Pharmacology/Drug Development

Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N,N'-dibenzyl tryptophan were prepared and characterized in vitro by Ca-imaging and patch-clamp electrophysiology assays. Molecular modeling studies led to identification of a broad and well-defined interaction network of these derivatives inside the TRPM8-agonist binding site, underlying their antagonist activity. The (5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione () emerged as a potent (IC50 4.10 ± 1.2 nM), selective, and metabolically stable TRPM8 antagonist. In vivo, showed significant target coverage in an icilin-induced WDS (at 11.5 mg/kg i.p.), an oxaliplatin-induced cold allodynia (at 10-30 μg s.c.), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg i.p.) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.

Latent sensitization is a model of chronic pain in which a persistent state of pain hypersensitivity is suppressed by opioid receptors, as evidenced by the ability of opioid antagonists to induce a period of mechanical allodynia. Our objective was to determine if substance P and its neurokinin 1 receptor (NK1R) mediate the maintenance of latent sensitization. Latent sensitization was induced by injecting rats in the hindpaw with complete Freund's adjuvant (CFA), or by tibial spared nerve injury (SNI). When responses to von Frey filaments returned to baseline (day 28), the rats were injected intrathecally with saline or the NK1R antagonist RP67580, followed 15 min later by intrathecal naltrexone. In both pain models, the saline-injected rats developed allodynia for 2 h after naltrexone, but not the RP67580-injected rats. Saline or RP67580 were injected daily for two more days. Five days later (day 35), naltrexone was injected intrathecally. Again, the saline-injected rats, but not the RP67580-injected rats, developed allodynia in response to naltrexone. To determine if there is sustained activation of NK1Rs during latent sensitization, NK1R internalization was measured in lamina I neurons in rats injected in the paw with saline or CFA, and then injected intrathecally with saline or naltrexone on day 28. The rats injected with CFA had a small amount of NK1R internalization that was significantly higher than in the saline-injected rats. Naltrexone increased NK1R internalization in the CFA-injected rats but nor in the saline-injected rats. Therefore, sustained activation of NK1Rs maintains pain hypersensitivity during latent sensitization.

25OHD is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. Our results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as Type 1 Diabetes.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial tissue, joint dysfunction, and damage. Epoxyeicosatrienoic acids (EETs) are endogenous anti-inflammatory compounds, which are quickly converted by the soluble epoxide hydrolase (sEH) enzyme into a less active form with decreased biological effects. The inhibition of the sEH enzyme has been used as a strategy to lower nociception and inflammation. The goal of this study was to investigate whether the peripheral treatment with the sEH enzyme inhibitor 1- trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) could prevent the hypernociception and inflammation in the albumin-induced arthritis model in rats' temporomandibular joint (TMJ). After the induction of experimental arthritis, animals were assessed for nociceptive behavior test, leukocyte infiltration counts and histologic analysis, ELISA to quantify several cytokines and Western blotting. The peripheral pretreatment with TPPU inhibited the arthritis-induced TMJ hypernociception and leukocyte migration. Moreover, the local concentrations of proinflammatory cytokines were diminished by TPPU, while the anti-inflammatory cytokine interleukin-10 was up-regulated in the TMJ tissue. Finally, TPPU significantly decreased protein expression of iNOS, while did not alter the expression of MRC1. This study provides evidence that the peripheral administration of TPPU reduces hypernociception and inflammation in TMJ experimental arthritis.

Prodrugs (MRS7422, MRS7476) of highly selective A adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively, were synthesized by succinylation of the 2' and 3' hydroxyl groups, and the parent, active drug was shown to be readily liberated upon incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury model, 3 μmol/kg, p.o.), a known AAR effect. MRS7476 (5 mg/kg, p.o., twice daily) was found to protect against non-alcoholic steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 production, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2 levels) or inflammation. Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls. Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body AAR knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables water solubility and in vivo activity of masked AAR agonists in models of two disease conditions.

Peripheral neurotoxicity is a debilitating condition that afflicts up to 90% of patients with colorectal cancer receiving oxaliplatin-containing therapy. Although emerging evidence has highlighted the importance of various solute carriers to the toxicity of anticancer drugs, the contribution of these proteins to oxaliplatin-induced peripheral neurotoxicity remains controversial. Among candidate transporters investigated in genetically engineered mouse models, we provide evidence for a critical role of the organic cation transporter 2 (OCT2) in satellite glial cells in oxaliplatin-induced neurotoxicity, and demonstrate that targeting OCT2 using genetic and pharmacological approaches ameliorates acute and chronic forms of neurotoxicity. The relevance of this transport system was verified in transporter-deficient rats as a secondary model organism, and translational significance of preventive strategies was demonstrated in preclinical models of colorectal cancer. These studies suggest that pharmacological targeting of OCT2 could be exploited to afford neuroprotection in cancer patients requiring treatment with oxaliplatin.

Sustained-release buprenorphine is widely used in mice with the intention of providing long-lasting analgesia. Statements about duration of therapeutic efficacy are based on persistence of serum buprenorphine levels over a minimum threshold, but behavioral data demonstrating sustained efficacy is not established. Additionally, chronic opioid exposure can induce tolerance and/or hyperalgesia; mice receiving sustained-release buprenorphine have not been evaluated for these effects. This study assessed clinical efficacy and duration of sustained-release buprenorphine in inflammatory, post-operative, and cancer pain; and screened for centrally-mediated opioid-induced hyperalgesia as well as opioid tolerance. At 1-2 mg/kg sustained-release buprenorphine, statistically significant analgesic efficacy occurred only at time points up to 2 h. These animals showed no changes in von Frey thresholds on the contralateral side, i.e. no centrally-mediated opioid hyperalgesia. To establish whether acute onset opioid tolerance resulted from a single sustained-release buprenorphine administration, we used the tail flick assay, exposing mice to sustained-release buprenorphine or saline on Day 1 and buprenorphine on Day 2. We measured duration and efficacy of 1 mg/kg buprenorphine after 1 mg/kg sustained-release buprenorphine, and also quantified a dose-response curve of buprenorphine (0.1-3 mg/kg) after 2 mg/kg sustained-release buprenorphine. Compared to control animals, mice previously exposed to sustained-release buprenorphine showed diminished analgesic response to buprenorphine; the resultant dose-response curve showed decreased efficacy. Pretreatment with naloxone, an opioid receptor antagonist, blocked sustained-release buprenorphine analgesic action. The short duration of antinociception following administration of sustained-release buprenorphine in mice is caused by the rapid development of tolerance.