WO2004072645A2 - Identification of organic compounds capable of modulating the activity of epithelial sodium ion channels - Google Patents

Identification of organic compounds capable of modulating the activity of epithelial sodium ion channels Download PDF

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Publication number
WO2004072645A2
WO2004072645A2 PCT/EP2004/001371 EP2004001371W WO2004072645A2 WO 2004072645 A2 WO2004072645 A2 WO 2004072645A2 EP 2004001371 W EP2004001371 W EP 2004001371W WO 2004072645 A2 WO2004072645 A2 WO 2004072645A2
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cells
dye
membrane potential
sodium ion
epithelial
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PCT/EP2004/001371
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French (fr)
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WO2004072645A3 (en
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Henry Luke Danahay
Martin Gosling
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Novartis Ag
Novartis Pharma Gmbh
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Publication of WO2004072645A3 publication Critical patent/WO2004072645A3/en

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • 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

Definitions

  • the present invention relates to the identification of substances or agents that modulate the activity of epithelial sodium ion channels (ENaCs) and the use of such substances in the treatment of inflammatory diseases, particularly those of the respiratory system.
  • ENaCs epithelial sodium ion channels
  • the epithelial cells that line the respiratory tract play a critical role in regulating the composition, volume and physical properties of the fluids that bathe the airways.
  • the plasma membranes of these cells contain channels that actively absorb sodium ions and thereby regulate fluid movement.
  • One of these channels called the epithelial sodium ion channel or ENaC, controls the fluid that is absorbed into the bloodstream and thus regulates the airway surface liquid volume. If these channels are blocked in some way, fluid will collect in the lumen, which encourages mucus precursors to hydrate and stimulate mucus clearance.
  • the present invention provides a fluorescence-based method for identifying ENaC blocking or activating molecules.
  • the method is suitable for use on a high throughput basis.
  • the present invention relates to a method of identifying a substance suitable for use in the treatment of an inflammatory disease which modulates the activity of a human epithelial sodium ion channel (ENaC), wherein the method comprises treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.
  • EaC human epithelial sodium ion channel
  • the epithelial cells are cultured for a time that is sufficient to provide a "dome-forming" transporting phenotype. This tends to be at least 5 days but can take 7 days.
  • the cells Once the cells exhibit the "dome-forming" transporting phenotype they are treated with the dye or the candidate substance.
  • the cells are preferably treated with the dye before being treated with the candidate substance.
  • the epithelial cells preferably functionally express high levels of ENaC.
  • ENaC should be the dominant regulator of membrane potential in the cell type.
  • Suitable epithelial cells include M-l murine cortical collecting duct cells.
  • the fluorescent membrane potential sensitive dye is preferably a lipophilic anionic dye.
  • Suitable dyes include Molecular Devices #R8034, which is commercially available from Molecular Devices, and fluorescence resonance energy transfer (FRET)-based voltage sensing probe sets DiSBAQ>/CC2-DMPE and DiSBAC 4 /CC2-DMPE, which are commercially available from Invitrogen Corporation.
  • the present invention relates to a method of identifying a substance suitable for use in the treatment of an inflammatory disease that modulates the activity of the epithelial sodium ion channel (ENaC).
  • This disease would suitably be one, for example, that is associated with the impairment of mucociliary clearance.
  • this method comprises treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.
  • ENaC In transporting epithelia, ENaC is constitutively open. The consequent influx of Na + plays a role in the regulation of membrane potential that is measurable using a membrane potential sensitive dye.
  • Fluorescent membrane potential sensitive dyes can provide a fast and simple method for detecting changes in membrane potential. Lipophilic anionic membrane potential dyes in particular partition into and out of cells depending upon the plasma membrane potential. The fluorescence intensity of the dye increases when it binds to cytoplasmic proteins. It has now been found that when ENaC is constitutively open and therefore contributing to membrane potential, adding an ENaC blocking compound induces a membrane hyperpolarisation that causes the membrane potential sensitive dye to partition out of the cells and the fluorescence signal to decrease.
  • Pyrazinecarboxamides such as amiloride, benzamil and dimethyl-amiloride (DMA) are known to block human epithelial sodium channels. Amiloride has been used clinically as a diuretic but its short half life makes it unsuitable for use in treating airway disease.
  • the method of the present invention can be used to identify alternative and preferably more potent ENaC inhibitors that are more suitable for use as pharmaceuticals. This method is amenable to high throughput screening.
  • Classical ion channel assay technologies such as patch clamp and short circuit current techniques are not suitable for high throughput screening and are therefore not suitable on a practical level for use in drug discovery programmes. Isotope flux cannot easily be used for the high throughput screening of sodium channels because of the high energy associated with 22 Na.
  • Epithelial cells for use in the method of the present invention preferably functionally express high numbers of epithelial sodium channels on their surfaces.
  • ENaC should preferably be the dominant regulator of membrane potential in the cell type.
  • M-l murine cortical collecting duct cells meet both criteria and show close structural and functional homology with human epithelial cells. M-l murine cortical collecting duct cells also surprisingly retain their ability to transport sodium ions and water through their plasma membrane (i.e. "polarise") when they have been cultured on a plastic surface. This characteristic makes them a particularly convenient choice for use in a plate-based assay system.
  • polarise plasma membrane
  • the epithelial cells are preferably loaded into the wells of a multi-welled cell plate, for example a plastic 96 well plate, with a suitable cell medium.
  • a multi-welled cell plate for example a plastic 96 well plate, with a suitable cell medium.
  • Each well should preferably contain an equal concentration of epithelial cells.
  • the epithelial cells are cultured for a time that is sufficient to provide a dome-forming phenotype that indicates that they are actively transporting sodium ions and water through their plasma membrane. This tends to take at least 5 days but can take up to 9 days. At that time the membrane potential of the cells will also be sensitive to ENaC inhibitors such as pyrazinecarboxamides.
  • the epithelial cells are treated with a fluorescent membrane potential sensitive dye and a candidate substance. If more than one candidate substance is to be tested each candidate substance is placed in a different well.
  • the cells are preferably treated with the dye as a control before being treated with the candidate substance(s) so that the conditions within the wells are substantially the same before the candidate compounds are introduced and one can more clearly monitor the effect of the candidate substance from the time it is used to treat the cells. However one could treat the cells with the candidate substance(s) before or at the same time as treating the cells with dye.
  • the dye treatment step suitably involves using appropriate automated means to deposit a predetermined volume of the dye from the same stock solution into each well.
  • the dye is preferably a lipophilic anionic dye as such dyes tend to be particularly suitable for use with epithelial cells.
  • Suitable dyes include Molecular Devices #R8034, which is commercially available from Molecular Devices Corporation and is understood to have a bis-oxanol-Hke structure.
  • the dye should preferably be left to partition between the intracellular and extracellular compartments. It will fluoresce when bound to intracellular proteins.
  • the cells are preferably incubated for a time that is suitable to allow this partitioning. This can take 10 minutes to an hour, for example about 30 minutes, at a suitable incubation temperature, for example about 37° C.
  • the step by which the epithelial cells are treated with a candidate substance or a number of candidate substances suitably involves using appropriate automated means to deposit a predetermined volume of a candidate substance in each well. If a multi-welled sample plate such as a plastic 96 well plate is used one can conveniently place a different candidate substance in each well. However for statistical purposes it is preferable to test each candidate in triplicate, for example 3 x 32 candidate substances on a plastic 96 well plate.
  • the fluorescence emitted by the contents of each well is measured using appropriate equipment before and after the addition of the candidate substances.
  • a FLIPR ® fluorescence imaging plate reader (ex Molecular Devices Corporation) can conveniently be used for this purpose but other suitable equipment is also commercially available.
  • Fluorescence measurements are monitored over time, preferably for several minutes, conveniently for 4 to 12 minutes.
  • a candidate substance that causes a significant reduction in fluorescence counts over time in comparison with the control counts can be identified as at least a potential ENaC inhibitor or antagonist.
  • a candidate compound that causes a significant increase in fluorescence counts over time can be identified as at least a potential ENaC activator or agonist.
  • ENaC inhibitors can enhance mucus clearance and thus may be used to treat diseases associated with the impairment of mucociliary clearance.
  • ENaC inhibitors/antagonists and ENaC activators/agonists are hereinafter alternatively referred to collectively as “agents of the invention” .
  • the present invention provides the use of an epithelial sodium ion channel inhibitor in the preparation of a pharmaceutical for the treatment of a condition mediated by epithelial sodium ion channels.
  • Conditions to which the present invention is applicable include inflammatory diseases such as 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), and eosinophil-associated inflammatory or obstructive airways diseases, particularly asthma and allergic rhinitis.
  • COPD chronic obstructive pulmonary disease
  • ARDS adult (or acute) respiratory distress syndrome
  • eosinophil-associated inflammatory or obstructive airways diseases particularly asthma and allergic rhinitis.
  • the present invention is also applicable to acute and chronic respiratory tract conditions including cough, colds, influenza, cystic fibrosis and cancer, all conditions with an associated decrease in mucociliary clearance.
  • 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, and one or more co-solvents known in the art such as ethanol (up to 20% by weight), any may contain one or more surfactants such as oleic acid or sorbitan trioleate, and one or more bulking agents such as lactose.
  • HFA hydro-fluoro-alkane
  • HFA227 hydro-fluoro-alkane
  • co-solvents known in the art
  • any may contain one or more surfactants such as oleic acid or sorbitan trioleate, and one or more 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 may contain 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.
  • 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.
  • 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.
  • M-l murine cortical collecting duct cells (ATCC #CRL-2038) are cultured in plastic flasks with PC-1 media supplemented with 5 mM glutamine. On reaching 80 - 90% confluence the cells are trypsinised and seeded into 96 well black cluster plates with clear bottoms and optical masking at 100,000 cells/well in 100 ⁇ l PC-1 medium. Cells are maintained in incubators at 37°C in 5% CO 2 at 100% humidity for up to 7 days. The medium is exchanged the day before the assay is conducted. Preparation of the membrane potential dye
  • Membrane potential dye (Molecular Devices #R8034) is prepared as per the manufacturer's instructions as a 2x stock concentration in HBSS (Hank's Balanced Salt Solution, GibcoBRL #14025-050) containing 20 mM HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2- ethanesulphonic acid] GibcoBRL #15630-056) at pH 7.4 (NaOH). The dye is stored at -20°C and defrosted immediately before use.
  • the M-l cells are washed 3 times with HBSS (containing 20 mM HEPES) using a LABSYSTEMSTM microplate washer, leaving the cells in a final volume of 100 ⁇ l. 100 ⁇ l of the membrane potential dye that has been prepared according to the manufacturer's instructions is added to the cells. The cells are incubated for 30 min at 37°C in 5% CO 2 at 100% humidity.
  • Candidate compounds amiloride, benzamil and dimethyl-amiloride are prepared in HBSS (containing 20 nxM HEPES) at 5x the final concentration and added on line in a volume of 50 ⁇ l. DMSO tolerance of the system is assessed separately. The wells are run in triplicate.
  • the fluorescence of the contents of each well is measured using 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 potential regulated by ENaC.
  • FLIPR ® Fluorescence Imaging Plate Reader
  • Cat # R8034 Molecular Devices proprietary FLIPR ® membrane potential assay kit
  • the cells form a confluent, 'cobblestone' epithelia.
  • the dye is loaded and amiloride and benzamil are added to the cells to induce a rapid decrease in the basal fluorescence.
  • the IGso values for amiloride and benzamil are 10 ⁇ M and 3 ⁇ M respectively.
  • the cells retain the confluent, 'cobblestone' appearance.
  • the dye is loaded and amiloride, benzamil and DMA are added to the cells again to induce a rapid decrease in the basal fluorescence.
  • the ICJO values for amiloride, benzamil and DMA i.e. the concentration of these compounds at which 50% inhibition occurs, are 2 ⁇ M, 50 nM and 8 ⁇ M respectively. The values are determined from concentration-inhibition curves in a conventional manner.
  • FLIPR ® traces illustrate that 30 ⁇ M amiloride, benzamil and dimethyl-amiloride (DMA) attenuate the basal fluorescence of membrane potential dye loaded cells indicative of membrane hyperpolarisation.
  • DMA dimethyl-amiloride
  • Table 1 shows sample time course data for three ENaC blockers, namely amiloride, benzamil and DMA, illustrating that at 30 ⁇ M each compound induces a decrease in fluorescence counts, indicative of membrane hyperpolarisation.
  • the inhibitor potency order for this effect is benzamil > amiloride > DMA. This is consistent with the established potency order for blockade of ENaC.
  • Example 1 The assay described in Example 1 is used to screen a library of potential ENaC inhibitors and activators. M-l cells are washed 3 times with HBSS (containing 20 mM HEPES) using a LABSYSTEMSTM microplate washer, leaving the cells in a final volume of 100 ⁇ l. 50 ⁇ l of MPD is added to the cells which are then incubated for 30 minutes at 37°C in 5% CO 2 at 100% humidity.
  • BIOMOLTM ion channel plate (BioMol #2805) containing candidate substances at 10 mM in DMSO is used to prepare an experimental plate containing each of the compounds diluted in HBSS (containing 20 mM HEPES) at a concentration of 40 ⁇ M. Compounds are added in 50 ⁇ l to achieve a final concentration of 10 ⁇ M. At the end of the experiment, amiloride (50 ⁇ l, 150 ⁇ M) is added to all wells as a positive control to achieve a final concentration of 30 ⁇ M. Wells are run as individual data points.
  • the assay gives hits for known ENaC inhibitors amiloride and benzamil as well as dichlorobenzamil and phenamil. Dichlorobenzamil and phenamil are therefore identified by the assay as potent ENaC inhibitors.
  • Known inhibitors of other cation channels such as flufenamic acid and verapamil that are present on the plate do not achieve hits. That indicates that observed hyperpolarisation is dominated by an ENaC-dependent mechanism and thus the assay is selective for ENaC.
  • Figure 2 shows sample raw data illustrating the effects of a library of 72 commercially available ion channel modulators on the basal membrane potential of M-l cells.
  • a test compound is added at 60 seconds (dotted line) followed by amiloride (30 ⁇ M) to each well.
  • "Hits" are clearly identified as compounds that induce a rapid reduction in fluorescence units upon addition (i.e. wells G6 [phenamil] and B8 [amiloride] and D8 [dichlorobenzamil]).
  • Each of the hits shown represents a recognised ENaC blocker.
  • Modulators of Ca 2+ , K + and Cl ⁇ channels do not show a rapid reduction in fluorescence units when added to the wells.

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Abstract

A method of identifying a substance that modulates the activity of epithelial sodium ion channels and as such may be suitable for use in the treatment of inflammatory diseases. The method involves treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.

Description

ORGANIC COMPOUNDS
The present invention relates to the identification of substances or agents that modulate the activity of epithelial sodium ion channels (ENaCs) and the use of such substances in the treatment of inflammatory diseases, particularly those of the respiratory system.
The epithelial cells that line the respiratory tract play a critical role in regulating the composition, volume and physical properties of the fluids that bathe the airways. The plasma membranes of these cells contain channels that actively absorb sodium ions and thereby regulate fluid movement. One of these channels, called the epithelial sodium ion channel or ENaC, controls the fluid that is absorbed into the bloodstream and thus regulates the airway surface liquid volume. If these channels are blocked in some way, fluid will collect in the lumen, which encourages mucus precursors to hydrate and stimulate mucus clearance.
The present invention provides a fluorescence-based method for identifying ENaC blocking or activating molecules. The method is suitable for use on a high throughput basis.
Accordingly, in a first aspect the present invention relates to a method of identifying a substance suitable for use in the treatment of an inflammatory disease which modulates the activity of a human epithelial sodium ion channel (ENaC), wherein the method comprises treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.
In general a decrease in fluorescence of the dye-treated cells in the presence of a candidate substance indicates the substance induces membrane hyperpolarisation by blocking ENaC.
The epithelial cells are cultured for a time that is sufficient to provide a "dome-forming" transporting phenotype. This tends to be at least 5 days but can take 7 days. Once the cells exhibit the "dome-forming" transporting phenotype they are treated with the dye or the candidate substance. The cells are preferably treated with the dye before being treated with the candidate substance.
The epithelial cells preferably functionally express high levels of ENaC. Ideally ENaC should be the dominant regulator of membrane potential in the cell type. Suitable epithelial cells include M-l murine cortical collecting duct cells. The fluorescent membrane potential sensitive dye is preferably a lipophilic anionic dye. Suitable dyes include Molecular Devices #R8034, which is commercially available from Molecular Devices, and fluorescence resonance energy transfer (FRET)-based voltage sensing probe sets DiSBAQ>/CC2-DMPE and DiSBAC4/CC2-DMPE, which are commercially available from Invitrogen Corporation.
Throughout this specification and in the claims that follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As mentioned above, in a first aspect the present invention relates to a method of identifying a substance suitable for use in the treatment of an inflammatory disease that modulates the activity of the epithelial sodium ion channel (ENaC). This disease would suitably be one, for example, that is associated with the impairment of mucociliary clearance.
In broad terms this method, or assay, comprises treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.
In transporting epithelia, ENaC is constitutively open. The consequent influx of Na+ plays a role in the regulation of membrane potential that is measurable using a membrane potential sensitive dye. Fluorescent membrane potential sensitive dyes can provide a fast and simple method for detecting changes in membrane potential. Lipophilic anionic membrane potential dyes in particular partition into and out of cells depending upon the plasma membrane potential. The fluorescence intensity of the dye increases when it binds to cytoplasmic proteins. It has now been found that when ENaC is constitutively open and therefore contributing to membrane potential, adding an ENaC blocking compound induces a membrane hyperpolarisation that causes the membrane potential sensitive dye to partition out of the cells and the fluorescence signal to decrease.
Pyrazinecarboxamides such as amiloride, benzamil and dimethyl-amiloride (DMA) are known to block human epithelial sodium channels. Amiloride has been used clinically as a diuretic but its short half life makes it unsuitable for use in treating airway disease. The method of the present invention can be used to identify alternative and preferably more potent ENaC inhibitors that are more suitable for use as pharmaceuticals. This method is amenable to high throughput screening. Classical ion channel assay technologies such as patch clamp and short circuit current techniques are not suitable for high throughput screening and are therefore not suitable on a practical level for use in drug discovery programmes. Isotope flux cannot easily be used for the high throughput screening of sodium channels because of the high energy associated with 22Na.
Epithelial cells for use in the method of the present invention preferably functionally express high numbers of epithelial sodium channels on their surfaces. ENaC should preferably be the dominant regulator of membrane potential in the cell type.
M-l murine cortical collecting duct cells meet both criteria and show close structural and functional homology with human epithelial cells. M-l murine cortical collecting duct cells also surprisingly retain their ability to transport sodium ions and water through their plasma membrane (i.e. "polarise") when they have been cultured on a plastic surface. This characteristic makes them a particularly convenient choice for use in a plate-based assay system.
The epithelial cells are preferably loaded into the wells of a multi-welled cell plate, for example a plastic 96 well plate, with a suitable cell medium. Each well should preferably contain an equal concentration of epithelial cells.
The epithelial cells are cultured for a time that is sufficient to provide a dome-forming phenotype that indicates that they are actively transporting sodium ions and water through their plasma membrane. This tends to take at least 5 days but can take up to 9 days. At that time the membrane potential of the cells will also be sensitive to ENaC inhibitors such as pyrazinecarboxamides.
The epithelial cells are treated with a fluorescent membrane potential sensitive dye and a candidate substance. If more than one candidate substance is to be tested each candidate substance is placed in a different well. The cells are preferably treated with the dye as a control before being treated with the candidate substance(s) so that the conditions within the wells are substantially the same before the candidate compounds are introduced and one can more clearly monitor the effect of the candidate substance from the time it is used to treat the cells. However one could treat the cells with the candidate substance(s) before or at the same time as treating the cells with dye. The dye treatment step suitably involves using appropriate automated means to deposit a predetermined volume of the dye from the same stock solution into each well. The dye is preferably a lipophilic anionic dye as such dyes tend to be particularly suitable for use with epithelial cells. Suitable dyes include Molecular Devices #R8034, which is commercially available from Molecular Devices Corporation and is understood to have a bis-oxanol-Hke structure.
The dye should preferably be left to partition between the intracellular and extracellular compartments. It will fluoresce when bound to intracellular proteins. The cells are preferably incubated for a time that is suitable to allow this partitioning. This can take 10 minutes to an hour, for example about 30 minutes, at a suitable incubation temperature, for example about 37° C.
The step by which the epithelial cells are treated with a candidate substance or a number of candidate substances suitably involves using appropriate automated means to deposit a predetermined volume of a candidate substance in each well. If a multi-welled sample plate such as a plastic 96 well plate is used one can conveniently place a different candidate substance in each well. However for statistical purposes it is preferable to test each candidate in triplicate, for example 3 x 32 candidate substances on a plastic 96 well plate.
The fluorescence emitted by the contents of each well is measured using appropriate equipment before and after the addition of the candidate substances. A FLIPR® fluorescence imaging plate reader (ex Molecular Devices Corporation) can conveniently be used for this purpose but other suitable equipment is also commercially available.
Fluorescence measurements are monitored over time, preferably for several minutes, conveniently for 4 to 12 minutes. A candidate substance that causes a significant reduction in fluorescence counts over time in comparison with the control counts can be identified as at least a potential ENaC inhibitor or antagonist. A candidate compound that causes a significant increase in fluorescence counts over time can be identified as at least a potential ENaC activator or agonist.
Further tests should preferably be conducted to confirm that a potential ENaC inhibitor or activator is directly responsible for modulating the activity of ENaC. One could for example confirm that a compound is an ENaC inhibitor by measuring a change in transepithelial short circuit current Baucher R. C. Am. J. Respir. Crit. Care Med. 150: 221-281 (1994). ENaC inhibitors can enhance mucus clearance and thus may be used to treat diseases associated with the impairment of mucociliary clearance.
ENaC inhibitors/antagonists and ENaC activators/agonists are hereinafter alternatively referred to collectively as "agents of the invention" .
Accordingly the present invention provides the use of an epithelial sodium ion channel inhibitor in the preparation of a pharmaceutical for the treatment of a condition mediated by epithelial sodium ion channels.
Conditions to which the present invention is applicable include inflammatory diseases such as 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), and eosinophil-associated inflammatory or obstructive airways diseases, particularly asthma and allergic rhinitis. The present invention is also applicable to acute and chronic respiratory tract conditions including cough, colds, influenza, cystic fibrosis and cancer, all conditions with an associated decrease in mucociliary clearance.
The effectiveness of an agent of the invention in enhancing clearance may be demonstrated, for example, in a model described in Yerxa, B. R. et al /. Pharm. Exp. Ther. 302: 871-880 (2002).
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, and one or more co-solvents known in the art such as ethanol (up to 20% by weight), any may contain 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 may contain 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.
EXAMPLES
Preparation of materials
Preparation of the M-l cell culture
M-l murine cortical collecting duct cells (ATCC #CRL-2038) are cultured in plastic flasks with PC-1 media supplemented with 5 mM glutamine. On reaching 80 - 90% confluence the cells are trypsinised and seeded into 96 well black cluster plates with clear bottoms and optical masking at 100,000 cells/well in 100 μl PC-1 medium. Cells are maintained in incubators at 37°C in 5% CO2 at 100% humidity for up to 7 days. The medium is exchanged the day before the assay is conducted. Preparation of the membrane potential dye
Membrane potential dye (Molecular Devices #R8034) is prepared as per the manufacturer's instructions as a 2x stock concentration in HBSS (Hank's Balanced Salt Solution, GibcoBRL #14025-050) containing 20 mM HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2- ethanesulphonic acid] GibcoBRL #15630-056) at pH 7.4 (NaOH). The dye is stored at -20°C and defrosted immediately before use.
Example 1
Epithelial sodium ion channel assay using known ENaC inhibitors
The M-l cells are washed 3 times with HBSS (containing 20 mM HEPES) using a LABSYSTEMS™ microplate washer, leaving the cells in a final volume of 100 μl. 100 μl of the membrane potential dye that has been prepared according to the manufacturer's instructions is added to the cells. The cells are incubated for 30 min at 37°C in 5% CO2 at 100% humidity.
Candidate compounds amiloride, benzamil and dimethyl-amiloride are prepared in HBSS (containing 20 nxM HEPES) at 5x the final concentration and added on line in a volume of 50 μl. DMSO tolerance of the system is assessed separately. The wells are run in triplicate.
The fluorescence of the contents of each well is measured using 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 potential regulated by ENaC. The effects of candidate substances are measured as the change (or mean change in the case of replicates) in basal fluorescence units following addition of the substances to the wells. This is expressed in either absolute units or as the % of the maximum decrease.
At 2 days after seeding, the cells form a confluent, 'cobblestone' epithelia. The dye is loaded and amiloride and benzamil are added to the cells to induce a rapid decrease in the basal fluorescence. The IGso values for amiloride and benzamil are 10 μM and 3 μM respectively. At 6 -7 days after seeding, the cells retain the confluent, 'cobblestone' appearance. The dye is loaded and amiloride, benzamil and DMA are added to the cells again to induce a rapid decrease in the basal fluorescence. The ICJO values for amiloride, benzamil and DMA i.e. the concentration of these compounds at which 50% inhibition occurs, are 2 μM, 50 nM and 8 μM respectively. The values are determined from concentration-inhibition curves in a conventional manner.
FLIPR® traces illustrate that 30 μM amiloride, benzamil and dimethyl-amiloride (DMA) attenuate the basal fluorescence of membrane potential dye loaded cells indicative of membrane hyperpolarisation.
Table 1 below shows sample time course data for three ENaC blockers, namely amiloride, benzamil and DMA, illustrating that at 30 μM each compound induces a decrease in fluorescence counts, indicative of membrane hyperpolarisation. At this time, the inhibitor potency order for this effect is benzamil > amiloride > DMA. This is consistent with the established potency order for blockade of ENaC.
TABLE 1
Figure imgf000009_0001
Figure 1 shows sample concentration-response data illustrating the inhibitory effect of amiloride and three analogues: benzamil, dimethylamiloride (DMA) and ethyl-isopropyl amiloride (EIPA), on the basal membrane potential of cultured M-l cells using the procedure described above. Changes in membrane potential are assessed using the same sensitive fluorescent dye but expressed as % inhibition of basal fluorescence. Mean data (± SEM) is shown (n=4).
Example 2
ENaC assay for high-throughput screening of candidate substances
The assay described in Example 1 is used to screen a library of potential ENaC inhibitors and activators. M-l cells are washed 3 times with HBSS (containing 20 mM HEPES) using a LABSYSTEMS™ microplate washer, leaving the cells in a final volume of 100 μl. 50 μl of MPD is added to the cells which are then incubated for 30 minutes at 37°C in 5% CO2 at 100% humidity.
A commercially prepared BIOMOL™ ion channel plate (BioMol #2805) containing candidate substances at 10 mM in DMSO is used to prepare an experimental plate containing each of the compounds diluted in HBSS (containing 20 mM HEPES) at a concentration of 40 μM. Compounds are added in 50 μl to achieve a final concentration of 10 μM. At the end of the experiment, amiloride (50 μl, 150 μM) is added to all wells as a positive control to achieve a final concentration of 30 μM. Wells are run as individual data points.
The assay gives hits for known ENaC inhibitors amiloride and benzamil as well as dichlorobenzamil and phenamil. Dichlorobenzamil and phenamil are therefore identified by the assay as potent ENaC inhibitors. Known inhibitors of other cation channels such as flufenamic acid and verapamil that are present on the plate do not achieve hits. That indicates that observed hyperpolarisation is dominated by an ENaC-dependent mechanism and thus the assay is selective for ENaC.
Figure 2 shows sample raw data illustrating the effects of a library of 72 commercially available ion channel modulators on the basal membrane potential of M-l cells. A test compound is added at 60 seconds (dotted line) followed by amiloride (30 μM) to each well. "Hits" are clearly identified as compounds that induce a rapid reduction in fluorescence units upon addition (i.e. wells G6 [phenamil] and B8 [amiloride] and D8 [dichlorobenzamil]). Each of the hits shown represents a recognised ENaC blocker. Modulators of Ca2+, K+ and Cl~ channels do not show a rapid reduction in fluorescence units when added to the wells.

Claims

1. A method of identifying a substance suitable for use in the treatment of an inflammatory disease which modulates the activity of epithelial sodium ion channels, wherein the method comprises treating a sample of epithelial cells with a candidate substance and a fluorescent membrane potential sensitive dye and measuring any changes in fluorescence.
2. A method according to claim 1, wherein the epithelial cells are cultured for 2 to 7 days before being treated with the dye or the candidate substance.
3. A method according to claim 1 or 2, wherein the epithelial cells are treated with the dye before being treated with the candidate substance.
4. A method according to any preceding claim, wherein the fluorescent membrane potential sensitive dye is a lipophilic anionic dye.
5. A method according to claim 4, wherein the fluorescent membrane potential sensitive dye is Molecular Devices #R8034.
6. A method according to any preceding claim, wherein the epithelial cells are M-l murine cortical collecting duct cells.
7. The use of an epithelial sodium ion channel inhibitor in the preparation of a pharmaceutical for the treatment of a condition mediated by epithelial sodium ion channels.
8. The use according to claim 7, wherein the condition is asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome, allergic rhinitis or cystic fibrosis.
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