WO2008141438A1 - Gabaergic modulators for treating airway conditions - Google Patents

Abstract

The present disclosure provides uses, methods and compositions comprising GABAergic modulators such as antisense nucleic acids, antibodies or drugs for treating airway conditions in an animal. Airway conditions include conditions such as asthma, or conditions comprising altered mucus production.

Description

Title. GABAergic modulators for treating airway conditions

Field of the Disclosure

[0001] The present disclosure relates to methods and compositions for treating airway conditions. In particular, the disclosure relates to methods and compositions of GABAergic modulators for regulating airway mucus production.

Background of the Disclosure

[0002] Asthma is a prevalent human disease worldwide. The prevalence of this disease has increased significantly over the past 2-3 decades. The most significant pathological changes in asthma are airway hyper-reactivity, airway and pulmonary inflammation and mucus overproduction. In particular, mucus overproduction is the primary cause of death in severe asthma attacks¹⁶'¹⁷. However, the currently available treatments for mucus hypersecretion have limited efficacy and are largely non-specific⁹. [0003] Gamma-aminobutyric acid (GABA) is a major neurotransmitter, which, via the subtype A GABA receptors (GABAAR), induces fast inhibition in the adult mammalian brain. GABAergic transmissions are known to play an essential role in brain function³ and neural diseases⁴. The knowledge regarding the role of this system outside of brain is very limited. Recent reports showed that subunits of GABA_AR are expressed in the type Il alveolar ECs (type Il ECs)². However, it was unknown whether GABA_AR are expressed in airway ECs.

Summary of the Disclosure

[0004] The present disclosure discloses the identification of a functional excitatory, rather than inhibitory, GABAergic system in airway epithelial cells (ECs) and that this system is critically involved in the mucus overproduction in allergic asthma.

[0005] The airway apical-membrane-located GABA_AR may serve as an easily accessible target for therapeutic reagents. Indeed, intranasal administration of GABA_AR antagonists reduced the allergen-induced airway mucus overproduction in the mouse model of asthma. The present data not only demonstrate an essential role for an epithelial GABAergic system in airway mucus production, but leads to new therapeutic strategies for the management of airway conditions, such as severe asthma.

[0006] Accordingly, in one embodiment, the present disclosure provides a method of treating an airway condition comprising administering an effective amount of a GABAergic modulator to an animal in need thereof. In another embodiment, the present disclosure provides the use of a GABAergic modulator for treating an airway condition. In yet another embodiment, the present disclosure provides the use of a GABAergic modulator in the preparation of a medicament for the treatment of an airway condition. In a further embodiment, the present disclosure provides a GABAergic modulator for use in treating an airway condition. [0007] In an embodiment, the airway condition comprises altered mucus production.

[0008] In one embodiment, the GABAergic modulator is an inhibitor. In another embodiment, the GABAergic modulator is an activator.

[0009] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

[0010] The disclosure will now be described in relation to the drawings in which:

[0011] Figure 1 shows an excitatory GABAergic system in lung ECs. a. lmmunoblot of human pulmonary EC lines (BEAS-2B and A549 cells), primary human small airway ECs (SAEC) and mouse lung for GAD65/67 and GABAAR subunits (as labeled at the left of each blot). b and c. lmmunohistochemistry reveals the cellular distribution of GABAergic molecules in the mouse lung. Mouse lung ECs were demonstrated by immunostaining with an antibody to pan-cytokeratin (green), lmmunostaining of GAD65/67 (red, in b); scale bar, 40 μm. Staining of GABA_AR β2/β3 subunits (red, in c); Scale bar, 15 μm. d. The representative trace of GABA- evoked currents (left panel) and GABA-induced depolarization (right panel) in primary human SAECs. e. The GABA-induced current was blocked by picrotoxin (PTXN) (left panel). Summary of effects of PTXN and bicuculline (Bicu) on GABA-evoked currents (right panel, in comparison to control, ^*** P < 0.001 ; ** P < 0.01). f. Representative traces of GABA-currents under different holding membrane potentials (from -60 to 40 mV, 20mV-step; left panel); and summary of the endogenous resting membrane potential (E_R) and the reversal potential of GABA-induced current (EGABA) (right panel), g. PTXN induced outward current (left panel) and hyperpolarization (right panel). These data indicate a "tonic" GABAergic activity in the pulmonary ECs. h. ELISA results of the effect of GABA on BrdU incorporation to SAEC. The unit of BrdU was a value of absorbance normalized to the control reading. ^* in comparison to all other groups, P < 0.05.

[0012] Figure 2 shows OVA-treatments increase the expression of airway GABAergic signaling components, a. Representative confocal images of immunostaining of GAD (red, middle panels) in the lung tissues from control (Ctrl) and OVA-treated (OVA) mice. Scale bar, 40 μm. b. Typical confocal images of immunostaining of GABAAR β2/β3 subunits (red, middle panels) in lung tissues from control (Ctrl) and OVA-treated (OVA) mice. Scale bar, 15 μm. The immunoflourescent staining is semi-quantitatively analyzed, showing as fluorescent pixels per image field. Summaries of the fluorescence density of the GAD (in C) and β2/β3 subunit (in d) in the airway epithelium of control mice and OVA-treated mice (n = 20 image fields of 8 lung slices from 4 mice; ^***, P < 0.00001). e. Representative confocal images of - A -

immunostaining of GAD in airway biopsies from a human asthmatic subject, who was consecutively treated by inhaling diluent (control) and combined allergens (challenge) with 24 h intervals. Scale bar, 20 μm. f. Summary of the GAD fluorescence density in the airway epithelium of human biopsies (/7 = 6 cases; ^* P <0.01). g. Typical confocal images of immunostaining of GABA_AR β2/β3 subunits in airway biopsies from the same subject described in e. Scale bar, 20 μm. Insets show the increased expression of GABA_ARS in the apical membrane of airway ECs after allergen inhalation challenge.

[0013] Figure 3 shows Pulmonary IL-13 increases during allergic asthma and stimulates the expression of GAD and GABA_ARs in airway ECs. a. ELISA revealed increases of IL-13 in BAL fluid (left), in the culture supernatants of spleen cells (meddle) and draining lymph node cells (right) from the OVA-treated mice. b. Representative confocal images of immunostaining of GAD65/67 (green) in control and IL-13 (5 ng/mL for 6 d)- treated SAECs. The nuclei were stained with propidium iodide (red). Fluorescent assay showed an increase in the intensity of GAD65/67 in the IL- 13 treated cells (control 39.9 ± 5.3 pixels/225 μm², n = 63 cells; IL-13 86.2 ± 4.9 pixels/225 μm², n = 89 cells in 2 experiments; P< 0.01). c. lmmunoblot of β2-subunits in control and IL-13 treated SAECs. d. Representative confocal images show immunostaining of GAD65/67 (red) in lung tissues from the control and intranasal (i.n.) IL-13-treated mice. e. Summary of the immunofluorescence density of GAD in lung tissues from the control and IL- 13-treated mice (* P < 0.01). f. Typical confocal images show the immunofluorescent staining of GABA_AR β2 and β3 subunits (red) in lung tissues from control and IL-13-treated mice. Scale bar in d and f, 20 μm. g. Summary of the immunofluorescence density of β2 and β3 subunits in lung tissues from the control and IL-13-treated mice (* P < 0.01).

[0014] Figure 4 shows GABAergic blockade decreases OVA-induced airway goblet cell hyperplasia and mucus overproduction, a. Typical histological images (H & E staining) of lung sections from a control mouse, an

OVA-challenged (OVA) mouse, and an OVA-challenged mouse that was treated with i.n. picrotoxin (OVA+PTXN). The insets show the inflammatory cell infiltrations surrounding the airway, b. Typical PAS-staining of lung tissues from the control, OVA-treated and OVA+PTXN-treated mice. Scale bar, 60 μm. c. Representative images of immunostaining of mucin (green) with an antibody to MUC5AC. Scale bar, 50 μm. d. Summary of histological mucus index (HMI) (which indicates the proportion of mucus-producing epithelium in the bronchial epithelium) obtained from the OVA-treated and OVA+inhibitor- treated mice. Note that i.p. or i.n. application of PTXN₁ or bicuculline (Bicu) significantly reduced mucus production (^** P < 0.001 , in comparison to OVA- challenged mice; n = 3 mice), e. Summary of HMI from the IL-13-treated and IL-13+PTXN-treated mouse lung tissues (* P < 0.05, n = 3 mice), f. Summary of ELISA of IL-13 in BAL from mice in different testing groups.

[0015] Figure 5 shows RT-PCR assays of GAD and GABA_AR subunits in BEAS-2B cells. [0016] Figure 6 shows the selective GABA_AR antagonist bicuculline blocks the GABA-induced current in pulmonary EC cells. The current evoked by GABA in A549 cells was blocked by the competitive GABA_AR antagonist bicuculline methobromide (100 μmol/L).

[0017] Figure 7 shows the intracellular and extracellular alcian blue staining increase in the SAEC after GABA treatment. Shown are representative pictures of alcian blue staining (arrow) of SAECs grown under control conditions and treated with GABA or GABA plus PTXN. The enclosed square area in each picture in the upper panel is enlarged and shown in the lower panel. Scale bar, 15 μm. [0018] Figure 8 shows the expression of GAD and GABA_AR subunits increases in the lung of OVA-treated mice, lmmunoblotting assays show expression of GAD65/67 and two GABA_AR subunits in lung tissues from control (Ctrl) and OVA-treated mice. The number under each immunoblot band is the density normalized to that of β-actin. [0019] Figure 9 shows GABA_AR β2 and β3 subunits are not expressed in the airway smooth muscle cells. Confocal microscopy images show double immunostaining of a lung tissue slice from an OVA-treated mouse, with an antibody against α-smooth muscle actin (green) and the antibody to the β2 and β3 subunits of GABA_ARs (red). Note that no GABA_AR stain is co-localized with smooth muscle staining, indicative of that GABA_ARS are not expressed in airway smooth muscle cells. Scale bar, 30 μm.

Detailed description of the disclosure

[0020] In one embodiment, the present disclosure provides a method of treating an airway condition in an animal comprising administering an effective amount of a GABAergic modulator to the animal in need thereof. In another embodiment, the disclosure provides the use of a GABAergic modulator for treating an airway condition. In yet another embodiment, the disclosure provides use of a GABAergic modulator for preparing a medicament for the treatment of an airway condition. In a further embodiment, the disclosure provides a GABAergic modulator for use in treating an airway condition.

[0021] The term "GABAergic modulator" as used herein means any substance that can modulate a GABAergic system including, without limitation, a substance that inhibits the GABAergic system, a substance that activates the GABAergic system or a substance that regulates the level of this system. Such modulators can act at any step in the GABAergic pathway.

[0022] The term "effective amount" as used herein means a quantity sufficient to, when administered to an animal, effect beneficial or desired results, including clinical results, and, a such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of inhibiting the GABAergic system, for xample, it is an amoun of the GABAergic modulator sufficient to achieve such an inhibition of the GABAergic system as compared to the respond obtained without administration of the GABAergic modulator. [0023] The term "airway condition" as used herein means any condition or disease in which the airway is compromised. In one embodiment, the airway condition has altered mucus production.

[0024] The term "treating an airway condition" as used herein means improving or enhancing the clearance of the airway or improving or enhancing the breathing of the animal.

[0025] The clinical indication of mucus overproduction is high sputum production and the observation of mucus plugs which block the airway. Furthermore, not only the amount, but also the composition of mucus may be regulated. Normal mucus contains 90% water and 10% proteins as well as carbohydrates and lipids which is the adhesive partition of mucus. In some pathological conditions, including asthma, the protein portion (mucins) can increase significantly, thus the sputum becomes more sticky and more difficult to be cleared. Activation of the GABAergic system may increase mucin production, water production or increase both water and mucins. Enhancing water secretion in pathological conditions in which higher mucins exist in the mucus, such as cystic fibrosis, may help clear the mucus plug in such diseases. Moreover, since mucus production is a physiological process which is important for trapping and clearance of foreign bodies from the airway, a decrease in mucus production may be detrimental and activation of the GABAergic system could increase mucus production.

[0026] Airway conditions characterized by increased mucus production include, without limitation, asthma, bronchitis, bronchiectasis, bronchiolitis, cystic fibrosis and chronic airway inflammatory conditions, such as chronic obstructive pulmonary disease. In one embodiment, the airway condition is asthma.

[0027] The term "asthma" as used herein includes, without limitation, conditions characterized by airway hyperreactivity, inflammation and mucus overproduction. Asthma attacks typically are induced by an allergen, an infection, inhaled pollutants and even exercise. [0028] It may also be desirable to reduce the amount of water in the mucus in some disease conditions, for example, in cystic fibrosis patients.

[0029] Mucus overproduction can occur in almost all airway inflammatory conditions. Thus, in another embodiment the airway condition is a chronic airway inflammatory condition. In a particular embodiment, the airway condition is chronic obstructive pulmonary disease.

[0030] The term "animal" includes all members of the animal kingdom, preferably a mammal and more preferably a human. Accordingly, in one embodiment, the animal is a mammal. In a particular embodiment, the animal is a human.

[0031] In an embodiment, the GABAergic modulator modulates a

GABA receptor or the GAD enzyme.

[0032] The term "GABA receptor" as used herein means a member of the class of receptors that respond to gamma-aminobutyric acid. Examples of GABA receptor subtypes are a subtype GABA A receptor (GABA_AR), subtype GABA B receptor (GABA_BR) or a subtype GABA C receptor (GABA_CR).

[0033] In one embodiment, the modulator is an inhibitor of GABAAR,

GABABR, GABA_CR or GAD. In another embodiment, the modulator is an activator of GABA_AR, GABA_BR, GABA_CR or GAD. [0034] A "GABAAR activator" or "GABA_BR activator" or "GABA_CR activator" as used herein includes any substance that is capable of increasing the expression or activity of the receptor and includes, without limitation, substances that activate the receptor or the interaction of GABA with the receptor. Such activators optionally include exogenous DNA that express GABA receptors, substances that cause overexpression of GABA receptors, small molecule activators and other substances directed at the GABA receptor. In a preferred embodiment, the GABAAR actvator or GABA₆R activator or GABA_CR activator is targeted to the airway epithelium.

[0035] A "GABA_AR inhibitor" or "GABA_BR inhibitor" or "GABA_CR inhibitor" as used herein includes any substance that is capable of inhibiting the expression or activity of the receptor and includes, without limitation, substances that inhibit the receptor or the interaction of GABA with the receptor. Such inhibitors optionally include antisense nucleic acid molecules, proteins, antibodies (and fragments thereof), small molecule inhibitors, and other substances directed at the GABA receptor or one of its subunits. In a preferred embodiment, the GABA_AR inhibitor or GABA_BR inhibitor or GABA_CR inhibitor is targeted to the airway epithelium.

[0036] Accordingly, in one embodiment, the GABA_AR inhibitor is an antisense nucleic acid of a subunit of a GABA_AR, said subunit having a nucleic acid sequence shown in Table 1 (SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 51 , 53 or 55). The present inventors have confirmed 5 GABA_AR subunits that are expressed in airway epithelial cells. They are: α2-subunits, Official Symbol: GABRA2, gene ID# 2555; β1- subunit, Official Symbol: GABRB1 , gene ID# 2560; β2-subunit, Official Symbol: GABRB2, gene ID# 2561 ; δ-subunit, Official Symbol: GABRD, gene ID# 2563; π-subunit, Official Symbol: GABRP, genelD#2568). In another embodiment, the GABA_BR inhibitor is an antisense nucleic acid of a subunit of a GABABR, said subunit having a nucleic acid sequence shown in Table 2 (SEQ ID NOs: 35, 37 or 39). In yet another embodiment, the GABA₀R inhibitor is an antisense nucleic acid of a subunit of a GABA₀R, said subunit having a nucleic acid sequence shown in Table 3 (SEQ ID NOs: 41 , 43 or 45).

[0037] The term "antisense nucleic acid" as used herein means a nucleic acid that is produced from a sequence that is inverted relative to its normal presentation for transcription. Antisense nucleic acid molecules may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

[0038] In a further embodiment, the GABA_AR modulator is an antibody that binds to a subunit of a GABAAR, said subunit having the amino acid sequence as shown in Table 1 (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 52, 54 or 56). In another embodiment, the GABA_BR modulator is an antibody that binds to a subunit of a GABABR, said subunit having the amino acid sequence as shown in Table 2 (SEQ ID NOs: 36, 38 or 40). In yet another embodiment, the GABAcR modulator is an antibody that binds to a subunit of a GABAcR, said subunit having the amino acid sequence as shown in Table 3 (SEQ ID NOs: 42, 44 or 46). The sequences listed in the Tables are shown in the Appendix. The term "antibody" as used herein also includes smaller portions or fragments of the complete antibody sequence that may contain the binding portions of a given antibody sequence. The antibody can be an activator if it is stimulatory, causing GABAergic signaling through the receptor or it can be an inhibitor if it is inhibitory, blocking ligand stimulation of the receptor or altering the configuration of the receptor leading to lower infinity to GABA. [0039] The term "antibody" as used herein is intended to include fragments thereof which also specifically react with a GABA receptor or GAD, including, without limitation, Fab, F(ab)'₂ and scFv fragments. Antibodies can be prepared recombinantly as fragments or fragmented using conventional techniques and the fragments screened for utility in the same manner as described below. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

[0040] Conventional methods can be used to prepare antibodies. For example, by using a GABA receptor or GAD or fragment thereof, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the protein or fragment thereof which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a protein or fragment thereof include conjugation to carriers or other techniques well known in the art. For example, the protein or fragment thereof can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera. [0041] To produce monoclonal antibodies, antibody producing cells

(lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor, D, and Roder, J: The production of monoclonal antibodies from human lymphocytes. Immunology Today 4:3 72-79, 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96) and screening of combinatorial antibody libraries (Huse,W, Sastry.L, Iverson.S, Kang.A, Alting-Mees,M, Burton, D, Benkovic.S, and Lerner.R: Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246:4935 1275-1282, 1989). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the protein or fragment thereof and the monoclonal antibodies can be isolated. Therefore, the disclosure also contemplates hybridoma cells secreting monoclonal antibodies with specificity for a GABA receptor or GAD or fragment thereof. [0042] Chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the disclosure. Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes a GABA receptor or GAD or fragment thereof (See, for example, Morrison et al. (Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains with Human Constant Region Domains. PNAS 81 :21 6851-6855, 1984), and Takeda et al. (Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences. Nature 314:452- 454), and the patents of Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom patent GB 2177096B).

[0043] Monoclonal or chimeric antibodies specifically reactive with a a

GABA receptor or GAD or fragment thereof as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al. (Construction and Testing of Mouse-Human Heteromyelomas for Human Monoclonal Antibody Production. PNAS 80:12 7308-7312, 1983), Kozbor et al., supra; Olsson et al. (Methods in Enzymol, 92:3-16 1982) and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)

[0044] Specific antibodies, or antibody fragments, reactive against a a GABA receptor or GAD or fragment thereof may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules encoding a a GABA receptor or GAD or fragment thereof. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al. (Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 348:544-546, 1989), Huse et al., supra and McCafferty et al (Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552-555, 1989)).

[0045] Antibodies may also be prepared using DNA immunization. For example, an expression vector containing a nucleic acid encoding a a GABA receptor or GAD or fragment thereof may be injected into a suitable animal such as mouse. The protein will therefore be expressed in vivo and antibodies will be induced. The antibodies can be isolated and prepared as described above for protein immunization. [0046] A person skilled in the art could also readily make a soluble version of the GABA_AR receptor or GABA_BR receptor or GABAcR receptor, which would be expected to bind to ligand but be unable to provide GABAergic signaling, thus acting as GABAAR inhibitor or GABA_BR inhibitor or GABAcR receptor, respectively. [0047] The term "GAD enzyme" or "GAD" as used herein means glutamic acid decarboxylase which is involved in the production of GABA in the body.

[0048] A "GAD activator" as used herein includes any substance that is capable of activating the expression or activity of the GAD enzyme. Such activators include, without limitation, exogenous DNA that express GAD, substances that cause overexpression of GAD protein, small molecule activators and other substances directed at the GAD enzyme. In a preferred embodiment, the GAD activator is targeted to the airway epithelium.

[0049] A "GAD inhibitor" as used herein includes any substance that is capable of inhibiting the expression or activity of the GAD enzyme. Such inhibitors include, without limitation, antisense nucleic acid molecules, proteins, antibodies (and fragments thereof), small molecule inhibitors and other substances directed at the GAD enzyme. In a preferred embodiment, the GAD inhibitor is targeted to the airway epithelium. [0050] Accordingly, in one embodiment, the GAD inhibitor is an antisense nucleic acid of GAD having a nucleic acid sequence shown in Table 4 (SEQ ID NO: 47 or 49).

[0051] In another embodiment, the GAD inhibitor is an antibody that binds to GAD having the amino acid sequence as shown in Table 4 (SEQ ID NO: 48 or 50). In this embodiment, the term antibody also includes smaller portions or fragments of the complete antibody sequence that may contain the binding portions of a given antibody sequence.

[0052] In another embodiment, the GABAergic activator is additional

GAD enzyme targeted to the airway. In addition, since airway epithelial cells can be easily accessed, inhaling GAD siRNA may cause a limited effect localized in the airway. Accordingly, in another embodiment, the GABAergic modulator is siRNA.

[0053] The GABAergic modulators may also contain or be used to obtain or design "peptide mimetics". For example, a peptide mimetic may be made to mimic the function of a GABAergic modulator. "Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367).

[0054] Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of the secondary structures of the proteins described herein. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

[0055] In a further embodiment, a GABAergic modulating drug is used.

In one embodiment, a GABAergic inhibitor drug is used. In another embodiment, a GABAergic activator is used. [0056] In an embodiment, the GABAergic inhibitor is a GABA receptor antagonist. The term "receptor antagonist" as used herein means any molecule that blocks or decreases the amount of ligand binding to the receptor, or a molecule that binds to the ligand such that signaling through the receptor is diminished or abolished. There are many GABA_AR antagonist drugs that are known to fully or partially block different GABA A receptors and therefore can be used or administered individually or in combination to modulate or maximize the effect. Well known GABA_AR antagonist drugs include, without limitation, bicuculline, clozapine, flumazenil (Anexate) and picrotoxin. Stresam (etifoxine), Ulcon (Chlordiazepoxide) and Dehydroepiandrosterone Neurosteroid (DHEA) are also known and currently marketed GABA_AR antagonist drugs. Other known GABAAR antagonists have been described in Squires and Saederup, Neurochemical Research, Vol. 23, No. 10, 1998, pp. 1283-1290, incorporated herein by reference and also shown in Table 5. Many of these drugs are known to be clinically effective in other indications and include, without limitation, Chlorprothixene, Clomacran, Clopipazan, Fluotracen, Sulforidazine, Thioproperazine, cis- Thiothixene, Amoxapine, Clothiapine, Dibenzepine, lnkasan (Metralindole), Metiapine, Zimelidine, Bathophenanthroline disulfonate, and Isocarboxazid. Other classical antagonists are Pitrazepine, R5135, Securinine, Strychnine, Theophylline, d-Tubocurarine, cicutoxin and oenanthotoxin. In addition, many endogenous molecules such as Cortisol, and exogenous compounds such as anesthetics and alcohols can modulate the functions of GABA_ARs. Known GABABR antagonists include, without limitation, AVE1876, Inovelon, Rufinamide, SGS742, SYN111 , saclofen, phaclofen, SCH50911 , CGP35348, CGP56433A1 CGP55845A and CGP 36742. Gamma-amino butyric acid type A (GABAA) receptor inverse agonists bind to the GiABA A receptor but produce effects opposite to that of the natural ligand GABA. Known GABAAR inverse agonists include, without limitation, NGD97-1 and Suritozole.

[0057] Accordingly, in one embodiment, the GABAAR inhibitor is bicuculline, clozapine, flumazenil (Anexate) or picrotoxin, and or a pharmaceutically acceptable salt thereof or derivative thereof. In another embodiment, the GABAAR inhibitor is bicuculline, clozapine, Dehydroepiandrosterone Neurosteroid (DHEA), flumazenil (Anexate), Stresam (etifoxine), picrotoxin, Ulcon (Chordiazepoxide) or picrotoxin, and or a pharmaceutically acceptable salt thereof or derivative thereof. In a further embodiment, the GABA_AR inhibitor is a drug listed in Table 5 and or a pharmaceutically acceptable salt thereof or derivative thereof.

[0058] In another embodiment, the GABAergic activator is a GABA receptor agonist. The term "receptor agonist" as used herein means any molecule that increases receptor signaling or ligand binding to the receptor. Known GABABR agonist drugs include, without limitation, Acamprol, Acamprosate Calcium, ADX71441 , AGI006, Backen, Baclan, Baclofen, Befon, Campral, Clofen, DL404, Kemstro, Liofen, Lioresal, Muscimol, NS-11, Riclofen, SKF97541 , Stelax, Tefsole, XP19986 and Xyrem (sodium oxybate). Partial GABAAR agonists include, without limitation, adipiplon (NG2-73). [0059] The term "pharmaceutically acceptable" as used herein means compatible with the treatment of animals, in particular, humans. [0060] The term "pharmaceutically acceptable salt" as used herein means an acid addition salt or base addition salt which is suitable for or compatible with the treatment of patients. The selection of appropriate salts that maintain activity will be known to one skilled in the art. [0061] The term "derivative" as used herein refers to a modification, for example, a chemical modification to the drug. The selection of appropriate derivatives that maintain activity will be known to one skilled in the art.

[0062] Conventional treatment may also be used in combination with the methods and uses of the disclosure. [0063] The currently used agents used for treatment of mucus hypersecretion in asthma include, without limitation, bronchodilators such as β-Adrenergic agonists that can increase ciliary best frequency thus helpful for clearing the mucus, anti-inflammatory agents such as inhaled corticosteroids that can inhibit inflammation thus reducing cytokine/factor production that promote mucus production, mucolytic agents such as N-acetylcystein and S- carboxymethyl cysteine that can break the disulfide bonds bridging mucin chains thus reducing the viscosity of mucus, and expectorants such as ammonium chloride that can make the sputum to be easier coughing up. Accordingly, in another embodiment, the methods and uses of the disclosure further comprise at least one convential asthma treatment selected from the group consisting of brochodilators, anti-inflammatory agents, mucolytic agents and expectorants.

[0064] The disclosure also provides a pharmaceutical composition for treating an airway condition in an animal in need thereof comprising a GABAergic modulator and a pharmaceutically acceptable carrier, diluent or excipient.

[0065] The disclosure further provides a pharmaceutical composition for modulating airway mucus overproduction in an animal comprising a GABAergic modulator and a pharmaceutically acceptable carrier, diluent or excipient. [0066] The GABAergic modulators may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals. Administration of a therapeutically active amount of the pharmaceutical compositions of the present applicataion is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of protein to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0067] The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, intramuscular, etc.), oral administration, inhalation, intranasal, transdermal administration (such as topical cream or ointment, etc.), or suppository applications. In one embodiment, the active substance is administered by inhalation or intranasally. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. The active substance may be formulated into delayed release formulations such that mucus overproduction can be prevented for longer periods of time than a conventional formulation.

[0068] The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences (2000 - 20th edition) Mack Publishing Company). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0069] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0070] The following non-limiting examples are illustrative of the present disclosure:

Examples Results: [0071] lmmunoblot assays were performed to examine the expression of GABA_AR subunits and GAD65 and 67 (GAD65/67) in pulmonary ECs. The results showed that GAD65/67 and several types of GABAAR subunits were distinctively expressed in the human bronchial EC line (BEAS-2B cells) and human alveolar type Il EC line (A549 cells), as well as in primary human small airway ECs (SAEC) and mouse lung tissues (Fig. 1a). Notably, the expression profiles of GAD and GABA_ARS in BEAS-2B cells were similar to that in mouse lung tissues. In line with the immunoblotting results, reverse transcription polymerase chain reaction (RT-PCR) assays detected the mRNAs encoding GAD67 and α2, β2 and π subunits of GABA_ARs in BEAS-2B cells (Fig. 5). These results suggested that GABAergic signaling -related molecules are expressed in airway ECs. To investigate the cellular localization of the GABAergic system in the lung, immunoflourescent staining of GAD65/67 and GABAARS was carried out in the naive mouse lung slices. Since immunoblot revealed a higher level of the GABAAR β2 subunit in the mouse lung (Fig. 1a), and since in neurons β2- or β3-subunits are required for most of functional GABAARS⁵, GABAARS were stained using an antibody recognizing both the β2 and β3 subunits. Confocal microscopy of the stained tissues revealed that GAD65/67 was expressed in all ECs in the bronchial airway (Fig. 1b), but in only a small proportion of the alveolar ECs (Fig. 1b, inset). The GABAAR β2 or β3 subunit was stained on the apical membrane of a small proportion of airway ECs (Fig. 1c), and certain alveolar ECs (Fig. Ic, inset). These results showed that under normal conditions, GABA_ARs and the GABA-production mechanism are indeed present, albeit at low levels, in bronchial airway ECs, thus forming a complete GABAergic system. [0072] GABAARS are pentameric Cl^" channels. Perforated patch recordings were performed in widely-used primary human SAECs⁶ and primary human type Il ECs⁷. Under voltage-clamp mode at a holding potential of -60 mV, application of GABA (100 μmol/L) evoked rapid inward current in 4 of 26 tested SAECs (Fig. 1d, left), whereas 4 of 4 tested type Il ECs generated inward currents in response to GABA (data not shown). Under current-clamp mode, GABA induced a membrane depolarization in these cells (Fig. 1d, right). The GABA-evoked currents were effectively blocked by the GABAAR antagonists picrotoxin (50 μmol/L) (Fig. 1e), or bicuculline (100 μmol/L) (Fig. 6 and Fig. 1e, right). Further studies revealed that the reversal potential of GABA-currents (EQ_AB_A) in A549 cells (Fig. 1f) was -12 ± 2.4 mV (n = 7), and the "resting" potential (E_R) of these cells was -43 ± 2.6 mV (n = 9) (Fig. 1f, right). These analyses predict that GABA_ARs in lung ECs mediate an anionic efflux. Moreover, application of picrotoxin to A549 cells generated an outward current under voltage-clamp mode (Fig. 1g left, n = 6) and induced hyperpolarization under current-clamp conditions (Fig. 1g, right, n = 5). These results suggested that the autocrine or paracrine GABA system persistently maintains pulmonary ECs in a depolarized state.

[0073] Whether the airway epithelial GABAergic system plays a role in physiological or pathological processes was then studied. It has been well established that Cl^~ transport in airway ECs crucially regulates the cell proliferation and mucus production. Using the bromdeoxyuridine (BrdU) cell proliferation assay, the present inventors found that treating the cultured primary SAECs with GABA increased the BrdU incorporation, while addition of picrotoxin reduced the action of GABA (Fig. 1h), In addition, treating SAECs with GABA increased both intracellular and extracellular stains of alcian blue (Fig. 7), which implied an increased production and secretion of mucin-like glycoprotein⁸. These results imply that GABA_AR activation enhances the proliferation of airway ECs and increases the production of mucus in the airway epithelium. [0074] Airway goblet cell hyperplasia and mucus overproduction are prominent pathological changes of the exacerbation of asthma⁹. To investigate whether the GABA signaling contributes to the pathogenesis of asthma, mice were sensitized and then challenged with OVA, a widely-used approach for inducing allergic asthma-like reactions in animals¹⁰. Remarkably, the expression of GAD in the cytosol (Fig. 2a, c) and GABA_AR subunits in the apical membrane of mouse airway ECs (Fig. 2b, d) increased significantly following the allergen challenge, lmmunoblots of the mouse lung tissues confirmed an increase in the expression of GAD and GABA_ARs (Fig. 8). Similarly, immunohistochemistry of human biopsies revealed that the expression of GAD in the cytosol (Fig. 2e,f, n = 6) and GABAAR subunits in the apical membrane (Fig. 2g) of the airway ECs increased significantly in the tissues taken from asthmatic subjects 24 h after allergen inhalation challenge, in comparison to the samples collected from the same subject before the allergen challenge. These results indicate that the GABAergic system in the airway ECs is indeed up-regulated during asthmatic reactions. Of note, the GABAAR subunits were not stained in the smooth muscles of airways in naϊve or OVA-treated mice (Fig. 9), nor in human airway smooth muscle cells (not shown), which implied that the GABA signaling is selectively associated with epithelial cells.

[0075] We then determined the factor(s) that mediate the allergen- induced increase of GABA signaling molecules in airway ECs. IL-13, a classical TH2 cytokine, could be one of the factors, because it is a key regulator of allergic asthma¹¹'¹². Using previously described assays¹³, we confirmed that the levels of IL-13 in the bronchoalveolar lavage (BAL) and the production of IL-13 by lymphocytes were elevated in OVA-treated mice (Fig. 3a). Treating the cultured SAECs with IL-13 increased the expression of GAD (Fig. 3b) and GABAAR β2 subunits (Fig. 3c). In addition, 8 out of 12 (67%) cells in the IL-13-treated dishes exhibited currents (9.2 ± 3.0 pA, n = 8) in response to application of GABA, whereas only 2 out of 10 (20%) cells in control dishes did so. In line with these in vitro results, intranasal (i.n.) application of IL-13 to mice significantly enhanced the expression of GAD (Fig. 3d,e) and GABA_AR β2 and β3 subunits (Fig. 3f,g) in bronchial ECs. These data indicate that IL-13 plays a crucial role in initiating the airway epithelial GABA signaling in allergic asthma.

[0076] To confirm the causal relationship between the epithelial GABA signaling and the hyperplasia of goblet cells in asthmatic reactions, the OVA- sensitized/challenged mice were treated with picrotoxin or bicuculline via intraperitoneal (i.p.) or i.n. application. Blocking GABA signaling lessened the airway epithelium swelling (Fig. 4a, b) and goblet cells hyperplasia (Fig. 4c). Importantly, the OVA-induced mucus overproduction by airway ECs was significantly reduced by picrotoxin (Figs. 4b,c,d), or by bicuculline (Fig. 4d). The IL-13-induced mucus overproduction was also suppressed by i.n picrotoxin (Fig. 4e). However, blocking GABA_AR failed to affect the OVA- induced increase of IL-13 in the lung (Fig. 4f). In addition, i.n. or i.p. GABA_AR inhibitors failed to block the OVA-induced inflammatory cell infiltrations in the sub-epithelial interstitial tissue of the airway wall (Fig. 4a inset) or in BAL. Considering that IL-13 is produced primarily by T_H2 cells after allergen challenge, we propose that up-regulation of the epithelial GABAergic system is down-stream of IL-13 receptor activation, and that this GABAergic system plays a selective role in goblet cell metaplasia and mucus overproduction.

[0077] This study demonstrates that a novel autocrine/paracrine GABAergic system in the airway ECs plays an essential role in the process of goblet cell hyperplasia and mucus overproduction. In animal asthmatic models and human asthmatic subjects, the expression of GABAergic signaling molecules was up-regulated following allergen challenge, demonstrating a true relevance of this GABAergic system in asthma. IL-13 plays a critical role in regulating the airway epithelial GABA signaling. In this regard, previous studies have shown that cytokines enhance the expression of GABAARS in neurons¹⁴, and GABA_AR activation induces neural progenitor differentiation¹⁵.

[0078] Indeed, intranasal administration of GABA_ARs antagonists reduced the allergen-induced airway mucus overproduction in the mouse model of asthma.

METHODS

[0079] Mouse models of allergic asthmatic reactions. Allergic asthmatic reactions were induced in mice using two methods. With the first method as previously described¹³, female BALB/c mice (6 to 8 weeks old, from Charles River Laboratories) were initially sensitized with 2 μg OVA (ICN Biomedicals) in 2 mg AI(OH)₃, via i.p. injection. Two weeks after sensitization, the mice were challenged with 50 μg of OVA (40 μL i.n.). With the second method, recombinant IL-13 (purchased from eBioscience) was administered via i.n. application to female BALB/c mice at 0.5 μg/40μL, on the 1^st, 3^rd and 5^th d. Starting from the day of intranasal OVA challenge, or intranasal administration of IL-13, mice in one group were treated daily with picrotoxin (PTXN, i.p., 0.2 μg/g body weight in 200 μL, or i.n., 0.2 μg/g body weight in 50 μL), or by bicuculline (i.n. 2 μg/g body weight in 40 μL). Mice were sacrificed at day 6 after the OVA challenge, or the IL-13 treatment. All mice used in this study were housed in the Central Animal Care Facility of the University of Manitoba, and the experimental protocols were approved by the Animal Use Committee of the University of Manitoba.

[0080] Mice assessed for the development of AHR subjected to a model of OVA challenge, where the total respiratory system resistance (RRS) is a prominent outcome. Female BALB/c mice, aged 10 to 12 weeks, were purchased from Harlan Sprague Dawley. All mice were housed in environmentally controlled, specific pathogen-free conditions for a one-week acclimatization period and throughout the duration of the studies. All procedures were approved by the Animal Research Ethics Board at McMaster University, and conformed to the NIH guidelines for experimental use of animals. Mice were sensitized as previously described¹⁸. Briefly, all mice received i.p. injections of OVA conjugated to Al (OH)3 on Days 1 and 11 , and i.n. OVA on Day 11. At day 29 and 30, the sensitized mice were subjected to i.n administration of OVA (100 μg in 25 μl_ saline). In a subgroup of these OVA-challenged mice, bicuculline (2 μg/g body weight in 40 μl_) was given i.n. RRS responses to intravenous saline and increasing doses of methacholine (MCh) were performed at the 24^th h after the second OVA challenge using the FlexiVent ventilator system (SCIREQ)¹⁸

[0081] BAL analyses. As previously described¹⁹, the trachea of each mouse was cannulated after euthanasia, and the lungs were washed twice with 1 mL phosphate buffer solution (PBS). Cells in the fluid samples were counted, and the samples were then spun down. The pellets were re- suspended with saline, and slides were prepared for differential cell counting. The cells on the slides were stained with Fisher Leukostat Stain Kit (Fisher Scientific). The numbers of monocytes, lymphocytes and eosinophils (identified by morphology and staining characteristics) in a total of 200 cells on each slide were counted. The level of IL-13 in the BAL fluid samples was measured using enzyme linked immunosorbent assay (ELISA) as previously described¹³. [0082] Western blotting. Cultured lung ECs and mouse lung tissues were lysed in ice-cold PBS with 1% Triton X-100 and 0.5% sodium deoxycholate supplemented with protease inhibitors. The general procedures of Western blotting were the same as previously described²⁰. The antibodies to GAD 65/67, GABA_AR-α5 and β-actin were purchased from Sigma. The antibody to GABA_AR-α2 was from Alomone Labs. The antibodies to GABA_AR- β1 and β3 were from Affinity Bioreagents. The antibodies to GABA_AR-β2 and - δ were from Chemicon, and the antibody to GABA_AR-π was from Abeam. For quantification, the blotting films were scanned by means of a GS800 densitometer (Bio-Rad), and the band densities were calculated using the Quantity One program (Bio-Rad). The blotting assays were repeated at least 3 times with lung tissue samples from 3 mice. For blotting assays of GAD and most GABA_AR subunits, the mouse cerebral cortex was used as the positive control. For the π subunit, the Jacket cell lysate was used as the positive control.

[0083] Human airway biopsies. Airway biopsies were obtained from six subjects with mild asthma and using no medication other than infrequent (< 5 times weekly) inhaled β2-agonists to treat their symptoms. The subjects had not had an asthma exacerbation or a respiratory tract infection for at least 4 weeks before the study. The diagnosis of asthma was based on the presence of variable airflow limitation and airway hyperresponsiveness²¹. All subjects were nonsmokers and demonstrated an allergen-induced early and late asthmatic response²². These subjects underwent sequential diluent (control) and allergens inhalation challenges as described previously²³'²⁴, separated by a period of at least 3 weeks. Fibreoptic bronchoscopy and endobronchial biopsy was performed according to the recommendations of the U.S. National Institutes of Health²⁵, 24 h after challenge. Mucosal biopsies were taken from the segmental and subsegmental carinae of the lung and fixed in 10% buffered formalin for 24 h. The study of allergen induced airway responses in mild asthmatic subjects was reviewed and approved by the Human Research Ethics Board of McMaster University before the study began and all subjects gave informed consent before being enrolled into the study. [0084] lmmunohistochemistry and confocal microscopy. Paraffin sections of mouse lung tissue and human bronchial airway biopsy were deparaffinized with xylene and then dehydrated in 100%, 95%, and 70% ethanol. Epitopes were unmasked by heating the tissue sections in citrate buffer at pH 6 in a microwave. The tissues were permeabilized with 0.1% Triton X-100 and blocked with 10% normal goat serum for 1 h. The slices were incubated overnight with primary antibodies (antibody to GAD 65/67, 1 :800 dilution; antibody to GABA_AR β2 and β3, 1 :100 dilution; Upstate; antibody to MUC5AC/clone 45M1 , 2 μg/ml; Lab Vision Corp; antibody to α smooth muscle actin, 1 :1,000 dilution; Abeam), and subsequently with CY3- conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. An FITC-conjugated antibody to pan-cytokeratin antibody (1 :100 dilution; Sigma) was used to visualize ECs in the lung. When mouse monoclonal antibodies were used on mouse sections, immunofluorescence was performed using mouse on mouse (M. O. M.) kit (Vector Laboratories). Controls were performed either without primary antibodies or incubated in mouse IgG (Santa Cruz Biotechnology) to ensure stain specificity. The immunohistochemistry of each protein was repeated in lung tissue slices of 3 to 6 mice, lmmunocytochemistry of cultured cells was performed as previously described²⁰²⁶. Confocal images of stained lung tissue or lung ECs were studied via an inverted microscope (Carl Zeiss) using the Zeiss LSM program. The fluorescence intensity for a specific protein stain was set below the threshold for the negative control. Digital images of lung tissues containing small airways and/or alveolar structures were obtained for analysis. [0085] H & E staining and mucus analysis. Lung tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, stained by hematoxylin and eosin (H & E) and examined for pathological changes under light microscopy. Mucus and mucus-containing goblet cells within the bronchial epithelium were stained with a periodic acid-Schiff (PAS) staining kit (Sigma). The histological mucus index (HMI), the percentage of mucus- positive area of the whole bronchial epithelium¹³, was determined by Image- Pro Plus software (Media Cybernetics). [0086] Cell culture. Lung ECs were plated in culture dishes (Nunc) or on glass coverslips and incubated at 37⁰C in a humidified atmosphere of 5% CO₂. Isolated primary human type Il ECs²⁷, BEAS-2B cells and A549 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. The primary human SAECs were purchased from Cambrex Bio Science Walkersville, Inc., and were cultured in small-airway growth media (SAGM, Cambrex Bio Science Walkersville, Inc.) on dishes coated with collagen I (BD Biosciences). Procedures of culturing lymphocytes from spleen and draining lymph nodes of OVA-treated mice and analyzing allergen-driven IL-13 production by these cells were performed as previously described¹³²⁸.

[0087] BrdU Assay. The SAEC proliferation was quantified by measuring the ability of cells to incorporate bromdeoxyuridine (BrdU) using ELISA. For the assay, SAEC were seeded on to 96-well plates at a density of 3x10³/well. Twenty four hours after seeding, triplicate wells were treated with GABA (10 μmol/L), picrotoxin (PTXN, 25 μmol/L), or GABA plus PTXN for 24 h. BrdU (10 μmol/L) was added in the cultures 6h before assay, which was performed in accordance with the manufacture's instructions provided with the BrdU ELISA Kit (Roche Applied Science). [0088] Electrophysiology. After removal of the culture media, lung

ECs were bathed in a solution that contained (in mmol/L): 155 NaCI, 1.3 CaCI₂, 5.4 KCI, 25 HEPES, and 33 glucose, at pH 7.4 and osmolarity about 315 mOsm. An Axopatch-1 D amplifier (Axon Instruments) was used to make perforated patch recordings at room temperature. The patch electrodes were filled with a solution that contained (in mmol/L) 155 KCI, 15 KOH, 10 HEPES, 2 MgCI₂, 1 CaCI₂, and 2 tetraethylammonium, at pH 7.35 and osmolarity 315 mOsm. Gramicidin (15-20 μg/mL)²⁹ was included in the electrode solution for membrane perforation. Application of the GABA_AR agonist and/or antagonist was achieved via a computer-controlled multibarrel perfusion system (SF- 77B, Warner Instruments). Electrical signals were digitized and filtered (1-2 kHz). Transmembrane currents were acquired on-line by means of Clampex (Axon Instruments), and the data were analyzed off-line using Clampfit (Axon Instruments).

[0089] Statistical Analysis. Statistical analyses were performed with

Sigmaplot software (SPSS). Data are expressed as mean ± standard error of the mean (s.e.m.) and were examined with Student's unpaired or paired t tests when appropriate. A p value less than 0.05 was considered significant.

[0090] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0091] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1: Human GABA-A Receptor Subunits

Table 2: Human GABA-B Receptor Subunits

Table 3: Human GABA-C Receptor Subunits

Table 4: Human GAD enzymes

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Claims

Claims:

1. A use of an effective amount of a GABAergic modulator for treating an airway condition in an animal in need thereof.

2. The use of claim 1 , wherein the airway condition comprises altered mucus production.

3. The use of claim 1 or 2, wherein the GABAergic modulator is a GABAergic activator.

4. The use of claim 3, wherein the GABAergic activator activates GABAAR, GABA_BR, GABA_CR or GAD.

5. The use of claim 1 or 2, wherein the GABAergic modulator is a GABAergic inhibitor.

6. The use of claim 5, wherein the airway condition is asthma.

7. The use of claim 5 or 6, wherein the GABAergic inhibitor is a GABA_AR inhibitor, a GABA₈R inhibitor, a GABAcR inhibitor or a GAD inhibitor.

8. The use of claim 7, wherein the GABAAR inhibitor is an antisense nucleic acid sequence of a subunit of GABAAR, said subunit having a sequence as shown in SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 51 , 53 or 55.

9. The use of claim 7, wherein the GABA_AR inhibitor is a blocking antibody against the GABA_AR.

10. The use of claim 9, wherein the blocking antibody against the GABA_AR binds to a subunit of GABAAR, said subunit having a sequence as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 52, 54 or 56.

11. The use of claim 7, wherein the GABA_AR inhibitor is bicuculline, clozapine, flumazenil (Anexate) or picrotoxin, and or a pharmaceutically acceptable salt thereof.

12. The use of claim 7, wherein the GABA_AR inhibitor is bicuculline, clozapine, Dehydroepiandrosterone Neurosteroid (DHEA), flumazenil

(Anexate), Stresam (etifoxine), picrotoxin, Ulcon (Chordiazepoxide) or picrotoxin, and or a pharmaceutically acceptable salt thereof.

13. The use of claim 7, wherein the GABA_AR inhibitor is a drug listed in Table 5 and or a pharmaceutically acceptable salt thereof.

14. The use of claim 7, wherein the GABABR inhibitor is an antisense nucleic acid sequence of a subunit of GABA_BR, said subunit having a sequence as shown in SEQ ID NOs: 35, 37 or 39.

15. The use of claim 7, wherein the GABABR inhibitor is a blocking antibody against the GABABR.

16. The use of claim 15, wherein the blocking antibody against the GABABR binds to a subunit of GABA_BR, said subunit having a sequence as shown in SEQ ID NOs: 36, 38 or 40.

17. The use of claim 7, wherein the GABAcR inhibitor is an antisense nucleic acid sequence of a subunit of GABAcR₁ said subunit having a sequence as shown in SEQ ID NOs: 41 , 43 or 45.

18. The use of claim 7, wherein the GABAcR inhibitor is a blocking antibody against the GABAcR.

19. The use of claim 18, wherein the blocking antibody against the GABAcR binds to a subunit of GABA_CR, said subunit having a sequence as shown in SEQ ID NOs: 42, 44 or 46.

20. The use of claim 7, wherein the GAD inhibitor is an antisense nucleic acid sequence of a subunit of GAD, said subunit having a sequence as shown in SEQ ID NOs: 47 or 49.

21. The use of claim 7, wherein the GAD inhibitor is a blocking antibody against GAD.

22. The use of claim 21 , wherein the blocking antibody against GAD binds to a subunit of GAD, said subunit having a sequence as shown in SEQ ID NOs: 48 or 50.

23. The use of any one of claims 1-22, wherein the animal is a mammal.

24. The use of claim 23, wherein the animal is a human.

25. The use of any one of claims 1-24 wherein the GABAergic modulator is administered by inhalation or intranasally.

26. A pharmaceutical composition comprising a GABAergic modulator and a pharmaceutically acceptable carrier for modulating mucus production in an animal.

27. A pharmaceutical composition comprising a GABAergic modulator and a pharmaceutically acceptable carrier for treating an airway condition.

28. The composition of claims 26 or 27, wherein the GABAergic modulator is a GABAergic activator.

29. The composition of claims 26 or 27, wherein the GABAergic modulator is a GABAergic inhibitor.