Pharmacology/Drug Development

Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins (PGs) and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids (PUFAs) are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Interestingly, diol levels are increased in the synovial fluid of humans with OA, warranting further research on the biological role of this lipid pathway in the progression of OA. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides, resulting in analgesia in both neuropathic, and inflammatory pain conditions. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models, which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEHI in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for 5 days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After 5 days, EC1728 significantly reduced pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-α, and reduced cytotoxicity in canine chondrocytes challenged with IL1β to simulate an arthritic environment. These results provide the first example of altering lipid epoxides as a therapeutic target for OA potentially acting by protecting chondrocytes from inflammatory induced cytotoxicity. Considering the challenges and high variability of naturally occurring disease in aged dogs, these data provide initial proof of concept justification that inhibiting the sEH is a non-NSAID, non-opioid, disease altering strategy for treating OA, and warrants further investigation.

We investigated whether administration of the δ-opioid receptor (DOR) agonist H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), which also activates nuclear factor erythroid 2-related factor 2 (Nrf2), alleviated chronic inflammatory and/or neuropathic pain and inhibited the depressive-like behaviors associated with persistent neuropathic pain. The possible mechanisms implicated were also assessed. We evaluated the following effects in male C57BL/6J mice with inflammatory pain induced by or neuropathic pain caused by the chronic constriction of sciatic nerve: (1) the antinociceptive effects of UFP-512; (2) the effects of UFP-512 on the expression of Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), inducible nitric oxide synthase, DOR, and mitogen-activated protein kinases (MAPK) in the spinal cord of animals with inflammatory or neuropathic pain; (3) the antinociceptive effects of the coadministration of UFP-512 with the Nrf2 activator sulforaphane (SFN); and (4) the antidepressant effects of UFP-512 in animals with depressive-like behaviors associated with neuropathic pain. Our results demonstrated that the intraperitoneal administration of UFP-512 inhibited chronic inflammatory and neuropathic pain and reduced the depressive-like behaviors associated with persistent neuropathic pain. The antiallodynic effects of UFP-512 were significantly augmented when it was coadministered with SFN in both types of chronic pain. The administration of UFP-512 increased/reestablished the spinal cord protein levels of Nrf2 and HO-1 in mice with inflammatory or neuropathic pain. However, while during inflammatory pain UFP-512 inhibited spinal c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation induced by peripheral inflammation. This DOR agonist blocked the spinal activated PI3K/Akt signaling pathway under chronic neuropathic pain conditions, but it did not alter the enhanced protein levels of p-JNK or p-ERK1/2 induced by sciatic nerve injury. These results revealed the antinociceptive and antidepressant effects of UFP-512 in animals with chronic pain and the different mechanism of action of this DOR agonist in the presence of inflammatory or neuropathic pain. Our data also suggest the administration of UFP-512 as an alternative for the treatment of chronic pain and the depressive-like behaviors associated with neuropathic pain.

Enhancement of endocannabinoid signaling has emerged as an attractive strategy for the treatment of pain. In addition to the well-characterized hydrolytic pathways, cyclooxygenase-2 (COX-2) mediated oxygenation is thought to be an alternative route for endocannabinoid metabolism and therefore provides a new avenue for drug intervention. In this study, we examined the therapeutic effect of indomethacin morpholinamide (IMMA), a novel substrate-selective COX-2 inhibitor, in the chronic constriction injury (CCI) mouse model. Treatment with IMMA significantly alleviated hyperalgesia and mechanical allodynia demonstrated by increased thermal withdrawal latency in Hargreaves test and tactile thresholds in Von Frey test. Accumulation of astrocytes and microglia in spinal cord dorsal horn and infiltration of macrophages into the dorsal root ganglion and sciatic nerve were reduced by drug treatment. Co-administration of the CB2 receptor antagonist, but not the CB1 receptor antagonist partially reversed the inhibitory effect of IMMA on pain sensitivity and inflammatory infiltrates. IMMA downregulated the mRNA expression of TNF-α and IL-1β and the production of IL-6 and MCP-1 proteins in the ipsilateral sciatic nerve. The enhanced NF-κB DNA binding activity in the CCI mouse dorsal spinal cord was also significantly reduced, suggesting that inactivation of NF-κB contributes to the anti-inflammatory property of IMMA. However, different from the previous reports showing that IMMA can increase endocannabinoids without interfering with arachidonic acid metabolism, treatment with IMMA failed to elevate the endogenous levels of AEA and 2-AG, but significantly reduced the production of prostaglandin E (PGE). Furthermore, the mRNA expression of enzymes involved in PGE production, COX-2 and prostaglandin E synthase 2 in the ipsilateral sciatic nerve was also suppressed by IMMA treatment. Taken together, these results suggested that IMMA might exert anti-nociceptive effects through multiple mechanisms which include, but are not limited to, CB2 receptor activation and reduced PGE production.

Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective μ-opioid receptor (MOR) agonist [D-Ala, -Me-Phe, Gly-ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 ± 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 ± 36-207 ± 56 pA) in 15-20% of recorded DRG neurons from naïve mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naïve and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia.

A growing body of evidence has indicated that the release of nociceptive factors, such as interleukins and chemokines, by activated immune and glial cells has crucial significance for neuropathic pain generation and maintenance. Moreover, changes in the production of nociceptive immune factors are associated with low opioid efficacy in the treatment of neuropathy. Recently, it has been suggested that CC chemokine receptor type 1 (CCR1) signaling is important for nociception. Our study provides evidence that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with significant upregulation of endogenous CCR1 ligands, namely, CCL2, CCL3, CCL4, CCL6, CCL7 and CCL9 in the spinal cord and CCL2, CCL6, CCL7 and CCL9 in dorsal root ganglia (DRG). We showed that single and repeated intrathecal administration of J113863 (an antagonist of CCR1) attenuated mechanical and thermal hypersensitivity. Moreover, repeated administration of a CCR1 antagonist enhanced the analgesic properties of morphine and buprenorphine after CCI. Simultaneously, repeated administration of J113863 reduced the protein levels of IBA-1 in the spinal cord and MPO and CD4 in the DRG and, as a consequence, the level of pronociceptive factors, such as IL-1beta, IL-6, and IL-18. The obtained data provide evidence that CCR1 blockade reduces hypersensitivity and increases opioid-induced analgesia through the modulation of neuroimmune interactions.

Potent opioid-based therapies are often unsuccessful in promoting satisfactory analgesia in neuropathic pain. Moreover, the side-effects associated with opioid therapy are still manifested in neuropathy-like diseases, including tolerance, abuse, addiction and hyperalgesia, although the mechanisms underlying these effects remain unclear. Studies in the spinal cord and periphery indicate that neuropathy alters the expression of mu-[MOR], delta-[DOR] or kappa-[KOR] opioid receptors, interfering with their activity. However, there is no consensus as to the supraspinal opioidergic modulation provoked by neuropathy, the structures where the sensory and affective-related pain components are processed. In this study we explored the effect of chronic constriction of the sciatic nerve over 7 and 30 days (CCI-7d and CCI-30d, respectively) on MOR, DOR and KOR mRNAs expression, using in situ hybridization, and the efficacy of G-protein stimulation by DAMGO, DPDPE and U-69593 (MOR, DOR and KOR specific agonists, respectively), using [35S]GTPγS binding, within opioid-sensitive brain structures. After CCI-7d, CCI-30d or both, opioid receptor mRNAs expression was altered throughout the brain: MOR – in the paracentral/centrolateral thalamic nuclei, ventral posteromedial thalamic nuclei, superior olivary complex, parabrachial nucleus and posterodorsal tegmental nucleus; DOR – in the somatosensory cortex [SSC], ventral tegmental area, caudate putamen [CPu], nucleus accumbens [NAcc], raphe magnus [RMg] and PB; and KOR – in the locus coeruleus. Agonist-stimulated [35S]GTPγS binding was altered following CCI: MOR – CPu and RMg; DOR – prefrontal cortex [PFC], SSC, RMg and NAcc; and KOR -PFC and SSC. Thus, this study shows that several opioidergic circuits in the brain are recruited and modified following neuropathy.