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A bifunctional peptide containing an opioid and nociceptin receptor-binding pharmacophore, H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (KGNOP1), was tested for its analgesic properties when administered intrathecally in naïve and chronic constriction injury (CCI)-exposed rats with neuropathy-like symptoms. KGNOP1 significantly increased the acute pain threshold, as measured by the tail-flick test, and also increased the threshold of a painful reaction to mechanical and thermal stimuli in CCI-exposed rats. Both of the effects could be blocked by pre-administration of [Nphe1]-Nociceptin (1-13)-NH 2 (NPhe) or naloxone, antagonists for nociceptin and opioid receptors, respectively. This led us to conclude that KGNOP1 acts as a dual opioid and nociceptin receptor agonist in vivo. The analgesic effect of KGNOP1 proved to be more powerful than clinical drugs such as morphine and buprenorphine. Repeated daily intrathecal injections of KGNOP1 led to the development of analgesic tolerance, with the antiallodynic action being completely abolished on day 6. Nevertheless, the development of tolerance to the antihyperalgesic effect was delayed in comparison to morphine, which lost its efficacy as measured by the cold plate test after 3 days of daily intrathecal administration, whereas KGNOP1 was efficient up to day 6. A single intrathecal injection of morphine to KGNOP1-tolerant rats did not raise the pain threshold in any of the behavioural tests; in contrast, a single intrathecal dose of KGNOP1 significantly suppressed allodynia and hyperalgesia in morphine-tolerant rats.

Background and aims: Recent experimental and clinical studies provide evidence for important role of chemokine receptors CCR2 and CCR5 in neuropathic pain pathomechanism. The aim was to compare the behavioural and molecular effects of maraviroc (CCR5 antagonist), RS504393 (CCR2 antagonist) and cenicriviroc (dual CCR2/CCR5 antagonist) and determine if the simultaneous blockade of both CCR2 and CCR5 is better than blocking only one of these receptors.Methods: Wistar rats and Swiss Albino mice were subjected to chronic constriction injury (CCI) of sciatic nerve. Examined substances were injected intrathecally, repeatedly, and preemptively, then once per day for 7 days (rats) or once 9 days after CCI (mice). The neuropathic pain-related symptoms were assessed by von Frey and cold plate tests. The RT-qPCR was used for biochemical analysis. Significant difference were evaluated using ANOVA with Bonferroni correction. Results: The repeated administration of each of examined substances attenuated neuropathic pain in rats after CCI. It was correlated with reduction in spinal microglial activation. We observed that maraviroc reduced CCI-elevated level of pronociceptive CCL3-4. Cenicriviroc significantly lowered the spinal level of CCL2-4, and CCL7. In contrast, RS504393 did not influence the mRNA level of any of them. Finally, we demonstrated that single injection of cenicriviroc was the most effective in CCI-exposed mice.Conclusion: Based on our results we suggest that blocking CCR2/CCR5 simultaneously brings more beneficial effects on both behavioral and molecular level. Acknowledgments: Supported by National Science Centre Poland grants: OPUS-2016/21/B/NZ4/00128, PRELUDIUM-2018/29/N/NZ7/00287 and statutory funds of Institute of Pharmacology, Polish Academy of Sciences.

Background and aims: Chemokine signaling has been implicated in the pathogenesis of neuropathic pain. Thus, the aim of our study was to investigate the roles of CXCR2 and CXCR3 antagonists on neuropathic pain symptoms and opioid effectiveness.Methods: Chronic Constriction Injury (CCI) model of neuropathic pain was used. The pharmacological tools were injected intrathecally, and pain behavior was evaluated by von Frey/cold plate tests. Analysis of mRNA/protein expression was performed by qRT-PCR/Western blotting. The co-localization of CXCR2/CXCR3 with neural/glial cells was visualized by immunofluorescence. Results: Our results demonstrated that each of the CXCR2 (CXCL1-3) and CXCR3 (CXCL4, CXCL9-11, CCL21) ligands induced hypersensitivity reactions in naive animals and the effect occurring shortly after administration is associated with the neural location of CXCR2 and CXCR3, as confirmed by immunofluorescence. Furthermore, the neutralizing antibodies against CCI-upregulated CXCL3, CXCL9, CXCL10, CCL21 and a selective antagonists of the CXCR2 (NVP-CXCR2-20) and CXCR3 (NBI-74330) reduced the neuropathic pain-related behavior. Interestingly, in contrast to NVP-CXCR2-20, the NBI-74330 also enhanced morphine analgesic potency, diminished the spinal microglial markers and pronociceptive factors in CCI-exposed animals. Conclusions: Our data provide new evidence that CXCR2 and CXCR3 are a promising target for diminishing neuropathic pain. Importantly, the pharmacological modulation of neuroimmunological interactions via CXCR3 may represent a new strategy for effective polytherapy with opioids. Acknowledgments: Supported by the National Science Centre, Poland OPUS-11-2016/21/B/NZ4/00128, PRELUDIUM-12-2016/23/N/NZ7/00356, statutory funds of the Institute of Pharmacology PAS Department of Pain Pharmacology. Piotrowska supported by the Foundation for Polish Sciences, the L`Oreal-UNESCO For Women in Science program in Poland.

Background and aims: Unsatisfactory management of chronic pain significantly impairs patients' quality of life. Recent studies focused on the search for new analgesics indicated the key role of CCR2 and CCR5 chemokine receptors in neuropathy. Therefore, the aim was to investigate the effects of the dual CCR2/CCR5 antagonist, cenicriviroc, on pain-related behavior and morphine effectiveness in neuropathic pain. To define mechanisms underlying observed effects, we studied changes in the level of microglial activation and important pronociceptive cytokines.Methods: Wistar rats were implanted with intrathecal catheters. Afterwards, chronic constriction injury of sciatic nerve was performed. Cenicriviroc (CVC) was administered intrathecally, preemptively, and then once per day for 7 days. The last day rats received a single dose of morphine. Pain-related behaviors were measured using von Frey and cold plate test. Biochemical analysis was performed using RT-qPCR. Results: Our studies showed that CVC attenuated mechanical and thermal hypersensitivity and enhanced morphine-induced analgesia in a rat neuropathic pain model. Simultaneously, CVC diminished the activation of microglia/macrophages and prevented the CCI-induced mRNA upregulation of IL-1beta, NOS2, CCL2, CCL3 and CCL7 in the spinal cord and/or dorsal root ganglia. However, do not influence the level of IL-18. Conclusion: We demonstrated that beneficial effects of CVC result from reduction in the level of important pronociceptive factors. It suggests that pharmacological modulation of CCR2/CCR5 may serve as an innovative strategy for neuropathic pain treatment. Acknowledgments: National Science Centre grants: OPUS-2016/21/B/NZ4/00128, PRELUDIUM-2018/29/N/NZ7/00287 and statutory funds of Institute of Pharmacology, PAS.