Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, Its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects

Abstract

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial μ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y^7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.

Keywords: antipruritic effect; conditioned place aversion; motor incoordination; nalfurafine; sedation; κ opioid receptor.

Figure 1.

The chemical structures of nalfurafine (a) and 42B (b) with atom notations.

Figure 2.. Stimulation of [³⁵S]GTPγS binding to membranes of CHO cells stably transfected with the MOR, DOR, KOR or NOR by nalfurafine, 42B, and a reference full agonist for each receptor.

[³⁵S]GTPγS binding to membranes was performed with various concentrations of each compound in 50 mM Tris buffer (pH 7.4) in the presence of 50 mM (for the MOR, DOR or NOR) or 100 mM NaCl (for the KOR), 5 mM MgCl₂, 15 μM GDP, 1 mM EDTA with ~10 μg membrane proteins and ~80 pM [³⁵S]GTPγS at 30°C for 60 min. Nonspecific binding, determined using 10 μM cold GTPγS, was ~500 dpm. Data were normalized and expressed as percentage of the maximal [³⁵S]GTPγS binding of the respective reference full agonist. Basal [³⁵S]GTPγS binding in the absence of added compounds was ~2000 dpm and maximal binding (with basal binding subtracted) was ~2500 dpm for the MOR and DOR, ~5000 dpm for the KOR, ~1500 dpm for the NOR. Each value represents the mean ± SEM of at least three independent experiments performed in duplicate. EC₅₀ values and maximal responses are shown in Table 1. Each value is mean ± SEM (n=3–4).

Figure 3.. KOR agonists-induced G protein activation (GloSensor cAMP assay) and β-arrestin recruitment (Tango assay).

GloSensor cAMP and Tango assays were performed by PDSP of the NIMH, which is directed by Dr. Bryan Roth of University of North Carolina (Web site: https://pdsp.unc.edu/ims/investigator/web/). EC₅₀ values and maximal responses are shown in Table 2. Each value is mean ± SEM (n=3).

Figure 4.. Binding poses of nalfurafine and 42B with the active MOR and KOR from molecular docking studies.

(a) nalfurafine_MOR^active, (b) nalfurafine_KOR^active, (c) 42B_MOR^active, (d) 42B_KOR^active. The MOR and KOR shown as cartoon models in light-blue and light-pink, respectively. Nalfurafine, 42B, and key amino acid residues shown as stick models. Carbon atoms: nalfurafine in green; 42B in cyan; key amino acid residues of the MOR in yellow, KOR in gray. The red dashed line represented possible hydrogen bonds. The water molecules shown as sphere models in magenta.

Figure 5.. 42B produced antinociceptive and anti-scratching effects in mice, like nalfurafine.

(a) and (b) 42B inhibited formalin-induced pain behaviors in mice like nalfurafine. Saline or one of several doses of 42B or nalfurafine was injected (s.c.) 5 min before formalin and the amount of time the animal spent licking the injected paw was counted for 20 min starting at 15 min after formalin injection. A₅₀ doses were determined as described. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test. Results of one-way ANOVA are: 42B, F(3,24) = 41.63, p < 0.001; nalfurafine, F(4, 41) = 18.65, p < 0.0001. Significance levels are *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared to saline control, by Dunnett’s post-hoc test (mean ± SEM, n = 6–10 animals/group). Data on nalfurafine were from Liu, et al. and are shown for comparison. (c) and (d) 42B induced inhibition of scratching behavior induced by compound 48/80, like nalfurafine. Saline or one of the different doses of 42B or nalfurafine was injected (s.c.) 20 min before compound 48/80 and the bouts of scratching were counted for 30 min. A₅₀ doses were determined as described previously. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test. Results of one-way ANOVA were: 42B, F(3,28) = 11.68, p < 0.001; nalfurafine, F(5,44) = 31.04, p < 0.0001. Significance levels are **p < 0.01, ***p < 0.001, ****p < 0.0001, compared to saline control by Dunnett’s post-hoc test (mean ± SEM, n = 6–12 animals/group). Data on nalfurafine were from Liu, et al. and are shown for comparison. (e) The selective KOR antagonist norBNI blocked anti-scratching effects of 42B in compound 48/80 scratching test. Saline or norBNI was injected (20 mg/kg, i.p.) 20 h before saline or 42B, 20 min later compound 48/80 was injected and the bouts of scratching were counted for 30 min. Data were analyzed with two-way ANOVA followed by Sidak’s post-hoc test. Results of two-way ANOVA showed a significant main effects of 42B [F(1,21) = 13.26, p < 0.01], a significant main effects of norBNI [F(1,21) = 7.47, p < 0.05] and a significant interaction [F (1,21) = 5.39, p < 0.05]. Significance levels are **p < 0.01, compared to saline+saline group; ^##p < 0.01, compared to saline+42B group, by Sidak’s post-hoc test (mean ± SEM, n = 5–8 animals/group).

Figure 6.. Comparison of 42B and nalfurafine in KOR agonist-induced adverse behaviors: motor incoordination, sedation, and conditioned place aversion (CPA) in mice.

(a) and (b) unlike nalfurafine, 42B caused motor incoordination in the rotarod test in mice. After training the previous day, mice were injected s.c. with saline, U50,488H (2, 5 mg/kg), 42B (1, 3, 5 mg/kg) or nalfurafine (20 μg/kg) and tested on the rotarods 10, 20, 30, and 40 min after injection. The time each stayed on the rods was recorded and normalized against the baseline. Data were analyzed with two-way ANOVA followed by Dunnett’s post-hoc test (mean ± SEM, n= 9–12/group). 42B: Results of two-way ANOVA showed a significant main effect of treatment [F (3,36) = 10.48, p < 0.0001], a significant main effect of time [F (4,144) = 32.33, p < 0.0001] and a significant interaction [F (12,199) = 4.65, p < 0.0001]. Nalfurafine: Results of two-way ANOVA showed a significant main effect of treatment [F (3,36) = 15.43, p < 0.0001], a significant main effect of time [F (4,144) = 24.14, p < 0.0001] and a significant interaction [F (12,199) = 5.46, p < 0.0001]. *p < 0.05, **p < 0.01, ****p < 0.0001, compared with 0 min of each group; ^#p < 0.05, ^##p < 0.01, ####p < 0.0001, compared with the saline group at the same time, by Dunnett’s post-hoc test. Data on nalfurafine and 5 mg/kg U50,488H were from Liu, et al. and are shown for comparison. (c) and (d) Unlike nalfurafine, 42B caused inhibition of novelty-induced locomotor activity in mice. Mice were treated s.c. with saline, 42B (1, 3, 5 mg/kg), U50,488H (5 mg/kg), or nalfurafine (20 μg/kg) (2.5x A₅₀ values in the anti-scratching test) and locomotor activities were monitored. Cumulative data between 0–30 min post-injection are shown here. Each value represents mean ± SEM (n = 8–14). (c) Ambulatory activity of 42B: ***p < 0.001, compared with the saline group by one-way ANOVA [F (3,34) = 7.59, p < 0.001] followed by Dunnett’s post-hoc test. (d) Ambulatory activity of U50,488H and nalfurafine: *p < 0.05, compared with the saline group by one-way ANOVA [F (2,27) = 6.254, p < 0.01] followed by Dunnett’s post-hoc test. Data on nalfurafine and 5 mg/kg U50,488H were from Liu, et al. and are shown for comparison. (e) and (f) 42B did not cause CPA in mice, like nalfurafine. On Day 0, mice were subject to pre-test. On Days 1–6, Mice were injected with saline or one of the various doses of U50,488H, 42B or nalfurafine before each 30-min conditioning session (1 saline session and 1 drug session/day) for 6 days. On Day 7 (post-test), the length of time the animal spent on the drug-paired side was measured. The graph shows the time the animal spent during the post-test subtracting the amount of time spent during the pre-test. Data were analyzed with one-way ANOVA followed by Dunnett’s post-hoc test (for the 42B and nalfurafine experiments) and unpaired t-test (for U50,488H) (mean ± SEM, n = 9–10/group). 42B, F(3,36) = 0.45, p > 0.05; nalfurafine, F(4,47) = 0.38, p > 0.05. Significance levels are **p < 0.01, compared to saline control by unpaired t-test. Data on nalfurafine and U50,488H were from Liu, et al. and are shown for comparison. (g) Naloxone at a dose (1 mg/kg) selective for the MOR did not alter lack of CPA of 42B. On Day 0, mice were subject to pre-test. On Days 1–6, saline or naloxone was injected 25 min before saline or 42B, then 30-min conditioning session began immediately (1 saline session and 1 drug session/day) for 6 days. U50,488H alone was used as the CPA positive control. On Day 7 (post-test), the length of time the animal spent on the drug-paired side was measured. The graph shows the time the animal spent during the post-test subtracting the amount of time spent during the pre-test. Data were analyzed with two-way ANOVA followed by Sidak’s post-hoc test (for the 42B-naloxone experiments) and unpaired t-test (for U50,488H) (mean ± SEM, n = 10/group). Results of two-way ANOVA showed no significant main effects of 42B [F(1,36) = 0.13, p > 0.05], naloxone [F(1,36) = 0.004, p > 0.05] or interaction [F (1,36) = 1.16, p > 0.05]. Significance levels are **p < 0.01, compared to saline + saline by unpaired t-test.