JP2005534311A - Screening for suitable agents for the treatment of leukocyte-related inflammatory diseases - Google Patents

Abstract

The present invention relates to a method for assaying a substance that modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6). The assay method consists of combining a candidate substance and the polypeptide, and then measuring the effect of the candidate substance on the polypeptide. Such substances can be used in the manufacture of a medicament for the treatment of inflammatory diseases, particularly those associated with leukocytes.

Description

  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, in particular respiratory inflammatory diseases.

  During inflammation, various cells are drawn into the tissue. These cells include various leukocytes, especially inflammatory phagocytes such as neutral eosinophils, eosinophilic granulocytes and monocytes. Neutrophils are chronic obstructive pulmonary disease (COPD), for example, chronic bronchitis and its associated pulmonary emphysema, respiratory diseases such as adult respiratory distress syndrome (ARDS), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis , And involved in inflammation and tissue destruction in rheumatoid arthritis. Eosinophils are implicated in respiratory diseases such as asthma and allergic rhinitis.

  The key steps in which leukocytes act in inflammatory conditions are the transition of these cells into tissues, for example the transition to the respiratory tract in respiratory inflammation or the joint in rheumatoid arthritis, cell activation and a range of inflammatory Release of mediators, leukotrienes, oxygen radicals, proteases, etc. The signal that leukocyte migration and activation is required is often transmitted through the receptor in response to an increase in cytosolic calcium levels. Ins (1,4,5) P3 receptors are known to release calcium ions from intracellular stores, but little is known about the channels of the plasma membrane through which those ions pass.

The Drosophila transient receptor potential or “trp” gene is known to encode a Ca ⁺⁺ selective storage-acting Ca ⁺⁺ uptake (SOC), which is a human homolog (TRPC). And was used to find related genes. This led to the discovery of a protein family that functions as ion channels, ie TRPC1-6.

  Boulay et al. Cloned and sequenced mouse TRPC6, “transiently receiving Drosophila transiently involved in calcium uptake for activation of receptors coupled to the G protein of the Gq class. Cloning and expression of a novel mammalian homologue of container potential (trp) "(J. Biol. Chem. Vol.272, 29672-29680 (1997)). Mouse TRPC6 is available from GenBank as deposit number NP_0388866.

  Human TRPC6, also known as classical TRP6 or TRPC6, is available from Genbank as deposit number NP_004612.

  D'Esposito et al., “Identification and assignment of the human transient receptor potential channel for the chromosome 11q21-> q22, the transient receptor potential channel 6 gene TRPC6. 6 gene TRPC6 to chromosome 11q21-> q22) ”(Cytogenet. Cell Cenet. 83 (1-2), 46-47 (1998)).

  Hofmann et al. In “Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol” (Nature Vol. 397, 21 January 1999), CHO-K1 cells. Expresses functional TRPC6 channels as heterologous (proven electrophysiologically), indicating that histamine stimulates large Ca ++ influx in CHO-K1 cells. They also demonstrate that TRPC6 channel activity can be measured as well as Na + influx.

  TRPC6 has been found to be present in cells attracted to tissues during inflammation. TRPC6 mRNA has been shown to be present in isolated primary human neutrophils and also in human lung macrophages isolated from smokers and COPD patients by bronchoalveolar lavage. Expression of TRPC6 mRNA has also been found in cultured human airway smooth muscle cells and fully differentiated human bronchial epithelial cells. TRPC6 protein is human T-lymphocytes, cultured human airway smooth muscle cells, fully differentiated human bronchial epithelial cells, bronchial airway smooth muscle, airway epithelial cells, submucosal gland and tissue leukocytes, ie, neutrophils It has been shown to be expressed in spheres, macrophages and lymphocytes.

  Substances that reduce the activation of leukocytes such as neutrophils or eosinophils by inhibiting the influx of calcium ions into the cells are useful in the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Something is proposed by the present invention. Thus, the present invention provides TRPC6 as a therapeutic target for such diseases and also modulators that stimulate or inhibit channel activity formed by the gene product of human TRPC6, ie peptides, peptidomimetics, small molecules or other drugs A “screening assay” method for identifying candidate compounds or agents, such as

  Accordingly, in a first aspect, the present invention is a method for identifying a substance suitable for use in the treatment of leukocyte-related inflammatory diseases that modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6). A candidate substance and the polypeptide, and then measuring an effect of the candidate substance on the polypeptide.

  In a second aspect, the present invention relates to a pharmaceutical composition comprising a compound that inhibits calcium ion influx through a human TRPC6 ion channel and a pharmaceutically acceptable carrier.

  In the third aspect, the present invention relates to the use of a polypeptide encoded by the human TRPC6 gene, an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding the polypeptide. Use or use of an antibody that immunoreacts with a polynucleotide probe comprising at least 15 contiguous nucleotides of the polynucleotide, wherein the use is in the manufacture of a medicament that inhibits leukocyte accumulation in human tissue.

  In the fourth aspect, the present invention relates to the use of a polypeptide encoded by the human TRPC6 gene, and an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding the polypeptide. The use or use of an antibody that immunoreacts with a polynucleotide probe comprising at least 15 contiguous nucleotides of the polynucleotide, the use in the manufacture of a medicament for the treatment of leukocyte-related inflammatory diseases.

  In a fifth aspect, the present invention relates to the use of a TRPC6 inhibitor in the manufacture of a medicament for the treatment of leukocyte related inflammatory diseases.

  As described above, in the first aspect, the present invention provides a method for identifying a substance suitable for use in the treatment of leukocyte-related inflammatory disease that modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene. About. In a broad sense, the method or assay method comprises combining a candidate substance with a polypeptide encoded by a transient receptor potential channel 6 gene, and then measuring the effect of the candidate substance on the polypeptide. .

  Substances suitable for use in the treatment of leukocyte-related inflammatory diseases serve as enhancers of the activity (herein “human TRPC6 agonists”) or inhibitors (herein “human TRPC6 antagonists”).

  The TRPC6 gene product, i.e., the polypeptide forming the TRPC6 ion channel, can be obtained by, for example, cell-based or screening assays that identify compounds that exhibit a stimulatory or inhibitory effect on the activity of the human TRPC6 channel, or an appropriate reporter. It can be measured by genetic assay.

  The screening method described above is performed by, for example, preparing a cell that expresses a TRPC6 polypeptide on its surface, for example, an insect, mammal, or yeast cell, and then incubating the resulting cell with a candidate substance to obtain the function of the TRPC6 polypeptide. It can be carried out by measuring whether the active activity is increased or inhibited.

  In an appropriate test of TRPC6 channel activation, candidate substances are stably transfected with TRPC6 and combined with cells expressing a functional TRPC6 channel, and membrane depolarization is measured to show TRPC6-mediated Na + influx.

  Appropriate testing of TRPC6 channel inhibition uses cells expressing endogenous G protein couple-bound calcium mobile receptors, such as muscarinic acetylcholine receptors capable of activating TRPC6 channels, and receptor agonists such as muscarinic acetylcholine receptor agonists. In addition, it stimulates the TRPC6 channel. The cells are treated with a candidate substance prior to addition of the receptor agonist. This treatment should result in an increase in fluorescence, but the increase is diminished if the candidate compound inhibits the TRPC6 channel. Any change in fluorescence can be measured by a suitable device, such as a fluorescence image plate reader.

  Using this method, it was found that 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl inhibits the activity of the human TRPC6 ion channel.

  1- [b- [3- (4-Methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl has been disclosed to inhibit receptor-mediated calcium uptake (RMCE) (Merritt et al in Biochem. J. (1990) 271: 515-522). Inoue et al. Also disclosed blocking the TRPC6 ion channel in mice (Inoue et al in Circ. Res. (2001) 88: 325-332).

  The present invention also provides compounds that inhibit calcium ion influx through the human TRPC6 ion channel, such as 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl. And a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

  In order to produce a pharmaceutical that inhibits leukocyte accumulation in human tissue, an antibody that immunoreacts with a polypeptide encoded by the human TRPC6 gene (referred to herein as “human TRPC6 antibody”), or a nucleotide encoding the polypeptide An antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the sequence (referred to herein as a “human TRPC6 antisense oligonucleotide”) can be used.

  The above human TRPC6 antibodies and antisense oligonucleotides can be used to treat leukocyte-related inflammatory diseases.

  Human TRPC6 agonists, human TRPC6 antagonists, human TRPC6 antibodies and human TRPC6 antisense oligonucleotides are collectively referred to herein as “agents of the invention”.

  Neutrophil-related inflammatory diseases to which the present invention can be applied include neutrophil-related inflammation or obstructive airway diseases, especially chronic obstructive pulmonary disease (COPD), such as chronic bronchitis and emphysema, and adults (or acute) ) Respiratory Distress Syndrome (ARDS), rheumatoid arthritis, and inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Eosinophil-related inflammatory diseases to which the present invention can be applied are eosinophil-related inflammation or obstructive airway diseases, particularly asthma and allergic rhinitis.

  Among peripheral tissues, the lung expresses TRPC6 at the highest level with the placenta. TRPC6 is present in many resident cells of the lung, such as epithelial cells and airway smooth muscle cells, but is also present in pulmonary artery smooth muscle. TRPC6 is upregulated in these smooth muscle cells when stimulated by platelet derived growth factor (PDGF). Increased PDGF is associated with pulmonary hypertension and arterial smooth muscle cell proliferation. Inhibiting TRPC6 expression, for example, with antisense, suppresses the proliferation of PDGF-stimulated arterial smooth muscle cells. Therefore, a medicament that inhibits pulmonary artery smooth muscle TRPC6 is useful for the treatment of pulmonary hypertension.

  Human TRPC6 polypeptide can be isolated by appropriate conventional methods. Since the sequence of the human TRPC6 gene is known, specific primers may be used as a convenient option. For example, (SEQ01) 5'-gcaatgaaagctttggaccc-3 'as a forward primer and (SEQ02) 5'-atcgtataactatagactccat-3' as a reverse primer produces a 300 base pair polymerase chain reaction (PCR) product.

  The human TRPC6 polynucleotide is cDNA, genomic DNA or RNA and can be obtained by an appropriate conventional method. For example, it can be prepared by chemical synthesis from the constituent nucleotides, for example, by automated solid phase synthesis using known techniques and equipment.

  The human TRPC6 antibody may be a polyclonal antibody or a monoclonal antibody. Such antibodies can be prepared by conventional techniques. Methods for producing polyclonal antibodies to purified antigens are well established (reference: Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11). Human TRPC6 antibodies can be used for the detection or quantification of expression levels of human TRPC6 polypeptide, or for inhibition of the ligand / antiligand binding activity of human TRPC6 polypeptide.

  The human TRPC6 antisense oligonucleotide may be DNA, a DNA analog such as a phosphorothioate or methylphosphonate analog of DNA, RNA, an RNA analog, or a peptide nucleic acid (PNA). Antisense oligonucleotides can be synthesized, for example, by conventional methods using automated solid phase methods. This can be used to inhibit expression of the human TRPC6 gene if it is desired. Alternatively, short interfering RNA (siRNA) can be used as a specific means of target gene knockdown. RNA interference inhibits gene expression and can therefore be used to explore gene function. This technique is described by Elvasia et al. (S.M. Elbashir et al in Methods 26 (2002) 199-213).

  The human TRPC6 polynucleotide probe consists of at least 15 contiguous nucleotides of the above polynucleotide or its complement. The probe is cDNA, genomic DNA or RNA, and can be synthesized by a conventional method. It is usually a synthetic oligonucleotide consisting of 15 to 50 nucleotides, and can be labeled with a fluorescent group to produce a detectable signal, for example. Nucleotides encoding the first fifty amino acids of human TRPC6 can be used for this purpose. The human TRPC6 polynucleotide probe can be used to detect the presence or absence of the human TRPC6 gene, that is, to detect a genetic abnormality.

  The effectiveness of the agent of the present invention in inhibiting or reversing leukocyte-related inflammatory diseases can be attributed to models of the disease, such as the rat or mouse lipopolysaccharide-induced lung inflammation model, or the literature (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 present invention may be administered by any suitable route, eg, orally, eg, in the form of a tablet or capsule; parenterally, eg, intravenously; topically, eg, as an ointment or cream; For example, as a patch; by inhalation; or nasally.

  A pharmaceutical composition containing the agent of the present invention can be produced using conventional diluents or additives and techniques known in the formulation art. Thus, oral dosage forms are tablets and capsules, and inhalable compositions can include aerosols or other sprayable or dry powder formulations.

  Where the composition comprises an aerosol formulation, the formulation is preferably, for example, a hydro-fluoro-alkane (HFA) propellant, such as HFA134a or HFA227 or mixtures thereof, one or more co-solvents known in the art, For example, ethanol (up to 20% by weight), one or more surfactants such as oleic acid or sorbitan trioleate, and one or more fillers such as lactose.

  When the composition consists of a dry powder formulation, the active ingredient is preferably of a particle size up to 10 microns, and the formulation reduces product performance degradation due to diluents or carriers such as lactose and moisture. Contains compounds that help to prevent.

  Where the composition consists of a spray formulation, the formulation preferably comprises, for example, a dissolved or suspended active ingredient, water, ethanol or propylene glycol as a co-solvent and a stabilizer that is also a surfactant. Containing. The present invention includes (i) an inhalable form, such as an aerosol or other nebulizable composition or inhalable particulate, such as a micronized form of the agent of the invention; (ii) an inhalable form of the invention. An inhalable medicament comprising an agent; (iii) a pharmaceutical product comprising such an agent of the present invention in inhalable form combined with an inhaler; and (iv) an agent of the present invention in inhalable form An inhalation device containing.

  The dosage of the agent of the present invention employed in practicing the present invention can, of course, vary depending on, for example, the particular condition to be treated, the desired effect and the dosage form. In general, a suitable daily dose for inhalation administration is in the range of 1 μg to 10 mg / kg, whereas a suitable daily dose for oral administration is in the range of 0.1 mg to 1000 mg / kg.

  The invention is illustrated by the following examples.

Assays suitable for high-throughput screening Assays for screening candidate or test compounds are those that stably transduce TRPC6 and express functional TRPC6 channels, eg, the mammalian cell line HEK293, which is capable of activating TRPC6 channels. Which expresses the sex muscarinic acetylcholine receptor). The assay uses a 96-well FLIPR (registered trademark) (Fluorescence Imaging Plate Reader) (Molecular Devices Corporation) and a molecular device exclusive FLIPR (registered trademark) membrane potential assay kit (catalog # R8034). And measure membrane depolarization from TRPC6-mediated Na + influx. The specificity of this TRPC6-mediated response is that if Na + is replaced by an impermeable monovalent cation NMDG + in the extracellular buffer, it is not observed in TRPC6-expressing cells, and Na + influx-dependent membrane detachment. It can be shown by demonstrating that no polarization is observed in control cells not transfected with TRPC6. TRPC6-expressing cells are seeded in 96-well black-walled flat-bottomed microplates at a coating volume of 100 μl and grown to closeness for the assay. To prepare the cells for assay, remove the cell culture medium and add 100 μl of loading buffer to each well. The loading buffer has a pH of 7.4 and consists of 140 mM NaCl, 0.15 mM CaCl ₂ , 3.3 mM KH ₂ PO ₄ , 1.2 mM MgCl ₂ , 10 mM D-glucose and 20 mM HEPES. Consists of FLIPR® membrane potential dye diluted to the required assay concentration in a buffer containing 03 mM BAPTA-AM. Plates are incubated at 37 ° C. for 20 minutes before starting the assay. To detect an inhibitor of TRPC6, a test agent is added to each well before or after stimulation with the muscarinic acetylcholine receptor agonist carbachol (which activates the TRPC6 channel). The expected effect of the inhibitor should be a decrease in fluorescence increase due to carbachol stimulation. An activator of TRPC6 can be detected by replacing the test agent with carbachol, in which case the activator stimulates an increase in fluorescence.

Detection of 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxy TRPC6 clone 14 cells pre-treated with phenethyl] -1H-imidazole.HCl for 10 minutes are stimulated with carbachol (10 μM) according to the assay described in Example 1. Fluorescence is measured before carbachol is added (minimum response, ie Min), and when the fluorescence increase upon carbachol stimulation reaches a maximum (maximum response, ie Max). As can be seen in Table 1 below, a reduced fluorescence response is observed in compound treated cells compared to the control response in the absence of compound.

Data is calculated as (Max-Min) / Min response. Each concentration of 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole · HCl is the mean ± SEM (standard error of the mean value) of n = 10 wells. . The average IC ₅₀ value calculated from the n = 4 experiments is 4.9 ± 0.8 μM (mean ± SEM).

This result indicates that the assay described in Example 1 detects 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl. Yes.

[Sequence Listing]

Claims (12)

  A method for identifying a substance suitable for use in the treatment of leukocyte-related inflammatory disease that modulates the activity of a polypeptide encoded by the human transient receptor potential 6 gene (TRPC6), comprising combining a candidate substance with the polypeptide, Next, the method of measuring the effect of the candidate substance on the polypeptide.   2. The candidate substance and a cell that is stably transduced by TRPC6 and that expresses a functional TRPC6 channel is combined, and any membrane depolarization exhibiting TRPC6-mediated Na + influx is measured. Method.   An endogenous calcium-mobile G protein-coupled receptor capable of activating TRPC6 channel by agonist stimulation is expressed by the cell, a receptor agonist is added following a candidate substance for stimulating TRPC6 channel, and inhibition measurement is performed. The method of claim 1 enabling.   A pharmaceutical composition comprising a compound that inhibits the inflow of calcium ions through a human TRPC6 ion channel and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 4, wherein the compound is 1- [b- [3- (4-methoxyphenyl) propoxy] -4-methoxyphenethyl] -1H-imidazole.HCl.   Use of an antibody that immunoreacts with a polypeptide encoded by the human TRPC6 gene, or use of an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding the polypeptide. Use in the manufacture of a medicament that inhibits leukocyte accumulation in human tissues.   Use of an antibody that immunoreacts with a polypeptide encoded by the human TRPC6 gene, or use of an antisense oligonucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a nucleotide sequence encoding the polypeptide. Use in the manufacture of a medicament for the treatment of leukocyte-related inflammatory diseases.   The use according to claim 7, wherein the disease is a neutrophil-related disease such as chronic obstructive pulmonary disease, adult respiratory distress syndrome, rheumatoid arthritis or inflammatory bowel disease.   The use according to claim 7, wherein the disease is an eosinophil-related disease such as asthma and allergic rhinitis.   Use of a TRPC6 inhibitor in the manufacture of a medicament for the treatment of leukocyte related inflammatory diseases.   Use of a TRPC6 inhibitor in the manufacture of a medicament for the treatment of pulmonary hypertension. Methods for identifying substances suitable for use in the treatment of leukocyte-related inflammatory diseases that modulate the activity of a polypeptide encoded by the human transient receptor potential 6 gene substantially as described herein in connection with the Examples .