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

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5ß3γ2 selective GABAA receptor (GABAAR) 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 α1-3,5ß3γ2 GABAARs 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 GABAAR ligands.

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

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α4ß3γ2 GABAAR selective compound 1 and acidic α5ß3γ2 selective GABAAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAARs. 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 GABAARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAAR ligands.

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by ß2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR 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 [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR 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.

ß2-Adrenoceptor agonists are required for development of the asthma phenotype in a murine model.

ß(2)-Adrenoceptor (ß2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent ß2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the ß2AR, epinephrine. In this study, we demonstrate that activation of the ß2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting ß2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase-null mice restored the asthma phenotype. We conclude that ß2AR agonist-induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the ß2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of ß2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain "ß-blockers."

Optimization of substituted imidazobenzodiazepines as novel asthma treatments.

We describe the synthesis of analogs of XHE-III-74, a selective α4ß3γ2 GABAAR ligand, shown to relax airway smooth muscle ex vivo and reduce airway hyperresponsiveness in a murine asthma model. To improve properties of this compound as an asthma therapeutic, a series of analogs with a deuterated methoxy group in place of methoxy group at C-8 position was evaluated for isotope effects in preclinical assays; including microsomal stability, cytotoxicity, and sensorimotor impairment. The deuterated compounds were equally or more metabolically stable than the corresponding non-deuterated analogs and increased sensorimotor impairment was observed for some deuterated compounds. Thioesters were more cytotoxic in comparison to other carboxylic acid derivatives of this compound series. The most promising compound 16 identified from the in vitro screens also strongly inhibited smooth muscle constriction in ex vivo guinea pig tracheal rings. Smooth muscle relaxation, determined by reduction of airway hyperresponsiveness with a murine ovalbumin sensitized and challenged model, showed that 16 was efficacious at low methacholine concentrations. However, this effect was limited due to suboptimal pharmacokinetics of 16. Based on these findings, further analogs of XHE-III-74 will be investigated to improve in vivo metabolic stability while retaining the efficacy at lung tissues involved in asthma pathology.

Ligand bias prevents class equality among beta-blockers.

ß-Blockers are used for a wide range of diseases from hypertension to glaucoma. In some diseases/conditions all ß-blockers are effective, while in others only certain subgroups are therapeutically beneficial. The best-documented example for only a subset of ß-blockers showing clinical efficacy is in heart failure, where members of the class have ranged from completely ineffective, to drugs of choice for treating the disease. Similarly, ß-blockers were tested in murine asthma models and two pilot clinical studies. A different subset was found to be effective for this clinical indication. These findings call into question the current system of classifying these drugs. To consider 'ß-blockers', as a single class is misleading when considering their rigorous pharmacological definition and their appropriate clinical application.

Chronic treatment with DCPCX, an adenosine A(1) antagonist, worsens long-term memory.

Alzheimer's disease is characterized by progressive cognitive disturbances and neurotransmitter dysfunction. Previous studies targeting the adrenergic A1 pathway suggest that this plays a role in cognitive impairment in Alzheimer's disease. Previous studies have reported that acute treatment with A1 antagonists appears to improve behavioral deficits in rodent models of memory and behavioral impairment. In this study, we addressed whether the chronic administration of 8-cyclopentyl-1,3-dipropylxanthine, a potent and selective adenosine A1 antagonist, could reverse the memory deficits found in aged APPswe/PS1dE9 mice. Chronic treatment did not improve memory in the APPswe/PS1dE9 mouse model and resulted in reduced exploratory behavior, suggestive of reduced anxiety, and a worsening of long-term memory in nontransgenic mice. These results have important implications for understanding the mechanisms of A1 receptor modulation as a target in Alzheimer's disease therapy.