WO2005023305A2 - Modulation de l'activite cellulaire au moyen d'un agent diminuant le taux de cholesterol au sein d'une cellule - Google Patents

Modulation de l'activite cellulaire au moyen d'un agent diminuant le taux de cholesterol au sein d'une cellule Download PDF

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WO2005023305A2
WO2005023305A2 PCT/GB2004/003875 GB2004003875W WO2005023305A2 WO 2005023305 A2 WO2005023305 A2 WO 2005023305A2 GB 2004003875 W GB2004003875 W GB 2004003875W WO 2005023305 A2 WO2005023305 A2 WO 2005023305A2
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cell
cholesterol
acid
cells
level
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WO2005023305A3 (fr
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Janet Marjorie Allen
John Paul Overington
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Inpharmatica Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • 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
    • 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
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to methods for modulating the activity of cells, and compositions useful in such methods.
  • the invention relates to the use of an agent that reduces the level of cholesterol within a cell to modulate the activity of the cell, and to methods involving such use.
  • Elevated plasma levels of cholesterol are associated with an increased risk of various cardiovascular diseases such as atherosclerosis, myocardial infarction, angina pectoris, stroke and intermittent claudication (Oxford Textbook of Medicine 4 th edition, Editors: Warrell, Cox, Firth and Benz, Oxford University Press or Concise Oxford Textbook of Medicine, Editors: Ledingham and Warrell, Oxford University Press).
  • cardiovascular diseases such as atherosclerosis, myocardial infarction, angina pectoris, stroke and intermittent claudication
  • Bile acid sequestrants such as Cholestyramine resin, Colesevelam HC1, Colestipol, and Polidexide
  • Fibrates such as Bezafibrate, Binifibrate, Ciprofibrate, Clinofibrate, Clofibrate, Clofibric acid, Etofibrate, Fenofibrate, Gemfibrozil, Pirifibrate, Ronifibrate, Simfibrate and Theofibrate
  • 3-hydroxy-3 methylglutaryl Coenzyme A reductase (HMG CoA reductase) inhibitors including statins such as Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitivastatin, Pravastatin, Rosuvastatin, and Simvastatin); various Nicotinic acid derivatives (such as Acipimox, Aluminium nicotinate, Nicert
  • HMG CoA reductase inhibitors have established themselves as safe, efficacious and highly successful drugs, particularly for the treatment of hypercholestemia.
  • HMG CoA reductase is found in both eukaryotes and prokaryotes and converts 3-hydroxy-3 methylglutaryl CoA to mevalonate, which is a key precursor for the synthesis of sterols and isoprenoids in humans (Annual Reviews in Biochemistry, 1981, 50, 585-621).
  • the HMG CoA reductase enzyme catalyses a key rate-limiting step in isoprenoid and sterol biosynthesis.
  • Drugs that target and inhibit the HMG CoA reductase enzyme include a family of related compounds collectively called 'statins', which includes those statins listed above (http://www.nlm.nih.gov/medlineplus/druginfo/uspdi/202284.html and http://www.drugstore.com/qxal 026 33318 I sespiderwhat is_an hmg_coa_reductase_inhi bitor.htm).
  • statins The primary therapeutic indication for the statins is in lowering plasma cholesterol levels, particularly LDL-cholesterol, an important risk factor in coronary heart disease (Igel et al, J. Clin. Pharmacol., 42, 835-845, 2002). Plasma levels of cholesterol are reduced in patients treated with statins (Isles and Paterson. Quarterly Journal of Medicine, 2000, 93, 567-74; Izzat et al. Journal of Pharmacology and Experimental Therapeutics, 2000, 293, 315-320; Watts and Burke, Curr Opin Lipidol, 1996, 7, 341-55; Shviro and Leitersdorf. Br J.
  • Statin drugs are generally typified by the presence of the mevalonate group (or a masked form thereof), mimicking the natural substrate for the HMG CoA reductase enzyme (and the essential precursor for de novo biosynthesis of cholesterol). For this therapeutic indication, the statins are orally administered and reach their primary site of action, the liver, via the circulation.
  • Statin drugs can be either in the free-acid form (as in Atorvastatin, Pravastatin, Fluvastatin, Rosuvastatin, Pitivastatin and Cervistatin) or in the cyclic lactone form (as in Mevastatin, Simvastatin and Lovastatin).
  • These free acid and lactone forms are facilely interconverted, both in vitro and in vivo, and following oral dosing and absorption the inactive lactone form is converted to the active free acid (Corsini, Maggi, and Catapano, Pharmacol. Res., 31, 9-, 1995).
  • the mode of binding of these drugs to their target enzyme is well known and established through published studies (Istvan and Deisenhofer, Science, 292, 1160-1164, 2001).
  • statin compounds are, to varying degrees, concentrated in the liver relative to other tissues (Koga et al, Eur. J Biochem., 209, 315-319, 1992; Nezasa et al, Xenobiotica, 33, 379-388, 2002), distribution to other organs increases the risk of adverse events (Guillot et al, J. Cardiovasc. Pharmacol, 21, 339-346, 1993; Lennernas and Fager, Clin. Pharmacokinet, 32, 403-425, 1997).
  • statins The main limiting adverse effect of statins is myopathy (of either cardiac muscle or skeletal muscle), that can lead to rhabdomyolysis and renal failure (see, for example, Davidson Expert Opin Drug Safety, 2002, 1, 207-12; Moghadasian. Expert Opin Drug Saf. 2002 Sep;l(3):269-74; Gotto. Clin Cardiol. 2003 Apr;26(4 Suppl 3):III3-12; Thompson et al. JAMA. 2003 Apr 2;289(13):1681-90; Ballntyne et al. Arch Intern Med. 2003 Mar 10;163(5):553-64; Bolego et al. Curr Opin Lipidol. 2002, 13, 637-44; Evans and Rees.
  • statin pharmacokinetics has focussed on the identification and optimisation of statins that are optimally targeted to the liver or are characterised by slow release of the active ingredient in order to produce statin drugs that effectively reduce plasma cholesterol whilst minimising the risk of adverse events (see, for example, Davidson, Expert Opin. Investig. Drugs. 2002 Mar;l 1(3):125-41 or WO 98/15264).
  • statin drugs that do not suffer from the side-effects described above, in particular the side-effect of rhabdomyolysis.
  • Cholesterol is also synthesised de novo within cells, since it is an essential component of cellular membranes, in particular the lipid rafts of the plasma membrane.
  • statins control only the plasma level of cholesterol, and statins are not currently employed to manipulate intracellular levels of cholesterol.
  • Inflammatory disorders arise when the host's immune system mounts an inappropriate inflammatory response. Inflammatory disorders include allergies leading to clinical features such as allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria, gastro-intestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crolin's disease, ulcerative colitis, autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease, gastric and duodenal ulceration, multiple sclerosis, wound healing, inflammatory bowel disease, type I diabetes mellitus, liver hepatitis and cirrhosis, chronic obstructive airways disease (COPD), emphysema and chronic bronchitis, atherosclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, degenerative
  • allergies include asthma, house dust mite allergies, food allergies, pet allergies, pollen allergies and insect sting allergies. Allergies are currently treated by drugs that block the effect of histamine, such as anti-histamines. However, drugs that act as anti- histamines are frequently limited in use by side-effects of sedation and drowsiness. In addition, these drugs have other side effects including palpitations and arrhythmias, hypotension, hypersensitivity reactions, rashes and photosensitivity reactions, extrapyramidal effects, confusion, tremor and depression.
  • Corticosteroids remain the mainstay of treatment of various allergic conditions whether administered using local application (for example, inhalers for asthma, dermal application for eczema) or, in severe cases, corticosteroids are given orally at high dose. Corticosteroids have adverse effects and must be used with caution. For example, corticosteroids can cause immunosuppression, osteoporosis, diabetes and hypertension. An additional problem with the use of corticosteroids to treat allergic conditions is that these diseases frequently affect paediatric populations. Corticosteroids are well-recognised to impair normal skeletal growth and their use in children is not recommended. There are even concerns over the use of inhaled corticosteroids in children. Dermal application of corticosteroids results in thinning of skin.
  • bronchodilators for asthma, such as ⁇ adrenoceptor agonists or anti-muscarinic bronchodilators. Both these treatments relieve symptoms but have potential serious effects on the cardiovascular system.
  • Mast cells are implicated in a number of the above inflammatory disorders, particularly the allergic disorders.
  • Mast cells express the high affinity receptor for immunoglobulin E (Fc ⁇ RI) on their cell surface. Aggregation of this receptor on mast cells results in the activation of intracellular signalling cascades which ultimately lead to mast cell activation and degranulation, which in turn results in the release of mediators such as histamine and serotonin together with pro-inflammatory cytokines and metalloproteinases.
  • Fc ⁇ RI immunoglobulin E
  • mast cells are also localised in the gastrointestinal tract and those cells are considered to be important in the pathophysiology of various gastro-intestinal diseases, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • Mast cell activation has also been implicated in a variety of other diseases such as autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease and gastric and duodenal ulceration.
  • Fc ⁇ RI is a multi-subunit receptor comprising an ⁇ chain ligand-binding domain, which binds to IgE, a ⁇ chain homo-dimer, required to mediate signal transduction, and a ⁇ chain, which plays a role in amplification of the signal (Turner and Kinet, Nature 1999, 402, B24- 30).
  • the intracellular signalling pathways activated by Fc ⁇ RJ involve the sequential activation of tyrosine kinases including Lyn and Syk, and phospholipase C leading to the generation of inositol 1,4,5, trisphosphate (IP 3 ).
  • IP 3 The generation of IP 3 leads to the subsequent elevation of intracellular calcium, by release of calcium from intracellular stores and influx of extracellular calcium (through the activation of calcium channels on the plasma membrane). Signalling via the Fc ⁇ RI receptor is implicated in those diseases and conditions in which mast cells are implicated.
  • lipid rafts provide discrete microdomains within the membrane that allow the efficient coupling of ligand binding on the extracellular face of the membrane to the initiation of intracellular signalling cascades that cause cell activation.
  • the lipid rafts appear to co-ordinate and focus the signal transduction coupling from extracellular ligands to intracellular signalling cascades. This has been well-characterised for a number of immune cell receptors, notably the T cell receptor and the IgE receptor, Fc ⁇ RI.
  • Disruption of lipid rafts results in an uncoupling of efficient signal transduction through receptors such as GPCRs, the T cell receptor and the high affinity IgE receptor, and will thus be useful in the treatment or prophylaxis of a wide variety of diseases and conditions, including but not limited to allergies, such as asthma, house dust mite allergies, food allergies, pet allergies, pollen allergies and insect sting allergies which lead to allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria, gastro-intestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease, gastric and duodenal ulceration, multiple sclerosis, wound healing, inflammatory bowel disease, liver hepatitis and cirrhosis, chronic obstructive airways disease (COPD), e
  • lipid rafts as structures are exploited by various infective agents either to get into cells or to exit cells. Accordingly, lipid rafts are implicated in other diseases and conditions, including but not limited to, intracellular pathogens including bacteria such as Salmonella, Chlamydiae, listeria, Mycobacteria tuberculosis, viruses such as HIV, Measles virus, Papilloma viruses, Epstein-Barr virus, Respiratory Syncytial Virus (RSV), Hepatitis, Herpes viruses, Influenza virus, Ebola and Marburg viruses, parasites such as, Plasmodium (malaria), leishmania, Trypanosoma (sleeping sickness), Toxoplasma gondii, bacterial infections including Shigella, Escherichia Coli (including 0157), Campylobacter, Vibrio cholerae, Clostridium difficile and Clostridium tetani.
  • intracellular pathogens including bacteria such as Salmonella, Chlamyd
  • the invention is based on the discovery that agents that reduce the level of cholesterol within cells may be used to modulate the activity of a wide variety of cells via disruption of membrane signalling pathways.
  • the invention is also based on the discovery of the mechanism by which such agents lead to the disruption of membrane signalling pathways and the associated discovery that such agents can be used to modulate the level of cholesterol present in the lipid rafts of the cell membrane.
  • the PharmaCartaTM platform is the Applicant's proprietary integrated large-scale discovery informatics platform that enables the simultaneous exploration of bioinformatics, chemogenomics, disease indication and pharmacology data sets.
  • the PharmaCartaTM platform incorporates bioinformatic tools and data sets which when combined are capable of gene prediction, EST and oligonucleotide probe mapping, structural and functional annotation of protein sequences, analysis of target draggability and selectivity, conformationally biased protein modelling, active site mapping, analysis of compound structure-activity relationship (SAR) and target data and in s ⁇ lico ADME optimisation.
  • PharmaCartaTM takes a proteome-wide view, providing for the selection of draggable disease-pathway linked targets and corresponding selective chemical leads. Linking the small molecule, target and disease domains, PharmaCartaTM provides a general and systematic approach to target prioritisation and subsequent lead discovery. PharmaCartaTM can rapidly extract either compounds known to be active against a novel target or, given a compound as a query, extract targets likely to interact with the query compound.
  • the PharmaCarta M platform is a powerful bioinformatics tool for drag discovery purposes, and allows rapid and effective in s ⁇ lico target selection, lead identification and lead optimisation by simultaneously searching, comparing and analysing the available data sets.
  • the use of the PharmaCartaTM platform for drag discovery may follow the following steps:
  • proteins associated with a phenotype of interest e.g. a disease
  • proteins over-expressed or over-active in a diseased cell line may be identified by analysis of differential microarray data or comparative 2D-PAGE coupled to mass spectroscopy;
  • the lead compounds or other potential actives are subjected to in s ⁇ lico predictive ADME analysis (carried out by the PharmaCarta 1 platform) in order to optimise the lead compounds or other potential actives.
  • the lead compounds or other potential actives may be tested in simple in vitro assays.
  • the PharmaCartaTM platform was used to analyse a public-domain gene expression dataset derived from an analysis of DNA microarray data (Riley et al., 2002, Proc. Natl. Acad. Sci. USA, 99, pp. 11,790-5). A number of nucleotide sequences observed to be upregulated upon cell activation were assessed for draggability by the PharmaCartaTM platform.
  • the PharmaCartaTM platform identified a number of drug target candidates. A subset of these drag target candidates were specifically selected for more detailed investigation and further prioritised by the PharmaCartaTM platform according to selectivity and compound ADMET criteria. One of the genes in this prioritised subset, HMG CoA reductase, was then specifically selected as a candidate for further analysis. The subsequent Examples (Examples 2-7) describe this further analysis.
  • Examples 2-7 disclose that inhibition of HMG CoA reductase in mast cells results in decoupling of the IgE receptor (Fc ⁇ RI) from the relevant intracellular signalling cascades. It is thus disclosed in Examples 2-7 that treatment of mast cells with HMG CoA reductase inhibitors results in inhibition of a number of the activities and functions of mast cells. These Examples demonstrate that the uncoupling of the receptor from the signalling cascades by HMG CoA reductase inhibitors relates directly to the inhibition of intracellular HMG CoA reductase, since co-treatment of cells with HMG CoA reductase inhibitors and mevalonate rescues the activities and functions.
  • methyl ⁇ cyclodextrin has been shown also to inhibit the activities and functions investigated. Accordingly, these Examples disclose that a number of agents that reduce the level of cholesterol within cells may be used to modulate the activity of a variety of cell types via disruption of membrane signalling pathways, since the effects on mast cells are not specific to HMG CoA reductase inhibitors.
  • agents that lower intracellular cholesterol via diverse mechanisms will disrupt lipid rafts.
  • such agents may disrupt lipid rafts by preventing the de novo synthesis of cholesterol for insertion into the plasma membrane (e.g. HMG CoA reductase inhibitors) or may disrupt the trafficking of cholesterol into the plasma membrane by reducing the intracellular pool available for trafficking (e.g. PPAR ⁇ agonists or LXR agonists).
  • a method of modulating the activity of a cell comprising exposing the cell to an agent that reduces the level of cholesterol within the cell.
  • agents that reduce the level of cholesterol within a cell lead to a reduction in the level of cholesterol in the cell membrane of that cell. More particularly, agents that reduce the level of cholesterol within a cell lead to a reduction in the level of cholesterol within the lipid rafts of the cell membrane. Furthermore, a reduction in the level of cholesterol in the lipid rafts of the cell membrane results in the modulation of the activity of membrane signalling pathways, provided that the membrane signalling pathways comprise at least one signalling component associated with the lipid rafts of the cell membrane.
  • the present invention provides simple methods for the disraption of lipid raft micro- domains, via the use of agents that reduce the level of intracellular cholesterol.
  • the disruption of the lipid raft micro-domains by these methods results in the uncoupling of efficient signal transduction for a number of receptors localised in the lipid raft micro- domains.
  • the method of the first aspect of the invention allows the modulation of the activity of a cell using an agent that reduces the level of cholesterol within the cell, because a reduction in the level of cholesterol within the cell leads to disraption of signalling via lipid raft associated signalling pathways.
  • disraption of lipid rafts will be useful in the treatment of a wide range of diseases.
  • Such diseases include, but are not limited to, allergies, such as asthma, house dust mite allergies, food allergies, pet allergies, pollen allergies and insect sting allergies which lead to allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria, gastro-intestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease, gastric and duodenal ulceration, multiple sclerosis, wound healing, inflammatory bowel disease, liver hepatitis and cirrhosis, chronic obstructive airways disease (COPD), emphysema and chronic bronchitis, atherosclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, degenerative joint
  • An advantage of the methods of the invention in the treatment of inflammation is that such methods of treatment remove the need to treat atopy using steroids.
  • These methods of the first aspect of the invention provide the use of an agent that reduces the level of cholesterol within a cell to modulate the activity of that cell.
  • the term "modulating the activity of a cell” refers to the modulation of any cellular activity of interest.
  • Preferred activities that are modulated include cellular growth, cellular proliferation, cellular differentiation and cellular effector function (for example, degranulation of mast cells).
  • the modulation may be a decrease in the level of the activity of interest, an increase in the level of the activity of interest, or the maintenance of a specific level of activity.
  • the cell or cells whose activity is modulated may be any suitable type of cell, as discussed below.
  • agent refers to any suitable molecule, compound, nucleic acid, polypeptide or other moiety. Preferred agents are described in detail herein.
  • the term “reduces the level of cholesterol” refers to any reduction in the level of cholesterol present within the cell, within the cell membrane or within the lipid rafts of the cell membrane.
  • the reduction in the level of cholesterol may be achieved.
  • the cellular cholesterol pool may be lowered by disrupting de novo cholesterol biosynthesis, or the trafficking of cholesterol into the plasma membrane may be disrupted by reducing the intracellular cholesterol pool available for trafficking.
  • These preferred embodiments of the first aspect of the invention thus provide the use of an agent that reduces the level of cholesterol within a cell for the modulation of the activity of a membrane signalling pathway in a cell, wherein the signalling pathway comprises at least one signalling component associated with a lipid raft of the cell membrane.
  • membrane signalling pathway refers to any signalling pathway in which at least one signalling component is associated with the cell membrane.
  • the modulation of the activity of a membrane signalling pathway may be the modulation of any activity of a membrane signalling pathway of interest.
  • the term “signalling component” refers to any polypeptide or other moiety that is involved in signal transduction in a signalling pathway. Accordingly, this term includes polypeptides, small molecules (e.g. calcium ions and inositol triphosphate) and other signal transduction agents, such as hormones.
  • the term “lipid raft” refers to the cholesterol and sphingolipid rich lipid micro-domains present in the cell membrane of certain eukaryotic cells. Lipid rafts were reviewed in London E, Curr. Opin. Struct. Biol, 2002, Aug;12(4):480-6.
  • a membrane signalling pathway may contain a number of types of signalling component associated with a lipid raft of the cell membrane.
  • signalling components may be transmembrane proteins or proteins that partially traverse the cell membrane.
  • Transmembrane proteins may be single pass or multipass membrane proteins, such as 7TM G-protein linked receptors or ion channels.
  • signalling components may be polypeptides anchored to the lipid bilayer via a lipid anchor (e.g. a GPI anchor) or polypeptides associated with the cell membrane via interaction with other polypeptides located at the cell membrane.
  • the signalling component associated with a lipid raft of the cell membrane may be the FcE RI receptor complex or the Icrac calcium channel.
  • the modulation of the activity of a membrane signalling pathway may be the disraption of FcE RI signalling or the modulation of calcium influx via the Icrac calcium channel, respectively.
  • agents that reduce the level of cholesterol within a cell lead to a reduction in the level of cholesterol in the cell membrane of that cell. More particularly, agents that reduce the level of cholesterol within a cell lead to a reduction in the level of cholesterol within the lipid rafts of the cell membrane. Furthermore, a reduction in the level of cholesterol in the lipid rafts of the cell membrane results in the modulation of the activity of membrane signalling pathways, provided that the membrane signalling pathways comprise at least one signalling component associated with the lipid rafts of the cell membrane.
  • the reduction in the level of cholesterol within the cell leads to a reduction in the level of cholesterol in the cell membrane.
  • the reduction in the level of cholesterol within the cell leads to a reduction in the level of cholesterol in the lipid rafts of the cell membrane.
  • the level of cholesterol present in the cell may readily be determined by the Amplex Red Cholesterol Assay (Molecular Probes). Further information on this assay may be found at http://www.probes.com/media/pis/mpl2216.pdf. This assay may also be used for the determination of the level of cholesterol present in the cell membrane, in which case the plasma membrane would be separated from the cell contents following lysis of the cell before the assay protocol is followed.
  • lipid rafts present in the cell membrane may readily be investigated by a number of methods known to those of skill in the art. For example, it is known that lipid rafts are insoluble in TritonX-100, and a number of experimental protocols have been developed that use detergent resistance as a way of monitoring rafts and the proteins associated with rafts (see, for example, London and Brown. Biochim BioPhys Acta 2000, 1508, 182-195 and Brown and London. J. Biol. Chem. 2000, 275, 17221-17224). Alternatively, the cholera toxin beta subunit can be used to visualise the rafts, since it recognises GM1 gangliosides that are enriched in the rafts (see http://www.
  • the methods of the first and second aspects of the invention are applicable to a broad range of cell types.
  • T lymphocytes, B lymphocytes, mast cells, monocytes, macrophages, neuronal cells, glial cells, muscle cells, vascular endothelial cells and other cell types are known to contain lipid rafts in their cell membranes and are therefore susceptible to the method and uses of these aspects of the invention.
  • the cell or cells whose activity is modulated may be any suitable cell, wherein suitable cells are any cells which possess lipid rafts in their cell membranes.
  • the cell is of hematopoetic lineage.
  • Cells of hematopoetic lineage possess a large number of important membrane signalling networks and are therefore susceptible to the method of the first aspect of the invention.
  • Cells of hematopoetic lineage include B- lymphocytes, T-lymphocytes, natural killer cells, dendritic cells, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, monocytes, macrophages, and erythrocytes.
  • one aspect of the present invention provides a method for modulation of the immune response by modulating the activity of the membrane signalling networks within cells of hematopoetic lineage.
  • the cell of hematopoetic lineage is a mast cell.
  • mast cells are implicated in a number of important pathophysiological diseases and conditions. Accordingly, safe and effective methods for the modulation of the activity of membrane signalling networks in mast cells are highly desirable. Such methods are provided by the present invention.
  • treatment of mast cells with agents that reduce the level of intracellular cholesterol, particularly statins results in the inhibition of the Fc ⁇ RI-mediated signalling pathway and subsequent degranulation of the mast cell.
  • agents that reduce the level of intracellular cholesterol can be used to modulate the activity of mast cells.
  • agents that reduce the level of intracellular cholesterol can be used to suppress the function of mast cells.
  • the methods and uses may be used to inhibit activation of mast cells. Inhibition of mast cell activation is of great importance in the treatment of inflammatory disorders, particularly allergies, for the reasons described above.
  • a method for the prophylaxis or treatment of a disease or condition caused by the inappropriate activity of a membrane signalling pathway comprising exposing cells that possess said membrane signalling pathway to an agent that reduces the level of cholesterol within those cells.
  • the agent should be administered in an amount sufficient to reduce the level of cholesterol within the cells.
  • a third aspect of the invention there is provided the use of an agent that reduces the level of cholesterol within a cell in the manufacture of a medicament for the treatment or prophylaxis of a disease or condition in which inappropriate activity of a membrane signalling pathway present in said cell is implicated, wherein said medicament reduces the level of cholesterol within that cell.
  • the membrane signalling pathway comprises at least one signalling component associated with the lipid rafts of the cell membrane.
  • the inappropriate activity of a membrane signalling pathway present in a cell particularly the inappropriate activity of a membrane signalling pathway comprising at least one signalling component associated with the lipid rafts of the cell membrane, is implicated.
  • Such diseases include, but are not limited to, allergies, such as asthma, house dust mite allergies, food allergies, pet allergies, pollen allergies and insect sting allergies which lead to allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria, gastro-intestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease, gastric and duodenal ulceration, multiple sclerosis, wound healing, inflammatory bowel disease, liver hepatitis and cirrhosis, chronic obstructive airways disease (COPD), emphysema and chronic bronchitis, atherosclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, degenerative joint
  • a membrane signalling pathway present in mast cells includes, but are not limited to allergies, such as food allergies, pet allergies, pollen allergies insect sting allergies and other allergies leading to allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria, gastro-intestinal diseases, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, autoimmune diseases, nasal polyps, Fabry's disease, Kimura's disease and gastric and duodenal ulceration.
  • allergies such as food allergies, pet allergies, pollen allergies insect sting allergies and other allergies leading to allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria
  • gastro-intestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis
  • the disease or condition is preferably an inflammatory disorder, such as an inflammatory disorder selected from those described herein.
  • inflammatory disorders are caused by the over-activation of cells of hematopoetic lineage, including mast cells, which release large quantities of pro-inflammatory molecules.
  • the methods and uses of the present invention are particularly suited to the treatment and prophylaxis of mammalian allergic disorders. Mammalian allergic disorders that may be treated or prevented using the methods of the present invention include those allergies listed above.
  • the agent may be selected from the known beneficial cholesterol-lowering compounds, including Bile acid sequestrants (such as Cholestyramine resin, Colesevelam HC1, Colestipol, and Polidexide); Fibrates (such as Bezafibrate, Binifibrate, Ciprofibrate, Clinofibrate, Clofibrate, Clofibric acid, Etofibrate, Fenofibrate, Gemfibrozil, Pirifibrate, Ronifibrate, Simfibrate and Theofibrate); 3-hydroxy-3 methylglutaryl Coenzyme A reductase (HMG CoA reductase) inhibitors (including statins such as Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitivastatin, Pravastatin, Rosuvastatin, and Simvastatin); various Nicotinic acid derivatives, including statins such as Atorvastatin, Cerivastatin, Fluvastatin
  • the agent that reduces the level of cholesterol may be an inhibitor of de novo cholesterol biosynthesis.
  • the agent that reduces the level of cholesterol employed in the methods of the present invention may also reduce the level of cholesterol within the cell by other mechanisms.
  • the agents may be PPAR ⁇ antagonists, PPAR ⁇ agonists or LXR agonists.
  • the agent that reduces the level of cholesterol is an inhibitor of de novo cholesterol biosynthesis.
  • the present invention provides the use of an inhibitor of de novo cholesterol biosynthesis in the methods and uses described above.
  • the agent that reduces the level of cholesterol is a HMG CoA reductase inhibitor.
  • HMG CoA reductase inhibitors include the statins and derivatives or functional equivalents thereof.
  • HMG CoA reductase inhibitors are of special interest in the present invention, due to their direct inhibition of a key rate-limiting step in cholesterol biosynthesis.
  • the present invention provides the use of HMG CoA reductase inhibitors in the methods and uses described above.
  • the HMG CoA reductase enzyme is found in both eukaryotes and prokaryotes.
  • the HMG CoA reductase enzyme to which inhibitors of the invention are directed is generally the enzyme present in eukaryotes, for example, the HMG CoA reductase enzyme present in mammals and, in particular, the HMG CoA reductase enzyme present in primates, including humans.
  • the HMG CoA reductase inhibitor is a statin.
  • the statins have acceptable pha ⁇ nacodynamic and pharmacokinetic properties.
  • Particularly preferred compounds for use in accordance with the methods and uses of the present invention include Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitivastatin, Pravastatin, Rosuvastatin, and Simvastatin and derivatives or functional equivalents thereof.
  • the present invention provides the use of a statin or derivative or functional equivalent thereof in the methods and uses described above.
  • the side effects associated with systemic administration of cholesterol-lowering drugs, in particular HMG CoA reductase inhibitors, such as statins, are mentioned above.
  • the methods and uses of the present invention overcome these side-effects via the provision of agents that reduce the level of cholesterol within cells that are characterised by rapid clearance from, or rapid inactivation in, the systemic circulation.
  • the agent that reduces the level of cholesterol within the relevant cells is characterised by rapid clearance from, or rapid inactivation in, the systemic circulation.
  • specifically modified 'soft' forms of current cholesterol-lowering agents are employed, such as 'soft' statins.
  • the term 'soft' in relation to a cholesterol-lowering agent refers to a detuned or de-stabilised cholesterol-lowering agent that has been specifically selected, designed or engineered to ensure that it is rapidly cleared from, or rapidly inactivated in, the systemic circulation.
  • an agent needs to be absorbed at the site of delivery (e.g. topical, intranasal, intraoccular, pulmonary), but rapidly cleared or inactivated upon reaching the systemic circulation.
  • the present invention provides such agents via the provision of specifically selected, designed and engineered agents with intentionally rapid metabolic clearance and inactivation characteristics.
  • the required rapid metabolic clearance and inactivation may be achieved through the action of those metabolic enzymes primarily responsible for the metabolism of current cholesterol-lowering agents.
  • the cytochromes P450 (Scripture and Pieper, Clin. Pharmacokinetic., 40, 263-281, 2001; Evans and Rees, Drug Saf., 25, 649- 663, 2002) are primarily responsible for the metabolism of statins, the class of compounds that is preferred for use in the present invention.
  • soft agents that reduce the level of cholesterol within cells and that are characterised by rapid clearance from, or rapid inactivation in, the systemic circulation may include a number of known cholesterol-lowering agents.
  • Preferred soft agents that reduce the level of cholesterol within cells include prodrugs of known cholesterol-lowering agents.
  • the present invention uses specifically selected, designed and engineered HMG CoA reductase inhibitor compounds with intentionally rapid metabolic clearance and inactivation characteristics.
  • statins examples of which include Atorvastatin, Cerivastatin, Mevastatin, Pravastatin, Fluvastatin, Simvastatin, Lovastatin, Rosuvastatin, and Pitavastatin.
  • statins examples of which include Atorvastatin, Cerivastatin, Mevastatin, Pravastatin, Fluvastatin, Simvastatin, Lovastatin, Rosuvastatin, and Pitavastatin.
  • Figure 5 Extensive metabolic clearance in fact occurs for most statins (Igel et al, Eur. J. Clin.
  • the soft cholesterol-lowering agent may comprise a specifically modified form of a cholesterol-lowering agent, wherein sites for metabolism by certain enzymes are introduced in order to shorten the plasma half-life and corresponding systemic exposure.
  • the rapid metabolic clearance and inactivation could be achieved by generating cholesterol-lowering agents, such as HMG CoA reductase inhibitors, which are inactivated by drug metabolising enzymes such as esterases (Satoh and Hosokawa, Annu. Rev. Pharmacol. Toxicol., 38, 257-288, 1998). More particularly, HMG CoA reductase inhibitors may be generated which are inactivated by plasma esterases (Williams, Clin. Pharmacokinet, 10, 392-403,1985).
  • a class of particularly preferred soft agents are lipophilic ester prodrags of the statins. These prodrags are formed between the active statin drug and a suitable lipophilic alcohol. These compounds have specific physicochemical and metabolic properties that enhance their utility for topical application and delivery.
  • these compounds comprise an ester prodrag linkage to the HMG-like moiety of the statin drug, which will be endogenously hydrolysed at various rates releasing the active substance, thus displaying beneficial sustained release and delivery properties.
  • these compounds comprise a lipophilic group reducing partitioning into the plasma and subsequent distribution to other compartments.
  • these compounds represent specifically optimised statin prodrags for use in the methods of the present invention.
  • statin prodrags there are several pharmaceutically acceptable lipophilic alcohols available which may be used to form such statin prodrags, including, but not limited to, methanol, ethanol, propan- l-ol, propan-2-ol, butan-1-ol, butan-2-ol, pentan-1-ol, hexan-1 -ol, heptan-1-ol, octan-1-ol, nonan-1-ol, decan-1-ol, 2-ethyl-hexan-l-ol, 3,3,5-trimethyl-cyclohexanol, 2-ethoxy- ethanol, and menthol.
  • each of these lipophilic alcohols may be of use in combination with the known statins in the production of statin prodrags of utility in the methods of the present invention.
  • the present invention provides the lipophilic ester statin prodrags produced by esterification of a statin using any one of these lipophilic alcohols, and the use of any of these compounds in the methods of the present invention.
  • the present invention provides a compound of the general formula X-Y, where X is selected from the group consisting of Atorvastatin, Fluvastatin, Rosuvastatin, Pitavastatin, Cerivastatin, Pravastatin, Simvastatin (free acid form), Lovastatin (free acid form) and Mevastatin (free acid form), and Y is selected from the group consisting of methanol, ethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, pentan-1-ol, hexan-1- ol, heptan-1-ol, octan-1-ol, nonan-1-ol, decan-1-ol, 2-ethyl-hexan-l-ol, 3,3,5-trimethyl- cyclohexanol, 2-ethoxy-ethanol and menthol.
  • X is selected from the group consisting of Atorvastatin, Fluvastatin, Rosuvastatin
  • the soft cholesterol-lowering agent comprises a specifically engineered statin, wherein sites for metabolism by a specific enzyme have been introduced in order to shorten the plasma half-life of the statin and the corresponding level of systemic exposure.
  • de-tuned (or destabilised) statins are shown in Figure 8, in which the fluorine atom has been substituted with a hydrogen atom to de-tune the statin.
  • Compounds provided according to this embodiment of the invention include (3R,5R)-3,5- Dihydroxy-7-(2-isopropyl-4,5-diphenyl-3 -phenylcarbamoyl-pyrrol- 1 -yl)-heptanoic acid, (E)-(3R, 5 S)-3 , 5 -Dihydroxy-7-( 1 -isopropyl-3 -phenyl- 1 H-indol-2-yl)-hept-6-enoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[4-isopropyl-2-(methanesulfonyl-methyl-amino)-6-phenyl- pyrimidin-5-y ⁇ ]-hept-6-enoic acid, (E)-(3R,5S)-7-(2-Cyclopropyl-4-phenyl-quinolin-3-y ⁇ )- 3,5-dihydroxy-hept-6-enoic acid and (
  • Preferred soft statin compounds include hydroxylated forms of the statins. These soft agents have the benefit of being available to phase II drug metabolism enzymes (for example those involved in glucuronidation, sulphation, etc) and are thus primed for rapid and safe clearance.
  • Compounds provided according to this embodiment of the invention include (3R,5R)-3-,5- Dihydroxy-7-[2-(4-hydroxy-phenyl)-5-isopropyl-3-phenyl-4-phenylcarbamoyl-pyrrol-l- yl]-heptanoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[3-(4-hydroxy-phenyl)-l-isopropyl-lH- indol-2-yl]-hept-6-enoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[4-(4-hydroxy-phenyl)-6- isopropyl-2-(methanesulfonyl-methyl-amino)-pyrimidin-5-yl]-hept-6-enoic acid, (E)- (3R,5S)-7-[2-Cyclopropyl-4-(4-hydroxy-phenyl)-quinolin
  • Preferred compounds according to this embodiment of the invention include ester prodrags of the hydroxylated statins, formed by an ester bond linking the hydroxyl of the active ingredient and a pharmaceutically acceptable acid.
  • Pharmaceutically acceptable carboxylic acids suitable for the formation of such esters may be selected from the pharmaceutically acceptable organic acids listed herein. Illustrative examples of such esters are shown in Figure 10.
  • X is a hydroxylated statin selected from the groups consisting of (3R,5R)-3-,5-Dihydroxy-7-[2-(4-hydroxy-phenyl)-5-isopropyl-3-phenyl-4- phenylcarbamoyl-pyrrol-1 -yl]-heptanoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[3-(4- hydroxy-phenyl)-l-isopropyl-lH-indol-2-yl]-hept-6-enoic acid, (E)-(3R,5S)-3,5- Dihydroxy-7-[4-(4-hydroxy-phenyl)-6-isopropyl-2-(methanesulfonyl-methyl-amino)- pyrimidin-5-yl]-hept-6-enoic acid, (E)-(3
  • the agents that reduce the level of cholesterol within cells and that are characterised by rapid clearance from, or rapid inactivation in, the systemic circulation may be soft cholesterol-lowering agents, in particular soft statins, and prodrags of soft cholesterol-lowering agents, in particular prodrags of soft statins.
  • agents that reduce the level of cholesterol within cells and that are characterised by rapid clearance from, or rapid inactivation in, the systemic circulation may also include the metabolites of these soft drags, and prodrags thereof.
  • statins refer to both the free acid and the lactone forms of the statins.
  • the present invention provides methods for the treatment and prophylaxis of a variety of localised diseases or conditions (for example, allergies as described herein), comprising delivering the agent locally to sites in need thereof (for example, sites of allergic response), whilst minimising systemic exposure to the agent by ensuring that the agent benefits from rapid clearance from, or rapid inactivation in, the systemic circulation.
  • localised diseases or conditions for example, allergies as described herein
  • sites in need thereof for example, sites of allergic response
  • specific treatment of allergic conditions may be achieved by delivering agents locally to sites of allergic response, whilst minimising systemic exposure to the agent by ensuring that the agent benefits from rapid clearance from, or rapid inactivation in, the systemic circulation.
  • This serves to minimise effects on serum cholesterol and the potential for harmful side-effects, in particular Rhabdomyolysis.
  • metliods of treatment and prophylaxis will also benefit from the advantage of "steroid sparing" for children, when used for asthma or dermatitis.
  • the agent may be applied specifically to the topologically exterior surface of a mammal.
  • the agent may be applied specifically to a certain region of the topologically exterior surface of a mammal, such as the skin, the nasal mucosa, the ocular mucosa or the respiratory tract.
  • agent to the skin, or a region thereof, is preferred for the treatment or prophylaxis of dermatitis, eczema, wound healing or psoriasis.
  • agent to the respiratory tract, or a region thereof, is preferred for the treatment or prophylaxis of asthma or allergic rhinitis, allergic conjunctivitis or COPD (emphysema and chronic bronchitis).
  • a pharmaceutical composition comprising an agent that reduces the level of cholesterol in a cell and a pharmaceutically acceptable carrier, wherein the agent that reduces the level of cholesterol in the cell is characterised by rapid clearance from, or rapid inactivation in, the systemic circulation.
  • a pharmaceutically acceptable carrier wherein the agent that reduces the level of cholesterol in the cell is characterised by rapid clearance from, or rapid inactivation in, the systemic circulation.
  • Suitable agents that reduce the level of cholesterol in cells and are characterised by rapid clearance from, or rapid inactivation in, the systemic circulation are described herein.
  • the agent that reduces the level of cholesterol is a statin, or derivative thereof, as described above.
  • agents of the present invention can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions, pharmaceuticals and medicaments contemplated by the present invention may be formulated in various ways well-known to one of skill and administered by similarly well-known methods.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions can be used therein.
  • pharmaceutically acceptable salt' refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids and bases.
  • inorganic acids suitable for use in this invention include, but are not limited to hydrochloric, hydrobromic, hydroiodic, sulfuric, and phosphoric acids.
  • Appropriate organic acids for use in this invention include, but are not limited to aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic, stearic, sulfanilic, algenic, and galacturonic.
  • inorganic bases suitable for use in this invention include metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium, and zinc.
  • Appropriate organic bases may be selected, for example, from N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine (N- methylglucamine), and procaine.
  • the agent of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, or controlled-release such as sustained-, dual-, or pulsatile delivery applications.
  • the compound may also be administered via fast dispersing or fast dissolving dosage forms or in the form of a high-energy dispersion or as coated particles. Suitable pharmaceutical formulations of the compound may be in coated or un-coated form as desired.
  • Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and gran
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compound may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device.
  • Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof.
  • Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients.
  • Release rate-modifying excipients maybe present both within the dosage form i.e. within the matrix, and/or on the dosage form i.e. upon the surface or coating.
  • Fast dispersing or dissolving dosage formulations may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.
  • dispersing or dissolving as used herein to describe FDDFs are dependent upon the solubility of the drag substance used i.e. where the drag substance is insoluble a fast dispersing dosage form can be prepared and where the drag substance is soluble a fast dissolving dosage form can be prepared.
  • the compound can also be administered parenterally, for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion or needleless injection techniques.
  • parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drags or hormones.
  • the daily dosage level of the compound will usually be from 10 to 500 mg (in single or divided doses).
  • tablets or capsules of the compound may contain from 5mg to 250mg of active compound for administration singly or two or more at a time, as appropriate.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
  • the compound may be taken as a single dose on an "as required" basis (i.e. as needed or desired).
  • the compound can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM or 1,1,1,2,3,3,3- heptafluoropropane (HFA 227EATM), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM or 1,1,1,2,3,3,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
  • Aerosol or dry powder formulations are preferably arranged so that each metered dose 10 or "puff contains from 1 to 50 mg of a compound of the invention for delivery to the patient.
  • the overall daily dose with an aerosol will be in the range of from 1 to 50 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the compound may also be formulated for delivery via an atomiser.
  • Formulations for atomiser devices may contain the following ingredients as solubilisers, emulsifiers or suspending agents: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycols, sodium chloride, fluorocarbons, polyethylene glycol ethers, sorbitan trioleate, oleic acid.
  • the compound can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the compound may also be dermaliy administered.
  • the compound may also be transdermaliy administered, for example, by the use of a skin patch.
  • the compound may also be administered by the ocular, pulmonary or rectal routes.
  • the compound can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • the compound may be formulated in an ointment such as petrolatum.
  • the compound of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, 5 emulsifying wax and water.
  • it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the compound may also be used in combination with a cyclodextrin.
  • Cyclodextrins are known to form inclusion and non-inclusion complexes with drag molecules. Formation of a drag-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drag-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drag the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A-91/11172, WO-A-94/02518 and WO- A-98/55148.
  • a preferred oral dosing regimen in allergic asthma, allergic conjunctivitis, allergic rhinitis or atopic dermatitis for a typical adult male is from 25 to 250 mg of compound when required.
  • the drug may be administered parenterally, sublingually or buccally.
  • particularly preferred formulations are topical and inhaled. Oral dosing is also a preferred formulation.
  • the compound, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate or pro-drag thereof is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • the identification of the mechanism by which the activity of cells can be modulated allows for the design of screening methods capable of identifying compounds that are effective in the lowering of intracellular cholesterol.
  • a method of screening for agents useful for the modulation of the activity of a cell comprising assessing the ability of a selected moiety to reduce the level of cholesterol within said cell.
  • a method of screening for agents useful for the modulation of the activity of a cell of hematopoetic lineage comprising assessing the ability of a selected moiety to reduce the level of cholesterol within cells of hematopoetic lineage.
  • a method of screening for agents useful for the modulation of mast cell activation comprising assessing the ability of a selected moiety to reduce the level of cholesterol within mast cells.
  • the methods may include one or more steps in which calcium mobilisation, kinase phosphorylation, TNF ⁇ release, degranulation, proliferation and adhesion are assayed, as described in Examples 2-7.
  • statins to reduce the level of cholesterol in mast cells.
  • Figure 1 Measurement of calcium transients in RBL-2H3 cells.
  • A Control compared to cells treated for 2 days with fluvastatin, lovastatin or pravastatin (lO ⁇ M).
  • B Peak calcium following Fc ⁇ RI aggregation in control cells compared to cells treated with either 3 ⁇ M or 10 ⁇ iM fluvastatin or lovastatin for two days. Additional cells were treated with methyl ⁇ cyclodextrin dissolved in DMSO for one hour at 37°C. cells were treated with DMSO alone to act as a control.
  • C Peak calcium following Fc ⁇ RI aggregation in control cells compared to cells treated for two days with either 10 ⁇ M fluvastatin or lovastatin supplemented or not with lOOmM mevalonate.
  • Figure 2 Measurement of ⁇ hexosaminidase in the supernatant of RBL-2H3 cells following FceRI aggregation. Release from control cells was compared to cells treated with fluvastatin, lovastatin or pravastatin (3 or 1 ⁇ M) for two days and from cells treated with fluvastatin, lovastatin or pravastatin (3 ⁇ M) supplemented with mevalonate (lOOmM) and from cells treated with methyl ⁇ cyclodextrin for one hour.
  • Figure 3 Measurement of RBL-2H3 cell proliferation using the Cy Quant assay (Molecular Probes) over 5 days. The rate of cell proliferation for control cells was compared to that for cells treated with fluvastatin (3 ⁇ M).
  • Figure 4 Measurement of Jurkat cell proliferation using the CyQuant assay (Molecular Probes) over 5 days. The rate of cell proliferation for control cells was compared to that for cells treated with fluvastatin (lO ⁇ M), lovastatin (lO ⁇ M) or pravastatin (lO ⁇ M).
  • Figure 6 Molecular structures of current statin drugs.
  • Figure 7 Molecular structures of representative soft statin compounds.
  • Figure 8 Molecular stractures of representative soft statin compounds.
  • Figure 9 Molecular stractures of representative soft statin compounds.
  • Figure 10 Molecular structures of representative soft statin compounds.
  • the present invention is based on the discovery that agents that reduce the level of cholesterol within cells may be used to modulate the activity of a wide variety of cells via disruption of membrane signalling pathways.
  • the invention is also based on the discovery of the mechanism by which such agents lead to the disraption of membrane signalling pathways, and the associated discovery that such agents can be used to modulate the level of cholesterol present in the lipid rafts of the cell membrane.
  • Example 1 describes the use of the PharmaCartaTM platform to identify draggable targets and lead compounds.
  • Example 2 describes the validation of the findings made using the PharmaCartaTM platform in mast cells and T cells, and explain why the results generated by the PharmaCartaTM platform are broadly applicable to a wide range of cholesterol-lowering agents, a wide range of cell types and a wide range of signalling components associated with the lipid rafts of the cell membrane.
  • Example 1 Use of PharmaCartaTM to identify draggable targets and lead compounds
  • this enzyme is the target for HMG CoA reductase inhibitor drugs termed 'statins' on the market.
  • the PharmaCartaTM platform was able to identify known drag compounds that might be useful in novel therapeutic settings.
  • TCR engagement results in the activation of a Src-like kinase, ZAP-70, PI3 kinase, small molecular weight GTPase, Tec-family kinase and phospholipase C.
  • the outcome is an increase in inositol trisphosphate IP 3 which binds to the IP 3 receptor and causes a rise in intracellular calcium and activation of calcium influx through the opening of the calcium channel, Icrac.
  • the components are frequently cell type specific but the overall pathway is similar.
  • An analogous pathway is responsible for activation of mast cells following IgE binding to Fc ⁇ RI and for the activation of B cells following engagement for the B cell receptor (BCR).
  • HMG CoA reductase inhibitors have a similar effect on IgE:FceRI mediated signalling cascades in mast cells as is observed for the TCR in T cells (see below).
  • RBL-2H3 cells were maintained in a 40:10 mix of Modified Eagle medium (MEM):RPMI1640 supplemented with 10% foetal bovine serum (FBS), 5 mM glutamine, penicillin and streptomycin. Cells were sensitised with 1 ⁇ g/ml dinitrophenol specific rat IgE for 12 hours prior to all experiments.
  • MEM Modified Eagle medium
  • FBS foetal bovine serum
  • FBS foetal bovine serum
  • penicillin and streptomycin penicillin and streptomycin.
  • lovastatin (10 or 3 ⁇ M), fluvastatin (10 or 3 ⁇ M) or pravastatin (10 or 3 ⁇ M) for 24 or 48 hours prior to experiments.
  • Drags were dissolved in DMSO and diluted at least 1000 fold directly into the tissue culture medium.
  • HBS HEPES buffered saline
  • 2 mM calcium chloride 2 mM calcium chloride
  • 2 mg/ml glucose 2 mg/ml bovine serum albumin
  • Fura-2 AM Molecular Probes
  • cells were resuspended in HBS supplemented with 1 mM Ca2+ and 1.8 mg/ml glucose.
  • Cells were added to a stirred cuvette containing 1.4 ml of HBS 1 mM Ca2+ at 37C in a Cairn spectrophotometer.
  • Excitation wavelengths of 340, 360 and 380nm were provided by a filter wheel rotating at 35 Hz in the light path. Emitted light was filtered by a 485 nm long pass filter and samples were averaged every 500 ms.
  • the background-corrected 340/380 ratio was calibrated using the method of Grynkiewicz et al, (J. Biol. Chem., 1985, 260, 3440-3450). Following 5 minutes equilibration at 37°C, cell surface receptor bound IgE was aggregated by the addition of the antigen DNP-conjugated albumin (1 ⁇ g/ml) (Sigma, Aldrich).
  • statin treatment (lO ⁇ M lovastatin and fluvastatin) was supplemented with addition of lOOmM mevalonate.
  • calcium transients were restored to normal. Peak calcium after Fc ⁇ RI aggregation is shown in Figure lc for cells treated with either lovastatin or fluvastatin (lO ⁇ M) and compared to cells treated with the same dose of lovastatin or fluvastatin but the medium was supplemented with 100 mM mevalonate.
  • this example demonstrates that inhibition of HMG CoA reductase in mast cells with either lovastatin or fluvastatin inhibits intracellular calcium transients in vitro in response to IgE activation of mast cells.
  • this effect can be reversed by addition of mevalonate to the cells.
  • Pravastatin which does not cross the plasma membrane, had little effect on the IgE mediated calcium transients.
  • the effect of lovastatin and fluvastatin is a direct result of inhibition of the enzyme HMG CoA reductase as supplying the cells with exogenous mevalonate, the enzyme product, overcomes the inhibitory effect of both fluvastain and lovastatin.
  • HMG CoA reductase is required within the cell for the de novo synthesis of cholesterol, which is required for the formation of lipid rafts.
  • statin treatment on peak calcium was compared to the depletion of plasma membrane cholesterol directly using methyl ⁇ cyclodextrin.
  • Cells were treated with methyl ⁇ cyclodextrin (lOmM) for one hour at 37°C prior to loading with Fura-2 and completing calcium transient recordings as detailed above. Peak calcium after aggregation of FceRI in these cells was inhibited to the same extent as fluvastatin (lO ⁇ M) treatment of the cells ( Figure lb).
  • RBL-2H3 cells were treated with lovastatin (3 or 1 ⁇ M), fluvastatin (3 or 1 ⁇ M) or pravastatin (3 ⁇ iM) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOO ⁇ M) in combination with either lovastatin or fluvastatin. The cells were primed with 1 ⁇ g/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 ⁇ g/ml DNP-BSA, the cells were incubated for 2 mins at 37°C. The cells were then lysed in Laemelli buffer and lysates were stored at -20°C prior to running on SDS/PAGE.
  • lovastatin 3 or 1 ⁇ M
  • fluvastatin 3 or 1 ⁇ M
  • pravastatin 3 ⁇ iM
  • Proteins were then blotted on to nitrocellulose and these were then probed using specific monoclonal antibodies to phosphotyrosine using the monoclonal antibody 4G10 (Upstate Biotechnology), to Syk and Lyn kinase using specific monoclonal antibodies (Upstate Biotechnology).
  • the specific bands were visualised using standard technologies and the relevant bands imaged using an imaging system from Alphalnnotech. Digital images were recorded using a CCD camera and stored electronically.
  • Receptor coupled tyrosine phosphorylation was identical to the control for cells treated with pravastatin (3 ⁇ M) or co-treated with mevalonate (100 ⁇ M) together with either lovastatin (3 ⁇ M) or fluvastatin (3 ⁇ M).
  • This data indicates that receptor coupled tyrosine phosphorylation is inhibited in the presence of active HMG CoA reductase inhibitors such as lovastatin or fluvastatin and that this effect is through the inhibition of cellular HMG CoA reductase.
  • HMG CoA reductase inhibitors such as lovastatin or fluvastatin
  • cell surface plasma membrane cholesterol was depleted using the drag, methyl ⁇ cyclodextrin (Fisher, de Hoog and Mann, PNAS, 2003, 100, 5813-8). Following priming of the cells with IgE, cells were incubated for 1 hour with methyl ⁇ cyclodextrin (10 mM). Receptor coupled tyrosine phosphorylation was inhibited in cells pretreated with methyl ⁇ cyclodextrin.
  • this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits Fc ⁇ RI coupling to signal transduction pathways in mast cells as measured by IgE receptor activated (a) tyrosine phosphorylation of Syk kinase and (b) dual phosphorylation of ERK.
  • This effect of lovastatin or fluvastatin was a result of the inhibition of HMG CoA reductase as provision of mevalonate to the cells overcomes the inhibitory effect of the statins.
  • methyl ⁇ cyclodextrin inhibited receptor coupled tyrosine phosphorylation. Methyl ⁇ cyclodextrin acts to deplete the plasma membrane of cholesterol and thereby disrupt lipid rafts directly, and thus these data show that the effects on Fc ⁇ RI signalling are not statin-specific.
  • RBL-2H3 cells were treated with lovastatin (3 or 1 ⁇ M), fluvastatin (3 or 1 ⁇ M) or pravastatin (3 ⁇ iM) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOO ⁇ M) in combination with either lovastatin or fluvastatin. The cells were primed with 1 ⁇ g/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 ⁇ g/ml DNP-BSA, the cells were incubated for a further 30 - 60 mins at 37°C.
  • the supernatant was harvested and after removal of cell debris by centrifugation, ⁇ - hexosaminidase activity was measured in the supernatant using a standard colorimetric assay. Briefly, 50 ⁇ l supernatant was incubated with 200 ⁇ l of lmM jp-nitrophenyl N- acetyl- ⁇ -D-glucosamine for 60 mins at 37°C. Reactions were quenched by addition of 0.1 M sodium carbonate buffer. The enzyme activity was measured by absorbance at 400nm.
  • cell surface plasma membrane cholesterol was depleted using the drug, methyl ⁇ cyclodextrin (Fisher, de Hoog and Mann, P ⁇ AS, 2003, 100, 5813-8).
  • methyl ⁇ cyclodextrin 10 mM
  • the receptor was aggregated by the addition of D ⁇ P-BSA and hexosaminidase release measured.
  • receptor coupled hexosaminidase release was abolished in cells treated with methyl ⁇ cyclodextrin.
  • disruption of the lipid rafts directly by depletion of plasma membrane cholesterol using methyl ⁇ cyclodextrin results in inhibition of Fc ⁇ RI triggered degranulation.
  • this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits Fc ⁇ RI mediated degranulation of mast cells as measured by release of ⁇ hexosaminidase.
  • methyl ⁇ cyclodextrin inhibited release of ⁇ hexosaminidase indicating that the integrity of the lipid rafts on the plasma membrane are a key feature to maintain receptor coupled degranulation.
  • Example 5 Effect of statins on the Fc ⁇ RI mediated release of the proinflammatorv cytokine, TNF ⁇
  • RBL-2H3 cells were treated with lovastatin (3 or 1 ⁇ M), fluvastatin (3 or 1 ⁇ M) or pravastatin (3 ⁇ M) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOO ⁇ M) in combination with either lovastatin or fluvastatin (3 ⁇ M). The cells were primed with 1 ⁇ g/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 ⁇ g/ml DNP-BSA, the cells were incubated for a further 120 mins at 37C. The supernatant was harvested and, after removal of cell debris by centrifugation, the levels of TNF ⁇ were measured in the cell supernatants using a standard ELIS A assay (manufacturer) .
  • Receptor coupled release of TNF ⁇ is inhibited by treatment of cells with either lovastatin or fluvastatin. This inhibition by the statin was abolished in cells co-treated with mevalonate (100 ⁇ M) indicating that the action of the statin is a feature of the inhibition of HMG CoA reductase.
  • this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits Fc ⁇ RI mediated release of the proinflammatory cytokine, TNF ⁇ .
  • RBL-2H3 cells Triplicate wells of RBL-2H3 cells were treated with lovastatin (3, 1, 0.3 ⁇ M), fluvastatin (3, 1, 0.3 ⁇ M) or pravastatin (3, 1, 0.3 ⁇ M) for 5 days. Cells were harvested each day for 5 days and the cell number quantified using the CY-Quant assay (Molecular Probes).
  • Jurkat cells were treated with lovastatin (10 ⁇ M), fluvastatin (10 ⁇ M) or pravastatin (10 ⁇ M) for 5 days. Cells were harvested each day for 5 days and the cell number quantified using the CY-Quant assay (Molecular Probes).
  • the rate of proliferation of the Jurkat cells was inhibited by treatment of cells with either lovastatin or fluvastatin.
  • Pravastatin had no effect on the rate of proliferation of the cells over the 5 day period.
  • this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits the proliferation of the Jurkat cell line.
  • RBL-2H3 cells grow as adherent cells in the 40:10 mix of MEM:RPMI1640.
  • Treatment of cells with lovastatin or fluvastatin (3 ⁇ M) results in a change in the morphology of the cells. This change takes place within 6-8 hours of culture. The cells become rounded and no longer adhere tightly to the tissue culture dish.
  • Pretreating cells with mevalonate (100 ⁇ M) at the same time as the lovastatin or fluvastatin (3 ⁇ M) prevented the statin induced change in morphology.
  • Pravastatin (3 ⁇ M) had no effect on the appearance of the cells in culture.
  • Example 10 Effect of statins on store operated calcium influx in RBL-2H3 cells
  • lovastatin and fluvastatin on store operated calcium entry was determined and compared to the effect of disrupting lipid rafts using methyl ⁇ cyclodextrin. Briefly, RBL- 2H3 cells were maintained in a 40:10 mix of Modified Eagle medium (MEM) :RPMI 1640 supplemented with 10% foetal bovine serum (FBS), 5 mM glutamine, penicillin and streptomycin.
  • MEM Modified Eagle medium
  • FBS foetal bovine serum
  • penicillin and streptomycin penicillin and streptomycin.
  • Cells were treated with lovastatin (10 ⁇ M), fluvastatin (10 ⁇ M) for 24 or 48 hours prior to experiments. Drugs were dissolved in DMSO and diluted at least 1000 fold directly into the tissue culture medium. Cells treated with methyl ⁇ cyclodextrin were incubated with a 20mM solution of methyl ⁇ cyclodextrin for one hour at 37°C immediately prior to loading with Fura 2-AM. An identical set of cells were incubated with an equivalent volume of DMSO to control for any effects of the solvent.
  • HBS HEPES buffered saline
  • 2 mM calcium chloride 2 mM calcium chloride
  • 2 mg/ml glucose 2 mg/ml bovine serum albumin
  • Fura-2 AM Molecular Probes
  • cells were resuspended in HBS supplemented with 1 mM Ca2+ and 1.8 mg/ml glucose.
  • Cells were added to a stirred cuvette containing 1.4 ml of HBS that was nominally calcium free at 37 C in a Cairn spectrophotometer.
  • Excitation wavelengths of 340, 360 and 380nm were provided by a filter wheel rotating at 35 Hz in the light path. Emitted light was filtered by a 485 nm long pass filter and samples were averaged every 500 ms. The background-corrected 340/380 ratio was calibrated using the method of Grynkiewicz et al, (J. Biol. Chem., 1985, 260, 3440-3450).
  • Intracellular calcium stores were depleted by the addition of thapsigargin and the intracellular calcium transients monitored. This demonstrated that the intracellular calcium release by thapsigargin was identical between all the treatments examined. This data demonstrates that the intracellular calcium stores were intact and unaffected by treatment of the cells with statins or by cholesterol depletion using methyl ⁇ cyclodextrin ( Figure 5). Following store depletion, calcium entry was then measured by adding calcium to the cuvette (final concentration 2mM). Following addition of extracellular calcium, the intracellular calcium levels rose rapidly in the control cells. The rate of entry was significantly delayed and the peak calcium was significantly lower in the cells treated with fluvatsatin and lovastatin.
  • statins were similar to that observed for depletion of cholesterol at the plasma membrane with methyl ⁇ cyclodextrin.
  • HMG CoA reductase inhibition results in decoupling of the IgE receptor (Fc ⁇ RI) from the intracellular signalling cascades.
  • Inhibitors of HMG CoA reductase are shown to inhibit the normal functioning of Fc ⁇ RI, the high affinity receptor for IgE, on the surface of mast cells.
  • Treatment of mast cells with HMG CoA reductase inhibitors is shown to result in an inliibition of IgE receptor stimulated degranulation as measured by the release of ⁇ hexosaminidase and the pro-inflammatory cytokine TNF ⁇ .
  • methyl ⁇ cyclodextrin has been shown to also inhibit the activities and functions of mast cells investigated.
  • the effects on the lipid raft micro-domains are not specific to HMG CoA reductase inhibitors.
  • agents that lower intracellular cholesterol through diverse mechanisms will disrupt lipid rafts, for example, by preventing the de novo synthesis of cholesterol for insertion into the plasma membrane (e.g. HMG CoA reductase inhibitors) or the trafficking of cholesterol into the plasma membrane by reducing the intracellular pool available for trafficking (e.g. PPAR ⁇ agonists or LXR agonists).
  • agents will be useful for modulating the activity of a range of cell types and of a range of membrane signalling networks.
  • disruption of lipid rafts will be beneficial in the treatment or prophylaxis of a number of diseases or conditions, including those described above.
  • Example 1 the findings made using the PharmaCartaTM platform in Example 1 were successfully confirmed in the other Examples above. Accordingly, these Examples confirm the utility of the PharmaCartaTM platform for the accurate identification of draggable targets and associated lead compounds, which can then be simply and rapidly validated at the laboratory bench.

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Abstract

L'invention concerne des méthodes de modulation de l'activité de cellules et des compositions utilisées dans de telles méthodes. Notamment, ladite invention a pour objet l'utilisation d'un agent qui permet de diminuer le taux de cholestérol au sein d'une cellule de manière à moduler l'activité de la cellule, ainsi que des méthodes impliquant une telle utilisation.
PCT/GB2004/003875 2003-09-10 2004-09-10 Modulation de l'activite cellulaire au moyen d'un agent diminuant le taux de cholesterol au sein d'une cellule WO2005023305A2 (fr)

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WO2007088123A3 (fr) * 2006-02-03 2007-09-20 Nicox Sa Utilisation de derives nitro-oxydes de medicaments pour le traitement de dystrophies musculaires
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US7846946B2 (en) 2005-06-20 2010-12-07 Schering Plough Corporation Heteroatom-linked substituted piperidines and derivatives thereof useful as histamine H3 antagonists
US7635705B2 (en) 2005-06-20 2009-12-22 Schering Corporation Heteroatom-linked substituted piperidines and derivatives thereof useful as histamine H3 antagonists
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