Pharmacology/Drug Development

Chronic constriction injury of the sciatic nerve in rats causes peripheral neuropathy leading to pain-like behaviors commonly seen in humans. Neuropathy is a leading cause of neuropathic pain, which involves a complex cellular and molecular response in the peripheral nervous system with interactions between neurons, glia, and infiltrating immune cells. In this study, we utilize a nonsteroidal anti-inflammatory drug -loaded nanoemulsion to deliver the cyclooxygenase-2 inhibitor, Celecoxib, directly to circulating monocytes following nerve injury, which provides long-lasting pain relief. However, it is not fully understood how cyclooxygenase-2 inhibition in a macrophage traveling to the site of injury impacts gene expression in the dorsal root ganglia. To elucidate aspects of the molecular mechanisms underlying pain-like behavior in chronic constriction injury, as well as subsequent pain relief with treatment, we employ RNAseq transcriptome profiling of the dorsal root ganglia associated with the injured sciatic nerve in rats. Using high throughput RNA sequencing in this way provides insight into the molecular mechanisms involved in this neuroinflammatory response. We compare the transcriptome from the dorsal root ganglias of the following study groups: chronic constriction injury animals administered with cyclooxygenase-2 inhibiting celecoxib-loaded nanoemulsion, chronic constriction injury animals administered with vehicle treatment, a drug-free nanoemulsion, and a group of naïve, unoperated and untreated rats. The results show an extensive differential expression of 115 genes. Using the protein annotation through evolutionary relationship classification system, we have revealed pain-related signaling pathways and underlying biological mechanisms involved in the neuroinflammatory response. Quantitative polymerase chain reaction validation confirms expression changes for several genes. This study shows that by directly inhibiting cyclooxygenase-2 activity in infiltrating macrophages at the injured sciatic nerve, there is an associated change in the transcriptome in the cell bodies of the dorsal root ganglia.

Increased mRNA translation in sensory neurons following peripheral nerve injury contributes to the induction and maintenance of chronic neuropathic pain. Metformin, a common anti-diabetic drug and an activator of AMP-activated protein kinase (AMPK), inhibits cap-dependent mRNA translation and reverses mechanical hypersensitivity caused by a neuropathic injury in both mice and rats. P-bodies are RNA granules that comprise sites for metabolizing mRNA through the process of de-capping followed by RNA decay. These RNA granules may also sequester mRNAs for storage. We have previously demonstrated that induction of cap-dependent translation in cultured trigeminal ganglion (TG) neurons decreases P-body formation and AMPK activators increase P-body formation. Here we examined the impact of AMPK activation on protein synthesis and P-body formation and on mouse dorsal root ganglion (DRG) neurons. We demonstrate that AMPK activators inhibit nascent protein synthesis and increase P-body formation in DRG neurons. We also demonstrate that mice with a spared-nerve injury (SNI) show decreased P-bodies in the DRG, consistent with increased mRNA translation resulting from injury. Metformin treatment normalizes this effect in SNI mice and increases P-body formation in sham animals. These findings indicate that P-bodies are dynamically regulated by nerve injury and this effect can be regulated via AMPK activation.

The development of multitarget opioid drugs has emerged as an attractive therapeutic strategy to eliminate opioid-related side effects. Our previous study developed a series of opioid and neuropeptide FF (NPFF) pharmacophore-containing chimeric peptides, including DN-9 (Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH), which produced potent nontolerance forming analgesia at the supraspinal level. In the present study, the antinociceptive effects of DN-9 in a series of preclinical pain models and the potential side-effects were investigated at the spinal level in mice. In the tail-flick test, intrathecal injection of DN-9 produced potent analgesia with an ED value at 1.33 pmol, and the spinal antinociception of DN-9 was mainly mediated by μ- and κ-opioid receptors. In addition, DN-9-induced spinal antinociception was augmented by the NPFF receptors antagonist. Furthermore, DN-9 could decrease both the frequency and amplitude of sEPSCs in lamina IIo neurons of the spinal cord, which were mediated by opioid receptors. In contrast to morphine, chronic intrathecal treatments with DN-9 did not induce analgesic tolerance, c-Fos expression or microglial activation. Intrathecal injection of DN-9 showed potent analgesia with antinociceptive ED values between 0.66 and 55.04 pmol in different pain models, including the formalin test, acetic acid-induced writhing test, carrageen-induced inflammatory pain and neuropathic pain. Moreover, DN-9 did not show side effects in locomotor function and coordination, gastrointestinal transit inhibition, the cardiovascular system, and body temperature regulation at antinociceptive doses. Taken together, the present study showed DN-9 produced effective, nontolerance forming analgesia with reduced side effects at the spinal level. DN-9 might be a promising compound for developing multifunctional opioid analgesics with limited adverse effects. Perspective: This article presents the potent and nontolerance forming analgesia effects of DN-9 in a series of preclinical pain models with less opioid related adverse effects at the spinal level in mice. This study also demonstrates that DN-9 has translational potential into an intrathecal analgesic.

Early life administration of vincristine (VNC), commonly used to treat pediatric leukemia, evokes peripheral neuropathy and mechanical pain hypersensitivity in rats that lasts into adolescence. However, the degree to which VNC-evoked neuropathic pain persists throughout adulthood has yet to be examined. It also remains unclear if pediatric VNC exposure can 'prime' developing nociceptive pathways and thereby exacerbate chronic pain following subsequent trauma later in life. To address these issues, rats received five total doses of VNC (60 µg/kg; or vehicle) on postnatal days (P) 11, 13, 17, 19 and 21 followed by a hindpaw surgical incision during adulthood. In addition, in order to model the clinical scenario where cancer relapse necessitates another round of chemotherapy, separate groups of rats that had been treated with VNC (or vehicle) as neonates were subsequently administered VNC as adults (five injections at 100 µg/kg). Intraepidermal nerve fiber density and baseline mechanical pain sensitivity were similar between the neonatal VNC and vehicle-treated littermate controls at 13-15 weeks of age, suggesting that the peripheral neuropathy, and resulting chronic pain, had resolved by adulthood. Importantly, there was no significant overall effect of early life VNC on the severity of post-operative pain following adult incision. Similarly, prior VNC exposure did not significantly influence the degree of mechanical pain hypersensitivity produced by adult VNC treatment. Collectively, these findings suggest that early life VNC administration does not increase the susceptibility to develop chronic pain as adults.