Eldecalcitol Induces Minimodeling-Based Bone Formation and Inhibits Sclerostin Synthesis Preferentially in the Epiphyses Rather than the Metaphyses of the Long Bones in Rats.

This study aimed to examine minimodeling-based bone formation between the epiphyses and metaphyses of the long bones of eldecalcitol (ELD)-administered ovariectomized rats. Sixteen-week-old female rats were divided into four groups: sham-operated rats receiving vehicle (Sham group), ovariectomized (OVX) rats receiving vehicle (Vehicle group), or ELDs (30 or 90 ng/kg BW, respectively; ELD30 and ELD90 groups). ELD administration increased bone volume and trabecular thickness, reducing the number of osteoclasts in both the epiphyses and metaphyses of OVX rats. The Sham and Vehicle groups exhibited mainly remodeling-based bone formation in both regions. The epiphyses of the ELD groups showed a significantly higher frequency of minimodeling-based bone formation than remodeling-based bone formation. In contrast, the metaphyses exhibited significantly more minimodeling-based bone formation in the ELD90 group compared with the ELD30 group. However, there was no significant difference between minimodeling-based bone formation and remodeling-based bone formation in the ELD90 group. While the minimodeling-induced new bone contained few sclerostin-immunoreactive osteocytes, the underlying pre-existing bone harbored many. The percentage of sclerostin-positive osteocytes was significantly reduced in the minimodeling-induced bone in the epiphyses but not in the metaphyses of the ELD groups. Thus, it seems likely that ELD could induce minimodeling-based bone formation in the epiphyses rather than in the metaphyses, and that ELD-driven minimodeling may be associated with the inhibition of sclerostin synthesis.

Prolonged Duration of Erythropoiesis-Stimulating Agents' Action Delays Disease Progression in Anti-Thy 1 Antibody-Induced Chronic Glomerulonephritis Rats.

BACKGROUND: Although erythropoiesis-stimulating agents (ESAs) exert renoprotective effects in renal disease models, it has not been revealed whether the prolonged duration of action of ESAs contributes to their renoprotective effects. OBJECTIVE: We examined whether the prolonged duration of ESAs' action contributes to their renoprotective effects by comparing a divided administration of a short-acting ESA, epoetin beta (EPO), or a single administration of a long-acting ESA, epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), to a single administration of EPO in chronic glomerulonephritis (GN) rats. MATERIALS AND METHODS: Chronic GN was induced by intravenous injection of anti-Thy 1.1 antibody (0.6 mg/kg) into uninephrectomized rats (day 0). Chronic GN rats were intravenously injected once with vehicle (disease control; DC), EPO 5,000 IU/kg (single EPO), or C.E.R.A. 25 µg/kg (single C.E.R.A.) on day 1; or 3 times during the first week with EPO 1,667 IU/kg from day 1 (divided EPO; total 5,000 IU/kg). Hemoglobin (Hb) level and urinary total protein (U-TP) level which are the indexes of hematopoiesis and renoprotective effects, respectively, were measured several times over 8 weeks. RESULTS: Divided EPO and single C.E.R.A. increased Hb levels more greatly than did single EPO. In all chronic GN rats, elevated U-TP levels decreased transiently 2 weeks after chronic GN induction and then flared again. Single EPO significantly suppressed this exacerbation of U-TP levels compared to DC. Divided EPO and single C.E.R.A. each significantly suppressed the exacerbation of U-TP levels compared to single EPO. CONCLUSION: Prolonged duration of ESAs' action contributed significantly to their renoprotective effects.

Sequential Treatment with Eldecalcitol After PTH Improves Bone Mechanical Properties of Lumbar Spine and Femur in Aged Ovariectomized Rats.

Parathyroid hormone (PTH) analogs have a powerful anabolic effect on bone and are used in the treatment of patients with severe osteoporosis. However, there are limitations to how long they can be safely administered. Withdrawal of PTH results in the cancelation of its effects, necessitating subsequent treatment to maintain the bone quantity and quality. This study assessed the effects of Eldecalcitol (ELD), an active vitamin D3 derivative, after PTH in estrogen-deficient osteoporotic rats. Six-month-old female rats were ovariectomized, and PTH administration was started 7 weeks later. After 4 weeks of PTH treatment, the animals were divided into three groups and either continued to receive PTH (PTH-PTH), or were switched to ELD (PTH-ELD) or vehicle (PTH-Veh) for an additional 4 weeks. In the femur, increased BMD by 4 weeks treatment of PTH was significantly reduced in PTH-Veh but not in PTH-PTH and PTH-ELD. The same tendency was observed in the lumbar vertebrae. MicroCT imaging and histomorphometry analysis revealed that the favorable bone structure changes by PTH administration were also maintained in the femurs and tibias of the PTH-PTH and PTH-ELD groups. Increased bone strength by 4-week treatment of PTH in lumber also maintained in PTH-ELD. Furthermore, minimodeling was observed in the PTH-ELD group. These results demonstrate that treatment with ELD sequentially following PTH prevented the bone quantity and strength reduction that accompanies PTH withdrawal in estrogen-deficient rats.

Eldecalcitol, an Active Vitamin D3 Derivative, Prevents Trabecular Bone Loss and Bone Fragility in Type I Diabetic Model Rats.

Diabetes mellitus is known to adversely affect the bones and be associated with increased fracture risk. We examined whether eldecalcitol (ELD), an active vitamin D3 derivative, could inhibit the diabetic bone loss in streptozotocin-induced type I diabetic rats. ELD (10, 20, or 40 ng/kg), alfacalcidol (ALF; 25, 50, or 100 ng/kg), or vehicle was administered 5 times per week for 12 weeks from 1 week after diabetes induction. Normal control rats received the vehicle. Bone turnover markers, bone mineral density (BMD), and biomechanical strength of the lumbar spine and femur were measured, and bone histomorphometry was performed. Content of advanced glycation end products (AGEs) in the femoral shaft was also determined. In diabetic rats, serum osteocalcin (OC) concentration was lower and urinary excretion of deoxypyridinoline (DPD) tended to be higher than in normal rats. Areal BMD and maximum load of the lumbar vertebrae and femoral shaft were lower in diabetic rats than in normal rats. All doses of ELD and the highest dose of ALF reduced urinary DPD excretion, but had no effect on serum OC. The 20 and 40 ng/kg doses of ELD prevented decreases in BMD and the highest dose of ELD prevented the reduction in maximum load of the lumbar vertebrae, while ALF did not change these parameters. ELD and ALF did not affect areal BMD or biomechanical strength of the femoral shaft. In diabetic rats, bone volume and trabecular thickness in the trabecular bone of the lumbar vertebrae decreased and trabecular separation increased compared to normal rats. ELD and ALF prevented diabetes-induced deterioration of trabecular microstructure. AGE content in the femoral cortical bone increased in the diabetic rats, and ELD and ALF did not change AGE content compared to the diabetic rats. These results indicated that ELD suppressed bone resorption and prevented trabecular bone loss and deterioration of trabecular microstructure, resulting in prevention of reduction in biomechanical strength in type I diabetic rats.

Intermittent Ibandronate Maintains Bone Mass, Bone Structure, and Biomechanical Strength of Trabecular and Cortical Bone After Discontinuation of Parathyroid Hormone Treatment in Ovariectomized Rats.

Although parathyroid hormone (PTH) expresses an anabolic effect on bone mass, the increased bone mass disappears once PTH treatment is withdrawn. Therefore, sequential treatment with anti-bone-resorptive agents is required to maintain bone mass after PTH treatment. We examined the effect of sequential treatment with ibandronate (IBN), a nitrogen-containing bisphosphonate, following PTH in ovariectomized (OVX) rats. Wistar-Imamichi rats (27 weeks old) were ovariectomized and treated with PTH (10 µg/kg, s.c.; 5 times/week; PTH group) for 8 weeks from 8 weeks after OVX. Thereafter, PTH was withdrawn and rats were administered IBN (10 µg/kg, s.c.; every 4 weeks; PTH-IBN group) or vehicle (PTH-Veh group) for another 8 weeks. PTH increased bone mineral density (BMD) measured by dual-energy X-ray absorptiometry and biomechanical strength in the lumbar spine and femur as compared to the disease control rats. BMD and biomechanical strength in the PTH-Veh group were lower than in the PTH group, whereas in the PTH-IBN group they were maintained at the level of the PTH group. Microstructure of the trabecular and cortical bone in the PTH-IBN group was not significantly different from that in the PTH group. In histomorphometric analysis of the lumbar vertebra, eroded surface and osteoclast surface in the PTH-Veh group were no different from those in the PTH group, whereas they were lower in the PTH-IBN group. Osteoid surface, osteoblast surface, and mineralize surface decreased in both PTH-IBN and PTH-Veh groups compared to the PTH group, and these parameters in the PTH-IBN group were lower than in the PTH-Veh group. These results indicated that intermittent IBN after PTH treatment suppressed bone turnover and maintained BMD, biomechanical strength, and microstructure in the lumbar spine and femur of OVX rats.

Obesity does not diminish the efficacy of IL-6 signalling blockade in mice with collagen-induced arthritis.

OBJECTIVES: Obese rheumatoid arthritis patients often have higher disease activity and a poorer response to treatment than do non-obese patients. The present study aims to clarify the influence of obesity on the action of IL-6 and to evaluate the efficacy of IL-6 signalling blockade in arthritis with obesity. METHODS: Mice were fed a high-fat diet for 5 weeks, and the influence of this diet on macrophages and type II collagen-induced arthritis was investigated. RESULTS: The mice fed the high-fat diet showed greater expression of macrophage marker F4/80, not only in subcutaneous fat but also in knee synovium and the calcaneal region, than did the mice fed a normal diet. Furthermore, macrophages isolated from mice on the high-fat diet tended to show higher expression of cyclooxygenase-2 following IL-6 stimulation than did macrophages from mice fed the normal diet. Moreover, mice fed the normal or high-fat diet were immunised with type II collagen, and were treated with anti-mouse IL-6 receptor antibody (MR16-1). The anti-arthritis effect of MR16-1 was not reduced in mice fed the high-fat diet compared to mice fed the normal diet (inhibition ratio: 87% vs. 62%). Furthermore, at the peak of arthritis, cyclooxygenase-2 expression in the calcaneal region of mice fed the high-fat diet was higher than that in the mice fed the normal diet. CONCLUSIONS: These results suggested that a high-fat diet induces inflammatory changes in the synovium. We demonstrated that IL-6 signalling blockade by an anti-IL-6 receptor antibody can be effective in treating arthritis, even with obesity.

Nicorandil prevents sirolimus-induced production of reactive oxygen species, endothelial dysfunction, and thrombus formation.

Sirolimus (SRL) is widely used to prevent restenosis after percutaneous coronary intervention. However, its beneficial effect is hampered by complications of thrombosis. Several studies imply that reactive oxygen species (ROS) play a critical role in endothelial dysfunction and thrombus formation. The present study investigated the protective effect of nicorandil (NIC), an anti-angina agent, on SRL-associated thrombosis. In human coronary artery endothelial cells (HCAECs), SRL stimulated ROS production, which was prevented by co-treatment with NIC. The preventive effect of NIC on ROS was abolished by 5-hydroxydecanoate but not by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. NIC also inhibited SRL-induced up-regulation of NADPH oxidase subunit p22(phox) mRNA. Co-treatment with NIC and SRL significantly up-regulated superoxide dismutase 2. NIC treatment significantly improved SRL-induced decrease in viability of HCAECs. The functional relevance of the preventive effects of NIC on SRL-induced ROS production and impairment of endothelial viability was investigated in a mouse model of thrombosis. Pretreatment with NIC inhibited the SRL-induced acceleration of FeCl3-initiated thrombus formation and ROS production in the testicular arteries of mice. In conclusion, NIC prevented SRL-induced thrombus formation, presumably due to the reduction of ROS and to endothelial protection. The therapeutic efficacy of NIC could represent an additional option in the prevention of SRL-related thrombosis.

Nicorandil suppresses urinary protein excretion and activates eNOS in Dahl salt-sensitive hypertensive rats.

BACKGROUND: Hypertension is a risk factor common to both chronic kidney disease and cardiovascular disease. Nicorandil is widely used for the treatment of angina. We investigated the benefits of nicorandil with respect to renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. METHOD: DS rats were fed a high-salt (HS) diet and nicorandil was administered via the drinking water. Blood pressure and renal function were measured for 4 weeks after starting the rats on the HS diet. RESULTS: In rats fed the HS diet, renal dysfunction was manifested by an increase in urinary protein and N-acetyl-ß-D-glucosaminidase excretion. Nicorandil ameliorated renal function with a concomitant reduction in urinary 8-hydroxy-2'-deoxyguanosine and an increase in urinary NOx. Significant upregulation of endothelial nitric oxide synthase (eNOS) expression and an increase in the eNOS dimer/monomer ratio (reduction of eNOS uncoupling) was demonstrated in glomeruli following nicorandil treatment. The blood pressure of DS rats was increased by salt loading; however, no significant change in blood pressure was observed with nicorandil treatment. CONCLUSION: In DS rats fed a HS diet, nicorandil prevented the development of renal dysfunction, which was accompanied by an increase in eNOS expression in the kidneys.

GATA-4 transcription factor regulates cardiac COX-2 expression induced by nicorandil in left ventricle of rats.

BACKGROUND AND AIMS: Cardioprotective effects induced by delayed ischemic preconditioning and by nicorandil are mediated via expression of cardioprotective factors such as COX-2. The present study was undertaken to evaluate whether nicorandil could induce COX-2 in rats and to elucidate its mode of induction pharmacologically. METHODS AND RESULTS: Three hours after administration of nicorandil (10 mg/kg, p.o.), COX-2 mRNA and protein were significantly increased in the left ventricle, although other cardioprotective factors (Bcl-2, eNOS, hexokinase, HSP, and iNOS) were not increased. This COX-2 induction in the left ventricle was preceded by induction of GATA-4, which was significant from 1 h after administration. Ventricular levels of 6-keto-prostaglandin F1α were increased 6 h after administration. Although pinacidil or isosorbide dinitrate alone did not increase COX-2 mRNA, their combined application significantly increased COX-2 mRNA. Moreover, although glibenclamide or ODQ each partly inhibited the induction of COX-2 mRNA by nicorandil, their combined application significantly inhibited it. These results suggest that nicorandil induces COX-2 protein through both the activation of KATP channels and guanylate cyclase. CONCLUSION: The present study demonstrated that nicorandil induces COX-2 via GATA-4 induction in the heart through both KATP channel activation and its nitrate-like properties.

22-Oxacalcitriol prevents progression of endothelial dysfunction through antioxidative effects in rats with type 2 diabetes and early-stage nephropathy.

BACKGROUND: Vitamin D deficiency is associated with endothelial dysfunction in type 2 diabetes patients, but the effectiveness of vitamin D supplementation remains controversial. We assessed whether 22-oxacalcitriol (OCT) could prevent endothelial dysfunction in type 2 diabetes mellitus (DM) rats. METHODS: DM rats with early-stage nephropathy were treated for 10 weeks with OCT (0.2 µg/kg) three times per week or by an implanted insulin pellet. Endothelial dysfunction was assessed by femoral flow-mediated dilation (FMD). RESULTS: Insulin significantly improved FMD as blood glucose levels normalized. OCT also improved FMD without hypercalcemia or hyperphosphatemia and without affecting blood glucose or blood pressure. In femoral arteries, OCT significantly suppressed the elevated expression of p22(phox), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, and improved the endothelial nitric oxide synthase (eNOS) dimer-to-monomer ratio. In cultured endothelial cells, OCT significantly inhibited high-glucose (HG)-induced reactive oxygen species (ROS) production. Simultaneously, OCT significantly suppressed HG-induced p22(phox) expression and improved eNOS uncoupling as was observed in the in vivo study. CONCLUSION: In DM rats, OCT improved endothelial dysfunction, at least in part, by suppressing ROS generation through p22(phox) expression, which might contribute to improving eNOS uncoupling.

Nicorandil ameliorated hypertensive renal injury without lowering blood pressure in spontaneously hypertensive rats.

Proteinuria, a symptom of hypertensive renal injury, is a powerful predictor of mortality in chronic kidney disease patients with hypertension. The present study investigated whether a nonhypotensive dose of nicorandil could decrease hypertensive renal injury in male spontaneously hypertensive rats (SHR). Nicorandil (15 mg/kg/day, for 20 weeks) was administered in the drinking water to rats from 11 weeks old. Heart size, kidney size, and ß(2)-microglobulin occurring with tubular histopathological damage were each significantly greater in SHR than in Wistar-Kyoto (WKY) rats, as was 24-hour excretion of urinary protein (SHR: 33.1 ± 3.5 mg/day, WKY: 5.4 ± 0.3 mg/day). Nicorandil significantly decreased urinary protein (21.7 ± 2.8 mg/day), glomerular cell density, and histopathological score without affecting systolic blood pressure. Nicorandil increased expression of endothelial nitric oxide synthase (eNOS) protein in the renal cortex in SHR without affecting expressions of mRNA for endothelin or genes involved in tissue damage or fibrosis. eNOS expression was negatively correlated with glomerular cell density. In addition, nicorandil increased urinary excretion of NOx, but did not change the eNOS dimer-to-monomer ratio or the decreased level of renal heparan sulfate in SHR. In conclusion, in SHR, long-term administration of nicorandil can ameliorate hypertensive proteinuria, without lowering blood pressure, possibly through an increase in eNOS expression.

Paclitaxel-induced endothelial dysfunction in living rats is prevented by nicorandil via reduction of oxidative stress.

Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel-induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was measured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly reduced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect nitroglycerin-induced vasodilation. Diazoxide and tempol, but not isosorbide dinitrate, had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduction in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47(phox), gp91(phox) mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced nitric oxide (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)-sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxidative stress via K(ATP) channel activation.

Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats.

BACKGROUND: Nicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes. METHODS: Male Sprague-Dawley rats (6 weeks old) were intraperitoneally injected with streptozotocin (STZ, 40 mg/kg, once a day for 3 days) to induce diabetes. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day, superoxide dismutase mimetic) were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs) were treated with high glucose (35.6 mM, 24 h) and reactive oxygen species (ROS) production with or without L-NAME (300 µM), apocynin (100 µM) or nicorandil (100 µM) was measured using fluorescent probes. RESULTS: Endothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 ± 1.4%; normal, 19.5 ± 1.7%; n = 6-7). There was a 2.4-fold increase in p47phox expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 ± 2.6%; tempol, 13.3 ± 1.4%; n = 6). Nicorandil significantly inhibited the increased expressions of p47phox and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil suggesting that eNOS itself might serve as a superoxide source under high-glucose conditions and that nicorandil might prevent ROS production from eNOS. CONCLUSIONS: These results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling.

Interleukin-6: evolving role in the management of neuropathic pain in neuroimmunological disorders.

BACKGROUND: Neuropathic pain in neuroimmunological disorders refers to pain caused by a lesion or disease of the somatosensory system such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MS and NMOSD are autoimmune disorders of the central nervous system, and ≥ 50% of patients with these disorders experience chronic neuropathic pain. The currently available medications for the management of neuropathic pain have limited effectiveness in patients with MS and NMOSD, and there is an unmet medical need to identify novel therapies for the management of chronic neuropathic pain in these patients. In this review article, we summarize the role of interleukin-6 (IL-6) in the pathogenesis of MS and NMOSD and the ameliorative effects of anti-IL-6 therapies in mouse models of experimental autoimmune encephalomyelitis (EAE). MAIN BODY: Intraperitoneal injection of MR16-1, an anti-IL-6 receptor (IL-6R) antibody, reduced mechanical allodynia and spontaneous pain in EAE mice, which was attributed to a reduction in microglial activation and inhibition of the descending pain inhibitory system. The effect of anti-IL-6 therapies in ameliorating neuropathic pain in the clinical setting is controversial; a reduction in pain intensity has been reported with an anti-IL-6 antibody in four studies, namely a case report, a pilot study, a retrospective observational study, and a case series. Pain intensity was evaluated using a numerical rating scale (NRS), with a lower score indicating lesser pain. A reduction in the NRS score was reported in all four studies. However, in two randomized controlled trials of another anti-IL-6R antibody, the change in the visual analog scale pain score was not statistically significantly different when compared with placebo. This was attributed to the low mean pain score at baseline in both the trials and the concomitant use of medications for pain in one of the trials, which may have masked the effects of the anti-IL-6R antibody on neuropathic pain. CONCLUSION: Thus, anti-IL-6 therapies might have a potential to reduce neuropathic pain, but further investigations are warranted to clarify the effect of inhibition of IL-6 signaling on neuropathic pain associated with MS and NMOSD.

Anti-IL-6 Receptor Antibody Inhibits Spontaneous Pain at the Pre-onset of Experimental Autoimmune Encephalomyelitis in Mice.

Chronic pain is a significant symptom in patients with autoimmune encephalomyelitis, such as multiple sclerosis and neuromyelitis optica. The most commonly used animal model of these diseases is experimental autoimmune encephalomyelitis (EAE). We previously reported that evoked pain, such as mechanical allodynia, was improved by an anti-IL-6 receptor antibody in EAE mice. However, few reports have evaluated spontaneous pain in EAE mice. Here, we assessed spontaneous pain in EAE mice by utilizing the Mouse Grimace Scale (MGS, a standardized murine facial expression-based coding system) and evaluated the influence of an anti-IL-6 receptor antibody (MR16-1). EAE was induced in female C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55 emulsified in adjuvant and administration of pertussis toxin. Mice were placed individually in cubicles and filmed for about 10 min. Ten clear head shots per mouse from the video recording were given a score of 0, 1, or 2 for each of three facial action units: orbital tightening, nose bulge, and ear position. Clinical symptoms of EAE were also scored. Measurement of 5-HT in the spinal cord and functional imaging of the periaqueductal gray (PAG) were also performed. Compared with control mice, MGS score was significantly higher in EAE mice. MR16-1 prevented this increase, especially in pre-onset EAE mice. Promotion of spinal 5-HT turnover and reduction of PAG activity were observed in pre-onset EAE mice. These results suggest that MR16-1 prevented spontaneous pain developed before EAE onset.

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits.

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.

Oral mitemcinal (GM-611), an erythromycin-derived prokinetic, accelerates normal and experimentally delayed gastric emptying in conscious dogs.

1. We examined effects of orally administered mitemcinal, an erythromycin-derived motilin agonist, on gastric emptying and antroduodenal motility in conscious normal dogs and conscious dogs with experimentally delayed gastric emptying. For comparison, we also examined the effects of orally administered cisapride. 2. Gastric emptying was assessed by adding paracetamol to the test meal and determining three of its pharmacokinetic parameters as indices of gastric emptying. Antroduodenal motility was assessed from the output of force transducers chronically implanted in the gastric antrum and duodenum. 3. In normal dogs, mitemcinal (0.25, 0.5 and 1 mg/kg) dose-dependently accelerated gastric emptying, significantly increasing all three indices at doses of 0.5 and 1 mg/kg; cisapride (1, 3 and 10 mg/kg) had no significant effect. Mitemcinal also dose-dependently stimulated antroduodenal motility in the interdigestive and digestive states. Cisapride, at 100-fold the dose, produced similar effects in the interdigestive state, but mixed results in the digestive state. 4. In dogs with delayed gastric emptying induced by subcutaneous clonidine (0.03 mg/kg), mitemcinal (0.25, 0.5 and 1 mg/kg) dose-dependently improved delayed gastric emptying, significantly increasing two of three indices at a dose of 1 mg/kg. Cisapride (1, 3 and 10 mg/kg) caused non-significant increases in the indices of gastric emptying, with roughly bell-shaped dose-response curves. The highest dose of mitemcinal (1 mg/kg) also stimulated antroduodenal motility. 5. In dogs with delayed gastric emptying induced by vagotomy, mitemcinal (0.125, 0.25 and 0.5 mg/kg) dose-dependently improved delayed gastric emptying, significantly increasing all three indices at doses of 0.25 and 0.5 mg/kg. Cisapride (3 mg/kg) restored the indices to roughly prevagotomy levels, but none of the increases was significant. Mitemcinal, at a dose of 0.25 mg/kg, also stimulated antroduodenal motility. 6. Because delayed gastric emptying is the basic characteristic of gastroparesis, the fact that mitemcinal accelerated gastric emptying in dogs with normal and delayed gastric emptying much more robustly than cisapride adds to the evidence that mitemcinal is likely to be useful for the treatment of patients with gastroparesis.

Mitemcinal (GM-611), an orally active motilin receptor agonist, improves delayed gastric emptying in a canine model of diabetic gastroparesis.

1. The aim of the present study was to evaluate the effects of mitemcinal (GM-611), an orally active motilin receptor agonist, on delayed gastric emptying in a canine model of diabetic gastroparesis and to compare these effects with those of cisapride. 2. Moderate hyperglycaemia was induced by a single intravenous injection of a mixture of streptozotocin (30 mg/kg) and alloxan (50 mg/kg). Dogs that maintained moderate hyperglycaemia (fasting plasma glucose 200-300 mg/dL) without insulin treatment were selected and gastric emptying in these dogs was determined by the paracetamol method. 3. One year after the onset of diabetes, there was no difference in the gastric emptying of normal and diabetic dogs. However, after 5 years, the diabetic dogs showed delayed gastric emptying. The motor nerve conduction velocity of the tibial nerve was significantly lower in diabetic dogs compared with normal dogs at both time points. 4. Histopathological examination at the end of the study showed that there were fewer nerve fibres in both dorsal vagal and tibial nerves of diabetic dogs compared with normal dogs. The onset of delayed gastric emptying is thought to have occurred gradually, in parallel with abnormal autonomic nerve function induced by the long period of moderate hyperglycaemia. 5. Oral administration of mitemcinal (0.125, 0.25 or 0.5 mg/kg) dose-dependently accelerated delayed gastric emptying, significant at 0.5 mg/kg, in diabetic dogs, whereas cisapride (1, 3 or 10 mg/kg) had no significant effect. These results add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Anti-IL-6 receptor antibody improves pain symptoms in mice with experimental autoimmune encephalomyelitis.

BACKGROUND AND AIMS: Chronic pain is a prevalent symptom in patients with autoimmune encephalomyelitis such as multiple sclerosis and neuromyelitis optica. Although IL-6 is involved in various inflammatory and immune diseases, the roles of IL-6 in autoimmune-related pain have not been clarified. Therefore, we examined the effect of anti-IL-6 receptor antibody (MR16-1) on the pain sensitivity of experimental autoimmune encephalomyelitis (EAE) mice. MATERIALS AND METHODS: EAE was induced in female C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55 emulsified in adjuvant (Day 0). Pertussis toxin was intravenously administered at Days 0 and 2. Mice were sequentially scored for clinical symptoms of EAE. [Exp. 1] MR16-1 was intraperitoneally administered on Days 0 or 3. Sensitivity to pain was measured by the von Frey test (Days 7, 14, 20). The spinal cord was isolated and assessed by immunohistochemistry. [Exp. 2] MR16-1 was intraperitoneally administered on Day 12 when significant pain had already occurred. Pain assessment was conducted before the immunization, on Day 12 and after EAE onset. And then, spinal cord was isolated and flow cytometry was performed. RESULTS: [Exp. 1] MR16-1 prevented the increase in clinical score and sensitivity to pain in EAE mice. Immunohistochemical analysis showed that Iba1+ microglia were increased in the spinal cord of EAE mice, and were reduced by MR16-1. [Exp. 2] Administration of MR16-1 on Day 12 also reduced sensitivity to pain under EAE onset. Flow cytometry showed that CD45lowCD11b+ microglia were increased in the spinal cord of EAE mice, and that this increase was inhibited by MR16-1. CONCLUSION: These findings suggest that MR16-1 can decrease mechanical allodynia in EAE mice through inhibition of microglial activation and proliferation in the spinal cord.

Epoetin beta pegol ameliorates flow-mediated dilation with improving endothelial nitric oxide synthase coupling state in nonobese diabetic rats.

BACKGROUND/AIMS: Patients with diabetic nephropathy have a high cardiovascular mortality. Epoetin beta pegol (continuous erythropoietin receptor activator, C.E.R.A.) is a drug for the treatment of renal anemia. In this study, we investigated the effect of C.E.R.A. on vascular endothelial function as evaluated by flow-mediated dilation (FMD) and the relationship between hematopoiesis and FMD in diabetic nephropathy rats. METHODS: Male Spontaneously Diabetic Torii rats (SDT, 22 weeks old) were used. C.E.R.A. (0.6, 1.2 µg/kg) was administered subcutaneously once every 2 weeks for 8 weeks. At 1 week after last administration (31 weeks old), we assessed FMD in the femoral arteries of anesthetized rats using a high-resolution ultrasound system. FMD was also measured 1 week after single C.E.R.A. treatment (5.0 µg/kg) to examine the influence of hematopoiesis. RESULTS: Flow-mediated dilation was significantly decreased in SDT rats before the start of C.E.R.A. treatment (22 weeks old). Repeated administration of C.E.R.A. dose-dependently improved FMD in SDT rats (31 weeks old) without changing blood glucose, nitroglycerin-induced vasodilation, or kidney function. Long-term administration of C.E.R.A. improved the state of endothelial nitric oxide synthase uncoupling in the femoral arteries of SDT rats, which showed a positive correlation with FMD. On the other hand, there was no correlation between FMD and Hb or Hct in SDT rats. Furthermore, at 1 week after single administration of C.E.R.A., FMD was not significantly improved although hemoglobin levels were comparable with levels following long-term C.E.R.A. CONCLUSION: Long-term treatment with C.E.R.A. improved FMD in SDT rats even after onset of endothelial dysfunction.