MA-5 ameliorates autism-like behavior in mice prenatally exposed to valproic acid.

Indole-3-acetic acid is a common naturally occurring auxin in plants. A synthesized derivative of this compound, 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid also called mitochonic acid 5 (MA-5), has shown to increase the survival ratio of fibroblasts from patients with mitochondrial disease under stress-induced conditions. Further studies verified its efficacy in pathological models, such as an ischemia-reperfusion model, possibly by increasing ATP production. However, the efficacy of MA-5 in mental disorders, such as anxiety, schizophrenia, and autism spectrum disorders (ASD), has not been investigated. Our study focused on examining the effect of MA-5 in a mouse model of ASD induced by prenatal exposure to valproic acid (VPA). VPA exposure significantly deteriorated the level of anxiety and exploratory behavior in an open field test. We fed mice an MA-5-containing diet for 5 weeks and observed an improvement in the above behavior in the MA-5-fed groups. The efficacy of MA-5 was also observed in the elevated plus maze and three-chambered tests. These findings suggest that MA-5 could potentially be used to treat ASD, especially in patients with mitochondrial dysfunction.

Discovery of DS-3801b, a non-macrolide GPR38 agonist with N-methylanilide structure.

Motilin is a 22-amino-acid gastrointestinal (GI) hormone and is involved in the regulation of GI motility through binding to GPR38, the motilin receptor which is expressed on smooth muscle cells in the GI tract. Therefore, GPR38 agonists are expected to be novel gastrointestinal prokinetic agents for the treatment of functional gastrointestinal disorders such as gastroparesis and chronic constipation. We identified a series of N-methylanilide derivatives as novel non-macrolide GPR38 agonists. Among them, 12 di-l-tartrate (DS-3801b) was selected as a clinical candidate for further evaluation.

RS9, a novel Nrf2 activator, attenuates light-induced death of cells of photoreceptor cells and Müller glia cells.

The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure.

The protein disulfide isomerase AGR2 is essential for production of intestinal mucus.

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.

The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model.

Asthma exacerbations can be triggered by viral infections or allergens. The Th2 cytokines IL-13 and IL-4 are produced during allergic responses and cause increases in airway epithelial cell mucus and electrolyte and water secretion into the airway surface liquid (ASL). Since ASL dehydration can cause airway inflammation and obstruction, ion transporters could play a role in pathogenesis of asthma exacerbations. We previously reported that expression of the epithelial cell anion transporter pendrin is markedly increased in response to IL-13. Herein we show that pendrin plays a role in allergic airway disease and in regulation of ASL thickness. Pendrin-deficient mice had less allergen-induced airway hyperreactivity and inflammation than did control mice, although other aspects of the Th2 response were preserved. In cultures of IL-13-stimulated mouse tracheal epithelial cells, pendrin deficiency caused an increase in ASL thickness, suggesting that reductions in allergen-induced hyperreactivity and inflammation in pendrin-deficient mice result from improved ASL hydration. To determine whether pendrin might also play a role in virus-induced exacerbations of asthma, we measured pendrin mRNA expression in human subjects with naturally occurring common colds caused by rhinovirus and found a 4.9-fold increase in mean expression during colds. Studies of cultured human bronchial epithelial cells indicated that this increase could be explained by the combined effects of rhinovirus and IFN-gamma, a Th1 cytokine induced during virus infection. We conclude that pendrin regulates ASL thickness and may be an important contributor to asthma exacerbations induced by viral infections or allergens.

RS-1748, a novel CC chemokine receptor 4 antagonist, inhibits ovalbumin-induced airway inflammation in guinea pigs.

CC chemokine receptor 4 (CCR4) is generally recognized as a preferential marker for T helper 2 cells, and we have previously reported morpholine-derivative CCR4 antagonists, RS-1154 and RS-1269. Here, we investigate the pharmacological profiles of a novel pyrimidine-derivative CCR4 antagonist, 2-{4-[2-(diethylamino)ethoxy]phenyl}-N-(2,4-difluorobenzyl)-5-fluoropyrimidin-4-amine (RS-1748), which showed potency to inhibit the bindings of [(125)I]CCL17 and [(35)S]GTPgammaS to human CCR4-expressing Chinese hamster ovary (CHO) cells with IC(50) values of 59.9 nM and 18.4 nM, respectively. Furthermore, RS-1748 inhibited ovalbumin-induced airway inflammation in guinea pigs at a dose of 10 mg/kg. These results indicate that RS-1748 would be a promising lead compound for developing a therapeutic agent against asthma.

Distinct roles of FOXA2 and FOXA3 in allergic airway disease and asthma.

RATIONALE: Increased production of mucus is a prominent feature of asthma. IL-13-driven mucous cell metaplasia is associated with decreased expression of the transcription factor FOXA2 and increased expression of the related transcription factor FOXA3 in animal and cell culture models. OBJECTIVES: Establish how changes in FOXA2 and FOXA3 expression contribute to mucous metaplasia and determine whether FOXA2 and FOXA3 expression is altered in asthma. METHODS: Mice expressing a Foxa2 transgene in airway epithelial cells and mice deficient in Foxa3 were analyzed after allergen sensitization and challenge. Expression of FOXA2, FOXA3, MUC5AC, and the highly IL-13-inducible gene CLCA1 was analyzed in airway biopsies from subjects with asthma and control subjects. MEASUREMENTS AND MAIN RESULTS: Expression of a Foxa2 transgene reduced allergen-induced mucous metaplasia by 45% compared with control transgenic mice (P < 0.05) whereas inactivation of Foxa3 had no detectable effects on mucous metaplasia. Expression of FOXA2 was reduced in subjects with asthma and was negatively correlated with MUC5AC and CLCA1 levels in subjects with asthma. In contrast, FOXA3 expression was not significantly correlated with MUC5AC and was positively correlated with CLCA1. CONCLUSIONS: Increasing Foxa2 expression reduced mucous metaplasia in an allergic mouse model. Subjects with asthma had decreased FOXA2 expression, suggesting that therapeutic approaches that increase FOXA2 expression or function could be beneficial for reducing mucus production in asthma. Unlike FOXA2, FOXA3 did not regulate mucous metaplasia.

NFE2L2 activator RS9 protects against corneal epithelial cell damage in dry eye models.

Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.

A triterpenoid Nrf2 activator, RS9, promotes LC3-associated phagocytosis of photoreceptor outer segments in a p62-independent manner.

Daily phagocytosis of shed photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is required to sustain the visual function. Recent reports revealed that POS phagocytosis is progressed with LC3-associated manner. Patients with age-related macular degeneration (AMD) had impaired autophagic degradation in the RPE. Nrf2 is a key antioxidant transcriptional regulator that ameliorates oxidative stress which is another contributor to AMD pathogenesis. Nrf2 activation also induces the autophagy receptor protein, p62. However, the role of the Nrf2-p62 pathway in LC3-associated phagocytosis of POS is poorly understood. Here, we investigated the relationships between Nrf2 activation and POS phagocytosis progression. A triterpenoid Nrf2 activator, RS9, facilitated POS uptake into phagolysosomes in RPE cells. RS9 also induced the expression of the autophagy-related proteins, LC3-II and p62, as well as phase-2 antioxidant enzymes. The effect of RS9 on POS phagocytosis was abolished by autophagy inhibition. Unexpectedly, p62 knockdown did not inhibit the effect of RS9 on POS phagocytosis, although, RS9-mediated LC3-II induction by RS9 was inhibited in p62 knockdown RPE cells. We also found that RS9 activated the AMPKα-mTOR signaling pathway earlier than p62 induction. Knockdown of AMPKα1, but not α2, inhibited the RS9-mediated activation of LC3-associated phagocytosis and RS9-mediated induction of LC3-II. Furthermore, intravitreal treatment of RS9 to adult mice decreased the size of POS phagolysosomes after light exposure. Collectively, these results showed that RS9-mediated activation of POS phagocytosis was mainly ascribed to the enhancement of autophagy via AMPKα1 activation. Our findings reveal novel effects of Nrf2 and AMPK α1 activation that contribute to the maintenance of the RPE function via LC3-associated POS phagocytosis.

An anti-PLVAP antibody suppresses laser-induced choroidal neovascularization in monkeys.

Plasmalemma vesicle-associated protein (PLVAP, also called PV-1) is the only protein that forms endothelial diaphragms. PLVAP expression is very low in the normal blood-retinal barrier; however, pathological factors such as high glucose and vascular endothelial growth factor (VEGF) induce its expression, leading to the exacerbation of cellular permeability. Because the new blood vessels are fragile and leaky, PLVAP could possibly be considered a therapeutic target against retinovascular diseases. VEGF inhibitors are commonly used for the treatment of such diseases; however, there are several concerns associated with their use, especially in the case of chronic suppression of VEGF. In this study, we investigated the expressional level of PLVAP mRNA in VEGF-treated endothelial cells and the retinas of 2 animal models: streptozotocin-induced diabetic Brown Norway rats and Sprague-Dawley rats with oxygen-induced retinopathy. Among transcellular transport-related genes, the induction of PLVAP mRNA is the most apparent; the increase of PLVAP mRNA levels in the retina is evident during pathological progression. Furthermore, anti-PLVAP antibodies were generated, and their efficacy against laser-induced choroidal neovascularization was tested in cynomolgus monkeys. Although the leakage was exacerbated in the saline-injected group during the progression of neovascularization, the intravitreal injection of anti-PLVAP antibodies significantly ameliorated the exudation. These data imply that the PLVAP inhibition is a promising therapeutic approach against retinal diseases such as diabetic macular edema, retinopathy of prematurity, and wet age-related macular degeneration.

Nrf2 Activator RS9 Suppresses Pathological Ocular Angiogenesis and Hyperpermeability.

Purpose: Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods: The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results: Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-ß, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions: These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.

Nrf2 Is an Attractive Therapeutic Target for Retinal Diseases.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many species. The retina is constantly exposed to reactive oxygen species, and oxidative stress is a major contributor to age-related macular diseases. Moreover, the resulting inflammation and neuronal degeneration are also related to other retinal diseases. The well-known Nrf2 activators, bardoxolone methyl and its derivatives, have been the subject of a number of clinical trials, including those aimed at treating chronic kidney disease, pulmonary arterial hypertension, and mitochondrial myopathies. Recent studies suggest that Nrf2 activation protects the retina from retinal diseases. In particular, this is supported by the finding that Nrf2 knockout mice display age-related retinal degeneration. Moreover, the concept has been validated by the efficacy of Nrf2 activators in a number of retinal pathological models. We have also recently succeeded in generating a novel Nrf2 activator, RS9, using a biotransformation technique. This review discusses current links between retinal diseases and Nrf2 and the possibility of treating retinal diseases by activating the Nrf2 signaling pathway.

A novel Nrf2 activator from microbial transformation inhibits radiation-induced dermatitis in mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that regulates many antioxidants, and we have recently succeeded in obtaining a novel Nrf2 activator, RS9, from microbial transformation. RS9 is categorized as a triterpenoid, and well-known triterpenoids such as RTA 402 (bardoxolone methyl) and RTA 408 have been tested in clinical trials. RTA 408 lotion is currently being tested in patients at risk for radiation dermatitis. This prompted us to study the profiles of RS9 in the skin. All the above triterpenoids increased the level of an Nrf2-targeted gene, NADPH:quinone oxidoreductase-1, in normal human epidermal keratinocytes. Among them, the activity of RS9 was prominent; furthermore, the cellular toxicity was less compared with RTA compounds. BALB/c mice were irradiated with 30 Gy/day on Day 0, and compounds were topically applied on the back once daily from Day 1 to Day 30. Dermatitis scores peaked on Day 18, with a score of 2.6 in vehicle-treated mice, and topical applications of 0.1% RTA 402, RTA 408 and RS9 reduced the scores to 1.8, 2.0 and 1.4, respectively. Moreover, the percentage of animals with scores ≥2 was analyzed, and 0.1% RS9 suppressed the percentage from 100% to 47%. These results imply that RS9 has potential efficacy for treating radiation dermatitis.

Cytoprotective Effects of a Novel Nrf2 Activator, RS9, in Rhodopsin Pro347Leu Rabbits.

PURPOSE: Rhodopsin Pro347Leu transgenic rabbits were previously generated as models of retinitis pigmentosa (RP). While the mechanism underlying the retinal deterioration in these rabbits remains unresolved, it is likely that oxidative stress is one of the factors triggering cellular loss. We have recently succeeded in obtaining a novel activator (RS9) of nuclear factor erythroid 2-related factor (Nrf2, also known as NFE2L2), which regulates antioxidant transcriptional factors. The purpose of this study was to investigate whether RS9 delays progressive retinal degeneration in the transgenic rabbits. METHODS: RS9 microspheres (3 mM, 50 µL) were injected into the vitreous of rhodopsin Pro347Leu transgenic rabbits at 6 weeks, after which outer nuclear layer (ONL) thickness was measured by optical coherence tomography. Rabbits were sacrificed at 15 weeks. RESULTS: After intravitreal injection of RS9 microspheres, the concentration of RS9 in the vitreous was maintained at 1 nM for 2 weeks. At a concentration of 0.3 mM and 50 µL, RS9 significantly inhibited thinning of the ONL in transgenic rabbits compared to vehicle-injected transgenic rabbits. In RS9-injected transgenic rabbits, Nrf2-targeted genes had increased significantly, and levels of interleukin-6 mRNA decreased. CONCLUSIONS: Activation of Nrf2 signaling has potential as a novel approach for the prevention and treatment of RP, not only by driving intrinsic antioxidant enzymes, but also by inhibiting inflammatory responses. Although microspheres were employed in this study, small implants that release more compounds might be a realistic method for clinical trials.

Oral administration of a T cell epitope inhibits symptoms and reactions of allergic rhinitis in Japanese cedar pollen allergen-sensitized mice.

Although the concept of a T cell epitope in specific immunoprophylaxis was proposed more than a decade ago, it had not been well demonstrated since then that a T cell epitope inhibits symptoms and reactions of allergic disease in animal models. In this study, we have established a system to evaluate symptoms and reactions of allergic rhinitis in mice, and investigated whether oral administration of a T cell epitope relieves sensitized mice of allergic rhinitis. P2-246-259 (RAEVSYVHVNGAKF) is a BALB/c mouse T-cell epitope of Cry j 2, which is a major Japanese cedar (Cryptomeria japonica) pollen allergen. Mice were administered orally with 200 microg/animal of P2-246-259 four times within 2 weeks before sensitization, and sensitized intranasally with Cry j 2 twice. Of the cardinal symptoms of allergic rhinitis, we assessed sneezing and airway obstruction, but could not estimate rhinorrhea or pruritus. Sneezing frequency was significantly increased by challenge with Cry j 2. Concerning allergic reactions, vascular permeability of the nasal mucosa in the early phase and hyperreactivity to histamine in the late phase were also exacerbated by the challenge. These symptoms and reactions of allergic rhinitis were significantly inhibited by oral administration of P2-246-259. These results indicate utility of mice as models for allergic rhinitis; furthermore, the effects of P2-246-259 on allergic rhinitis imply that oral administration of a T cell epitope is a promising approach for specific immunoprophylaxis.

Novel Nrf2 activators from microbial transformation products inhibit blood-retinal barrier permeability in rabbits.

BACKGROUND AND PURPOSE: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. EXPERIMENTAL APPROACH: The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood-retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. KEY RESULTS: Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. CONCLUSION AND IMPLICATIONS: Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease.

A novel beta-sheet breaker, RS-0406, reverses amyloid beta-induced cytotoxicity and impairment of long-term potentiation in vitro.

Fibril formation of amyloid beta peptide (Abeta) is considered to be responsible for the pathology of Alzheimer's disease (AD). The Abeta fibril is formed by a protein misfolding process in which intermolecular beta-sheet interactions become stabilized abnormally. Thus, to develop potential anti-AD drugs, we screened an in-house library to find compounds which have a profile as a beta-sheet breaker. We searched for a beta-sheet breaker profile in an in-house library of approximately 113,000 compounds. From among the screening hits, we focused on N,N'-bis(3-hydroxyphenyl)pyridazine-3,6-diamine (named RS-0406), which had been newly synthesized in our laboratory. This compound (10-100 microg ml(-1)) was found to be capable of significantly inhibiting 25 microM Abeta(1-42) fibrillogenesis and, furthermore, disassembling preformed Abeta(1-42) fibrils in vitro. 3 We then investigated the effect of RS-0406 on 111 nM Abeta(1-42)-induced cytotoxicity in primary hippocampal neurons, and found that 0.3-3 microg ml(-1) RS-0406 ameliorates the cytotoxicity. Moreover, 3 microg ml(-1) RS-0406 reversed 1 micro M Abeta(1-42)-induced impairment of long-term potentiation in hippocampal slices. 4 In this study, we have succeeded in identifying RS-0406 which has potential to inhibit Abeta(1-42) fibrillogenesis, and to protect neurons against Abeta(1-42)-induced biological toxicity in vitro. These results suggest that RS-0406 or one of the derivatives could become a therapeutic agent for AD patients.

Inhibitors beta-amyloid-induced toxicity by modulating the Akt signaling pathway.

The Akt signaling pathway plays a crucial role in neuronal survival, leading to inhibition of apoptosis. Many stimulants including neurotrophins are reported to activate this pathway in preclinical studies; however, there are no drugs for neurodegenerative diseases adopting such a concept on the market so far. Among neurodegenerative diseases, Alzheimer's disease is the most common and characterized by senile plaques and neurofibrillary tangles, which consist of beta-amyloid and hyperphosphorylated tau, respectively. Recent studies suggest that activation of Akt inhibits toxicity of beta-amyloid and formation of neurofibrillary tangles, leading to protection of neurons against apoptosis. This review discusses the possibility of treatment of Alzheimer's disease by activating the Akt signaling pathway.

A novel compound RS-0466 reverses beta-amyloid-induced cytotoxicity through the Akt signaling pathway in vitro.

beta-Amyloid peptide is the principal protein in the senile plaques of Alzheimer's disease and is considered to be responsible for the pathology of Alzheimer's disease. Several studies have shown that beta-amyloid is cytotoxic, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of viability in cells. Utilizing the MTT assay, we screened an in-house library to find compounds that suppress beta-amyloid-induced inhibition of MTT reduction. From among the screening hits, we focused on 6-ethyl-N,N'-bis(3-hydroxyphenyl)[1,3,5]triazine-2,4-diamine (named RS-0466), which had been newly synthesized in our laboratory. This compound was found to be capable of significantly inhibiting beta-amyloid-induced cytotoxicity in HeLa cells and of reversing the decrease of phosphorylated Akt induced by beta-amyloid. Furthermore, RS-0466 reversed the beta-amyloid-induced impairment of long-term potentiation in rat hippocampal slices. These results raise the possibility that RS-0466 or its derivatives have potential as a therapeutic agent for Alzheimer's disease patients, and its effect is at least in part mediated by activation of Akt.

A novel CC chemokine receptor 4 antagonist RS-1269 inhibits ovalbumin-induced ear swelling and lipopolysaccharide-induced endotoxic shock in mice.

There is growing evidence that chemokines recruit leukocytes in allergic, inflammatory and immune responses. CC chemokine receptor 4 (CCR4) is implicated as a preferential marker for T helper 2 cells, and the cells selectively respond to CC chemokine ligand 17 (CCL17) and CCL22. We searched for compounds having a profile as a CCR4 antagonist from an in-house library and have previously reported that 3-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}quinazoline-2,4(1H,3H)-dione (named RS-1154) was capable of significantly inhibiting the binding of [(125) I]CCL17 to human CCR4-expressing CHO cells. From further synthesis of its derivatives, we newly focused on 3-(isobutyrylamino)-N-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}benzamide (RS-1269), which showed potency comparable to RS-1154 in inhibiting CCL17-induced migration of DO11.10 mice-derived T helper 2 cells with an IC(50) value of 5.5 nM in vitro. We then investigated the pharmacological effects of RS-1269 on ovalbumin-induced ear swelling and lipopolysaccharide-induced endotoxic shock in mice. The ear thickness was significantly decreased by oral administration of RS-1269 at the dose of 30 mg/kg. Treatment with lipopolysaccharide significantly increased the serum level of tumour necrosis factor-α. Compared with an anti-CCL17 antibody, RS-1269 significantly inhibited the production at the dose of 100 mg/kg. These results raise the possibility that RS-1269 or one of its derivatives has potential to serve as a prototype compound to develop therapeutic agents for atopic dermatitis and inflammatory diseases.