Pharmacological Profile of Naldemedine, a Peripherally Acting μ-Opioid Receptor Antagonist: Comparison with Naloxone and Naloxegol.

Opioid-induced constipation (OIC), a typical side effect of opioids, is due to activation of the μ-opioid receptors in the enteric nervous system. Peripherally acting μ-opioid receptor antagonists (PAMORAs) can reverse OIC by inhibiting the peripheral action of opioids without affecting centrally mediated analgesia. Naldemedine is a PAMORA with potent antagonist activity against μ-, δ-, and κ-opioid receptors. In this study, the pharmacological profiles of naldemedine, compared to those of naloxone and naloxegol, were evaluated. In vitro, Schild plot analysis indicated that naldemedine was a non-competitive antagonist of μ-opioid receptors, while other compounds were competitive antagonists. Also, naldemedine showed slower association and dissociation kinetics than the other compounds. In vivo, naldemedine dose-dependently ameliorated morphine-induced inhibition of small intestinal transit (SIT). The dose response curve was not sifted at 1 and 3 mg/kg morphine. On the contrary, that of naloxegol was significantly shifted to the right from 1 to 3 mg/kg morphine. In morphine-dependent rats, naldemedine caused peripheral withdrawal symptoms (diarrhea) at doses higher than 1 mg/kg, while the dose that produced half the maximal preventive effect (ED50) against constipation was 0.03 mg/kg. Naldemedine showed slower onset and a lesser severity of diarrhea than the other compounds at close to the ED50 value in the SIT model. Our results reveal that naldemedine has different pharmacological profiles (type of antagonism and binding kinetics) to the other compounds. This might explain the differential inhibition of morphine-induced SIT and withdrawal symptoms among the three antagonist compounds. SIGNIFICANCE STATEMENT: Naldemedine is a novel PAMORA with potent antagonist activity against μ-, δ-, and κ-opioid receptors. Naldemedine showed a non-competitive antagonism and slower association and dissociation kinetics against μ opioid receptors than naloxone and naloxegol. Naldemedine showed insurmountable antagonism of morphine-induced SIT inhibition and lower and slower peripheral withdrawal symptoms (diarrhea) than the other compounds. Therefore, naldemedine has a different pharmacological profile (the type of antagonism and binding kinetics) to the other compounds.


Introduction
Opioid analgesics are the standard therapeutic agents for the management of moderate-to-severe pain and alleviate pain predominantly by stimulating the μ-opioid receptors in the central nervous system. However, their clinical use is limited by three major adverse effects (nausea/vomiting, constipation, and drowsiness), mediated primarily via μ-opioid receptors (Benyamin R et al., 2008;Swegle JM et al., 2006). Opioid-induced constipation (OIC) is one of the most common and debilitating side effects of opioids and occurs primarily via activation of enteric μ-opioid receptors, which results in a reduction in the frequency of small and large intestinal movement, development or worsening of straining, and hardstool formation after initiation of opioid therapy (Bell T et al., 2009). OIC occurs in 40-60% and 60-90% of patients who take opioids for non-cancer and cancer pain, respectively (Bruner HC et al., 2015).
The American Gastroenterological Association (AGA) has issued new guidelines on the medical management of OIC (Crockett SD et al., 2018). According to the guidelines, traditional laxatives are recommended as first-line agents to treat patients with OIC. If an adequate trial of laxatives results in suboptimal symptom control, the AGA recommends escalation of therapy to peripherally acting μopioid receptor antagonists (PAMORAs). PAMORAs aim to reverse OIC by blocking opioid actions at peripheral μ-opioid receptors in the gastrointestinal tract without adversely affecting analgesia.
Currently three PAMORAs (naldemedine, naloxegol and methylnaltrexone) are approved in the United States (US) for OIC, and one (alvimopan) is approved for post-operative ileus. AGA strongly recommends naldemedine and naloxegol for OIC as second-line drugs because of high-and moderatequality evidence of efficacy, respectively. Oxycodone/naloxone prolonged-release tablets are also approved in the European Union (EU) to treat severe pain and were designed to address opioid-induced bowel dysfunction, including OIC, by combining the analgesic efficacy of oxycodone with selective blockade of enteric μ-opioid receptors by naloxone (Morlion BJ et al., 2017).
Naldemedine is a 7-carboxamide morphinan derivative of naltrexone (Inagaki et al., 2018): 17-(cyclopropylmethyl)-6,7-didehydro-4,5α-epoxy-3,6,14-trihydroxy-N-[2-(3-phenyl-1,2,4-oxadiazol-5yl)propan-2-yl]morphinan-7-carboxamide 4-methylbenzenesulfonic acid. Naldemedine shows antagonist activity to μ-, δ-, and κ-opioid receptors (Kanemasa T et al., 2019) and does not easily pass the blood-brain barrier (Watari R et al., 2018). Naldemedine decreased OIC in several in vivo rat models This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on March 13, 2020as DOI: 10.1124 at ASPET Journals on May 7, 2020 jpet.aspetjournals.org Downloaded from JPET # 264515 6 at doses that did not compromise opioid analgesia. Naldemedine is indicated for the treatment of OIC, as a once-daily oral drug at a dose of 0.2 mg, in adult patients with chronic non-cancer pain in US, in patients with chronic non-cancer pain and cancer in Japan, and in adult patients who have previously been treated with a laxative in the EU. The effectiveness of naldemedine was established in seven phase III randomized, double-blind and placebo-controlled trials of 0.2 mg once-daily treatment for patients with non-cancer pain or cancer pain (Hale M et al., 2017;Webster LR et al., 2018;Katakami N et al., 2017;Saito Y et al., 2018).
We evaluated the in vitro and in vivo pharmacological profiles of naldemedine compared to those of naloxone, a central-acting opioid antagonist, and naloxegol, another PAMORA. Briefly, the objective was to assess 1) the binding and antagonist activities to μ-, δ-, and κ-opioid receptors and the types of antagonism to μ-opioid receptors, 2) the binding kinetics to μ-opioid receptors, 3) the effects on morphine-induced SIT inhibition caused by different morphine doses, and 4) the potential of withdrawal symptoms in morphine-dependent rats. The results reveal that naldemedine had different antagonism and binding kinetics from the other compounds studied. This might explain the differential inhibition of morphine-induced SIT and withdrawal symptoms among the three μ-opioid receptor antagonists.
JPET # 264515 8 The in vitro binding affinities and antagonist activities of three μ-opioid receptor antagonists for recombinant human μ-, δ-, and κ-opioid receptors were determined as described previously (Suzuki T et al., 2018).
In binding assays, the inhibition constant (Ki) value for each sample was calculated using the following equation, and the concentration that inhibited 50% of specific binding (IC50) was obtained: Data are presented as means ± standard errorin three independent experiments.

In Vitro Functional Assays of μ-Opioid Receptor using Guinea-Pig Ileum
The ileum was prepared from guinea pig and suspended with an approximately 1 g load in an organ bath.
The contractile responses were evoked by square-wave stimulation via a ring platinum electrode. After confirmation of a stable contractile response, the antagonist was added for 20 min, and DAMGO was cumulatively added to examine the effects on the contractile responses. The antagonist, at one concentration at a time, was administered to one ileum preparation, and three animals were used for the ileum preparations.
The IC50 value in the presence and absence of antagonist was obtained by calculating the linear regression line based on the least-squares method using SAS and its cooperative system, EXAS ver. 6.10 (Arm Systex Co., Ltd). The concentration ratio (CR) was calculated using the following equation: CR = IC50 in the presence of test compound ÷ IC50 in the absence of test compound This article has not been copyedited and formatted. The final version may differ from this version. For each antagonist, the pA2 values were obtained from a Schild plot of the CR values. In the case that the slope of the line obtained by Schild plot was ≤ 0.8 or ≥ 1.2, the pA2 value was not determined.
Data were expressed as means ± standard error of three independent preparations.

Type of Antagonism to Human μ-Opioid Receptor
The type of antagonism of the three antagonists against clinically used opioids was evaluated by [ 35 S]-GTPγS binding assay. Morphine-, oxycodone-, hydrocodone-or fentanyl-induced [ 35 S]-GTPγS binding was determined in the absence and presence of three antagonists (for naloxegol, hydrocodone and fentanyl were not used). To identify the antagonism, the slope of the Schild regression and its 95% confidence interval were calculated. The observed association rate constant (Kobs) was determined from nonlinear regression fits of the data to a one-phase exponential association model: B = Beq (1−e (−Kobs*t) ), in which Beq is the specific binding (scintillation count) of radiolabeled ligand to the receptors at equilibrium, and B is the specific binding of radiolabeled ligand at a given incubation time t. The dissociation rate constant (Koff) was calculated from nonlinear regression fits of the data to a one-phase exponential decay model using the following formula: Koff = 0.693 ÷ t1/2, in which t1/2 is the time until the binding of radiolabeled ligand to the receptor decreases to 50% after the addition of excess unlabeled ligand. The association rate constant (Kon) was calculated using the following formula: Kon = Koff ÷ Kd.

Small Intestinal Transit Test
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Downloaded from
The antagonistic effect of the three antagonists on constipation caused by morphine-induced inhibition of SIT were determined as described previously (Suzuki T et al. Life Sci. 2018). Briefly, 6-week-old Crlj: WI male rats were allocated into groups (10-12 per group) based on body weight, and administered naldemedine (orally), naloxone (subcutaneously), naloxegol (orally), or vehicle, followed by morphine (15 min later; 1 or 3 mg/kg subcutaneously). Evans Blue dye was administered intragastrically (0.5% in 2 mL) 45 min post-dose. Rats were euthanized 15 min later by cervical dislocation, and the stomach and small intestine were quickly removed; the mesentery was completely separated to avoid circling. The distance traveled by the Evans Blue dye relative to the total length of the small intestine was measured.
Small intestinal transition was calculated as: small intestine transition (%) = (distance moved by Evans Blue dye [cm] ÷ total length of small intestine [cm]) × 100. The effect of the three antagonists or vehicle on morphine-induced inhibition of SIT was assumed to be the percentage maximal possible effect (MPE).

The percentage MPEij for the jth individual in
Yij is the small intestine transition for the jth individual in Group i, and Ȳ1 is the average small intestine transition in the vehicle control group (Group 1) and Ȳ2 is the average small intestine transition in the morphine control group (Group 2). Dunnett's test for the anti-constipation effect was conducted among the morphine control group and antagonist groups. The ED50 values of each antagonist were estimated using a sigmoid Emax model.

Morphine-Withdrawal Model
The effects of the three compounds on withdrawal symptoms in morphine-dependent rats were evaluated as described previously (Kanemasa T et al., 2019).
Withdrawal signs were observed immediately, and 1, 2, 4, 6, and/or 8 hours, after dosing. Central withdrawal signs (jumping, wet-dog shakes, and teeth chattering) and peripheral withdrawal signs (diarrhea) were recorded. The number of times a rat jumped or had wet-dog shakes was counted for 20 min during each observation period. Diarrhea and teeth chattering were scored as follows: 0 = normal, JPET # 264515 11 naloxegol at each time point were compared with the vehicle control by Wilcoxon rank-sum test.
Statistical significance was set at the p < 0.05 level. Statistical analysis was performed using SAS software (version 9.1, SAS Institute, Japan) for Microsoft Windows. Statistical significance was set at p < 0.05. This article has not been copyedited and formatted. The final version may differ from this version.

Binding Affinities and Antagonist Activities Against Human μ-Opioid Receptor
The binding affinities and antagonist activities of naldemedine, naloxone, and naloxegol against recombinant human μ-, δ-, and κ-opioid receptors are shown in Table 1. Naloxone and naloxegol showed μ-opioid receptor-selective binding affinities and antagonist activities. However, naldemedine showed high affinity and potent antagonist activity for human μ-, δ-, and κ-opioid receptors. The binding affinities and antagonist activities for human μ-opioid receptor were similar among the three compounds.

Type of Antagonism of μ-Opioid Receptors
For competitive assays, the antagonistic activities of the three compounds were examined using isolated guinea pig ileum. The antagonism of the compounds against DAMGO-induced attenuation of electrical field stimulation-evoked contractions was evaluated using the slope of a Schild regression. The slopes of the Schild regression for naldemedine, naloxone, and naloxegol antagonism were 1.42, 0.88, and 0.93, respectively ( Fig. 1) The pA2 values of naloxone and naloxegol were 8.89 and 8.34, respectively. The pA2 value of naldemedine was not calculated because the slope of the Schild regression was > 1.2.
Furthermore, the antagonistic activities of the three compounds against commonly prescribed µ-opioid receptor agonists were examined. Naldemedine and naloxone concentration-dependently blocked [ 35 S]-GTPγS binding induced by morphine, oxycodone, hydrocodone, and fentanyl. Naloxegol also concentration-dependently blocked [35S]-GTPγS binding by morphine and oxycodone. The slopes of the Schild regression for naldemedine, naloxone, and naloxegol antagonism are shown in Table 2. The 95% confidence intervals of the slope of Schild regression for naloxone and naloxegol against all opioid agonists contained unity. However, those for naldemedine against all opioid agonists did not contain unity. Therefore, naldemedine, but not naloxone and naloxegol, possessed non-competitive antagonist activity against μ-opioid receptor versus several commonly prescribed opioids.

Binding Kinetics to Human μ-Opioid Receptor
It has been reported that many non-competitive antagonisms of small-molecule G-protein-coupled receptors are associated with slow dissociation of antagonist from the receptor (Swinney DC, 2004 H]-naloxegol were used in the association and dissociation studies at 0.5, 0.5, and 7.0 nM, respectively. Naldemedine showed slower association and dissociation rates for binding to human μ-opioid receptor than naloxone and naloxegol (Fig. 2). Also, the Kon and Koff values of naldemedine were lower than those of naloxone. The Kon value of naldemedine was similar to that of naloxegol; however, the Koff value of naldemedine was lower than that of naloxegol (Table 3). These results suggest that naldemedine has slow association and dissociation binding kinetics, which are likely to be due to non-competitive antagonism of the μ-opioid receptor.

Effects on Morphine-Induced Small Intestinal Transit Inhibition
Because insurmountable antagonism is associated with slow dissociation of the antagonist from the receptor (Swinney DC, 2004), the effects of the three compounds on morphine-induced SIT inhibition at high and low doses were evaluated. The three compounds showed dose-dependent suppression of subcutaneously administered 1 and 3 mg/kg morphine-induced inhibition of rat SIT, with ED 50 values (95% confidence interval) of 0.027 (0.017, 0.037) mg/kg and 0.034 (0.012, 0.055) mg/kg for naldemedine, 0.052 (0.012, 0.092) mg/kg and 0.124 (0.074, 0.174) mg/kg for naloxone, and 1.240 (−0.103, 2.582) mg/kg and 3.786 (0.613, 6.958) mg/kg for naloxegol, respectively (Fig. 3). Naldemedine showed a 1.26-fold shift in the ED50 value from 1 to 3 mg/kg morphine and the shift in the dose-response curve of the two doses was not significant (p = 0.267). In contrast to naldemedine, naloxone and naloxegol showed 2.38-and 3.05-fold shifts, respectively, in the ED50 value from 1 to 3 mg/kg morphine.
Naloxegol, but not naloxone, showed a significant shift in the dose-response curves at the two morphine doses (p = 0.19 for naloxone and p = 0.015 for naloxegol). Therefore, naldemedine showed insurmountable antagonism of morphine-induced inhibition of SIT.

Withdrawal Symptoms in Morphine-Dependent Rats
Naldemedine at > 1 mg/kg caused a peripherally mediated withdrawal symptom (diarrhea) 1 or 2 hours after dosing, and a significant centrally mediated withdrawal symptom at 3 mg/kg 1 and 4 hours after dosing in morphine-dependent rats (Supplemental Table 1A). The dose levels of naldemedine that did not cause peripherally or centrally mediated withdrawal symptoms were 0.3 and 1 mg/kg, respectively. This article has not been copyedited and formatted. The final version may differ from this version. These dose levels were about 10-or 30-fold, respectively, the ED50 value (0.03 mg/kg) for anticonstipation activity (3 mg/kg morphine-induced SIT inhibition). Naloxegol and naloxone caused both peripherally and centrally mediated withdrawal symptoms at the doses close to ED50 values (0.1 mg/kg and 3 mg/kg) for anti-constipation activity (3 mg/kg morphine-induced SIT inhibition, Supplemental Table 1B and 1C, Fig. 4A and 4B). Naldemedine at 0.03 mg/kg caused weak diarrhea (without statistical significance: p=0.82 and 0.82) at 2 and 4 hours after administration, whereas naloxegol 3 mg/kg and naloxone 0.1 mg/kg caused significant diarrhea immediately and 1 hour after dosing in morphinedependent rats, respectively. Therefore, naldemedine showed a slower onset of peripheral withdrawal symptom compared with naloxone and naloxegol. (Fig. 4A).
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Discussion
We investigated the pharmacological profiles of naldemedine compared to those of naloxone and naloxegol. All three compounds are μ-opioid receptor antagonists; however, each possessed distinct antagonist type and binding kinetics. Naldemedine showed non-competitive antagonism and slower binding kinetics, while naloxone and naloxegol showed competitive antagonism and faster binding kinetics under controlled conditions. This unique profile of naldemedine may explain its differential in vivo pharmacological effects versus naloxone and naloxegol. Naldemedine reversed the inhibition of SIT independently of the morphine dose. Also, in morphine-dependent rats, the peripheral withdrawal symptoms of naldemedine were milder and of slower onset than those of naloxone and naloxegol. Thus, naldemedine, naloxone, and naloxegol exhibited unique antagonist/binding profiles, resulting in different in vivo pharmacological effects, under controlled conditions.
In in vitro functional assays using isolated ileum preparations in guinea pig (Fig. 1), the three antagonists produced concentration-dependent rightward shifts in the agonist (DAMGO) concentration response curve relative to vehicle. The rightward shifts of naloxone and naloxegol were parallel, but higher concentrations of naldemedine caused flattening of the dose-response curve. The slopes of the Schild regression for naloxone and naloxegol antagonism (0.88 and 0.93, respectively) were closer to unity than that for naldemedine (1.4). These results show that naloxone and naloxegol are competitive antagonists, whereas naldemedine is a non-competitive antagonist, because the slopes of the Schild regression of competitive antagonists are from 0.8 to 1.2 (Janković SM et al., 1999). In vitro functional assays confirmed that naloxone and naloxegol are competitive antagonists, whereas naldemedine is a non-competitive antagonist. To clarify the difference among the types of antagonism in vivo, the effects of the three antagonists on morphine-induced SIT inhibition caused by two doses of morphine were assessed. Although the three compounds showed dose-dependent suppression of subcutaneously administered morphine-induced inhibition of rat SIT at 1 and 3 mg/kg morphine (Fig. 3), naldemedine showed a 1.26-fold shift in the ED 50 value at 1 and 3 mg/kg morphine; the shift in the dose-response curves at 1 and 3 mg/kg morphine was not significant. In contrast, naloxone and naloxegol showed a 2.38-fold and 3.05-fold shift in the ED50 value at 1 and 3 mg/kg morphine. The shift for naldemedine and naloxone was not significant whereas that for naloxegol was significant. In clinical practice, there This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on March 13, 2020 as DOI: 10.1124/jpet.119.264515 at ASPET Journals on May 7, 2020 jpet.aspetjournals.org Downloaded from is wide variation in the doses of opioids prescribed to patients. For instance, the total daily dose of opioid in a phase 3 trial of naldemedine (Hale M et al., 2017) ranged from less than 30 mg to more than 400 mg oral morphine equivalent. To avoid dose adjustment in patients prescribed opioids, the noncompetitive antagonism of naldemedine is preferable. The differences in the proportion of spontaneous bowel movement (SBM) responders between naldemedine and placebo groups were similar across baseline opioid dose strata in a phase 3 trial (Hale M et al., 2017). An efficacy analysis based on opioid dose groups in a phase 3 trial showed that the proportion of SBM responders was higher in the naldemedine group relative to the placebo group for all opioid dose strata (Hale M., 2019). The percentage increases in responders for the naldemedine groups compared with the placebo groups in the pooled population were as follows: 30 to ≤ 100 morphine milligram equivalent (MME), 14.7%; > 100 to ≤ 200 MME, 14.9%; > 200 to ≤ 400 MME, 18.5%; and ≥ 400 MME, 37.1%. Therefore, there is no attenuation of the proportion of SBM responders with increasing opioid dose.
In the binding kinetics study, naldemedine showed slower association and dissociation kinetics compared with naloxone and naloxegol. Non-competitive/insurmountable antagonism has been observed for many of the marketed small-molecule G-protein-coupled receptor antagonists, including the angiotensin-2 receptor-1 antagonists, the muscarinic M3 receptor antagonists, and the histamine H1 receptor antagonists. The insurmountable antagonism of these drugs is associated with very slow dissociation of the antagonist from the receptor (Swinney DC., 2004). In the non-peptide corticotropin releasing factor type-1 (CRF1) receptor antagonists, non-competitive antagonist behavior appeared to be correlated to the CRF1 receptor off-rate kinetics (Ramsey SJ et al., 2011). The slow dissociation kinetics profile of naldemedine might be associated with non-competitive antagonism. Although the three compounds each have a morphinan scaffold, only naldemedine exhibited slow binding kinetics.
The cause of the difference in binding kinetics requires further investigation, such as co-crystal structure analysis.
Regarding withdrawal symptoms in morphine-dependent rats, naldemedine showed a wider margin between the ED 50 values of morphine-induced SIT inhibition and lowest severity of central withdrawal symptom compared with naloxone and naloxegol. This might be because of the difference in the brainto-plasma concentration ratio (brain Kp). The brain Kp value of naldemedine is low (0.0184) in mice This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on March 13, 2020as DOI: 10.1124 at ASPET Journals on May 7, 2020 jpet.aspetjournals.org Downloaded from (Watari R et al., 2019). Although brain Kp values of naloxone and naloxegol have not been reported, the ED50 values for the anti-constipation activity (intravenous 10 mg/kg morphine-induced gastrointestinal transit inhibition in rats) of oral naloxone and naloxegol were 0.69 and 23.1 mg/kg, whereas those for the anti-analgesic activity (intravenous 10 mg/kg morphine-induced nociception in the hot-plate model in rats) were 1.14 and 55.4 mg/kg, respectively (Floettmann E et al., 2017). The margins between the ED50 values of the anti-constipation and anti-analgesic effects were 1.65 and 2.40, respectively. In contrast, the ED50 values for the anti-constipation effect (subcutaneous 3 mg/kg morphine-induced SIT inhibition in rats) of oral naldemedine was 0.03 mg/kg, whereas naldemedine did not affect the subcutaneous 6 mg/kg morphine-induced analgesic effect in a rat tail-flick test up to 7 mg/kg. The highest dose of naldemedine without an anti-analgesic effect was 233-fold higher than the ED50 value of naldemedine in the SIT study (Kanemasa et al., 2019). These data suggest the brain Kp value of naldemedine to be lower than that of naloxone and naloxegol. In addition to the difference in central withdrawal symptoms, naldemedine (oral 0.03 mg/kg) showed a lower incidence and slower onset of a peripheral withdrawal symptom (diarrhea) compared with naloxone (subcutaneous 0.1 mg/kg) and naloxegol (oral 3 mg/kg) at a dose close to the ED50 value in the SIT study (Fig. 4).
Administration of opioid antagonist while receptors are still occupied by an agonist displaces opioids from their receptors and results in the sudden onset of withdrawal (Sigmon SC et al., 2012). To alleviate a withdrawal symptom, a "soft landing" while allowing underlying neuroadaptations to revert gradually to their normal state is important except for emergency case such as rapid withdrawal in the opioid-overdose situation. This suggests that the displacement rate of opioid agonist by opioid antagonist from μ-opioid receptor contributes to the severity of withdrawal symptoms. Because naldemedine showed a slower association rate compared with naloxone and naloxegol, the rate of displacement by naldemedine might be slower than that by naloxone and naloxegol. As a result, naldemedine might show slower onset and milder peripheral withdrawal symptoms.
In an in vitro functional assay (Table 1), naldemedine, naloxone, and naloxegol showed similar antagonist activities to human μ-opioid receptor. However, naldemedine showed more potent antagonist activity to human δ-and κ-opioid receptors than naloxone and naloxegol.
Methylnaltrexone, another clinically available PAMORA also showed human μ-opioid receptor-This article has not been copyedited and formatted. The final version may differ from this version.  (Kanemasa et al., 2019). The δ-and κ-opioid receptor antagonist activities of naldemedine might not contribute to improvement of opioid-induced gastrointestinal transit inhibition, because the morphine-induced inhibition on small and large intestinal transit was blocked by a μ-opioid receptor selective antagonist, but not by δ-and κ-opioid receptor selective antagonists (Matsumoto K et al., 2016). However, δ-opioid receptor antagonist activity contributes to improvement of opioid-induced nausea and vomiting, major adverse effects of opioids, because the δopioid receptor selective antagonist, TAN-452, attenuated morphine-induce nausea and vomiting in ferrets (Suzuki T et al., 2018). Therefore, naldemedine might influence opioid-induced nausea and vomiting via the δ-opioid receptor.
In conclusion, we showed that naldemedine, naloxone, and naloxegol exert unique pharmacological effects due to their different modes of antagonism and binding kinetics. Although the pharmacological effects in the present study are not directly representative of the clinical pharmacological effects, our results provide important information for the clinical use of PAMORAs to manage the adverse effects of opioids. This article has not been copyedited and formatted. The final version may differ from this version.     Statistical significance was determined using Steel's multiple comparison test (naldemedine) or Wilcoxon rank-sum test (naloxone and naloxegol) with respect to the corresponding vehicle control (*P < 0.05; **P < 0.01).

Figure 4b. Precipitated central withdrawal symptoms (teeth chattering) of naldemedine, naloxone,
and naloxegol in morphine-dependent rats. Data are means ± standard error of 8-10 rats per group.
Statistical significance was determined using Steel's multiple comparison test (naldemedine) or Wilcoxon rank-sum test (naloxone and naloxegol) with respect to the corresponding vehicle control (*P < 0.05; **P < 0.01). This article has not been copyedited and formatted. The final version may differ from this version. Values are means ± standard error Mean of three independent experiments carried out in duplicate.
Binding and functional activities of naldemedine were from Kanemasa et al., 2019.
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