WO2005023305A2

WO2005023305A2 - Modulating cell activity by using an agent that reduces the level of cholesterol within a cell

Info

Publication number: WO2005023305A2
Application number: PCT/GB2004/003875
Authority: WO
Inventors: Janet Marjorie Allen; John Paul Overington
Original assignee: Inpharmatica Limited
Priority date: 2003-09-10
Filing date: 2004-09-10
Publication date: 2005-03-17
Also published as: WO2005023305A3; GB0321228D0

Abstract

This invention relates to methods for modulating the activity of cells, and compositions useful in such methods. In particular, 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.

Description

MODULATING CELL ACTIVITY BY USING AN AGENT THAT REDUCES THE LEVEL OF CHOLESTEROL WITHIN A CELL

All publications, patents and patent applications cited herein are incorporated in full by reference.

Introduction to HMG CoA Reductase and HMG CoA Reductase Inhibitors

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). Thus, a number of methods and compositions for reducing the plasma level of cholesterol have been employed to treat such cardiovascular diseases.

Compounds that beneficially modulate levels of cholesterol include several known drug classes, for instance 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, Nicertirol, Nicoclonate, Nicomol, and Oxiniacic acid); Thyroid Hormones/Analogues (such as Dextrothyroxine, Eitroxate, and Thyropropic acid) and a wide variety of other agents (such as Acifran, Benfluorex, β-Benzylbutyraimde, Carnitine, Chondrotin sulphate, Colmestrone, Detaxtran, Dextran sulphate Sodium, Eicosopentanoic acid, Eritadenine, Ezetimibe, Furazabol, Meglutol, Melinamide, γ-Oryzanol, Pantethine, Pentaerythritol tetraacetate, α-Phenylbutyramide, Pirozadil, Probcuol, β-Sitosterol, Sultosilic acid, Tiadenol, Triparanol, and Xenbucin). Notably, 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). Thus, 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).

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. Clin Practice Supplement 1996, 77 A, 24-7; see also http://www.nlm.nih.gov/medlineplus/druginfo/uspdi/202284.html and http://www. drugstore.com/qxal 026 33318 l_sespiderwhat_is_an_hmg_coa reductase inhibitor.htm). 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). However, whilst the 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). 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. Curr Opin Lipidol. 2002 Aug;13(4):415-20). Rhabdomyolysis is a dose-related, and rarely fatal side effect (although there have been reported cases of irreversible rhabdomyolysis) generally seen for statin drugs, and it was the occurrence of this side effect that led to the withdrawal of Cerivastatin from the market. However, the effect is not restricted to cerivastatin and appears to be related to the systemic levels of exposure to statins. As a result of these adverse effects, all the current statin drugs are targeted for clearance by the liver which (i) targets the drug to the main site of action to lower plasma cholesterol and (ii) reduces systemic exposure.

Recent research into 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).

There remains a need for safe and effective methods for the administration of 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. The known therapeutic indications for statins control only the plasma level of cholesterol, and statins are not currently employed to manipulate intracellular levels of cholesterol. Introduction to Inflammatory and Allergic Disorders

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 joint disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomerulonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementia, toothache, sprains, strains, myositis, burns, injuries, and pain and inflammation that follows surgical and dental procedures in a patient.

Common 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.

Other treatments for allergic conditions are symptomatic such as the use of bronchodilators for asthma, such as β 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 (FcεRI) 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.

Release of these mediators from mast cells in response to IgE-coated allergens in atopic individuals results in clinical features of allergies, such as allergic asthma, allergic conjunctivitis, allergic rhinitis, atopic dermatitis, eczema and acute and chronic urticaria.

In addition, 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. 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.

FcεRI is a multi-subunit receptor comprising an α chain ligand-binding domain, which binds to IgE, a γ chain homo-dimer, required to mediate signal transduction, and a β chain, which plays a role in amplification of the signal (Turner and Kinet, Nature 1999, 402, B24- 30). The intracellular signalling pathways activated by FcεRJ involve the sequential activation of tyrosine kinases including Lyn and Syk, and phospholipase C leading to the generation of inositol 1,4,5, trisphosphate (IP₃). The generation of IP₃ 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 FcεRI receptor is implicated in those diseases and conditions in which mast cells are implicated.

For example, the central role of FcεRI in mediating atopy is evidenced by the clear genetic association studies that have consistently mapped to the β chain (Clin. Exp. Allergy, 1999, 29, 1555-62). Allergen coated with IgE results in the aggregation of FcεRI on the surface of mast cells (Turner and Kinet, Nature 1999, 402, B24-30). This aggregation, as described above, results in the activation of intracellular signalling cascades which ultimately lead to degranulation of the mast cell and the release of inflammatory mediators. It is the release of inflammatory mediators that precipitates atopic responses to allergens in sensitised individuals, such as those listed above.

There remains a need for new safe and effective methods and drugs for the modulation of the activity of cells. For example, there remains a need for new safe and effective methods and drugs for the modulation of the immune response. In particular, there remains a great need for new safe and effective methods and drugs for the treatment and prevention of inflammatory disorders. More particularly, there remains a great need for new safe and effective methods and drugs for the treatment of allergic conditions in mammals. In particular, there is a need for a treatment that inhibits allergen-stimulated mast cell degranulation, which is well-recognised to be the precipitating event underlying atopic conditions.

Introduction to Lipid Rafts

In recent years, it has become clear that the plasma membrane of cells is not uniform but that there are discrete microdomains in the membrane which are rich in sphingolipids and cholesterol. These specialised micro-domains contain ordered cholesterol and have become known as 'lipid rafts' (Field et al., J. Biol. Chem., 1997, 272, 4276-4280). In a number of cells, it has become clear that certain membrane associated proteins preferentially partition into these lipid rafts (Foster, de Hoog and Mann, PNAS, 2003, 100, 5813-8). These include various seven transmembrane domain receptors and their associated G proteins and various proteins that are attached to the inner membrane leaflet through lipid moieties such as prenylation, including small molecular weight G proteins, such as Ras, Rac, cdc42 and Rho. The 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, FcεRI. Investigators in this area have used various agents to disrupt the lipid rafts, and have shown that the efficient coupling of signal transduction from ligand engagement of the receptor to intracellular signalling cascades is lost. To disrupt the rafts in vitro, various agents have been used such as filipin and nystatin (see Foster, de Hoog and Mann, PNAS, 2003, 100, 5813-8).

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), emphysema and chronic bronchitis, atherosclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, degenerative joint disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomerulonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementias, toothache, sprains, strains, myositis, burns, injuries, pain and inflammation that follows surgical and dental procedures in a patient, Alzhemiers disease, Parkinsons disease, muscular dystrophy, polyneuropathies (demyelinating diseases), neoplasia, hypertension, diabetes, hyperparathyroidism, sepsis and septic shock.

In addition, 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.

Thus, there is a need for new methods and compositions that are useful in the modulation of lipid raft structure. Such methods are likely to be applicable to a broad range of cell types, and will therefore be of use in the treatment or prophylaxis of a wide range of diseases and conditions, including those listed above.

THE INVENTION

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.

These discoveries all resulted from the use of the Applicant's proprietary informatics platform, PharmaCarta™, which is described briefly below.

All known drug active ingredients act via only a few hundred distinct molecular targets, and these targets fall within a smaller number of different structural families. Thus, selection of a potentially draggable target is of crucial importance in the process of drug discovery. In addition, even once a draggable target has been identified, the next challenge is to identify the best lead compounds from an almost infinite range of chemistry possibilities. Once lead compounds have been selected, it is then necessary to optimise those leads by balancing adsorption, distribution, metabolism and excretion (ADME) properties and potency properties to create promising development candidates.

The PharmaCarta™ 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. In order to achieve this, the PharmaCarta™ 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 sϊlico ADME optimisation.

PharmaCarta™ 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, PharmaCarta™ provides a general and systematic approach to target prioritisation and subsequent lead discovery. PharmaCarta™ 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.

As a result, the PharmaCarta ^M platform is a powerful bioinformatics tool for drag discovery purposes, and allows rapid and effective in sϊlico target selection, lead identification and lead optimisation by simultaneously searching, comparing and analysing the available data sets. For example, the use of the PharmaCarta™ platform for drag discovery may follow the following steps:

A. Identification of proteins associated with a phenotype of interest (e.g. a disease). For example, 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;

B. Analysis of the draggability of the proteins identified in step A by PharmaCarta™. The draggability analysis is based on known drag interactions and also employs proprietary structure-based draggability rules. Where one or more of the proteins identified in step A is not already annotated, automated annotation of sequences can be carried out at this stage by PharmaCarta™. As a result of this analysis, the PharmaCarta™ platform prioritises the initial targets for further analysis; C. A number of the prioritised targets are selected, and the sequence data for those targets used to build homology models, based upon known structures for proteins of the same structural family. Pharmacophores are then identified using the homology models for modelling, and the pharmacophores identified then used to search a proprietary database of chemical structures. The construction of the homology model and the search of chemical structures are both carried out by the PharmaCarta™ platform, providing a number of lead compounds or other potential actives;

D. The lead compounds or other potential actives are subjected to in sϊlico predictive ADME analysis (carried out by the PharmaCarta¹ platform) in order to optimise the lead compounds or other potential actives. In addition, the lead compounds or other potential actives may be tested in simple in vitro assays.

Of course, there are many other ways in which the PharmaCarta™ platform may be used to provide valuable data suitable for in vitro verification. Further information concerning the PharmaCarta™ platform is provided at http:Wwww.inpharmatica.com. Example 1 describes in detail the role of the PharmaCarta™ platform in the discoveries underlying the present invention.

Briefly, the PharmaCarta™ 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 PharmaCarta™ platform. The PharmaCarta™ 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 PharmaCarta™ 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.

Briefly, Examples 2-7 disclose that inhibition of HMG CoA reductase in mast cells results in decoupling of the IgE receptor (FcεRI) 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. Notably, methyl β 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. Thus, agents that lower intracellular cholesterol via diverse mechanisms will disrupt lipid rafts. For example, 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. PPARα agonists or LXR agonists).

Accordingly, in a first aspect of the invention, there is provided 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. Thus, 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. As noted above, 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 disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomeralonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementias, toothache, sprains, strains, myositis, burns, injuries, and pain and inflammation that follows surgical and dental procedures in a patient, Alzhemiers disease, Parkinsons disease, muscular dystrophy, polyneuropathies (demyelinating diseases), neoplasia, hypertension, diabetes, hyperparathyroidism, sepsis and septic shock, 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.

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.

As used herein, 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.

As used herein, the term "agent" refers to any suitable molecule, compound, nucleic acid, polypeptide or other moiety. Preferred agents are described in detail herein.

As used 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. There are a number of different mechanisms by which the reduction in the level of cholesterol may be achieved. For example, 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.

In preferred embodiments of the first aspect of the invention, there is provided a method wherein the activity of a membrane signalling pathway is modulated and wherein the membrane signalling pathway comprises at least one signalling component associated with a lipid raft of the cell membrane.

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.

As used herein, the term "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.

As used herein, 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. As used herein, 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. For example, such 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. Alternatively, such 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.

For example, in these preferred embodiments the signalling component associated with a lipid raft of the cell membrane may be the FcE RI receptor complex or the Icrac calcium channel. Thus, 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.

As described above, 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.

Accordingly, in preferred embodiments of the first aspect of the invention, the reduction in the level of cholesterol within the cell leads to a reduction in the level of cholesterol in the cell membrane. In more preferred embodiments, 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. The 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. probes.com/media pis/mp34775.pdf or Harder et al. J. Cell Sci. 1998, 141, 929-942). Other alternative methods include freeze fracture of the plasma membrane followed by electron microscopy (see, for example, Prior et al. Sci STKE. 2003 Apr 8;2003(177):PL9 or Field et al J. Biol. Chem. 1999, 274, 1753).

As noted above, the methods of the first and second aspects of the invention are applicable to a broad range of cell types. For example, 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. As a result, as mentioned above, 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.

Preferably, 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.

Thus, 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. In a preferred embodiment, the cell of hematopoetic lineage is a mast cell. As described above, 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. In the present application, it is disclosed that treatment of mast cells with agents that reduce the level of intracellular cholesterol, particularly statins, results in the inhibition of the FcεRI-mediated signalling pathway and subsequent degranulation of the mast cell. Therefore, it is disclosed herein that agents that reduce the level of intracellular cholesterol, particularly statins, can be used to modulate the activity of mast cells. In particular, it is disclosed that such agents can be used to suppress the function of mast cells. Accordingly, in these aspects of the invention, 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.

As noted above, the methods and uses of the present invention have utility as novel, safe and tolerable treatments for a number of serious conditions.

According to a second aspect of the invention, there is provided 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. In such methods, the agent should be administered in an amount sufficient to reduce the level of cholesterol within the cells.

According to 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.

In preferred embodiments of the second and third aspects of the invention, the membrane signalling pathway comprises at least one signalling component associated with the lipid rafts of the cell membrane. There are a wide variety of diseases in which 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 disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomerulonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementias, toothache, sprains, strains, myositis, burns, injuries, and pain and inflammation that follows surgical and dental procedures in a patient, Alzhemiers disease, Parkinsons disease, muscular dystrophy, polyneuropathies (demyelinating diseases), neoplasia, hypertension, diabetes, hyperparathyroidism, sepsis and septic shock, intracellular pathogens including bacteria such as Salmonella, Chlamydiae, listeria, Mycobacteria tuberculosis, viruses such as HIV, Measles virus, Papilloma virases, Epstein-Barr virus, Respiratory Syncytial Virus (RSV), Hepatitis, Herpes virases, Influenza virus, Ebola and Marburg virases, 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.

Diseases in which the inappropriate activity of a membrane signalling pathway present in mast cells is implicated include, 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.

Diseases in which the inappropriate activity of a membrane signalling pathway present in mast cells is especially implicated include those leading to allergic asthma, allergic conjunctivitis, allergic rhinitis and atopic dermatitis.

In these methods and uses of the second and third aspects of the invention, the disease or condition is preferably an inflammatory disorder, such as an inflammatory disorder selected from those described herein. As described above, 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.

A number of suitable agents that reduce the level of cholesterol within cells are envisaged for use in the present invention. 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 (such as Acipimox, Aluminium nicotinate, Nicertirol, Nicoclonate, Nicomol, and Oxiniacic acid); Thyroid Hormones/ Analogues (such as Dextrothyroxine, Eitroxate, and Thyropropic acid) and a wide variety of other agents (such as Acifran, Benfluorex, β-Benzylbutyraimde, Carnitine, Chondrotin sulphate, Colmestrone, Detaxtran, Dextran sulphate Sodium, Eicosopentanoic acid, Eritadenine, Ezetimibe, Furazabol, Meglutol, Melinamide, γ- Oryzanol, Pantethine, Pentaerythritol tetraacetate, α-Phenylbutyramide, Pirozadil, Probcuol, β-Sitosterol, Sultosilic acid, Tiadenol, Triparanol, and Xenbucin). 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. For example, the agents may be PPARγ antagonists, PPARα agonists or LXR agonists.

In preferred embodiments, the agent that reduces the level of cholesterol is an inhibitor of de novo cholesterol biosynthesis.

Accordingly, the present invention provides the use of an inhibitor of de novo cholesterol biosynthesis in the methods and uses described above.

In more preferred embodiments, 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.

Accordingly, the present invention provides the use of HMG CoA reductase inhibitors in the methods and uses described above.

As previously noted, 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.

In more preferred embodiments, the HMG CoA reductase inhibitor is a statin. Many of the statins have acceptable phaπnacodynamic 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.

Accordingly, 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.

Accordingly, it is preferred in the methods and uses of the invention that 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.

Furthermore, in preferred embodiments of the invention, specifically modified 'soft' forms of current cholesterol-lowering agents are employed, such as 'soft' statins.

As used herein, 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.

In order to be effective in this way, 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.

For example, 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. For example, 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.

Accordingly, 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. In particular, the present invention uses specifically selected, designed and engineered HMG CoA reductase inhibitor compounds with intentionally rapid metabolic clearance and inactivation characteristics.

There are a large number of known HMG CoA reductase inhibitors, many of which have acceptable pharmacodynamic and pharmacokinetic properties for safe oral dosing in humans. Due to the known and safe nature of certain of these agents as anti- hypercholestemia agents, preferred compounds for use in the methods of the present invention include statins, examples of which include Atorvastatin, Cerivastatin, Mevastatin, Pravastatin, Fluvastatin, Simvastatin, Lovastatin, Rosuvastatin, and Pitavastatin. The molecular structures of these compounds are shown in Figure 5. Extensive metabolic clearance in fact occurs for most statins (Igel et al, Eur. J. Clin. Pharmacol., 57, 357-364, 2001), in some cases limiting their oral bioavailability and resulting in a short plasma half-life (Smith et al, Am. J. Hypertens., 6, 375S-382S). Indeed, a number of the compounds specifically listed above already benefit from intrinsic metabolic clearance and inactivation, but are optimised for the treatment of hypercholestemia, and therefore further optimisation is preferred prior to use in the methods of the present invention. Thus, when current cholesterol-lowering agents, such as statins, are employed in the methods of the present invention, it is preferred that they are selected or optimised to ensure that they benefit from rapid clearance from, or rapid inactivation in, the systemic circulation.

For example, 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. Thus, 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). The advantage of the latter is their potential to metabolise compounds immediately upon entry into the systemic circulation, further reducing the risk of exposure to organs remote from the site of application, including the liver. As a result of these characteristics, such agents will be especially suitable for the modulation of the activity of specific regions of target cells of an organism. 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. Specifically, 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. In addition, these compounds comprise a lipophilic group reducing partitioning into the plasma and subsequent distribution to other compartments. Thus, these compounds represent specifically optimised statin prodrags for use in the methods of the present invention.

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. It is envisaged by the Applicant that 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.

Accordingly, 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. In these specific compounds, the linkage between the two constituents is therefore a labile ester group between the acid moiety of the statin active ingredient and the hydroxyl moiety of the alcohol.

Representative compounds of this embodiment of the invention are shown in Figure 7. In preferred embodiments, 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. Specific examples of 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-yι]-hept-6-enoic acid, (E)-(3R,5S)-7-(2-Cyclopropyl-4-phenyl-quinolin-3-yι)- 3,5-dihydroxy-hept-6-enoic acid and (E)-(3R,5S)-7-(2,6-Diisopropyl-5-methoxymethyl-4- phenyl-pyridin-3-yl)-3,5-dihydroxy-hept-6-enoic acid. Each of these compounds is provided in both their free acid and pharmaceutically acceptable salt forms.

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-3-yl]-3,5-dihydroxy-hept-6- enoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[4-(4-hydroxy-phenyl)-2,6-diisopropyl-5- methoxymethyl-pyridin-3-yl]-hept-6-enoic acid. Each of these compounds is provided in both their free acid and pharmaceutically acceptable salt forms.

The molecular structures of particularly preferred soft agents of this type are shown in Figure 9.

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.

Compounds provided according to this embodiment of the invention are of the general formula X-Y, where 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)-(3R,5S)-7-[2-Cyclopropyl-4-(4-hydroxy-phenyl)- quinolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid and (E)-(3R,5S)-3,5-Dihydroxy-7-[4-(4- hydroxy-phenyl)-2,6-diisopropyl-5-methoxymethyl-pyridin-3-yl]-hept-6-enoic acid, and Y is selected from the group consisting of formic acid, acetic acid, propan-1-oic acid, propan- 2-oic acid, butan-1-oic acid, butan-2-oic acid, pentan-1-oic acid, hexan-1 -oic acid, heptan- 1-oic acid, octan-1-oic acid, nonan-1-oic acid, decan-1-oic acid, benzoic acid, cinnamic acid and 1-hydroxy-benzoic acid. In these specific compounds, the linkage between the two constituents is therefore a labile ester group between the alcohol moiety of the statin active ingredient and the carboxylate moiety of the organic acid.

Accordingly, in these embodiments of the present invention, 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.

In addition, 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.

As noted above, in vivo and in vitro conversion between the free acid and the lactone forms of the statin drags occurs readily. Accordingly, in the methods of the present invention both the free acid or the lactone form of the statin compounds described above may be employed. Accordingly, references to statins herein refer to both the free acid and the lactone forms of the statins.

Methods of treatment and prophylaxis as described above, in which the agent is administered only to a specific region of target cells are also included in the present invention. Thus, 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.

Accordingly, using the present invention 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. Such metliods of treatment and prophylaxis will also benefit from the advantage of "steroid sparing" for children, when used for asthma or dermatitis.

In the methods of the invention, the agent may be applied specifically to the topologically exterior surface of a mammal. For example, 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.

Application of the agent to the skin, or a region thereof, is preferred for the treatment or prophylaxis of dermatitis, eczema, wound healing or psoriasis.

Application of the 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).

In a fourth aspect of the invention, there is provided 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. 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.

In preferred embodiments according to this fourth aspect of the invention, the agent that reduces the level of cholesterol is a statin, or derivative thereof, as described above.

The 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. Thus, 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. 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.

Pharmaceutically acceptable salts can be used therein. The term 'pharmaceutically acceptable salt', as used herein, refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids and bases.

Examples of 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. Examples of 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.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ. 1991).

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.

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. For aqueous suspensions and/or elixirs, 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 (FDDFs) 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. The terms 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. For such 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.

The precise 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.

For oral and parenteral administration to human patients, the daily dosage level of the compound will usually be from 10 to 500 mg (in single or divided doses).

Thus, for example, 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 skilled person will also appreciate that, in the treatment of certain conditions (including allergic asthma, allergic conjunctivitis, allergic rhinitis and atopic dermatitis), 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 134A™ or 1,1,1,2,3,3,3- heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, 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. 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.

Alternatively, 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.

For ophthalmic use, 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. Alternatively, the compound may be formulated in an ointment such as petrolatum.

For application topically to the skin, 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. Alternatively, 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. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drag absorption after oral administration, the drug may be administered parenterally, sublingually or buccally.

For the present invention, particularly preferred formulations are topical and inhaled. Oral dosing is also a preferred formulation.

For veterinary use, 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.

Importantly, 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.

According to a fifth aspect of the invention, there is provided 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.

Persons skilled in the art will be able to devise screening assays suitable for identifying agents that reduce the level of intracellular cholesterol.

According to a further embodiment of this fifth aspect of the invention, there is provided 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.

According to a further embodiment of this fifth aspect of the invention, there is provided 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. In these embodiments, the methods may include one or more steps in which calcium mobilisation, kinase phosphorylation, TNFα release, degranulation, proliferation and adhesion are assayed, as described in Examples 2-7.

Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the use of statins to reduce the level of cholesterol in mast cells.

It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Brief description of the Figures

Figure 1: Measurement of calcium transients in RBL-2H3 cells. (A) Control compared to cells treated for 2 days with fluvastatin, lovastatin or pravastatin (lOμM). (B) Peak calcium following FcεRI aggregation in control cells compared to cells treated with either 3μM or 10 μiM fluvastatin or lovastatin for two days. Additional cells were treated with methyl β 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 FcεRI aggregation in control cells compared to cells treated for two days with either 10 μM fluvastatin or lovastatin supplemented or not with lOOmM mevalonate.

Figure 2: Measurement of β 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 μM) for two days and from cells treated with fluvastatin, lovastatin or pravastatin (3 μM) supplemented with mevalonate (lOOmM) and from cells treated with methyl β 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 (3μM).

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 (lOμM), lovastatin (lOμM) or pravastatin (lOμM).

Figure 5: Intracellular calcium concentrations in RBL-2H3 cells in zero calcium treated with thapsigargin (TG) and addition back of extracellular calcium as indicated, a = resting basal levels, b = peak calcium following release from stores, c = peak calcium following addition of extracellular calcium to the cuvette. A = control cells, B = cells treated with methyl β cyclodextrin, C = lovastatin treated cells, D = fluvastatin treated cells.

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.

Examples

As described above, 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.

These discoveries all resulted from the use of the Applicant's proprietary informatics platform, PharmaCarta™, which has been described above. Example 1 below describes the use of the PharmaCarta™ platform to identify draggable targets and lead compounds. The subsequent Examples (Examples 2-10) describe the validation of the findings made using the PharmaCarta™ platform in mast cells and T cells, and explain why the results generated by the PharmaCarta™ 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 PharmaCarta™ to identify draggable targets and lead compounds

To harness the PharmaCarta™ pipeline, a high-quality public-domain gene expression dataset derived from an analysis of T-cell activation using a DNA microarray was analysed (Riley et al, 2002, Proc. Nafl. Acad. Sci. USA, 99, pp. 11,790-5). Of the initial 25,000 oligonucleotide probes in the dataset, 2,300 were found to be significantly regulated during T-cell activation. These were then assessed by the PharmaCarta™ platform for draggability. Of 149 drug target candidates identified by the PharmaCarta™ platform (covering 40 protein families), 21 were selected for more detailed investigation due to the nature and temporal pattern of their expression profiles. This subset was further prioritised by the PharmaCarta™ platform according to selectivity and compound ADMET criteria. Of the 2, 3000 genes in the Riley dataset whose level of transcription was observed to be increased after T cell activation HMG CoA reductase was specifically selected as a candidate for further analysis.

As described above, this enzyme is the target for HMG CoA reductase inhibitor drugs termed 'statins' on the market. Thus, the PharmaCarta™ platform was able to identify known drag compounds that might be useful in novel therapeutic settings.

Notably, this observation made using the PharmaCarta™ platform is supported by previous experiments, which had shown that treatment of T cells with statins in vitro results in inhibition of TCR stimulated proliferation and release of pro-inflammatory cytokines. This action of HMG CoA reductase inhibitors has been proposed to result from a failure to prenylate various key proteins required to trigger the intracellular signalling pathways - including the Src-like kinases and the small molecular weight GTPases, both of which are localised to the inner surface of the plasma membrane by their prenylation. In the immune system, the signalling cascades initiated by the ligand binding receptor are very similar. Thus, in T cells, 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 IP₃ which binds to the IP₃ 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 FcεRI and for the activation of B cells following engagement for the B cell receptor (BCR).

The inventors have noted the similarity in this pattern and have discovered that 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).

Example 2: Effect of statins on FcεRI mediated calcium transients in mast cells

The effect of lovastatin and fluvastatin on intracellular calcium transients was determined. Briefly, 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 μg/ml dinitrophenol specific rat IgE for 12 hours prior to all experiments.

Cells were treated with lovastatin (10 or 3 μM), fluvastatin (10 or 3 μM) or pravastatin (10 or 3 μM) 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.

For measurement of cytosolic calcium levels, cells were harvested and suspended in HEPES buffered saline (HBS) supplemented with 2 mM calcium chloride, 1.8 mg/ml glucose and 2 mg/ml bovine serum albumin. Cells were loaded with Fura-2 AM (Molecular Probes) in pluronate at room temperature in the dark. After removal of excess Fura-2 by dilution and centrifugation, 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 μg/ml) (Sigma, Aldrich). As shown in Figure la, in untreated cells, aggregation of FcεRI resulted in a transient increase in cytosolic calcium followed by a more sustained elevation in intracellular calcium. In cells treated with either lovastatin or fluvastatin (10 μM), both the initial peak calcium was reduced and the sustained elevated intracellular calcium levels were reduced or abolished. Peak calcium was reduced to 39.7 ± 17.5 % in cells treated with lOμM fluvastatin compared to control untreated cells. In cells treated with pravastatin (a drug that does not cross the plasma membrane), the calcium transients were very similar to the untreated control cells.

The effect of fluvastatin and lovastatin was dose dependent. In additional experiments, the effect of 3 μM lovastatin and fluvastatin on peak calcium was compared to the effect of lOμM lovastatin and fluvastatin. Figure lb demonstrates that for both drugs, the lower concentration significantly reduced peak calcium after FcεRI aggregation but that the reduction was smaller than that observed for 1 OμM treatment.

In some cells, statin treatment (lOμM lovastatin and fluvastatin) was supplemented with addition of lOOmM mevalonate. In these cells, calcium transients were restored to normal. Peak calcium after FcεRI aggregation is shown in Figure lc for cells treated with either lovastatin or fluvastatin (lOμM) and compared to cells treated with the same dose of lovastatin or fluvastatin but the medium was supplemented with 100 mM mevalonate.

Therefore, 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. In addition, it has been shown that 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. This indicates that 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. These data indicate that active inliibition of HMG CoA reductase results in the inhibition of IgE-receptor coupling to cytosolic calcium transients in mast cells. This effect on mast cells is dependent on the inhibition of the enzyme, since provision of mevalonate to the cells restores the response.

The effect of statin treatment on peak calcium was compared to the depletion of plasma membrane cholesterol directly using methyl β cyclodextrin. Cells were treated with methyl β 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 (lOμM) treatment of the cells (Figure lb). These data indicate that the effects caused by modulation of the level of cholesterol in the cell membrane are not statin-specific.

Example 3: Effect of statins on FcεRI mediated signalling pathways in mast cells

RBL-2H3 cells were treated with lovastatin (3 or 1 μM), fluvastatin (3 or 1 μM) or pravastatin (3 μiM) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOOμM) in combination with either lovastatin or fluvastatin. The cells were primed with 1 μg/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 μg/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. 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.

Aggregation of cell surface bound IgE resulted in the rapid appearance of phosphorylated tyrosine proteins in the cell lysates. The predominant band appeared at a molecular weight around 70 kDa and this band was shown to be Syk kinase. Receptor coupled activation of tyrosine phosphorylation was abolished in cells pretreated with either fluvastatin or lovastatin (3 μM). At lower concentrations (lμM), the intensity of the phosphorylated bands was diminished. Receptor coupled tyrosine phosphorylation was identical to the control for cells treated with pravastatin (3 μM) or co-treated with mevalonate (100 μM) together with either lovastatin (3 μM) or fluvastatin (3 μM). 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. This demonstrates that the effect of the statins on the receptor coupled events occurs as a result of their activity against the HMG CoA reductase.

In additional experiments, cell surface plasma membrane cholesterol was depleted using the drag, methyl β 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 β cyclodextrin (10 mM). Receptor coupled tyrosine phosphorylation was inhibited in cells pretreated with methyl β cyclodextrin.

Similar experiments were completed using specific antibodies to dual phosphorylated ERK (data not shown).

Therefore, this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits FcεRI 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. In addition, methyl β cyclodextrin inhibited receptor coupled tyrosine phosphorylation. Methyl β cyclodextrin acts to deplete the plasma membrane of cholesterol and thereby disrupt lipid rafts directly, and thus these data show that the effects on FcεRI signalling are not statin-specific.

Example 4: Effect of statins on FcεRI coupled degranulation of mast cells

RBL-2H3 cells were treated with lovastatin (3 or 1 μM), fluvastatin (3 or 1 μM) or pravastatin (3 μiM) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOOμM) in combination with either lovastatin or fluvastatin. The cells were primed with 1 μg/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 μg/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, β- hexosaminidase activity was measured in the supernatant using a standard colorimetric assay. Briefly, 50μl supernatant was incubated with 200 μl of lmM jp-nitrophenyl N- acetyl-β-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. To ensure the hexosaminidase release was a measure of receptor mediated degranulation, an equivalent set of cells were incubated with the relevant drags but the receptor was not aggregated to measure spontaneous release from these cells and act as a no cross-link control.

As shown in Figure 2, aggregation of cell surface IgE resulted in the release of β hexosaminidase. This release was unaffected by incubation of the cells with pravastatin (3 μM). Treatment of the cells with either fluvastatin or lovastatin inhibited IgE stimulated release of β hexosaminidase. At a concentration of 3 μM, receptor stimulated release was abolished whereas at 1 μM release was inhibited by about 80%. Hexosaminidase release was unaffected in cells treated with mevalonate (100 μM) in combination with either lovastatin (3 μM) or fluvastatin (3 μM). This indicates that the effect of the statin on the receptor triggered response is mediated through the inhibition of HMG CoA reductase.

In additional experiments, cell surface plasma membrane cholesterol was depleted using the drug, methyl β cyclodextrin (Fisher, de Hoog and Mann, PΝAS, 2003, 100, 5813-8). Following priming of the cells with IgE, cells were incubated for 1 hour with methyl βcyclodextrin (10 mM). The receptor was aggregated by the addition of DΝP-BSA and hexosaminidase release measured. As shown in Figure 2, receptor coupled hexosaminidase release was abolished in cells treated with methyl β cyclodextrin. Thus, disruption of the lipid rafts directly by depletion of plasma membrane cholesterol using methyl β cyclodextrin results in inhibition of FcεRI triggered degranulation.

Therefore, this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits FcεRI mediated degranulation of mast cells as measured by release of β hexosaminidase. In addition, methyl β cyclodextrin inhibited release of β 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 FcεRI mediated release of the proinflammatorv cytokine, TNFα

RBL-2H3 cells were treated with lovastatin (3 or 1 μM), fluvastatin (3 or 1 μM) or pravastatin (3 μM) for 1 or 2 days. Some cells were, in addition, treated with mevalonate (lOOμM) in combination with either lovastatin or fluvastatin (3 μM). The cells were primed with 1 μg/ml dinitrophenol specific IgE for 12 hours prior to all experiments. Following addition of 1 μg/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 TNFα were measured in the cell supernatants using a standard ELIS A assay (manufacturer) .

Receptor coupled release of TNFα 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 μM) indicating that the action of the statin is a feature of the inhibition of HMG CoA reductase. Treatment of IgE sensitised cells with methyl β cyclodextrin for one hour prior to receptor aggregation, inhibited the release of TNFα into the supernatant indicating that cholesterol depletion of the plasma membrane and disruption of the lipid rafts of the plasma membrane inhibits the receptor coupled release of TNFα.

Therefore, this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits FcεRI mediated release of the proinflammatory cytokine, TNFα.

Example 6: Effect of statins on mast cell proliferation

Triplicate wells of RBL-2H3 cells were treated with lovastatin (3, 1, 0.3 μM), fluvastatin (3, 1, 0.3 μM) or pravastatin (3, 1, 0.3 μM) for 5 days. Cells were harvested each day for 5 days and the cell number quantified using the CY-Quant assay (Molecular Probes).

As shown in Figure 3, the rate of proliferation of the RBL-2H3 cells was inhibited by treatment of cells with 3μM fluvastatin. Proliferation was slowed in cells treated with 1 μM fluvastatin (data not shown). Lovastatin had similar effects at 3μM (data not shown). Pravastatin had no effect on the rate of proliferation of the cells over the 5 day period. Therefore, this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits the proliferation of the RBL-2H3 mast cell line.

Example 7: Effect of statins on Jurkat cell proliferation

Jurkat cells were treated with lovastatin (10 μM), fluvastatin (10 μM) or pravastatin (10 μM) for 5 days. Cells were harvested each day for 5 days and the cell number quantified using the CY-Quant assay (Molecular Probes).

As shown in Figure 4, 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.

Therefore, this example demonstrates that inhibition of HMG CoA reductase with either lovastatin and fluvastatin inhibits the proliferation of the Jurkat cell line.

Example 8: Effect of statins on mast cell adhesion

RBL-2H3 cells grow as adherent cells in the 40:10 mix of MEM:RPMI1640. Treatment of cells with lovastatin or fluvastatin (3 μM) 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 μM) at the same time as the lovastatin or fluvastatin (3 μM) prevented the statin induced change in morphology. Pravastatin (3 μM) had no effect on the appearance of the cells in culture.

These data indicate that either lovastatin or fluvastatin inhibits the adhesion of RBL-2H3 cells. Therefore, this example demonstrates that active inliibition of HMG CoA reductase results in reduced adhesion of the mast cells to the tissue culture dish.

Example 9: Measurement of cellular cholesterol

The effect of treatment of cells with fluvastatin on total cellular cholesterol was measured. Cells were treated for two days with fluvastatin (lOμM). Control cells were treated in the same way. Additional cells were treated with methyl β cyclodextrin (lOmM) for one hour. Cells were harvested and after extensive washing in phosphate buffered saline, the cells were extracted with chloroform:methanol. The organic phase was collected and dried down in glass vials. The lipids in the glass vial were dissolved in ethanol and the cholesterol measured using the Amplex Red Cholesterol Assay kit (Molecular Probes). Results demonstrate that total cellular cholesterol was reduced by ~15% in the cell treated with fluvastatin compared to the control cells.

Example 10: Effect of statins on store operated calcium influx in RBL-2H3 cells

The effect of lovastatin and fluvastatin on store operated calcium entry was determined and compared to the effect of disrupting lipid rafts using methyl β 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.

Cells were treated with lovastatin (10 μM), fluvastatin (10 μM) 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 β cyclodextrin were incubated with a 20mM solution of methyl β 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.

For measurement of cytosolic calcium levels, cells were harvested and suspended in HEPES buffered saline (HBS) supplemented with 2 mM calcium chloride, 1.8 mg/ml glucose and 2 mg/ml bovine serum albumin. Cells were loaded with Fura-2 AM (Molecular Probes) in pluronate at room temperature in the dark. After removal of excess Fura-2 by dilution and centrifugation, 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 β 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. In keeping with the previous dose response observations, the effect was more marked in the cells treated with fluvastatin compared to lovastatin. The effect of the statins was similar to that observed for depletion of cholesterol at the plasma membrane with methyl β cyclodextrin.

This data demonstrates that the intracellular calcium stores are normal following both statin treatment and treatment with methyl β cyclodextrin but that store operate calcium entry is impaired following either treatment. Store operated calcium entry is mediated through the activation of ion channel, Icrac, in these cells and this data indicates that treatment of cells with statins or methyl β cyclodextrin impairs the normal operation of this channel. As channels are normally localised in lipid rafts and both methyl β cyclodextrin and statins are known to reduce the level of cholesterol within cells, the findings for the statin treated cells and the methyl β cyclodextrin treated cells supports the concept that the cholesterol-lowering agents are acting through the disraption of lipid rafts in the plasma membrane.

Summary of Examples 1-10

In these Examples, it has been described that inhibition of HMG CoA reductase in mast cells results in decoupling of the IgE receptor (FcεRI) from the intracellular signalling cascades. Inhibitors of HMG CoA reductase are shown to inhibit the normal functioning of FcεRI, 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 β hexosaminidase and the pro-inflammatory cytokine TNFα. Furthermore, it is shown that inhibition of HMG CoA reductase inhibits the proliferation of mast cells and the adhesion of mast cells to tissue culture dishes. These Examples demonstrate that the uncoupling of the receptor from the signalling cascades and degranulation by statins relates directly to the inhibition of intracellular HMG CoA reductase, since co-treatment of cells with statins and mevalonate rescues the response (in terms of signalling pathways, degranulation, morphological appearance and proliferation).

Importantly, methyl β cyclodextrin has been shown to also inhibit the activities and functions of mast cells investigated. Thus, the effects on the lipid raft micro-domains are not specific to HMG CoA reductase inhibitors.

In addition, the effect of reducing the level of cholesterol within the Jurkat T-cell line using statins was also shown to lead to inhibition of the proliferation of those cells. Thus, the effects on the lipid raft micro-domains are not specific to mast cells.

Furthermore, it has been shown that agents that reduce the level of cholesterol within the lipid bilayer, and thus disrupt lipid rafts, impair the normal operation of the Icrac calcium channel. Thus, the effects on the signalling components associated with the lipid raft micro-domains are not limited to the FcE RI receptor complex.

Thus, 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. PPARα agonists or LXR agonists). Such agents will be useful for modulating the activity of a range of cell types and of a range of membrane signalling networks.

As noted above, disruption of lipid rafts will be beneficial in the treatment or prophylaxis of a number of diseases or conditions, including those described above.

Notably, the findings made using the PharmaCarta™ platform in Example 1 were successfully confirmed in the other Examples above. Accordingly, these Examples confirm the utility of the PharmaCarta™ platform for the accurate identification of draggable targets and associated lead compounds, which can then be simply and rapidly validated at the laboratory bench.

Claims

CLAIMS:

1. A method of modulating the activity of a cell, comprising exposing the cell to an agent that reduces the level of cholesterol within said cell.

2. A method according to claim 1 , wherein the activity of a membrane signalling pathway is modulated, and wherein the membrane signalling pathway comprises at least one signalling component associated with a lipid raft of the cell membrane.

3. A method according to claim 1 or claim 2, wherein the reduction in the level of cholesterol within the cell leads to a reduction in the level of cholesterol in the cell membrane.

4. A method according to any one of claims 1-3, wherein 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.

5. A method according to any one of the preceding claims, wherein said cell is a cell of hematopoetic lineage.

6. The method of claim 5, wherein the cell of the hematopoetic lineage is a mast cell.

7. The method of claim 6, wherein the activity of a mast cell is modulated.

8. The method according to any one of the preceding claims, wherein the activity of the FcεRI-mediated IgE antigen-signalling pathway is modulated.

9. The method of any one of the preceding claims, wherein the activity that is modulated is involved in the immune response.

10. A method for the prophylaxis or treatment of a disease or condition in which the inappropriate activity of a membrane signalling pathway is implicated, comprising exposing cells that possess said membrane signalling pathway to an agent that reduces the level of cholesterol within said cells in an amount sufficient to reduce the level of cholesterol within the cells.

11. 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 the cell.

12. The method according to claim 10 or the use according to claim 11, wherein said disease or condition is selected from the group consisting of 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 (LBS) 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 disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomerulonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementias, toothache, sprains, strains, myositis, burns, injuries, pain and inflammation that follows surgical and dental procedures in a patient, Alzhemiers disease, Parkinsons disease, muscular dystrophy, polyneuropathies (demyelinating diseases), neoplasia, hypertension, diabetes, hyperparathyroidism, sepsis and septic shock, and infections by intracellular pathogens, including bacteria such as Salmonella, Chlamydiae, listeria, Mycobacteria tuberculosis, viruses such as HIV, Measles virus, Papilloma virases, Epstein-Barr virus, Respiratory Syncytial Virus (RSV), Hepatitis, Herpes virases, Influenza virus, Ebola and Marburg viruses, parasites such as, Plasmodium (malaria), leishmania, Trypanosoma (sleeping sickness), Toxoplasma gondii, and bacterial infections including Shigella, Escherichia Coli (including 0157), Campylobacter, Vibrio cholerae, Clostridium difficile and Clostridium tetani.

13. A method or use according to any one of claims 10-12, wherein said disease or condition is an inflammatory disorder selected from the group consisting of 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), 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, 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 joint disease, connective tissue diseases, ankylosing spondylitis, bursitis, Sjogren's syndrome, psoriasis, psoriatic arthritis, neuralgia, synovitis, glomerulonephritis, vasculitis, sacoidosis, inflammations that occur as sequellae to influenza, the common cold and other viral infections, gout, contact dermatitis, low back and neck pain, dysmenorrhea, headache, dementia, toothache, sprains, strains, myositis, burns, injuries, and pain and inflammation that follows surgical and dental procedures in a patient.

14. A method according to claim 13, wherein said inflammatory disorder is a mammalian allergic disorder.

15. A method according to claim 14, wherein said mammalian allergic disorder is selected from the group consisting of asthma, house dust mite allergies, food allergies, pet allergies, pollen allergies and insect sting allergies.

16. A method or use according to any of the preceding claims, wherein said agent that reduces the level of cholesterol is selected from the group consisting of 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), Nicotinic acid derivatives (such as Acipimox, Aluminium nicotinate, Nicertirol, Nicoclonate, Nicomol, and Oxiniacic acid), Thyroid Hormones/Analogues (such as Dextrothyroxine, Eitroxate, and Thyropropic acid) and other agents (such as Acifran, Benfluorex, β-Benzylbutyraimde, Carnitine, Chondrotin sulphate, Colmestrone, Detaxtran, Dextran sulphate Sodium, Eicosopentanoic acid, Eritadenine, Ezetimibe, Furazabol, Meglutol, Melinamide, γ-Oryzanol, Pantethine, Pentaerythritol tetraacetate, α-Phenylbutyramide, Pirozadil, Probcuol, β-Sitosterol, Sultosilic acid, Tiadenol, Triparanol, and Xenbucin).

17. A method or use according to claim 16, wherein said agent that reduces the level of cholesterol is an inhibitor of de novo cholesterol biosynthesis.

18. A method according to claim 17 wherein the agent is a HMG CoA reductase inhibitor.

19. A method according to claim 18, wherein said HMG CoA reductase inhibitor is a statin or a derivative or functional equivalent thereof.

20. A method according to claim 19, wherein the statin is selected from the group consisting of Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitivastatin, Pravastatin, Rosuvastatin, and Simvastatin, and derivatives or functional equivalents thereof.

21. The method or use according to any one of claims 10-20, wherein said agent is characterised by rapid clearance from, or inactivation in, the systemic circulation.

22. The method or use according to claim 21, wherein said agent is a soft agent.

23. The method or use according to claim 22, wherein said soft agent is a soft statin.

24. The method or use according to claim 23, wherein said soft statin is a statin prodrag.

25. The method or use according to claim 25, wherein said statin prodrag is a lipophilic ester prodrag.

26. The method or use according to claim 24, wherein said prodrag is 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-1-ol, 3,3,5-trimethyl-cyclohexanol, 2-ethoxy-ethanol and menthol.

27. The method or use according to claim 23, wherein said soft statin comprises a statin engineered to possess a site for metabolism of the drug by a plasma enzyme.

28. The method or use according to claim 27, wherein said plasma enzyme is a plasma esterase.

29. The method or use of claim 27, wherein said soft statin is selected from the group consisting of (3R,5R)-3,5-Dihydroxy-7-(2-isopropyl-4,5-diphenyl-3-phenylcarbamoyl- pyrrol-l-yl)-heptanoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-(l-isopropyl-3-phenyl-lH- indol-2-yl)-hept-6-enoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7-[4-isopropyl-2- (methanesulfonyl-methyl-amino)-6-phenyl-pyrimidin-5-yl]-hept-6-enoic acid, (E)- (3R,5S)-7-(2-Cyclopropyl-4-phenyl-quinolin-3-yl)-3,5-dihydroxy-hept-6-enoic acid and (E)-(3R,5S)-7-(2,6-Diisopropyl-5-methoxymethyl-4-phenyl-pyridin-3-yl)-3,5- dihydroxy-hept-6-enoic acid.

30. The method or use of claim 27, wherein said soft statin is selected from the group consisting of (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,5 S)-7-[2-Cyclopropyl-4-(4-hydroxy- phenyl)-quinolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid, (E)-(3R,5S)-3,5-Dihydroxy-7- [4-(4-hydroxy-phenyl)-2,6-diisopropyl-5-methoxymethyl-pyridin-3-yl]-hept-6-enoic acid.

31. The method or use according to claim 21, wherein said agent is a prodrag or a metabolite of a soft statin, or a prodrag of a metabolite of a soft statin.

32. The method or use of claim 31, wherein said agent is a prodrag of a soft statin of the general formula X-Y, where 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-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-3-yl]-3,5-dihydroxy-hept-6-enoic acid and (E)-(3R,5S)-3,5- Dihydroxy-7-[4-(4-hydroxy-phenyl)-2,6-diisopropyl-5-methoxymethyl-pyridin-3-yl]- hept-6-enoic acid, and Y is selected from the group consisting of formic acid, acetic acid, propan-1-oic acid, propan-2-oic acid, butan-1-oic acid, butan-2-oic acid, pentan- 1-oic acid, hexan-1 -oic acid, heptan-1-oic acid, octan-1-oic acid, nonan-1-oic acid, decan-1-oic acid, benzoic acid, cinnamic acid and 1-hydroxy-benzoic acid.

33. A method or use according to any one of claims 21-32, wherein said agent is applied to a topologically exterior surface of the mammal.

34. A method or use according to claim 33, wherein the topologically exterior surface is the skin.

35. A method or use according to 34, wherein the topologically exterior surface is the respiratory tract.

36. A pharmaceutical composition comprising an agent that reduces the level of cholesterol in a cell in conjunction with a pharmaceutically acceptable carrier, wherein said agent is rapidly cleared from, or rapidly inactivated in, the systemic circulation.

37. A pharmaceutical composition according to claim 36, wherein said agent is selected from the agents claimed in claims 22-32 above.

38. 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.

39. 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 said cell of hematopoetic lineage.

40. 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 said mast cell.

41. A method according to claim 40, wherein the screening method involves an assay of mast cell calcium mobilisation, kinase phosphorylation, TNFα release, degranulation, proliferation or adhesion are assayed.

42. A method of screening for agents useful for the modulation of T cell activation, comprising assessing the ability of a selected moiety to reduce the level of cholesterol within said T cell.

43. A method according to claim 40, wherein the screening method involves an assay of T cell proliferation.

44. A method of screening for agents useful for the modulation of the activity of the Icrac calcium channel, comprising assessing the ability of a selected moiety to reduce the level of cholesterol within the cell membrane with which the Icrac calcium channel is associated.

45. A method according to claim 40, wherein the screening method involves an assay of calcium influx via the Icrac calcium channel.

46. A method according to claim 2, wherein said signalling component associated with a lipid raft of the cell membrane is the Icrac calcium channel.

47. A method according to claim 46, wherein the modulation of the activity of a membrane signalling pathway is the modulation of calcium influx via the Icrac calcium channel.

48. The use of an agent that reduces the level of cholesterol within a cell to modulate the activity of said cell.

49. 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 said cell, wherein the signalling pathway comprises at least one signalling component associated with a lipid raft of the cell membrane.