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Pharmacological Characterization of TASP0412098, A Selective CRTH2 (Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells) Antagonist
AB48 Abstracts
SATURDAY
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
181
183
182
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Controlled Allergen Exposure in the Environmental Exposure Chamber (EEC) Results in a Late Phase Inflammatory Response Evidenced by Increased Eosinophils in the Upper Airway of Allergic Patients N. Camuso, S. Zafar, N. Najera, A. Liac, G. Aguirre, A. Salapatek; Cetero Research, Mississauga, ON, CANADA. RATIONALE: An increase in eosinophils is an important inflammatory biomarker in the respiratory airways of allergic patients upon allergen exposure. Historically, nasal lavage results have proved difficult to interpret due to high eosinophil cell count variability. Therefore, the ability of controlled and extended allergen exposure in the EEC to induce a detectable increase in eosinophils in the upper airway of allergic patients was investigated. METHODS: Patients with a positive SPT to ragweed and a history of seasonal allergic rhinitis during the last two ragweed seasons were exposed to aerosolized ragweed allergen (35006500 grains/m3) for 8 hours in the EEC. Nasal lavage samples were collected from 40 subjects pre-and post-EEC as part of a larger study. Samples were processed and cytospin slides were prepared. Eosinophils were counted and expressed as a percentage of leukoctyes in the sample. RESULTS: A significant increase (p50.0005) was observed in the mean percentage of eosinophils in the nasal lavage sample after an 8 hour allergen EEC exposure (12.37%62.980%) compared to 0.85%60.432% prior to EEC entry. Thirteen of the 40 subjects showed a >10% increase in percentage of eosinophils with a mean value of 32.90%66.261% and a coefficient of variation of 68.61%. CONCLUSIONS: Extended allergen exposure in the EEC elicited a significant inflammatory response, characterized by an increase in eosinophils in the nasal cavity of allergic patients. The EEC provides an effective screening tool for identifying patients in late phase allergy, thereby reducing variability when using eosinophil counts as a an end point and providing further mechanistic insights for drug action. Mold and House Dust Mite Allergens Activate and Sensitize Sensory Neurons Innervating Airways M. J. Patil1, M. A. Henry2, A. N. Akopian2; 1Pharmacology Dept, Graduate School, UTHSCSA, San Antonio, TX, 2Dental School, UTHSCSA, San Antonio, TX. RATIONAL: The peripheral nervous system can contribute to the initiation and maintenance of chronic airway inflammation, a hallmark of several pulmonary diseases. We hypothesize that certain mold and house dust mite (HDM) allergens belonging to the protease family activate and sensitize sensory neurons innervating airways. These actions on sensory neurons lead to the release of neuropeptides and could initiate of neurogenic inflammation in airways. METHODS: Neuronal cultures were generated from rat T1-T3 dorsal root (DRG) and jugular-nodose ganglia (NG). Activation and sensitization of neurons were measured using Ca2+ imaging and patch-clamp electrophysiology. Neuropeptide release from airways was assessed using radio-immuno assay. Sensory neurons innervating airways were identified by back labeling from airways. Data were analyzed with one or two-way ANOVA. RESULTS: Asthma-inducing HDM and mold allergens DerP3&9, PenC13 and DerP1 belonging to the protease family generate Ca2+ influx and currents in distinct subsets of T1-T3 DRG and NG neurons. A set of responsive sensory neurons innervate airways. These allergens also sensitize the action of acrolein - an irritant from cigarette smoke and an environmental pollutant. Importantly, the allergens are capable of inducing and sensitizing the release of the vasoactive calcitonin-gene related peptide (CGRP) from bronchi preparations. CONCLUSION: HDM and mold allergens attributed to the induction of asthma and belonging to the protease family activate and sensitize sensory neurons innervating airways. The activation of these sensory neurons leads to the release of CGRP, possibly contributing to the initiation of airway inflammation.
Peripheral Blood Mononuclear cells from Patients with Bronchial Asthma show impaired regulatory responses to Rhinovirus in vitro K. Iikura; Sagamihara National Hospital, Kanagawa, JAPAN. RATIONALE: Asthmatic subjects tend to have impaired antiviral immune responses to rhinovirus (RV) whereas they are likely to exhibit more severe and prolonged symptoms upon RV infection. To clarify the mechanisms how inflammatory reactions in asthmatic subjects are exaggerated, cytokine and chemokine secretion profiles in PBMCs after exposure to RV were examined. METHODS: Thirty-five currently asthmatic subjects (BA) and 36 agematched control subjects (nBA) were recruited. PBMCs obtained from BA without clinical exacerbation within 2 weeks before the phlebotomy and from nBA were stimulated with RV-14 for 72h. The concentrations of interferon-gamma-inducible protein-10 (IP-10), IFN-gamma, IL-4, IL-13, and IL-10 in the supernatants were measured by ELISA. RESULTS: The levels of IFN-gamma and IP-10 were correlated positively (r50.34, p50.004). The concentrations of IL-10 were significantly higher in nBA (221.7 6 37.1 vs. 670.8 6 95.3 pg/ml, p<0.0001), but there were no statistically significant differences in the levels of IFN-gamma and IP-10 between BA and nBA (229.9 6 38.1 vs. 417.2 6 96.3 pg/ml, p50.07; 5151 6 1237 vs. 2994 6 616.6 pg/ml, p50.4, respectively). Concentrations of IL-4 and IL-13 were below the detection limits. The ratio of IFN-gamma/IL-10 and IP-10/IL-10 were significantly higher in BA than nBA (p50.009 and p50.01, respectively). CONCLUSIONS: Our results suggest that asthmatic subjects are likely to have comparable ability to attract CXCR3-positive cells with normal subjects, whereas they have impaired regulatory response which presumably leads to exhibit more severe and prolonged symptoms upon RV infection.
Pharmacological Characterization of TASP0412098, A Selective CRTH2 (Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells) Antagonist M. Kawanishi1, K. Watanabe1, Y. Asakura1, A. Oka1, T. Naruse1, R. Nishikawa2, T. Takayama2, T. Koami2, D. Wakasugi2, M. Kawamura2, A. Takaoka1, S. Tokita1; 1Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama-shi, Saitama, JAPAN, 2 Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama-shi, Saitama, JAPAN. RATIONALE: Chemoattractant receptor-homologous molecule expressed on Th2 (CRTH2) is a G protein-coupled receptor (GPCR) which expresses on Th2 cells, eosinophils and basophils. Prostaglandin D2 (PGD2), an arachidonic acid metabolite produced by mast cells, acts as the ligand of CRTH2. It has been suggested that the interaction between CRTH2 and PGD2 might have important role(s) in allergic diseases such as bronchial asthma, allergic rhinitis and atopic dermatitis. We succeeded in optimization of a novel CRTH2 antagonist, the isoquinoline derivative TASP0412098, and examined biological activities and pharmacological efficacies on allergic responses. METHODS: Receptor binding assay was performed by CRTH2 transfected cells. Antagonistic activity was determined by intracellular Ca2+ influx using CRTH2 transfected cells, migration assay and IL-13 production assay performed by Th2 cells derived from healthy adult volunteer. Eosinophil shape change assay was performed by eosinophil derived from peripheral blood cells of guinea pig. In vivo efficacy were examined using guinea pig asthma model and murine contact allergic dermatitis model. RESULTS: TASP0412098 exhibited strong inhibitory effects on CRTH2-PGD2 interaction (IC50: 2.1nM) in highly specific manner.In cellular studies, TSAP0412098 also inhibited PGD2-induced intracellular Ca2+ influx, Th2 cells migration and IL-13 production from Th2 cells. Administration of TASP0412098 attenuated eosinophil shape change reaction at ex vivo, suppressed bronchial asthmatic responses in guinea pigs and inflammatory responses in contact allergic dermatitis model in mice. CONCLUSIONS: CRTH2 plays important roles in allergic diseases, and TASP0412098 is a useful compound for the treatment of allergic diseases.
SATURDAY
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
181
183
182
184
Controlled Allergen Exposure in the Environmental Exposure Chamber (EEC) Results in a Late Phase Inflammatory Response Evidenced by Increased Eosinophils in the Upper Airway of Allergic Patients N. Camuso, S. Zafar, N. Najera, A. Liac, G. Aguirre, A. Salapatek; Cetero Research, Mississauga, ON, CANADA. RATIONALE: An increase in eosinophils is an important inflammatory biomarker in the respiratory airways of allergic patients upon allergen exposure. Historically, nasal lavage results have proved difficult to interpret due to high eosinophil cell count variability. Therefore, the ability of controlled and extended allergen exposure in the EEC to induce a detectable increase in eosinophils in the upper airway of allergic patients was investigated. METHODS: Patients with a positive SPT to ragweed and a history of seasonal allergic rhinitis during the last two ragweed seasons were exposed to aerosolized ragweed allergen (35006500 grains/m3) for 8 hours in the EEC. Nasal lavage samples were collected from 40 subjects pre-and post-EEC as part of a larger study. Samples were processed and cytospin slides were prepared. Eosinophils were counted and expressed as a percentage of leukoctyes in the sample. RESULTS: A significant increase (p50.0005) was observed in the mean percentage of eosinophils in the nasal lavage sample after an 8 hour allergen EEC exposure (12.37%62.980%) compared to 0.85%60.432% prior to EEC entry. Thirteen of the 40 subjects showed a >10% increase in percentage of eosinophils with a mean value of 32.90%66.261% and a coefficient of variation of 68.61%. CONCLUSIONS: Extended allergen exposure in the EEC elicited a significant inflammatory response, characterized by an increase in eosinophils in the nasal cavity of allergic patients. The EEC provides an effective screening tool for identifying patients in late phase allergy, thereby reducing variability when using eosinophil counts as a an end point and providing further mechanistic insights for drug action. Mold and House Dust Mite Allergens Activate and Sensitize Sensory Neurons Innervating Airways M. J. Patil1, M. A. Henry2, A. N. Akopian2; 1Pharmacology Dept, Graduate School, UTHSCSA, San Antonio, TX, 2Dental School, UTHSCSA, San Antonio, TX. RATIONAL: The peripheral nervous system can contribute to the initiation and maintenance of chronic airway inflammation, a hallmark of several pulmonary diseases. We hypothesize that certain mold and house dust mite (HDM) allergens belonging to the protease family activate and sensitize sensory neurons innervating airways. These actions on sensory neurons lead to the release of neuropeptides and could initiate of neurogenic inflammation in airways. METHODS: Neuronal cultures were generated from rat T1-T3 dorsal root (DRG) and jugular-nodose ganglia (NG). Activation and sensitization of neurons were measured using Ca2+ imaging and patch-clamp electrophysiology. Neuropeptide release from airways was assessed using radio-immuno assay. Sensory neurons innervating airways were identified by back labeling from airways. Data were analyzed with one or two-way ANOVA. RESULTS: Asthma-inducing HDM and mold allergens DerP3&9, PenC13 and DerP1 belonging to the protease family generate Ca2+ influx and currents in distinct subsets of T1-T3 DRG and NG neurons. A set of responsive sensory neurons innervate airways. These allergens also sensitize the action of acrolein - an irritant from cigarette smoke and an environmental pollutant. Importantly, the allergens are capable of inducing and sensitizing the release of the vasoactive calcitonin-gene related peptide (CGRP) from bronchi preparations. CONCLUSION: HDM and mold allergens attributed to the induction of asthma and belonging to the protease family activate and sensitize sensory neurons innervating airways. The activation of these sensory neurons leads to the release of CGRP, possibly contributing to the initiation of airway inflammation.
Peripheral Blood Mononuclear cells from Patients with Bronchial Asthma show impaired regulatory responses to Rhinovirus in vitro K. Iikura; Sagamihara National Hospital, Kanagawa, JAPAN. RATIONALE: Asthmatic subjects tend to have impaired antiviral immune responses to rhinovirus (RV) whereas they are likely to exhibit more severe and prolonged symptoms upon RV infection. To clarify the mechanisms how inflammatory reactions in asthmatic subjects are exaggerated, cytokine and chemokine secretion profiles in PBMCs after exposure to RV were examined. METHODS: Thirty-five currently asthmatic subjects (BA) and 36 agematched control subjects (nBA) were recruited. PBMCs obtained from BA without clinical exacerbation within 2 weeks before the phlebotomy and from nBA were stimulated with RV-14 for 72h. The concentrations of interferon-gamma-inducible protein-10 (IP-10), IFN-gamma, IL-4, IL-13, and IL-10 in the supernatants were measured by ELISA. RESULTS: The levels of IFN-gamma and IP-10 were correlated positively (r50.34, p50.004). The concentrations of IL-10 were significantly higher in nBA (221.7 6 37.1 vs. 670.8 6 95.3 pg/ml, p<0.0001), but there were no statistically significant differences in the levels of IFN-gamma and IP-10 between BA and nBA (229.9 6 38.1 vs. 417.2 6 96.3 pg/ml, p50.07; 5151 6 1237 vs. 2994 6 616.6 pg/ml, p50.4, respectively). Concentrations of IL-4 and IL-13 were below the detection limits. The ratio of IFN-gamma/IL-10 and IP-10/IL-10 were significantly higher in BA than nBA (p50.009 and p50.01, respectively). CONCLUSIONS: Our results suggest that asthmatic subjects are likely to have comparable ability to attract CXCR3-positive cells with normal subjects, whereas they have impaired regulatory response which presumably leads to exhibit more severe and prolonged symptoms upon RV infection.
Pharmacological Characterization of TASP0412098, A Selective CRTH2 (Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells) Antagonist M. Kawanishi1, K. Watanabe1, Y. Asakura1, A. Oka1, T. Naruse1, R. Nishikawa2, T. Takayama2, T. Koami2, D. Wakasugi2, M. Kawamura2, A. Takaoka1, S. Tokita1; 1Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama-shi, Saitama, JAPAN, 2 Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama-shi, Saitama, JAPAN. RATIONALE: Chemoattractant receptor-homologous molecule expressed on Th2 (CRTH2) is a G protein-coupled receptor (GPCR) which expresses on Th2 cells, eosinophils and basophils. Prostaglandin D2 (PGD2), an arachidonic acid metabolite produced by mast cells, acts as the ligand of CRTH2. It has been suggested that the interaction between CRTH2 and PGD2 might have important role(s) in allergic diseases such as bronchial asthma, allergic rhinitis and atopic dermatitis. We succeeded in optimization of a novel CRTH2 antagonist, the isoquinoline derivative TASP0412098, and examined biological activities and pharmacological efficacies on allergic responses. METHODS: Receptor binding assay was performed by CRTH2 transfected cells. Antagonistic activity was determined by intracellular Ca2+ influx using CRTH2 transfected cells, migration assay and IL-13 production assay performed by Th2 cells derived from healthy adult volunteer. Eosinophil shape change assay was performed by eosinophil derived from peripheral blood cells of guinea pig. In vivo efficacy were examined using guinea pig asthma model and murine contact allergic dermatitis model. RESULTS: TASP0412098 exhibited strong inhibitory effects on CRTH2-PGD2 interaction (IC50: 2.1nM) in highly specific manner.In cellular studies, TSAP0412098 also inhibited PGD2-induced intracellular Ca2+ influx, Th2 cells migration and IL-13 production from Th2 cells. Administration of TASP0412098 attenuated eosinophil shape change reaction at ex vivo, suppressed bronchial asthmatic responses in guinea pigs and inflammatory responses in contact allergic dermatitis model in mice. CONCLUSIONS: CRTH2 plays important roles in allergic diseases, and TASP0412098 is a useful compound for the treatment of allergic diseases.