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

Abstract

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.

Keywords: GABAA receptor; MIDD0301; airway hyperresponsiveness; airway inflammation; asthma; imidazobenzodiazepine.

Figure 1

Structures of α₅β₃γ₂ GABA_AR selective positive allosteric modulators

Figure 2

Muscle force in guinea pig airway smooth muscle contracted with 1 μM substance P. MIDD0301 (0–100 μM) induced a significant relaxation of substance P-contracted guinea pig tracheal rings compared to vehicle (0.1% DMSO). Muscle force is expressed as a percent of the initial muscle remaining at various time points. A two-way ANOVA repeated measures analysis was used to determine significance with *, **, and *** equals p < 0.05, 0.01, or 0.001, respectively, compared to vehicle control (n = 33).

Figure 3

Effect of orally administrated MIDD0301 and salmeterol on airway hyperresponsiveness. Specific airway resistance (sRaw) was measured at increasing nebulized dosages of methacholine by DSI’s Buxco FinePointe noninvasive airway mechanics instrument (NAM). Ova s/c mice were administered vehicle or (A) 100 mg/kg, (B) 50 mg/kg, (C) 20 mg/kg of MIDD0301 via oral gavage b.i.d. for 5 days or (D) salmeterol also via oral gavage at 1 mg/kg b.i.d. for 5 days. Means ± SEM are presented for groups of 10 BALB/c mice. * and ** indicate p < 0.05 and p < 0.01 significance for the MIDD0301 group and •, ••, and ••• indicate p < 0.05, p < 0.01, and p < 0.001 significance between control mice compared to ova s/c mice using a two-way ANOVA repeated measures.

Figure 4

Pharmacokinetic profile of MIDD0301 in mouse blood, lungs, and brain. A) Time-dependent systemic distribution of MIDD0301 (25 mg/kg via oral gavage, vehicle 2% hydroxypropyl methylcellulose and 2.5% polyethylene glycol) and summary of calculated pharmacokinetic parameters; B) Sensorimotor coordination study using a rotarod apparatus with mice treated orally with 100 mg/kg MIDD0301 (vehicle 2% hydroxypropyl methylcellulose and 2.5% polyethylene glycol) (n = 9). 5 mg/kg diazepam was administered i.p. (vehicle 5% DMSO, 35% propylene glycol and 60% PBS) as control. The time that each treated mouse remained balanced on the rotating rod (15 rpm for up to 3 minutes) was recorded.

Figure 5

Induced electrophysiological responses by MIDD0301. A) Current responses of CD4⁺ T lymphocytes isolated from ova s/c mice (n = 12) in the presence of 600 nM GABA and increasing concentration of MIDD0301 applied for 3 seconds, as determined by automated patch clamp. B) Normalized current responses of isolated CD4⁺ T lymphocytes (ova s/c mice) in the presence of 600 nM GABA (100 %) and increasing concentrations of MIDD03101 for eight independent measurements with an n = 12. Data was fitted to a Y=Bottom + (Top-Bottom)/(1+10^((LogIC₅₀-X)*HillSlope)) to determine EC₅₀ and top of the curve (potentiation); C) Average enhancement of current evoked to GABA by 0.1 μM or 1 μM of MIDD0301 using transiently transfected cells with α GABA_AR subunits, as indicated, along with β₃ and γ₂L subunits measured by patch clamp. Data represent mean ± SEM with an n = 5.

Figure 6. Effect of MIDD0301 and salmeterol on airway inflammatory cells

Groups of ova s/c mice were administered vehicle, MIDD0301 (20, 50 or 100 mg/kg), or salmeterol (1 mg/kg) via oral gavage b.i.d. for 5 days. BALF was harvested from each animal and used for (A) quantification of total inflammatory cells using anti-CD45 APC antibody and flow cytometry. (B) CD4⁺ T cell, (C) F4/80⁺ cell, and (D) Siglec F⁺ cell populations were quantified by flow cytometry. Data represent mean ± SEM from 10 mice in each group. One way ANOVA was used to calculate significance indicated as *, **, and *** for p < 0.05, p < 0.01, and p < 0.001 compared to vehicle treated ova s/c mice. The gated positive events are depicted in the Supporting Information.

Figure 7. Cellular changes of the asthmatic mouse lung due to MIDD0301 treatment

Representative images of lungs from mice that were injected i.p. with EdU, a thymidine analog, and harvested four hours later. After standard histology processing, sections were treated with a fluorescent azide under “Click” chemistry conditions enabling conjugation of incorporated EdU to visualize cells that underwent the S phase during a four hour period (column 1). Slides were counterstained with Hoechst 33342 (column 2) and superimposed images are presented in column 3. Row 1 presents lung images of control mice. Row 2 depicts lung images of vehicle-treated ova s/c mice and row 3 images of MIDD0301 (100 mg/kg) treated ova s/c mice.

Figure 8. Effect of MIDD0301 and salmeterol on mucin production

A) Representative images of lung section of control mice (non-asthmatic) and ova s/c mice treated orally with vehicle, MIDD0301 (100 mg/kg, b.i.d. 5 days) or salmeterol (1 mg/kg, b.i.d. 5 days). Scale bar represents 100 μm. Slices were stained with periodic acid fluorescent Schiff’s stain coloring airway epithelium green and mucin red. B) Morphometric quantification of mucin volume density. Data represent mean ± SEM mucin volume density from six mice in each group. One-way ANOVA was used for the analysis.

Figure 9. Effects of MIDD0301 on cytokine expression in the lung

Mouse Th1/Th2/Th17 cytokines were quantified in mouse tissue homogenates using the BD mouse Th1/Th2/Th17 cytometric bead array kit. Ova s/c mice were administered vehicle or 100 mg/kg MIDD0301 (vehicle 2% hydroxypropyl methylcellulose and 2.5% polyethylene glycol) via oral gavage twice daily for 5 days. Data represent mean ± SEM from 10 mice in each group. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001 significance determined by one-way ANOVA, compared to vehicle treated ova s/c mice.