Mirogabalin Decreases Pain-like Behaviors by Inhibiting the Microglial/Macrophage Activation, p38MAPK Signaling, and Pronociceptive CCL2 and CCL5 Release in a Mouse Model of Neuropathic Pain.

Neuropathic pain is a chronic condition that significantly reduces the quality of life of many patients as a result of ineffective pain relief therapy. For that reason, looking for new analgesics remains an important issue. Mirogabalin is a new gabapentinoid that is a specific ligand for the α2σ-1 and α2σ-2 subunits of voltage-gated calcium channels. In the present study, we compared the analgesic effect of pregabalin and mirogabalin in a neuropathic pain chronic constriction injury (CCI) of the sciatic nerve in a mouse model. The main purpose of our study was to determine the effectiveness of mirogabalin administered both once and repeatedly and to explain how the drug influences highly activated cells at the spinal cord level in neuropathy. We also sought to understand whether mirogabalin modulates the selected intracellular pathways (p38MAPK, ERK, JNK) and chemokines (CCL2, CCL5) important for nociceptive transmission, which is crucial information from a clinical perspective. First, our study provides evidence that a single mirogabalin administration diminishes tactile hypersensitivity more effectively than pregabalin. Second, research shows that several indirect mechanisms may be responsible for the beneficial analgesic effect of mirogabalin. This study reports that repeated intraperitoneally (i.p.) mirogabalin administration strongly prevents spinal microglia/macrophage activation evoked by nerve injury, slightly suppresses astroglia and neutrophil infiltration, and reduces the p38MAPK levels associated with neuropathic pain, as measured on Day 7. Moreover, mirogabalin strongly diminished the levels of the pronociceptive chemokines CCL2 and CCL5. Our results indicate that mirogabalin may represent a new strategy for the effective pharmacotherapy of neuropathic pain.

Analgesic Effects of Fisetin, Peimine, Astaxanthin, Artemisinin, Bardoxolone Methyl and 740 Y-P and Their Influence on Opioid Analgesia in a Mouse Model of Neuropathic Pain.

Treatment of neuropathic pain remains a challenge for modern medicine due to the insufficiently understood molecular mechanisms of its development and maintenance. One of the most important cascades that modulate the nociceptive response is the family of mitogen-activated protein (MAP) kinases and phosphatidylinositol-3-kinase (PI3K), as well as nuclear factor erythroid 2-related factor 2 (Nrf2). The aim of this study was to determine the effect of nonselective modulators of MAP kinases-fisetin (ERK1/2 and NFκB inhibitor, PI3K activator), peimine (MAPK inhibitor), astaxanthin (MAPK inhibitor, Nrf2 activator) and artemisinin (MAPK inhibitor, NFκB activator), as well as bardoxolone methyl (selective activator of Nrf2) and 740 Y-P (selective activator of PI3K)-in mice with peripheral neuropathy and to compare their antinociceptive potency and examine their effect on analgesia induced by opioids. The study was performed using albino Swiss male mice that were exposed to chronic constriction injury of the sciatic nerve (CCI model). Tactile and thermal hypersensitivity was measured using von Frey and cold plate tests, respectively. Single doses of substances were administered intrathecally on day 7 after CCI. Among the tested substances, fisetin, peimine, and astaxanthin effectively diminished tactile and thermal hypersensitivity in mice after CCI, while artemisinin did not exhibit analgesic potency in this model of neuropathic pain. Additionally, both of the activators tested, bardoxolone methyl and 740 Y-P, also showed analgesic effects after intrathecal administration in mice exposed to CCI. In the case of astaxanthin and bardoxolone methyl, an increase in analgesia after combined administration with morphine, buprenorphine, and/or oxycodone was observed. Fisetin and peimine induced a similar effect on tactile hypersensitivity, where analgesia was enhanced after administration of morphine or oxycodone. In the case of 740 Y-P, the effects of combined administration with each opioid were observed only in the case of thermal hypersensitivity. The results of our research clearly indicate that substances that inhibit all three MAPKs provide pain relief and improve opioid effectiveness, especially if they additionally block NF-κB, such as peimine, inhibit NF-κB and activate PI3K, such as fisetin, or activate Nrf2, such as astaxanthin. In light of our research, Nrf2 activation appears to be particularly beneficial. The abovementioned substances bring promising results, and further research on them will broaden our knowledge regarding the mechanisms of neuropathy and perhaps contribute to the development of more effective therapy in the future.

Effect of pharmacological modulation of the kynurenine pathway on pain-related behavior and opioid analgesia in a mouse model of neuropathic pain.

Dysfunction of the central nervous system are accompanied by changes in tryptophan metabolism, with the kynurenine pathway (KP) being the main route of its catabolism. Recently, KP metabolites, which are collectively called kynurenines, have become an area of intense research due to their ability to directly and indirectly affect a variety of classic neurotransmitter systems. However, the significance of KP in neuropathic pain is still poorly understood. Therefore, we designed several experiments to verify changes in the mRNA levels of KP enzymes in parallel with other factors related to this metabolic route after chronic constriction injury of the sciatic nerve (CCI model) in mice. The analysis revealed an increase in, Kmo, Kynu and Haoo mRNA levels in the spinal cord on the 7th day after CCI, while Kat1, Kat2, Tdo2, Ido2 and Qprt mRNA levels remain unchanged. Subsequent pharmacological studies provided evidence that modulation of KP by single intrathecal administration of 1-D-MT, UPF468 or L-kynurenine attenuates mechanical and thermal hypersensitivity and increases the effectiveness of selected opioids in mice as measured on day 7 after CCI. Moreover, our results provide the first evidence that the injection of L-kynurenine preceded by UPF468 (KMO inhibitor) is more effective at reducing hypersensitivity in animals with neuropathic pain. Importantly, L-kynurenine also exerts an analgesic effect after intravenous injections, which is enhanced by the administration of minocycline, an inhibitor of microglial activation. Additionally, L-kynurenine administered intrathecally and intravenously enhances analgesia evoked by all tested opioids (morphine, buprenorphine and oxycodone). Overall, our results indicate that the modulation of KP at different levels might be a new pharmacological tool in neuropathy management.

Mirogabalin Decreases Pain-like Behaviours and Improves Opioid and Ketamine Antinociception in a Mouse Model of Neuropathic Pain.

Neuropathic pain remains a difficult clinical challenge due to its diverse aetiology and complex pathomechanisms, which are yet to be fully understood. Despite the variety of available therapies, many patients suffer from ineffective pain relief; hence, the search for more efficacious treatments continues. The new gabapentinoid, mirogabalin has recently been approved for clinical use. Although its main mechanism of action occurs at the α2σ-1 and α2σ-2 subunits of calcium channels and is well documented, how the drug affects the disturbed neuropathic interactions at the spinal cord level has not been clarified, which is crucial information from a clinical perspective. The findings of our study suggest that several indirect mechanisms may be responsible for the beneficial analgesic effect of mirogabalin. This is the first study to report that mirogabalin enhances the mRNA expression of spinal antinociceptive factors, such as IL-10 and IL-18BP, and reduces the concentration of the pronociceptive substance P. Importantly, mirogabalin improves the morphine-, buprenorphine-, oxycodone-, and ketamine-induced antinociceptive effects in a neuropathic pain model. Our findings support the hypothesis that enhancing opioid and ketamine analgesia by combining these drugs with mirogabalin may represent a new strategy for the effective pharmacotherapy of neuropathic pain.

New insights into the analgesic properties of the XCL1/XCR1 and XCL1/ITGA9 axes modulation under neuropathic pain conditions - evidence from animal studies.

Recent studies have indicated the involvement of chemokine-C-motif ligand 1 (XCL1) in nociceptive transmission; however, the participation of its two receptors, canonical chemokine-C-motif receptor 1 (XCR1) and integrin alpha-9 (ITGA9), recently recognized as a second receptor, has not been clarified to date. The aim was to explore by which of these receptors XCL1 reveals its pronociceptive properties and how the XCL1-XCR1 and XCL1-ITGA9 axes blockade/neutralization influence on pain-related behavior and opioid analgesia in the model of neuropathic pain. In our studies we used Albino Swiss mice which were exposed to the unilateral sciatic nerve chronic constriction injury (CCI) as a neuropathic pain model. Animals received single intrathecal (i.t.) injection of XCL1, XCL1 neutralizing antibodies, antagonist of XCR1 (vMIP-II) and neutralizing antibodies of ITGA9 (YA4), using lumbar puncture technique. Additionally we performed i.t. co-administration of abovementioned neutralizing antibodies and antagonists with single dose of morphine/buprenorphine. To assess pain-related behavior the von Frey and cold plate tests were used. To measure mRNA and protein level the RT-qPCR and Western Blot/Elisa/immunofluorescence techniques were performed, respectively. Statistical analysis was conducted using ANOVA with a Bonferroni correction. Presented studies have shown time-dependent upregulation of the mRNA and/or protein expression of XCL1 in the spinal cord after nerve injury as measured on day 1, 4, 7, 14, and 35. Our immunofluorescence study showed that XCL1 is released by astroglial cells located in the spinal cord, despite the neural localization of its receptors. Our results also provided the first evidence that the blockade/neutralization of both receptors, XCR1 and ITGA9, reversed hypersensitivity after intrathecal XCL1 administration in naive mice; however, neutralization of ITGA9 was more effective. In addition, the results proved that the XCL1 neutralizing antibody and, similarly, the blockade of XCR1 and neutralization of ITGA9 diminished thermal and mechanical hypersensitivity in nerve injury-exposed mice after 7 days. Additionally, neutralization of XCL1 improves morphine analgesia. Moreover, blockade of XCR1 positively influences buprenorphine effectiveness, and neutralization of ITGA9 enhances not only buprenorphine but also morphine analgesia. Therefore, blockade of the XCL1-ITGA9 interaction may serve as an innovative strategy for the polypharmacotherapy of neuropathic pain in combination with opioids.

Blockade of CC Chemokine Receptor Type 3 Diminishes Pain and Enhances Opioid Analgesic Potency in a Model of Neuropathic Pain.

Neuropathic pain is a serious clinical issue, and its treatment remains a challenge in contemporary medicine. Thus, dynamic development in the area of animal and clinical studies has been observed. The mechanisms of neuropathic pain are still not fully understood; therefore, studies investigating these mechanisms are extremely important. However, much evidence indicates that changes in the activation and infiltration of immune cells cause the release of pronociceptive cytokines and contribute to neuropathic pain development and maintenance. Moreover, these changes are associated with low efficacy of opioids used to treat neuropathy. To date, the role of CC chemokine receptor type 3 (CCR3) in nociception has not been studied. Similarly, little is known about its endogenous ligands (C-C motif ligand; CCL), namely, CCL5, CCL7, CCL11, CCL24, CCL26, and CCL28. Our research showed that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with upregulation of CCL7 and CCL11 in the spinal cord and dorsal root ganglia (DRG). Moreover, our results provide the first evidence that single and repeated intrathecal administration of the CCR3 antagonist SB328437 diminishes mechanical and thermal hypersensitivity. Additionally, repeated administration enhances the analgesic properties of morphine and buprenorphine following nerve injury. Simultaneously, the injection of SB328437 reduces the protein levels of some pronociceptive cytokines, such as IL-6, CCL7, and CCL11, in parallel with a reduction in the activation and influx of GFAP-, CD4- and MPO-positive cells in the spinal cord and/or DRG. Moreover, we have shown for the first time that an inhibitor of myeloperoxidase-4-aminobenzoic hydrazide may relieve pain and simultaneously enhance morphine and buprenorphine efficacy. The obtained results indicate the important role of CCR3 and its modulation in neuropathic pain treatment and suggest that it represents an interesting target for future investigations.

The Kynurenine Pathway as a Potential Target for Neuropathic Pain Therapy Design: From Basic Research to Clinical Perspectives.

Accumulating evidence suggests the key role of the kynurenine pathway (KP) of the tryptophan metabolism in the pathogenesis of several diseases. Despite extensive research aimed at clarifying the mechanisms underlying the development and maintenance of neuropathic pain, the roles of KP metabolites in this process are still not fully known. Although the function of the peripheral KP has been known for several years, it has only recently been acknowledged that its metabolites within the central nervous system have remarkable consequences related to physiology and behavior. Both the products and metabolites of the KP are involved in the pathogenesis of pain conditions. Apart from the neuroactive properties of kynurenines, the KP regulates several neurotransmitter systems in direct or indirect ways. Some neuroactive metabolites are known to have neuroprotective properties (kynurenic acid, nicotinamide adenine dinucleotide cofactor), while others are toxic (3-hydroxykynurenine, quinolinic acid). Numerous animal models show that modulation of the KP may turn out to be a viable target for the treatment of diseases. Importantly, some compounds that affect KP enzymes are currently described to possess analgesic properties. Additionally, kynurenine metabolites may be useful for assessing response to therapy or as biomarkers in therapeutic monitoring. The following review describes the molecular site of action and changes in the levels of metabolites of the kynurenine pathway in the pathogenesis of various conditions, with a particular emphasis on their involvement in neuropathy. Moreover, the potential clinical implications of KP modulation in chronic pain therapy as well as the directions of new research initiatives are discussed.

Comparison of the Effects of Chemokine Receptors CXCR2 and CXCR3 Pharmacological Modulation in Neuropathic Pain Model-In Vivo and In Vitro Study.

Recent findings have highlighted the roles of CXC chemokine family in the mechanisms of neuropathic pain. Our studies provide evidence that single/repeated intrathecal administration of CXCR2 (NVP-CXCR2-20) and CXCR3 ((±)-NBI-74330) antagonists explicitly attenuated mechanical/thermal hypersensitivity in rats after chronic constriction injury of the sciatic nerve. After repeated administration, both antagonists showed strong analgesic activity toward thermal hypersensitivity; however, (±)-NBI-74330 was more effective at reducing mechanical hypersensitivity. Interestingly, repeated intrathecal administration of both antagonists decreased the mRNA and/or protein levels of pronociceptive interleukins (i.e., IL-1beta, IL-6, IL-18) in the spinal cord, but only (±)-NBI-74330 decreased their levels in the dorsal root ganglia after nerve injury. Furthermore, only the CXCR3 antagonist influenced the spinal mRNA levels of antinociceptive factors (i.e., IL-1RA, IL-10). Additionally, antagonists effectively reduced the mRNA levels of pronociceptive chemokines; NVP-CXCR2-20 decreased the levels of CCL2, CCL6, CCL7, and CXCL4, while (±)-NBI-74330 reduced the levels of CCL3, CCL6, CXCL4, and CXCL9. Importantly, the results obtained from the primary microglial and astroglial cell cultures clearly suggest that both antagonists can directly affect the release of these ligands, mainly in microglia. Interestingly, NVP-CXCR2-20 induced analgesic effects after intraperitoneal administration. Our research revealed important roles for CXCR2 and CXCR3 in nociceptive transmission, especially in neuropathic pain.

Metamizole relieves pain by influencing cytokine levels in dorsal root ganglia in a rat model of neuropathic pain.

BACKGROUND: Treatment of neuropathic pain is still challenging. Recent studies have suggested that dorsal root ganglia (DRG), which carry sensory neural signals from the peripheral nervous system to the central nervous system, are important for pathological nociception. A proper understanding of the significance and function of DRG and their role in pharmacotherapy can help to improve the treatment of neuropathic pain. Metamizole, also known as sulpyrine or dipyrone, is a non-opioid analgesic commonly used in clinical practice, but it is not used for neuropathic pain treatment. METHODS: Chronic constriction injury (CCI) of the sciatic nerve was induced in Wistar rats. Metamizole was administered intraperitoneally (ip) preemptively at 16 and 1 h before CCI and then twice a day for 7 days. To evaluate tactile and thermal hypersensitivity, von Frey and cold plate tests were conducted, respectively. RESULTS: Our behavioral results provide evidence that repeated intraperitoneal administration of metamizole diminishes the development of neuropathic pain symptoms in rats. Simultaneously, our findings provide evidence that metamizole diminishes the expression of pronociceptive interleukins (IL-1beta, IL-6, and IL-18) and chemokines (CCL2, CCL4, and CCL7) in DRG measured 7 days after sciatic nerve injury. These assays indicate, for the first time, that metamizole exerts antinociceptive effects on nerve injury-induced neuropathic pain at the DRG level. CONCLUSIONS: Finally, we indicate that metamizole-induced analgesia in neuropathy is associated with silencing of a broad spectrum of cytokines in DRG. Our results also suggest that metamizole is likely to be an effective medication for neuropathic pain.

Comparison of the beneficial effects of RS504393, maraviroc and cenicriviroc on neuropathic pain-related symptoms in rodents: behavioral and biochemical analyses.

The latest research highlights the role of chemokine signaling pathways in the development of nerve injury-induced pain. Recent studies have provided evidence for the involvement of CCR2 and CCR5 in the pathomechanism underlying neuropathy. Thus, the aim of our study was to compare the effects of a selective CCR2 antagonist (RS504393), selective CCR5 antagonist (maraviroc) and dual CCR2/CCR5 antagonist (cenicriviroc) and determine whether the simultaneous blockade of both receptors is better than blocking only one of them selectively. All experiments were performed using Wistar rats/Swiss albino mice subjected to chronic constriction injury (CCI) of the sciatic nerve. To assess pain-related reactions, the von Frey and cold plate tests were used. The mRNA analysis was performed using RT-qPCR. We demonstrated that repeated intrathecal administration of the examined antagonists attenuated neuropathic pain in rats 7 days post-CCI. mRNA analysis showed that RS504393 did not modulate the spinal expression of the examined chemokines, whereas maraviroc reduced the CCI-induced elevation of CCL4 level. Cenicriviroc significantly lowered the spinal levels of CCL2-4 and CCL7. At the dorsal root ganglia, strong impacts of RS504393 and cenicriviroc on chemokine expression were observed; both reduced the CCI-induced elevation of CCL2-5 and CCL7 levels, whereas maraviroc decreased only the CCL5 level. Importantly, we demonstrated that a single intrathecal/intraperitoneal injection of cenicriviroc had greater analgesic properties than RS504393 or maraviroc in neuropathic mice. Additionally, we demonstrated that cenicriviroc enhanced opioid-induced analgesia. Based on our results, we suggest that targeting CCR2 and CCR5 simultaneously, is an interesting alternative for neuropathic pain pharmacotherapy.

CCR4 antagonist (C021) influences the level of nociceptive factors and enhances the analgesic potency of morphine in a rat model of neuropathic pain.

Neuropathic pain is a chronic condition which significantly reduces the quality of life and serious clinical issue that is in general resistant to available therapies. Therefore looking for new analgesics is still critical issue. Recent, studies have indicated that chemokine signaling pathways are crucial for the development of neuropathy; however, the role of CC chemokine receptor 4 (CCR4) in this process has not yet been studied. Therefore, the aim of our research was to investigate the influence of C021 (a CCR4 antagonist) and CCR4 CC chemokine ligands 17 and 22 (CCL17 and CCL22) on the development of hypersensitivity and the effectiveness of morphine induced analgesia in naive animals and/or animals exposed to chronic constriction injury (CCI) of the sciatic nerve. Firstly, we demonstrated that the intrathecal administration of CCL17 and CCL22 induced pain-related behavior in naive mice. Secondly, we revealed that the intrathecal injection of C021 significantly reduced CCI-induced hypersensitivity after nerve injury. In parallel, C021 reduced microglia/macrophages activation and the level of some pronociceptive interleukins (IL-1beta; IL-18) in the spinal cord 8 days after CCI. Moreover, C021 not only attenuated mechanical and thermal hypersensitivity but also enhanced the analgesic properties of morphine. Our research indicates that CCR4 ligands might be important factors in the early stages of neuropathy, when we observe intense microglia/macrophages activation. Moreover, pharmacological blockade of CCR4 may serve as a potential new target for better understanding the mechanisms of neuropathic pain development.

Neuropathic Pain Dysregulates Gene Expression of the Forebrain Opioid and Dopamine Systems.

Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury.

Recently, the role of CXCR2 in nociception has been noted. Our studies provide new evidence that the intrathecal administration of its CINC ligands (Cytokine-Induced Neutrophil Chemoattractant; CXCL1-3) induces pain-like behavior in naïve mice, and the effect occurring shortly after administration is associated with the neural location of CXCR2, as confirmed by immunofluorescence. RT-qPCR analysis showed, for the first time, raised levels of spinal CXCR2 after chronic constriction injury (CCI) of the sciatic nerve in rats. Originally, on day 2, we detected escalated levels of the spinal mRNA of all CINCs associated with enhancement of the protein level of CXCL3 lasting until day 7. Intrathecal administration of CXCL3 neutralizing antibody diminished neuropathic pain on day 7 after CCI. Interestingly, CXCL3 is produced in lipopolysaccharide-stimulated microglial, but not astroglial, primary cell cultures. We present the first evidence that chronic intrathecal administrations of the selective CXCR2 antagonist, NVP CXCR2 20, attenuate neuropathic pain symptoms and CXCL3 expression after CCI. Moreover, in naïve mice, this antagonist prevented CXCL3-induced hypersensitivity. However, NVP CXCR2 20 did not diminish glial activation, thus not enhancing morphine/buprenorphine analgesia. These results provide novel insight into the crucial role of CXCR2 in neuropathy based on CXCL3 modulation, which may become a potential therapeutic target in pain treatment.

Chemokines CCL2 and CCL7, but not CCL12, play a significant role in the development of pain-related behavior and opioid-induced analgesia.

The complex neuroimmunological interactions mediated by chemokines are suggested to be responsible for the development of neuropathic pain. The lack of knowledge regarding the detailed pathomechanism of neuropathy is one reason for the lack of optimally efficient therapies. Recently, several lines of evidence indicated that expression of CCR2 is increased in spinal cord neurons and microglial cells after peripheral nerve injury. It was previously shown that administration of CCR2 antagonists induces analgesic effects; however, the role of CCR2 ligands in neuropathic pain still needs to be explained. Thus, the goal of our studies was to investigate the roles of CCL2, CCL7, and CCL12 in neuropathic pain development and opioid effectiveness. The experiments were conducted on primary glial cell cultures and two groups of mice: naive and neuropathic. We used chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model. Mice intrathecally received chemokines (CCL2, CCL7, CCL12) at a dose of 10, 100 or 500 ng, neutralizing antibodies (anti-CCL2, anti-CCL7) at a dose of 1, 4 or 8 µg, and opioids (morphine, buprenorphine) at a dose of 1 µg. The pain-related behaviors were assessed using the von Frey and cold plate tests. The biochemical analysis of mRNA expression of glial markers, CCL2, CCL7 and CCL12 was performed using quantitative reverse transcriptase real-time PCR. We demonstrated that CCI of the sciatic nerve elevated spinal expression of CCL2, CCL7 and CCL12 in mice, in parallel with microglia and astroglial activation markers. Moreover, intrathecal injection of CCL2 and CCL7 induced pain-related behavior in naive mice in a dose-dependent manner. Surprisingly, intrathecal injection of CCL12 did not influence nociceptive transmission in naive or neuropathic mice. Additionally, we showed for the first time that intrathecal injection of CCL2 and CCL7 neutralizing antibodies not only attenuated CCI-induced pain-related behaviors in mice but also augmented the analgesia induced by morphine and buprenorphine. In vitro studies suggest that both microglia and astrocytes are an important cellular sources of the examined chemokines. Our results revealed the crucial roles of CCL2 and CCL7, but not CCL12, in neuropathic pain development and indicated that pharmacological modulation of these factors may serve as a potential therapeutic target for new (co)analgesics.

Kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviour and enhanced morphine analgesia in a mouse neuropathic pain model.

BACKGROUND: The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naïve and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain. METHODS: The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively. RESULTS: Our results have shown, for the first time, that administration of CXCL17 in naïve mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy. CONCLUSIONS: Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain.

Dataset of (±)-NBI-74330 (CXCR3 antagonist) influence on chemokines under neuropathic pain.

Our data give evidence that CXCR3 ligands exhibit pronociceptive properties and play an important role in the initiation, development and maintenance of neuropathic pain. Moreover, intrathecal administration of each CXCR3 ligand induced hypersensitivity reactions in naive mice and of its neutralizing antibodies diminished neuropathic pain syndrome in CCI-exposed mice. Furthermore, our results indicate that selective CXCR3 antagonist (±)-NBI-74330 reduced the neuropathic pain-related behaviour and also enhanced morphine analgesic potency in CCI-exposed rats. Interestingly, our data show that (±)-NBI-74330 administration diminished the spinal IBA1 and, in parallel, downregulated CXCL4, CXCL9 and CXCL10. In addition, CXCR3 antagonist increased the spinal GFAP, what correlates with upregulation of CXCR3 and CXCL11. Moreover, in DRG (±)-NBI-74330 did not change IBA1 and GFAP positive cells activation, however downregulated also CXCL9. CXCR3 and CXCL10 were co-localized predominantly with neuronal marker in the spinal cord. Summing up, chronic (±)-NBI-74330 intrathecal injection promotes beneficial analgesic effects in rat neuropathic pain model, as described in details in "Pharmacological blockade of CXCR3 by (±)-NBI-74330 reduces neuropathic pain and enhances opioid effectiveness - evidence from in vivo and in vitro studies" (Piotrowska et al., 2018).

Alterations in the Activity of Spinal and Thalamic Opioid Systems in a Mice Neuropathic Pain Model.

Clinical studies have reported lower effectivity of opioid drugs in therapy of neuropathic pain. Therefore, to determine the changes in endogenous opioid systems in this pain more precisely, we have studied the changes in the pain-related behavior on days 1, 14, and 28 following a chronic constriction injury (CCI) to the sciatic nerve in mice. In parallel, we have studied the changes of µ-(MOP), δ-(DOP) and κ-(KOP) receptors, proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels, as well as GTPγS binding of opioid receptors on the ipsi- and contralateral parts of the spinal cord and thalamus on the 14th day following CCI, as on this day the greatest manifestation of pain-related behavior was observed. On ipsilateral spinal cord, the decrease in MOP/DOP/KOP receptor and increase in PDYN/PENK mRNA expression was observed. In thalamus, MOP/DOP/KOP receptor expression decreased contralaterally. On ipsilateral side, there were no changes in PDYN/PENK or DOP/KOP receptor expression, but MOP mRNA decreased. The spinal GTPγS binding of MOP/DOP/KOP receptor ligands decreased on the ipsilateral side, yet the effect was less pronounced for DOP receptor ligands. In thalamus, a decrease was observed on the contralateral side for all opioid receptor ligands, especially for DOP ligand. A less pronounced decrease in GTPγS binding of spinal DOP ligands may indicate a weaker stimulation of ascending nociceptive pathways, which could explain the absence of decreased activity of DOP receptor ligands in neuropathy. These findings may suggest that drugs with a higher affinity for the DOP receptor will perform better in neuropathic pain.

Zaprinast diminished pain and enhanced opioid analgesia in a rat neuropathic pain model.

The mechanism of neuropathic pain is complex and unclear. Based on our results, we postulate that an intensification of the kynurenine pathway occurs as a consequence of nerve injury. The G protein-coupled receptor 35 (GPR35) is important for kynurenine pathway activation. Cyclic GMP-specific phosphodiesterase inhibitors have also been shown to have beneficial effects on neuropathic pain. Therefore, the aims of our research were to elucidate how a substance that acts as both an agonist of GPR35 and an inhibitor of phosphodiesterase influences neuropathic pain in a rat model. Here, we demonstrated that preemptive and repeated intrathecal (i.t.) administration (16 h and 1 h before injury and then after nerve ligation daily for 7 days) of zaprinast (1 µg/5 µl) significantly attenuated mechanical (von Frey test) and thermal (cold plate test) hypersensitivity measured on day 7 after chronic constriction injury, and the effect of even a single injection lasted up to 24 h. Our data indicate that zaprinast diminished the number of IBA1-positive cells and consequently attenuated the levels of IL-1beta, IL-6, IL-18, and NOS2 in the lumbar spinal cord and/or dorsal root ganglia. Our results also demonstrated that zaprinast potentiated the analgesic properties of morphine and buprenorphine. In summary, in a neuropathic pain model, zaprinast significantly reduced pain symptoms and enhanced the effectiveness of opioids. Our data provide new evidence that modulation of both GPR35 and phosphodiesterase could be an important strategy for innovative pharmacological treatments designed to decrease hypersensitivity evoked by nerve injury.

Pharmacological blockade of CXCR3 by (±)-NBI-74330 reduces neuropathic pain and enhances opioid effectiveness - Evidence from in vivo and in vitro studies.

It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work.

Pharmacological Inhibition of Indoleamine 2,3-Dioxygenase-2 and Kynurenine 3-Monooxygenase, Enzymes of the Kynurenine Pathway, Significantly Diminishes Neuropathic Pain in a Rat Model.

Neuropathic pain caused by a primary injury or dysfunction in the peripheral or central nervous system is a tremendous therapeutic challenge. Here, we have collected the first evidence from a single study on the potential contributions to neuropathic pain development by enzymes in the kynurenine pathway [tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO1/2), kynurenine 3-monooxygenase (KMO); kynureninase, 3-hydroxyanthranilate-3,4-dioxygenase (HAOO)] at the spinal cord and dorsal root ganglia (DRG) levels. At the spinal cord, mRNA levels of IDO2, KMO, and HAOO were elevated as measured on day 7 after chronic constriction injury in a rat model, parallel to the C1q-positive cell activation. According to our data obtained from primary microglial cell cultures, all enzymes of the kynurenine pathway except TDO were derived from these cells; however, the activation of microglia induced stronger changes in IDO2 and KMO. Our pharmacological studies gave evidence that the repeated intraperitoneal administration of minocycline, a microglia/macrophage inhibitor, not only attenuated tactile and thermal hypersensitivity but also diminished the levels of IDO2 and KMO mRNA. Our further pharmacological studies confirmed that IDO2 and KMO enzymes take part in the development of neuropathic pain, since we observed that the repeated administration of IDO2 (1-methyl-D-tryptophan) and KMO [UPF 648 - (1S,2S)-2-(3,4-dichlorobenzoyl)cyclopropanecarboxylic acid] inhibitors diminished hypersensitivity development as measured on days 2 and 7. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology in rats and indicate that IDO2 and KMO represent novel pharmacological targets for treating neuropathy.