The multifunctional peptide DN-9 produced peripherally acting antinociception in inflammatory and neuropathic pain via μ- and κ-opioid receptors

Abstract

Background and purpose: Considerable effort has recently been directed at developing multifunctional opioid drugs to minimize the unwanted side effects of opioid analgesics. We have developed a novel multifunctional opioid agonist, DN-9. Here, we studied the analgesic profiles and related side effects of peripheral DN-9 in various pain models.

Experimental approach: Antinociceptive effects of DN-9 were assessed in nociceptive, inflammatory, and neuropathic pain. Whole-cell patch-clamp and calcium imaging assays were used to evaluate the inhibitory effects of DN-9 to calcium current and high-K⁺ -induced intracellular calcium ([Ca²⁺ ]_i ) on dorsal root ganglion (DRG) neurons respectively. Side effects of DN-9 were evaluated in antinociceptive tolerance, abuse, gastrointestinal transit, and rotarod tests.

Key results: DN-9, given subcutaneously, dose-dependently produced antinociception via peripheral opioid receptors in different pain models without sex difference. In addition, DN-9 exhibited more potent ability than morphine to inhibit calcium current and high-K⁺ -induced [Ca²⁺ ]_i in DRG neurons. Repeated treatment with DN-9 produced equivalent antinociception for 8 days in multiple pain models, and DN-9 also maintained potent analgesia in morphine-tolerant mice. Furthermore, chronic DN-9 administration had no apparent effect on the microglial activation of spinal cord. After subcutaneous injection, DN-9 exhibited less abuse potential than morphine, as was gastroparesis and effects on motor coordination.

Conclusions and implications: DN-9 produces potent analgesia with minimal side effects, which strengthen the candidacy of peripherally acting opioids with multifunctional agonistic properties to enter human studies to alleviate the current highly problematic misuse of classic opioids on a large scale.

Figure 1

Antinociceptive effects of DN‐9 and morphine in the mouse tail–flick test. Time‐response curves for the antinociception induced by 0.095, 0.95, and 9.48 μmol·kg⁻¹ of DN‐9 (a), 3.11, 9.32, and 31.08 μmol·kg⁻¹ of morphine (b) in male mice, and 0.095, 0.95, and 9.48 μmol·kg⁻¹ of DN‐9 in female mice (c) after s.c. administration. The AUC values of MPE % during the observed period from these data were statistically analysed and are presented in the insert. ^* P < .05, significantly different from saline group; one‐way ANOVA followed by Dunnett's post hoc test. (d) Effects of peripheral administration of the opioid receptor antagonists naloxone (Nal, 10 mg·kg⁻¹) and naloxone methiodide (NALM, 10 mg·kg⁻¹) and NPFF receptor antagonist RF9 (5 mg·kg⁻¹) on the antinociception induced by DN‐9 and morphine. Effect of i.c.v. (e) and i.t. (f) administration of the opioid receptor antagonists naloxone (5 nmol) and NALM (5 nmol) and NPFF receptor antagonist RF9 (10 nmol) on the antinociception induced by DN‐9. (g) Effects of peripheral administration of the selective opioid receptor antagonists β‐FNA (1 mg·kg⁻¹), NTI (1 mg·kg⁻¹), and nor‐BNI (1 mg·kg⁻¹) on the antinociception induced by DN‐9, morphine, and CR845.Each data point represents the mean ± SEM, n = 7–10 mice per group. ^* P < .05, significantly different from saline + saline group, ^# P < .05, significantly different from saline + DN‐9 group, ^@ P < .05, significantly different from saline + CR845 group; one‐way ANOVA followed by Bonferroni's post hoc test. (h) in vitro plasma stability assessment of DN‐9 in mouse plasma. Each data point represents the mean ± SEM, n = 4 independent experiments. RF9, 1‐adamantanecarbonyl‐RF‐NH₂

Figure 2

Effects of s.c. injection of DN‐9 and morphine on carrageenan‐induced inflammatory pain in mice. Time‐response curves for the anti‐allodynic effects induced by 1.19, 2.37, 4.74, and 9.48 μmol·kg⁻¹ of DN‐9 (a), 3.11, 15.54, and 31.08 μmol·kg⁻¹ of morphine (b) in male mice, and 2.37, 4.74, and 9.48 μmol·kg⁻¹ DN‐9 in female mice (c). The AUC values of MPE % during the observed period from these data were statistically analysed and presented in the insert. ^* P < .05, significantly different from saline group; one‐way ANOVA followed by Dunnett's post hoc test. (d) Effects of peripheral administration of the opioid receptor antagonists naloxone (Nal, 10 mg·kg⁻¹) and naloxone methiodide (NALM, 10 mg·kg⁻¹) and NPFF receptor antagonist RF9 (5 mg·kg⁻¹) on the anti‐allodynic effects of DN‐9 and morphine. ^* P < .05, significantly different from saline + saline group, ^# P < .05, significantly different from saline + DN‐9 group; one‐way ANOVA followed by Bonferroni's post hoc test. (e) The different anti‐allodynic effects between WT and MOR^−/− mice. ^# P < .05, significantly different from WT mice; unpaired t test. Each data point represents the mean ± SEM, n = 6–8 mice per group

Figure 3

Effects of s.c. injection of DN‐9 and morphine on CCI‐induced neuropathic pain in mice. Time‐response curves for the anti‐allodynic effects induced by 2.37, 4.74, and 9.48 μmol·kg⁻¹ of DN‐9 in male (a) and female (c) mice and 3.11, 9.32, and 31.08 μmol·kg⁻¹ of morphine in male mice (b); anti‐hyperalgesic effects induced by 1.19, 2.37, 4.74, and 9.48 μmol·kg⁻¹ of DN‐9 (d) and 3.11, 9.32, and 31.08 μmol·kg⁻¹ of morphine (e) in male mice and 2.37, 4.74, and 9.48 μmol·kg⁻¹ of DN‐9 in female mice (f). The AUC values of MPE % during the observed period from these data were statistically analysed and presented in the insert. The anti‐allodynic effects induced by 0.095, 0.95, and 9.48 μmol·kg⁻¹ of DN‐9 in male (g) and female mice (h) and 3.11, 6.22, 9.32, and 31.08 μmol·kg⁻¹ of morphine in male mice (g) in an acetone evaporation assay. ^* P < .05, significantly different from saline group; one‐way ANOVA followed by Dunnett's post hoc test. Effects of peripheral administrations of the opioid receptor antagonists naloxone (Nal, 10 mg·kg⁻¹) and naloxone methiodide (NALM, 10 mg·kg⁻¹) and NPFF receptor antagonist RF9 (5 mg·kg⁻¹) on the anti‐allodynic [(i) for mechanical stimuli and (k) for cold stimuli] and anti‐hyperalgesic effects (j) induced by DN‐9. ^* P < .05, significantly different from saline + saline group, ^# P < .05, significantly different from saline + DN‐9 group; one‐way ANOVA followed by Bonferroni's post hoc test, n = 6–10 mice per group

Figure 4

Effects of DN‐9 and morphine on calcium currents and [Ca²⁺]_i in DRG neurons. (a) Trace of calcium currents before and after morphine (10 μM) or DN‐9 (0.1 μM) treatment in small‐sized DRG neurons from WT mice. (b) Time‐course of relative calcium currents before and after morphine (10 μM) or DN‐9 (0.1 μM) perfusion. Each data point represents the mean ± SEM, n = 5–6 neurons per group. ^* P < .05, significantly different from control group; one‐way ANOVA, followed by Bonferroni's post hoc test. (c) Calcium responses of DRG neurons (as Δ F/Fo) to 50 mM of high K⁺ after 1 min of DN‐9 (0.01, 0.1, and 1 μM) or morphine (10 μM) perfusion. Calcium responses of DRG neurons to 50‐mM high K⁺ after 2 min of β‐FNA (1 μM), NTI (1 μM), and nor‐BNI (1 μM) plus 1 min of DN‐9 (1 μM) perfusion (d) and after 2 min of RF9 (1 μM) plus 1 min of DN‐9 (0.1 μM) perfusion. Each data point represents the mean ± SEM, n > 50 neurons per group. ^* P < .05, significantly different from control group, ^# P < .05, significantly different from DN‐9 + KCl group; one‐way ANOVA, followed by Bonferroni's post hoc test

Figure 5

Tolerance evaluation of DN‐9 and morphine in different pain models. Antinociceptive effects of repeated administration of DN‐9 (9.48 μmol·kg⁻¹, s.c.) and morphine (31.08 μmol·kg⁻¹, s.c.) on acute (a), inflammatory (b), and neuropathic pain models [(c) anti‐allodynia; (d) anti‐hyperalgesia]. To evaluate the role of NPFF receptor on the tolerance development of DN‐9, RF9 was administrated prior to DN‐9 daily treatment in acute (a) and inflammatory (b) pain models. ^* P < .05, significantly different from the nociceptive latency on Day 1; one‐way ANOVA followed by Tukey's HSD post hoc test. To evaluate the effects of DN‐9 on the nociceptive latency in mice tolerance to morphine (31.08 μmol·kg⁻¹, once daily for 7 days), DN‐9 (9.48 μmol·kg⁻¹) was s.c. administered on Day 8 in inflammatory (b) and neuropathic (c,d) pain models. Each data point represents the mean ± SEM, n = 7–8 mice per group. ^# P < .05, indicates significant difference between latencies on Day 7 and Day 8; paired t test. (e,f) Effects of chronic DN‐9 and morphine administration on the expression of microglial cells in spinal cord. Each data point represents the mean ± SEM, n = 5, 5–6 images per animal. ^* P < .05, significantly different from saline group; one‐way ANOVA followed by Bonferroni's post hoc test. Scale bar equals 50 μm

Figure 6

Effects of DN‐9 and morphine on locomotor activity in mice. (a) Time‐response curve of DN‐9 (9.48 μmol·kg⁻¹) and morphine (31.08 μmol·kg⁻¹) after s.c. administration. (b) The total distance after drug injection. Each data point represents the mean ± SEM, n = 8 mice per group. ^* P < .05, significantly different from saline group; one‐way ANOVA followed by Bonferroni's post hoc test. (c) Morphine, but not DN‐9, caused stereotypical circling after s.c. injection

Figure 7

Dependence evaluation of DN‐9 and morphine in mice. (a) Effects of s.c. administration of DN‐9 (9.48 μmol·kg⁻¹) and morphine (31.08 μmol·kg⁻¹) on place conditioning in mice. The results are expressed as CPP score (time spent in drug‐paired side on post‐conditioning day minus the time on preconditioning day). ^* P < .05, significantly different from saline group, ^# P < .05, significant difference between DN‐9 group and morphine group; unpaired t test. (b) The jumping counts of naloxone‐precipitated withdrawal after DN‐9 and morphine treatment in mice. Each data point represents the mean ± SEM, n = 8–10 mice per group. ^* P < .05, significantly different from saline group, ^# P < .05, significant difference between DN‐9 group and morphine group; one‐way ANOVA followed by Dunnett's post hoc test

Figure 8

Effects of s.c. administration of DN‐9 and morphine on gastrointestinal transit and motor coordination. (a) Effects of s.c. administration of DN‐9 (0.95, 9.48, 28.45, and 94.83 μmol·kg⁻¹) and morphine (31.08 μmol·kg⁻¹) on gastrointestinal transit in mice. ^* P < .05, significantly different from saline group, ^# P < .05, significantly different from morphine group; one‐way ANOVA followed by Dunnett's post hoc test. (b) Effects of s.c. injection of 9.48 μmol·kg⁻¹ of DN‐9 and 31.08 μmol·kg⁻¹ of morphine on motor coordination in mice. Each data point represents the mean ± SEM, n = 8–9 mice per group. No significant difference between drug group and saline group; one‐way ANOVA followed by Dunnett's post hoc test