Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-015-0286-1

ORIGINAL PAPER

Drug–Drug Interactions of a Novel j-Opioid Receptor Agonist, Nalfurafine Hydrochloride, Involving the P-Glycoprotein Akihiro Ando1 • Shinichiro Sasago2 • Yoshihiro Ohzone3 • Yohei Miyamoto1

Ó Springer International Publishing Switzerland 2015

Abstract Background and Objective Nalfurafine hydrochloride (TRK-820), which exhibits strong j-opioid agonistic activity, has an antipruritic effect on uremic pruritus. The permeability of nalfurafine across human P-glycoprotein (P-gp)-expressing LLC-PK1 cells was investigated to evaluate drug–drug interactions (DDI) involving the P-gp efflux transporter of nalfurafine. Furthermore, we assessed the ratio of brain/plasma concentrations (Kp) as an indicator to investigate the changes in the blood–brain barrier (BBB) transport through P-gp when digoxin or verapamil was concomitantly administered with nalfurafine in mice. Methods All samples were analyzed by liquid chromatography–tandem mass spectrometry or a liquid scintillation counter. Results The cleared volume ratio (cleared volume from basal to apical/cleared volume from apical to basal) of nalfurafine in P-gp-expressing cells was higher than that in the control cells; however, no concentration-dependent decrease in the cleared volume ratio of digoxin was observed in the presence of nalfurafine. The Kp value in mice showed similar profiles to those observed with

& Akihiro Ando [email protected] 1

Pharmaceutical Clinical Research Department, Toray Industries, Inc., 1-1, Nihonbashi-muromachi 2-chome, Chuoku, Tokyo 103-8666, Japan

2

Department of Bio Research, Kamakura Techno-Science, Inc., 6-10-1, Tebiro, Kamakura, Kanagawa 248-0036, Japan

3

ADME & Tox. Research Institute, Sekisui Medical Co., Ltd., 2117 Muramatsu, Tokai-mura, Naka-gun, Ibaraki 319-1182, Japan

nalfurafine alone and when co-administered with digoxin or verapamil. Conclusions From these results, nalfurafine was found to be a substrate for P-gp, but had no inhibitory effect on P-gp-mediated transport. Furthermore, it is unlikely that nalfurafine transport via the BBB is affected by P-gp substrates in humans.

1 Introduction Three types of opioid receptors (l, d, and j) have been well established by pharmacological and biological studies [9, 22]. The activation of j-opioid receptors produces analgesia with minimum physical dependence and respiratory depression [12], and its agonists have antipruritic activity. The activation of j-opioid receptors is also effective against antihistamine-sensitive pruritus [2, 4]. The selective j-opioid receptor agonist nalfurafine hydrochloride (TRK-820) [13, 17] has been confirmed to be clinically tolerated, efficacious, and safe in the treatment of hemodialysis patients with uremic pruritus resistant to conventional treatment, especially in severely ill patients [10, 11, 14, 23]. Nalfurafine was shown to have a marked effect on intractable pruritus in previous studies using animal models by a mechanism of action on j-opioid receptors in the central nervous system (CNS) [19, 21]. P-glycoprotein (P-gp) is a kind of human ATP-binding cassette (ABC) protein superfamily, a large group of proteins comprised of membrane transporters, ion channels, and receptors, and is encoded in humans by the multidrug resistance gene MDR1 (ABCB1) [1, 3]. P-gp was initially discovered in multidrug-resistant tumor cell lines [7]. However, subsequent studies have shown that P-gp is ubiquitously expressed in healthy tissues, including the

A. Ando et al.

small intestine, blood–brain barrier (BBB), liver, and kidney [1, 3, 20]. P-gp has very broad substrate specificity, with a tendency towards lipophilic, cationic compounds [16, 18]. The number of substrates and/or inhibitors of P-gp is continually increasing and includes anticancer agents, antibiotics, antivirals, calcium channel blockers, and immunosuppressive agents [1]. P-gp-mediated drug–drug interactions (DDIs) attributable to alterations in the disposition of drugs have been widely recognized and affect their bioavailability and effectiveness [3, 5]. A drug can affect the distribution of another drug into the brain via BBB transport, especially in drugs acting on the CNS, which can result in side effects [3]. In this study, we investigated the potential involvement of P-gp in nalfurafine cell permeation using human P-gpexpressing LLC-PK1 cells. Moreover, we determined nalfurafine concentrations in the plasma and brain when digoxin or verapamil was concomitantly administered as a P-gp substrate with nalfurafine in mice to confirm changes in BBB transport through P-gp.

2 Materials and Methods 2.1 Chemicals Nalfurafine hydrochloride, (2E)-N-[(5R,6R)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]3-(furan-3-yl)-N-methylprop-2-enamide monohydrochloride, and deuterated nalfurafine hydrochloride (TRK-820d4) were synthesized by Toray Industries, Inc (Tokyo, Japan). 3H-Digoxin and 14C-mannitol were purchased from PerkinElmerÒ (Waltham, MA, USA). Digoxin, verapamil, Medium 199, gentamicin, hygromycin B, and bovine serum albumin (BSA) were purchased from SigmaAldrichÒ (St. Louis, MO, USA). Hanks’ Balanced Salt Solution (109 concentrated, 109 HBSS) were purchased from Sigma-AldrichTM (Carlsbad, CA, USA). Sodium hydrogencarbonate, 1 M sodium hydroxide, 0.1 M sodium hydroxide, distilled water, methanol, formic acid, dimethyl sulfoxide (DMSO), sodium dihydrogen phosphate, and trifluoroacetic acid obtained commercially were of special grade or analytical grade. Milli-Q water was water purified with an ultra-pure water purification system (Millipore, Billerica, MA, USA). 2.2 Cells P-gp-expressing cells (porcine kidney epithelial LLC-PK1 cells with transfection vectors containing human P-gp cDNA) and control cells (LLC-PK1 cells with transfection vectors only) were used under sublicense from Discovery Labware, Inc., BD Biosciences (Franklin Lakes, NJ, USA).

The control cells and P-gp-expressing cells were seeded at a density of 4 9 104 cells/insert in the plates (culture insert, PET porous filter, pore size 3 lm, area 0.3 cm2; and FalconTM cell culture insert companion plate, Franklin Lakes, NJ, USA), and were incubated in a CO2 incubator (37 °C, 5 % CO2 in air) for 8 days while the culture medium was changed every 2 or 3 days to prepare cell monolayers for the determination of cellular transport activity (cleared volume). The medium for passage was composed of Medium 199 containing 9 % fetal bovine serum (FBS), 50 lg/mL gentamicin, and 100 lg/mL hygromycin B. The medium for differentiation was composed of Medium 199 containing 9 % FBS and 50 lg/mL gentamicin. 2.3 Animals Male Crlj:CD1(ICR) mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All animals were acclimated to the experimental conditions 7 days before use. Animals were housed in an airconditioned room at a temperature of 22 ± 3 °C. Food and water were supplied ad libitum throughout the acclimatization and experimental period; however, the animals were fasted overnight before administration, except for water, and were fed 4–5 h post dose. At the commencement of this study, the chairperson and members of the Animal Experiment Committee (Kamakura, Kamakura TechnoScience, Inc.) approved (permit number AC-037 M) all procedures corresponding to the animal experiment according to the ‘‘Guidelines for Animal Experiments in Kamakura Techno-Science, Inc.’’ based on Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan, 2006. 2.4 Transport Study of Nalfurafine Using Human P-gp-Expressing LLC-PK1 Cells The medium in the culture insert and the plate was removed by aspiration and replaced with HBSS, and confluent cells (P-gp-expressing and control cell) were preincubated at 37 °C for 1 h. The experiment was started by replacing the buffer at either apical or basal side of the cell monolayer with HBSS solution of 1 lM nalfurafine, 1 lM 3 H-digoxin, or 1 lM 14C-mannitol. Cells were incubated at 37 °C, and an aliquot of medium (70 lL of HBSS) was collected from the opposite side (receiver compartment) after incubation for 0.5, 1, and 2 h. To compensate for the collection volume, 70 lL of HBSS pre-warmed at 37 °C was added immediately after the collection. Samples were prepared and incubated in triplicate. The appearance of radioactivity in the acceptor compartment was monitored. The positive control of P-gp-mediated transport and

Drug–Drug Interactions of Nalfurafine Involving the P-gp

intercellular flux were monitored as the appearance of 3Hdigoxin and 14C-mannitol in the acceptor compartment, respectively. To determine the radiolabeled compounds (3H-digoxin and 14C-mannitol), the collected HBSS, 70 lL, was mixed with 10 mL of scintillator (Hionic-FluorTM, PerkinElmerÒ, Waltham, MA), and radioactivity was measured using a liquid scintillation counter (2500TR, PerkinElmerÒ, Waltham, MA). Non-radiolabeled nalfurafine hydrochloride was determined using chromatography–tandem mass spectrometry (LC/MS/MS) systems. The calibration samples prepared with HBSS and analytical samples, 70 lL, were mixed with 30 lL of the internal standard solution, and the mixtures were stirred with a vortex mixer. The whole quantity of each mixture was transferred into a sample tube for LC/MS/MS and injected into the LC/MS/MS port. Nalfurafine was analyzed using a high-performance liquid chromatography (HPLC) system (Agilent 1100 series, Agilent Techonologies, Santa Clara, CA, USA), a tandem mass spectrometer equipped with an electrospray interface (API4000, Applied Biosystems, MDS SCIEX, Warrington, UK), and an analytical column, a CAPCELL PAK C18 UG-120 (2.0 mm I.D. 9 50 mm L, 3 lm, Shiseido, Tokyo, Japan) maintained at 40 °C. The mobile phase consisted of 0.1 % formic acid and acetonitrile (84:16, v/v). The flow rate was 0.2 mL/min, and the injection volume was 5 lL. Conditions at the electrospray ionization (ESI; ionization mode, turbo ionspray) inlet were as follows: ion spray voltage, 4000 V; probe temperature, 650 °C. Positive selected-ion monitoring and multiple reaction monitoring (MRM) were employed for quantification, and the transitions (m/z) chosen were 477.0 ? 308.5 for nalfurafine and 481.1 ? 309.5 for TRK-820-d4. A calibration curve was constructed using six concentrations of calibration samples. The regression equation of the calibration curve was determined by linear regression. The calibration curve (1–300 nM) was backcalculated and the acceptance criterion was that the accuracy was within the range 85.0–115 % [80.0–120 % at the lower limit of quantification (LLOQ)]. 2.5 Inhibitory Effect of Nalfurafine on Digoxin Transport Using Human P-gp-Expressing LLCPK1 Cells The inhibitory effect of nalfurafine on digoxin transport was evaluated in a manner similar to the transport study of nalfurafine. 3H-Digoxin, verapamil, and 14C-mannitol were used as the P-pg substrate, positive control of the inhibitor, and intercellular transport marker, respectively. The medium in the culture insert and the plate was removed by aspiration, and HBSS was added, with or without an appropriate concentration of inhibitors (nalfurafine and verapamil), to both the apical and basal sides of the cell

monolayer. The concentrations of the inhibitors in incubation mixture were 0 (vehicle), 0.001, 0.01, 0.1, and 1 lM for nalfurafine, and 30 lM for verapamil. After preincubation at 37 °C for 1 h, the experiment was started by replacing the HBSS at either the apical or basal side of the cell monolayer with HBSS containing 3H-digoxin and nalfurafine (A), 3H-digoxin and verapamil (B), or 14Cmannitol (C). Following the incubation at 37 °C for a further 2 h, an aliquot of medium (HBSS) was withdrawn from each compartment, and the radioactivity in the opposite compartment (receiver) to that initially containing 3 H-digoxin and 14C-mannitol was determined. 2.6 Drug–Drug Interactions at P-gp in the BBB Between Nalfurafine and P-gp Substrates Nalfurafine and the P-gp substrate (digoxin or verapamil) were co-administered by oral gavage to three parallel groups (group A, 1 mg/kg nalfurafine; group B, 1 mg/kg nalfurafine and 8.8 mg/kg digoxin; group C, 1 mg/kg nalfurafine and 17 mg/kg verapamil, 6 weeks, 22.4–30.1 g, n = 3 for each group) in CD-1 (ICR) male mice. At 0.25, 0.5, 0.75, 1, 2, 4, 6, and 24 h after the administration, whole blood was collected from the abdominal aorta of three mice for each group at each time point under ether anesthesia through a 1-mL disposable syringe to which 5 lL of heparin sodium (Novo-Heparin, 10,000 units/ 10 mL, Mochida Pharmaceutical, Tokyo, Japan) was added. After blood collection, the skull was opened to obtain the cerebrum and cerebellum together. The brain was washed thoroughly with physiological saline, weighed, and stored at -30 °C until analysis. Plasma was obtained by centrifugation (Centrifuge 5417R, Eppendorf, Hamburg, Germany) at 4 °C, 68009g, for 5 min and stored at 30 °C until analysis. The brain was thawed at room temperature then and weighed after an approximately equal amount of distilled water was added; the brain was then homogenized using a Polytron homogenizer (KINEMATICA, Littau, Switzerland). Twenty microliters of internal standard solution (distilled water for blank) and 200 lL 100 mM sodium dihydrogen phosphate were added to 100 lL of the collected plasma or brain homogenate sample to which 20 lL 50 vol% methanol was added. All contents of each sample mixture were then transferred to solid-phase columns (OASISÒ HLB 1 mL, Waters, Milford, MA, USA) that had been conditioned in advance with 2 mL methanol and 2 mL distilled water. After washing with 2 mL distilled water and 2 mL 50 vol% methanol, each column was eluted with 2 mL formic acid/methanol (1:99, v/v). The eluted solutions were dried under flowing nitrogen gas, and then 150 lL 0.1 vol% formic acid/ methanol (80:20, v/v) was added to redissolve the residue using a Vortex mixer. The solutions were centrifuged at

A. Ando et al.

4 °C, 10,6009g for 10 min using a centrifuge filter (UltrafreeÒ MC 0.45 lm, Millipore Billerica, MA, USA) to obtain a filtrate used as the sample for analysis. Nalfurafine was analyzed using an HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA), a tandem mass spectrometer equipped with an electrospray interface (API4000, Applied Biosystems MDS SCIEX, Warrington, UK), and a CAPCELL PAK C18 MGII (2.0 mm I.D. 9 50 mm L, 5 lm, Shiseido, Tokyo, Japan) maintained at 40 °C. The mobile phase consisted of 0.02 vol% trifluoroacetic acid (TFA) in distilled water (A) and 0.02 vol% TFA in methanol (B). The organic modifier content B was isocratic 50 % over 2.5 min for the plasma, and over 5 min for the brain. The flow rate was 0.2 mL/ min and the injection volume was 10 lL. Conditions at the ESI (ionization mode, turbo spray) inlet were as follows: spray voltage, 5000 V; probe temperature, 700 °C. Positive selected-ion monitoring and multiple reaction monitoring (MRM) were employed for quantification, and the transitions (m/z) chosen were 477.2 ? 308.2 for nalfurafine and 419.3 ? 200.5 for TRK-820-d4. Measurement of the concentration of concomitant drugs, digoxin and verapamil, was performed as follows. Pretreatment of the measurement samples was performed with methanol for deproteinization. Methanol was added to the plasma sample, the samples were mixed for 1 min, and then centrifuged at 4 °C 10,6009g for 2 min. Distilled water was added to the supernatant after centrifugation and the mixture was used as the sample for analysis. Digoxin and verapamil were analyzed using an HPLC system and tandem mass spectrometer equipped with an electrospray interface, the same as that used to analyze nalfurafine. Analytical columns were SunfireTM C18 (2.1 mm I.D. 9 20 mm L, 3.5 lm, Waters, Milford, MA, USA) for digoxin and CAPCELL PAK C18 MGII (2.0 mm I.D. 9 50 mm, 5 lm, Shiseido, Tokyo, Japan) for verapamil, and were maintained at 40 °C. The mobile phase consisted of 0.1 vol% formic acid (A) and methanol (B). The organic modifier content B was isocratic 70 % over 2.5 min for digoxin and isocratic 50 % over 2.5 min for verapamil. The flow rate was 0.2 mL/min. Conditions at the ESI (ionization mode, turbo spray) inlet were as follows: spray voltage, 5500 V; probe temperature, 500 °C (digoxin) and 650 °C (verapamil). Positive selected-ion monitoring and MRM were employed for quantification, and the transitions (m/z) chosen were 781.4 ? 651.3 for digoxin and 455.3 ? 165.0 for verapamil. A calibration curve was created with a minimum of six concentration points, excluding the blank, that cover all ranges of measurements. Accuracy was within the range 85.0–115 % (LLOQ 80.0–120 %) for at least 75 % of all points used for the calibration curve, or for a minimum of six points.

2.7 Data Analysis The permeability of nalfurafine, 3H-digoxin, and 14Cmannitol (cleared volume, lL/well) from apical to basal (A to B) and basal to apical (B to A) was calculated by dividing the amount permeated by the initial concentration in the donor compartment. The cleared volume ratio was calculated by dividing the cleared volume of B to A by that of A to B. In the inhibitory effect study, the cleared volumes of 3H-digoxin from the A to B side and the B to A, respectively, were statistically compared between the verapamil or nalfurafine group and the vehicle group. These data were analyzed by Tukey’s honestly significant difference (HSD) test at the significance level P \ 0.05 or P \ 0.01. The analysis was performed using Statistica ver. 5.1 (StatSoft, Inc., Tulsa, OK, USA). The concentrations of measured plasma and brain samples were calculated using AnalystÒ 1.4 software (Applied Biosystems/ MDS Sciex). Pharmacokinetic parameters were calculated from the mean values of the plasma and brain concentrations at each time point. A non-linear regression data analysis program (WinNonlin Professional version 5.0.1, Pharsight) was used to calculate the pharmacokinetic parameters [maximum concentration (Cmax), time to reach Cmax (Tmax), area under the concentration–time curve from time zero to 24 h (AUC0–24 h), area under the concentration–time curve from time zero to infinity (AUC0–?), mean residence time from time zero to infinity (MRT0–?), and elimination half-life (t1/2)] in the plasma. A noncompartmental analysis model was used as the analysis model. AUC0–24 h of brain concentrations was calculated using the trapezoidal method. The ratio of brain/plasma concentrations, the Kp value, was calculated by dividing the brain concentration by the plasma concentration.

3 Results 3.1 Transport Study of Nalfurafine Using Human P-gp-Expressing LLC-PK1 Cells The cellular transport (cleared volume) and cleared volume ratio of nalfurafine, 3H-digoxin, and 14C-mannitol across the monolayers of control and P-gp-expressing cells are shown in Fig. 1 and Table 1. After the incubation of nalfurafine at 1 lM for 0.5, 1, and 2 h, the cleared volumes from A to B in the control cells were 8.48, 22.0, and 45.0 lL/well, respectively, and the cleared volumes from B to A were 7.58, 21.0, and 41.1 lL/well, respectively. The cleared volume ratios were 0.9, 1.0, and 0.9, respectively. The cleared volumes from A to B in P-gp-expressing cells were 3.81, 8.37, and 17.1 lL/

Drug–Drug Interactions of Nalfurafine Involving the P-gp

(a) Control cells

Fig. 1 Cellular transport of nalfurafine, 3H-digoxin, and 14 C-mannitol across the monolayers of control (a) and P-gp-expressing (b) cells. Each point represents the mean ± SD of three experiments. Data are the cleared volume from A to B (filled squares), and the cleared volume from B to A (open circles)

(b) P-gp-expressing cells

Cleared volume (µL/well)

Nalfurafine 100

100

50

50

0

0 0

0.5

1

1.5

2

0

0.5

1

1.5

2

0

0.5

1

1.5

2

0

0.5

1

1.5

2

Cleared volume (µL/well)

3H-Digoxin

30

30

20

20

10

10

0

0 0

0.5

1

1.5

2

Cleared volume (µL/well)

14C-Mannitol

10

10

5

5

0

0 0

0.5

1

Time (h)

Table 1 Cleared volume ratio in cellular transport across the monolayers Incubation time (h)

Nalfurafine

3

H-Digoxin

14

C-Mannitol

Cleared volume ratio Control cells

P-gp-expressing cells

0.5

0.9

5.7

1

1.0

5.7

2

0.9

5.6

0.5

1.2

10.8

1

1.5

12.6

2

1.8

15.1

0.5

0.3

0.6

1

0.8

0.8

2

0.7

1.4

The cleared volume ratio was calculated by using the mean cleared volume value of three experiments. The initial concentration in the donor compartment was 1 lM for each compound

1.5

2

Time (h)

well, respectively, and the cleared volumes from B to A were 21.8, 47.3, and 95.3 lL/well, respectively. The cleared volume ratios were 5.7, 5.7, and 5.6, respectively. After the incubation of the positive control substance 3 H-digoxin at 1 lM for 0.5, 1, and 2 h, the cleared volumes from A to B in the control cells were 0.615, 1.08, and 2.10 lL/well, respectively, and the cleared volumes from B to A were 0.710, 1.63, and 3.84 lL/well, respectively. The cleared volume ratios were 1.2, 1.5, and 1.8, respectively. The cleared volumes from A to B in P-gp-expressing cells were 0.499, 0.910, and 1.58 lL/well, respectively, and the cleared volumes from B to A were 5.37, 11.5, and 23.9 lL/ well, respectively. The cleared volume ratios were 10.8, 12.6, and 15.1, respectively. After the incubation of 14C-mannitol for 0.5, 1, and 2 h, the cleared volumes from A to B in the control cells were 1.01, 1.11, and 2.84 lL/well, respectively, and the cleared volumes from B to A were 0.335, 0.893, and 2.00 lL/well, respectively. The cleared volume ratios were 0.3, 0.8, and

A. Ando et al.

0.7, respectively. The cleared volumes from A to B in P-gp-expressing cells were 1.13, 2.29, and 4.02 lL/well, respectively, and the cleared volumes from B to A were 0.662, 1.94, and 5.50 lL/well, respectively. The cleared volume ratios were 0.6, 0.8, and 1.4, respectively. 3.2 Inhibitory Effect of Nalfurafine on Digoxin Transport Using Human P-gp-Expressing LLCPK1 Cells

Cleared volume (µL/well)

Fig. 2 Inhibitory effects of nalfurafine on 3H-digoxin transport across the monolayers of a control and b P-gpexpressing cells. Each column and bar represents the mean ± SD of three experiments. Closed columns are the cleared volumes from A to B, and open columns are the cleared volumes from B to A. Control and P-gp-expressing cells were preincubated in the presence of nalfurafine (0.001, 0.01, 0.1, and 1 lM), verapamil (30 lM), or without either for 1 h. *P \ 0.05; **P \ 0.01

Cleared volume (µL/well)

The inhibitory effects of nalfurafine on 3H-digoxin transport across the monolayers of control and P-gp-expressing cells are shown in Fig. 2 and the cleared volume ratio obtained in this experiment is shown in Table 2. In the presence of a typical inhibitor, verapamil (30 lM), the cleared volumes of digoxin in the control cells were 5.08 lL/well from the A to B side and 5.66 lL/ well from the B to A side, and the cleared volume ratio was 1.1. In the presence of verapamil, the cleared volumes of digoxin in P-gp-expressing cells were 6.13 lL/well from the A to B side and 9.93 lL/well from the B to A side, and the cleared volume ratio was 1.6. The cleared volumes of 14 C-mannitol in the control cells were 2.84 lL/well from the A to B side and 2.40 lL/well from the B to A side, and the cleared volume ratio was 0.8. The cleared volumes of 14 C-mannitol in P-gp-expressing cells were 4.28 lL/well from the A to B side and 4.27 lL/well from the B to A side, and the cleared volume ratio was 1.0. At nalfurafine concentrations from 0.001 to 1 lM, the cleared volumes of 3H-digoxin in the control cells were

3.26–3.46 lL/well from the A to B side, and 3.83–4.18 lL/ well from the B to A side. The cleared volume ratios were 1.2. The cleared volumes of digoxin in P-gp-expressing cells were 1.35–1.63 lL/well from the A to B side, and 14.4–17.9 lL/well from the B to A side. The cleared volume ratios were from 10.7 to 11.7. No differences between each group were detected in the control cells. In P-gp-expressing cells, verapamil inhibited significantly the cleared volumes of 3H-digoxin from the A to B side (P \ 0.01) and the B to A side (P \ 0.05), respectively as compared with vehicle. Nalfurafine (0.001–1 lM) did not inhibit the cleared volumes of 3Hdigoxin from both the A to B side and the B to A side as compared with vehicle. 3.3 Drug–Drug Interactions at P-gp in the BBB Between Nalfurafine and P-gp Substrates Digoxin 8.8 mg/kg or verapamil 17 mg/kg was orally coadministered with nalfurafine 1 mg/kg to Crlj:CD1(ICR) male mice. The plasma concentrations of digoxin and verapamil are shown in Fig. 3. Nalfurafine concentrations in the plasma and brain are shown in Fig. 4, and pharmacokinetic parameters are shown in Table 3. The Kp values (ratio of brain/plasma concentrations) of nalfurafine are shown in Fig. 5. The plasma concentrations of digoxin were in the range 8.13–711 ng/mL in the digoxin 8.8 mg/kg co-administration group (group B) up to 24 h after its oral administration, and the plasma concentrations of verapamil were in

(a) Control cells

25 20 15 10 5 0

(b) P-gp-expressing cells

25 20 15

*

10 ** 5 0 0 (Vehicle)

0.001

0.01

0.1

Nalfurafine (µM) 3H-Digoxin

1

Verapamil 30 µM (Vehicle) 14C-Mannitol

Drug–Drug Interactions of Nalfurafine Involving the P-gp Table 2 Cleared volume ratio for the inhibitory effects of nalfurafine and verapamil on 3 H-digoxin transport across the monolayers, and the cleared volume ratio for 14C-mannitol transport

Inhibitor Compound 3

H-Digoxin

14

C-Mannitol

Cleared volume ratio Concentration (lM)

Control cells

P-gp-expressing cells

(Vehicle)

0

1.3

12.2

Nalfurafine

0.001

1.2

11.0

0.01

1.2

11.7

0.1

1.2

11.0

1

1.2

10.7

Verapamil

30

1.1

1.6

–a

–a

0.8

1.0

The cleared volume ratio was calculated by using the mean cleared volume value of three experiments. The initial concentration in the donor compartment of 3H-digoxin and 14C-mannitol was 1 lM a

Transport of

14

C-mannitol across the monolayers without an inhibitor

the range 2.00–92.6 ng/mL in the verapamil 17 mg/kg coadministration group (group C) up to 6 h after its oral administration. The Cmax values of nalfurafine in the plasma and brain in the digoxin co-administration group (group B) were 15.7 ng/mL and 3.45 ng/g, respectively, which were 0.78 and 0.79 times the Cmax found in the single administration group (group A; 20.0 ng/mL and 4.45 ng/g, respectively). While the Tmax was 0.5 h in the plasma and 0.75 h in the brain in the single administration group, it was 0.25 h in the plasma and 0.5 h in the brain in the digoxin co-administration group. The AUC0–24 h values of nalfurafine in the plasma and brain were 33.5 ng
h/mL and 3.16 ng
h/g, respectively, in the digoxin co-administration group, which 1000

Plasma concentration (ng/mL)

Digoxin Verapamil

100 #

10

1 0

3

6

9

12

15

18

21

24

Time (h) Fig. 3 Time profiles of the plasma concentrations of digoxin and verapamil in male mice after the single oral administration of nalfurafine or co-administration with a P-gp substrate (digoxin or verapamil). Each point and bar represents the mean ± SD of three animals. #This point represents the mean of two animals. Group B (open circles), 1 mg/kg nalfurafine and 8.8 mg/kg digoxin; group C (open squares), 1 mg/kg nalfurafine and 17 mg/kg verapamil. The plasma concentrations of verapamil were below the lower limit of quantification (1 ng/mL) in all three animals at 24 h

were 1.1 and 1.2 times the AUC0–24 h found in the single administration group (31.3 ng
h/mL and 2.66 ng
h/g, respectively). The MRT0–? and t1/2 in the plasma were 1.66 and 1.01 h in the digoxin co-administration group, and 1.29 and 0.922 h in the single administration group, respectively. The plasma and brain concentrations of nalfurafine in the digoxin co-administration group ranged from 0.52 to 2.1 times and from 0.49 to 1.5 times, respectively, those of nalfurafine in the single nalfurafine administration group at the same time points. Kp values ranged from 0.41 to 2.4 times. The Cmax of nalfurafine in the plasma was 99.7 ng/mL and in the brain was 12.0 ng/g in the verapamil co-administration group (group C), which were 5.0 and 2.7 times the Cmax found in the single administration group (group A). While the Tmax in the single administration group was 0.5 h in the plasma and 0.75 h in the brain, it was 0.25 h in the plasma and brain in the verapamil coadministration group. The AUC0–24 h values of nalfurafine in the plasma and brain in the verapamil co-administration group were 81.9 ng
h/mL and 6.30 ng
h/g, respectively, which were 2.6 and 2.4 times the AUC0–24 h found in the single administration group. The MRT0–? and t1/2 in the plasma were 1.15 and 0.875 h in the verapamil co-administration group, and 1.29 and 0.922 h in the single administration group, respectively. The concentrations of nalfurafine in the plasma and brain in the verapamil coadministration group were 1.1–5.7 times and 1.1–5.6 times those in the single administration group at the same time points. The Kp values ranged from 0.66 to 1.3 times.

4 Discussion To assess the involvement of P-gp in the membrane permeation of nalfurafine and the inhibitory effect of nalfurafine on P-gp-mediated transport, the cellular transport activities of nalfurafine and 3H-digoxin with or without

A. Ando et al.

(a) Plasma

(a) Brain

Concentration (ng/mL or g)

1000

1000

Nalfurafine Nalfurafine + Digoxin Nalfurafine + Verapamil

100

100

#

10

10

1

1

0.1 1

2

3

4

5

Time (h)

Table 3 Pharmacokinetic parameters of the plasma and brain in male mice after the single oral administration of nalfurafine or co-administration with a P-gp substrate Group A (nalfurafine)

Group B (?digoxin)

Group C (?verapamil)

Plasma Cmax (ng/mL)

20.0

15.7 (0.79)

99.7 (5.0)

Tmax (h)

0.5

0.25 (0.5)

0.25 (0.5)

31.3

33.5 (1.1)

81.9 (2.6)

AUC0–? (ng
h/mL)

28.4

30.3 (1.1)

78.3 (2.8)

MRT0–? (h)

1.29

1.66 (1.3)

1.15 (0.89)

t1/2 (h)

0.922

1.01 (1.1)

0.875 (0.95)

Cmax (ng/g)

4.45

3.45 (0.78)

12.0 (2.7)

Tmax (h)

0.75

0.5 (0.67)

h

(ng
h/mL)

Brain

AUC0–24

h

(ng
h/g)

2.66

3.16 (1.2)

6

0

1

2

3

4

5

6

Time (h)

Fig. 4 Time profiles of the plasma (a) and brain (b) concentrations of nalfurafine in male mice after the single oral administration of nalfurafine or co-administration with a P-gp substrate (digoxin or verapamil). Each point and bar represents the mean ± SD of three animals. #This point represents the mean of two animals. Group A (open diamonds), 1 mg/kg nalfurafine; group B (filled circles), 1 mg/ kg nalfurafine and 8.8 mg/kg digoxin; group C (filled squares), 1 mg/

AUC0–24

#

0.1 0

Parameter

Nalfurafine Nalfurafine + Digoxin Nalfurafine + Verapamil

0.25 (0.33) 6.30 (2.4)

Data were calculated from the mean nalfurafine concentration in the plasma and brain at each time point (n = 3, n = 2 for the nalfurafine ? verapamil treatment at 0.75 h post dose). The value in parentheses is the ratio of the co-administration group to group A (nalfurafine). Group A, 1 mg/kg nalfurafine; group B, 1 mg/kg nalfurafine and 8.8 mg/kg digoxin; group C, 1 mg/kg nalfurafine and 17 mg/kg verapamil

nalfurafine were investigated using monolayers of P-gpexpressing cells. Since P-gp is expressed on the apical surface of P-gp-expressing cells, the cleared volume from B to A was higher than that from A to B. As a known characteristic index of the cellular transport of P-pg substrates, the cellular transport activity ratio in P-gp-

kg nalfurafine and 17 mg/kg verapamil. The plasma concentrations of nalfurafine at 24 h in each group were below the lower limit of quantification (0.2 ng/mL) in all three animals. The brain concentrations of nalfurafine at 4, 6, and 24 h in group A, and at 6 and 24 h in group B and group C were below the lower limit of quantification (0.2 ng/g) in all three animals

expressing cells (cleared volume ratio, i.e., the ratio of the cleared volume from B to A to that from the A to B) was significantly higher than the cleared volume ratio in control cells [8]. The cleared volume ratios of 3H-digoxin in the control cells were below 1.8, while the cleared volume ratios of 3H-digoxin in P-gp-expressing cells were over 10.8. Hence, the distinct P-gp-mediated transport of 3Hdigoxin was observed. In the inhibitory experiment, the cleared volume ratio of digoxin in P-gp-expressing cells in the absence of verapamil was 12.2, while the cleared volume ratio in the presence of verapamil was 1.6, which indicates a distinct inhibitory effect on P-gp. Furthermore, the adequate effluxes of the intercellular transport marker 14 C-mannitol were observed in control and P-gp-expressing cells. From these results, this assay system was judged to be appropriate for the evaluation of cellular transport activity. The cleared volume from B to A was approximately 5.7 times higher than that from A to B in the transport of nalfurafine in P-gp-expressing cells. The results revealed a distinct involvement of P-gp in the membrane permeation of nalfurafine, which suggests that nalfurafine is a substrate for P-gp. It is known that the B to A cleared volume of digoxin in P-gp-expressing cells is equal to the A to B cleared volume in those cases when digoxin transport mediated by P-gp is inhibited [8]. In the inhibitory experiment, there was no concentration-dependent decrease in the cleared volume ratio of digoxin in the nalfurafine concentration range of 0.001–1 lM. The results revealed that nalfurafine (up to 1 lM) had no inhibitory effect on the

Drug–Drug Interactions of Nalfurafine Involving the P-gp 10

Nalfurafine Nalfurafine + Digoxin Nalfurafine + Verapamil

Kp value

1

#

0.1

0.01 0

1

2

3

4

5

6

Time (h) Fig. 5 Ratio of brain/plasma concentrations (Kp) in male mice after the single oral administration of nalfurafine or co-administration with a P-gp substrate (digoxin or verapamil). Each point and bar represents the mean ± SD of three animals. #This point represents the mean of two animals. Group A (open diamonds), 1 mg/kg nalfurafine; group B (filled circles), 1 mg/kg nalfurafine and 8.8 mg/kg digoxin; group C (filled squares), 1 mg/kg nalfurafine and 17 mg/kg verapamil

P-gp-mediated transport of digoxin. Nalfurafine is orally administered at 2.5 lg or 5 lg once daily for the treatment of uremic pruritus [15]. Following repeated administrations of 5 lg/day of nalfurafine for 12 days, maximum plasma concentration of nalfurafine was 10.25 pg/mL [6], and maximum intestinal exposure of nalfurafine is 20 ng/mL calculated by dividing 5 lg (dose) by 250 mL. Nalfurafine concentrations in the systemic circulation and intestine do not reach 1 lM (ca. 500 ng/mL). Thus, nalfurafine has a low potential inhibitory effect on the P-gp-mediated transport of another drug in clinical use. When nalfurafine was co-administered with digoxin or verapamil in mice, the plasma concentrations of digoxin and verapamil were in the ranges of 8.13–711 and 2.00–92.6 ng/mL, respectively, which demonstrated that mice were exposed to both drugs. Nalfurafine was already confirmed as a P-gp substrate from the results of another experimental study using knockout mice (data not shown). When co-administered with digoxin, nalfurafine concentrations in the plasma and brain were similar to those for nalfurafine alone, since each pharmacokinetic parameter, except for Tmax, was only 0.78–1.3 times higher than that for nalfurafine alone. On the other hand, when co-administered with verapamil, nalfurafine concentrations in the plasma and brain increased equally at each time point with those when nalfurafine was administered alone. Cmax, AUC0–24 h, and AUC0–? values increased by 2.4–5.0 times. Other variables of nalfurafine assessed as MRT0–? and t1/2 were 0.89 and 0.95 times higher, respectively. When co-administered with digoxin or verapamil, the Kp value showed similar changes to those with

nalfurafine alone. From these results, it was suggested that increases in nalfurafine concentrations in the plasma and brain when co-administered with verapamil were not caused by changes in nalfurafine in the BBB transport, but by increases in nalfurafine concentrations in the brain associated with an increase in the plasma concentration by the verapamil co-administration. In our experiments, the effect of concomitant drugs on the metabolism of nalfuranine was investigated using human liver microsomes. Nalfurafine (0.2, 1, and 5 lM), each concomitant drug (0, 0.1 to 100 lM), and human liver microsomes (0.5 mg protein/mL) were incubated at 37 °C for 60 min and the concentrations of metabolite catalyzed mainly by CYP3A4 were determined. The inhibition constant was calculated from Dixon plots. As a result, digoxin did not inhibit the metabolism of nalfurafine (formation of metabolite). However, verapamil showed an inhibitory effect; its inhibition constant was 11.2 lM. Therefore, an increase in plasma concentrations was induced by DDI during metabolism. Neither digoxin nor verapamil is considered to influence membrane permeability in the BBB transport. Additionally, in the transport experiment using human LLC-PK1 cells, the cleared volume (8.48–45.0 lL/ well) of nalfurafine from A to B across monolayers in the control cells was significantly higher than to that of 3H14 digoxin (0.615–2.10 lL/well) or C-mannitol (1.01–2.84 lL/well), which suggests that nalfurafine is highly permeable. Nalfurafine transport across the membrane is unlikely to be affected by P-gp substrates because of its high permeability.

5 Conclusions From these results, nalfurafine was found to be a substrate for P-gp, but had no inhibitory effect on P-gp-mediated transport. Furthermore, P-gp substrates were considered to have no influence on the BBB transport of nalfurafine. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this paper.

References 1. Chan LM, Lowes S, Hirst BH. The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci. 2004;21:25–51. 2. Cowan A, Kehner GB. Antagonism by opioids of compound 48/80-induced scratching in mice. Br J Pharmacol. 1997;122 (Suppl):169P. 3. Eyal S, Hsiao P, Unadkat JD. Drug interactions at the blood– brain barrier: fact or fantasy? Pharmacol Ther. 2009;123:80–104. 4. Gmerek DE, Cowan A. Role of opioid receptors in bombesininduced grooming. Ann N Y Acad Sci. 1988;525:291–300.

A. Ando et al. 5. Han HK. Role of transporters in drug interactions. Arch Pharm Res. 2011;34:1865–77. 6. Inui S. Nalfurafine hydrochloride for the treatment of pruritus. Expert Opin Pharmacother. 2012;13:1507–13. 7. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976;455:152–62. 8. Kawahara I, Kato Y, Suzuki H, Achira M, Ito K, Crespi CL, Sugiyama Y. Selective inhibition of human cytochrome P450 3A4 by N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-b]pyridin-5-yl)methyl]piperazin1-yl]-4(S)-hydroxy-2(R)-phenylmethy lpentanamide and P-glycoprotein by valspodar in gene transfectant systems. Drug Metab Dispos. 2000;28:1238–43. 9. Knapp RJ, Malatynska E, Fang L, Li X, Babin E, Nguyen M, Santoro G, Varga EV, Hruby VJ, Roeske WR, Yamamura HI. Identification of a human delta opioid receptor: cloning and expression. Life Sci. 1994;54:463–9. 10. Kumagai H, Ebata T, Takamori K, Miyasato K, Muramatsu T, Nakamoto H, Kurihara M, Yanagita T, Suzuki H. Efficacy and safety of a novel j-agonist for managing intractable pruritus in dialysis patients. Am J Nephrol. 2012;36:175–83. 11. Kumagai H, Ebata T, Takamori K, Muramatsu T, Nakamoto H, Suzuki H. Effect of a novel kappa-receptor agonist, nalfurafine hydrochloride, on severe itch in 337 haemodialysis patients: a phase III, randomized, double-blind, placebo-controlled study. Nephrol Dial Transplant. 2010;25:1251–7. 12. Millan MJ. Kappa-opioid receptors and analgesia. Trends Pharmacol Sci. 1990;11:70–6. 13. Nagase H, Hayakawa J, Kawamura K, Kawai K, Takezawa Y, Matsuura H, Tajima C, Endo T. Discovery of a structurally novel opioid kappa-agonist derived from 4,5-epoxymorphinan. Chem Pharm Bull. 1998;46:366–9. 14. Nakao K, Mochizuki H. Nalfurafine hydrochloride: a new drug for the treatment of uremic pruritus in hemodialysis patients. Drugs Today. 2009;45:323–9.

15. Phan NQ, Lotts T, Antal A, Bernhard JD, Sta¨nder S. Systemic kappa opioid receptor agonists in the treatment of chronic pruritus: a literature review. Acta Derm Venereol. 2012;92:555–60. 16. Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev. 2003;55:3–29. 17. Seki T, Awamura S, Kimura C, Ide S, Sakano K, Minami M, Nagase H, Satoh M. Pharmacological properties of TRK-820 on cloned l-, d- and j-opioid receptors and nociceptin receptor. Eur J Pharmacol. 1999;376:159–67. 18. Smit JW, Duin E, Steen H, Oosting R, Roggeveld J, Meijer DK. Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level. Br J Pharmacol. 1998;123: 361–70. 19. Togashi Y, Umeuchi H, Okano K, Ando N, Yoshizawa Y, Honda T, Kawamura K, Endoh T, Utsumi J, Kamei J, Tanaka T, Nagase H. Antipruritic activity of the kappa-opioid receptor agonist, TRK-820. Eur J Pharmacol. 2002;435:259–64. 20. Tsuji A. Transporter-mediated drug interactions. Drug Metab Pharmacokinet. 2002;17:253–74. 21. Umeuchi H, Togashi Y, Honda T, Nakao K, Okano K, Tanaka T, Nagase H. Involvement of central mu-opioid system in the scratching behavior in mice, and the suppression of it by the activation of kappa-opioid system. Eur J Pharmacol. 2003;477: 29–35. 22. Wang JB, Johnson PS, Persico AM, Hawkins AL, Griffin CA, Uhl GR. Human mu opiate receptor. cDNA and genomic clones, pharmacologic characterization and chromosomal assignment. FEBS Lett. 1994;338:217–22. 23. Wikstro¨m B, Gellert R, Ladefoged SD, Danda Y, Akai M, Ide K, Ogasawara M, Kawashima Y, Ueno K, Mori A, Ueno Y. jOpioid system in uremic pruritus: multicenter, randomized, double-blind, placebo-controlled clinical studies. J Am Soc Nephrol. 2005;16:3742–7.