DS-8500a, an Orally Available G Protein-Coupled Receptor 119 Agonist, Upregulates Glucagon-Like Peptide-1 and Enhances Glucose-Dependent Insulin Secretion and Improves Glucose Homeostasis in Type 2 Diabetic Rats.

G protein-coupled receptor 119 (GPR119) has been shown to be highly expressed in small intestinal L-cells and pancreatic ß-cells and mediates intracellular cAMP concentration, glucagon-like peptide (GLP-1) secretion, and glucose-stimulated insulin secretion (GSIS). This study examined the pharmacological effects of 4-(5-{(1R)-1-[4-(cyclopropylcarbonyl) phenoxy]propyl}-1,2,4-oxadiazol-3-yl)-2-fluoro-N-[(2R)-1-hydroxypropan-2-yl]benzamide (DS-8500a), a novel, orally available, selective GPR119 agonist. In in vitro studies, DS-8500a increased intracellular cAMP in a concentration-dependent manner in human, rat, and mouse GPR119-expressing Chinese hamster ovary (CHO)-K1 cells, with EC50 values of 51.5, 98.4, and 108.1 nmol/l, respectively. DS-8500a had no effect on intracellular cAMP in pcDNA3.1/CHO-K1 cells. In in vivo studies, DS-8500a augmented plasma GLP-1 concentration in Zucker fatty (ZF) rats, and enhanced GSIS and did not change plasma glucose concentration in fasted Sprague-Dawley (SD) rats. A single dose of DS-8500a showed dose-dependent glucose-lowering effects at oral glucose tolerance test (OGTT) in ZF rats. In a repeat-dosing study, DS-8500a had statistically significant glucose-lowering effects at OGTT performed at the 1st day and after 2 weeks of treatment in neonatal streptozotocin-treated (nSTZ) rats, and the efficacy levels of DS-8500a in each test were greater than those of GSK1292263 or MBX-2982, which had been clinically tested previously as GPR119 agonists. Through pharmacokinetics and pharmacodynamics assessment, the high intrinsic activity of DS-8500a was suggested to be one of the reasons for the greater glucose lowering effect in the nSTZ rats. DS-8500a is a useful compound among GPR119 agonists that can maximize the potential benefit of GPR119 in type 2 diabetes.

[The role of pharmacology to produce firuglipel (DS-8500a), an orally available GPR119 agonist for type 2 diabetes mellitus].

GPR119 (G-protein coupled receptor 119) has been shown to be highly expressed in the lower small intestinal and colorectal L-cells and pancreatic ß-cells, and mediates intracellular cAMP concentration, glucagon like peptide (GLP-1) secretion, and glucose stimulated insulin secretion (GSIS). As the next generation for the treatment of type 2 diabetes mellitus (T2DM), GPR119 agonist has been intensively studied by pharmaceutical companies and a lot of patents have been applied by them. In such highly competitive condition, biological differentiation and to find an advantage among GPR119 agonists were necessary to proceed the candidate compound in further clinical investigation. Firuglipel (DS-8500a) is an orally available GPR119 agonist synthesized in DAIICHI SANKYO CO., LTD (DS). It was originated from DS-chemical library and optimized in the aspect of bioavailability and safety. Firuglipel had a higher intrinsic activity (IA) of the production of intracellular cAMP in human GPR119 expressing CHO-K1 cells than those of other GPR119 agonists studied. The level of IA in each GPR119 agonist was correlated with the existence of agonist conformer. In parallel with the study for the differentiation from other GPR119 agonists, we compared firuglipel with dipeptidyl peptide-4 (DPP-4) inhibitor in NONcNZO10/LtJ mice and evaluated their combination in streptozotocin (STZ) treated C57BL/6J mice to clarify future positioning among anti-diabetics therapy. These pharmacological studies illustrated here can draw out a clinical value of compound and expected to lead the production of first-in-class agent in pharmaceutical companies.

Pathological and gene expression analysis of a polygenic diabetes model, NONcNZO10/LtJ mice.

The NONcNZO10/LtJ mouse is a polygenic model of type-2 diabetes (T2D) that shows moderate obesity and diabetes, and is regarded as a good model reflective of the conditions of human T2D. In this study, we analyzed pathological changes of pancreases with the progress of time by using histopathology and gene expression analysis, including microRNA. A number of gene expression changes associated with decreased insulin secretion (possibly regulated by miR-29a/b) were observed, and zinc homeostasis (Slc30a8, Mt1 and Mt2) or glucose metabolism (Slc2a2) was suggested as being the candidate mechanism of pancreas failure in NONcNZO10/LtJ mice. These results demonstrate NONcNZO10/LtJ mice have a complex pathogenic mechanism of diabetes, and moreover, this fundamental information of NONcNZO10/LtJ mice would offer the opportunity for research and development of a novel antidiabetic drug.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Identification of the 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives as highly selective PDE4B inhibitors.

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC50 value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.

Intraoral administration of a T-cell epitope peptide induces immunological tolerance in Cry j 2-sensitized mice.

Sublingual immunotherapy using allergen-derived peptides is feasible as a novel specific immunotherapy, but its efficacy has not yet been demonstrated in either humans or animals. In addition, it remains obscure whether the oral immune system is involved in the mechanism of sublingual immunotherapy. Here, we show that the intraoral administration of the T-cell epitope peptide P2-246-259 derived from Cry j 2, a major Japanese cedar (Cryptomeria japonica) pollen allergen, to Cry j 2-sensitized mice induces immunological tolerance, and that ex vivo lymph node cell proliferation to P2-246-259 and Cry j 2 was inhibited. In addition, intraoral administration was shown to be superior to intragastric administration in terms of tolerance induction, suggesting that the oral immune system contributes to the induction of immunological tolerance. Therefore, the significant efficacy of sublingual immunotherapy using a peptide on allergen-specific T-cells was demonstrated in animals, and this may be potentiated by the oral mucosal immune system.

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells.

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.

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.

Ketotic hypoglycemia in patients with allergic diseases.

BACKGROUND: Ketotic hypoglycemia is the most common cause of recurrent hypoglycemia in early childhood but its etiology is poorly understood. Elimination diets have been used for the prevention or treatment of some allergic diseases; however, these diets entail a risk to the normal nutrition and growth of children. The present study investigated whether elimination diets are related to the occurrence of ketotic hypoglycemia. METHODS: The prevalence of allergy treated with elimination diet therapy was retrospectively investigated in 18 patients with ketotic hypoglycemia seen in Yamaguchi University Hospital between January 1995 and September 1999. Data were gathered by reviewing the patients' medical charts. RESULTS: Ten (55.6%) of 18 patients with ketotic hypoglycemia had allergic diseases. Six (60%) of the patients had been treated with strict elimination diets and the others were on incomplete elimination diets for the prevention of allergy. The ketotic hypoglycemia patients with allergic diseases had experienced fewer pre-existing infectious diseases than those without allergic diseases. CONCLUSIONS: Patients with allergic diseases treated with elimination diets have a relatively high tendency towards developing ketotic hypoglycemia. They might have a lower capacity to tolerate fasting due to their excessive avoidance of many foods, even during periods when they are not undergoing strict elimination diets.

Chronic inflammation of the skin can be induced in IgE transgenic mice by means of a single challenge of multivalent antigen.

BACKGROUND: It is now widely accepted that IgE mediates immediate-type allergic response. However, the pathologic role of IgE is controversial in the chronic allergic inflammation observed in atopic diseases, such as asthma and atopic dermatitis. OBJECTIVE: We investigated the role of IgE in cutaneous allergic reactions by using 2 newly developed lines of antigen-specific IgE transgenic mice. METHODS: IgE transgenic mice were administered subcutaneously with corresponding antigens, and the subsequent ear swelling was measured. RESULTS: A single subcutaneous administration of TNP-conjugated ovalbumin (OVA) into the ears of nonimmunized mice carrying the TNP-specific IgE transgene elicited immediate-phase and late-phase ear swelling as expected, which peaked at 20 minutes and 8 hours later, respectively. Interestingly, however, 2 to 3 days after the antigen challenge, more intense ear swelling appeared. Its magnitude and duration were dependent on the valency of TNP in OVA, as well as the dose of TNP-OVA, and it lasted over 1 month when 100 microg of OVA conjugated with 11 molecules of TNP was given. Interestingly, administration of OVA to OVA-specific IgE transgenic mice elicited immediate-phase and late-phase ear swelling but not third-phase ear swelling. Massive infiltration of inflammatory cells was observed in the third-phase ear swelling of TNP-specific IgE transgenic mice. Cyclosporine A almost completely inhibited the third-phase ear swelling and cellular infiltration, whereas an antihistamine, cyproheptadine, did not show any significant effect on the third-phase reaction. CONCLUSION: These results indicate that IgE can trigger not only immediate-type hypersensitivity but also chronic allergic inflammation. Our findings highlight a novel immunopathologic role of IgE in chronic atopic disorders.

Three T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, retain their immunogenicity and tolerogenicity in a linked peptide.

It has been demonstrated in detail that administration of a dominant T-cell determinant to animals induces activation or immunological tolerance of T cells. However, it has not been determined whether multiple T-cell determinants, when integrated into a single peptide, retain their potential to induce T-cell activation and tolerance. We prepared a synthetic peptide comprising three T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, and investigated the immunogenicity and tolerogenicity of each T-cell determinant in the linked peptide by means of lymph node cell proliferation assays using mice. Lymph node cells from mice immunized with each of the three T-cell determinants proliferated against the linked peptide in a dose-dependent manner, similar to that of the immunized peptide. Lymph node cells from mice immunized with the linked peptide proliferated against all of the three T-cell determinants. In addition, the degree of proliferation against the three T-cell determinants occurred according to their original immunogenicity, as observed in the native protein antigens. Oral administration of the linked peptide to mice before they were immunized with Cry j 1 and Cry j 2 inhibited lymph node cell proliferation against the three T-cell determinants, depending on the dose of the linked peptide administered. In conclusion, it was demonstrated that three T-cell determinants retain their original immunogenicity and tolerogenicity in a linked peptide comprising them.

NF-kappaB activation in cerebrospinal fluid cells from patients with meningitis.

We examined whether or not NF-kappaB, a factor that regulates expression of the genes that code for pro-inflammatory cytokines, is activated in cerebrospinal fluid (CSF) cells to investigate the production of pro-inflammatory cytokines by CSF cells in patients with meningitis. Western blotting demonstrated that NF-kappaB was more activated in CSF cells of patients with bacterial meningitis than in those of patients with aseptic meningitis. NF-kappaB was hardly activated in carcinomatous meningitis. The NF-kappaB activation in CSF cells of patients with meningitis tended to be correlated with the CSF interleukin-6 concentration. Our data suggested that CSF cells produce pro-inflammatory cytokines through NF-kappaB activation in meningitis, and that increased NF-kappaB activation in CSF cells indicate infectious meningitis rather than carcinomatous meningitis.