Potentiation of morphine antinociception and inhibition of diabetic neuropathic pain by the multi-chemokine receptor antagonist peptide RAP-103

Abstract

Aims: We determined the ability of the multi-chemokine receptor (CCR2/CCR5/CCR8) antagonist RAP-103 to modulate pain behaviors in an acute model of surgical pain, with and without an added opioid (morphine), and by itself in a chronic model of Streptozotocin (STZ)-induced diabetic peripheral neuropathy (DPN).

Materials and methods: Pain behaviors were assessed by mechanical and thermal tests in rats. Cytokine and chemokine biomarkers in sciatic nerve and spinal cord were assessed by in situ qPCR.

Key findings: In the incisional pain assay, RAP-103 (0.01-1 mg/kg, i.p.) alone had no antiallodynic effect post-surgery. RAP-103 (0.5 mg/kg) when co-administered with morphine (0.5-5 mg/kg), reduced the ED₅₀ of morphine from 3.19 mg/kg to 1.42 mg/kg. In a DPN model, rats exhibited persistent mechanical and cold allodynia. Oral administration of RAP-103 (0.5-0.02 mg/kg/day) resulted in a complete reversal of established hypersensitivity in DPN rats (P < .001), which gradually returned to pain hypersensitivity after the cessation of the treatment. The mRNA expression of cytokines, IL-1β, TNFα; chemokines CCL2, CCL3; and chemokine receptors CCR2 and CCR5 in DPN rat sciatic nerve, but not spinal cord, were significantly increased. RAP-103 resulted in significant reductions in sciatic nerve expression of IL-1β, TNFα and CCL3 in STZ-induced diabetic rats with trends toward lower levels for CCL2 and CCR5, while CCR2 was unchanged.

Significance: In acute pain, co-administration of RAP-103 with morphine provided the same antinociceptive effect with a reduced dose of morphine, reducing opioid side-effects and risks. RAP-103 by itself is an effective non-opioid antinociceptive treatment for diabetic neuropathic pain.

Keywords: Antagonist; CCR5; Chemokine; Neuropathy; Opioid; Pain.

Fig. 1.

RAP-103 time- and dose-response on acute post-incisional pain. After baseline measurements, rats underwent surgery (t = 0). At 15 min, post-surgery baseline was recorded. Animals were injected with either water or RAP-103 i.p. at t = 25 min and paw withdrawal thresholds were measured at 30, 60, 120, 240, and 360 min. Mechanical allodynia (paw withdrawal threshold) was calculated at the indicated times. No change in antinociception (paw withdrawal thresholds) by RAP-103 compared to vehicle was observed. Paw withdrawal thresholds measured at 24, 48, and 72 h. also did not show a change in antinociception (data not shown). Data were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test, significance P < .05.

Fig. 2.

A) Comparison of morphine alone at different doses with morphine plus RAP-103 on acute post-incisional pain. Percent reversal of mechanical allodynia was calculated using data from t = 60 min. % reversal = [(60 min threshold − pre-dose threshold) / (baseline threshold predose threshold)] × 100. Data were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test, **P < .01. B) Dose-response curves for morphine alone and morphine in combination with a fixed dose (0.5 mg/kg) of the chemokine receptor antagonist, RAP-103 are shown. The percent reversal of mechanical allodynia was calculated using data from t = 60 min. The ED₅₀ of morphine alone is 3.19 mg/kg; the ED₅₀ of morphine plus RAP-103 is 1.42 mg/kg. Data were analyzed using the Student’s unpaired one-tailed t-test, P < .05.

Fig. 3.

Daily oral administration of RAP-103 significantly attenuated mechanical allodynia in streptozocin-induced diabetic rats. (N = 5–7 per dose). Data are presented as the mean ± SEM; *P < .05, **P < .01, ***P < .001.

Fig. 4.

Daily oral administration of RAP-103 significantly attenuated cold allodynia in diabetic rats. (N = 5–7 per dose). Data are presented as the mean ± SEM; *P < .05, **P < .01, ***P < .001.

Fig. 5.

Oral administration of RAP-103, for 7 days inhibited DPN-associated inflammation in sciatic nerve. In STZ-treated rats the levels of mRNA for proinflammatory mediators IL1β, TNFα; CCR5 chemokine ligand CCL3; and chemokine receptor CCR5 were substantially increased in sciatic nerves compared to controls as determined by qRT-PCR assays. RAP-103 significantly reduced levels of these biomarkers. Levels of target mRNAs were normalized to the housekeeping gene GAPDH. Fold changes vs naive animals were presented. Statistical analysis was performed by unpaired t-tests. N = 3/group. The criterion for statistical significance was *P < .05, **P < .01, P < .001.

Fig. 6.

mRNA expression of inflammatory mediators in the spinal cord (L4-L6) of STZ induced DPN rats. Neuroinflammation in the spinal cord of DPN rats was not prominent. However, RAP-103 reduced the levels of TNFα and CCR5 as determined by qRT-PCR. Statistical analysis was performed by unpaired t-tests. N = 3/group. The criterion for statistical significance was *P < .05, **P < .01.