Pharmacology/Drug Development

AMP activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates mechanistic target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling, two signaling pathways that are linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators as well as indirectly through the upregulation of upstream kinases such as liver kinase B1 (LKB1) which is a mechanism of action of metformin. Metformin's anti-hyperalgesic effects have been shown to occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin as well as other structurally distinct AMPK activators narciclasine, ZLN-024 and MK8722 to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. We found that metformin was the only AMPK activator to have sex specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming while direct AMPK activators, ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree. However, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they are able to drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment. SIGNIFICANCE STATEMENT: Non-opioid treatments for post-surgical pain are needed. Our work focused on whether direct or indirect AMPK activators would show greater efficacy for inhibiting incisional pain and also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for post-surgical pain because they inhibit acute pain caused by incision and also prevent long-term neuronal plasticity that is involved in persistent post-surgical pain. Our work points to the natural product, indirect AMPK activator, narciclasine, as an excellent starting point for development of therapeutics.

ProSAAS is one of the most widely expressed proteins throughout the brain and has recently been found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor, GPR171. While BigLEN- GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray (PAG), which is a key brain area involved in pain modulation and opioid functions. We also found that although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces MOPr signaling and morphine induced-antinociception, whereas the GPR171 agonist enhances morphine antinociception suggesting that GPR171 may be a novel target towards the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.

Fibromyalgia is a disease characterised as generalised chronic primary pain that causes functional disability and a reduction in patients' quality of life, without specific pathophysiology or appropriate treatment. Previous studies have shown that kinins and their B and B receptors contribute to chronic painful conditions. Thus, we investigated the involvement of kinins and their B and B receptors in a fibromyalgia-like pain model induced by reserpine in mice. Nociceptive parameters (mechanical allodynia, cold sensitivity and overt nociception) and behaviours of burrowing, thigmotaxis, and forced swimming were evaluated after reserpine administration in mice. The role of kinin B and B receptors was investigated using knockout mice or pharmacological antagonism. The protein expression of kinin B and B receptors and the levels of bradykinin and monoamines were measured in the sciatic nerve, spinal cord and cerebral cortex of the animals. Knockout mice for the kinin B and B receptor reduced reserpine-induced mechanical allodynia. Antagonism of B and B receptors also reduced mechanical allodynia, cold sensitivity and overt nociception reserpine-induced. Reserpine altered thigmotaxis, forced swimming and burrowing behaviour in the animals; with the latter being reversed by antagonism of kinin B receptor. Moreover, reserpine increased the protein expression of kinin B and B receptors and levels of kinin, as well as reduced the levels of monoamines in peripheral and central structures. Kinins and its B and B receptors are involved in fibromyalgia-like pain symptoms. B or B receptors might represent a potential target for the relief of fibromyalgia-like pain symptoms.

Chronic low back pain due to lumbar spinal stenosis (LSS) is common, costly, mechanistically complex, and clinically challenging. However, the factors and mechanisms causing and mediating chronic pain induced by cauda equina compression remain unclear. Here, we examined the role of cyclooxygenase (COX)-2 in infiltrated macrophages, a key mediator of inflammation, in chronic neuropathic pain by LSS using an animal model. LSS was induced in adult male rats by cauda equina compression procedure using a silicone block within the epidural spaces of L5-L6 vertebrae. Locomotor deficit was observed after compression and mechanical allodynia was developed progressively for 4 weeks after injury. A number of macrophage were also infiltrated into the spinal parenchyma and cauda equina and COX-2 was expressed in infiltrated macrophages at 28 days after cauda equina compression. The administration of COX-2 inhibitors, celecoxib and MPO-0029, significantly alleviated LSS-induced chronic mechanical allodynia and inhibited the mRNA expression of inflammatory mediators such as tnf-α, Il-1β, il-6, and inos. Furthermore, COX-2 inhibitors significantly reduced prostaglandin E2 production. These results demonstrated the role of COX-2 in LSS-induced chronic neuropathic pain and suggest that the regulation of COX-2 can be considered as a therapeutic target to relive neuropathic pain.