Regorafenib Is Transported by the Organic Anion Transporter 1B1 and the Multidrug Resistance Protein 2

Abstract

Regorafenib is a small molecule inhibitor of tyrosine kinases, and has been shown to improve the outcomes of patients with advanced colorectal cancer and advanced gastrointestinal stromal tumors. The transport profiles of regorafenib by various transporters were evaluated. HEK293/organic anion transporting polypeptide 1B1 (OATP1B1) cells exhibited increased drug sensitivity to regorafenib. Regorafenib inhibited the uptake of ³H-estrone sulfate by HEK293/OATP1B1 cells in a dose-dependent manner, but did not affect its elimination by P-glycoproteins. The concentration of regorafenib was significantly lower in LLC-PK1/multidrug resistance protein 2 (MRP2) cells than in LLC-PK1 cells treated with the MRP2 inhibitor, MK571. MK571 abolished the inhibitory effects of regorafenib on intracellular accumulation in LLC-PK1/MRP2 cells. The uptake of regorafenib was significantly higher in HEK293/OATP1B1 cells than in OATP1B1-mock cells. Transport kinetics values were estimated to be K_m=15.9 µM and V_max=1.24 nmol/mg/min. No significant difference was observed in regorafenib concentrations between HEK293/OATP1B3 and OATP1B3-mock cells. These results indicated that regorafenib is a substrate for MRP2 and OATP1B1, and also suggest that the substrate preference of regorafenib may implicate the pharmacokinetic profiles of regorafenib.

Regorafenib has been developed as a molecular targeted medicine for patients with metastatic colorectal cancer and advanced gastrointestinal stromal tumors, and inhibits certain tyrosine kinases. The biological effects of receptor tyrosine kinase activation are mediated by a complex cascade of intracellular signaling molecules that are potential targets for therapy, including the Raf, mitogen-activated protein extracellular kinase (MEK), and extracellular signal-regulated kinase (ERK) pathways.¹⁾ Regorafenib is a multikinase inhibitor that targets Raf serine/threonine kinases and the vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor (PDGF) receptor-β, c-Kit, Flt3, and p38 tyrosine kinases, which blocks VEGF- and PDGF-dependent angiogenesis.²⁾

The family of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 2 (MRP2, ABCC2), plays important roles in the detoxification and excretion of xenobiotics.³⁾ MRP2, an MRP isoform, is a clinically important transporter that has been shown to function in the terminal excretion of cytotoxic and carcinogenic substances.⁴⁾ MRP2 is important clinically because it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain drugs and disease states.⁵⁾ Organic anion transporting polypeptides (OATPs) belong to the superfamily of solute carrier transporters and are classified within the solute carrier of the OATPs (SLCO) gene family. OATPs function in the uptake of a wide range of amphipathic compounds by cells, including numerous endo- and xenobiotics. OATPs are known to be critically involved in the absorption, distribution, and excretion of drugs and other xenobiotics. Two members of the OATP1 family, OATP1B1 and OATP1B3, are expressed in the basolateral membrane of the liver. The expression of these transporters was previously shown to be in cancer tissues has been identified. Evidence to suggest that the expression of OATPs affect the development of cancer development is increasing. Expression levels of OATPs are altered in many different types of cancers; these altered expression levels have been correlated with cancer stage. OATPs are capable of transporting multiple compounds that affect cancer cell growth and survival, including hormones, hormone precursors, and anticancer drugs.⁶⁾ Some tyrosine kinase inhibitors are substrates or inhibitors of drug transporters. It was recently reported that nilotinib and vandetanib were transported by OATP1B1 and OATP1B3.^7,8) Sorafenib was identified as potent inhibitors of OATP1B1 in vitro.⁹⁾ The substrate preferences of transporters can lead to differences in pharmacokinetic profiles and drug-interaction characteristics.¹⁰⁾ However, detailed substrate preferences of regorafenib on P-gp, MRP2, OATP1B1 and OATP1B3 were not clear. In this study, we report the substrate preferences for the transporters about regorafenib.

MATERIALS AND METHODS

Chemicals

Regorafenib was purchased from CHEMSCENE, LLC. (Monmouth Junction, NJ, U.S.A.). Sorafenib was purchased from Toronto Research Chemicals Inc. (North York, Canada). Dulbecco’s modified Eagle’s medium (DMEM) and collagen (C9791) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Tritium-labeled [6,7-³H]-estrone sulfate, ammonium salt (2.12 TBq/mmol), and [³H]-taurocholic acid (0.185 TBq/mmol) were purchased from PerkinElmer, Inc. (Waltham, MA, U.S.A.). All other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell Culture

The human epidermoid carcinoma cell lines, KB-31 and KB-G2 (MDR1-transfected KB-31 cells),¹¹⁾ and pig kidney LLC-PK1 and LLC-PK1/MRP2 cell lines¹²⁾ were kindly provided by Dr. Furukawa (Department of Molecular Oncology, Kagoshima University, Japan). LLC-PK1/MRP2 was generated by transfecting human MRP2 cDNA into parent LLC-PK1 cells. The OATP1B1/HEK293 and OATP1B3/HEK293 cell lines¹³⁾ were kindly provided by Dr. Yamaguchi (Department of Pharmacy, Tohoku University, Japan). Cells were grown in DMEM, supplemented with 10% fetal bovine serum and 2 mM glutamine, at 37°C in a 5% CO₂ humidified atmosphere. Cells were seeded at a concentration of 2×10⁵ cells/mL on the day before the regorafenib treatment.

Cell Growth Assays

The inhibition concentration of 50% (IC₅₀) cell growth was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were incubated in culture medium with various concentrations of drugs in a final volume of 100 µL. After 3 d, 20 µL of MTT (2.5 mg/mL) was added to each well and the plates were incubated for an additional 3 h. The resulting formazan was dissolved in 100 µL of 20% sodium dodecyl sulfate solution.¹⁴⁾ The plates were read at 590 nm after overnight shaking using the iMark Micro Plate Reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Drug Concentration Assay

The ultra pressure liquid chromatography (UPLC) analysis was conducted to evaluate the accumulation of regorafenib in cells. The UPLC system consisted of a QSM pump, TUV detector, CHA column heater and SM-FTN auto sampler (Waters, Milford, U.S.A.). The analysis conditions used for the UPLC analysis were modified from van Erp et al.¹⁵⁾ The column temperature was maintained at 50°C in the CHA column heater. Two microliter samples were injected onto a reverse-phase column (ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 1 mm×50 mm, Waters). The operation conditions were as follows: Detector, ultraviolet absorption photometer wavelength of 254 nm; mobile phases, 0.1% formic acid and 2 mM ammonium acetate in water/0.1% formic acid and 2 mM ammonium acetate in methanol (35 : 65); flow rate of 0.4 mL/min. The retention times of regorafenib and the internal standard were 2.65 min and 2.18 min, respectively (Fig. 1). Washed cells were lysed with methanol containing an internal standard, 0.5 µM sorafenib. The lysis solution was centrifuged at 12000×g for 15 min. Methanol was removed by evaporation from the supernatant, and residue was then deproteinized with the mobile-phase solution. The solution was filtrated through a 0.2-µm filter (Advantec Toyo Kaisha, Ltd., Hydrophobic PTFE Membrane Filter). Protein concentrations were determined using a protein assay (Bio-Rad Laboratories).

Fig. 1. A Typical Chromatogram from the Assay for Intracellular Regorafenib

Cells were incubated in KH buffer with regorafenib for 3 min. They were then deproteinized with methanol and the mobile-phase solution. The retention times of regorafenib and the internal standard were 2.65 min and 2.18 min, respectively. The column temperature was maintained at 50°C in the column heater. Two microliter samples were injected onto a reverse-phase column (ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 1 mm×50 mm). Flow rate: 0.4 mL/min. Analysis conditions were described in detail in Materials and Methods.

Drug Accumulation Assay

Cell culture dishes were coated with collagen solution (50 µg/mL) before seeding cells. Seeded cells on dishes were washed with KH buffer (pH 7.4, 37°C, 118 mM NaCl, 5 mM D-glucose, 4.83 mM KCl, 0.96 mM KH₂PO₄, 23.8 mM NaHCO₃, 1.53 mM CaCl₂, 1.2 mM MgSO₄, 12.5 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)), and were then incubated for 10 min. These cells were incubated for 3 min at doses of 1, 3, 5, 10, 30, and 50 µM of regorarafenib in KH buffer, and then washed with ice-cold KH buffer containing 1% (bovine serum albumin (BSA) for 30 s. The cells were washed three times with ice-cold KH buffer and incubated with ³H-estrone sulfate (10 nM) or ³H-taurocholic acid (50 nM) for 2 min. The accumulation of radio activity in the cells was evaluated by liquid scintillation counting using TRI-CARB 1600TR (PerkinElmer, Inc., Waltham, MA, U.S.A.).

RESULTS

Inhibitory Effect of Regorafenib on Cell Growth

Cell viability was evaluated by the MTT assay. Etoposide was employed as the positive control for drug resistance against MDR1. KB-G2 cells exhibited drug resistance to regorafenib and etoposide. Methotrexate was also employed as the positive control for drug resistance against MRP2. LLC-PK1/MRP2 cells showed drug resistance to MTX. HEK293/OATP1B1 cells exhibited increased drug sensitivity to regorafenib, that of LLC-PK1/MRP2 cells was increased (Table 1).

Table 1. Concentration of Regorafenib Required to Inhibit Cell Growth by 50% and Resistance Ratios

	KB-31	KB-G2	Ratio
Regorafenib	5.5±0.3	9.1±0.1**	1.65
Etoposide	3.9±0.2	219.5±3.6***	56.2
	LLC-PK1	LLC-PK1/MRP2
Regorafenib	42.0±3.2	82.4±2.7***	1.96
Methotraxate	181±19	33154±2394***	183.2
	HEK293	HEK293/OATP1B1
Regorafenib	11.0±1.2	6.2±0.3**	0.56

The concentration of regorafenib needed to inhibit cell growth by 50% was evaluated by the MTT assay. Each value indicates means±S.E. of the mean (nM) from 3 independent experiments. Resistant ratios are shown in the ratio column. KB-G2 is a human MDR1 gene-transfected cell line with the human epidermoid carcinoma cell line KB-31. Statistical analyses for paired samples were performed by the two-tailed Student’s t-test; ** p<0.01, *** p<0.001.

Accumulation of Regorafenib in Cells

The concentration of regorafenib in cells was determined by the UPLC analysis (Fig. 1). Cells were incubated for 60 min at a dose of 1 µM of regorafenib. The concentration of regorafenib was significantly lowered in LLC-PK1/MRP2 cells than in LLC-PK1 cells those treated with MK571. MK571 returned to regorafenib concentration in the LLC-PK1/MRP2 cells. The P-gp inhibitor, cyclosporin A did not affect the elimination of regorafenib from KB-31 and KB-G2 cells (Table 2). HEK293/OATP1B1 cells were incubated at doses of 1, 3, 5, 10, 30, and 50 µM of regorafenib for 3 min. The concentration of regorafenib was significantly higher in HEK293/OATP1B1 cells than in OATP1B1-mock cells. Transport kinetics values were estimated to be K_m=15.9 µM and V_max=1.24 nmol/mg/min (Fig. 2b). No significant difference was observed in regorafenib concentrations between HEK293/OATP1B3 cells and OATP1B3-mock cells (Fig. 3).

Table 2. Concentration of Regorafenib in Cells after a 60-min Incubation

	Vehicle	Cyclosporin A
KB-31	1.26±0.08	0.93±0.06
KB-G2	1.27±0.08	1.09±0.09
	Vehicle	MK571
LLC-PK1	1.40±0.06	1.36±0.09
LLC-PK1/MRP2	0.97±0.02*	1.39±0.05

Intracellular concentrations of regorafenib after the incubation of cells at a dose of 1 µM of regorafenib for 60 min. Regorafenib concentrations were shown as per the amount of whole cell homogenate protein. Each value indicates means±S.E. of the mean (nmol/µg protein) from triplicate measurements. Differences between the treatments were evaluated using the Tukey–Kramer test; * p<0.05, significantly different from LLC-PK1 cells or those treated with MK571.

Fig. 2. (a) Concentration of Regorafenib in HEK293/OATP1B1 Cells; (b) Eadie–Hofstee Plot Analysis or Regorafenib Uptake; (c) Inhibitory Effects of Regorafenib on the Uptake of ³H-Estrone Sulfate by HEK293/OATP1B1 Cells

(a) Cells were incubated at doses of 1, 3, 5, 10, 30, and 50 µM of regorafenib for 3 min. The concentration of regorafenib was shown as per the amount of whole cell homogenate protein. Each value indicates means±S.E. of the mean (nmol/µg protein) from triplicate measurements. Differences between individual groups of cells were evaluated using the Tukey–Kramer post hoc test, ** p<0.01 significantly different from OATP1B1-mock cells. (b) Cells were incubated for 3 min at a dose of 1–50 µM; K_m=15.9 µM, V_max=1.24 nmol/mg/min. (c) Cells were incubated with ³H-estrone sulfate at a dose of 10 nM for 2 min. Regorafenib was added to the medium at doses of 0.5, 1, 5, 10, 50, and 100 µM. The relative uptake of ³H-estrone sulfate was inhibited by the addition of regorafenib in a dose-dependent manner.

Fig. 3. Uptake of Regorafenib by HEK293/OATP1B3 Cells

Cells were incubated at a dose of 1 µM of regorafenib. The concentration of regorafenib was shown as per the amount of whole cell homogenate protein. Each value indicates means±S.E. of the mean (pmol/µg protein) from triplicate measurements. Comparisons between 3 groups were performed with the Tukey–Kramer test. No significant differences were observed between the groups.

Inhibition Assay

The inhibitory effects of regorafenib on OATP1B1 and OATP1B3 were evaluated. HEK293/OATP1B1 cells significantly accumulated ³H-estrone sulfate, a substrate for OATP1B1 (HEK293/OATP1B1, 0.183±0.011 pmol/mg protein; HEK293/MOCK, 0.031±0.005 pmol/mg protein; means±standard error (S.E.), p=3×10⁻¹¹, Student’s t-test). Regorafenib inhibited the uptake of ³H-estrone sulfate by HEK293/OATP1B1 cells in a dose-dependent manner (Fig. 2c). HEK293/OATP1B3 cells significantly accumulated ³H-taurocholic acid, a substrate for OATP1B3 (HEK293/OATP1B3, 0.253±0.026 pmol/mg protein; HEK293/MOCK, 0.145±0.005 pmol/mg protein; means±S.E., p=0.002, Student’s t-test). Regorafenib did not inhibit the transport of ³H-taurocholic acid in HEK293/OATP1B3 cells (data not shown).

DISCUSSION

We here in demonstrated that regorafenib was transported by MRP2 and OATP1B1. Evidences to suggest that tyrosine kinase inhibitors (TKIs) are transported by the ATP-binding cassette (ABC) transporters is increasing. P-gp, MRP2, MRP1 and the breast cancer resistance protein (BCRP, ABCG2) have been shown to transport TKIs, such as imatinib, gefitinib, and sorafenib.^14,16) Khurana et al. recently reported that nilotinib and vandetanib were transported by OATP1B1 and OATP1B3.^7,8) A member of the OATPs family, OATP1A2 was previously reported to transport imatinib, a drug used to treat certain types of leukemias.¹⁷⁾ The substrate specificity of OATPs for TKIs has not yet been elucidated in detail. The present study is the first report to show that the tyrosine kinase inhibitor, regorarafenib was a substrate for MRP2 and OATP1B1. A summary of the pharmacokinetics of regorafenib (Stivarga tablets®, BAY73-4506) showed the efflux ratios of regorafenib to be 0.30 and 0.093, for LLC-PK1/MDR1 and LLC/PK1, respectively and also that regorafenib did not inhibit the basal to apical transport of dipyridamole (1 µM) when used at a dose of 10 µM. This summary also reported that 10 µM of regorafenib did not inhibit the uptake of pravastatin, a substrate for OATP, by HEK293/OATP1B1 cell, while 0.1 µM of regorafenib did not inhibit the uptake of pravastatin by HEK293/OATP1B3 cells.¹⁸⁾ These findings indicated that regorafenib was not a substrate for P-gp, OATP1B1, or OATP1B3. In the present study, KB-G2 cells showed negligible drug resistance to regorafenib, and the inhibitory treatment with cyclosporine A for P-gp did not affect the accumulation of regorafenib (Table 2). The results of the present study also suggested that regorafenib was not a substrate of P-gp. MK571 inhibited the accumulation of regorafenib, and the IC₅₀ of regorafenib was increased in LLC-PK1/MRP2 cells (Tables 1, 2). These results indicated that regorafenib was transported by MRP2. We also demonstrated that regorafenib inhibited the transport of ³H-estrone sulfate and accumulation of regorafenib in HEK293/OATP1B1 cells; therefore, regorafenib may be transported by OATP1B1. However, the summary of the pharmacokinetics of BAY73-4506 reported that 10 µM of regorafenib did not inhibit the uptake of pravastatin, suggesting that regorafenib was not a substrate for OATP1B1. The present study showed that the K_m value was 15.9 µM, indicating that 10 µM of regorafenib could not inhibit the uptake of pravastatin.

Polarized tissues directly involved in drug disposition (the intestines, kidney, and liver) asymmetrically express various drug transporters on their apical and basolateral sides to allow for vectorial drug transport. The OATPs family on the basolateral membrane and MRP2 on the apical membrane of hepatocytes have been shown to take up and excrete organic anionic compounds.¹⁹⁾ Vectorial drug transport plays a crucial role in the elimination of organic anionic compounds from the blood to bile. For example, the markedly higher transcellular flux of enalapril and estradiol-17β-D-glucuronide from the basolateral to apical membrane was observed in double-transfected OATP1B1/MRP2/Madin–Darby canine kidney (MDCK) cells than in single-transfected OATP1B1/MDCK cells.²⁰⁾ A phase I study also reported that the pharmacokinetics of regorafenib showed a bimodal pattern for its concentrations in the plasma (t_max, 3.6 and 48 h); suggesting the disposition of regorafenib for enterohepatic circulation.²¹⁾ The vectorial drug transport of OATP1B1 on the basolateral membrane and MRP2 on the apical membrane may play a role in the enterohepatic circulation of regorafenib.

In conclusion, the results of the present study suggests that regorafenib is a substrate for MRP2 and OATP1B1, and also that the substrate preference of regorafenib may affect the pharmacokinetic profiles and drug resistance to anticancer drugs.

Acknowledgment

The present study was supported by the Oncology Pharmacy Specialists of Oncology Professionals in Hokkaido, who were supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, Schreck RE, Abrams TJ, Ngai TJ, Lee LB, Murray LJ, Carver J, Chan E, Moss KG, Haznedar JO, Sukbuntherng J, Blake RA, Sun L, Tang C, Miller T, Shirazian S, McMahon G, Cherrington JM. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin. Cancer Res., 9, 327–337 (2003).
• 2) Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M, Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollag G, Trail PA. BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res., 64, 7099–7109 (2004).
• 3) Dietrich CG, Geier A, Oude Elferink RP. ABC of oral bioavailability: transporters as gatekeepers in the gut. Gut, 52, 1788–1795 (2003).
• 4) König J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim. Biophys. Acta, 1461, 377–394 (1999).
• 5) Gerk PM, Vore M. Regulation of expression of the multidrug resistance-associated protein 2 (MRP2) and its role in drug disposition. J. Pharmacol. Exp. Ther., 302, 407–415 (2002).
• 6) Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer. Annu. Rev. Pharmacol. Toxicol., 52, 135–151 (2012).
• 7) Khurana V, Minocha M, Pal D, Mitra AK. Role of OATP-1B1 and/or OATP-1B3 in hepatic disposition of tyrosine kinase inhibitors. Drug Metabol. Drug Interact., 29, 179–190 (2014).
• 8) Khurana V, Minocha M, Pal D, Mitra AK. Inhibition of OATP-1B1 and OATP-1B3 by tyrosine kinase inhibitors. Drug Metabol. Drug Interact., 29, 249–259 (2014).
• 9) Hu S, Mathijssen RH, de Bruijn P, Baker SD, Sparreboom A. Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro–in vivo correlations. Br. J. Cancer, 110, 894–898 (2014).
• 10) Thomas-Schoemann A, Blanchet B, Bardin C, Noé G, Boudou-Rouquette P, Vidal M, Goldwasser F. Drug interactions with solid tumor-targeted therapies. Crit. Rev. Oncol. Hematol., 89, 179–196 (2014).
• 11) Mukai M, Che XF, Furukawa T, Sumizawa T, Aoki S, Ren XQ, Haraguchi M, Sugimoto Y, Kobayashi M, Takamatsu H, Akiyama S. Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells. Cancer Sci., 94, 557–563 (2003).
• 12) Chen ZS, Kawabe T, Ono M, Aoki S, Sumizawa T, Furukawa T, Uchiumi T, Wada M, Kuwano M, Akiyama S. Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter. Mol. Pharmacol., 56, 1219–1228 (1999).
• 13) Yamaguchi H, Takeuchi T, Okada M, Kobayashi M, Unno M, Abe T, Goto J, Hishinuma T, Shimada M, Mano N. Screening of antibiotics that interact with organic anion-transporting polypeptides 1B1 and 1B3 using fluorescent probes. Biol. Pharm. Bull., 34, 389–395 (2011).
• 14) Shibayama Y, Nakano K, Maeda H, Taguchi M, Ikeda R, Sugawara M, Iseki K, Takeda Y, Yamada K. Multidrug resistance protein 2 implicates anticancer drug-resistance to sorafenib. Biol. Pharm. Bull., 34, 433–435 (2011).
• 15) van Erp NP, de Wit D, Guchelaar HJ, Gelderblom H, Hessing TJ, Hartigh J. Hartigh Jd. A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci., 937, 33–43 (2013).
• 16) He M, Wei MJ. Reversing multidrug resistance by tyrosine kinase inhibitors. Chin. J. Cancer, 31, 126–133 (2012).
• 17) Hu S, Franke RM, Filipski KK, Hu C, Orwick SJ, de Bruijn EA, Burger H, Baker SD, Sparreboom A. Interaction of imatinib with human organic ion carriers. Clin. Cancer Res., 14, 3141–3148 (2008).
• 18) Pharmaceutical and Medical Devices Agency, Japan. “The review report of stivarga tablets 40 mg.”: ‹http://www.pmda.go.jp/english/service/pdf/drugs/stivarga_mar2010_e.pdf›, cited 15 March, 2013.
• 19) Ito K, Suzuki H, Horie T, Sugiyama Y. Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport. Pharm. Res., 22, 1559–1577 (2005).
• 20) Liu L, Cui Y, Chung AY, Shitara Y, Sugiyama Y, Keppler D, Pang KS. Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers. J. Pharmacol. Exp. Ther., 318, 395–402 (2006).
• 21) Sunakawa Y, Furuse J, Okusaka T, Ikeda M, Nagashima F, Ueno H, Mitsunaga S, Hashizume K, Ito Y, Sasaki Y. Regorafenib in Japanese patients with solid tumors: phase I study of safety, efficacy, and pharmacokinetics. Invest. New Drugs, 32, 104–112 (2014).