WO2001028584A1 - Treatment of respiratory diseases and infections - Google Patents

Treatment of respiratory diseases and infections Download PDF

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Publication number
WO2001028584A1
WO2001028584A1 PCT/AU2000/001254 AU0001254W WO0128584A1 WO 2001028584 A1 WO2001028584 A1 WO 2001028584A1 AU 0001254 W AU0001254 W AU 0001254W WO 0128584 A1 WO0128584 A1 WO 0128584A1
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Prior art keywords
virus
ion channels
parainfluenza virus
protein
human parainfluenza
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PCT/AU2000/001254
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French (fr)
Inventor
David Ian Cook
Karl Kunzelmann
Alex Howard Beesley
John Atherton Young
Nicholas King
Guna Karupiah
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The University Of Sydney
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Priority to AU10074/01A priority Critical patent/AU1007401A/en
Publication of WO2001028584A1 publication Critical patent/WO2001028584A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • 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
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus

Definitions

  • the present invention relates to the treatment of respiratory diseases and infections, particularly cystic fibrosis (CF) and influenza.
  • CF cystic fibrosis
  • Cystic fibrosis a common inherited respiratory disease, for example, is characterised by excessive sodium absorption and deficient chloride secretion by respiratory epithelium (1, 2). These abnormalities lead to increased water absorption from the airways resulting in the formation of thick mucous which can cause severe obstruction of bronchi.
  • many respiratory infections particularly those produced by viruses such as influenza viruses, parainfluenza viruses and rhinoviruses, cause abnormal production of fluid in airways which can lead to rhinorrhea (3), accumulation of fluid in the eustachian tubes and middle ear with subsequent development of otitis media (3, 4, 5), and pulmonary oedema (6, 7).
  • viruses, or components derived therefrom, which inhibit Na + ion channels in respiratory epithelium may be used in novel alternative therapies of other diseases which are characterised by overactivity of epithelial Na + ion channels, for example, the autosomal dominant form of hypertension, Liddle's syndrome (12).
  • the present invention provides a method of treating or preventing the symptoms of a viral or bacterial infection, said symptoms being those caused by abnormal production of fluid by epithelia, wherein said method comprises administering to a subject a composition comprising an agent which inhibits a protein involved in a signal transduction mechanism regulating epithelial Na + ion channels, especially amiloride-sensitive Na + ion channels, and wherein said signal transduction mechanism is one which is activated by haemagglutinin.
  • the method of the first aspect may be used for the treatment or prevention of symptoms of a viral infection caused by abnormal production of fluid by respiratory epithelia or epthelium of the eustachian tubes and middle ear. Accordingly, the method of the first aspect is preferably used for the treatment or prevention of rhinorrhea, otitis media and/or pulmonary oedema. As indicated above, such conditions are typical symptoms of infection by orthomyxoviridae (e.g. influenza viruses A, B and C), and paramyxoviridae (e.g. parainfluenza viruses such as human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, and murine parainfluenza virus 1 (i.e.
  • orthomyxoviridae e.g. influenza viruses A, B and C
  • paramyxoviridae e.g. parainfluenza viruses such as human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, and
  • the method of the first aspect is primarily intended for human therapeutic use but may also be useful in veterinary applications, particularly in dogs, cats, horses, cattle and birds (e.g. chickens and turkeys).
  • composition used in the method of the first aspect preferably comprises an agent for inhibiting either or both of the intracellular signal transduction proteins, phospholipase C and protein kinase C (which the applicant has found become activated during inhibition of Na + ion channels by influenza haemagglutinin) .
  • an agent for inhibiting phospholipase C is U-73122 (Calbiochem).
  • An example of a suitable agent which inhibits protein kinase C is bisindolylmaleimide I (Calbiochem).
  • a suitable agent is an antibody or fragment thereof (or other suitable binding partner of haemagglutinin) that binds to haemagglutinin such that interaction of the haemagglutinin with the epithelial cell surface receptor is prevented.
  • the agent may be present in the composition in admixture with a suitable pharmacologically-acceptable carrier.
  • the composition may be administered by any suitable route but, preferably, orally, nasally, topically, intramuscularly or intravenuously.
  • the preferred route of administration is nasally or orally by way of an aerosolised composition.
  • composition used in the method of the first aspect will be preferably administered to provide a dosage of the agent in the range of 0.01 to 2.0 mg/kg/day for a topical agent administered by inhalation and of 0.1 to 20.0 mg/kg/day for an agent that is administered systemically.
  • the present invention provides a method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising a viral or bacterial preparation which inhibits Na + ion channels in respiratory epithelium, in an amount effective to cause hydration of lung mucous.
  • the method of the second aspect is primarily intended for human therapeutic use but the method may also be useful in veterinary applications, particularly in the treatment of airway mucous obstructions in dogs, cats, horses, cattle and birds.
  • the airway mucous obstructions may be associated with cystic fibrosis, asthma, bronchitis or bronchiectasis.
  • composition used in the method of the second aspect may comprise a viral preparation such as a virus or a component derived therefrom which inhibits Na + ion channels, especially amiloride-sensitive Na + ion channels, in respiratory epithelium.
  • a composition comprising a viral preparation may further comprise a pharmacologically-acceptable carrier, particularly of the kind suitable for aerosolisation (e.g. saline-based compositions (13, 14)).
  • Preferred viruses are selected from orthomyxoviridae (e.g. influenza viruses A, B and C), paramyxoviridae (e.g parainfluenza viruses, newcastle disease virus, viruses causitive of mumps, etc.) and other viruses which include a haemagglutinating protein (e.g. rhinoviruses). They are preferably rendered non-infectious (e.g. cold-adapted viruses (15) and "split virus" preparations) .
  • orthomyxoviridae e.g. influenza viruses A, B and C
  • paramyxoviridae e.g parainfluenza viruses, newcastle disease virus, viruses causitive of mumps, etc.
  • viruses which include a haemagglutinating protein e.g. rhinoviruses
  • They are preferably rendered non-infectious (e.g. cold-adapted viruses (15) and "split virus" preparations) .
  • Preferred virally-derived components are derived from viruses selected from those mentioned in the preceding paragraph.
  • the virally-derived components may be in the form of a crude fraction from disrupted virus preparations or, alternatively, may be in the form of a pure preparation of an isolated viral protein or proteins. They may also take the form of a peptide or polypeptide fragment of a viral protein.
  • Most preferred virally-derived components consist of or comprise a haemagglutinating protein, especially haemagglutinin, or a fragment or analog thereof which is capable of inhibiting Na + ion channels, especially amiloride-sensitive Na + ion channels, in respiratory epithelium.
  • composition used in the method of the second aspect may comprise a bacterial preparation such as a bacteria or a component derived therefrom which inhibits Na + ion channels, especially amiloride-sensitive Na + ion channels, in respiratory epithelium.
  • a composition comprising a bacterial preparation may further comprise a pharmacologically-acceptable carrier.
  • Preferred bacteria are selected from bacteria including a haemagglutinating protein (e.g. Bordatella pertussis) .
  • the bacteria are preferably rendered non-infectious by, for example, treating the bacteria with solubilizing agents such as detergents and, optionally, treatment by chromatography or other method of purification to remove interfering pharmacological activities.
  • Preferred bacterially-derived components are derived from bacteria selected from those mentioned in the preceding paragraph.
  • the bacterially- derived components may be in the form of cell membrane fractions, whole cell lysates or, alternatively, may be in the form of a pure preparation of an isolated bacterial protein or proteins. They may also take the form of a peptide or polypeptide fragment of a bacterial protein.
  • Most preferred bacterially-derived components consist of or comprise a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na + ion channels, especially amiloride-sensitive Na + ion channels, in respiratory epithelium.
  • the applicant has found that the inhibition of Na + ion channels by influenza haemagglutinin involves activation of intracellular signal transduction proteins, phospholipase C and protein kinase C. Accordingly, it is further proposed that agents which stimulate these enzymes may also be used in novel alternative therapies of respiratory diseases such as cystic fibrosis.
  • the present invention provides a method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating Na + ion channels, especially amiloride-sensitive Na + ion channels, in respiratory epithelium, in an amount effective to cause hydration of lung mucous, wherein said signal transduction mechanism is one which is activated by haemagglutinin.
  • the method of the third aspect is primarily intended for human therapeutic use but may also be useful in veterinary applications.
  • the method of the third aspect is preferably used for treatment or prevention of airway mucous obstructions associated with cystic fibrosis, asthma, bronchitis or bronchiectasis.
  • the composition used in the method of the third aspect preferably comprises an agent for stimulating phospholipase C (e.g. G-protein stimulators which, in turn, stimulate phospholipase C) or an agent which stimulates protein kinase C such as 1,2-dioctanoyl-sn-glycerol and phorbol esters.
  • the composition may further comprise a pharmacologically- acceptable carrier, particularly of the kind suitable for aerosolisation.
  • the composition may be administered orally, topically, intramuscularly or intravenously, but is preferably administered nasally.
  • viruses and bacteria, or components derived from those viruses or bacteria, which inhibit Na + ion channels in respiratory epithelium may be useful in the treatment of diseases which are characterised by overactivity of Na + ion channels in other types of epithelia (e.g. renal epithelia).
  • the present invention provides a method of treating a disease characterised by overactivity of epithelial Na + ion channels, said method comprising administering to said subject an effective amount of a composition comprising a viral or bacterial preparation which causes inhibition of epithelial Na + ion channels, especially amiloride-sensitive Na + ion channels.
  • the viral or bacterial preparation may be replaced with a compound which binds and stimulates the epithelial cell receptor recognised by influenza haemagglutinin.
  • Such compounds may mimic the receptor-binding region of influenza haemagglutinin or may otherwise bind to the receptor to bring about the stimulation of phospholipase C and/or protein kinase C activity.
  • the present invention extends to methods involving the administration of compositions comprising such compounds.
  • the present invention provides a method of treating a disease characterised by overactivity of epithelial Na + ion channels, said method comprising administering to said subject an effective amount of a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating epithelial Na + ion channels, especially amiloride-sensitive Na + ion channels, wherein said signal transduction mechanism is one which is activated by haemagglutinin.
  • haemagglutinating protein refers to a protein capable of agglutination of the red blood cells of an appropriate species.
  • Haemagglutinating activity may be measured by any of the known methods in the art, for example by adding a 0.5% v/v chicken red blood cell suspension to a diluted stock of the virus, bacteria or component thereof being tested as contained in a 96-well U-bottom cluster plate (26, 27). The results are measured in haemagglutinating units (HAU), wherein one HAU corresponds to the highest dilution of influenza virus stock at which complete agglutination of a 0.5% v/v chicken red blood cell suspension occurs.
  • HAU haemagglutinating units
  • Figure 1 Panel A. Original recordings showing the response of the tracheal epithelium to 10 mmol/1 amiloride under (i) control conditions, (ii) following 1 h exposure of the apical membrane to UV-inactivated PR8 (10 6 pfu/ml prior to inactivation) and (iii) following 1 h apical exposure to active PR8 virus (10 6 pfu/ml).
  • Panel B Concentration-response curve for amiloride.
  • Panel C Panel C.
  • Panel A Original recordings showing the effects of 10 ⁇ mol/1 forskolin plus 100 ⁇ mol/1 IBMX on tracheal epithelium, before and after 1 h apical exposure to 10 ⁇ pfu/ml PR8.
  • Panel B Original recordings showing the effects of 100 ⁇ mol/1 carbachol on tracheal epithelium, before and after 1 h apical exposure to 10 6 pfu/ml PR8.
  • Panel C The effects of 1 h apical exposure to 10 6 pfu/ml PR8 virus on the current activated by 10 ⁇ mol/1 forskolin plus 100 ⁇ mol/1 IBMX and by 100 ⁇ mol/1 carbachol.
  • Figure 3. Panel A.
  • DOG (10 ⁇ mol/1; DOG) and BIM (0.1 ⁇ mol/1; BIM) on the amiloride-sensitive short circuit current.
  • Example 1 Inhibition of Na + ion channels by influenza virus and parainfluenza virus
  • M-l mouse cortical collecting duct cells were provided by Dr C. Korbmacher (Oxford University, UK). The cells were grown for three days on permeable supports (Transwell - Coll, Costar, Cambridge MA) in DMEM/F12 media containing: 10% fetal calf serum, glutamine (2 mmol/1), penicillin (100,000 U/l), streptomycin (100,000 U/l) and dexamethasone (0.1 ⁇ rnol/1).
  • IBMX 3-isobutyl-l-methylxanthine
  • DOG 1,2- dioctanoyl-sn-glycerol
  • BIM bisindolylmaleimide I
  • staurosporine pertussis toxin
  • cytochalasin D carbachol and chloroquine
  • U-73122 and Go-6983 were from Calbiochem
  • the apical membrane of the epithelium was exposed to UV-inactivated PR8 influenza virus for 1 hour. This had no effect on the electrical properties of the epithelium (Figs lA(ii) and lC). Similarly, exposure to virus-free allantoic fluid was without effect (Fig. IC).
  • the inhibition was not therefore a non-specific consequence of endocytosis of the virus (see also Fig. 4A).
  • Studies on the time-course of the inhibition revealed a linear decline in the amiloride- sensitive short circuit current which was 50% complete after approximately 60 minutes (Fig. ID).
  • the dependence of the inhibition on virus concentration is shown in Fig. IE.
  • the Sendai virus (2.5 x 10 7 pfu/ml) was also found to be inhibitory; reducing the amiloride-sensitive current from 31.7 ⁇
  • These early steps include: (i) the binding of haemagglutinin in the viral coat to sialic acid residues on a receptor protein in the apical membrane, which can be inhibited by pre-treatment with neuraminidase (17); (ii) the endocytosis of the virus-receptor complex, which can be blocked by cvtochalasin D (18); and (iii) uncoating of the virus due to the movement of H + through the M2 protein in the viral coat, which can blocked by amantidine (19, 20) or by chloroquine, which dissipates the endosomal pH gradient (21).
  • Staurosporine a non-selective inhibitor of serine and threonine kinases, partially prevented the inhibitory effect of the virus, but also itself significantly reduced the amiloride-sensitive current (Fig. 4C). Since Na + ion channels in respiratory epithelium have not previously been shown to be regulated by protein kinase C, it was shown that the activator of protein kinase C, 1,2-dioctanoyl-sn-glycerol (DOG), inhibits the amiloride-sensitive current (Fig. 4D), whereas inhibition of protein kinase C with BIM stimulates it (Fig. 4D). Pertussis toxin (300 ng/ml for 3 hours) did not prevent the inhibitory effect of the virus, although, like staurosporine, it reduced the base-line amiloride-sensitive current (data not shown).
  • DOG 1,2-dioctanoyl-sn-glycerol
  • HN protein which exhibits minimal amino acid sequence homology to influenza haemagglutinin (25), indicates that similar results may be achieved with any viral or bacterial pathogen having haemagglutinating activity. This is further supported by the results achieved with Concanavalin A and pertussis toxin.

Abstract

It has been found that influenza virus, particularly its haemagglutinin protein, causes inhibition of Na+ ion channels in respiratory epithelium. This observation has allowed for the development of novel methods for (i) treating and/or preventing symptoms associated with viral or bacterial infection (e.g. rhinnorhea, otitis media and/or pulmonary oedema), (ii) treating and/or preventing airway mucous obstructions (e.g. such as those associated with cystic fibrosis, asthma, bronchitis or bronchiecstasis), and (iii) treating diseases characterised by overactivity of epithelial Na+ ion channels (e.g. hypertension).

Description

TREATMENT OF RESPIRATORY DISEASES AND INFECTIONS
Field of the Invention:
The present invention relates to the treatment of respiratory diseases and infections, particularly cystic fibrosis (CF) and influenza.
Background to the Invention:
Abnormal production of fluid by the respiratory epithelium has been implicated in many respiratory diseases. Cystic fibrosis, a common inherited respiratory disease, for example, is characterised by excessive sodium absorption and deficient chloride secretion by respiratory epithelium (1, 2). These abnormalities lead to increased water absorption from the airways resulting in the formation of thick mucous which can cause severe obstruction of bronchi. In addition, many respiratory infections, particularly those produced by viruses such as influenza viruses, parainfluenza viruses and rhinoviruses, cause abnormal production of fluid in airways which can lead to rhinorrhea (3), accumulation of fluid in the eustachian tubes and middle ear with subsequent development of otitis media (3, 4, 5), and pulmonary oedema (6, 7). Current pharmacological therapies of cystic fibrosis aim to hydrate the lung mucous, so that the mucous may be more readily removed from the airways by mucociliary clearance and/or coughing. For example, aerosolised amiloride has been used to inhibit Na+ ion absorption from airway surfaces thereby augmenting hydration of lung mucous (8). Similarly, lung mucous may be hydrated through the use of ATP or UTP which appear to stimulate
Cl" ion secretion from respiratory epithelium (9).
There are few therapies for viral respiratory infections. Those that are available for influenza include agents that block the viral neuraminidase such as zanamivir, and agents that block the M2 ion channel such as amantidine and ramantidine. Of these, only zanamivir has been shown to reduce the incidence of otological complications following influenza infection (5, 10). Use of decongestants such as oxymetazoline, which can be beneficial in reducing rhinorrhea, also does not appear to reduce the incidence of otological complications (11). In work leading to the present invention, the present applicant discovered that influenza virus causes inhibition of Na+ ion channels in murine respiratory epithelium. In subsequent investigations, it was found that this inactivation was highly specific and appeared to be due to the binding of influenza haemagglutinin to an epithelial cell surface receptor. The applicant proposes that symptoms of viral infections caused by abnormal production and/or accumulation of fluid in the airways, eustachian tubes and the middle ear, and/or by pulmonary oedema, may be treated by blocking the mechanisms by which viruses down-regulate Na+ ion channels. The applicant further proposes that certain viruses, or components derived therefrom, which inhibit Na h ion channels in respiratory epithelium may be used in novel alternative therapies for cystic fibrosis and other respiratory diseases which are characterised by abnormal production of fluid by respiratory epithelium. Still further, the applicant proposes that certain viruses, or components derived therefrom, which inhibit Na+ ion channels in respiratory epithelium may be used in novel alternative therapies of other diseases which are characterised by overactivity of epithelial Na+ ion channels, for example, the autosomal dominant form of hypertension, Liddle's syndrome (12).
Disclosure of the Invention: Thus, in a first aspect, the present invention provides a method of treating or preventing the symptoms of a viral or bacterial infection, said symptoms being those caused by abnormal production of fluid by epithelia, wherein said method comprises administering to a subject a composition comprising an agent which inhibits a protein involved in a signal transduction mechanism regulating epithelial Na+ ion channels, especially amiloride-sensitive Na+ ion channels, and wherein said signal transduction mechanism is one which is activated by haemagglutinin.
The method of the first aspect may be used for the treatment or prevention of symptoms of a viral infection caused by abnormal production of fluid by respiratory epithelia or epthelium of the eustachian tubes and middle ear. Accordingly, the method of the first aspect is preferably used for the treatment or prevention of rhinorrhea, otitis media and/or pulmonary oedema. As indicated above, such conditions are typical symptoms of infection by orthomyxoviridae (e.g. influenza viruses A, B and C), and paramyxoviridae (e.g. parainfluenza viruses such as human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, and murine parainfluenza virus 1 (i.e. Sendai virus) and rubulaviruses such as human parainfluenza viruses 2, 4a and 4b, mumps, newcastle disease virus and canine parainfluenza virus 2). The method, however, may also be applicable to the treatment or prevention of symptoms associated with other types of viruses, especially those which include a haemagglutinating protein
(e.g. rhinoviruses (27).
The method of the first aspect may also be used for the treatment or prevention of symptoms of a bacterial infection caused by abnormal production of fluid by epithelia. The method of the first aspect may, therefore, be suitable for the treatment or prevention of symptoms caused by infection by Bordatella pertussis, which causes whooping cough, or other infective bacteria of the respiratory tract which include a haemagglutinating protein.
The method of the first aspect is primarily intended for human therapeutic use but may also be useful in veterinary applications, particularly in dogs, cats, horses, cattle and birds (e.g. chickens and turkeys).
The composition used in the method of the first aspect preferably comprises an agent for inhibiting either or both of the intracellular signal transduction proteins, phospholipase C and protein kinase C (which the applicant has found become activated during inhibition of Na+ ion channels by influenza haemagglutinin) . An example of a suitable agent for inhibiting phospholipase C is U-73122 (Calbiochem). An example of a suitable agent which inhibits protein kinase C is bisindolylmaleimide I (Calbiochem). Another example of a suitable agent is an antibody or fragment thereof (or other suitable binding partner of haemagglutinin) that binds to haemagglutinin such that interaction of the haemagglutinin with the epithelial cell surface receptor is prevented. The agent may be present in the composition in admixture with a suitable pharmacologically-acceptable carrier. The composition may be administered by any suitable route but, preferably, orally, nasally, topically, intramuscularly or intravenuously. For treatment of symptoms caused by abnormal production of fluid by respiratory epithelium, the preferred route of administration is nasally or orally by way of an aerosolised composition. The composition used in the method of the first aspect will be preferably administered to provide a dosage of the agent in the range of 0.01 to 2.0 mg/kg/day for a topical agent administered by inhalation and of 0.1 to 20.0 mg/kg/day for an agent that is administered systemically.
In a second aspect, the present invention provides a method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising a viral or bacterial preparation which inhibits Na+ ion channels in respiratory epithelium, in an amount effective to cause hydration of lung mucous.
The method of the second aspect is primarily intended for human therapeutic use but the method may also be useful in veterinary applications, particularly in the treatment of airway mucous obstructions in dogs, cats, horses, cattle and birds. When used in the treatment of a human subject, the airway mucous obstructions may be associated with cystic fibrosis, asthma, bronchitis or bronchiectasis.
The composition used in the method of the second aspect may comprise a viral preparation such as a virus or a component derived therefrom which inhibits Na+ ion channels, especially amiloride-sensitive Na+ ion channels, in respiratory epithelium. A composition comprising a viral preparation may further comprise a pharmacologically-acceptable carrier, particularly of the kind suitable for aerosolisation (e.g. saline-based compositions (13, 14)).
Preferred viruses are selected from orthomyxoviridae (e.g. influenza viruses A, B and C), paramyxoviridae (e.g parainfluenza viruses, newcastle disease virus, viruses causitive of mumps, etc.) and other viruses which include a haemagglutinating protein (e.g. rhinoviruses). They are preferably rendered non-infectious (e.g. cold-adapted viruses (15) and "split virus" preparations) .
Preferred virally-derived components are derived from viruses selected from those mentioned in the preceding paragraph. The virally-derived components may be in the form of a crude fraction from disrupted virus preparations or, alternatively, may be in the form of a pure preparation of an isolated viral protein or proteins. They may also take the form of a peptide or polypeptide fragment of a viral protein. Most preferred virally-derived components consist of or comprise a haemagglutinating protein, especially haemagglutinin, or a fragment or analog thereof which is capable of inhibiting Na+ ion channels, especially amiloride-sensitive Na+ ion channels, in respiratory epithelium. The composition used in the method of the second aspect may comprise a bacterial preparation such as a bacteria or a component derived therefrom which inhibits Na+ ion channels, especially amiloride-sensitive Na+ ion channels, in respiratory epithelium. A composition comprising a bacterial preparation may further comprise a pharmacologically-acceptable carrier.
Preferred bacteria are selected from bacteria including a haemagglutinating protein (e.g. Bordatella pertussis) . The bacteria are preferably rendered non-infectious by, for example, treating the bacteria with solubilizing agents such as detergents and, optionally, treatment by chromatography or other method of purification to remove interfering pharmacological activities.
Preferred bacterially-derived components are derived from bacteria selected from those mentioned in the preceding paragraph. The bacterially- derived components may be in the form of cell membrane fractions, whole cell lysates or, alternatively, may be in the form of a pure preparation of an isolated bacterial protein or proteins. They may also take the form of a peptide or polypeptide fragment of a bacterial protein. Most preferred bacterially-derived components consist of or comprise a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na+ ion channels, especially amiloride-sensitive Na+ ion channels, in respiratory epithelium.
The composition used in the method of the second aspect will be preferably administered by inhalation to provide a haemagglutinating effect of the viral or bacterial preparation in the range of 24000 to 240000 HAU per day, as may be achieved by a dose of influenza A virus (PR8) haemagglutinin of 0.003 to 0.003 mg/kg/day administered by inhalation.
The applicant has found that the inhibition of Na+ ion channels by influenza haemagglutinin involves activation of intracellular signal transduction proteins, phospholipase C and protein kinase C. Accordingly, it is further proposed that agents which stimulate these enzymes may also be used in novel alternative therapies of respiratory diseases such as cystic fibrosis.
Thus, in a third aspect, the present invention provides a method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating Na+ ion channels, especially amiloride-sensitive Na+ ion channels, in respiratory epithelium, in an amount effective to cause hydration of lung mucous, wherein said signal transduction mechanism is one which is activated by haemagglutinin.
As with the methods of the first and second aspects, the method of the third aspect is primarily intended for human therapeutic use but may also be useful in veterinary applications. The method of the third aspect is preferably used for treatment or prevention of airway mucous obstructions associated with cystic fibrosis, asthma, bronchitis or bronchiectasis.
The composition used in the method of the third aspect preferably comprises an agent for stimulating phospholipase C (e.g. G-protein stimulators which, in turn, stimulate phospholipase C) or an agent which stimulates protein kinase C such as 1,2-dioctanoyl-sn-glycerol and phorbol esters. The composition may further comprise a pharmacologically- acceptable carrier, particularly of the kind suitable for aerosolisation. The composition may be administered orally, topically, intramuscularly or intravenously, but is preferably administered nasally.
The composition used on the method of the third aspect will be preferably administered to provide a dosage of the agent in the range of 0.01 to 2.0 mg/kg/day for a topical agent administered by inhalation and of 0.1 to 20 mg/kg/day for an agent that is administered systemically.
The applicant further proposes that certain viruses and bacteria, or components derived from those viruses or bacteria, which inhibit Na+ ion channels in respiratory epithelium may be useful in the treatment of diseases which are characterised by overactivity of Na+ ion channels in other types of epithelia (e.g. renal epithelia).
Thus, in a fourth aspect, the present invention provides a method of treating a disease characterised by overactivity of epithelial Na+ ion channels, said method comprising administering to said subject an effective amount of a composition comprising a viral or bacterial preparation which causes inhibition of epithelial Na+ ion channels, especially amiloride-sensitive Na+ ion channels.
With regard to the methods of the second and fourth aspects it shall be readily appreciated that the viral or bacterial preparation may be replaced with a compound which binds and stimulates the epithelial cell receptor recognised by influenza haemagglutinin. Such compounds may mimic the receptor-binding region of influenza haemagglutinin or may otherwise bind to the receptor to bring about the stimulation of phospholipase C and/or protein kinase C activity. It is to be understood that the present invention extends to methods involving the administration of compositions comprising such compounds.
The composition used in the method of the fourth aspect will be preferably administered to provide a dosage of the viral or bacterial preparation in the range of 240000 to 2400000 HAU per day. In a fifth aspect, the present invention provides a method of treating a disease characterised by overactivity of epithelial Na+ ion channels, said method comprising administering to said subject an effective amount of a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating epithelial Na+ ion channels, especially amiloride-sensitive Na+ ion channels, wherein said signal transduction mechanism is one which is activated by haemagglutinin. The composition used on the method of the fifth aspect will be preferably administered to provide a dosage of the agent in the range of 0.1 to 20.0 mg/kg/day. The methods of the fourth and fifth aspects may be useful in both human therapeutic and veterinary applications. When used in the treatment of a human subject, the method is preferably used for the treatment of a disease characterised by overactivity of Na+ ion channels in renal epithelia (e.g. hypertension, particularly hypertension associated with the autosomal dominant form of Liddle's syndrome).
As used herein, "haemagglutinating protein" refers to a protein capable of agglutination of the red blood cells of an appropriate species. "Haemagglutinating activity" may be measured by any of the known methods in the art, for example by adding a 0.5% v/v chicken red blood cell suspension to a diluted stock of the virus, bacteria or component thereof being tested as contained in a 96-well U-bottom cluster plate (26, 27). The results are measured in haemagglutinating units (HAU), wherein one HAU corresponds to the highest dilution of influenza virus stock at which complete agglutination of a 0.5% v/v chicken red blood cell suspension occurs. The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.
The invention will hereinafter be further described by way of the following non-limiting example and accompanying figures.
Brief description of the accompanying figures: Figure 1. Panel A. Original recordings showing the response of the tracheal epithelium to 10 mmol/1 amiloride under (i) control conditions, (ii) following 1 h exposure of the apical membrane to UV-inactivated PR8 (106 pfu/ml prior to inactivation) and (iii) following 1 h apical exposure to active PR8 virus (106 pfu/ml). Panel B. Concentration-response curve for amiloride. Panel C. The effects on amiloride-sensitive current of 1 h apical treatment with: (i) active PR8 (106 pfu/ml); (ii) active WSN33 influenza virus (106 pfu/ml); (iii) UV-inactivated PR8 (106 pfu/ml); (iv) replication-deficient adenovirus, MX17 (106 pfu/ml); and (v) allantoic fluid not infected with any virus. Panel D. Time course of the decline in the amiloride-sensitive current produced by 10^ pfu/ml PR8. Panel E. Concentration- response curve for inhibition of the amiloride-sensitive current following apical exposure to purified PR8 for 1 h.
Figure 2. Panel A. Original recordings showing the effects of 10 μmol/1 forskolin plus 100 μmol/1 IBMX on tracheal epithelium, before and after 1 h apical exposure to 10^ pfu/ml PR8. Panel B. Original recordings showing the effects of 100 μmol/1 carbachol on tracheal epithelium, before and after 1 h apical exposure to 106 pfu/ml PR8. Panel C. The effects of 1 h apical exposure to 106 pfu/ml PR8 virus on the current activated by 10 μmol/1 forskolin plus 100 μmol/1 IBMX and by 100 μmol/1 carbachol. Figure 3. Panel A. Original recordings showing the effect of 10 μmol/1 amiloride on Ml mouse collecting duct cells before and after 1 h apical exposure to 106 pfu/ml PR8. Panel B. Original recordings showing the effect of 10 μmol/1 amiloride on mouse colon before and after 1 h apical exposure to 106 pfu/ml PR8. Panel C. Effect of 106 pfu/ml PR8 on the amiloride-sensitive current in Ml cells and mouse colon. Figure 4. Panel A. Effect on the PR8-induced inhibition of the amiloride-sensitive current of cytochalasin D (30 μg/ml; CD); chloroquine (500 μmol/1; CQ); amantidine (200 μmol/1; AM) and pretreatment of the apical membrane with neuraminidase (0.1 U/ml) for 30 minutes at 37°C (NA). Panel B. Effect of "split virus" (48 mg total protein/1) on the amiloride sensitive current and its neutralization by anti-haemagglutinin antibody (1:100 dilution: HA-antibody) . Panel C. Effects of staurosporine (2 μmol/1; Stauro), bisindolylmaleimide I (0.1 μmol/1; BIM), Go-6983 (20 nmol/1; Go) and U- 73122 (10 μmol/1; U-73122) on the inhibition by 106 pfu/ml PR8 of the amiloride-sensitive current. Panel D. Effect of 1,2-dioctanoyl-sϋ-glycerol
DOG (10 μmol/1; DOG) and BIM (0.1 μmol/1; BIM) on the amiloride-sensitive short circuit current.
Example 1: Inhibition of Na+ ion channels by influenza virus and parainfluenza virus
MATERIALS AND METHODS.
Viruses. The pneumotropic influenza virus A/PR/8/34 (PR8; HlNl), and the neuro tropic influenza A virus, A/WSN/33 (WSN33; HlNl), were grown for 2 days in the allantoic cavity of 10-day embryonated hens' eggs. Aliquots of allantoic fluid containing the virus were stored at -70°C. The purified virus used in Fig. IE was prepared by sucrose gradient centrifugation and stored in phosphate-buffered saline. Infectivity of the influenza viruses was tittered on monolayers of MDCK cells. HA activity was measured with chicken erythrocytes using standard methods. PR8 "split virus", prepared by the Commonwealth Serum Laboratories
(Melbourne), was the gift of Dr L.E. Brown (Microbiology and Immunology, University of Melbourne). It was produced in allantoic fluid as described above and was purified by zonal centrifugation. The purified virus (adjusted to pH 8-8.5 with NaOH) was inactivated by incubation with 0.1% (v/v) beta- propiolactone and disrupted with 1.6% (w/v) sodium taurodeoxycholate for 2 h at 37°C after which it was dialysed against phosphate buffered saline (pH 7.2) with sodium azide added as a preservative (total protein 9.3 μg/μl, haemagglutinin 2.9 μg/μl). This solution was then dialysed against phosphate buffered saline to produce the final azide-free "split- virus" stock solution (4.8 μg/μl total protein, 1.5 μg/μl haemagglutinin). The replication-deficient adenovirus, MX-17, the gift of Dr G.W. Both (CSIRO Division of Molecular Sciences, North Ryde, NSW), was grown and tittered as previously described (16). It contains a null insert and does not express a transgene. The murine parainfluenza type I virus, Sendai virus was grown, purified and tittered as described above for the influenza viruses.
Cell culture. M-l mouse cortical collecting duct cells were provided by Dr C. Korbmacher (Oxford University, UK). The cells were grown for three days on permeable supports (Transwell - Coll, Costar, Cambridge MA) in DMEM/F12 media containing: 10% fetal calf serum, glutamine (2 mmol/1), penicillin (100,000 U/l), streptomycin (100,000 U/l) and dexamethasone (0.1 μrnol/1).
Ussing chamber experiments. Mice were killed by cervical dislocation. The trachea was then removed, freed of connective tissue, opened longitudinally and divided into two or three pieces which were then put into a chilled solution containing (mmol/1): NaCl 145, KC1 3.8, D-glucose 5, MgCl2 1, HEPES 5, Ca-gluconate 1.3, pH 7.4. The tissue pieces were mounted in an Ussing chamber with a circular aperture of 0.95 mm2 (17). The luminal and basolateral surfaces of the epithelium were perfused continuously at a rate of 10 to 20 ml/min (chamber volume 1 ml). The bath solution contained
(mmol/1): NaCl 145, KH2P04 0.4, K2HP04 1.6, D-glucose 5, MgCl2 1, Ca- gluconate 1.3, pH 7.4. Bath solutions were heated to 37°C. Experiments were carried out under open-circuit conditions. Values for transepithelial potential differences (Vt were referred to the serosal side of the epithelium. Transepithelial resistance (Rt was determined by applying short (1 s) current pulses (ΔI = 0.5 μA) (see (17)).
Chemicals. 3-isobutyl-l-methylxanthine (IBMX), forskolin, 1,2- dioctanoyl-sn-glycerol (DOG), bisindolylmaleimide I (BIM), staurosporine, pertussis toxin, cytochalasin D, carbachol and chloroquine were obtained from Sigma; U-73122 and Go-6983 were from Calbiochem (Alexandria,
NSW); neuraminidase was from Boehringer (Mannheim, Germany); monoclonal anti-haemagglutinin antibody was a gift of Dr L.E. Brown (Microbiology and Immunology, University of Melbourne). The haemagglutinating lectin, Concanavalin A, was obtained from Sigma. Pertussis toxin was obtained from Calbiochem. Statistical Methods. Statistical significance was assessed using Student's unpaired Mest. In the figures, * indicates a statistically significant effect.
RESULTS AND DISCUSSION. The baseline characteristics of the mouse tracheal epithelium studied in an Ussing chamber under open circuit conditions were determined. The baseline transepithelial potential difference and transepithelial resistance were -2.65 ± 0.28 mV (n = 20) and 77.0 ± 9.2 Ωcm2 (n = 20), respectively, corresponding to an equivalent short circuit current of -37.4 ± 3.4 μAcπr2 (n = 20). As shown in Fig. lA(i), exposure of the apical membrane to a maximal concentration of amiloride (10 μmol/1, Fig. IB) reduced the transepithelial potential difference (-0.77 ± 0.13 mV, n = 20; P < 0.05), and the calculated short circuit current (-10.7 ± 1.9 μAcπr2, n = 20; P < 0.05), whilst increasing the transepithelial resistance (82.8 ± 10.2 Ωcm2, n = 20; P < 0.05). It thus was possible to calculate that the amiloride-sensitive current, which is a measure of the activity of epithelial Na+ ion channels (18), was 26.7 ± 2.6 μAcπr2 (n = 20; Fig. IC).
Subsequently, the apical membrane of the epithelium was exposed to UV-inactivated PR8 influenza virus for 1 hour. This had no effect on the electrical properties of the epithelium (Figs lA(ii) and lC). Similarly, exposure to virus-free allantoic fluid was without effect (Fig. IC). Exposure of the apical membrane to active PR8 influenza virus for 1 hour (Figs lA(iii) and IC), however, decreased the transepithelial potential difference (-1.92 ± 0.35 mV, n = 20; P < 0.05), the total short circuit current (-25.2 ± 4.1 μAcπr2 n = 20; P < 0.05) and the amiloride-sensitive current (-7.3 ± 1.2 uAcnr2, n =
20; P < 0.05). The neurotropic influenza virus, WSN33 (10θ pfu/ml; Fig. IC), was also inhibitory, but the replication-deficient type-5 adeno virus, MX-17, was not (106 pfu/ml; Fig. IC). The inhibition was not therefore a non-specific consequence of endocytosis of the virus (see also Fig. 4A). Studies on the time-course of the inhibition revealed a linear decline in the amiloride- sensitive short circuit current which was 50% complete after approximately 60 minutes (Fig. ID). The dependence of the inhibition on virus concentration is shown in Fig. IE.
In a separate experiment, the Sendai virus (2.5 x 107 pfu/ml) was also found to be inhibitory; reducing the amiloride-sensitive current from 31.7 ±
10.3 μAcm"2 (n=5) to 13.3 ± 3.3 μAcm"2 (n=5) after 1 hour. Additionally, an experiment was conducted to examine whether Concanavalin A, a haemagglutinating lectin from jack bean, could inhibit the amiloride-sensitive current. It was found that Concanavalin A could indeed inhibit the amiloride-sensitive current, and that the time course of this inhibition was similar to that observed with influenza virus. Specifically, the
Concanavalin A (50 μg/ml) reduced the amiloride-sensitive current from 35.5 ± 5.3 μAcm"2 (n=5) to 16.5 ± 6.7 μAcm"2 (n=5) after 1 hour.
Exposure of the respiratory epithelium to a mixture of forskolin (10 μmol/1) and IBMX (100 μmol/1; Figs 2A and 2C), which increases intracellular cyclic AMP, or to carbachol (100 μmol/1; Figs 2B and 2D), which increases intracellular Ca2+, has been shown to increase transepithelial potential difference and short circuit current by activation of Cl" secretion (3, 6). PR8 virus had no effect on the time-course (Figs 2A and 2B) or the size of these responses (Figs 2C and 2D). An examination was then made to determine whether other amiloride- sensitive Na+ transporting epithelia were also sensitive to influenza virus. It was found that PR8 virus inhibited amiloride-sensitive currents in both the Ml mouse collecting duct cell line (Fig. 3C) and in mouse colon (Fig. 3C). The time-course of the inhibition (Fig. ID) suggested that it was a consequence of one of the early steps of the infection process (4). These early steps include: (i) the binding of haemagglutinin in the viral coat to sialic acid residues on a receptor protein in the apical membrane, which can be inhibited by pre-treatment with neuraminidase (17); (ii) the endocytosis of the virus-receptor complex, which can be blocked by cvtochalasin D (18); and (iii) uncoating of the virus due to the movement of H+ through the M2 protein in the viral coat, which can blocked by amantidine (19, 20) or by chloroquine, which dissipates the endosomal pH gradient (21). Of the treatments that block these early steps of viral infection, only neuraminidase prevented the inhibition of the amiloride-sensitive current by PR8 virus (Fig. 4A). Furthermore, a "split-virus" preparation, in which the virus had been disrupted with detergent (see Methods), was inhibitory (Fig. 4B). This inhibition could be prevented by a monoclonal antibody directed against haemagglutinin (Fig. 4B). Thus the inhibition produced by influenza virus seems to have been due to haemagglutinin binding to a receptor in the apical membrane of the respiratory epithelium. Finally, experiments were conducted to examine the intracellular mechanisms of the inhibition produced by the influenza virus (Fig. 4C). The inhibitor of phospholipase Cβ, U-73122, blocked the response, as did a broad- spectrum inhibitor of protein kinase C, bisindolylmaleimide I (BIM), and the selective inhibitor of the α and β isoforms of protein kinase C, Go-6983.
Staurosporine, a non-selective inhibitor of serine and threonine kinases, partially prevented the inhibitory effect of the virus, but also itself significantly reduced the amiloride-sensitive current (Fig. 4C). Since Na+ ion channels in respiratory epithelium have not previously been shown to be regulated by protein kinase C, it was shown that the activator of protein kinase C, 1,2-dioctanoyl-sn-glycerol (DOG), inhibits the amiloride-sensitive current (Fig. 4D), whereas inhibition of protein kinase C with BIM stimulates it (Fig. 4D). Pertussis toxin (300 ng/ml for 3 hours) did not prevent the inhibitory effect of the virus, although, like staurosporine, it reduced the base-line amiloride-sensitive current (data not shown).
Pertussis toxin (300ng/ml) reduced the amiloride sensitive sodium current from 40.7 ± 4.1 μA/cm2 to 18.8 ± 2.1 μA/cm2 (n=5). This effect was completely inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM; 100 nmol/1; control amiloride-sensitive current + BIM: 30.0 ± 5.4 μA cm2 (n=5) vs pertussis toxin (330 ng/ml) + BIM: 27.0 ± 3.9 μA/cm2
(n=5)). The effect of pertussis toxin was completely reproduced by the B oligomer of the toxin which has haemagglutinating activity (26) (control amiloride-sensitive current: 42.8 ± 9.0 μA cm2 (n=5) vs amiloride-sensitive current after exposure to pertussis toxin B oligomer (300 ng/ml): 23.0 ± 4.6 μA/cm2 (n=5)).
This is the first report of any viral or bacterial pathogen that regulates amiloride-sensitive Na+ ion channels. Given the role of amiloride-sensitive Na+ ion channels in controlling the amount of fluid in the respiratory tract (22, 23), the observation that influenza haemagglutinin inhibits Na+ ion channel activity offers a novel alternative to the use of Na+ ion channel blockers such as amiloride in the treatment of the Na+ ion channel overactivity seen in cystic fibrosis (24). In addition, the observation that amiloride-sensitive Na+ ion channels are also inhibited by Sendai virus, which includes a haemagglutinating protein (i.e. HN protein) which exhibits minimal amino acid sequence homology to influenza haemagglutinin (25), indicates that similar results may be achieved with any viral or bacterial pathogen having haemagglutinating activity. This is further supported by the results achieved with Concanavalin A and pertussis toxin.
References:
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It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:
1. A method of treating or preventing the symptoms of viral or bacterial infection, said symptoms being those caused by abnormal production of fluid by epithelia, wherein said method comprises administering to a subject a composition comprising an agent which inhibits a protein involved in a signal transduction mechanism regulating epithelial Na+ ion channels, and wherein said signal transduction mechanism is one which is activated by haemagglutinin.
2. A method according to claim 1, wherein the epithelial Na+ ion channels are amiloride-sensitive Na+ ion channels.
3. A method according to claim 1 or 2, wherein the epithelial Na+ ion channels are Na+ ion channels of respiratory epithelia.
4. A method according to any one of claims 1 to 3, wherein the method is used for the treatment or prevention of rhinnorhea, otitis media and/or pulmonary oedema.
5. A method according to any one of claims 1 to 4, wherein the method is used for treating or preventing symptoms of infection by a virus selected from those having haemagglutinating activity.
6. A method according to any one of claims 1 to 4, wherein the method is used for treating or preventing symptoms of infection by a virus selected from the group consisting of orthomyxoviridae and paramyxoviridae.
7. A method according to claim 6, wherein the virus is selected from the group consisting of: influenza virus A, influenza virus B, influenza virus C, human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, human parainfluenza virus 2, human parainfluenza virus 4a, human parainfluenza virus 4b, mumps, newcastle disease virus and canine parainfluenza virus 2.
8. A method according to any one of claims 1 to 4, wherein the method is used for treating or preventing symptoms of infection by a virus selected from the group consisting of rhinoviruses.
9. A method according to any one of claims 1 to 4, wherein the method is used for treating or preventing symptoms of infection by an infective bacteria.
10. A method according to claim 9, wherein the infective bacteria includes a haemagglutinating protein.
11. A method according to claim 9 or 10, wherein the infective bacteria is infective of respiratory epithelium.
12. A method according to claim 9, wherein the infective bacteria is Bordatella pertussis.
13. A method according to any of claims 1 to 12, wherein the agent inhibits phospholipase C and/or protein kinase C.
14. A method according to any one of claims 1 to 13, wherein the composition is administered to provide a dosage of the said agent in the range of 0.01 to 20.0 mg/kg/day.
15. A method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising a viral or bacterial preparation which inhibits Na+ ion channels in respiratory epithelium, in an amount effective to cause hydration of lung mucous.
16. A method according to claim 15, wherein the epithelial Na+ ion channels are amiloride-sensitive Na+ ion channels.
17. A method according to claim 15 or 16, wherein the method is used for treating or preventing airway mucous obstructions associated with cystic fibrosis, asthma, bronchitis or bronchiectasis.
18. A method according to any one of claims 13 to 16, wherein said composition comprises a viral preparation, said viral preparation being selected from viruses and virally-derived components having haemagglutinating activity.
19. A method according to claim 18, wherein said composition comprises a virus selected from the group consisting of orthomyxoviridae and paramyxoviridae.
20. A method according to claim 19, wherein the virus is selected from the group consisting of: influenza virus A, influenza virus B, influenza virus C, human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, human parainfluenza virus 2, human parainfluenza virus 4a, human parainfluenza virus 4b, mumps, newcastle disease virus and canine parainfluenza virus 2.
21. A method according to claims 18, wherein said composition comprises a virus selected from the group consisting of rhinoviruses.
22. A method according to any one of claims 18 to 21, wherein the virus has been rendered non-infectious.
23. A method according to claim 18, wherein said composition comprises a virally-derived component, said virally-derived component consisting of or comprising a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
24. A method according to claim 23, wherein the haemagglutinating protein or fragment thereof is from a virus selected from the group consisting of orthomyxoviridae and paramyxoviridae.
25. A method according to claim 24, wherein the virus is selected from the group consisting of: influenza virus A, influenza virus B, influenza virus C, human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, human parainfluenza virus 2, human parainfluenza virus 4a, human parainfluenza virus 4b, mumps, newcastle disease virus and canine parainfluenza virus 2.
26. A method according to claims 23, wherein the haemagglutinating protein or fragment thereof is from a virus selected from the group consisting of rhinoviruses.
27. A method according to claim 23, wherein the haemagglutinating protein is haemagglutinin.
28. A method according to any one of claims 14 to 17, wherein said composition comprises a bacterial preparation, said bacterial preparation being selected from bacteria and bacterially-derived components.
29. A method according to claim 28, wherein said composition comprises a bacteria which includes a haemagglutinating protein.
30. A method according to claim 29, wherein the bacteria is Bordatella pertussis.
31. A method according to claim 29 or 30, wherein the bacteria has been rendered non-infectious.
32. A method according to claim 28, wherein said composition comprises a bacterially-derived component, said bacterially-derived component consisting of or comprising a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
33. A method according to claim 32, wherein the haemagglutinating protein or fragment thereof is pertussis toxin or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
34. A method according to any one of claims 15 to 33, wherein the composition is administered to provide a dosage of the viral or bacterial preparation in the range of 24000 to 240000 HAU per day.
35. A method of treating or preventing mucous obstruction of airways in a subject, said method comprising administering to said subject a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating Na+ ion channels in respiratory epithelium, in an amount effective to cause hydration of lung mucous, wherein said signal transduction mechanism is one which is activated by haemagglutinin.
36. A method according to claim 35, wherein the epithelial Na+ ion channels are amiloride-sensitive Na+ ion channels.
37. A method according to claim 35 or 36, wherein the method is used for treating or preventing airway mucous obstructions associated with cystic fibrosis, asthma, bronchitis or brochiectasis.
38. A method according to any of claims 35 to 37, wherein the agent stimulates phospholipase C and/or protein kinase C.
39. A method according to any one of claims 35 to 38, wherein the composition is administered to provide a dosage of the said agent in the range of 0.01 to 2.0 mg/kg/day.
40. A method of treating a disease characterised by overactivity of epithelial Na+ ion channels, said method comprising administering to said subject an effective amount of a composition comprising a viral or bacterial preparation which causes inhibition of epithelial Na+ ion channels.
41. A method according to claim 40, wherein the epithelial Na+ ion channels are amiloride-sensitive Na+ ion channels.
42. A method according to claim 40 or 41, wherein the method is used for treating hypertension.
43. A method according to any one of claims 40 to 42, wherein said composition comprises a viral preparation, said viral preparation being selected from viruses and virally-derived components havinh haemagglutinating activity.
44. A method according to claim 43, wherein said composition comprises a virus selected from the group consisting of orthomyxoviridae and paramyxoviridae .
45. A method according to claim 44, wherein the virus is selected from the group consisting of: influenza virus A, influenza virus B, influenza virus C, human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, human parainfluenza virus 2, human parainfluenza virus 4a, human parainfluenza virus 4b, mumps, newcastle disease virus and canine parainfluenza virus 2.
46. A method according to claims 43, wherein said composition comprises a virus selected from the group consisting of rhinoviruses.
47. A method according to any one of claims 44 to 46, wherein the virus has been rendered non-infectious.
48. A method according to claim 43, wherein said composition comprises a virally-derived component, said virally-derived component consisting of or comprising a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
49. A method according to claim 48, wherein the haemagglutinating protein or fragment or analog thereof is from a virus selected from the group consisting of orthomyxoviridae and paramyxoviridae.
50. A method according to claim 49, wherein the virus is selected from the group consisting of: influenza virus A, influenza virus B, influenza virus C, human parainfluenza virus 1, human parainfluenza virus 3, bovine parainfluenza virus 3, human parainfluenza virus 2, human parainfluenza virus 4a, human parainfluenza virus 4b, mumps, newcastle disease virus and canine parainfluenza virus 2.
51. A method according to claims 49, wherein the haemagglutinating protein or fragment or analog thereof is from a virus selected from the group consisting of rhinoviruses.
52. A method according to claim 51, wherein the haemagglutinating protein is haemagglutinin.
53. A method according to any one of claims 40 to 42, wherein said composition comprises a bacterial preparation, said bacterial preparation being selected from bacteria and bacterially-derived components.
54. A method according to claim 53, wherein said composition comprises a bacteria which includes a haemagglutinating protein.
55. A method according to claim 54, wherein the bacteria is Bordatella pertussis.
56. A method according to claim 54 or 55, wherein the bacteria has been rendered non-infectious.
57. A method according to claim 53, wherein said composition comprises a bacterially-derived component, said bacterially-derived component consisting of or comprising a haemagglutinating protein or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
58. A method according to claim 57, wherein the haemagglutinating protein or fragment thereof is pertussis toxin or a fragment or analog thereof which is capable of inhibiting Na+ ion channels in respiratory epithelium.
59. A method according to any one of claims 40 to 59, wherein the composition is administered to provide a dosage of the viral or bacterial preparation in the range of 24000 to 2400000 HAU per day.
60. A method of treating a disease characterised by overactivity of epithelial Na+ ion channels, said method comprising administering to said subject an effective amount of a composition comprising an agent which stimulates a protein involved in a signal transduction mechanism regulating epithelial Na+ ion channels, wherein said signal transduction mechanism is one which is activated by haemagglutinin.
61. A method according to claim 60, wherein the epithelial Na+ ion channels are amiloride-sensitive Na+ ion channels.
62. A method according to claim 60 or 61, wherein the method is used for treating hypertension.
63. A method according to any of claims 60 to 62, wherein the agent stimulates phospholipase C and/or protein kinase C.
64. A method according to any one of claims 60 to 63, wherein the composition is administered to provide a dosage of the said agent in the range of 0.1 to 20.0 mg/kg/day.
PCT/AU2000/001254 1999-10-15 2000-10-16 Treatment of respiratory diseases and infections WO2001028584A1 (en)

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US9586910B2 (en) 2011-06-27 2017-03-07 Parion Sciences, Inc. 3,5-diamino-6-chloro-N-(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide
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US10752597B2 (en) 2011-06-27 2020-08-25 Parion Sciences, Inc. 3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide
US10071970B2 (en) 2012-12-17 2018-09-11 Parion Sciences, Inc. Chloro-pyrazine carboxamide derivatives with epithelial sodium channel blocking activity
US9695134B2 (en) 2012-12-17 2017-07-04 Parion Sciences, Inc. 3,5-diamino-6-chloro-N-(n-(4-phenylbutyl)carbamimidoyl)pyrazine-2-carboxamide compounds
US10246425B2 (en) 2012-12-17 2019-04-02 Parion Sciences, Inc. 3,5-diamino-6-chloro-N-(N-(4-phenylbutyl)carbamimidoyl) pyrazine-2-carboxamide compounds
US9593084B2 (en) 2012-12-17 2017-03-14 Parion Sciences, Inc. Chloro-pyrazine carboxamide derivatives with epithelial sodium channel blocking activity
US9957238B2 (en) 2013-12-13 2018-05-01 Parion Sciences, Inc. Arylalkyl-and aryloxyalkyl-substituted epithelial sodium channel blocking compounds
US10233158B2 (en) 2013-12-13 2019-03-19 Parion Sciences, Inc. Arylalkyl- and aryloxyalkyl-substituted epithelial sodium channel blocking compounds
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