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1 Journal of Pharmacological Sciences 1 (16) 8e14 HOSTED B Contents lists available at ScienceDirect Journal of Pharmacological Sciences journal homepage: Full paper Nalfurafine hydrochloride, a selective k opioid receptor agonist, has no reinforcing effect on intravenous self-administration in rhesus monkeys Kaoru Nakao a, *, Mikito Hirakata a, ohei Miyamoto a, Mie Kainoh a, oshio Wakasa b, Tomoji anagita c a Pharmaceutical Research Laboratories, Toray Industries, Inc., -1, Tebiro 6-chome, Kamakura, Kanagawa , Japan b Marketing Department, Ina Research Inc., Nishiminowa, Ina-shi, Nagano , Japan c Scientific Advisor, Ina Research Inc., Nishiminowa, Ina-shi, Nagano , Japan article info abstract Article history: Received 15 June 15 Received in revised form November 15 Accepted 24 November 15 Available online 2 December 15 Keywords: Nalfurafine hydrochloride TRK-8 k opioid receptor Self-administration Monkey Nalfurafine hydrochloride [(E)-N-[17-(cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-6b-yl]- 3-(furan-3-yl)-N-methylprop-2-enamide monohydrochloride; nalfurafine] is used in Japan as an antipruritic for the treatment of intractable pruritus in patients undergoing hemodialysis or with chronic liver disease. It is a potent and selective agonist at the k opioid receptor, but also has weak and partial agonist activity at m opioid receptors. Opioids, especially those acting at m receptors, carry a risk of abuse. This is an important factor in the consideration of therapeutic risk vs. benefit in clinical use and the potential for misuse as a public health problem. It is therefore necessary to carefully evaluate the reinforcing effects of nalfurafine. To this end, we investigated intravenous self-administration of nalfurafine in rhesus monkeys. The number of self-administration of nalfurafine at doses of.625,.125 and.25 mg/kg/infusion was not higher than that of saline in rhesus monkeys that frequently selfadministered pentazocine (.25 mg/kg/infusion). These results indicate that nalfurafine has no reinforcing effect in rhesus monkeys in the intravenous self-administration paradigm. 15 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access article under the CC B-NC-ND license ( 1. Introduction The selective k opioid receptor agonist nalfurafine hydrochloride [(E)-N-[17-(cyclopropylmethyl)-4,5a-epoxy-3,14- dihydroxymorphinan-6b-yl]-3-(furan-3-yl)-n-methylprop-2- enamide monohydrochloride; nalfurafine] is a well-tolerated and effective treatment for intractable pruritus in hemodialysis patients (1e4). It was approved for clinical use in January 9 by the Japanese Ministry of Health, Labour and Welfare (1,3). Receptor binding and functional studies of recombinant human and rat opioid receptors have shown nalfurafine to be a more potent and selective agonist at k receptors than m and d opioid receptors (3,5). Opioids, such as morphine, carry a risk of abuse mediated by their activation of m receptors (6). However, unlike m receptor agonists, which play a major role in reward and place preference * Corresponding author. Tel.: þ ; fax: þ address: Kaoru_Nakao@nts.toray.co.jp (K. Nakao). Peer review under responsibility of Japanese Pharmacological Society. conditioning in animals, selective k agonists generally lack a reinforcing effect (7), and are considered not to have the potential to become drugs of abuse in humans. Although nalfurafine is a selective k agonist, forskolin-stimulated camp accumulation studies have shown that the drug also possesses weak partial agonist activity at m receptors (3). Its potential risk of abuse must therefore be carefully evaluated. No evidence was found of psychological or physical dependence on nalfurafine in a clinical study in Japanese hemodialysis patients with severe pruritus (2). However, animal studies of abuse liability have the advantages of using a wider range of doses and including a positive control drug. Nalfurafine does not induce conditioned place preference in mice (8) or rats (9), indicating that the drug does not produce a rewarding effect; however, no studies of nalfurafine self-administration in rats or monkeys have been reported to date, despite the usefulness of such studies in evaluating potential abuse liability in humans (7,e13). Therefore, the present study was designed to investigate the reinforcing effect of nalfurafine using intravenous self-administration in rhesus monkeys / 15 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access article under the CC B-NC-ND license ( creativecommons.org/licenses/by-nc-nd/4./).

2 K. Nakao et al. / Journal of Pharmacological Sciences 1 (16) 8e Materials and methods 2.1. Animals Six male and four female adult rhesus monkeys (Macaca mulatta) were used. The animals were drug-free for 6e18 months and weighed 3.9e6.7 kg. The monkeys were housed individually at 22e28 C, e8% humidity, and under a 12-hour light/dark cycle (lights on at 7: a.m.). The monkeys received approximately g of solid feed (PS, Oriental east Co., Ltd., Tokyo, Japan) once a day in Experiment 1, and about 1 g in Experiment 2. Additionally, half a banana was occasionally given to each monkey in both experiments. The monkeys were allowed free access to drinking water. All experiments were conducted in accordance with the principles specified by the Institutional Animal Care and Use Committee of Ina Research Inc Experiment 1: gross behavioral observation after a single intravenous dose Six monkeys (three male, three female) were used in a series of experiments comprising three trials conducted at 1-week intervals (Table 1). In each trial, the general condition of the animals was observed prior to drug administration. Animals were assessed for the following signs, in accordance with the observation criteria of the test facility: salivation, retching, vomiting, continuous movement, hypoactivity, crouching, prone posture, aggression towards the observer, reactivity to the observer, reaction towards the observer by grimacing (a sign of fear), eye closing, pupil dilation or contraction, slow movement, and ataxia. The animals' general condition was also observed after drug administration at, 15 and min, and 1, 2, 3, 4 and 5 h. Where changes in general condition persisted for 5 h, those animals were assessed again 24 h after administration. Food consumption was observed on the day of administration and the following day, and any changes were recorded. The diameter of each pupil was estimated by eye; a change in diameter of approximately 2 mm or more compared with the pre-administration diameter was recorded as dilation or contraction Experiment 2: intravenous self-administration Four monkeys (three male, one female) that had previously selfadministered pentobarbital sodium and/or cocaine intravenously were used in a series of self-administration experiments. In each animal, a silicon catheter (outer diameter: 2 mm; inner diameter: 1 mm) was implanted into the jugular or femoral vein under pentobarbital anesthesia and connected to an automatic drug infusion apparatus. A red light approximately 5 cm above the selfadministration lever was illuminated when drug solution was available, and turned off during infusion. Table 2 shows the Table 1 Administration schedules for gross behavioral observation of rhesus monkeys. Animal ID No. (Sex) Dose of nalfurafine (mg/kg) 1st trial 2nd trial 3rd trial #1333 (male) a.5 e b #1376 (female) 1. a.25 #1381 (female) e b.5 a #1 (male) a #14 (male) e b 2. e b #15 (female) 1. a a a Administration of 5% mannitol solution. b No assignment in corresponding trial. schedule for Experiment 2. In the first study period, the selfadministration solution was saline (.25 ml/kg/infusion). For each monkey, when self-administration did not exceed infusions/day for 7 consecutive days, the monkey was allowed to self-administer pentazocine freely (.25 mg/kg/infusion). When pentazocine selfadministration reached 35 infusions/day or more for 3 consecutive days, it was replaced with 5% mannitol solution (.125 ml/kg/ infusion), until self-administration decreased to infusions/day or fewer for 7 consecutive days. Monkeys were then allowed free access to nalfurafine self-administration (.625,.125 or.25 mg/ kg/infusion) for 2 weeks at each dose, after which all monkeys received scheduled intravenous infusions of nalfurafine (.25 mg/ kg/infusion) at 6-hour intervals for 2 weeks. During this repeated infusion period, the monkeys were still allowed free access to selfadministered nalfurafine at the same unit dose. When selfadministration occurred, scheduled intravenous infusion of nalfurafine was postponed by 6 h from the self-administered infusion. After a 2-week period of scheduled infusions, self-administration of nalfurafine (.25 mg/kg/infusion) was observed for an additional 2 weeks. In the final study period, nalfurafine was replaced with 5% mannitol (.25 ml/kg/infusion) for 1 week of self-administration. During the pentazocine and nalfurafine study periods, raisins were attached to the self-administration lever when selfadministration fell to three infusions/day or fewer for 3 consecutive days, to encourage pressing of the lever and maintenance of self-administration behavior; these data were excluded from the analysis, and the self-administration periods were subsequently extended by the number of days that were excluded. Animals were assessed for hypoactivity and slow movement between 9: and : during each study period Administration In Experiment 1, each animal was seated on a chair designed for a monkey and the test solution was administered intravenously into the forelimb. The injection volume and rate were.25 ml/kg and 1 ml/23 s, respectively. In Experiment 2, the monkeys selfadministered the solution intravenously (14) by pressing the lever of the self-administration apparatus. Drug or control solution was infused through a catheter inserted into the vein under a fixed ratio schedule in which every lever press resulted in an infusion (FR1). Infusion volume and rate were.125 or.25 ml/kg and 1 ml/ 23 s, respectively. There was no limit to the number of infusions that could be received. Daily self-administration was recorded for 24 h (9:e9: a.m.). On the days in which the test solution was replaced, observations were interrupted for 1 h (9:e: a.m.) Drugs Nalfurafine hydrochloride (Toray Industries, Inc., Tokyo, Japan) was dissolved in 5% mannitol solution to ensure stability of the drug. Pentazocine (in ampoules of mg/ml; Sosegon Inj. mg, amanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) was diluted with saline Statistical analysis No statistical analyses were performed in Experiment 1. For data obtained in Experiment 2, means and standard errors were calculated for the number of infusions self-administered by each animal per day, for each solution. A repeated measures analysis of variance (ANOVA), in which the error terms were assumed to be correlated within an animal, was used to estimate the mean number of selfadministered infusions per day for study periods 1e9, and to test the period effects of study periods 2e9 vs. study period 1. The

3 K. Nakao et al. / Journal of Pharmacological Sciences 1 (16) 8e14 Table 2 Schedule for intravenous self-administration in rhesus monkeys. Study period response variable was the number of self-administrations per day, and the explanatory variable was the study period as a fixed effect. The repeated measures ANOVA was carried out using the MIXED procedure in SAS 9.4 (SAS Institute Inc., Cary, North Carolina, USA) and P <.5 was considered statistically significant. 3. Results Drug Unit dose (/kg/infusion) Termination criteria 1 Saline.25 ml infusions/day, 7 consecutive days 2 Pentazocine.25 mg 35 infusions/day, 3 consecutive days 3 5% mannitol.125 ml infusions/day, 7 consecutive days 4 Nalfurafine.625 mg 2 weeks 5 Nalfurafine.125 mg 2 weeks 6 Nalfurafine.25 mg 2 weeks 7 Nalfurafine.25 mg, 2 weeks 6-hour interval a 8 Nalfurafine.25 mg 2 weeks 9 5% mannitol.25 ml 1 week a Scheduled repeated infusions at 6-hour intervals when not self-administering Gross behavioral observations after a single intravenous dose (Experiment 1) Changes in general condition after administration of 5% mannitol or nalfurafine are summarized in Table 3. After 5% mannitol administration, increased and/or decreased aggression towards the observer was noted in five out of the six monkeys, and decreased grimacing at the observer was noted in one of these five animals. However, most of these signs were transient. No abnormal Table 3 Gross behavioral manifestations after intravenous single-dose administration of nalfurafine and 5% mannitol in rhesus monkeys. Signs Number of animals showing each sign 5% Mannitol Nalfurafine (mg/kg).25 ml/kg (6) (2) (2) (2) (2) Increase in aggressive behavior 3 2 towards observer Decrease in aggressive behavior towards observer Decrease in grimacing at observer 1 1 Prone posture Slow movement Hypoactivity Crouching Eye closing 1 2 Ataxia 1 Jaw slackening 2 Salivation 2 No changes 1 2 Parentheses indicate total number of animals observed. changes in general condition were observed in the sixth animal. No changes were observed in the two monkeys that received nalfurafine at.25 mg/kg. Animals that received.5, 1., or 2. mg/kg nalfurafine showed increased or decreased aggression towards the observer and/or decreased grimacing at the observer, similar to the behavior observed after 5% mannitol administration. These changes were not dose-dependent. Additionally, prone position, slow movement, hypoactivity, and crouching were observed in animals that received nalfurafine at a dose of.5 mg/kg or higher. Eye closing was seen at 1 mg/kg or higher, and ataxia, jaw slackening and salivation were observed at 2 mg/kg. Most of the changes observed at.5 and 1. mg/kg appeared immediately after administration or within min and persisted for 2e3 h. Conversely, most changes at 2. mg/kg appeared 15e min after administration and could still be observed 5 h after administration, but had disappeared by 24 h post-dose Intravenous self-administration (Experiment 2) The number of infusions self-administered per day during each study period are shown in Figs. 1 and 2. During the saline selfadministration period, the means were.3e3.1 infusions/day for the last 7 consecutive days (point estimate of mean (PE) [95% confidence interval (CI)]:.99 [.44, 1.54] infusions/day). When saline was replaced with pentazocine, self-administration increased in all monkeys within 2e4 days after substitution, with daily means of 41.3e44. infusions for the last 3 consecutive days (PE [95% CI]: [41.42, 43.] infusions/day, P <.1 vs. saline). Next, pentazocine was replaced with 5% mannitol, resulting in gradual decreases in self-administration in all monkeys, with daily means of 3.6e6.6 infusions for the last 7 consecutive days (PE [95% CI]: 5.3 [4.48, 5.57] infusions/day, P <.1 vs. saline). When these monkeys were subsequently allowed free access to selfadministered nalfurafine at.625,.125 and.25 mg/kg/infusion for 2 weeks at each dose, the mean number of self-administration was.e3.1 infusions/day (PE [95% CI] infusions/day, respectively: 1.42 [1.3, 1.81], P ¼.212 vs. saline;.53 [.14,.91], P ¼.173 vs. saline;.42 [.4,.81], P ¼.96 vs. saline) and did not exceed that of saline or 5% mannitol. In addition to having free access to self-administered nalfurafine, the animals also received scheduled infusions of nalfurafine (.25 mg/kg/infusion) at 6-hour intervals. During this study period, nalfurafine selfadministration decreased significantly (PE [95% CI]:.21 [.81,.59] infusions/day, P ¼.22 vs. saline). Furthermore, no increase in daily nalfurafine self-administration was observed after scheduled infusions (PE [95% CI]:.35 [.4,.74] infusions/day, P ¼.6 vs. saline). In the final period, nalfurafine was replaced with 5% mannitol, but no changes in self-administration were observed (PE [95% CI]:.36 [.19,.91] infusions/day, P ¼.8 vs. saline). When self-administration of pentazocine increased, hypoactivity (in all animals) and slow movement (in two out of four animals) were observed. Nalfurafine at.125 and.25 mg/kg/infusion (study periods 5 and 6, respectively) produced hypoactivity in one and two out of the four animals, respectively. However, these signs were observed after self-administration of nalfurafine induced by raisins attached to the self-administration lever (Table 4). 4. Discussion Depression-like symptoms including prone posture, slow movement, hypoactivity, crouching, eye closing, and ataxia were noted after acute intravenous administration of nalfurafine at doses of.5e2. mg/kg, but not.25 mg/kg. This is in agreement with previous studies showing that the k agonists GR89,696 and

4 K. Nakao et al. / Journal of Pharmacological Sciences 1 (16) 8e14 11 Mean daily number of self-administration Animal ID No. :#1375 (female) :#1392 (male) :#12 (male) :#13 (male) Study Period Test substance Saline Pentazocine 5% mannitol Nalfurafine 5%mannitol Unit dose -.25 mg/kg μg/kg.125 μg/kg.25 μg/kg.25 μg/kg Scheduled inj..25 μg/kg - Fig. 1. Study period means for continuous intravenous self-administration of nalfurafine and pentazocine in rhesus monkeys. Study periods: 1, saline self-administration at.25 ml/ kg/infusion; 2, pentazocine self-administration at.25 mg/kg/infusion; 3, 5% mannitol solution self-administration at.125 ml/kg/infusion; 4, 5 and 6, nalfurafine selfadministration at.625,.125 and.25 mg/kg/infusion, respectively; 7, nalfurafine self-administration at.25 mg/kg/infusion with scheduled infusions of the drug at 6-hour intervals at the same unit dose (scheduled infusions postponed by 6 h from the time of each self-administered infusion); 8, nalfurafine self-administration at.25 mg/kg/infusion without scheduled infusions; 9, 5% mannitol solution self-administration at.25 ml/kg/infusion. Data are the mean and standard error of daily infusions self-administered by each animal. U69,593 dose-dependently elevate sedation and muscle relaxation scores in monkeys (15). The reinforcing effects of dependenceproducing drugs may be detected at lower than the minimum effective dose for gross behavioral changes (16), so we set the unit dose range for the self-administration part of the study to.625e.25 mg/kg/infusion. In a series of intravenous self-administration experiments, four monkeys were allowed to self-administer nalfurafine at.625,.125 and.25 mg/kg/infusion, after frequent self-administration of pentazocine, which has mixed m and k agonist properties in receptor binding studies and [ 35 S]GTPgS binding studies (17). However, no difference was observed between self-administration of nalfurafine and that of saline in any animals. Furthermore, no increase in nalfurafine self-administration was observed during or after scheduled nalfurafine infusions. Self-administration did not increase even when nalfurafine was replaced with 5% mannitol in the final study period in this experiment. Retching and vomiting were observed in two monkeys following several self-administered infusions of nalfurafine at.25 mg/kg/infusion, so the unit dose range used in the present study was considered sufficient to examine the drug's reinforcing effects. Moreover, nalfurafine produced hypoactivity at.125 and.25 mg/kg/infusion in study periods 5 and 6, respectively. Although pentazocine also produced hypoactivity and slow movement, robust self-administration of this drug was observed, indicating that the lack of nalfurafine selfadministration was not due to its non-specific suppression of motor function. Together, these results indicate that nalfurafine has no reinforcing effects in monkeys in the intravenous selfadministration paradigm. Intravenous injection of.25 mg/kg nalfurafine inhibits scratching behavior induced by intravenous morphine in rhesus monkeys, indicative of its antipruritic effect (18). This also indicates that nalfurafine does not show a reinforcing effect at doses sufficient to produce an antipruritic effect in monkeys. Furthermore, because nalfurafine is clinically taken orally at doses of 2.5 or 5 mg (approximately.5 or.1 mg/kg, respectively, for a kg person) to relieve intractable pruritus by patients undergoing hemodialysis (1e3) or with chronic liver disease, the unit dose range of nalfurafine used in this study is considered to be equivalent to, or higher than, that used in clinical practice. The mesolimbic dopamine system from the ventral tegmental area to the nucleus accumbens (NAcc), a subregion of the ventral striatum, plays a critical role in drug dependence (19). In vivo microdialysis studies have shown that systemic administration of commonly abused drugs such as amphetamine (), cannabinoids (21), heroin (21), cocaine (22), ethanol (23) and nicotine (24), increases extracellular dopamine levels in the NAcc. Dynorphin A and U-,488H (endogenous and exogenous selective k agonists, respectively) decrease extracellular dopamine levels in the NAcc (25). The effect of nalfurafine on NAcc dopamine levels has not been reported, but it has been shown that nalfurafine decreases extracellular dopamine levels in the striatum of a rat model of dyskinesia (26) and in the prefrontal cortex of a rat model of schizophrenia (27). This suggests that the reason nalfurafine does not induce selfadministration in monkeys is because it does not stimulate the mesolimbic dopamine system, which is involved in reinforcement. Nalfurafine has been characterized in vitro as a potent and selective full agonist of the k opioid receptor, with weak partial agonist activity at the m receptor (3,5).Ina[ 35 S]GTPgS binding assay using a mouse pons/medulla membrane preparation, a much higher concentration of nalfurafine antagonized the increase of [ 35 S]GTPgS binding induced by the m agonist DAMGO (28). However, when co-administered subcutaneously with morphine, nalfurafine augmented morphine-induced antinociception in mice, in

5 12 K. Nakao et al. / Journal of Pharmacological Sciences 1 (16) 8e14 A B C D #1375 #1392 #12 #13 Period Mean Study period 1 period 2 period period 4 period period 6 period period 8 period 9 Fig. 2. Daily means of continuous intravenous self-administration of nalfurafine and pentazocine in rhesus monkeys. Solid lines represent the time course of the number of selfadministered infusions per day for each animal. Dashed lines represent means for each period estimated by repeated measures of analysis of variance.

6 K. Nakao et al. / Journal of Pharmacological Sciences 1 (16) 8e14 13 Table 4 Summary of hypoactivity and slow movement during each intravenous selfadministration period. Study period Number of animals (total, 4) showing each sign Hypoactivity Slow movement a 6 2 a a Signs observed when raisins were attached to the self-administration lever for maintenance of self-administration behavior. the hot-plate test (29) and acetic acid writhing test (). Furthermore, subcutaneous co-administration of nalfurafine and morphine produced an additive antinociceptive effect in the warm water tailwithdrawal assay in rhesus monkeys (31). Together, this indicates that systemic administration of nalfurafine does not act as an antagonist at m receptors, despite its partial agonist activity. In contrast, pentazocine is a non-selective partial agonist at m and k receptors (17), and inhibits morphine-induced antinociception in the mouse hot-plate test when co-administered subcutaneously (29), suggesting that systemically administered pentazocine acts as an antagonist at the m receptor. The difference in properties between nalfurafine and pentazocine may contribute to their different effects on reinforcement. The selective full k agonist U69,593 does not induce self-administration alone, and a combination of U69,593 and fentanyl, a selective m agonist, produces a proportion-dependent decrease in fentanyl self-administration (7), suggesting that abuse liability is influenced by the balance of m and k activities in a mixed m/k agonist such as pentazocine. It has been reported that nalfurafine has a discriminative stimulus effect in rats. However, whereas U-,488H generalized to the discriminative stimulus effects of nalfurafine, nalfurafine did not generalize completely to those of U-,488H, indicating that the subjective effects of nalfurafine and U-,488H are somewhat different (32). In contrast, salvinorin A, a selective k agonist and constituent of the hallucinogenic plant Salvia divinorum, generalized completely to the discriminative stimulus effects of U-,488H, indicating that the two drugs have similar subjective effects (33). Moreover, in long-term clinical trials of intravenously or orally administered nalfurafine, conducted for up to 52 weeks in hemodialysis patients, there was no evidence of rewarding effects such as drug-seeking behavior or physical dependence (2,34). Together, these findings suggest that nalfurafine has minimal abuse liability. 5. Conclusions We have shown that nalfurafine does not produce reinforcing effects in monkeys at a range of doses including those equivalent to its antipruritic dose. 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