Tomonari Watabiki

Amelioration of Neuropathic Pain by Novel Transient Receptor Potential Vanilloid 1 Antagonist AS1928370 in Rats without Hyperthermic Effect

Transient receptor potential vanilloid 1 (TRPV1) is activated by a variety of stimulations, such as endogenous ligands and low pH, and is believed to play a role in pain transmission. TRPV1 antagonists have been reported to be effective in several animal pain models; however, some compounds induce hyperthermia in animals and humans. We discovered the novel TRPV1 antagonist (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide (AS1928370) in our laboratory. AS1928370 bound to the resiniferatoxin-binding site on TRPV1 and inhibited capsaicin-mediated inward currents with an IC50 value of 32.5 nM. Although AS1928370 inhibited the capsaicin-induced Ca2+ flux in human and rat TRPV1-expressing cells, the inhibitory effect on proton-induced Ca2+ flux was extremely small. In addition, AS1928370 showed no inhibitory effects on transient receptor potential vanilloid 4, transient receptor potential ankyrin 1, and transient receptor potential melastatin 8 in concentrations up to 10 μM. AS1928370 improved capsaicin-induced secondary hyperalgesia and mechanical allodynia in an L5/L6 spinal nerve ligation model in rats with respective ED50 values of 0.17 and 0.26 mg/kg p.o. Furthermore, AS1928370 alleviated inflammatory pain in a complete Freunds adjuvant model at 10 mg/kg p.o. AS1928370 had no effect on rectal body temperature up to 10 mg/kg p.o., although a significant hypothermic effect was noted at 30 mg/kg p.o. In addition, AS1928370 showed no significant effect on motor coordination. These results suggest that blockage of the TRPV1 receptor without affecting the proton-mediated TRPV1 activation is a promising approach to treating neuropathic pain because of the potential wide safety margin against hyperthermic effects. As such, compounds such as ASP1928370 may have potential as new analgesic agents for treating neuropathic pain.

Pharmacological characterization and gene expression profiling of an L5/L6 spinal nerve ligation model for neuropathic pain in mice

L5/L6 spinal nerve ligation (SNL) in rodents induces behavioral signs similar to the symptoms of neuropathic pain in humans. L5/L6 SNL in rats has been well characterized so far, but there have been few studies using mice. In this study, we established an L5/L6 SNL model in mice and examined the effects of known antinociceptive drugs in the model. We also analyzed the changes in gene expression in dorsal root ganglions with special reference to those which are known to change in a neuropathic pain state to validate the model. Mechanical allodynia in the ipsilateral side paw was observed beginning on day 1 and lasted for at least 2 months following surgery. Diclofenac showed no significant effect on the mechanical allodynia. Gabapentin and pregabalin completely reversed allodynia, but they also caused a decrease in locomotor activity. Duloxetine caused a partial recovery of the threshold. Mexiletine completely reversed allodynia, but it also caused sedation or motor impairment. Morphine caused a partial recovery of the threshold and hyper-locomotion. This mouse L5/L6 SNL model represents a robust mechanical allodynia, which shows a similar pharmacological response to that reported in rats and human patients with neuropathic pain. The pattern changes in gene expression also resembled those reported in rats. This model will therefore be useful for investigation of the effects of novel antinociceptive compounds and the mechanisms of neuropathic pain.

Minodronic acid, a third-generation bisphosphonate, antagonizes purinergic P2X2/3 receptor function and exerts an analgesic effect in pain models

The P2X(2/3) receptor has an important role in the nociceptive transmission. Minodronic acid is a third third-generation bisphosphonate and a potent inhibitor of bone resorption. We found that minodronic acid inhibited alpha,beta-methylene ATP-induced cation uptake with the potency higher than that of suramin in the P2X(2/3) receptor receptor-expressing cells. Other bisphosphonates did not show such activity. Subcutaneously administered (10-50 mg/kg) minodronic acid significantly inhibited the alpha,beta-methylene ATP-, acetic acid- and formalin-induced nociceptive behaviors in mice. These unique effects of minodronic acid would be beneficial for the treatment of accelerated bone turnover diseases accompanied by bone pain, including bone metastases.

Systemic administration of 5-HT2C receptor agonists attenuates muscular hyperalgesia in reserpine-induced myalgia model

Fibromyalgia is a prevalent musculoskeletal disorder characterized by chronic widespread pain that significantly reduces quality of life in patients. Due to the lack of consistently effective treatment, the development of improved therapies for treating fibromyalgia is necessary. As dysfunction of serotonergic analgesic control appears to be involved in the pathophysiology of fibromyalgia, the present study explored the potential of 5-HT(2C) receptor agonists as novel therapies for treating this disease. Three 5-HT(2C) receptor agonists (lorcaserin, vabicaserin and YM348) that have been suggested to be useful in the treatment of several central nervous system diseases, including obesity and schizophrenia, were used. The effect of systemic administration of these agents on the muscular hyperalgesia that develops in the reserpine-induced myalgia (RIM) rat, a putative animal model of fibromyalgia, was investigated. RIM rats exhibited decreased muscle pressure thresholds. Microdialysis experiments showed that the concentration of serotonin (5-HT) in the spinal cord of RIM rats was significantly lower than that of controls. Lorcaserin (0.3-3 mg/kg p.o.), vabicaserin (0.3-3 mg/kg s.c.) and YM348 (0.03-0.3 mg/kg p.o.) recovered the muscle pressure threshold. The effect of lorcaserin was reversed by the pretreatment with SB242084, a 5-HT(2C) receptor antagonist. Our findings demonstrate that 5-HT(2C) receptors play a critical role in muscular hyperalgesia in RIM rats and suggest that 5-HT(2C) receptor agonists have therapeutic potential for treating chronic pain in patients with fibromyalgia although clinical extrapolation remains to be a future challenge.

Spontaneous and evoked pain-associated behaviors in a rat model of neuropathic pain respond differently to drugs with different mechanisms of action

Given that patients with neuropathic pain suffer a mixture of spontaneous and evoked pain symptoms, we assessed the effects of drugs with different mechanism of action on spontaneous and evoked pain-associated behaviors in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Frequent aberrant limb movement on the operated side was measured to assess spontaneous pain-associated behavior, and mechanical allodynia and thermal hyperalgesia were evaluated to assess evoked pain-associated behaviors. These three types of behavior were assessed after administration of the following drugs: pregabalin (α2δ-subunit ligand), morphine (μ-opioid receptor agonist), perampanel (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA] receptor antagonist), clonidine, dexmedetomidine (α2-adrenoceptor agonists), and diclofenac (non-steroidal anti-inflammatory drug [NSAID]). Pregabalin at an oral dose of 10 or 30mg/kg significantly alleviated frequent aberrant limb movement and mechanical allodynia, but not thermal hyperalgesia. Morphine at a subcutaneous dose of 1 or 3mg/kg significantly improved all three types of behavior. Perampanel at an oral dose of 1mg/kg attenuated only frequent aberrant limb movement. Intraperitoneal administration of clonidine (0.01 or 0.03mg/kg) and dexmedetomidine (0.03mg/kg) significantly improved all three types of behavior, while diclofenac did not relieve any of the behaviors. Pregabalin, clonidine, and dexmedetomidine significantly decreased motor performance at doses close to analgesic doses in the rotarod test. The present study demonstrates that responses to spontaneous and evoked pain symptoms in neuropathic pain condition differ depending on a drugs mechanism of action. The selection and application of drugs according to the specific symptoms would be considered for the medication of patients with neuropathic pain.

Analgesic effects of novel lysophosphatidic acid receptor 5 antagonist AS2717638 in rodents

&NA; Lysophosphatidic acid (LPA) is a bioactive lipid that acts via at least six G protein‐coupled receptors, LPA receptors 1–6 (LPA1‐6), for various physiological functions. We examined (1) whether LPA5 is involved in pain signaling in the spinal cord; and (2) the pharmacological effects of a novel LPA5 antagonist on intrathecal prostaglandin (PG)‐ and (S)‐&agr;‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐induced allodynia, and neuropathic and inflammatory pain in rodents. Intrathecal injection of a selective LPA5 agonist, geranylgeranyl diphosphate, and a non‐selective agonist, LPA, induced allodynia in wild type, but not in LPA5 knockout mice. These novel results suggest that LPA5 is important for pain signal transmission in the spinal cord. AS2717638 (6,7‐dimethoxy‐2‐(5‐methyl‐1,2‐benzoxazol‐3‐yl)‐4‐(piperidin‐1‐ylcarbonyl)isoquinolin‐1(2H)‐one) bound to the LPA‐binding site on LPA5 and selectively inhibited LPA‐induced cyclic adenosine monophosphate accumulation in human LPA5‐but not LPA1‐, 2‐, or 3‐expressing cells. Further, oral administration of AS2717638 inhibited LPA5 agonist‐induced allodynia in mice. AS2717638 also significantly improved PGE2‐, PGF2&agr;‐, and AMPA‐induced allodynia, while both pregabalin and duloxetine alleviated only PGE2‐induced allodynia in mice. Similarly, AS2717638 significantly ameliorated static mechanical allodynia and thermal hyperalgesia in rat models of chronic constriction injury (CCI)‐induced neuropathic pain. AS2717638 also showed analgesic effects in a rat model of inflammatory pain. These findings suggest that LPA5 antagonists elicit broad analgesic effects against both neuropathic and inflammatory pain. Accordingly, pharmacological LPA5 antagonists are attractive development candidates for potential novel pain therapies. HighlightsLysophosphatidic acid receptor 5 (LPA5) is involved in spinal pain signaling.A novel LPA5 antagonist shows broad analgesic effects in multiple animal pain models.Pharmacological antagonism of LPA5 is an attractive novel pain therapy.

Assessment of canine sensory function by using sine-wave electrical stimuli paradigm

The paradigm of sine-wave electrical stimuli has been used for sensory neurological assessment in humans. In the present study, we applied the paradigm to the dog for the quantitative assessment of sensory function. Sine-wave electrical current stimuli at frequencies of 2000, 250, and 5Hz were delivered to bipolar electrodes attached to the skin surface of the hind paws. The stimulation intensity was gradually increased, and the minimum intensity required to elicit the lifting behavior in the stimulated paw was determined as current threshold (CT) for each of the three frequencies. Dogs consistently showed the lifting behavior at CTs without showing aversive behaviors such as vocalization and wriggling. The baseline CTs (mean+/-SEM, n=12) were 4430+/-110microA for CT2000, 2215+/-173microA for CT250, and 2305+/-152microA for CT5. The CTs immediately increased after bolus intravenous injection of fentanyl at 10microg/kg, although the significant increase disappeared within 1h. The time course for the CTs was parallel to that of plasma fentanyl concentration. In conclusion, the present study applied the paradigm of transcutaneous sine-wave electrical stimuli to the dog, and used the hind paw lifting as endpoint behavior. This paradigm is simple, non-invasive, useful in the assessment of sensory function, and can be adapted to investigate the pharmacokinetics/pharmacodynamics relation of drugs. Further studies are needed to give the conclusive interpretation of the endpoint behavior.

In vitro and in vivo pharmacological characterization of ASP8477: A novel highly selective fatty acid amide hydrolase inhibitor

Abstract Although exogenous agonists for cannabinoid (CB) receptors are clinically effective for treating chronic pain, global activation of brain CB receptors causes frequent central nervous system (CNS) side‐effects. Fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme for anandamide (AEA), an endogenous CB. Recently, we discovered a novel FAAH inhibitor, 3‐pyridyl 4‐(phenylcarbamoyl)piperidine‐1‐carboxylate (ASP8477). In vitro studies demonstrated that ASP8477 inhibited human FAAH‐1, FAAH‐1 (P129T) and FAAH‐2 activity with IC50 values of 3.99, 1.65 and 57.3 nM, respectively. ASP8477 at 10 &mgr;M had no appreciable interactions with 65 different kinds of receptors, ion channels, transporters and enzymes, including CB1 and CB2 receptors and monoacylglycerol lipase. In adolescent rats, orally administered ASP8477 (0.3–10 mg/kg) elevated AEA concentrations in both plasma and brain. In a capsaicin‐induced secondary hyperalgesia model, a pretreatment with ASP8477 significantly improved mechanical allodynia and thermal hyperalgesia at 0.3–3 mg/kg p.o. ASP8477 also significantly improved mechanical allodynia in an L5/L6 spinal nerve ligation neuropathic pain model, with an ED50 value of 0.63 mg/kg, and in a streptozotocin‐induced diabetic neuropathy model at 3 and 10 mg/kg p.o. Furthermore, ASP8477 significantly attenuated the reduction in rearing events at 1 and 3 mg/kg p.o. in a monoiodoacetic acid‐induced osteoarthritis model. Importantly, ASP8477 had no significant effect on motor coordination up to 30 mg/kg p.o. These results indicate that ASP8477 is a potent, selective, and oral active FAAH inhibitor with activity in the CNS, with the potential to be a new analgesic agent with a wide safety margin.

211 ESTABLISHMENT OF L5/L6 SPINAL NERVE LIGATION MODEL OF NEUROPATHIC PAIN IN MICE AND PHARMACOLOGICAL CHARACTERIZATION

of protein gene product 9.5 (PGP 9.5), a marker of nerve tissue, was utilized to detect the nerve growth in scar tissue under electron microscopy. Results: Results illustrate that the whirl body and the detachment of plasmalemma could be found in Schwann cells of large myelinated fibers. Neurotubule disruption and neurofilament disorganization also developed in small myelinated or unmyelinated fibers. In addition, numerous nerve fibers labeled by PGP 9.5 were observed in scar tissue on light or electron microscopy after 3 months lumbar laminectomy. These small fibers frequently accompany vessels and spread from periphery to central connective tissue of scar. Conclusions: We conclude that the dysmyelination and axonopathy in roots, and the nerve ingrowth in scar tissue could induce neuropathic pain, which might be the main causes of pain syndrome after spine operation.