations of 6-hydroxy paclitaxel accounted for

one-tenth of those of paclitaxel (Figure 2). The fp of paclitaxel at 0 h and 7 h were determined in all the subjects. The mean values were 0.0158±0.0042 and 0.0354±0.0247, respectively, which were similar to the previously reported values (0.036~0.079) (Brouwer et al., 2000). The unbound

concentration of paclitaxel (Cu,max) was calculated using individual fp and Cmax at 0 h. It was somewhat Downloaded from

below the Ki for OATP1B1 with preincubation (Figure S5).

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Effect of paclitaxel administration on the plasma concentrations of the endogenous OATP1B biomarkers at ASPET Journals on September 26, 2021

Plasma concentrations of 10 endogenous biomarkers, three porphyrin metabolites (CPI, and

CPIII), and bile acid glucuronides or sulfate (GCDCA-S, GCDCA-G, GDCA-S, GDCA-G, LCA-S,

GLCA-S, TLCA-S, CDCA-24G), were determined on day 0 (baseline) and day 1 (before and after paclitaxel administration) (Figure 3). During the observed time (–3 to 7 h), there was no obvious change in the plasma concentrations on day 0. After a 3-h infusion of paclitaxel, the plasma concentrations of the endogenous biomarkers were higher than the predose level, or baseline (day 0).

The time to reach maximum concentration differed among the biomarkers; end of infusion, CPI, CPIII, and CDCA-24G; 0–2 h postinfusion, GCDCA-S, GDCA-S, LCA-S, GLCA-S, TLCA-S; 4 h postinfusion, GCDCA-G and GDCA-G. The increased plasma concentrations did not return to the

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corresponding baseline levels by 7 h postinfusion. Note that LCA-S, GLCA-S, and TLCA-S could not be quantified in one patient (PTX-5).

The interindividual difference in the response to paclitaxel administration is shown as AUC in

Figure 4. The mean and range of the AUC ratio are shown in Table 2. There were nonresponders

(AUC ratio < 1.25) to paclitaxel for LCA-S, GLCA-S, and TLCA-S; LCA-S, PTX-7 and PTX-10; Downloaded from

GLCA-S, PTX-7 and PTX-9; TLCA-S, PTX-7, PTX-8, and PTX-9. Because of these nonresponders,

the mean ratio of AUC was somewhat smaller for these metabolites. The mean ratio of AUC was dmd.aspetjournals.org similar among the other metabolites (Table 2).

at ASPET Journals on September 26, 2021

Effect of paclitaxel administration on the plasma concentrations of the intermediate metabolites of bile acid synthesis and bile acids

The plasma concentrations of C4 and bile acids were also measured in the patients with or without paclitaxel administration. The plasma concentrations of C4 on day 0 were similar throughout the study, whereas those on day 1 decreased significantly over time (Figure 5A). Plasma concentrations of bile acids including the precursor of the glucuronide and sulfate conjugates, such as GCDCA and GDCA, were also determined. The mean values increased at the end of infusion for TCA, GCA, GCDCA,

GDCA and TDCA followed by a rapid decline to the baseline levels, whereas there was no difference in the plasma concentrations of CDCA and DCA with and without paclitaxel administration (Figure

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5A). There was a clear interindividual difference in the baseline levels of bile acids, and effect of paclitaxel administration on AUC of bile acids (Figure 5B).

Discussion

DDI risk assessment of anticancer drugs remains mainly limited to in vitro or animal data because Downloaded from to elucidate the clinical relevance of the inhibition at their therapeutic dose ranges is ethically and

practically difficult. Paclitaxel is one of the anticancer drugs which were suggested to inhibit OATP1B dmd.aspetjournals.org by conventional prediction method (Marada et al., 2015; Murata et al., 2019). Taking advantage of endogenous biomarkers, circumventing the need for investigators to administer probe drugs, we at ASPET Journals on September 26, 2021 assessed OATP1B1 and OATP1B3 inhibition potency of paclitaxel at its therapeutic dose for the treatment of non-small lung cancer.

Consistent with previous reports, the in vitro inhibition study showed the concentration- dependent inhibition of OATP1B1 and OATP1B3 by paclitaxel (Figure 1). Competitive inhibition was confirmed for OATP1B1 inhibition (Figure S2). Whereas, absence of paclitaxel effect on renal organic anion transporters, OAT1 and OAT3 (Figure S4) excludes nonspecific effects, at least during the experiments. Furthermore, the inhibition of OATP1B1 and OATP1B3 by paclitaxel was time dependent (Figure 1; Figure S6), which had been reported for cyclosporin A (Shitara and Sugiyama,

2017; Tátrai et al., 2019). A metabolite of paclitaxel, 6-Hydroxy paclitaxel, retained a time- dependent effect on OATP1B1 with similar inhibition potency, whereas it no longer showed time- dependent inhibition of OATP1B3 (Figure S8). The time-dependence of OATP1B1 inhibition appears

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to be reversible because the activity partially recovered after incubation in the absence of paclitaxel

(Figure S6). The possibility that paclitaxel treatment decreased the expression of OATP1B1 and

OATP1B3 proteins in whole cells and on the cell surface was excluded (Figure S7). Shitara and

Sugiyama (2017) proposed a trans-inhibition model to account for the time-dependent effect of cyclosporin A (Shitara and Sugiyama, 2017). Paclitaxel may also have inhibitory effect on OATP1B1 in the plasma membrane or from the cytosolic compartment, where paclitaxel accumulated during Downloaded from incubation. It is also possible that paclitaxel inhibits the maintenance of the driving force for OATP1B1 and OATP1B3, which has not been identified, or post-translational modifications such as phosphorylation, as has been proposed for other drug transporters (Sprowl et al., 2016). It should be dmd.aspetjournals.org noted that, as pointed out for OATP1B1 (Izumi et al., 2015), the inhibitory effect of paclitaxel for

OATP1B3 was substrate dependent; the inhibitory effect of paclitaxel on OATP1B3-mediated

[3H]CCK-8 uptake was weaker than that on pitavastatin uptake (Figure S9). Careful selection of test at ASPET Journals on September 26, 2021

OATP1B3 probe is required for conservative DDI risk assessment.

Comparison of the unbound concentration of paclitaxel in the plasma, estimated as the product of fp and Cmax, with its Ki for OATP1B1 and OATP1B3 (Figure S5) suggests that at this dose regimen, paclitaxel inhibits OATP1B1 more potently than OATP1B3. The plasma concentrations of the endogenous OATP1B biomarkers were significantly higher at the end of paclitaxel infusion than the two control values; baselines determined on the day before paclitaxel administration, and values before starting the administration of paclitaxel (confirming the interday difference) (Figure 3). The magnitude of changes in CPI concentrations induced by paclitaxel administration was similar to that previously observed in the subjects with OATP1B1 *15 homozygotes (Mori, Kashihara, et al., 2019) and those who received 300 mg rifampicin (Takehara et al., 2018). Previously, the AUC ratio of

GCDCA-S (4.3) when 300 mg rifampicin was administered, which was greater than that of CPI (2.3)

(Takehara et al., 2018), whereas the AUC ratios of CPI and GCDCA-S were similar in this study

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(Table 2). Such a difference may arise from the in vivo inhibition potency of OATP1B1 and OATP1B3 between rifampicin and paclitaxel, or disease-related factors. The AUC ratio of LCA-S, GLCA-S, and

TLCA-S did not change in 2–3 patients in this study. Such nonresponse of sulfate conjugates of secondary bile acids to OATP1B inhibition was also observed in the rifampicin study (Mori, Kimoto, et al., 2019). The reason for this remains unknown, but using such metabolites as surrogate OATP1B probes should be avoided. Downloaded from The plasma concentrations of bile acids were also determined on both days. Those on day 1 at some time points were higher than the predose levels (Figure 5 and Table S1). One patient who dmd.aspetjournals.org showed a higher AUC ratio of GCDCA (6.8) than others (0.8–1.7) also showed the highest AUC ratio of GCDCA-S (13) and GCDCA-G (6.4) compared with others (2.4–6 and 1.2–3.1, respectively), but at ASPET Journals on September 26, 2021 the AUC ratio of CPI was similar. Therefore, the AUC ratio of GCDCA-S and GCDCA-G might be influenced not only by OATP1B inhibition but also by the interday variation or effect of paclitaxel and/or the concomitantly administered drug on GCDCA. The possibility that paclitaxel inhibits NTCP can be excluded because of absence of the effect on NTCP-mediated uptake of [3H]taurocholate at 10

M (data not shown). In addition, C4, an intermediate metabolite of bile acid synthesis from cholesterol, continued to decline on day 1 after paclitaxel administration (Figure 5). Paclitaxel therapy might affect bile acid synthesis.

Though in limited patient number, effect of OATP1B1 genotypes (wild-type homozygotes and

*15 heterozygotes) and biochemical test results were shown in Figure S10 and S11. A nonsynonymous single nucleotide polymorphism (SNP) of OATP1B1 (c. 521T>C, p.Val174Ala) is

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well-known to be associated with higher systemic exposure of OATP1B1 substrate drugs (Hannah H

Lee and Ho, 2017; Yee et al., 2018). In Japanese people, this mutation is associated in a haplotype

(*15) with another nonsynonymous mutation (c. 388A>G, p.Asn130Asp) (Nishizato et al., 2003).

Japanese individuals homozygous for the *15 allele showed higher plasma concentrations of CPI and

GCDCA-S than homozygotes of reference types and heterozygotes (Mori, Kashihara, et al., 2019). Downloaded from

Consistent with our previous report, individuals heterozygous for this mutation did not show any

difference in the plasma concentrations of these substrates (Figure S10). Whereas, the AUC ratio of dmd.aspetjournals.org

CPIII, but not CPI, appeared to be OATP1B1 genotype dependent (Figure S10) which required further confirmation in future studies. There was no significant effect of biochemical test results on the AUC at ASPET Journals on September 26, 2021 ratio of CPI (Figure S11), and other test compounds (data not shown).

Together with previously reported practices (Lai et al., 2016; Yee et al., 2016; Shen et al., 2017;

Takehara et al., 2017, 2018; King-Ahmad et al., 2018; Kunze et al., 2018; Liu et al., 2018; Müller et al., 2018; Cheung et al., 2019), this study provides more convincing evidence to expand application of the endogenous OATP1B biomarkers to assess in vivo OATP1B inhibition under conditions where administration of probe drugs is limited, or under those closer to actual clinical practice. In addition to paclitaxel, mitoxantrone and etoposide were suggested to cause significant inhibition of OATP1B3

(Marada et al., 2015), and tyrosine kinase inhibitors might cause OATP1B1 inhibition via inhibition of phosphorylation (Sprowl et al., 2016). Endogenous OATP1B biomarkers will be able to

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demonstrate their clinical relevance in future studies in patients. Whereas the patients generally received drugs other than the potential perpetrator drug to treat their complications, these could have included drugs for which the clinical risk of OATP1B-mediated DDI has not been adequately assessed.

In this study, drugs commonly used to treat all patients comprised dexamethasone, diphenhydramine and famotidine, which at least in vitro, did not inhibit OATP1B1 (Figure S3), but the possibility of Downloaded from

OATP1B-mediated DDIs with these drugs needs to be confirmed in further studies.

The changes in endogenous OATP1B biomarkers induced by perpetrator drugs must be dmd.aspetjournals.org extrapolated to the changes in the AUC of OATP1B substrate drugs to suggest the risk of OATP1B- mediated DDI. An important tool for this purpose is the use of a physiologically based at ASPET Journals on September 26, 2021 pharmacokinetic (PBPK) model. Yoshikado et al. constructed a PBPK model of CPI that could account for the plasma concentration–time profiles of CPI after rifampicin administration (Yoshikado et al.,

2018). Once an appropriate PBPK model has been constructed for paclitaxel, by analogy with that for

CPI, it could be used to help estimate the in vivo Ki of paclitaxel for OATP1B that would be required to simulate DDIs between paclitaxel and OATP1B substrate drugs, including in cases using other dose regimens for paclitaxel medication. In addition, because the plasma concentrations of CPI and CPIII showed the most rapid decline after the end of the infusion (within 2 h) of the endogenous substrates tested, PBPK model-based simulation will indicate whether OATP1B1 inhibition by paclitaxel is significant during infusion and for at least 2 h after the end of infusion, which would enable

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implementation of an appropriate dosing schedule to avoid significant DDIs.

In conclusion, the present study showed that paclitaxel could inhibit OATP1B1 and OATP1B3 at the dose for the non-small cell lung cancer treatment (200 mg/m2 by a 3-h infusion). This study increases the feasibility of transporter-mediated DDI risk assessment in clinical settings.

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Authorship Contributions

Participated in research design: Ishida, Mizuno, Maeda, Sasaki, Fujita, Kusuhara.

Conducted experiments: Mori, Ishida, Kusumoto, Kondo, Izumi, Nakada, Nozaki, Fujita.

Contributed new reagents or analytic tools: None

Performed data analysis: Mori, Mizuno, Maeda, Fujita, Kusuhara. Downloaded from Wrote or contributed to the writing of the manuscript: Mizuno, Fujita, Kusuhara.

dmd.aspetjournals.org

at ASPET Journals on September 26, 2021

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Footnotes

Grants-in-Aid for Scientific Research foundation B from the Japan Society for the Promotion of

Science [17H04100]

Grants-in-Aid for Clinical Pharmacology Research Foundation.

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021

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Legends for Figures

Figure 1 Inhibitory effect of paclitaxel on the OATP1B1- and OATP1B3-mediated uptake in

HEK293 cells.

The uptake of [3H]E217G by HEK293 cells stably expressing OATP1B1 and that of pitavastatin

(0.3 M) by HEK293 cells stably expressing OATP1B3 for 3 min with (○) or without (●) paclitaxel Downloaded from preincubation (15 min) was determined in the absence and presence of paclitaxel at the designated

concentrations or rifampicin (20 M). The solid line represents the fitted line obtained by nonlinear dmd.aspetjournals.org regression analysis as described in the Materials and Methods section. The dotted line represents the mean value of the uptake of [3H]E217G in the presence of rifampicin. Each symbol represents the at ASPET Journals on September 26, 2021 mean value with SEM (n = 3).

Figure 2 Plasma concentrations of paclitaxel and 6-hydroxy paclitaxel.

Plasma concentrations of paclitaxel (closed symbols) and 6-hydroxy paclitaxel (open symbols) were determined at designated time points in non-small cell lung cancer patients following a 3 h intravenous infusion of paclitaxel (200 mg/m2) from -3h to 0h. Each symbol represents the mean value, and error bars representing SEM (n = 10) were within the symbol.

Figure 3 Effect of paclitaxel on the plasma concentrations of endogenous OATP1B substrates.

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DMD # 89474

Plasma concentrations of CPI, CPIII, sulfate-conjugated bile acids (GCDCA-S, GDCA-S, LCA-S,

GLCA-S, TLCA-S), and glucuronide-conjugated bile acids (GCDCA-G, GDCA-G, CDCA-24G) were determined at designated time points in non-small cell lung cancer patients treated with or without an intravenous dose of paclitaxel (200 mg/m2). Each symbol represents the mean value, and error bars represent SEM (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001. Downloaded from

Figure 4 Area under the plasma concentration–time curves for endogenous OATP1B dmd.aspetjournals.org substrates with and without paclitaxel administration

The symbols represent AUC values in 10 patients calculated using the linear trapezoidal rule from at ASPET Journals on September 26, 2021 time –3 to 7 h. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5 Plasma concentrations of C4 and bile acids with and without paclitaxel administration.

(A) Plasma concentrations of C4 and bile acids were determined at designated time points in non- small cell lung cancer patients treated with or without an intravenous dose of paclitaxel (200 mg/m2).

Each symbol represents the mean value, and error bars represent SEM (n = 10). *p < 0.05, **p <

0.01, ***p < 0.001.

(B) Area under the plasma concentration–time curves for C4 and bile acids. The symbols represent

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DMD # 89474

individual values of AUC calculated using the trapezoidal rule from time –3 to 7 h. *p < 0.05, **p <

0.01

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021

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DMD # 89474

Table 1 Non-small cell lung cancer patient information dmd.aspetjournals.org

Patient information is summarized in the table. In subject PTX-6, paclitaxel administration was discontinued due to paclitaxel allergy.

Body Age Height OATP1B1 Alb AGP AST ALT LDH ALP T-Bil D-Bil SCr eGFR ID Sex Weight Complications Medications (years) (cm) haplotype (g/dL) (mg/dL) (IU/L) (IU/L) (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mL/min) (kg) at ASPET Journals on September 26, 2021 Alcoholic hepatitis, Livact, Feburic, PTX-1 M 74 159.5 54.3 *1b/*1b 4.3 52.0 37 21 200 354 1.3 0.1 0.81 71.0 hyperuricemia, Xyzal allergic rhinitis Dyslipidemia, Lipitor, Edirol, PTX-2 F 75 161.5 56.9 *1a/*1b 4.0 65.0 21 14 180 216 0.9 <0.1 0.68 63.3 osteoporosis Protecadin Tenormin, Atelec, Olmetec, Xarelto, Hypertension, PTX-3 M 58 169 73.2 *1b/*1b 3.3 144.0 32 24 269 216 0.5 <0.1 0.89 68.7 Tambocor, atrial fibrillation Eperisone, alprazolam Bezatol, Urso, Pinellia Heart- PTX-4 M 57 164.5 53.5 *1b/*15 4.2 73.0 12 6 115 135 1.6 0.1 0.72 87.1 Dyslipidemia Purging Decoction, Clostridium butyricum PTX-5 M 48 169 58.6 *1b/*15 4.0 76.0 28 25 162 - 0.9 0.1 0.89 72.9 Hypertension Preminent, Depas Hypertension, Herbesser R, PTX-7 M 66 165 63 *1b/*15 3.9 84.0 35 49 294 305 0.5 0.1 1.03 56.4 angina valsartan PTX-8 M 67 163.5 44.6 *1a/*1b 3.4 154.0 19 8 289 - 0.7 0.1 0.69 87.1 Hypertension Irbetan, famotidine Alositol, Takecab, PTX-9 M 57 171 72 *1a/*1b 4.4 96.0 18 13 219 219 0.8 0.1 0.76 82.1 Hyperuricemia Rebamipide PTX- Nexium M 68 170 63.9 *1b/*1b 3.9 188.0 16 9 344 416 0.4 <0.1 1.15 49.6 Reflux esophagitis 10 Takecab, PTX- M 52 160 53 *1a/*1b 3.7 156.0 16 10 156 225 0.5 <0.1 0.82 77.6 – loxoprofen, Tapenta, 11 Etizolam, Rivotril

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Table 2 Effect of paclitaxel on AUC–3–7h of Endogenous OATP1B Substrates

AUC–3–7h of the endogenous OATP1B substrates were calculated. AUC–3–7r are presented as mean ±

SEM. Fold changes are presented as GMR and 90% CI. (n = 10).

Compounds Control + Paclitaxel Fold change

CPI 22.1 ± 1.7 61.4 ± 4.3 (nM  h) 2.8 (2.5, 3.1) Downloaded from

CPIII (nM  h) 4.48 ± 0.34 14.1 ± 1.3 3.1 (2.6, 3.7)

GCDCA-S (M  h) 2.06 ± 0.45 5.71 ± 1.0 2.9 (2.2, 3.9) dmd.aspetjournals.org

GDCA-S (M  h) 0.501 ± 0.09 2.2 ± 0.45 4.0 (2.8, 5.8)

LCA-S (M  h) 0.694 ± 0.16 1.46 ± 0.38 2.0 (1.2, 3.5) at ASPET Journals on September 26, 2021

GLCA-S (M  h) 4.15 ± 1.4 10.4 ± 3.3 2.2 (1.4, 3.6)

TLCA-S (M  h) 0.857 ± 0.22 1.94 ± 0.57 2.0 (1.3, 3.0)

GCDCA-G (nM  h) 20.6 ± 4.1 52.2 ± 10 2.5 (1.8, 3.4)

GDCA-G (nM  h) 94.4 ± 29 211 ± 46 2.5 (1.8, 3.4)

CDCA-24G (nM  h) 141 ± 33 369 ± 79 3.0 (2.2, 4.0)

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Figure 1 at ASPET Journals on September 26, 2021

OATP1B1 OATP1B3

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Figure 2 at ASPET Journals on September 26, 2021

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Figure 3 dmd.aspetjournals.org

CP-I CP-III GCDCA-S GCDCA-G at ASPET Journals on September 26, 2021 GDCA-S

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P P 0 P 0.0 0.0 0.0 0 -4 -2 0 2 4 6 8 -4 -2 0 2 4 6 8 -4 -2 0 2 4 6 8 -4 -2 0 2 4 6 8 -4 -2 0 2 4 6 8 Time (hr) Time (hr) Time (hr) Time (hr) time (hr) DMD Fast Forward. Published on February 29, 2020 as DOI: 10.1124/dmd.119.089474 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from

Figure 4 dmd.aspetjournals.org

CP-I CP-III GCDCA-S GCDCA-G GDCA-S ✱✱✱ ✱✱✱ ✱✱ 100 25 15 ✱✱✱ 0.15 ✱✱ 5

at ASPET Journals on September 26, 2021

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Figure 5 dmd.aspetjournals.org

A B C4 CA GCA *C4 *CA *GCA

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at ASPET Journals on September 26, 2021

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Title

Alteration in the plasma concentrations of endogenous OATP1B-biomarkers in non-small cell lung cancer patients treated with paclitaxel

Authors

Daiki Mori1, Hiroo Ishida2, Tadahaya Mizuno1, Sojiro Kusumoto3, Yusuke Kondo1, Saki Izumi4, Genki

Nakata1, Yoshitane Nozaki4, Kazuya Maeda1, Yasutsuna Sasaki2, Ken-ichi Fujita5, Hiroyuki Kusuhara1

Affiliations

1 Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the

University of Tokyo, Tokyo, Japan

2 Division of Medical Oncology, Department of Medicine, Showa University School of Medicine,

Tokyo, Japan

3 Division of Respiratory Medicine and Allergology, Department of Medicine, Showa University

School of Medicine, Tokyo, Japan

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

Ibaraki, Japan.

5 Division of Cancer Genome and Pharmacotherapy, Department of Clinical Pharmacy, Showa

DMD # 89474

University School of Pharmacy, Tokyo, Japan

Figure S1 Schematic diagram of the clinical study protocol CPI CPIII UPI/III GCDCA-S 4 5 ** 4 10

4 8

3 3

o o

i i

t t

3 t 6

a a

r r

2 2

C C

U U

2 U 4

A A 1 1 1 2

0 0 0 0

b 5 b 5 b 5 b 5 1 1 1 1 1 1 1 1 /* /* * /* * /* * /* b b / / / 1 1 b b b b b b * * *1 *1 *1 *1 *1 *1 , , , , b b b b 1 1 1 1 /* * * * a / / / 1 a a a * *1 *1 *1

GCDCA-G GDCA-S GDCA-G LCA-S

8 15 8 8

6 6 6

o o

o 10

i i

t t

a a

r r

4 4 4

C C

U U

A A A 5 A 2 2 2

0 0 0 0

b 5 b 5 b 5 b 5 1 1 1 1 1 1 1 1 /* /* /* /* /* /* /* /* b b b b b b b b *1 *1 *1 *1 *1 *1 *1 *1 , , , , b b b b 1 1 1 1 /* /* /* /* a a a a *1 *1 *1 *1

GLCA-S TLCA-S CDCA-24G

6 5 6

o o

4 o 4

i i

t t

t 3

a a

r r

C C

U U

U 2

A A 2 A 2 1

0 0 0

b 5 b 5 b 5 1 1 1 1 1 1 /* /* /* /* /* /* b b b b b b *1 *1 *1 *1 *1 *1 , , , b b b 1 1 1 /* /* /* a a a *1 *1 *1

Figure S10 Effect of SLCO1B1 genotypes on the AUC ratio for the endogenous OATP1B substrates Mean and SD were shown together with individual data in OATP1B1 wild-type homozygotes (*1a/*/b or *1b/*1b, n=7), and *15 heterozygotes (*1b/*15, n=3). ** p<0.01 calculated using t-test.

4 4 4

o o

i i

i 3 3 3

t t

a a

r r

C C

U U

U 2 2 2

A A

I I

- -

P P

C C C 1 1 1

0 0 0 0 1 2 3 4 5 0 50 100 150 200 0 10 20 30 40 Alb (g/dL) AGP (mg/dL) AST (IU/K)

4 4 4

o o i

i 3 3

t t

i 3

a a

r r

C C

C U U 2 2

U 2

A A

I I

- -

P P

P C C 1 1

C 1

0 0 0 0 20 40 60 0 100 200 300 400 500 0.0 0.5 1.0 1.5 2.0 ALT (IU/L) ALP (IU/L) t-bilirubin

4 4 4

o o o

i i

i 3 3 3

t t t

a a a

r r r

C C C

U U

U 2 2 2

A A A

I I I

- - -

P P P

C C C 1 1 1

0 0 0 0 100 200 300 400 0.0 0.4 0.8 1.2 0 20 40 60 80 100 LDH (IU/L) SCr (md/dL) eGFR (ml/min)

Figure S11 Correlation between the biochemical test results, and AUC ratio of CPI Individual data was shown. Rigid line represents the correlation. None of the correlation reached the statistical significance. OATP1B1 30 [PTX] = 0

Optimized parameters

) e

n [PTX] = 1 M

k i

a [PTX]=0 [PTX]=1

p o

r 20 u

K (μM) 5.44±0.72 27.9±6.2

p m

G g  ± ±

m Vmax 116 14 211 55

/ 1

n (pmol/min/mg 2 i 10

E protein)

/ ]

L ± ±

H CLdif 1.14 0.23 0.366 0.406



( [

Km, Michaelis constant; Vmax, maximum uptake rate; CLdif, 0 clearance for non-saturable component. 0 50 100 150 200 250 v (pmol/min/mg protein)

Figure S2 Eadie-Hofstee plot for the OATP1B1-mediated uptake of [3H]E217βG in the absence and presence of paclitaxel, and fitted parameters The uptake of [3H]E217βG for 3 min was determined in HEK293 cells stably expressing OATP1B1 was determined in the absence and presence of paclitaxel (1 M). The solid line represents the fitted line obtained by nonlinear regression analysis using GraphPad prism 8 (GraphPad Software, San Diego, CA). Each symbol represents the mean value, and error bars represent S.E. (n = 3).

OATP1B1 OATP1B1 )

) 35 40

i m

m 30

k 30 r

25 r

n u

20 u

e 20

2 o

15 o

]

10 H

g 3

3 10

[

m /

5 /



 ( 0 ( 0 l l ro M M M M o M M t     tr   n 0 0 0 0 n 1 0 o 1 0 0 0 o 1 1 1 1 C in C c e e e i in in in p d d Dexamethasone m ti ti m a i o ra if n d R a m y R a h F n e h ip D OATP1B3 OATP1B3

) 100

) 90

n i

i 80

k 3

o p

60 f

i 8

i 50

K K

40 o

o C

r 40

30 ]

]

H 3

3 20 m

[ 20

[

/ L

L 10



 ( ( 0 0 l l o M M ro M M M M r   t     t n 0 0 0 0 n 1 0 o 1 0 0 0 o 1 1 1 1 C C in c e e e i in in in p d d Dexamethasone m ti ti m a i o ra if n d R a m y R a h F n e h ip D Figure S3 Inhibitory effect of concomitant drugs on the uptake by HEK293 cells stably expressing OATP1B1/1B3 The uptake of [3H]E217ΒG (0.3 µM) and [3H] CCK-8 (0.3 µM) by HEK293 cells stably expressing OATP1B1/1B3 respectively was determined with or without concomitant drugs. The cellular accumulation was determined following 3 min incubation at 37 C. Each bar represents the mean value, and error bars represent S.E. (n = 3)

OAT1 OAT1 OAT3

)

i i

i 15 15 6

10 10 4

5 5 t 2



(

( P ( 0 0 0 l l l o M M M o M M M o M M M r    r    r  u u t 0 t 0 t n 0 1 0 n 0 1 0 n 0 1 0 o 2 l 1 o 2 l 1 o 5 l 1 c e l c e l C 2 e l id x e id x e d x e c ta x c ta x i ta x e li ta e li ta c li ta n c li n c li e c li e a c e a c n a c b P a b P a e P a o P o P b P r r ro P P P

Figure S4 Inhibitory effect of paclitaxel on the uptake by HEK293 cells stably expressing hOAT1 and hOAT3 The uptake of pyridoxic acid (3 µM) and homovanillic acid (3 µM) by HEK293 cells stably expressing hOAT1 and that of pravastatin (0.3 µM) by HEK293 cells stably expressing hOAT3 was determined with or without probenecid (250 µM) and paclitaxel (1 and 10 µM). The cellular accumulation was determined following 3 min incubation at 37 C. Each bar represents the mean value, and error bars represent S.E. (n = 3) 6

) Ki w/o preincubation

M 5 for OATP1B3

 (

0.8

Ki w/ preincubation x

a for OATP1B3 m

, 0.6

u Ki w/o preincubation C

for OATP1B1 l

e 0.4

i l

c 0.2 Ki w/ preincubation

a for OATP1B1 P 0.0

Figure S5 Comparison of the unbound plasma concentration of paclitaxel with the Ki values for OATP1B1 and OATP1B3

The unbound plasma concentration of paclitaxel (Cu,max) was estimated as product of unbound fraction and Cmax. The Ki values determined with or without preincubation were indicated by rigid and dotted lines, respectively. Red and blue lines represent Ki values for OATP1B1, and OATP1B3, respectively. Geometric mean and 95% confidence of interval was shown. A B

) 0 min

)

n i n 200

i 150 5 min 10 min

m m 10 min 3

30 min

r r

30 min o 150 60 min

100 60 min n

e t

e 100

e g

e 50



U (

 0 U ( 0 0 0 0.1 1 10 100 0.1 1 10 100 Paclitaxel (M) Paclitaxel (M)

Figure S6 Effect of paclitaxel pre-incubation on the uptake of [3H]E217ΒG by HEK293 cells stably expressing OATP1B1 (A) The uptake of [3H]E217ΒG without paclitaxel for 3 min after paclitaxel pre-incubation for 5, 10, 30, and 60 min was determined in HEK293 cells stably expressing OATP1B1 in the absence and presence of paclitaxel at the designated concentrations. Each point represents the mean value, and error bars represent the S.E. (n = 3). (B) The uptake of [3H]E217ΒG for 3 min after paclitaxel pre-incubation for 30 min and incubation with Krebs- Henseleit buffer for 0, 10, 30, and 60 min was determined in HEK293 cells stably expressing OATP1B1 in the absence and presence of paclitaxel at the designated concentrations. Each symbol represents the mean value, and error bars represent S.E. (n = 3).

(A) PMA PTX (B) PMA PTX

control control

10 μM 10 μM 30 μM 10 μM 10 μM 30 μM 10

1 μM 1 μM 1

whole 150 kDa whole 100 OATP1B3 100 kDa OATP1B1 kDa cell surface 150 kDa 100 cell OATP1B1 100 kDa kDa surface OATP1B3 -actin 37 kDa -actin 37 kDa

Figure S7 Protein expression of OATP1B1 and OATP1B3 after incubation with PMA or paclitaxel Expression of OATP1B1 (A), OATP1B3 (B) and β-Actin after incubation with PMA or paclitaxel at the designated concentration for 30 min was detected by Western blotting as described in Materials and Methods. Protein of whole cell lysate (20 g) and plasma membrane fraction (30 g) was analyzed.

OATP1B1 OATP1B3

)

n i

125 i 100

Co Co r

100 r 80

o f

Pre + Co f Pre + Co

n i

75 i 60

o r

50 r 40

g k

25 k 20

 

U U ( 0 ( 0 0 0.1 1 10 100 0 0.1 1 10 100 6-OHP (M) 6-OHP (M)

Figure S8 Effect of 6-hydroxy paclitaxel on the uptake of OATP1B1- and OATP1B3- mediated [3H]E217ΒG uptake in HEK293 cells The uptake of [3H]E217ΒG and pitavastatin (0.3 M) for 3 min with (○) or without (●) 6-hydroxy paclitaxel pre-incubation was determined in HEK293 cells stably expressing OATP1B1 and OATP1B3, respectively, in the absence and presence of 6-hydroxy paclitaxel at the designated concentrations. The solid line represents the fitted line obtained by nonlinear regression analysis as described in the Materials and Methods section. Each symbol represents the mean value, and error bars represent S.E. (n = 3).

OATP1B3 )

n 100

k 3

n 60

K o

C 40

] g

H 20

[ L

 0 ( 0 0.1 1 10 Paclitaxel (M)

Figure S9 Effect of paclitaxel on the OATP1B3-mediated uptake in HEK293 cells The uptake of [3H]CCK-8 (0.3 M) by HEK293 cells stably expressing OATP1B3 for 3 min with without paclitaxel pre-incubation was determined in the absence and presence of paclitaxel at the designated concentrations. Each symbol represents the mean value, and error bars represent S.E. (n = 3). Supplemental Methods

Source of chemicals used in this study

Compound Purchase source 6-hydroxy Paclitaxel Cayman Chemical (Michigan, USA) Homovanillic Acid FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Paclitaxel FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Rifampicin FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Ranitidine hydrochloride FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Diphenhydramine hydrochloride FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Dexamethasone FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Phorbol 12-Myristate 13-Acetate FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Cholate FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) Pitavastatin Calcium LKT Laboratories ( Minnesota, USA) β-Estradiol 17-(β-D-glucuronide) Sigma-Aldrich (St. Louis, MO) 4-Pyridoxic acid Sigma-Aldrich (St. Louis, MO) Famotidine Sigma-Aldrich (St. Louis, MO) Probenecid Sigma-Aldrich (St. Louis, MO) Glycochenodeoxycholic acid Sigma-Aldrich (St. Louis, MO) Taurolithocholic acid 3-sulfate Sigma-Aldrich (St. Louis, MO) disodium salt Coproporphyrin I, Sigma-Aldrich (St. Louis, MO) Dihydrochloride Coproporphyrin I-15N4 Sodium Toronto Research Chemicals BIsulfate Salt Coproporphyrin III, Sigma-Aldrich (St. Louis, MO) Dihydrochloride CCK-Octapeptide (26-33) Peptide Institute. Inc. (Osaka, Japan) 7α-Hydroxy-4-cholesten-3-one Toronto Research Chemicals (North York, Canada) 7α-Hydroxy-4-cholesten-3-one- Toronto Research Chemicals (North York, Canada) d7 Chenodeoxycholic Acid 24-Acyl- Toronto Research Chemicals (North York, Canada) β-D-glucuronide Glycochenodeoxycholic Acid 3- Toronto Research Chemicals (North York, Canada) Sulfate Disodium Salt Sulfolithocholic Acid Toronto Research Chemicals (North York, Canada) Trimethylamine Salt 3-Sulfoglycolithocholic Acid Toronto Research Chemicals (North York, Canada) Disodium Salt Sodium Taurocholate (TCA)-d5 Toronto Research Chemicals (North York, Canada) (TCA-d5) 4-Pyridoxic acid Sigma-Aldrich (St. Louis, MO) 4-Hydroxy-3- FUJIFILM Wako Pure Chemical Corporation (Osaka, methoxyphenylacetic Acid Japan)

Analytical conditions for test compounds

No. Column Gradient condition Flow rate

Compounds Column temperature Mobile Phase (mL/min LC-MS/MS (B concentration %) (cent degree) )

1 CAPCELL PAK 40 0-0.5 min: 30% Prominence 0.5-3.0 min: 30-98% Paclitaxel C18 MG II (3 µm, A 5 mM ammonium acetate 3.0-3.5 min: 98% 0.4 UFLC system 6α hydroxy paclitaxel 2×50 mm; B methanol 3.5-3.7 min: 98-30%

3.7-4.5 min: 30% QTRAP5500 Shiseido)

2 ACQUITY UPLC 60 0-6.5 min: 35% CP-I Nexera X2 LC 6.5-6.8 min: 35-98% BEH C18 column A 0.1% formic acid in water 6.8-7.0 min: 98% CP-Ⅲ 0.6 system- (1.7 µm, 2.1×150 B 0.1% formic acid in acetonitrile 7.0-7.1 min: 98-35%

15 CP-I- N4 (IS) 7.1-10 min: 35% QTRAP5500 mm; Waters) 3 300 A: 10mM ammonium acetate in Nexera X2 LC PC HILIC (3 µm, Pyridoxic acid acetonitrile:water (20:80) 2.0×150 mm; 0-8 min: 95% 0.4 system- Homovanillic acidPho OSAKA SODA) B: 10mM ammonium acetate in QTRAP5500 acetonitrile:water (95:5) MRM conditions for drugs and endogenous compounds. DP; declustering potential,

CE; collision energy, CXP; collision cell exit potential.

Compound Ion Q1 [Da] Q3 DP CE CXP Retention

mode [Da] [V] [V] [V] time[m]

Paclitaxel Positive 854.2 286.0 111 17 24 2.58

6-αhydroxy Positive 870.2 286.0 156 17 16 2.40

paclitalxel

CP-I Positive 328.3 298.2 61 23 34 4.40

15 a CP-I- N4 Positive 330.1 300.1 76 23 36 4.40

3.00b

CP-III Positive 328.3 298.2 61 23 34 5.24

Pyridoxic acid Negative 182.0 138.0 - - - 1.41

40.00 30.00 10.00

0 0 0

Homovanillic acid Negative 180.9 135.9 - - - 5.71

35.00 10.00 21.00

0 0 0 a Analytical condition No.2 b Analytical condition No.3

Table S1 Effect of paclitaxel on AUC–3–7h of C4 and bile acids

AUC–3–7h of the C4 and bile acids were calculated. AUC–3–7r are presented as mean ± S.E. Fold changes are presented as GMR and 95% CI. (n = 10).

Compounds Control + Paclitaxel Fold change

C4 (M  h) 0.476 ± 0.073 0.210 ± 0.019 0.47 (0.39, 0.58)

CA (M  h) 1.87 ± 0.32 2.74 ± 0.79 1.3 (0.29, 3.)

GCA (M  h) 2.57±0.36 4.10±0.64 0.9 (0.83, 0.98)

TCA (M  h) 0.427±0.070 0.767±0.115 2.9 (2.2, 3.9)

CDCA (M  h) 19.2±4.4 32.9±11.0 4.0 (2.8, 5.8)

DCA (M  h) 10.7±1.8 11.7±2.2 2.5 (1.8, 3.4)

GCDCA (M  h) 15.2±2.6 21.4±3.1 2.5 (1.8, 3.4)

GDCA (M  h) 3.79±0.48 6.19±1.2 2.0 (1.2, 3.5)

TDCA (M  h) 0.691±0.098 1.18±0.19 2.2 (1.4, 3.6)