Yukinori Nagakura

Biogenic amine depletion causes chronic muscular pain and tactile allodynia accompanied by depression: A putative animal model of fibromyalgia.

ABSTRACT Fibromyalgia is a prevalent and burdensome disorder characterized by chronic widespread pain and complex comorbid symptoms. To develop better treatments for pain‐centered fibromyalgia symptoms, there is still a need for animal models which mimic the features of fibromyalgia patients. In the present study, we have established a fibromyalgia animal model by utilizing a never‐before‐published pharmacological effect of reserpine. Repeated administration of reserpine (1 mg/kg s.c., once daily, for three consecutive days) causes a significant decrease in the muscle pressure threshold and tactile allodynia, which are sustained for 1 week or more in both male and female rats. This treatment regimen decreases the amount of biogenic amines (dopamine, norepinephrine, and 5‐hydroxytryptamine) in the spinal cord, thalamus, and prefrontal cortex, which are deeply involved in pain signal processing. It also significantly increases immobility time in the forced swim test, which is indicative of depression, a common comorbid symptom of fibromyalgia. Pregabalin, duloxetine, and pramipexole significantly attenuated the reserpine‐induced decrease in muscle pressure threshold, but diclofenac did not. The validity of the use of this reserpinized animal as a fibromyalgia model is demonstrated from three different aspects, i.e., face validity (manifestation of chronic pain and comorbid symptoms), construct validity (dysfunction of biogenic amine‐mediated central nervous system pain control is involved), and predictive validity (similar responses to treatments used in fibromyalgia patients). This animal model is expected to contribute to the better understanding of fibromyalgia pathophysiology and the evaluation of drugs, especially those which would activate biogenic amine system.

Compounds possessing 5-HT3 receptor antagonistic activity inhibit intestinal propulsion in mice

The role of 5-HT3 receptors in the control of intestinal propulsive activity was investigated in mice by a simple method in which the time taken for excretion of the head of an orally administered non-absorbable marker (whole gut transit time) was measured. Selective 5-HT3 receptor antagonists ramosetron (YM060) at 0.01-0.3 mg/kg s. c. and ondansetron at 0.1-1 mg/kg s.c. dose-dependently prolonged the whole gut transit time. Prokinetic benzamides, such as renzapride (0.3-10 mg/kg s.c.), zacopride (0.01-0.3 mg/kg s.c.) and cisapride (0.1-3 mg/kg s.c.), which have been reported to possess 5-HT3 receptor blocking properties, also dose-dependently prolonged it. These results indicate that activation of 5-HT3 receptors seems to be one factor that underlies the physiological control of intestinal propulsive activity in mice. In contrast to their beneficial therapeutic effects on gastroduodenal dysmotility, prokinetic benzamides, at least those which have 5-HT3 receptor antagonistic activity, may be unsuitable in the treatment of impaired lower intestinal propulsive activity.

Mechanisms of cisplatin- and m-chlorophenylbiguinide-induced emesis in ferrets

We investigated the involvement of peripheral and central serotonin (5-HT)3 receptors in cisplatin- and 5-HT3 receptor agonist-induced emesis in ferrets. Cisplatin (10 mg/kg i.v.)-induced emesis was inhibited by intravenous YM060 (0.003-0.1 microgram/kg). A highly selective and potent 5-HT3 receptor agonist, m-chlorophenylbiguanide (1-10 mg/kg i.p.), dose dependently elicited emesis an effect which was inhibited by YM060 (0.003-0.3 microgram/kg i.v.). Vagotomy markedly reduced this emesis, and the combination of abdominal vagotomy and greater splanchnicectomy abolished emesis. Lesion of greater splanchnic nerves alone did not markedly inhibit emesis. Intracerebroventricularly (4th ventricle) administered YM060 inhibited cisplatin- and m-chlorophenylbiguanide-induced emesis only at higher doses (0.01-0.1 and 0.01-0.03 microgram, respectively). Intracerebroventricularly (4th ventricle) administered m-chlorophenylbiguanide (30-100 micrograms) produced only a weak retching response. These results indicate that stimulation of abdominal vagal afferent nerves via peripheral 5-HT3 receptors is important for triggering cisplatin- and m-chlorophenylbiguanide-induced emesis in ferrets.

Characterization of 5-hydroxytryptamine (5-HT) receptor subtypes influencing colonic motility in conscious dogs

We examined the effects of exogenous 5-hydroxytryptamine (5-HT) and selective 5-HT receptor agonists and antagonists on proximal, middle and distal colonic motility in conscious fasted dogs with extraluminal force transducers implanted chronically. 5-HT (0.003–0.1 mg/kg i.v.) dose-dependently enhanced motility along the entire length of the colon. The 5-HT (0.03 mg/kg i.v.)-induced response was inhibited by 0.1–1.0 mg/kg i.v. methysergide, a 5-HT/12 antagonist, at all recording sites and by 0.1–1.0 mg/kg i.v. ketanserin, a 5-HT2A antagonist, at the middle and distal sites only. At 1 mg/kg i.v., YM060, a 5-HT3 antagonist, reduced the amplitude of the initial transient high-amplitude contractions induced by 5-HT, but did not affect the tonic contraction induced by 5-HT. At doses up to 3 mg/kg i.v.,2-methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino) ethyl ester (SDZ205-557), a 5-HT4 antagonist, and hexamethonium (up to 10 mg/kg i.v.) did not affect 5-HT-induced responses at any recording site. Renzapride, a 5-HT4 agonist, also stimulated motility along the entire length of the colon at 0.3 mg/kg i.v.. The renzapride-induced response was inhibited by 1 mg/kg i.v. SDZ205-557 or 3 mg/kg i.v. hexamethouium. m-Chlorophenylbiguanide (m-CPBG), a 5-HT3 agonist, (1 mg/kg i.v.) produced a transient high-amplitude contraction at all recording sites and this contraction was eliminated by pretreatment with 0.03 mg/kg i.v. YM060. The contraction produced by m-CPBG declined rapidly, so the increase in the motility index by m-CPBG was not significant at any recording site. Of the antagonists tested, 0.1–1 mg/kg i.v. methysergide produced a delayed and prolonged contractile response at the middle and distal sites. The onset of the response was delayed about 20 min after application and the response was maintained over the subsequent 60-min observation period. The methysergide (1 mg/kg i.v.)-induced response was inhibited by 3 mg/kg i.v. hexamethonium. The other antagonists, ketanserin, YM060 and SDZ205-557, had no contractile effect at any recording site.These results indicate that exogenous 5-HT stimulates motility along the entire length of the fasted canine colon and that 5-HT-induced responses in the proximal colon are mediated mainly by 5-HT1, whereas those in the middle and distal colon are mediated by both 5-HT1 and 5-HT2 receptors. Renzapride and methysergide also stimulate colonic motility via additional mechanisms. The activation of 5-HT4 receptors and the blockade of endogenous 5-HT inhibitory regulation via 5-HT1 receptors may be involved in the action of renzapride and methysergide respectively.

EFFECTS OF GASTROPROKINETIC AGENTS ON GASTROPARESIS IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

Abstract The influence of diabetic hyperglycemia on solid gastric emptying in rats was examined. Diabetes was produced by streptozotocin (STZ, 40mg/kg i.v.), and diabetic hyperglycemia was observed from 1 day after the STZ injection. The gastric emptying of glass beads in the diabetic rats was significantly delayed compared with that in age-matched control rats at 1, 3 and 7 days after diabetes induction. A slight decrease in gastric emptying was observed in the diabetic rats from 2 to 52 weeks after the diabetes induction. We also investigated the influence of gastroprokinetic agents on STZ-induced diabetic gastroparesis and subdiaphragmatic vagotomy-induced gastroparesis in rats. The selective 5-HT3 receptor antagonists ramosetron (YM060), YM114 (KAE-393), granisetron and ondansetron, and the substituted benzamides (5-HT4 receptor agonist/5-HT3 receptor antagonists) cisapride mosapride and SC-53116 dose-dependently enhanced gastric emptying in normal rats. These compounds also reversed the impairment of diabetic gastroparesis rats at 7 days after the STZ injection, but higher doses were required. The solid gastric emptying in subdiaphragmatic vagotomized rats was also delayed. Ramosetron and the substituted benzamides cisapride and zacopride partially reversed the gastroparesis in the vagotomized rats. These results suggest that acute hyperglycemia is important mechanism for the delay of solid gastric emptying in diabetic rats. It is also suggested that selective 5-HT3 receptor antagonists and substituted benzamides enhance gastric emptying not only in normal rats but also in diabetic and vagotomized rats.

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.

Reserpine causes biphasic nociceptive sensitivity alteration in conjunction with brain biogenic amine tones in rats.

The present study investigated the precise relationship between brain biogenic amine (dopamine, noradrenaline, and serotonin) tones and nociception. Nociceptive sensitivities to multimodal (muscle pressure, tactile, cold, and heat) stimuli were assessed in acute phase (up to 24 h after reserpine or tetrabenazine injection) and chronic phase (on day 2 or later) in rats. A single injection of reserpine (3 mg/kg s.c.) significantly decreased biogenic amines in the spinal cord (SC), thalamus (THA), and prefrontal cortex (PFC) in both acute and chronic phases, but significantly increased a dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the SC and a serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the SC and THA in acute phase. The content of all biogenic amine metabolites was at low level in chronic phase. Animals exhibited hypersensitivities to tactile and heat stimuli and hyposensitivity to muscle pressure stimulus in acute phase. In chronic phase, they manifested hypersensitivities to all modes of stimuli. Tetrabenazine (20 mg/kg i.p.) significantly decreased brain biogenic amines for a short time, although it did not significantly affect the nociceptive sensitivities. In conclusion, a single injection of reserpine causes a biphasic alteration of nociceptive sensitivities, which is in conjunction with the dynamic change of brain biogenic amine tones, in rats. Cold and heat hypersensitivities in addition to mechanical ones are induced by the reserpine treatment. Sustained modification of brain biogenic amine tones would be critical to induce a robust change in nociceptive sensitivities based on the different effects between reserpine and tetrabenazine.

Efficacy of drugs with different mechanisms of action in relieving spontaneous pain at rest and during movement in a rat model of osteoarthritis.

Patients with osteoarthritis (OA) suffer from joint pain aggravated by movement, which affect their quality of life. In the present study, a weight bearing paradigm for pain at rest and a gait paradigm for pain during movement were tested in rats with unilateral knee arthritis induced by an intra-articular injection of sodium monoiodoacetate (MIA). At week 3 after MIA (1mg/knee) injection, animals developed pain-associated, right-left imbalances of weight distribution (weight bearing) or foot print parameters (gait). Diclofenac, at doses up to 30 mg/kg orally (p.o.), did not have a significant effect on either paradigm. Morphine rectified the weight bearing and gait imbalances at 1 and 3mg/kg subcutaneously, respectively. The weak opioid and serotonin/norepinephrine reuptake inhibitor (SNRI) tramadol also significantly corrected the indices at 10mg/kg (weight bearing) and 100mg/kg p.o. (gait). The SNRI duloxetine at 30 mg/kg p.o. corrected the weight bearing imbalance but not gait imbalance. We assessed the effect of different drugs on pain-induced disturbances in weight distribution and gait in MIA-induced arthritic rats. Analgesic drugs, each with different mechanisms of action, were less effective in rectifying the imbalance in gait than that in weight distribution. The assessment of the effect of analgesics on not only rest pain but pain during movement is valuable for the comprehensive examination of their therapeutic efficacies in OA.

Pharmacological profile of YM-31636, a novel 5-HT3 receptor agonist, in vitro.

We investigated the in vitro pharmacological profile of YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate). In cloned human 5-HT3A receptors, YM-31636 had a pKi value of 9.67 vs. ramosetron and pKi values for other 5-HT3 receptor agonists were less than 7. YM-31636 showed very low affinities for other receptors. YM-31636 induced contraction of isolated guinea pig distal colon. The intrinsic activity was approximately 0.90 compared with 5-hydroxytryptamines (5-HT) 1.0, and the potency was 26 times greater than that of 5-HT. YM-31636 increased short-circuit current (Isc) in the isolated guinea pig distal colon. In this case, the relative intrinsic activity was approximately 0.19. In isolated guinea pig right atrium, YM-31636 induced tachycardia with the relative intrinsic activity of approximately 0.23. All these effects of YM-31636 were antagonized by ramosetron, a selective 5-HT3 receptor antagonist. These results suggest that YM-31636 is a potent and selective 5-HT3 receptor agonist, preferentially acting on the contraction of the colon.

Automated measurement of spontaneous pain‐associated limb movement and drug efficacy evaluation in a rat model of neuropathic pain

The withdrawal response elicited by a nociceptive stimulus, i.e., evoked pain measure, is commonly used as an efficacy endpoint in neuropathic pain animal models. It, however, has several limitations, which highlight the importance of examining spontaneous pain. The present study describes an automated method for measuring spontaneous pain behaviour in a rat model of neuropathic pain caused by chronic constriction injury (CCI) of sciatic nerve.