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WO2008141438A1 - Modulateurs gabaergiques destinés au traitement d'affections des voies respiratoires - Google Patents

Modulateurs gabaergiques destinés au traitement d'affections des voies respiratoires Download PDF

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WO2008141438A1
WO2008141438A1 PCT/CA2008/000942 CA2008000942W WO2008141438A1 WO 2008141438 A1 WO2008141438 A1 WO 2008141438A1 CA 2008000942 W CA2008000942 W CA 2008000942W WO 2008141438 A1 WO2008141438 A1 WO 2008141438A1
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gaba
gabaergic
inhibitor
subunit
airway
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Wei-Yang Lu
Xi Yang
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Sunnybrook Health Sciences Centre
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Sunnybrook Health Sciences Centre
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/12—Mucolytics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present disclosure relates to methods and compositions for treating airway conditions.
  • the disclosure relates to methods and compositions of GABAergic modulators for regulating airway mucus production.
  • GABA Gamma-aminobutyric acid
  • GABAAR subtype A GABA receptors
  • 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.
  • ECs airway epithelial cells
  • the airway apical-membrane-located GABA A R may serve as an easily accessible target for therapeutic reagents. Indeed, intranasal administration of GABA A R 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.
  • 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.
  • the present disclosure provides the use of a GABAergic modulator for treating an airway condition.
  • the present disclosure provides the use of a GABAergic modulator in the preparation of a medicament for the treatment of an airway condition.
  • the present disclosure provides a GABAergic modulator for use in treating an airway condition.
  • the airway condition comprises altered mucus production.
  • the GABAergic modulator is an inhibitor. In another embodiment, the GABAergic modulator is an activator.
  • 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).
  • SAEC primary human small airway ECs
  • 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 ⁇ m.
  • FIG. 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 ⁇ m.
  • b Typical confocal images of immunostaining of GABAAR ⁇ 2/ ⁇ 3 subunits (red, middle panels) in lung tissues from control (Ctrl) and OVA-treated (OVA) mice. Scale bar, 15 ⁇ m.
  • the immunoflourescent staining is semi-quantitatively analyzed, showing as fluorescent pixels per image field.
  • FIG. 3 shows Pulmonary IL-13 increases during allergic asthma and stimulates the expression of GAD and GABA A Rs 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).
  • c lmmunoblot of ⁇ 2-subunits in control and IL-13 treated SAECs.
  • Representative confocal images show immunostaining of GAD65/67 (red) in lung tissues from the control and intranasal (i.n.) IL-13-treated mice.
  • Figure 4 shows GABAergic blockade decreases OVA-induced airway goblet cell hyperplasia and mucus overproduction, a.
  • OVA-challenged (OVA) mouse OVA-challenged mouse
  • OVA+PTXN OVA-challenged mouse that was treated with i.n. picrotoxin
  • 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 ⁇ m.
  • HMI histological mucus index
  • Figure 5 shows RT-PCR assays of GAD and GABA A R subunits in BEAS-2B cells.
  • Figure 6 shows the selective GABA A R 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 A R antagonist bicuculline methobromide (100 ⁇ mol/L).
  • 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 ⁇ m.
  • Figure 8 shows the expression of GAD and GABA A R subunits increases in the lung of OVA-treated mice, lmmunoblotting assays show expression of GAD65/67 and two GABA A R subunits in lung tissues from control (Ctrl) and OVA-treated mice.
  • FIG. 9 shows GABA A R ⁇ 2 and ⁇ 3 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 ⁇ -smooth muscle actin (green) and the antibody to the ⁇ 2 and ⁇ 3 subunits of GABA A Rs (red). Note that no GABA A R stain is co-localized with smooth muscle staining, indicative of that GABA A RS are not expressed in airway smooth muscle cells.
  • Scale bar 30 ⁇ m.
  • 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.
  • the disclosure provides the use of a GABAergic modulator for treating an airway condition.
  • the disclosure provides use of a GABAergic modulator for preparing a medicament for the treatment of an airway condition.
  • the disclosure provides a GABAergic modulator for use in treating an airway condition.
  • GABAergic modulator 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.
  • 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.
  • 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.
  • treating an airway condition means improving or enhancing the clearance of the airway or improving or enhancing the breathing of the animal.
  • 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.
  • 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.
  • 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.
  • the airway condition is asthma.
  • asthma 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.
  • the airway condition is a chronic airway inflammatory condition.
  • the airway condition is chronic obstructive pulmonary disease.
  • 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.
  • the GABAergic modulator modulates a
  • GABA receptor or the GAD enzyme.
  • GABA receptor means a member of the class of receptors that respond to gamma-aminobutyric acid.
  • GABA receptor subtypes are a subtype GABA A receptor (GABA A R), subtype GABA B receptor (GABA B R) or a subtype GABA C receptor (GABA C R).
  • the modulator is an inhibitor of GABAAR
  • GABABR GABA C R or GAD.
  • the modulator is an activator of GABA A R, GABA B R, GABA C R or GAD.
  • a "GABAAR activator” or “GABA B R activator” or “GABA C R 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.
  • the GABAAR actvator or GABA 6 R activator or GABA C R activator is targeted to the airway epithelium.
  • a "GABA A R inhibitor” or “GABA B R inhibitor” or “GABA C R 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 A R inhibitor or GABA B R inhibitor or GABA C R inhibitor is targeted to the airway epithelium.
  • the GABA A R inhibitor is an antisense nucleic acid of a subunit of a GABA A R, 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 A R subunits that are expressed in airway epithelial cells.
  • the GABA B R 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).
  • the GABA 0 R inhibitor is an antisense nucleic acid of a subunit of a GABA 0 R, said subunit having a nucleic acid sequence shown in Table 3 (SEQ ID NOs: 41 , 43 or 45).
  • antisense nucleic acid 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.
  • the GABA A R 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).
  • the GABA B R 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).
  • 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.
  • 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.
  • 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.
  • polyclonal antisera or monoclonal antibodies can be made using standard methods.
  • a mammal e.g., a mouse, hamster, or rabbit
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
  • 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.
  • 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)).
  • Antibodies may also be prepared using DNA immunization.
  • 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.
  • a person skilled in the art could also readily make a soluble version of the GABA A R receptor or GABA B R 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 B R inhibitor or GABAcR receptor, respectively.
  • GABA enzyme or "GAD” as used herein means glutamic acid decarboxylase which is involved in the production of GABA in the body.
  • 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.
  • the GAD activator is targeted to the airway epithelium.
  • 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).
  • 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).
  • 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.
  • the GABAergic activator is additional
  • the GABAergic modulator is siRNA.
  • the GABAergic modulators may also contain or be used to obtain or design "peptide mimetics".
  • 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).
  • 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.
  • 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.
  • a GABAergic modulating drug is used.
  • a GABAergic inhibitor drug is used.
  • a GABAergic activator is used.
  • 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.
  • GABA A R 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.
  • GABA A R 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 A R 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.
  • 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.
  • GABABR antagonists include, without limitation, AVE1876, Inovelon, Rufinamide, SGS742, SYN111 , saclofen, phaclofen, SCH50911 , CGP35348, CGP56433A1 CGP55845A and CGP 36742.
  • GABAA Gamma-amino butyric acid type A
  • Known GABAAR inverse agonists include, without limitation, NGD97-1 and Suritozole.
  • the GABAAR inhibitor is bicuculline, clozapine, flumazenil (Anexate) or picrotoxin, and or a pharmaceutically acceptable salt thereof or derivative thereof.
  • 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.
  • the GABA A R inhibitor is a drug listed in Table 5 and or a pharmaceutically acceptable salt thereof or derivative thereof.
  • 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).
  • pharmaceutically acceptable means compatible with the treatment of animals, in particular, humans.
  • pharmaceutically acceptable salt 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.
  • derivative 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.
  • the currently used agents used for treatment of mucus hypersecretion in asthma include, without limitation, bronchodilators such as ⁇ -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.
  • 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.
  • 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.
  • 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.
  • the GABAergic modulators may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo.
  • 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.
  • 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.
  • 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.
  • the active substance is administered by inhalation or intranasally.
  • 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.
  • 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).
  • 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.
  • RT-PCR reverse transcription polymerase chain reaction
  • GABAARS were stained using an antibody recognizing both the ⁇ 2 and ⁇ 3 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 ⁇ 2 or ⁇ 3 subunit was stained on the apical membrane of a small proportion of airway ECs (Fig. 1c), and certain alveolar ECs (Fig. Ic, inset).
  • GABAARS are pentameric Cl " channels.
  • Perforated patch recordings were performed in widely-used primary human SAECs 6 and primary human type Il ECs 7 . Under voltage-clamp mode at a holding potential of -60 mV, application of GABA (100 ⁇ mol/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).
  • GABAAR subunits were not stained in the smooth muscles of airways in na ⁇ ve 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.
  • IL-13 a classical TH2 cytokine
  • BAL bronchoalveolar lavage
  • IL-13 a classical TH2 cytokine
  • the IL-13-induced mucus overproduction was also suppressed by i.n picrotoxin (Fig. 4e).
  • blocking GABA A R failed to affect the OVA- induced increase of IL-13 in the lung (Fig. 4f).
  • GABA A R 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.
  • IL-13 is produced primarily by T H 2 cells after allergen challenge
  • 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.
  • mice Mouse models of allergic asthmatic reactions. Allergic asthmatic reactions were induced in mice using two methods. With the first method as previously described 13 , female BALB/c mice (6 to 8 weeks old, from Charles River Laboratories) were initially sensitized with 2 ⁇ g OVA (ICN Biomedicals) in 2 mg AI(OH) 3 , via i.p. injection. Two weeks after sensitization, the mice were challenged with 50 ⁇ g of OVA (40 ⁇ L 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 ⁇ g/40 ⁇ L, on the 1 st , 3 rd and 5 th d.
  • OVA ICN Biomedicals
  • mice in one group were treated daily with picrotoxin (PTXN, i.p., 0.2 ⁇ g/g body weight in 200 ⁇ L, or i.n., 0.2 ⁇ g/g body weight in 50 ⁇ L), or by bicuculline (i.n. 2 ⁇ g/g body weight in 40 ⁇ L).
  • PTXN picrotoxin
  • bicuculline i.n. 2 ⁇ g/g body weight in 40 ⁇ L
  • OH Al
  • mice were subjected to i.n administration of OVA (100 ⁇ g in 25 ⁇ l_ saline).
  • OVA 100 ⁇ g in 25 ⁇ l_ saline
  • bicuculline 2 ⁇ g/g body weight in 40 ⁇ l_
  • 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
  • BAL analyses As previously described 19 , 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.
  • PBS phosphate buffer solution
  • the level of IL-13 in the BAL fluid samples was measured using enzyme linked immunosorbent assay (ELISA) as previously described 13 .
  • ELISA enzyme linked immunosorbent assay
  • 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 described 20 .
  • the antibodies to GAD 65/67, GABA A R- ⁇ 5 and ⁇ -actin were purchased from Sigma.
  • the antibody to GABA A R- ⁇ 2 was from Alomone Labs.
  • the antibodies to GABA A R- ⁇ 1 and ⁇ 3 were from Affinity Bioreagents.
  • the antibodies to GABA A R- ⁇ 2 and - ⁇ were from Chemicon, and the antibody to GABA A R- ⁇ was from Abeam.
  • 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.
  • the mouse cerebral cortex was used as the positive control.
  • the Jacket cell lysate was used as the positive control.
  • Airway biopsies were obtained from six subjects with mild asthma and using no medication other than infrequent ( ⁇ 5 times weekly) inhaled ⁇ 2-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 hyperresponsiveness 21 . All subjects were nonsmokers and demonstrated an allergen-induced early and late asthmatic response 22 . These subjects underwent sequential diluent (control) and allergens inhalation challenges as described previously 23 ' 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 Health 25 , 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 A R ⁇ 2 and ⁇ 3, 1 :100 dilution; Upstate; antibody to MUC5AC/clone 45M1 , 2 ⁇ g/ml; Lab Vision Corp; antibody to ⁇ smooth muscle actin, 1 :1,000 dilution; Abeam), and subsequently with CY3- conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies.
  • FITC fluorescein isothiocyanate
  • FITC-conjugated antibody to pan-cytokeratin antibody (1 :100 dilution; Sigma) was used to visualize ECs in the lung.
  • 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 described 2026 . Confocal images of stained lung tissue or lung ECs were studied via an inverted microscope (Carl Zeiss) using the Zeiss LSM program.
  • 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).
  • PES periodic acid-Schiff
  • HMI histological mucus index
  • 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 described 1328 .
  • BrdU Assay The SAEC proliferation was quantified by measuring the ability of cells to incorporate bromdeoxyuridine (BrdU) using ELISA.
  • BrdU bromdeoxyuridine
  • SAEC were seeded on to 96-well plates at a density of 3x10 3 /well. Twenty four hours after seeding, triplicate wells were treated with GABA (10 ⁇ mol/L), picrotoxin (PTXN, 25 ⁇ mol/L), or GABA plus PTXN for 24 h.
  • BrdU (10 ⁇ mol/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).
  • Electrophysiology After removal of the culture media, lung
  • ECs were bathed in a solution that contained (in mmol/L): 155 NaCI, 1.3 CaCI 2 , 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 MgCI 2 , 1 CaCI 2 , and 2 tetraethylammonium, at pH 7.35 and osmolarity 315 mOsm.
  • Gramicidin (15-20 ⁇ g/mL) 29 was included in the electrode solution for membrane perforation.
  • Application of the GABA A R 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).

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Abstract

La présente invention a pour objet des utilisations, des procédés et des compositions comprenant des modulateurs GABAergiques tels que des acides nucléiques antisens, des anticorps ou des médicaments destinés à traiter des affections des voies respiratoires chez un animal. Les affections des voies respiratoires comprennent des affections telles que l'asthme ou des affections comprenant une production modifiée de mucus.
PCT/CA2008/000942 2007-05-17 2008-05-16 Modulateurs gabaergiques destinés au traitement d'affections des voies respiratoires Ceased WO2008141438A1 (fr)

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US20100022563A1 (en) * 2008-07-23 2010-01-28 Henkin Robert I Phosphodiesterase inhibitor treatment
US20130131108A1 (en) * 2010-08-09 2013-05-23 University Of Maryland, Baltimore Methods of treating obstructive lung diseases using bitter tastants
CN104306378A (zh) * 2014-09-30 2015-01-28 河南科技大学第一附属医院 氟马西尼的一种新用途
US8968706B2 (en) 2005-04-29 2015-03-03 Robert I. Henkin Methods for diagnosing and treating loss or distortion of taste
US9719988B2 (en) 2007-01-31 2017-08-01 Cyrano Therapeutics, Inc. Methods for detection of biological substances
CN108530480A (zh) * 2017-03-06 2018-09-14 中国科学院上海药物研究所 Gpr84受体拮抗剂及其应用
US10598672B2 (en) 2014-02-18 2020-03-24 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US10898449B2 (en) 2016-12-20 2021-01-26 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
WO2021023809A1 (fr) * 2019-08-06 2021-02-11 Mc Sciences Ug Étifoxine s'utilisant dans le traitement de maladies liées à des mastocytes activés
US11033512B2 (en) 2017-06-26 2021-06-15 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and silicone acrylic hybrid polymer
US11337932B2 (en) 2016-12-20 2022-05-24 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene
US11648213B2 (en) 2018-06-20 2023-05-16 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US12329862B2 (en) 2018-06-20 2025-06-17 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US12485099B2 (en) 2016-12-20 2025-12-02 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene

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US8968706B2 (en) 2005-04-29 2015-03-03 Robert I. Henkin Methods for diagnosing and treating loss or distortion of taste
US11389453B2 (en) 2005-04-29 2022-07-19 Cyrano Therapeutics, Inc. Compositions and methods for treating chemosensory dysfunction
US10206927B2 (en) 2005-04-29 2019-02-19 Cyrano Therapeutics, Inc. Compostions and methods for treating chemosensory dysfunction
US9719988B2 (en) 2007-01-31 2017-08-01 Cyrano Therapeutics, Inc. Methods for detection of biological substances
US20100022563A1 (en) * 2008-07-23 2010-01-28 Henkin Robert I Phosphodiesterase inhibitor treatment
US12329753B2 (en) 2008-07-23 2025-06-17 Cyrano Therapeutics, Inc. Phosphodiesterase inhibitor treatment
US8580801B2 (en) * 2008-07-23 2013-11-12 Robert I. Henkin Phosphodiesterase inhibitor treatment
US10555940B2 (en) 2008-07-23 2020-02-11 Robert I. Henkin Phosphodiesterase inhibitor treatment
US9579315B2 (en) * 2010-08-09 2017-02-28 University Of Maryland, Baltimore Methods of treating obstructive lung diseases using bitter tastants
US20130131108A1 (en) * 2010-08-09 2013-05-23 University Of Maryland, Baltimore Methods of treating obstructive lung diseases using bitter tastants
US11125760B2 (en) 2014-02-18 2021-09-21 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US10598672B2 (en) 2014-02-18 2020-03-24 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US12298316B2 (en) 2014-02-18 2025-05-13 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US11774458B2 (en) 2014-02-18 2023-10-03 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
CN104306378A (zh) * 2014-09-30 2015-01-28 河南科技大学第一附属医院 氟马西尼的一种新用途
US10980753B2 (en) 2016-12-20 2021-04-20 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US11337932B2 (en) 2016-12-20 2022-05-24 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene
US10898449B2 (en) 2016-12-20 2021-01-26 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US12138353B2 (en) 2016-12-20 2024-11-12 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US12485099B2 (en) 2016-12-20 2025-12-02 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene
JP2020510017A (ja) * 2017-03-06 2020-04-02 上海 インスティテュート オブ マテリア メディカ、チャイニーズ アカデミー オブ サイエンシーズＳｈａｎｇｈａｉ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｍａｔｅｒｉａ Ｍｅｄｉｃａ， Ｃｈｉｎｅｓｅ Ａｃａｄｅｍｙ Ｏｆ Ｓｃｉｅｎｃｅｓ Ｇｐｒ８４受容体拮抗剤およびその使用
CN108530480A (zh) * 2017-03-06 2018-09-14 中国科学院上海药物研究所 Gpr84受体拮抗剂及其应用
US11033512B2 (en) 2017-06-26 2021-06-15 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine and silicone acrylic hybrid polymer
US11648213B2 (en) 2018-06-20 2023-05-16 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
US12329862B2 (en) 2018-06-20 2025-06-17 Lts Lohmann Therapie-Systeme Ag Transdermal therapeutic system containing asenapine
WO2021023809A1 (fr) * 2019-08-06 2021-02-11 Mc Sciences Ug Étifoxine s'utilisant dans le traitement de maladies liées à des mastocytes activés
US20220265667A1 (en) * 2019-08-06 2022-08-25 Mc Sciences Ug Etifoxine for use in the treatment of diseases related to activated mast cells

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