Development of Inhaled GABA_A Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders

Abstract

We report the modification of MIDD0301, an imidazodiazepine GABA A receptor (GABA A R) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABA A R binding site of clonazepam, with IC 50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α 1 β 3 γ 2 GABA A R structural data, suggests binding of the diazepine core between the α1+/γ2− interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/ pharmacodynamic properties of PI320 when administered orally.

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators

“…Furthermore, hydrogen bond interactions between the carboxylate and Ser206 and Ser205 and the imine function were identified. We reported molecular docking poses for MIDD0301 bearing a (R) or (S) methyl substituent instead of the cyclopropyl substituent and observed the almost same docking poses . The stereochemistry of the methyl substituent did not significantly influence GABA A R binding ( MIDD0301 (IC 50 = 26 nM) and MIDD0301S (IC 50 = 25 nM)).…”

“…We previously reported that MIDD0301 potently relaxes constricted airway smooth muscle ex vivo and reduces AHR in vivo. ,,, For the ex vivo experiment, guinea pig tracheal rings were suspended in an organ bath, constricted with substance-P, and treated with 5a–j followed by recording of muscle force (Figure ).…”

“…We reported molecular docking poses for MIDD0301 bearing a (R) or (S) methyl substituent instead of the cyclopropyl substituent and observed the almost same docking poses. 29 The stereochemistry of the methyl substituent did not significantly influence GABA A R binding (MIDD0301 (IC 50 = 26 nM) and MIDD0301S (IC 50 = 25 nM)). Based on this knowledge, we used molecular operating environment (MOE) software to compute binding scores of other synthesized GABA A R ligands.…”