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

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1ß3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

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

We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting ß2-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 GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1ß3γ2 GABAAR 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.

Development of a Novel, Small-Molecule Brain-Penetrant Histone Deacetylase Inhibitor That Enhances Spatial Memory Formation in Mice.

Histone acetylation is a prominent epigenetic modification linked to the memory loss symptoms associated with neurodegenerative disease. The use of existing histone deacetylase inhibitor (HDACi) drugs for treatment is precluded by their weak blood-brain barrier (BBB) permeability and undesirable toxicity. Here, we address these shortcomings by developing a new class of disulfide-based compounds, inspired by the scaffold of the FDA-approved HDACi romidepsin (FK288). Our findings indicate that our novel compound MJM-1 increases the overall level of histone 3 (H3) acetylation in a prostate cancer cell line. In mice, MJM-1 injected intraperitoneally (i.p.) crossed the BBB and could be detected in the hippocampus, a brain region that mediates memory. Consistent with this finding, we found that the post-training i.p. administration of MJM-1 enhanced hippocampus-dependent spatial memory consolidation in male mice. Therefore, MJM-1 represents a potential lead for further optimization as a therapeutic strategy for ameliorating cognitive deficits in aging and neurodegenerative diseases.

Identification and Quantification of MIDD0301 Metabolites.

BACKGROUND: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. OBJECTIVE: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301. METHODS: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration. RESULTS: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma. CONCLUSION: MIDD0301 undergoes no phase I and moderate phase II metabolism.

Nebulized MIDD0301 Reduces Airway Hyperresponsiveness in Moderate and Severe Murine Asthma Models.

We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABAAR) 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 GABAAR without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation.

The Effects of pH on the Structure and Bioavailability of Imidazobenzodiazepine-3-Carboxylate MIDD0301.

We describe the effects of pH on the structure and bioavailability of MIDD0301, an oral lead compound for asthma. MIDD0301 interacts with peripheral GABAA 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 seven-membered 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 1H 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.