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EP1527343A2 - Organic compounds - Google Patents

Organic compounds

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Publication number
EP1527343A2
EP1527343A2 EP03766324A EP03766324A EP1527343A2 EP 1527343 A2 EP1527343 A2 EP 1527343A2 EP 03766324 A EP03766324 A EP 03766324A EP 03766324 A EP03766324 A EP 03766324A EP 1527343 A2 EP1527343 A2 EP 1527343A2
Authority
EP
European Patent Office
Prior art keywords
trpc6
human
disease
polypeptide
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03766324A
Other languages
German (de)
French (fr)
Inventor
Sau Wei Novartis Horsham Research Centre LI
Christopher T. Novartis Horsham Res. Ctr. POLL
John Novartis Horsham Research Centre WESTWICK
Martin Novartis Horsham Research Centre GOSLING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Pharma GmbH
Novartis AG
Original Assignee
Novartis Pharma GmbH Austria
Novartis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0217503.2A external-priority patent/GB0217503D0/en
Application filed by Novartis Pharma GmbH Austria, Novartis AG filed Critical Novartis Pharma GmbH Austria
Publication of EP1527343A2 publication Critical patent/EP1527343A2/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the identification of substances or agents that modulate the activity of the transient receptor potential 6 ion channel, TRPC6, and the use of such substances in the treatment of inflammatory diseases, particularly those of the respiratory system.
  • a variety of cells are attracted into tissues during inflammation. These include various leukocytes, particularly inflammatory phagocytes such as neutrophilic and eosinophilic granulocytes and monocytes. Neutrophils have been associated with inflammation and tissue destruction in respiratory diseases such as chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema associated therewith, and adult respiratory distress syndrome (ARDS), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and rheumatoid arthritis. Eosinophils have been associated with respiratory diseases such as asthma and allergic rhinitis.
  • COPD chronic obstructive pulmonary disease
  • ARDS adult respiratory distress syndrome
  • Eosinophils have been associated with respiratory diseases such as asthma and allergic rhinitis.
  • Critical steps in the action of leukocytes in inflammatory conditions include the migration of these cells into the tissues, e.g. into the airways in respiratory inflammations or to the joints in rheumatoid arthritis, cell activation and the release of a range of inflammatory mediators, leukotrienes, oxygen radicals, proteases.
  • Signals that are needed for leukocyte migration and activation are often communicated through receptors that respond to an increase in the level of cytosolic calcium. Ins(l,4,5)P3 receptors are known to release calcium ions from intracellular stores but less is known about the channels in the plasma membrane through which those ions pass.
  • TRPC human omologue
  • TRPC6 has been found to be present in cells attracted into tissues during inflammation.
  • TRPC6 mRNA has been shown to be present in isolated primary human neutrophils and in human lung macrophages isolated by bronchoalveolar lavages from smokers and COPD patients.
  • TRPC6 mRNA expression has also been found in cultured human airway smooth muscle cells and well-differentiated human bronchial epithelial cells.
  • TRPC6 proteins have been shown to be expressed in human T-lymphocytes, cultured human airway smooth muscle cells, well-differentiated human bronchial epithelial cells, bronchial airway smooth muscle, epithelial cells of the airway epithelium, submucosal glands and tissue leukocytes i.e. neutrophils, macrophages and lymphocytes.
  • TRPC6 as a therapeutic target for such diseases and a "screening assay" for identifying modulators i.e., candidate compounds or agents including peptides, peptidomimetics, small molecules or other drugs, which stimulate or inhibit the activity of the channel formed by the gene product of human TRPC6.
  • the present invention relates to a method of identifying a substance suitable for use in the treatment of a leukocyte-associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6), wherein the method comprises combining a candidate substance with said polypeptide and measuring the effect of the candidate substance on the activity of said polypeptide.
  • TRPC6 human transient receptor potential 6 gene
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound that inhibits the influx of calcium ions through a human TRPC6 ion channel and a pharmaceutically acceptable carrier.
  • the present invention relates to the use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene, an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, or a polynucleotide probe comprising at least 15 consecutive nucleotides of that polynucleotide, in the preparation of a pharmaceutical that inhibits the accumulation of leukocytes in human tissue.
  • the present invention relates to the use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene, an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, or a polynucleotide probe comprising at least 15 consecutive nucleotides of that polynucleotide, in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
  • the present invention relates to the use of a TRPC6 inhibitor in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
  • the present invention relates to a method of the present invention for identifying a substance suitable for use in the treatment of a leukocyte-associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene.
  • this method, or assay comprises combining a candidate substance with a polypeptide encoded by the transient receptor potential channel 6 gene and measuring the effect of the candidate substance on the activity of that polypeptide.
  • TRPC6 agonists e.g. the activity of a TRPC6 gene product, i.e. the polypeptide that forms the TRPC6 ion channel, may be measured, for example, by a cell-based method or screening assay that identifies compounds which have a stimulatory or inhibitory effect on the activity of the human TRPC6 channel, or by an appropriate reporter gene assay.
  • the abovementioned screening method may be carried out, for example, by preparing cells which express the TRPC6 polypeptide on their surfaces, e.g. insect, mammal or yeast cells and then incubating the resulting cells with the candidate substance to determine the enhancement or inhibition of a functional activity of the TRPC6 polypeptide.
  • the candidate substance is combined with cells that are stably transfected with TRPC6 and express a functional TRPC6 channel and membrane depolarisation is measured to indicate any TRPC6-mediated Na + influx.
  • a suitable test for any inhibition of the TRPC6 channel cells that express an endogenous G protein-coupled calcium mobilising receptor e.g. muscarinic acetylcholine receptor that can activate TRPC6 channels are used and a receptor agonist e.g. muscarinic acetylcholine receptor agonist is added to stimulate the TRPC6 channels.
  • the cells are treated with the candidate substance prior to addition of the receptor agonist. That should produce an increase in fluorescence, but the magnitude of the increase will be reduced if the candidate substance inhibits the TRPC6 channels. Any change in fluorescence can be measured using suitable equipment, for example a fluorescence imaging plate reader.
  • the present invention also relates to a pharmaceutical composition that comprises a compound that inhibits the influx of calcium ions through a TRPC6 channel, such as l-[b-[3- (4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl, and a pharmaceutically acceptable carrier.
  • a compound that inhibits the influx of calcium ions through a TRPC6 channel such as l-[b-[3- (4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl
  • a pharmaceutically acceptable carrier such as l-[b-[3- (4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl
  • an antibody which is immunoreactive with the polypeptide encoded by the human TRPC6 gene herein a "human TRPC6 antibody”
  • an antisense oligonucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising a nucleotide sequence encoding that polypeptide
  • the aforementioned human TRPC6 antibodies and antisense oligonucleotides may be used to treat leukocyte-associated inflammatory diseases.
  • Human TRPC6 agonists, human TRPC6 antagonists, human TRPC6 antibodies and human TRPC6 antisense oligonucleotides are hereinafter alternatively referred to collectively as " agents of the invention " .
  • Neutrophil-associated inflammatory diseases to which the present invention is applicable include neutrophil-associated inflammatory or obstructive airways diseases, particularly chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema, and adult (or acute) respiratory distress syndrome (ARDS), rheumatoid arthritis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.
  • neutrophil-associated inflammatory or obstructive airways diseases particularly chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema, and adult (or acute) respiratory distress syndrome (ARDS), rheumatoid arthritis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.
  • Eosinophil- associated inflammatory diseases to which the present invention is also applicable include eosinophil-associated inflammatory or obstructive airways diseases, particularly asthma and allergic rhinitis.
  • TRPC6 is present in many of the resident cells of the lung, for instance epithelial cells and airway smooth muscle cells, however TRPC6 is also present in smooth muscle in the pulmonary artery. TRPC6 is up-regulated in these smooth muscle cells when stimulated by platelet derived growth factor (PDGF). PDGF increases are associated with pulmonary hypertension and proliferation of artery smooth muscle cells. Inhibition of TRPC6 expression e.g. using antisense suppresses PDGF-stimulated pulmonary artery smooth muscle cell proliferation. Accordingly, pharmaceuticals that inhibit TRPC6 in smooth muscle in the pulmonary artery are useful in the treatment of pulmonary hypertension.
  • PDGF platelet derived growth factor
  • a human TRPC6 polypeptide can be isolated using any suitable conventional method. Since the sequence for the human TRPC6 gene is known, specific primers may be used as a convenient option. For example, (SEQ 01) 5'-gcaaatgaaagctttggacc-3' as the forward primer and (SEQ 02) 5'-atcgtaacattatagactccat-3' as the reverse primer gives a polymerase chain reaction (PCR) product of 300 base pairs.
  • PCR polymerase chain reaction
  • a human TRPC6 polynucleotide may be cDNA, genomic DNA or RNA and may be obtained using any suitable conventional method. For example it may be prepared from the nucleotides which it comprises by chemical synthesis, e.g. automated solid phase synthesis using known procedures and apparatus.
  • a human TRPC6 antibody may be a polyclonal or monoclonal antibody. Such antibodies may be prepared using conventional procedures. Methods for the production of polyclonal antibodies against purified antigen are well established (cf. Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11). Human TRPC6 antibodies may be used to detect, or determine the level of expression of, human TRPC6 polypeptides, or to inhibit ligand/anti-ligand binding activities of human TRPC6 polypeptides.
  • a human TRPC6 antisense oligonucleotide may be DNA, an analogue of DNA such as a phosphorothioate or methylphosphonate analogue of DNA, RNA, an analogue of RNA, or a peptide nucleic acid (PNA).
  • the antisense oligonucleotide may be synthesised by conventional methods, for example using automated solid phase techniques. It may be used to inhibit the expression of the human TRPC6 gene, where this is desired.
  • a short interfering RNA siRNA
  • RNA interference inhibits gene expression and can therefore be used to explore gene function. This technique is described by S. M. Elbashir et al in Methods 26 (2002) 199-213.
  • a human TRPC6 polynucleotide probe comprises at least 15 contiguous nucleotides of the aforementioned polynucleotide or a complement thereof.
  • the probe may be cDNA, genomic DNA or RNA and can be synthesised by conventional methods. Usually it is a synthetic oligonucleotide comprising 15 to 50 nucleotides, which can be labelled, e.g. with a fluorophore, to provide a detectable signal.
  • the nucleotides that encode the first 50 or so amino acids of human TRPC6 may be used for this purpose.
  • a human TRPC6 polynucleotide probe can be used to detect the presence or absence of the human TRPC6 gene, i.e. to detect genetic abnormality.
  • an agent of the invention in inhibiting or reversing a leukocyte -associated inflammatory disease may be demonstrated in a model of the disease, e.g. a lipopolysaccharide-induced lung inflammation model in rat or mouse or models described by Durie et al., Clin. Immunol. Immunopathol. (1994) 73: 11-18; and Williams et al, Proc. Natl. Acad. Sci. USA (1992) 89: 9784-9788.
  • a model of the disease e.g. a lipopolysaccharide-induced lung inflammation model in rat or mouse or models described by Durie et al., Clin. Immunol. Immunopathol. (1994) 73: 11-18; and Williams et al, Proc. Natl. Acad. Sci. USA (1992) 89: 9784-9788.
  • the agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.
  • any appropriate route e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.
  • compositions containing agents of the invention may be prepared using conventional diluents or excipients and techniques known in the galenic art.
  • oral dosage forms may include tablets and capsules, and compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.
  • the composition comprises an aerosol formulation
  • it preferably contains, for example, a hydro-fluoro-alkane (HFA) propellant such as HFA134a or HFA227 or a mixture of these, one or more co-solvents known in the art such as ethanol (up to 20% by weight), one or more surfactants such as oleic acid or sorbitan trioleate, and one or more bulking agents such as lactose.
  • HFA hydro-fluoro-alkane
  • co-solvents known in the art
  • surfactants such as oleic acid or sorbitan trioleate
  • bulking agents such as lactose.
  • the active ingredient preferably has a particle diameter up to 10 microns and the formulation includes a diluent or carrier, such as lactose, and a compound that helps to protect against product performance deterioration due to moisture.
  • a diluent or carrier such as lactose
  • the composition comprises a nebulised formulation
  • it preferably contains, for example, the active ingredient, which is either dissolved or suspended, in a vehicle containing water, a co-solvent such as ethanol or propylene glycol and a stabiliser, which may be a surfactant.
  • a co-solvent such as ethanol or propylene glycol
  • a stabiliser which may be a surfactant.
  • the invention includes (i) an agent of the invention in inhalable form, e.g. in an aerosol or other atomizable composition or in inhalable particulate, e.g.
  • an inhalable medicament comprising an agent of the invention in inhalable form
  • a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device
  • an inhalation device containing an agent of the invention in inhalable form.
  • Dosages of agents of the invention employed in practising the present invention may of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 1 ⁇ g to 10 mg/kg while for oral administration suitable daily doses are of the order of 0.1 mg to 1000 mg/kg.
  • An assay for screening candidate or test compounds is performed using cells stably-transfected with TRPC6 and expressing a functional TRPC6 channel e.g. the mammalian cell line HEK 293 which expresses an endogenous muscarinic acetylcholine receptor which can activate TRPC6 channels.
  • the assay is performed on a 96-well FLIPR ® (Fluorescence Imaging Plate Reader (Molecular Devices Corporation) using the Molecular Devices proprietary FLIPR ® membrane potential assay kit (cat # R8034) to measure membrane depolarisation resulting from TRPC6-mediated Na + influx.
  • the loading buffer consists of FLIPR ® membrane potential dye diluted to the required assay concentration in a buffer composed of 140 mM NaCl, 0.15 mM C Cli, 3.3 mM KH 2 PO4, 1.2 mM MgCl 2 , 10 mM D-glucose and 20m M HEPES, at pH7.4 and containing 0.03 mM BAPTA-AM.
  • the plates are incubated at 37° C for 20 minutes prior to commencing the assay.
  • test agents are added to each well either before or after stimulation with the muscarinic acetylcholine receptor agonist carbachol, which activates the TRPC6 channel.
  • the expected effect of an inhibitor would be to reduce the carbachol-stimulated increase in fluorescence.
  • Activators of TRPC6 may be detected by substituting carbachol for a test agent, where activators will stimulate an increase in fluorescence.
  • TRPC6 clone 14 cells pre-treated with l-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphen- ethyl]-lH-imidazole-HCl for 10 minutes are stimulated with carbachol (10 ⁇ M) in accordance with the assay described in Example 1.
  • carbachol (10 ⁇ M)
  • the fluorescence is measured prior to carbachol addition (minimum response, i.e. Min.) and when the carbachol-stimulated increase in fluorescence has reached a maximum (maximum response, i.e. Max.). It can be seen in table 1 below that the reduced fluorescence responses are observed in compound treated-cells compared to the control response in the absence of compound.
  • the data is calculated as a (Max-Min)/Min response.

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Abstract

The invention relates to an assay for substances that modulate the activity of a polypeptide encoded human transient receptor potential 6 gene (TRPC6). The assay involves combining a candidate substrate substance with the polypeptide and measuring the effect of the candidate substance on the activity of said polypeptide. Such substances may be used in the preparation of pharmaceuticals for treating inflammatory diseases, particularly those associated with leukocytes.

Description

ORGANIC COMPOUNDS
The present invention relates to the identification of substances or agents that modulate the activity of the transient receptor potential 6 ion channel, TRPC6, and the use of such substances in the treatment of inflammatory diseases, particularly those of the respiratory system.
A variety of cells are attracted into tissues during inflammation. These include various leukocytes, particularly inflammatory phagocytes such as neutrophilic and eosinophilic granulocytes and monocytes. Neutrophils have been associated with inflammation and tissue destruction in respiratory diseases such as chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema associated therewith, and adult respiratory distress syndrome (ARDS), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and rheumatoid arthritis. Eosinophils have been associated with respiratory diseases such as asthma and allergic rhinitis.
Critical steps in the action of leukocytes in inflammatory conditions include the migration of these cells into the tissues, e.g. into the airways in respiratory inflammations or to the joints in rheumatoid arthritis, cell activation and the release of a range of inflammatory mediators, leukotrienes, oxygen radicals, proteases. Signals that are needed for leukocyte migration and activation are often communicated through receptors that respond to an increase in the level of cytosolic calcium. Ins(l,4,5)P3 receptors are known to release calcium ions from intracellular stores but less is known about the channels in the plasma membrane through which those ions pass.
The transient receptor potential or "trp" gene of Drosopbila is known to encode a Ca++ selective store operated Ca++ entry (SOC) and this was used to find the human omologue (TRPC) and related genes. This led to the discovery of a family of proteins that function as ion channels i.e. TRPC1-6.
Boulay et al cloned and sequenced murine TRPC6 and described that in "Cloning and expression of a novel mammalian homologue of Drosopbila transient receptor potential (trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein" J. Biol. Chem. Vol. 272, 29672-29680 (1997). Murine TRPC6 is available from GenBank under accession number NP_038866. Human TRPC6, also known as classical TRP6 or TRPC6, is available from GenBank under accession number NP_004612.
D'Esposito et al published the sequence of the human TRPC6 gene in the "Identification and assignment of the human transient receptor potential channel 6 gene TRPC6 to chromosome I l q21->q22 " Cytogenet. Cell Genet. 83 (1-2), 46-47 (1998).
Hofmann et al showed in "Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol", Nature Vol.397, 21 January 1999 that histamine stimulated greater Ca++ influx in CHO-K1 cells if they heterologously expressed a functional TRPC6 channel (demonstrated electrophysiologically). They also demonstrated that TRPC6 channel activity could be measured as Na+ influx.
TRPC6 has been found to be present in cells attracted into tissues during inflammation. TRPC6 mRNA has been shown to be present in isolated primary human neutrophils and in human lung macrophages isolated by bronchoalveolar lavages from smokers and COPD patients. TRPC6 mRNA expression has also been found in cultured human airway smooth muscle cells and well-differentiated human bronchial epithelial cells. TRPC6 proteins have been shown to be expressed in human T-lymphocytes, cultured human airway smooth muscle cells, well-differentiated human bronchial epithelial cells, bronchial airway smooth muscle, epithelial cells of the airway epithelium, submucosal glands and tissue leukocytes i.e. neutrophils, macrophages and lymphocytes.
It is proposed in accordance with the present invention that substances that attenuate the activation of leukocytes such as neutrophils or eosinophils by inhibiting the influx of calcium ions into such cells are useful for treating respiratory diseases such as asthma and chronic obstructive pulmonary disease. The present invention thus provides TRPC6 as a therapeutic target for such diseases and a "screening assay" for identifying modulators i.e., candidate compounds or agents including peptides, peptidomimetics, small molecules or other drugs, which stimulate or inhibit the activity of the channel formed by the gene product of human TRPC6.
Accordingly, in a first aspect the present invention relates to a method of identifying a substance suitable for use in the treatment of a leukocyte-associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6), wherein the method comprises combining a candidate substance with said polypeptide and measuring the effect of the candidate substance on the activity of said polypeptide.
In a second aspect the present invention relates to a pharmaceutical composition comprising a compound that inhibits the influx of calcium ions through a human TRPC6 ion channel and a pharmaceutically acceptable carrier.
In a third aspect the present invention relates to the use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene, an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, or a polynucleotide probe comprising at least 15 consecutive nucleotides of that polynucleotide, in the preparation of a pharmaceutical that inhibits the accumulation of leukocytes in human tissue.
In a fourth aspect the present invention relates to the use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene, an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, or a polynucleotide probe comprising at least 15 consecutive nucleotides of that polynucleotide, in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
In a fifth aspect the present invention relates to the use of a TRPC6 inhibitor in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
As mentioned above, in a first aspect the present invention relates to a method of the present invention for identifying a substance suitable for use in the treatment of a leukocyte-associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene. In broad terms this method, or assay, comprises combining a candidate substance with a polypeptide encoded by the transient receptor potential channel 6 gene and measuring the effect of the candidate substance on the activity of that polypeptide.
Substances that are suitable for use in the treatment of a leukocyte-associated inflammatory disease will tend to be enhancers (herein "human TRPC6 agonists" ) or inhibitors (herein "human TRPC6 antagonists" ) of its activity. The activity of a TRPC6 gene product, i.e. the polypeptide that forms the TRPC6 ion channel, may be measured, for example, by a cell-based method or screening assay that identifies compounds which have a stimulatory or inhibitory effect on the activity of the human TRPC6 channel, or by an appropriate reporter gene assay.
The abovementioned screening method may be carried out, for example, by preparing cells which express the TRPC6 polypeptide on their surfaces, e.g. insect, mammal or yeast cells and then incubating the resulting cells with the candidate substance to determine the enhancement or inhibition of a functional activity of the TRPC6 polypeptide.
In a suitable test for any activation of the TRPC6 channel, the candidate substance is combined with cells that are stably transfected with TRPC6 and express a functional TRPC6 channel and membrane depolarisation is measured to indicate any TRPC6-mediated Na+ influx.
In a suitable test for any inhibition of the TRPC6 channel, cells that express an endogenous G protein-coupled calcium mobilising receptor e.g. muscarinic acetylcholine receptor that can activate TRPC6 channels are used and a receptor agonist e.g. muscarinic acetylcholine receptor agonist is added to stimulate the TRPC6 channels. The cells are treated with the candidate substance prior to addition of the receptor agonist. That should produce an increase in fluorescence, but the magnitude of the increase will be reduced if the candidate substance inhibits the TRPC6 channels. Any change in fluorescence can be measured using suitable equipment, for example a fluorescence imaging plate reader.
Using such methods it has been found that l-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxy- phenethyl]-lH-imidazole-HCl inhibits the activity of the human TRPC6 ion channel.
l-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl was disclosed by Merritt et al in Biochem. J. (1990) 271: 515-522 to inhibit receptor-mediated calcium entry (RMCE). It was also disclosed by Inoue et al in Circ. Res. (2001) 88: 325-332 to block the murine TRPC6 ion channel.
The present invention also relates to a pharmaceutical composition that comprises a compound that inhibits the influx of calcium ions through a TRPC6 channel, such as l-[b-[3- (4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl, and a pharmaceutically acceptable carrier.
One can use an antibody which is immunoreactive with the polypeptide encoded by the human TRPC6 gene (herein a "human TRPC6 antibody" ) or an antisense oligonucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising a nucleotide sequence encoding that polypeptide (herein a " human TRPC6 antisense oligonucleotide" ), to prepare pharmaceuticals that inhibit the accumulation of leukocytes in human tissue.
The aforementioned human TRPC6 antibodies and antisense oligonucleotides may be used to treat leukocyte-associated inflammatory diseases.
Human TRPC6 agonists, human TRPC6 antagonists, human TRPC6 antibodies and human TRPC6 antisense oligonucleotides are hereinafter alternatively referred to collectively as " agents of the invention " .
Neutrophil-associated inflammatory diseases to which the present invention is applicable include neutrophil-associated inflammatory or obstructive airways diseases, particularly chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema, and adult (or acute) respiratory distress syndrome (ARDS), rheumatoid arthritis and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Eosinophil- associated inflammatory diseases to which the present invention is also applicable include eosinophil-associated inflammatory or obstructive airways diseases, particularly asthma and allergic rhinitis.
Amongst peripheral tissues, the lung along with the placenta has the highest level of TRPC6 expression. TRPC6 is present in many of the resident cells of the lung, for instance epithelial cells and airway smooth muscle cells, however TRPC6 is also present in smooth muscle in the pulmonary artery. TRPC6 is up-regulated in these smooth muscle cells when stimulated by platelet derived growth factor (PDGF). PDGF increases are associated with pulmonary hypertension and proliferation of artery smooth muscle cells. Inhibition of TRPC6 expression e.g. using antisense suppresses PDGF-stimulated pulmonary artery smooth muscle cell proliferation. Accordingly, pharmaceuticals that inhibit TRPC6 in smooth muscle in the pulmonary artery are useful in the treatment of pulmonary hypertension. A human TRPC6 polypeptide can be isolated using any suitable conventional method. Since the sequence for the human TRPC6 gene is known, specific primers may be used as a convenient option. For example, (SEQ 01) 5'-gcaaatgaaagctttggacc-3' as the forward primer and (SEQ 02) 5'-atcgtaacattatagactccat-3' as the reverse primer gives a polymerase chain reaction (PCR) product of 300 base pairs.
A human TRPC6 polynucleotide may be cDNA, genomic DNA or RNA and may be obtained using any suitable conventional method. For example it may be prepared from the nucleotides which it comprises by chemical synthesis, e.g. automated solid phase synthesis using known procedures and apparatus.
A human TRPC6 antibody may be a polyclonal or monoclonal antibody. Such antibodies may be prepared using conventional procedures. Methods for the production of polyclonal antibodies against purified antigen are well established (cf. Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11). Human TRPC6 antibodies may be used to detect, or determine the level of expression of, human TRPC6 polypeptides, or to inhibit ligand/anti-ligand binding activities of human TRPC6 polypeptides.
A human TRPC6 antisense oligonucleotide may be DNA, an analogue of DNA such as a phosphorothioate or methylphosphonate analogue of DNA, RNA, an analogue of RNA, or a peptide nucleic acid (PNA). The antisense oligonucleotide may be synthesised by conventional methods, for example using automated solid phase techniques. It may be used to inhibit the expression of the human TRPC6 gene, where this is desired. Alternatively, a short interfering RNA (siRNA) can be used as a specific tool for targeted gene knockdown. RNA interference inhibits gene expression and can therefore be used to explore gene function. This technique is described by S. M. Elbashir et al in Methods 26 (2002) 199-213.
A human TRPC6 polynucleotide probe comprises at least 15 contiguous nucleotides of the aforementioned polynucleotide or a complement thereof. The probe may be cDNA, genomic DNA or RNA and can be synthesised by conventional methods. Usually it is a synthetic oligonucleotide comprising 15 to 50 nucleotides, which can be labelled, e.g. with a fluorophore, to provide a detectable signal. The nucleotides that encode the first 50 or so amino acids of human TRPC6 may be used for this purpose. A human TRPC6 polynucleotide probe can be used to detect the presence or absence of the human TRPC6 gene, i.e. to detect genetic abnormality. The effectiveness of an agent of the invention in inhibiting or reversing a leukocyte -associated inflammatory disease may be demonstrated in a model of the disease, e.g. a lipopolysaccharide-induced lung inflammation model in rat or mouse or models described by Durie et al., Clin. Immunol. Immunopathol. (1994) 73: 11-18; and Williams et al, Proc. Natl. Acad. Sci. USA (1992) 89: 9784-9788.
The agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.
Pharmaceutical compositions containing agents of the invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets and capsules, and compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.
When the composition comprises an aerosol formulation, it preferably contains, for example, a hydro-fluoro-alkane (HFA) propellant such as HFA134a or HFA227 or a mixture of these, one or more co-solvents known in the art such as ethanol (up to 20% by weight), one or more surfactants such as oleic acid or sorbitan trioleate, and one or more bulking agents such as lactose.
When the composition comprises a dry powder formulation, the active ingredient preferably has a particle diameter up to 10 microns and the formulation includes a diluent or carrier, such as lactose, and a compound that helps to protect against product performance deterioration due to moisture.
When the composition comprises a nebulised formulation, it preferably contains, for example, the active ingredient, which is either dissolved or suspended, in a vehicle containing water, a co-solvent such as ethanol or propylene glycol and a stabiliser, which may be a surfactant. The invention includes (i) an agent of the invention in inhalable form, e.g. in an aerosol or other atomizable composition or in inhalable particulate, e.g. micronised form, (ii) an inhalable medicament comprising an agent of the invention in inhalable form; (iii) a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device; and (iv) an inhalation device containing an agent of the invention in inhalable form. Dosages of agents of the invention employed in practising the present invention may of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 1 μg to 10 mg/kg while for oral administration suitable daily doses are of the order of 0.1 mg to 1000 mg/kg.
The invention is illustrated by the following Examples.
Example 1
Assay suitable for high-throughput screening
An assay for screening candidate or test compounds is performed using cells stably-transfected with TRPC6 and expressing a functional TRPC6 channel e.g. the mammalian cell line HEK 293 which expresses an endogenous muscarinic acetylcholine receptor which can activate TRPC6 channels. The assay is performed on a 96-well FLIPR® (Fluorescence Imaging Plate Reader (Molecular Devices Corporation) using the Molecular Devices proprietary FLIPR® membrane potential assay kit (cat # R8034) to measure membrane depolarisation resulting from TRPC6-mediated Na+ influx. The specificity of this TRPC6-mediated response may be shown by demonstrating that it is not observed in XRPC6-expressing cells if Na+ is replaced in the extracellular buffer by the impermeant monovalent cation NMDG+ and that Na+ influx- dependent membrane depolarisation is not observed in control cells that have not been transfected with TRPC6. The TRPC6-expressing cells are seeded in 96-well black -walled flat- bottomed microplates in a plating volume of 100 μl and grown to confluence for the assay. To prepare the cells for the assay, the cell culture medium is removed and 100 μl of loading buffer is added to each well. The loading buffer consists of FLIPR® membrane potential dye diluted to the required assay concentration in a buffer composed of 140 mM NaCl, 0.15 mM C Cli, 3.3 mM KH2PO4, 1.2 mM MgCl2, 10 mM D-glucose and 20m M HEPES, at pH7.4 and containing 0.03 mM BAPTA-AM. The plates are incubated at 37° C for 20 minutes prior to commencing the assay. For the detection of inhibitors of TRPC6, test agents are added to each well either before or after stimulation with the muscarinic acetylcholine receptor agonist carbachol, which activates the TRPC6 channel. The expected effect of an inhibitor would be to reduce the carbachol-stimulated increase in fluorescence. Activators of TRPC6 may be detected by substituting carbachol for a test agent, where activators will stimulate an increase in fluorescence. Example 2
Detection of l-rb-r3-(4-methoxyphenyl)propoxy1-4-methoxyphenethyll-lH-imidazole-HCl
TRPC6 clone 14 cells pre-treated with l-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphen- ethyl]-lH-imidazole-HCl for 10 minutes are stimulated with carbachol (10 μM) in accordance with the assay described in Example 1. The fluorescence is measured prior to carbachol addition (minimum response, i.e. Min.) and when the carbachol-stimulated increase in fluorescence has reached a maximum (maximum response, i.e. Max.). It can be seen in table 1 below that the reduced fluorescence responses are observed in compound treated-cells compared to the control response in the absence of compound.
TABLE 1
Inhibition of TRPC6 ion channels by l-[b-[3-(4-methoxy- phenyl)-propoxy]-4-methoxyphen-ethyl]-lH-imidazole-HCl
The data is calculated as a (Max-Min)/Min response. Each concentration of l-[b-[3-(4- methoxyphenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl is the mean ± SEM (standard error of the mean) of n=10 wells. The calculated mean ICJO value from n=4 experiments is 4.9 + 0.8 μM (mean + S.E.M. ).
This shows that the assay described in Example I detects l-[b-[3-(4-methoxyphenyl)propoxy]- 4-methoxyphenethyl]-lH-imidazoleΗCl.

Claims

1. A method of identifying a substance suitable for use in the treatment of a leukocyte- associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6), wherein the method comprises combining a candidate substance with said polypeptide; and measuring the effect of the candidate substance on the activity of said polypeptide.
2. A method according to claim 1 wherein the candidate substance is combined with cells that are stably-transfected with TRPC6 and express a functional TRPC6 channel and measuring any membrane depolarisation to indicate a TRPC6-mediated Na+ influx.
3. A method according to claim 1 wherein the cells express an endogenous calcium-mobilising G protein— coupled receptor that can activate TRPC6 channels by agonist stimulation and a receptor agonist is added after the candidate substance to stimulate the TRPC6 channels and allow for any inhibition to be measured.
4. A pharmaceutical composition comprising a compound that inhibits the influx of calcium ions through a human TRPC6 ion channel and a pharmaceutically acceptable carrier.
5. A pharmaceutical composition according to claim 4 wherein the compound is l-[b-[3-(4- methoxyphenyl)propoxy]-4-methoxyphenethyl]-lH-imidazole-HCl.
6. The use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene or an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, in the preparation of a pharmaceutical that inhibits the accumulation of leukocytes in human tissue.
7. The use of an antibody which is immunoreactive with a polypeptide encoded by the human TRPC6 gene or an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding that polypeptide, in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
8. The use according to claim 7, in which the disease is a neutrophil-associated disease such as chronic obstructive pulmonary disease, adult respiratory distress syndrome, rheumatoid arthritis or inflammatory bowel disease.
9. The use according to claim 7, in which the disease is an eosinophil-associated disease such as asthma or allergic rhinitis.
10. The use of a TRPC6 inhibitor in the preparation of a pharmaceutical for the treatment of a leukocyte-associated inflammatory disease.
11. The use of a TRPC6 inhibitor in the preparation of a pharmaceutical for the treatment of pulmonary hypertension.
12. A method of identifying a substance suitable for use in the treatment of a leukocyte- associated inflammatory disease which modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene substantially as herein described with reference to the Examples.
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