Masafumi Shibamori

Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

Background: IL-6 signaling is a key component of inflammatory diseases. Results: Modified DNA aptamers that inhibit IL-6 signaling were discovered and optimized. Conclusion: Modified aptamers are stable in serum and block the interaction of IL-6 with its receptor IL-6Rα. Significance: Modified aptamers are a new class of antagonist with properties potentially suitable for clinical treatment of inflammation. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2′-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

DNA binding - dependent glucocorticoid receptor activity promotes adipogenesis via krüppel-like factor 15 gene expression

Glucocorticoids, such as dexamethasone, have been used as in vitro inducers of adipogenesis. However, the roles of the glucocorticoid receptor (GR) in adipogenesis have not been well characterized yet. Here, we show that inhibition of GR activity using the GR antagonist RU486 prevents human mesenchymal stem cell and mouse embryonic fibroblast (MEF) differentiation into adipocytes. Moreover, in MEFs isolated from GR knockout (GRnull) and GRdim mice deficient in GR DNA-binding activity, adipogenesis was blocked. We identified glucocorticoid response element sites in the first intron of KLF15 by bioinformatical promoter analysis and confirmed their functional relevance by demonstrating GR interaction by chromatin immunoprecipitation. Moreover, transfection of MEFs with siRNA for KLF15 significantly attenuated the expressions of adipogenic-marker genes and the lipid accumulation. Our results provide a new mechanism for understanding glucocorticoids-dependent adipogenesis and that GR promotes adipogenesis via KLF15 gene expression as a transcriptional direct target.

Direct inhibition of arginase attenuated airway allergic reactions and inflammation in a Dermatophagoides farinae-induced NC/Nga mouse model

The expression of arginase I has been a focus of research into the pathogenesis of experimental asthma, because arginase deprives nitric oxide synthase (NOS) of arginine and therefore participates in the attenuation of bronchodilators such as nitric oxide (NO). The present study used an intranasal mite-induced NC/Nga mouse model of asthma to investigate the contribution of arginase to the asthma pathogenesis, using an arginase inhibitor, N(omega)-hydroxy-nor-l-arginine (nor-NOHA). The treatment with nor-NOHA inhibited the increase in airway hyperresponsiveness (AHR) and the number of eosinophils in bronchoalveolar lavage fluid. NOx levels in the lung were elevated despite suppressed NOS2 mRNA expression. Accompanied by the attenuated activity of arginase, the expression of arginase I at both the mRNA and protein level was downregulated. The levels of mRNA for T helper 2 cytokines such as IL-4, IL-5, and IL-13, and for chemotactants such as eotaxin-1 and eotaxin-2, were reduced. Moreover, the accumulation of inflammatory cells and the ratio of goblet cells in the bronchiole were decreased. The study concluded that the depletion of NO caused by arginase contributes to AHR and inflammation, and direct administration of an arginase inhibitor to the airway may be beneficial and could be of use in treating asthma due to its anti-inflammatory and airway-relaxing effects, although it is not clear whether the anti-inflammatory effect is direct or indirect.

Effects of Intranasal Administration of Recombinant Murine Interferon-γ on Murine Acute Myocarditis Caused by Encephalomyocarditis Virus

BACKGROUND Viral myocarditis has been strongly implicated in the pathogenesis of dilated cardiomyopathy as well as acute myocarditis. Among the antiviral therapies, interferons (IFNs) have been widely studied and become very important in clinical practice. METHODS AND RESULTS To investigate the possibilities of IFN therapy in viral myocarditis, we analyzed the effects of recombinant murine interferon (mIFN)-gamma and natural mIFN-alpha/beta by the intranasal and intramuscular routes on the development of acute murine myocarditis caused by encephalomyocarditis virus. Both mIFN-gamma and mIFN-alpha/beta treatment by either route significantly increased the survival rate; none of the mIFN-gamma-treated mice died. The effect of mIFN-gamma was significantly greater than that of mIFN-alpha/beta. Furthermore, intranasal administration of mIFN-gamma significantly suppressed virus replication and inflammation in the heart. CONCLUSIONS Our data demonstrate that IFN therapy, especially intranasal administration of IFN-gamma, dramatically improved the prognosis of acute murine viral myocarditis by suppressing virus replication and raises the possibility of antiviral therapy with IFN-gamma in patients with acute myocarditis.

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.

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.

Abstract 4430: Protective effects of rebamipide liquid on radiation-induced glositis in rats.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC [Background & Aims] Rebamipide is widely used for mucosal protection, healing of gastric ulcers and treatment of gastritis. Recently, it was reported that Rebamipide gargle inhibited oral mucositis induced by chemoradiotherapy in head and neck cancer patients. In this study, we examined protective effects of Rebamipide Liquid on radiation-induced glossitis rat model and analyzed the expression level of pro-inflammatory cytokines and chemokines in the injury area of tongue. [Materials & Methods] Rebamipide Liquid, comprising with Rebamipide crystals less than 500 nm particles, was prepared by a neutralizing crystallization technique. This formulation is a stable homogenous aqueous suspension provided with appropriate viscosity by adding viscosity enhanced agents to improve the retention in the oral cavity. The glossitis was induced by X-ray with 15Gy irradiation only around the snout (Day 0) in rats. Rebamipide Liquid was administered intraorally at doses of 5, 10 or 20 mg/kg (1, 2 or 4%, respectively), 6 times a day for 14 days from Day -7 to Day 6. These tongue tissue specimens were obtained at Day 7 and their images were recorded by a digital camera for efficacy evaluations. Gene expression analysis was performed with quantitative real-time PCR (ABI) and protein level was evaluated with Bio-Plex system(BioRad) or ELISA in a different study which was conducted at the two doses of 0 and 20 mg/kg (0 and 4%). [Result & Conclusions] The ulcer-like areas (10.8 ± 1.2, 7.9 ± 1.1 and 7.0 ± 0.7%) at doses of 5 - 20 mg/kg (1 - 4%) of Rebamipide Liquid administration, were statistically smaller than the vehicle control at 14.7±1.6%. Another study revealed that the expression of pro-inflammatory cytokines (TNF-α, IL-6 and Il-1β) and chemokines (MCP-1 and Gro-α) were dramatically elevated in the irradiated tongue at Day7, as compared to normal. But these elevated expressions were significantly suppressed by the 4% Rebamipide treatment. These immuoassays elucidated that the production of these cytokines and chemokines were significantly suppressed by the Rebamipide administration. These results demonstrated that the Rebamipide Liquid has a potent pharmacological action for the radiation-induced oral mucositis mediated by the suppression of pro-inflammatory cytokines (TNF-α, IL-6 and Il-1β) and chemokines (MCP-1 and Gro-α). Citation Format: Takako Nakashima, Naoya Uematsu, Masafumi Shibamori, Kazushi Sakurai, Masayuki Sato, Takakuni Matsuda, Nobutomo Sako. Protective effects of rebamipide liquid on radiation-induced glositis in rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4430. doi:10.1158/1538-7445.AM2013-4430