The Role of the Canonical Beta-2 Adrenoceptor Gs Pathway in Development of the Asthma Phenotype in Murine Models

Asthma is a chronic inflammatory disease of the airways characterized by variable degrees of inflammation, mucous metaplasia and airway hyperresponsiveness (AHR). Asthma causes over a quarter million deaths per year and affects 300 million people worldwide. Current treatment modalities including inhaled β2-adrenoceptor (β2AR) agonists, and inhaled corticosteroids, the main bronchodilating and anti-inflammatory agents respectively are associated with serious and adverse side effects. Therefore, there is the need to develop novel therapies for the management of asthma. Using pharmacologic studies, we have previously shown in an allergen driven murine model of asthma that, chronic administration of certain “β-blockers” with inverse agonist properties such as nadolol, metoprolol, and ICI 118,551, but not the antagonists alprenolol, significantly attenuated three cardinal features of asthma: airway inflammation, mucous metaplasia and AHR. Also, in genetic studies, we established the requirement of the β2AR in development of the asthma phenotype where ovalbumin sensitized and challenged β2AR null mice, exhibited an attenuation of the asthma phenotype. These studies established the requirement of β2AR signaling in the development of the asthma phenotype in murine models. In mild asthmatics, clinical studies have also shown that, chronic administration of nadolol dose-dependently increased the provocative concentration of methacholine causing a 20% fall in forced expiratory volume in 1 second (FEV1). The β2AR can signal in the presence of a ligand or spontaneously (constitutively active receptors) in the absence of a ligand. It can also signal through at least two major pathways: the cAMP pathway and the β-arrestin pathway, which can lead to MAPK activation. This project was designed to study the role of constitutive versus ligand activation of the β2AR in development of the asthma phenotype in murine asthma models. We also investigated the pathways downstream of the β2AR required for the development of the asthma phenotype. Using pharmacologic and genetic studies, we have shown that ligand activation of the β2AR is required for the development of the asthma phenotype in murine asthma models. We also observed that, the Gs-cAMP pathway may demonstrate beneficial effects in a murine model of asthma.Pharmacological and Pharmaceutical Sciences, Department o

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABA_A Receptors in the Lung

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α₅β₃γ₂ selective GABA_A receptor (GABA_AR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α_1–3,5β₃γ₂ GABA_ARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4⁺ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4⁺ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABA_AR ligands

Development of GABA_A Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments

Recent studies have demonstrated that subtype-selective GABA_A receptor modulators are able to relax precontracted human airway smooth muscle <i>ex vivo</i> and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABA_A receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABA_A receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABA_AR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca²⁺]_i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca²⁺]_i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings <i>ex vivo</i>. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABA_AR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects

Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABA_A Receptor Modulators

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α₄β₃γ₂ GABA_AR selective compound <b>1</b> and acidic α₅β₃γ₂ selective GABA_AR positive allosteric modulator compound <b>2</b> relaxed airway smooth muscle <i>ex vivo</i> and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound <b>2</b> relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds <b>1</b> and <b>2</b> decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound <b>1</b> reduced the number of lung CD4⁺ T lymphocytes and directly modulated their transmembrane currents by acting on GABA_ARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABA_ARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABA_AR ligands