We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric αβγ selective GABA receptor (GABAR) 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 αβγ GABARs 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 GABAR ligands.

Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6–9) and human ( P < 0.05, n = 6–10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.

We describe the effects of pH on the structure and bioavailability of MIDD0301, an oral lead compound for asthma. MIDD0301 interacts with peripheral GABA A receptors to reduce lung inflammation and airway smooth muscle constriction. The structure of MIDD0301 combines basic imidazole and carboxylic acid function in the same diazepine scaffold, resulting in high solubility at neutral pH. Furthermore, we demonstrated that MIDD0301 can interconvert between a sevenmembered ring structure at neutral pH and an acyclic compound at or below pH 3. Both structures have two stable conformers in solution that can be observed by 1 H NMR at room temperature. Kinetic analysis showed opening and closing of the seven-membered ring of MIDD0301 at gastric and intestinal pH, occurring with different rate constants. However, in vivo studies showed that the interconversion kinetics are fast enough to yield similar MIDD0301 blood and lung concentrations for neutral and acidic formulations. Importantly, acidic and neutral formulations of MIDD0301 exhibit high lung distribution with low concentrations in brain. These findings demonstrate that MIDD0301 interconverts between stable structures at neutral and acidic pH without changes in bioavailability, further supporting its formulation as an oral asthma medication.

We report a 28‐day repeat dose immunotoxicity evaluation of investigational drug MIDD0301, a novel oral asthma drug candidate that targets gamma amino butyric acid type A receptors (GABAAR) in the lung. The study design employed oral administration of mice twice daily throughout the study period with 100 mg/kg MIDD0301 mixed in peanut butter. Compound dosing did not reveal signs of general toxicity as determined by animal weight, organ weight or haematology. Peanut butter plus test drug (in addition to ad libitum standard rodent chow) did not affect weight gain in the adult mice, in contrast to weight loss in 5 mg/kg prednisone‐treated mice. Spleen and thymus weights were unchanged in MIDD0301‐treated mice, but prednisone significantly reduced the weight of those organs over the 28‐day dosing. Similarly, no differences in spleen or thymus histology were observed following MIDD0301 treatment, but prednisone treatment induced morphological changes in the spleen. The number of small intestine Peyer's patches was not affected by MIDD0301 treatment, an important factor for orally administered drugs. Circulating lymphocyte, monocyte and granulocyte numbers were unchanged in the MIDD0301‐treated animals, whereas differential lymphocyte numbers were reduced in prednisone‐treated animals. MIDD0301 treatment did not alter IgG antibody responses to dinitrophenyl following dinitrophenyl‐keyhole limpet haemocyanin immunization, indicating that systemic humoral immune function was not affected. Taken together, these studies show that repeated daily administration of MIDD0301 is safe and not associated with adverse immunotoxicological effects in mice.

We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABA A R) modulator. The therapeutic efficacy of nebulized MIDD0301 in reducing airway resistance was investigated in spontaneous breathing mice using a whole-body plethysmograph and in unconscious mice using a forced oscillation technique. Prophylactic nebulized MIDD0301 reduced subsequent methacholine-induced bronchoconstriction in ovalbumin and house dust mite allergic asthma models and in normal mice. Nebulized MIDD0301 exhibited comparable or better therapeutic potency compared to nebulized albuterol and oral montelukast. Prophylactic nebulized MIDD0301 was also effective in reducing bronchoconstriction, comparable to nebulized albuterol or fluticasone, in a steroid resistant asthma mouse model induced by intratracheal installation of lipopolysaccharide and interferon-gamma. Oral dexamethasone was ineffective in this model. Nebulized MIDD0301 was also effective in reversing bronchospasm when dosed after methacholine challenge comparable to albuterol. Pharmacokinetic studies showed that about 0.06% of nebulized MIDD0301 entered the mouse lung when using a whole body plethysmograph and therapeutic levels were sustained in the lung for at least 25 min. Consistent with previous reports on orally dosed MIDD0301, high doses of nebulized MIDD0301 resulted in minimal brain exposure and thus no observable adverse sensorimotor or respiratory depression effects occurred. In addition, no adverse cardiovascular effects were observed following 100 mg/kg i.p. dosing. These results further demonstrate that charged imidazodiazepine MIDD0301 can selectively target lung GABA A R without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation.