Kazushi Sakurai

Hydroxyl radical scavenging by rebamipide and related compounds: Electron paramagnetic resonance study

We studied the scavenging activity of rebamipide, a novel antipeptic ulcer agent, and seven related compounds against hydroxyl radicals using the electron paramagnetic resonance (EPR) spin trapping technique. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trapping agent. We estimated the second order rate constant for the reaction between the agents tested and hydroxyl radical at pH 7.8 by kinetic competition studies. All compounds tested scavenged the hydroxyl radicals with a certain relationship between concentration and scavenging efficacy. A structure-scavenging activity relationship was derived from the kinetic evidence available on the formation and inhibition of the DMPO spin adduct EPR signal of hydroxyl radicals (DMPO-OH). Important determinants for scavenging hydroxyl radicals were the 3,4-double bond of the quinolinone ring in conjunction with a 2-oxo function and the carbonyl portion of the amido group in conjunction with a para-chlorobenzoyl function.

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against •OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H2O2), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a •OH-trapping agent after UVB exposure (305 nm) to H2O2 for 1 min in the presence of rebamipide. The signal intensity of •OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 1010, 8.16 × 109 and 1.65 × 1010 M-1 s-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the •OH generated by UVB/H2O2. Specific formation of this product explained the molecular characteristics of rebamipide as a potential •OH scavenger.

Rebamipide Reduces Recurrence of Experimental Gastric Ulcers: Role of Free Radicals and Neutrophils

Mucosal inflammation is a crucial factor for the recurrence of peptic ulcer. In this study, we examined the effect of rebamipide on neutrophils infiltration, lipid peroxidation, and antioxidative enzyme activities in the recurrence of experimental gastric ulcer. Ulcer recurrence was examined at 60, 100, and 140 days after production of acetic acid-induced gastric ulcers in rats. Gastric neutrophil infiltration, lipid peroxidation, and antioxidative enzyme activities were determined by analyses of myeloperoxidase (MPO) activity, thiobarbituric acid reactive substance (TBARS) levels, and glutathione peroxidase (GSHpx) and superoxide dismutase (SOD) activities in the ulcer region, respectively. The effect of rebamipide, an antigastric-ulcer agent, on ulcer recurrence was assessed following oral administration at 60 mg/kg/day from day 20. In the control and rebamipide groups, gastric ulcer indices were reduced on day 100 compared with day 60; however, increases were observed on day 140, indicating ulcer recurrence. In the rebamipide group, the ulcer index was smaller than in the control group at each time point and the effect was significant on day 140. Although marked elevation of MPO activities was observed in the control group during the experiment, no significant elevations were seen in the rebamipide group on days 100 and 140. TBARS levels were significantly elevated in the control group on day 140, but not in the rebamipide group. Rebamipide suppressed the decrease in GSHpx activity on day 60. These results suggest that lipid peroxidation of gastric tissue mediated by free radicals from neutrophils is responsible for the recurrence of acetic acid-induced gastric ulcers in rats, and that the elimination of free radicals by rebamipide may contribute to the reduction of severity in ulcer recurrence.

The first observation of O2− generation at real time in vivo from non-Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication

Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2 − which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2 − in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol‐induced O2 − generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2 − generation was completely abolished by GdCl3, Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non‐Kupffer cell population, probably liver sinusoid endothelial cell to produce O2 −

Establishment of an X-ray irradiation-induced glossitis model in rats: biphasic elevation of proinflammatory cytokines and chemokines

Oral mucositis is a frequent and serious side effect in patients who receive radiotherapy for head and neck cancer. The purpose of this study was to develop a noninvasive and quantitative model of oral mucositis in rats, investigate the pathophysiology, and evaluate the efficacy of pharmacological interventions. Rats received a single dose of 15 Gy of X-rays to the snout after shielding of the remainder of the rat body with lead plates to protect the body from irradiation (day 0). After irradiation, the macroscopic area of tongue injury gradually increased. The total area of injury and the ulcer-like area reached a maximum on day 7 and then gradually decreased until disappearance on day 28. Expression of proinflammatory cytokines and chemokines occurred transiently within 1–4 hours after irradiation and returned to a normal level at 24 hours. This expression was again observed from days 3 to 5 and increased significantly on day 7, which approximately coincided with the histologic severity of tissue damage. Subcutaneous administration of palifermin at 3 mg/kg per day for 3 consecutive days before irradiation completely prevented ulcer formation in this model. In conclusion, we established a novel model of glossitis in rats, induced by X-ray irradiation, in which biphasic elevations of expression of proinflammatory cytokines and chemokines could be monitored. This model is considered useful to investigate the pathophysiology of oral mucositis and evaluate the preventive effect of pharmacological interventions on oral mucositis induced by X-ray irradiation.

Effects of OPC-6535 on lipopolysaccharide-induced acute liver injury in the rat: Involvement of superoxide and tumor necrosis factor-α from hepatic macrophages

BACKGROUND The objective of this study was to investigate the effects of OPC-6535 on Propionibacterium acnes-primed and lipopolysaccharide-induced liver injury in the rat. METHODS P. acnes was administered intravenously to the rat at 16 mg/kg 7 days before the experiments. In liver perfusion experiments, lipopolysaccharide was mixed in perfusion buffer at 2.5 microg/mL. The chemiluminescence method and histochemical reduction of nitro blue tetrazolium were used for detecting superoxide. Release of cytokines into the perfusate was examined. In in vivo experiments, lipopolysaccharide was administered intravenously to the rat at 200 microg/kg. Concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cytokines were determined in the plasma, and myeloperoxidase activity was measured in the liver tissue. OPC-6535 was given intravenously at 1 mg/kg 30 minutes before lipopolysaccharide challenge, and was then, in perfusion experiments, added to the buffer at 10 micromol/L. RESULTS In perfusion experiments, P. acnes and lipopolysaccharide caused dramatic production of superoxide, tumor necrosis factor-alpha (TNF-alpha) and growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1). Superoxide was mainly from hepatic macrophages. Treatment with OPC-6535 suppressed superoxide and TNF-alpha but did not affect GRO/CINC-1. In in vivo experiments, P. acnes and lipopolysaccharide increased the level of TNF-alpha, GRO/CINC-1, AST and ALT in the plasma, and myeloperoxidase activity in the liver. OPC-6535 reduced TNF-alpha, AST, and ALT, but did not affect GRO/CINC-1 or myeloperoxidase. CONCLUSION Attenuation of liver injury by OPC-6535 is believed to be due to its inhibitory effects on superoxide and TNF-alpha production by hepatic macrophages in P. acnes- and lipopolysaccharide-treated rats.

Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model

The effect of rebamipide, a mucosal protective drug, on small intestinal mucosal injury caused by indomethacin was examined using a rat model. Indomethacin administration (10 mg/kg, p.o.) induced intestinal mucosal injury was accompanied by an increase in the numbers of intestinal bacteria particularly Enterobacteriaceae in the jejunum and ileum. Rebamipide (30 and 100 mg/kg, p.o., given 5 times) was shown to inhibit the indomethacin-induced small intestinal mucosal injury and decreased the number of Enterococcaceae and Enterobacteriaceae in the jejunal mucosa to normal levels. It was also shown that the detection rate of segmented filamentous bacteria was increased by rebamipide. PCR array analysis of genes related to inflammation, oxidative stress and wound healing showed that indomethacin induced upregulation and downregulation of 14 and 3 genes, respectively in the rat jejunal mucosa by more than 5-fold compared to that of normal rats. Rebamipide suppressed the upregulated gene expression of TNFα and Duox2 in a dose-dependent manner. In conclusion, our study confirmed that disturbance of intestinal microbiota plays a crucial role in indomethacin-induced small intestinal mucosal injury, and suggests that rebamipide could be used as prophylaxis against non-steroidal anti-inflammatory drugs -induced gastrointestinal mucosal injury, by modulating microbiota and suppressing mucosal inflammation in the small intestine.

Role of lipid peroxidation in protection of rats by rebamipide against gastric mucosal lesions induced by stress plus indomethacin

Abstract The roles of lipid peroxidation and prostaglandins (PGs) in the protective effect of rebamipide against gastric lesions in rats induced by stress plus indomethacin were investigated. Lesions of the gastric mucosa were induced within 3 h by restraint in water (23°C) plus intraperitoneal injection of indomethacin (20 mg/kg) but not by either of these treatments alone. Lipid peroxidation, measured as TBA reactive substances, was increased by stress + indomethacin treatment and the increase in TBA reactive substances was closely related to the lesion score ( P 2 from the gastric mucosa, the reduction being related with the lesion score ( P 2 , did not inhibit the induction of gastric lesions by stress + indomethacin. Rebamipide injected subcutaneously dose-dependently inhibited the induction of lesions at doses of 3–30 mg/kg. At a dose of 30 mg/kg, it inhibited the increase of TBA reactants in the gastric mucosa, but did not completely restore PGE 2 release. In the rebamipide-treated group, a significant correlation ( P

Rebamipide protects against glaucoma eyedrop-induced ocular surface disorders in rabbits

Purpose This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops. Methods Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy. Results There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group. Conclusions Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface.

Novel mouse model of steroid-resistant allergic rhinitis by repeated intranasal administration of OVA

Results In the One-week-challenge arm ,t he sRAW in MF and DEX group were lower than VC group both in the early (inhibition rates: MF 73%, DEX 94%) and the late phase responses (MF 76%, DEX 84% inhibition). In the Fiveweeks-challenge arm ,M F and DEX reduced the nasal congestion in the early phase (MF 42%, DEX 58% inhibition), however, the inhibition rate of the late phase was significantly decreased in the MF group (MF 28%, DEX 50% inhibition). The epithelial hyperplasia and the inflammatory cells infiltration were observed in the nasal mucosa, and these grades were more remarkable for Five-week-challenge arm than One-week-challenge arm. Conclusions After five weeks OVA challenge, the nasal congestion remained partially even after the nasal corticosteroid treatment. This repeated OVA administration method would provide a quite useful novel disease model to elucidate the mechanisms of nasal steroid-resistant-AR and develop a new remedy.