Pharmacology/Drug Development

Adrenic acid (AdA, 22:4) is an ω-6 polyunsaturated fatty acid (PUFA), derived from arachidonic acid. Like other PUFAs, it is metabolized by cytochrome P450s to a group of epoxy fatty acids (EpFAs), epoxydocosatrienoic acids (EDTs). EpFAs are lipid mediators with various beneficial bioactivities, including exertion of analgesia and reduction of endoplasmic reticulum (ER) stress, that are degraded to dihydroxy fatty acids by the soluble epoxide hydrolase (sEH). However, the biological characteristics and activities of EDTs are relatively unexplored, and, alongside dihydroxydocosatrienoic acids (DHDTs), they had not been detected . Herein, EDT and DHDT regioisomers were synthesized, purified, and used as standards for analysis with a selective and quantitative high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Biological verification in AdA-rich tissues suggests that basal metabolite levels are highest in the liver, with 16,17-EDT concentrations consistently being the greatest across the analyzed tissues. Enzyme hydrolysis assessment revealed that EDTs are sEH substrates, with greatest relative rate preference for the 13,14-EDT regioisomer. Pretreatment with an EDT methyl ester regioisomer mixture significantly reduced the onset of tunicamycin-stimulated ER stress in human embryonic kidney cells. Finally, administration of the regioisomeric mixture effectively alleviated carrageenan-induced inflammatory pain in rats. This study indicates that EDTs and DHDTs are naturally occurring lipids, and EDTs could be another therapeutically relevant group of EpFAs.

The psychoactive and non-psychoactive constituents of cannabis, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have synergistic analgesic efficacy in animal models of neuropathic pain when injected systemically. However, the relevance of this preclinical synergy to clinical neuropathic pain studies is unclear because the many of the latter use oral administration. We therefore examined the oral effectiveness of these phytocannabinoids and their interactions in a mouse chronic constriction injury (CCI) model of neuropathic pain. THC produced a dose-dependent reduction in mechanical and cold allodynia, but also induced side-effects with similar potency. CBD also reduced allodynia, albeit with lower potency than THC, but did not produce cannabinoid-like side-effects at any dose tested. Combination THC:CBD produced a dose-dependent reduction in allodynia, however, it displayed little to no synergy. Combination THC:CBD produced substantial, synergistic side-effects which increased with the proportion of CBD. These findings demonstrate that oral THC and CBD, alone and in combination, have analgesic efficacy in an animal neuropathic pain model. Unlike prior systemic injection studies, combination THC:CBD lacks analgesic synergy when delivered orally. Furthermore, both THC and combination THC:CBD display a relatively poor therapeutic window when delivered orally. This suggests that CBD provides a safer, albeit lower efficacy, oral treatment for nerve injury induced neuropathic pain than THC-containing preparations.

An opioid epidemic is spreading in North America with millions of opioid overdoses annually. Opioid drugs, like fentanyl, target the mu opioid receptor system and induce potentially lethal respiratory depression. The challenge in opioid research is to find a safe pain therapy with analgesic properties but no respiratory depression. Current discoveries are limited by lack of amenable animal models to screen candidate drugs. Zebrafish () is an emerging animal model with high reproduction and fast development, which shares remarkable similarity in their physiology and genome to mammals. However, it is unknown whether zebrafish possesses similar opioid system, respiratory and analgesic responses to opioids than mammals. In freely-behaving larval zebrafish, fentanyl depresses the rate of respiratory mandible movements and induces analgesia, effects reversed by mu-opioid receptor antagonists. Zebrafish presents evolutionary conserved mechanisms of action of opioid drugs, also found in mammals, and constitute amenable models for phenotype-based drug discovery.

Inhibition of the pituitary adenylate cyclase 1 receptor (PAC1R) is a novel mechanism that could be used for abortive treatment of acute migraine. Our research began with comparative analysis of known PAC1R ligand scaffolds, PACAP38 and Maxadilan, which resulted in the selection of des(24-42) Maxadilan, , as a starting point. C-terminal modifications of improved the peptide metabolic stability and . SAR investigations identified synergistic combinations of amino acid replacements that significantly increased the PAC1R inhibitory activity of the analogs to the pM IC range. Our modifications further enabled deletion of up to six residues without impacting potency, thus improving peptide ligand binding efficiency. Analogs and exhibited robust efficacy in the rat Maxadilan-induced increase in blood flow (MIIBF) pharmacodynamic model at 0.3 mg/kg subcutaneous dosing. The first cocrystal structure of a PAC1R antagonist peptide () with PAC1R extracellular domain is reported.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

Inflammatory Bowel Disease (IBD) is a life-long disorder that often begins between the ages of 15 and 30. Anecdotal reports suggest cannabinoids may be an effective treatment. This study sought to determine whether home cage wheel running is an effective method to assess IBD, and whether THC, the primary psychoactive compound in cannabis, can restore wheel running depressed by IBD. Adolescent and adult female Sprague-Dawley rats were individually housed in a cage with a running wheel. Rats were injected with trinitrobenzene sulphonic acid (TNBS) into the rectum to induce IBD-like symptoms. One day later, both vehicle and TNBS treated rats were injected with a low dose of THC (0.32 mg/kg, s.c.) or vehicle. Administration of TNBS depressed wheel running in adolescent and adult rats. No antinociceptive effect of THC was evident when administered 1 day after TNBS. In fact, administration of THC prolonged TNBS-induced depression of wheel running for over 5 days in adolescent and adult rats. These results show that home cage wheel running is affected by TNBS-induced IBD, making it a useful tool to evaluate the behavioral consequences of IBD, and that administration of THC, instead of producing antinociception, exacerbates TNBS-induced IBD. Perspective This article advances research on inflammatory bowel disease in two important ways: 1) Home cage wheel running is a new and sensitive tool to assess the behavioral consequences of IBD in adolescent and adult rats; and 2) Administration of the cannabinoid THC exacerbates the negative behavioral effects of IBD.

Cisplatin plays an essential role in the treatment of various cancers. Cisplatin exhibits high efficacy, but it often leads to severe neurotoxic side effects, such as chemotherapy-induced polyneuropathy (CIPN). The pathophysiology of CIPN is not fully understood. There is increasing evidence for damage to satellite glial cells (SGC) and dorsal root ganglion (DRG) neurons. We investigated the influence of cisplatin on the function of SGCs and the direct influence on DRGs. Satellite glial cells were isolated from DRG and exposed to 0.1, 1, 10, or 100 μM cisplatin for 2 h, 4 h, and 24 h. Using immunocytochemical staining and Western blot analysis, the expression of the glial fibrillary acid protein (GFAP), reactive oxygen species (ROS), and inward rectifier potassium channel 4.1 (K) was determined. An increase in the immune reactivity (IR) and protein levels of GFAP and ROS was measured, and a reduction of IR and protein level of K was detected. A decrease in these channels' current density was observed using the whole-cell patch-clamp recording. The interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) release of SGCs increased after cisplatin exposure as measured using ELISA, and interleukin-1β (IL-1β) decreased. The SGC-secreted factors in the supernatant after cisplatin treatment led to a modulation of cultured DRG neurons' excitability. Taken together, the modulation and function of different SGC proteins could be linked to a direct impact of cisplatin. Further, SGC-secreted factors influenced the excitability of sensory neurons. Overall, SGCs could be a potential target in preventing and treating chemotherapy-induced neuropathic pain.