NCP, a Dual Kappa and Mu Opioid Receptor Agonist, Is a Potent Analgesic Against Inflammatory Pain without Reinforcing or Aversive Properties

Abstract

While agonists of μ (MOR) and κ (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-{[4'-(2'-cyanopyridyl)]carboxamido}morphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro. Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A₅₀ values of 47.6 and 14.4 μg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10∼100 μg/kg) inhibited gastrointestinal transit with a maximum of ∼40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following subcutanous injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1∼320 μg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as nonaddictive analgesics for inflammatory pain. SIGNIFICANCE STATEMENT: Developing nonaddictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, the pharmacology of a potential nonaddictive analgesic, NCP, is reported. NCP has full KOR agonist/partial MOR agonist activities with a 6.5x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Fig. 1.

Chemical structure of NCP.

Fig. 2.

NCP produced antinociceptive effects against inflammatory pain in CD-1 mice. (A) NCP inhibited formalin-induced pain behaviors in mice. Saline, U50,488H (2.5 mg/kg), one dose of NCP 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 (Liu et al., 2019a). Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (for NCP) or unpaired t test (for U50,488H). NCP: F_(3,33) = 9.04, P < 0.001. *P < 0.05, ***P < 0.001, compared with saline control, by Dunnett’s post hoc test (mean ± S.E.M., n = 7–11 animals/group). U50,488H: ****P < 0.0001, compared with saline control, by unpaired t test (mean ± S.E.M., n = 9–11 animals/group). A₅₀ dose of NCP is 47.6 μg/kg. A₅₀ dose of U50,488H is 0.58 mg/kg (Liu et al., 2019a). (B) NCP inhibited acetic acid-induced writhing in mice. Water, U50,488H (5 mg/kg), or one dose of NCP was injected (s.c.) 20 min before acetic acid was administered intraperitoneally and the number of writhes (abdominal stretches) was recorded for 15 min starting at 5 min after acetic acid injection. The writhing number for each animal was normalized by the average writhing number of the control group, which was approximately 30. Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (for NCP) or unpaired t test (for U50,488H). NCP: F_(4,44) = 9.74, P < 0.0001. **P < 0.01, ****P < 0.0001, compared with water control, by Dunnett’s post hoc test (mean ± S.E.M., n = 7–13 animals/group). U50,488H: **P < 0.01, compared with the water control, by unpaired t test (mean ± S.E.M., n = 13 animals/group for water and n = 4 for U50). A₅₀ dose of NCP is 14.4 μg/kg.

Fig. 3.

Analgesic effects of NCP in the acetic acid writhing test are mediated by KOR but not MOR. (A) Pretreatment with β-FNA + norBNI or norBNI but not β-FNA abolished or reduced antinociceptive effects of NCP in the acetic acid-induced writhing test in CD-1 mice. norBNI (32 mg/kg, i.p.), β-FNA (32 mg/kg, s.c.), or both were pretreated 24 h or 48 h before water or NCP (40 μg/kg, s.c.) injection, respectively. Twenty minutes later, acetic acid (0.6%, 10 µl/g, i.p.) was injected. Five minutes later, the number of writhes (abdominal stretches) was recorded for 15 min. Writhing number for each animal was normalized by the average writhing number (∼30) of the control group. Data were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. F(4, 46) = 8.817, P < 0.0001. *** P < 0.001, compared with control (column 1) (mean ± S.E.M., n = 9–13 animals/group). (B) NCP reduced acetic acid-induced writhing to similar extents in MOR(−/−) and WT mice (C57BL/6 background). Saline or NCP (80 μg/kg, s.c.) was injected 20 min before acetic acid administration and the number of writhes (abdominal stretches) was recorded for 15 min starting at 5 min after acetic acid injection. Data were analyzed using two-way ANOVA. Results showed a significant main effect of drug [F_(1,33) = 17.03, p < 0.001] but no significant main effects of genotype [F_(1,33) = 0.0096, P > 0.05] or interaction [F_(1,33) = 0.00016, P > 0.05]. *P < 0.05, compared with water control, Bonferroni post hoc test (mean ± S.E.M., n = 9–10 animals/group).

Fig. 4.

NCP did not cause motor incoordination or CPA in CD-1 mice. (A) NCP did not cause motor incoordination in the rotarod test in mice. After training the previous day, mice were injected subcutaneously with saline, U50,488H (2 or 5 mg/kg), or NCP (80 μg/kg) and tested on the rotarods 10, 20, 30, and 40 min after injection. The doses used produced maximal antinociception (Fig. 2). 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 ± S.E.M., n= 10–12/group). NCP: Results of two-way ANOVA showed no significant main effect of treatment [F_(1,18) = 1.23, P > 0.05] or time [F_(4,72) = 1.37, P > 0.05] and no significant interaction [F_(4,99) = 0.67, P > 0.05]. U50,488H: Results of two-way ANOVA showed a significant main effect of treatment [F_(2,28) = 16.12, P < 0.0001], a significant main effect of time [F_(4,112) = 21.09, P < 0.0001], and a significant interaction [F_(8,139) = 6.47, P < 0.0001]. *P < 0.05, ***P < 0.001, ****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 U50,488H were from Liu et al. (2019a) and are shown for comparison. (B) NCP did not cause CPA in mice at 80 or 800 μg/kg (s.c.). NCP at 80 μg/kg (s.c.) produced maximal antinociception (Fig. 2). On day 0, mice were subject to pre-test. On days 1 to 3, mice were injected with saline or one dose of U50,488H or NCP and stayed in home cages for 10 min before each 30-min conditioning session (one saline session and one drug session/day) for 3 days. On day 4 (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 (mean ± S.E.M., n = 9–10/group). NCP, F (3, 35) = 1.175, P = 0.3333; U50,488H, F_(2,26) = 13.55, P < 0.0001. ***P < 0.001, compared with saline control by Dunnett’s post hoc test. Data on U50,488H were from Liu et al. (2019a) and are shown for comparison.

Fig. 5.

NCP did not inhibit novelty-induced hyperlocomotion even at doses up to 56xA₅₀ in the acetic acid writhing test. (A) and (B) NCP did not cause inhibition of novelty-induced locomotor activity or enhancement of locomotor activity in CD-1 mice. Mice were treated subcutaneously. with saline, NCP (80 or 800 μg/kg), or U50,488H (5 mg/kg) and locomotor activities were monitored. Cumulative data between 0 and 30 min post-injection are shown here. Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (for NCP) or unpaired t test (for U50,488H). NCP: (A) F (2, 21) = 0.8544, P = 0.4398; (B) F (2, 21) = 0.08671, P = 0.9173 (mean ± S.E.M., n = 8–12 animals/group). U50,488H: (A) ***P < 0.001, (B) *P < 0.05, compared with water control, by unpaired t test (mean ± S.E.M., n = 8 animals/group). Data on U50,488H were from Liu et al. (2019a) and are shown for comparison.

Fig. 6.

NCP was not self-administered in rats trained to self-administer heroin. Rats were trained to self-administer heroin under an FR5 schedule of reinforcement. Heroin was then replaced with different doses of NCP (1.0, 3.2, 10, 32, 100, 320 μg/kg/infusion) or heroin (10, 32, 100 μg/kg/infusion). Filled symbols denote statistical significance (p < 0.05) compared with saline (S) training sessions before test sessions. Points above H represent heroin training sessions (32 μg/kg/infusion) before test sessions. All points represent the mean ± S.E.M. of six rats (three per sex).

Fig. 7.

NCP inhibited GI transit. Mice were injected with either saline, morphine (1 mg/kg, s.c.), or NCP (3, 10, 30, 80, 100 μg/kg, s.c.). Thirty minutes later, charcoal meal was administered through oral gavage and, 20 min later, animals were sacrificed. Small intestine was removed from the pylorus to the ileocecal junction. The charcoal’s travel distance and the total length of the intestine were measured by placing the intestine on a ruled template. Charcoal transit was calculated as a percent of the total intestinal length. Data were analyzed with one-way ANOVA followed by Dunnett’s post hoc test (for NCP) or unpaired t test (for morphine). NCP: F_(5,38) = 12.47, P < 0.0001. **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with saline control, by Dunnett’s post hoc test (mean ± S.E.M., n = 7–8 animals/group). Morphine: ***P < 0.001, compared with saline control, by unpaired t test (mean ± S.E.M., n = 7–8 animals/group).

Fig. 8.

NCP had no effects on (A) respiration rate or (B) oxygen saturation. Following acclimation in the individual observation boxes, mice were connected to collar sensor and injected with either saline, morphine (10 mg/kg, s.c.), or NCP (10, 20, 40, 80, 160 µg/kg, s.c.). Respiratory rate and SpO2 were recorded over 60 min. NCP up to 160 µg/kg did not have any effect on respiratory rate and SpO2; however, morphine significantly reduced respiratory rate compared with vehicle (****P < 0.0001, n = 6–7, by unpaired t test). Each column represents the group mean ± S.E.M. N = 6–7 for each group.

Fig. 9.

NCP induced CPA in MOR(−/−) but not in WT mice, both in C57BL/6 background. CPA was carried out following the procedure described in Fig. 4 (mean ± S.E.M. n = 10–11). (A) For MOR(−/−) mice, ** P < 0.01, versus saline group, by unpaired t test. (B) For WT mice, data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test: F (2, 28) = 0.1484, P = 0.8627.

Fig. 10.

NCP, even at doses up to 10 mg/kg (694xA50 in writhing test), did not precipitate withdrawal-associated jump in mice chronically treated with escalating doses of morphine. Mice were subjected to twice-daily treatments for 5 consecutive days with saline or increasing doses of morphine through subcutaneous injections (day 1: 20 mg/kg, day 2: 40 mg/kg, day 3: 60 mg/kg, day 4: 80 mg/kg, day 5: 100 mg/kg). On the morning of day 6, mice were administered an additional injection of either 100 mg/kg morphine or saline. Two hours later, withdrawal was triggered by a subcutaneous injection of 10 mg/kg naloxone or NCP (0.08, 0.8, or 10 mg/kg). Subsequently, mice were placed inside a transparent cylinder and observed for 30 min. The numbers of jumps (mean ± S.E.M., n = 4–7) were counted and analyzed by one-way ANOVA followed by Dunnett’s post hoc test: F (4, 18) = 51.41, P < 0.0001; ****P < 0.0001, versus the saline-naloxone group.

Fig. 11.

NCP did not inhibit scratching behavior induced by compound 48/80 in CD-1 (A) and MOR−/− mice (B). Saline, NCP at different doses, or U50,488H (2.5 or 5 mg/kg) was injected subcutaneously 20 min before compound 48/80 injection and the bouts of scratching were counted for 30 min. Bouts of scratching were recorded and normalized to the average of saline group, which was about ∼300 bouts for CD-1 mice (A) or ∼150 bouts for MOR(−/−) mice (B). Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (for NCP) or unpaired t test (for U50,488H). NCP: (A) F (3, 33) = 0.4798, P = 0.6986; (B) F (2, 21) = 1.061, P = 0.3641. U50,488H: (A) ***P < 0.001, (B) **P < 0.01, compared with saline control. Data were presented as mean ± S.E.M. [n = 8–10 for (A), n = 7–9 for (B)].