WO2009095719A2

WO2009095719A2 - Treatment of mast cell related disorders

Info

Publication number: WO2009095719A2
Application number: PCT/GB2009/050091
Authority: WO
Inventors: Anant Parekh
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2008-02-01
Filing date: 2009-01-30
Publication date: 2009-08-06
Anticipated expiration: 2010-08-01
Also published as: GB0801888D0; WO2009095719A3

Abstract

The present disclosure provides methods, uses and products for use in treating disorders associated with mast cell activation. The present disclosure includes for example combinations of agents which inhibit Calcium Release Activated Calcium (CRAC) channels and agents which inhibit leukotriene activation of mast cells for use in treating disorders including allergic rhinitis and nasal polyposis.

Description

TREATMENT OF MAST CELL RELATED DISORDERS

FIELD OF THE INVENTION

The present disclosure relates to the treatment of disorders related to mast cell activation. In some aspects of the invention, the disclosure relates to the treatment of such disorders using modulators of a component of a CRAC channel pathway, particularly in mast cells. Also included in the present disclosure is the use of combinations of said modulators with a second class of agents which inhibit leukotriene function. The present disclosure includes inter alia methods, products, uses and other subject matter for the treatment of mast cell associated disorders.

BACKGROUND

Mast cells are key components of the innate immune system, where they help to orchestrate the inflammatory response along with other cell types such as eosinophils, dendritic cells and monocytes/macrophages. In particular, mast cells are present in mucosal and epithelial tissues in the vicinity of small blood vessels and postcapillary venules. Mast cells are also present in subendothelial connective tissue. Aberrant mast cell activation is linked to a variety of allergic diseases including asthma, eczema, rhinitis and nasal polyposis, which in combination affect up to 20% of the population in industrialized countries (Robbie-Ryan, M., and Brown, M. (2002) Current Opinion in Immunology 14, 728-733; Leynaert, B. et al (1999) J. Allergy and Clinical Immunology 104, 301 -304 and Borish, L. (2003) J. Allergy and Clinical Immunology 1 12, 1021 -1031 ).

The clinical effects of allergic reactions vary according to the site of mast-cell activation. Inhalation is the most common route of allergen entry. Allergic rhinitis is a condition which results from the activation of mucosal mast cells beneath the nasal epithelium by allergens such as pollens. Allergic rhinitis is characterised by intense itching and sneezing, local oedema, nasal discharge and irritation of the nose as a result of histamine release.

Another disorder associated with mast cell activation is allergic asthma, which is triggered by allergen-induced activation of submucosal mast cells in the lower airways. This leads to bronchiola constriction and increased secretion of mucus and fluid, which makes breathing more difficult by trapping inhaled air in the lungs. Chronic inflammation of the airways is often a feature of asthma.

Upon activation, mast cells release a variety of signals that target the bronchi and vasculature and recruit other immune cells to the inflammatory site. Some of these signals are released from the preformed granules e.g. histamine. Others are synthesized after activation. Prominent amongst such signals are the cysteinyl leukotrienes, a family of potent pro-inflammatory lipid mediators (Funk, C. D. (2001 )

Science 294, 1871 -1875; 5; Peters-Golden et al. (2006) Clinical and Experimental Allergy 36, 689-703.). This family includes leukotriene C₄ (LTC₄), LTD₄ and LTE₄.

LTC₄ is secreted from mast cells following Ca²⁺ influx through store-operated Ca²⁺ release activated Ca²⁺ channels (CRAC) channels (Chang, W. -C et al. (2006) FASEB Journal 20, 2381 -2383). LTC₄ synthesis occurs following liberation of arachidonic acid and therefore, CRAC channels must first stimulate arachidonic acid generation. Subsequently, this molecule can be modified by two pathways to give rise to prostaglandins, thromboxanes, and leukotrienes. Arachidonic acid is subsequently metabolised by 5-lipoxygenase to produce LTC₄ which is converted from LTA₄ by LTC₄ synthase. LTC_4, along with LTD₄ and LTE_4, is referred to as cysteinyl leukotrienes.

Receptor stimulation of mast cells leads to Ca²⁺ release from endoplasmic reticulum stores. This release in turn leads to Ca²⁺ influx through CRAC channels in the plasma membrane. The CRAC channel is a store operated Ca²⁺ channel. The CRAC channel pathway is thought to include two proteins, CRACM1 (also known as Oraii ), a plasma membrane protein with four transmembrane segments and STIM1 , an endoplasmic reticulum membrane-spanning protein (Zhang et al, Proc. Natl. Acad. Sci USA 103, 9357-9362 (2006) and Parekh, Nature Cell Biol. 8, No. 7, 2006 655-656). The possibility of further components being part of the CRAC channel has not been ruled out.

It has been reported that the mediators synthesized by mast cells contribute to both the acute and the chronic inflammatory responses. In particular, the lipid mediators e.g. leukotrienes (including LTC_4, and LTD₄) act rapidly to cause smooth muscle contraction, increased vascular permeability and mucus secretion.

Current therapies for allergic disorders include antihistamines, such as loratadine and cetirizine, and nasal corticosteroid based drugs such as beclomethasone. Also used to treat disorders such as allergic rhinitis and asthma, are leukotriene receptor antagonists such as pranlukast, montelukast and zafirlukast. One of the main therapies for the treatment of asthma are bronchodilators such as β-agonists, which can be either short or long acting, and which are usually inhaled.

BRIEF SUMMARY OF THE DISCLOSURE

Embodiments of the present invention are based, at least in part, on the inventor's findings that there is an interaction between Ca²⁺ release activated Ca²⁺ channels (CRAC) channel activation in mast cells and the activity of leukotriene C₄ to act as a local paracrine signal to activate neighbouring mast cells. Embodiments of the present invention therefore aim to provide novel therapies to reduce or prevent mast cell activation by targeting one or both of these associated pathways and therefore control and/ or treat mast cell associated disorders. The present invention provides agents, methods and uses for the control of disorders caused by mast cell activation and methods of screening for agents which control the activation of mast cells.

Thus, in one aspect of the present invention there is provided a combination of at least two pharmaceutical agents, each of which targets a different component of the pathways involved in (a) activation of CRAC channels; (b) leukotriene synthesis by mast cells; (c) leukotriene activation of mast cells. In embodiments of the invention, the use of such a combination to treat mast cell related disorders allows a reduced dosage of each agent to be used, as compared to the dosage required to achieve the same effect when each agent is used in isolation.

In one aspect of the invention, there is provided a method of treating a disorder caused by mast cell activation comprising administering a therapeutically effective amount of an agent which is an inhibitor of a component of CRAC channel pathway in mast cells. Such agents are referred to herein as "first agent".

In an aspect of the invention, there is provided an agent which is an inhibitor of a component of CRAC channel pathway in mast cells for use in treating a disorder associated with activation of mast cells, wherein said agent is a first agent and is for use in combination with a second agent which is an inhibitor of activation of mast cells by a cysteinyl leukotriene. In one embodiment, the cysteinyl leukotriene is selected from leukotriene C4 (LTC₄) and leukotriene D4 (LTD₄). The components of the CRAC channel pathway include a CRACM1 protein and a STIM1 protein. In one embodiment, the first agent is a direct or indirect inhibitor of the CRACM1 protein. In one embodiment, the first agent is a direct or indirect inhibitor of the STIM1 protein.

An agent of the invention, e.g. the first agent and/or the second agent, may be independently selected from an antibody, a small molecule, a protein, a peptide and a nucleic acid. In one embodiment, an agent of the invention, e.g. the first agent and/or the second agent is a nucleic acid sequence, e.g. an siRNA. In one embodiment, the first agent is an siRNA comprising the nucleic acid sequence of GCAUGGAAGGCAUCAGAAGUGUAUA (SEQ ID. NO 1 ).

The first agent may be for simultaneous, separate or sequential use with the second agent. In one embodiment, the second agent is selected from a cysteinyl leukotriene type I receptor (cysLTI receptor) antagonist, an inhibitor of LTC₄ production by mast cells and an inhibitor of 5-lipoxygenase.

In one embodiment, there is provided a combination of a first agent and more than one second agent. In one embodiment, the combination is for treating a mast cell related disorder or to alleviate the symptoms of such a disorder. In one embodiment, the combination comprises a first agent and at least two second agents independently selected from a 5-lipoxygenase inhibitor and a cysLTI receptor antagonist.

In one embodiment, there is provided a combination of more than one first agent and at least one second agent e.g. for use in treating mast-cell associated disorders.

In one embodiment, the disorder is a disorder associated with or caused by local activation of mast cells e.g. an allergic disorder. In an embodiment, the disorder is selected from allergic rhinitis, nasal polyposis and asthma. In one embodiment, the disorder is allergic rhinitis.

In a further aspect of the invention, there is provided a pharmaceutical composition comprising (a) a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and (b) a second agent which is an inhibitor of activation of mast cells by a leukotriene e.g. a cysteinyl leukotriene. In a further aspect of the invention, there is provided a pharmaceutical product comprising a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by a leukotriene.

Also included in the present invention, is a kit comprising a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by a leukotriene e.g. a cysteinyl leukotriene.

A further aspect of the invention relates to a combination of a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene, for treating a disorder associated with mast cell activation.

In an embodiment, the second agent is selected from montelukast, zafirlukast, pranlukast and zileuton and pharmaceutically acceptable salts thereof. In one embodiment, the second agent is an siRNA molecule which suppresses or silences expression of a cysteinyl leukotriene type I receptor. In one embodiment, the siRNA molecule has a nucleic acid sequence selected from one or more of the following:

Strand 1 sense - UAUCAUAUUCAACGAAGCUAUU (SEQ ID. No. 2), antisense - 5'-P UAGCUCGUUGAAUAUGAUAUU (SEQ ID. No. 3);

Strand 2 sense - GUACAUUGCCUCUCCGUGUUU (SEQ ID No. 4); anti-sense - 5'-P ACACGGAGAGGCAAUGUACUU (SEQ ID No. 5);

Strand 3 sense - GUGGGUUUCUUUGGCAAUAUU (SEQ ID. No. 6); anti-sense 5'-P UAUUGCCAAAGAAACCCACUU (SEQ ID No. 7);

Strand 4 sense - ACCUAUGCCUUAUAUGUUAUU (SEQ ID No. 8); antisense - 5'-P UAACAUAUAAGGCAUAGGUUU (SEQ ID No. 9).

In one embodiment, the second agent comprises a mixture of more than one of SEQ. ID No. 2 to 9.

Further classes of first agents and second agents are described in more detail below.

In one aspect of the present invention, there is provided a method of reducing the probability of or treating a disorder associated with mast cell activation comprising; administering a therapeutically effective amount of (a) a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and (b) a therapeutically effective amount of a second agent which is an inhibitor of activation of mast cells by leukotriene, to a subject in need thereof.

In one embodiment, the disorder is caused by mast cell activation. In one embodiment, the second agent is an inhibitor of activation of mast cells by leukotriene C₄ (LTC₄). In one embodiment, the first agent is administered simultaneously, separately or sequentially to the second agent. In one embodiment, the method is for treating a disorder which is caused by local activation of mast cells. In one embodiment, the disorder is selected from asthma, allergic rhinitis and nasal polyposis. Other disorders are disclosed below.

In one embodiment, the second agent is a cysteinyl leukotriene type I receptor antagonist e.g. montelukast, zafirlukast, pranlukast or a pharmaceutically salt thereof. In one embodiment, the second agent is a 5-lipooxygenase inhibitor e.g. zileuton or a pharmaceutically acceptable salt thereof. In one embodiment, the subject is a human.

In one aspect of the invention, there is provided use of an agent which inhibits a component of a CRAC channel pathway in a mast cell for the manufacture of a medicament for the treatment of a disorder which is associated with mast cell activation, wherein the agent is a first agent and is for sequential, simultaneous or separate administration with a second agent which inhibits activation of mast cells by a leukotriene.

In one embodiment, the disorder is selected from allergic rhinitis, asthma, and nasal polyposis. In one embodiment, the medicament is for a human patient. In one aspect of the invention, there is provided use of an inhibitor of activation of mast cells by leukotrienes for the manufacture of a medicament for the treatment of a disorder associated with mast cell activation, wherein said inhibitor is for sequential, simultaneous or separate administration with an agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell.

In a further aspect of the invention, there is provided a pharmaceutical product comprising at least two agents, each of which targets a different component of the pathways involved in activation of CRAC channels and/ or leukotriene production and/ or activity. In one embodiment, the product comprises an agent which inhibits leukotriene synthesis in mast cells, for example an inhibitor of 5-lipoxygenase (5-LO) activity. 5- lipoxygenase is an enzyme which metabolises arachidonic acid produced in the mast cells to form leukotrienes.

Examples of 5-lipoxygenase inhibitors which may be used in the present invention include, for example, zileuton and NDGA (nordihydroguaiaretic acid) and pharmaceutically acceptable salts thereof. In combination with such an agent, the product may also contain an inhibitor of a different component of the pathway for leukotriene production and activation e.g. an agent which is capable of disrupting the association between arachidonic acid and 5-LO by inhibiting the 5-lipoxygenase- activating protein (FLAP) to inhibit leukotriene production. Alternatively, the product may comprise a cysteinyl leukotriene type I receptor antagonist e.g. montelukast, zafirlukast, pranlukast or a pharmaceutically salt thereof. It will be appreciated that this aspect of the invention involves the use of at least two agents which are referred to herein as "second agents". Details of exemplary agents are given below under the headings "Further exemplary agents" and "Second agent".

In a stimulated mast cell, activation of CRAC channels lead to leukotriene synthesis. The secreted leukotrienes then activate adjacent mast cells, to produce a positive feedback cycle. If an event downstream of CRAC channel activation in a stimulated cell is inhibited, this may have similar results as inhibition of CRAC channels to stop the positive feedback which leads to further mast cell activation. In one embodiment, there is provided a combination of two or more pharmaceutically acceptable agents for treating a mast cell-related disorder, wherein the agents each target a different component involved in leukotriene synthesis and/or mast cell activation by leukotrienes e.g. the cysLTI receptor. As described herein, leukotriene production and activity on mast cells is linked to CRAC channel activation and subsequently mast cell activation.

Thus, by targeting at least two components involved in the positive feedback cycle, effective inhibition of mast cell activation may result. This may have utility in treating disorders associated with inappropriate mast cell activations e.g. the inflammatory and/or allergic disorders disclosed herein. The combination may include for example an agent which is a cysteinyl leukotriene receptor antagonist and a 5-lipoxygenase inhibitor. Such combinations may be for use in the manufacture of a medicament for the treatment of a mast-cell related disorder. In addition, these combinations may be for use in a method for treating a mast cell related disorder e.g. an inflammatory disorder or an allergic disorder. Also included in the present invention are kits and products comprising such combination of pharmaceutical agents.

In one embodiment, the present invention provides a method of treating disorders caused by local activation of mast cells and agents for use in such methods.

In one embodiment, the present invention relates to the treatment of an allergic disorder e.g. allergic rhinitis. Patients with allergic rhinitis often experience increased incidence of acute sinusitis and otitis media, both of which can be regarded as causatively linked to nasal disease. Thus, in one embodiment, the present invention concerns the treatment of disorders e.g. acute sinusitis and otitis media which are linked to allergic rhinitis when caused, at least in part, by mast cell activation.

First Agent

In one embodiment, the present invention provides an agent which modulates the activity of a component of the Ca²⁺ release activated Ca²⁺ (CRAC) channel pathway (hereinafter referred to as a "first agent") for use in treating mast cell associated disorders. As described above under "Background", the CRAC channel includes at least two components: (1 ) a CRACM1 protein which is a plasma membrane protein which comprises four transmembrane segments and (2) a STIM1 protein, an endoplasmic reticulum membrane-spanning protein. It is believed that both proteins are essential for the CRAC channel to function and react to store depletion of Ca²⁺. Both proteins are expressed in human mast cells. As used herein the term "component of the CRAC channel" includes any component which is involved in and/or facilitates the influx of extracellular Ca²⁺ into a cell cytosol following endoplasmic reticulum depletion of Ca²⁺ in particular into mast cells. Such components include the STIM1 protein and the CRACM1 protein.

The human CRACM1 protein is published under Genbank accession number Q96D31 . The term "CRACM1 protein" as used herein encompasses naturally-occurring variant forms (e.g. alternatively spliced forms) and naturally-occurring allelic variants. The human STIM1 protein is published under Genbank accession number Q13586. The term "STIM1 protein" as used herein encompasses naturally-occurring variant forms (e.g. alternatively spliced forms) and naturally-occurring allelic variants.

In one embodiment, the first agent is a CRAC channel inhibitor, particularly an inhibitor of CRAC channel activity in mast cells. Thus, the first agent may inhibit or reduce the activity of one or more components of the CRAC channel. Such agents may include, although are not limited to, agents which have one or more of the following characteristics: (1 ) the ability to inhibit or reduce the influx of Ca²⁺ into mast cells via the CRAC channel; (2) the ability to inhibit or reduce binding of agonists to a CRACM1 protein expressed in the plasma membrane of a mast cell; (3) the ability to inhibit or reduce STIM1 activity; and (4) the ability to inhibit or reduce the effect of binding of allergens to mast cells.

The first agent may be a direct or indirect inhibitor of a component of the CRAC channel. In one embodiment, the first agent is a modulator, e.g. an inhibitor, of a CRACM1 protein expressed by a mast cell. In one embodiment, the first agent may be a modulator, e.g. an inhibitor, of a STIM1 protein. In one embodiment, the first agent binds to a CRAC channel protein e.g. a CRACM1 protein or a STIM1 cell expressed by a mast cell. The binding of the agent is optionally binding with an affinity of greater than 10⁷ M, 10⁸ M, 10 ⁹ M, 10¹⁰ M, 10¹¹ M or 10¹² M. The binding may be specific for the CRAC channel protein or non-specific, although in some instances there is a degree of lower affinity non-specific binding to certain other ligands unrelated to the CRAC channel protein.

In one embodiment, the first agent may be selected from a protein, a peptide, an antibody or antibody fragment, a small molecule, a compound and a nucleic acid.

In one embodiment, the first agent may be a pyrazole derivative or pharmaceutically acceptable salt thereof. For example, the first agent may be a 3,5- bis(trifluoromethyl)pyrazole derivative e.g. 4-methy-4'-[3,5-bis(trifluoromethyl)-1 H- pyrazol-1 -yl]-1 ,2,3-thiadiazole-5-carboxanilide, (known as YM-58483 or BTP-2) or a pharmaceutically acceptable salt, solvate, clathrate, or prodrug thereof. In one embodiment, the first agent is a compound disclosed in for example WO99/19303 (granted in the US under patent no. 6348480), the contents of which are incorporated herein in their entirety.

In one embodiment, the first agent is selected from diethylstilbestrol (Zaharov et al., 2004; Molecular Pharmacology, 66, 702-707) and 2-aminoethyl diphenylborinate analogues e.g. those disclosed in Zhou et al., 2007; Biochem. Biophys. Res. Communications, 352, 277-282. Another class of suitable molecules include thiazole and [1 ,3,4] thiadiazole derivatives as disclosed in WO2007/087427, incorporated herein by reference in its entirety.

Second Agent

Aspects of the present invention provide the use of a second agent in combination with the first agent or with at least one additional second agent to treat disorders caused by inappropriate mast cell activation. The second agent is a modulator, e.g. an inhibitor of mast cell activation by leukotrienes. In one embodiment, the second agent is selected from a protein, a peptide, an antibody or antibody fragment, a small molecule, a compound and a nucleic acid.

In one embodiment, the second agent may inhibit or reduce activation of mast cells by leukotrienes e.g. cysteinyl leukotrienes. In one embodiment, the second agent is an inhibitor of activation of mast cells by at least one of leukotriene C₄ and leukotriene D₄.

In an embodiment, the agent is a modulator, e.g. an inhibitor, of a leukotriene synthesis pathway in mast cells. Thus, a class of suitable second agents may include, although is not limited to, agents which inhibit the production of leukotrienes, e.g. LTC₄ and/ or LTD_4, by mast cells. In an embodiment, the second agent is an inhibitor of 5-lipoxygenase activity. 5-lipoxygenase is an enzyme which metabolises arachidonic acid produced in the mast cells to form leukotrienes. Examples of 5-lipoxygenase inhibitors which may be used in the present invention include, for example, zileuton and NDGA

(nordihydroguaiaretic acid) and pharmaceutically acceptable salts thereof. In one embodiment, the second agent is an inhibitor of cysteinyl leukotriene mediated activation of mast cells. In one embodiment, the second agent is an agent which is capable of disrupting the association between arachidonic acid and 5-LO by inhibiting the 5-lipoxygenase-activating protein (FLAP) to inhibit leukotriene production.

In one embodiment, the second agent is an antagonist of a leukotriene receptor, for example, a cysteinyl leukotriene type I receptor, a G protein coupled receptor expressed by mast cells. Thus, in one embodiment, the inhibitor is a cysteinyl leukotriene receptor antagonist. Examples of such cysteinyl leukotriene receptors antagonists include, although are not limited to, montelukast, zafirlukast and pranlukast and their pharmaceutically acceptable salts, esters and prodrugs. For example, in one embodiment, the second agent is montelukast sodium. Montelukast and montelukast sodium salt were first disclosed in EP480717. Processes for the manufacture of montelukast and montelukast salts, including montelukast sodium are described in for example US2005/0107612-A and WO2005/105750. Other inhibitors of the cysteinyl leukotriene type I receptor which may be used in the present invention include for example BAY u9773 described in Nothacker et al, 2000, MoI Pharmacol 58:1601 -1608.

In one aspect of the invention, the present invention provides a method of treating a disorder associated with mast cell activation which comprises administration of a first agent as described herein to a patient in need thereof.

In one embodiment, the method further comprises administering a second agent as described herein. The first agent and the second agent may be administered simultaneously, sequentially or separately. In one embodiment, the method of the present invention comprises inhibiting or reducing mast cell activation by inhibiting CRAC channel activation in conjunction with inhibition of activation of the mast cell by leukotrienes. The term "inhibit" as used herein need not necessarily mean total inhibition of the pathways. Instead, the term is taken to include a reduction in activity as well as total inhibition.

In one aspect of the invention there is provided a method of treating a disorder associated with mast cell activation comprising administering a therapeutically effective amount of a first agent as described herein and a therapeutically effective amount of a second agent wherein said administration is sequential, simultaneous or separate. Embodiments of the present invention which include administration of a combination of the first agent and second agent may require lower dosages of each of the first agent and the second agent than sole administration of either the first agent or the second agent. Lower dosages may be advantageous, as toxic side effects may be minimized. Other potential advantages of the combined administration may include an improvement in efficacy as compared to administration of either the first agent or the second agent in isolation.

Thus, in one embodiment, there is provided a combination of a first agent and a second agent for the treatment of a disorder associated with mast cell activation, wherein the combination comprises an amount of a first agent which is reduced as compared to the dosage used if the first agent is for administration without the second agent,

The first agent may be for simultaneous, sequential or separate administration with the second agent. In one embodiment, the agents are administered at a time interval which enables both agents to inhibit mast cell activation by inhibiting the positive feedback system described herein. In this embodiment, the agents are administered at substantially the same time e.g. in response to the same stimulus e.g. an allergen.

In one embodiment, the combination may include a dosage of the first agent which is at least 10% less than a therapeutically effective dosage of the first agent if used as a monotherapy. In one embodiment, the combination comprises the first agent in a dosage at least 15% less than a therapeutically effective dosage of the first agent if used as a monotherapy i.e. when administered alone.

In one embodiment, the combination may include a dosage of the second agent which is at least 10% less than a therapeutically effective amount of the second agent if used as a monotherapy. In one embodiment, the combination comprises the second agent in a dosage at least 15% less than a therapeutically effective amount of the second agent if used as a monotherapy i.e. when administered alone.

Without being bound by theory, it is believed that a positive feedback step underlies mast cell activation and inhibition of this positive feedback may have clinical benefit in the treatment of disorders associated with mast cell activation e.g. disorders caused by mast cell activation. The present invention is based at least in part on the inventor's findings that stimulation of mast cells results in secretion of leukotrienes which then feed back to activate cell-surface cysteinyl leukotriene type I receptors, resulting in a cytoplasmic Ca²⁺ rise and further LTC₄ secretion.

Leukotrienes are normally removed via the bloodstream but in pathological conditions such as nasal polyposis and rhinitis, the tissue is often almost avascular. As a result, the leukotrienes are not removed and thus sustain mast cell activation. Inhibition of mast cell activation by leukotrienes in the first instance, for example by inhibiting CRAC channel activation so as to inhibit leukotriene production as well as inhibiting activation of neighbouring mast cells by leukotrienes may reduce the length of time a patient suffers the symptoms of an allergic or inflammatory reaction.

In support of the positive feedback theory, the inventors have found that (1 ) LTC₄ generates arachidonic acid production (i.e. arachidonic acid to LTC₄ to arachidonic acid), (2) LTC₄ generation is reduced by inhibition of cysteinyl LT1 receptors, (3) application of supernatant which includes LTC₄ results in protein kinase Cβ1 translocation to the plasma membrane, which is a key step in LTC₄ synthesis and also (4) the supernatant stimulates mast cells through the CRAC channels.

Thus, as discussed above, this relationship between the CRAC channel and the positive feedback by leukotriene secretion may mean that therapy comprising administration of an agent which modulates the functioning of a CRAC channel with an agent which modulates the positive feedback cycle by LTC₄ has an additional effect.

Therefore, given the positive feedback system between the CRAC channel activity and leukotriene production, one aspect of the present invention provides a combination which combines at least two agents, each of which target, e.g. inhibit, a different component of the leukotriene synthesis and activation pathway.

In one embodiment, there is provided a combination of a first agent and more than one second agent. The combination may be for use in treating a disorder associated with mast cell activation e.g. inflammation or allergic reactions. Disorders associated with mast cell activation which may be treated by a combination of a first agent and at least two second agents as described here, e.g. under the heading "Methods".

In one embodiment, the first agent is for use with a 5-lipoxygenase inhibitor e.g. zileuton and a cysLTI antagonist e.g. montelukast. In one embodiment, the combination comprises a first agent, zileuton and montelukast. Such a combination may have utility in treating mast cell related disorders.

In one embodiment, the combination of the first agent and at least two second agents comprises each agent in a dosage at least 15% less than a therapeutically effective amount of the agents if used as a monotherapy, e.g. the dosages of the agents are independently reduced by at least 20%, 25%, 30%, 35%, 40%, 45% or 50%, 55%, 60% or more as compared to a therapeutically effective dosage of the agents when used as a monotherapy i.e. when either agent is administered alone.

In one embodiment, the present invention is directed towards the treatment of a disorder which is caused or exacerbated by impaired removal of leukotrienes from a tissue in which mast cells are activated. Such disorders include for example nasal polyposis and rhinitis. In one embodiment, the tissue is partially or wholly avascular.

Further details of the present invention are set out below.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Paracrine signalling in mast cells. Figure 1 A is a graph showing Ca²⁺ signalling in fura-2 loaded RBL-1 mast cells when challenged with (1 ) active supernatant (open circles - cells stimulated with thapsigargin which depletes intracellular Ca²⁺ stores and thus opens CRAC channels leading to secretion of LTC₄ ); (2) control supernatant (filled circles - CaCI₂ had been added to bring free Ca²⁺ back to 2mM); and (3) in the presence of SR-2640, a cysteinyl leukotriene receptor antagonist. Figure 1 A shows that treatment with SR-2640 (1 -1 OμM) abolishes the Ca²⁺ in response to the active supernatant.

Figure 1 B is a graph showing the inhibition of Ca²⁺ signalling in fura-2 loaded RBL-1 mast cells by the cysteinyl leukotriene type I receptor antagonist, montelukast, and the 5-lipooxygenase blocker, zileuton, when the mast cells are challenged by active supernatant.

Figure 1 C is a graph showing the ability of stimulated supernatant to evoke a Ca²⁺ response when mast cells are treated with either an si RNA against 5-lipoxygenase (n=68 cells) or by exposure to zileuton (n=91 cells).

Figure 1 D is a transillumination image of recording condition used to probe local actions of leukotrienes. CRAC channel activation was restricted to just one cell by dialysing it with lnsP₃via a patch pipette whilst recording Ca²⁺ signals in adjacent cells.

Figure 1 E is a graph which shows the results of activating the CRAC channel in the patched cell; the activation of I_CR_AC resulted in a cytoplasmic Ca²⁺ rise in the patched cell and this triggered a cytoplasmic Ca²⁺ rise in adjacent cells.

Figure 1 F is a graph which shows the results of activating the CRAC channel in the patched cell which was pre-treated with montelukast. Pre-treatment with montelukast had no effect on the cytoplasmic Ca²⁺ rise in the patched cell but suppressed the Ca²⁺ rise in adjacent ones. This shows that leukotriene secretion from one cell activates nearby cells, demonstrating a role as a local paracrine signal.

Figure 1 G and Figure 1 H are graphs showing the effect of ATP on the Ca²⁺ response. These experiments were designed to assess whether ATP might contribute to the Ca²⁺ response evoked by the active supernatant. Figure 1 G and Figure 1 H shows that pre- treating cells with 10μM SR-2640 had no effect on the Ca²⁺ response to either a maximal ATP concentration or one just above threshold (50μM). Figure 1 1 is a graph showing that there is a lack of cross de-sensitization between the ATP response and application of supernatant on mast cells.

Figure U shows the results of application of activated supernatant to HEK293 cells which express P2Y but not cysteinyl leukotriene receptors. The experiment was designed to determine whether RBL cell supernatant contained enough ATP to trigger Ca²⁺ signals. As Figure U shows, application of the supernatant to fura-2 loaded HEK293 cells failed to evoke a Ca²⁺ rise, although the cells subsequently responded to ATP.

Figure 2 -Leukotrienes as paracrine signals

Figure 2A is a graph showing the response of HEK293 cells transfected with the gene encoding cysteinyl leukotriene type I receptor to application of the active supernatant and the response of transfected cells pre-treated with montelukast to the supernatant.

Figure 2B is a graph showing the result of acute application of leukotrienes to resting mast cells. As shown in Figure 2B, the results mimic that of the results achieved using the supernatant.

Figure 2C is a graph showing that responses to cysteinyl leukotriene (LTC₄ ) is dose dependent and responses to LTC₄ were fully suppressed by montelukast.

Figure 2D is a graph showing that application of LTC₄ was able to evoke a prominent cytoplasmic Ca²⁺ rise following desensitization of the ATP response, thus ruling out action on and by P2Y receptors.

Figure 3

Figure 3A is a graph showing a clear Ca²⁺ rise when RBL-derived supernatant was applied to fura 2 loaded acutely isolated rat peritoneal mast cells. This Ca²⁺ rise was suppressed by SR-2640 and montelukast.

Figure 3B is a graph which shows the results of application of phospholipase C inhibitor U73122 to fura 2 loaded cells. Figure 3C is a chart showing the reduction of stored intracellular Ca²⁺ as a result of application of the activated supernatant. The Ca²⁺ content was assessed using the size of ionomycin-triggered Ca²⁺ signal (n=77 for control and 86 for supernatant).

Figure 3D shows the application of the active supernatant trigger an influx of store- operated Ba²⁺ which is consistent with the findings shown in Figure 3C.

Figure 3E shows the effect of thapsigargin and active supernatant on store depletion

Figure 4 - signalling by leukotrienes occurs with modest Ca²⁺ influx and lasts for tens of minutes.

Figure 4A-C: Cells were stimulated with thapsigargin in Ca²⁺- free solution for 4 minutes and then exposed to Ca²⁺ -free solution alone for a further 4 minutes. Thereafter, different concentrations of external Ca²⁺ were admitted for a further 4 minutes. Supernatant was then collected and applied to Fura-2 loaded cells. Supernatant was collected from cells stimulated in 2 mM Ca²⁺ (Figure 4A), 0.5mM Ca²⁺ (Figure 4B) and 0.25 mM Ca²⁺ (Figure 4C). After collection, the Ca²⁺ concentration in the supernatant was made up to 2mM.

Figures 4D to 4G show the time course of leukotriene production measured functionally. Cells were stimulated with thapsigargin in Ca²⁺ -free solution for 4 minutes and exposed to Ca²⁺ -free solution for a further 4 minutes; then 2mM Ca²⁺ was readmitted, and supernatant was collected after various times, stated in the Figures. Supernatant was then applied to Fura-2 loaded cells. In these experiments, cells were washed extensively every 5 minutes to prevent the accumulation of leuokotrienes; 20 minutes reflects washes at 5, 10 and 15 minutes (supernatant was discarded in each case). Collection then occurred over the 15-20 minute period.

Figure 5 Figures 5A-C are graphs showing RNAi knockdown of STIM1 resulted in a substantial reduction in I_CR_AC

Figure 5A. The time course of I_CR_AC development is compared between a control cell (filled circles) and one treated with RNAi to STIM1 (open circles.)

Figure 5B. I-V curves from panel A, taken once the currents had peaked, are depicted. Figure 5C. Aggregate Data from 6 control cells and 7 cells treated with RNAi to STIM1 are shown.

Figure 5D is a graph showing the reduction in store-operated Ca²⁺ signal as a result of RNAi knockdown of STIM1.

Figure 5E-H are graphs showing that the pattern of Ca²⁺ signal to supernatant was changed by knocking down STIM1 .

Figure 5E shows Ca²⁺ spikes evoked by active supernatant.

Figure 5F shows the number of Ca²⁺ spikes was reduced in cells treated with RNAi to STIM1 .

Figure 5G shows the number of spikes in a 400 second time frame in a control cell and cells in which STIM1 has been knocked down. The number of control spikes is taken as 100%.

Figure 5H shows a comparison of the time interval between the first and second Ca²⁺ spike following application of supernatant between control cells and those treated with RNAi to STIML

Figure 6 - Cysteinyl leukotriene type I receptor stimulation activated CRAC channels.

Figure 6A is a graph showing Ba²⁺ influx following stimulation with LTC₄ is prevented by 1 μM Gd³⁺ (a blocker of CRAC channels). The control was carried out in the absence of Gd³⁺ and Ba²⁺ .

Figure 6B is a graph showing aggregate data from several cells (each bar>50 cells).

Figure 6C is a graph showing LTC₄ activates /_CR_AC in whole cell recording. /_CRAC was measured in divalent-free external solution.

Figure 6D shows current-voltage relation for the LTC₄ induced current is compared with that evoked by thapsigargin. /_CRAcwas measured in divalent-free external solution. Figure 6E shows that, following maximum activation of /_CR_AC DV exposure to thapsigargin, LTC₄ failed to elicit a further current. /_CR_AC was measured in divalent-free external solution.

Figure 7. CRAC channels and cysteinyl leukotriene responses in acutely isolated human mast cells.

Figure 7A is a chart indicating positive feedback in LTC₄ production. Application of active supernatant (containing LTC₄) resulted in an increase in LTC₄ levels when applied to a second population of resting RBL cells (bar labelled active supernatant on resting cells) and this increase was prevented by pre-treating the second population of cells with montelukast (bar labelled active supernatant on resting cells + mont. (500 nM montelukast)

Figure 7B indicates that thapsigargin elicited Ca²⁺ influx in human mast cells and this was blocked by 1 mM Gd³⁺. The inset shows a human mast cell stained with the mast cell specific marker c-kit. The isolation method used led to around 3-7 human mast cells per field of view.

Figure 7C indicates that I_CR_AC is present in human mast cells. Stores were depleted with thapsigargin (2 μM). Pipette contained buffered Ca²⁺ (150 nM free Ca²⁺, 10 mM total EGTA). The bath solution contained 10 mM Ca²⁺. The inset shows the current-voltage- relationship taken at 134 seconds.

Figure 8

Figure 8A and Figure 8B are graphs which show that SR-2640 and montelukast both reduce LTC₄ secretion in a dose dependent way in acutely isolated human mast cells.

Figure 8C and 8D show the distribution of protein kinase C in resting RBL cells (C) and stimulation with active supernatant triggers translocation of protein kinase Ca to the cell periphery (D).

Figure 8E and F are graphs showing stimulation of thapsigargin evoked a significant increase in LTC₄ secretion in nasal polyp tissue and nasal tissue from a sufferer of allergic rhinitis (E) and polyposis (F). Figure 8G is a graph showing LTC₄ secretion in human polyps following thapsigargin was suppressed by removing external Ca²⁺ or by inhibiting protein kinase C with GO- 6983.

Figure 8H is a graph showing application of supernatant from RBL-1 cells evoked robust Ca²⁺ signals in fura 2-loaded human mast cells and these were suppressed by blocking cysteinyl leukotriene type I receptors with either montelukast or SR-2640.

Figure 9 Figures 9a and 9b are graphs showing the effect of siRNA knock-out of the cysteinyl leukotriene type I receptor and its response to active supernatant.

Figure 10 Mast cells in nasal polyps.

Figure 1 OA and 1 OB show sections cut from nasal polyps from two patients were stained with toluidine blue to identify mast cells (arrows). Scale bar in left-hand image is 100 μm.

Figure 1 OC is a transillumination image of an experiment to measure intercellular Ca²⁺ wave propagation in RBL cells.

Figure 10D is a graph showing the effect of activating I_CR_AC in the patched cell (by dialysis with lnsP₃ in weak Ca²⁺ buffer) and the rise in cytoplasmic Ca²⁺ in other cells, loaded with fura 2, within the field of view. The patched cell was clamped at + 50 mV and then hyperpolarised to -80 mV 4 minutes after the onset of whole cell recording (to ensure all CRAC channels have been activated prior to hyperpolarisation).

Figure 10E is a graph plotting the diffusion coefficient against molecular weight for a range of small molecules broadly similar to LTC₄. The arrow indicates the molecular weight of LTC₄.

Figures 10F-H show that stimulation with concentrations of LTC₄ down to 2 pM evokes Ca²⁺ signals in RBL cells, revealing high affinity for the ligand.

Figure 11 is a schematic representation of the protocol used to obtain control and active supernatant.

Figures 12 Figure 12A-D are a series of graphs showing the effect of differing concentrations of montelukast on mast cell activation in response to direct application of LTC₄. The figure shows that montelukast blocks responses to 160 nM LTC₄ in a concentration dependent manner. The IC₅₀ for montelukast is ca. 7nM.

Figure 13 - Effect of 50% blockade of CRAC channel and 50% blockade of CysLTI receptor

Figure 13A to C show the response of a population of mast cells loaded with fura 2-to measure cytoplasmic Ca²⁺ to the application of supernatant from a first population of mast cells treated with 1 μM La³⁺, a CRAC channel blocker in the presence of differing concentrations of montelukast. 1 μM La3+ blocks CRAC channels by 50%. Figure 13A shows the response of the mast cells to supernatant in the absence of montelukast and indicates that the supernatant is able to evoke a response.

Figures 13B and 13C show the response of the mast cells in the presence of increasing concentrations of montelukast. As shown in Figure 13C, the mast cell response is blocked by low concentrations of montelukast (5nM and 7.5nM).

Figures 13D to F show the results of a control in which the supernatant was derived from a dish of mast cells that were not treated with La³⁺. Therefore where there was no prior partial block of CRAC channel, a higher concentration of montelukast is required to block the responses of a second pool of fura 2-loaded mast cells.

Figure 14 Figure 14 shows the result of application of both montelukast and zileuton on a second pool of fura 2 loaded mast cells, treated as described above in relation to Figure 13. The Figure shows that the combination of zileuton and montelukast results in a lower % of cells responding to the active supernatant than montelukast alone. Figure 14A-C shows a control of montelukast application only. Figure 14D-F shows application of zileuton and montelukast.

Figure 14G is a histogram summarising the results of Figures A to F.

DETAILED DESCRIPTION The present invention provides inter alia methods, products, agents and uses for the treatment of disorders associated with mast cell activation e.g. disorders caused by mast cell activation.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. ,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed. ), Molecular Biology and Biotechnology : a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1 -56081 -569-8). Definitions and additional information known to one of skill in the art in immunology can be found, for example, in Fundamental Immunology, W. E. Paul, ed., fourth edition, Lippincott-Raven Publishers, 1999.

As described above, the present invention provides an agent which is an inhibitor of a component of CRAC channel pathway in mast cells for use in treating a disorder associated with activation of mast cells, wherein said agent is a first agent and is for use in combination with a second agent which is an inhibitor of activation of mast cells by a cysteinyl leukotriene.

Also provided by the present invention is a combination of a first agent which is an inhibitor of a component of CRAC channel pathway in mast cells and a second agent which is an inhibitor of activation of mast cells by a cysteinyl leukotriene for use in treating a disorder associated with activation of mast cells, as well as other subject matter.

Further Exemplary Agents

In one embodiment, the first agent is a CRAC channel inhibitor and the second agent preferably a modulator of mast cell activation by leukotrienes. Some examples of suitable first and second agents are described under the heading "Summary of the Invention" and exemplary classes of first agents and second agents are also described below, without limitation. Exemplary first and second agents provided under the heading "Summary of the Invention" are applicable to the following description of suitable agents. In one embodiment, the agents of the invention, e.g. the first agent and the second agent are independently selected from a protein, a peptide, an antibody, a compound, a peptibody, a carbohydrate, a small organic molecule and a nucleic acid.

Small Molecules

As described above, an agent of the invention, e.g. the first and/or second agents may be small molecule compounds. In one embodiment, the first agent is a CRAC channel inhibitor e.g. 4-methyl-4'-[3,5-bis(trifluoromethyl)-1 H-pyrazol-1 -yl]-1 ,2,3-thiadiazole-5- carboxanilide (known as BTP-2).

In one embodiment, the second agent is a small molecule antagonist of a leukotriene receptor e.g. cysteinyl leukotriene type I receptor. In one embodiment, the second agent is selected from montelukast (CAS 151767-02-1 ) and pharmaceutically acceptable salts thereof, zafirlukast (CAS 107753-78-6), Cyclopentyl-3-[2-methoxy4-[(o- tolylsulfonyl)carbamoyl]-benzyl]-1 -methylindole-5-carbamate and pharmaceutically acceptable salts thereof, which are described in EP 199,543, pranlukast (CAS 103177- 37-3) (N- [4-0X0-2- (1 H-tetrazol-5-yl)-4H-1 -benzopyran-8-yl]-p- (4-phenylbutoxy) benzamide and pharmaceutical acceptable salts thereof, which are described in EP 173,516 and combinations thereof. In one embodiment, the second agent is SR-2640 (available from Sigma-Aldrich). In one embodiment, the second agent is an inhibitor of 5-lipoxygenase. In one embodiment, the second agent is zileuton.

Antibodies

The agents (e.g. first and/or second) of the invention may be, for example, an antibody or fragment thereof, e.g. a Fab fragment. Naturally within the scope of the agents of the invention are antibodies or fragments which are monoclonal, polyclonal, chimeric, human, or humanized. Other agents are encompassed by the present invention.

Thus, in one embodiment, the first agent may be an antibody or antibody fragment as defined above which inhibits a component of the CRAC channel in mast cells. In one embodiment, the first agent is an antibody which binds to a CRACM1 protein expressed by mast cells. In one embodiment, the second agent may be an antibody or antibody fragment as defined above, which inhibits activation of mast cells by leukotrienes, e.g. by LTC₄ . In one embodiment, the second agent is an antibody or antibody fragment which binds to, and optionally, antagonises a cysteinyl leukotriene type I receptor expressed by mast cells.

An antibody and immunologically active portions thereof, for instance, are typically molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (for example, IgG) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length. Light chains are encoded by a variable region gene at the NH2-terminus (about 1 10 amino acids in length) and a kappa or lambda constant region gene at the COOH- terminus. Heavy chains are similarly encoded by a variable region gene (about 1 16 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')₂, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science

242:423-426, 1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16, 1986).

Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors. An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.

In one embodiment, the antibody (e.g. either the first and/or the second agent) is a monoclonal antibody. A monoclonal antibody is produced by a single clone of B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Generally, a monoclonal antibody is produced by a specific hybridoma cell, or a progeny of the hybridoma cell propaged in culture. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

A suitable class of first agents may be chimeric antibodies which bind to a protein which is involved in the CRAC channel pathway e.g. a CRACM1 protein expressed by a mast cell. A suitable class of second agents may be chimeric antibodies which prevent mast cell activation by leukotrienes e.g. an antibody which binds to a cysteinyl leukotriene type

I receptor in an antagonistic manner. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.

Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent

No. 5,807,715, which is herein incorporated by reference. In one embodiment, the agent may be a humanized antibody or fragment thereof. A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor" and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions can be found in for example U.S. Patent No. 5,585,089, which is incorporated herein by reference. Humanized immunoglobulins can be constructed by means of genetic engineering, e.g., see U.S. Patent No. 5,225,539 and U.S. Patent No. 5,585,089, which are herein incorporated by reference.

In one embodiment, the first and/or second agent is a human antibody. A human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., Dower et al., PCT Publication No. WO91/17271 ; McCafferty et al., PCT Publication No. WO92/001047; and Winter, PCT Publication No. WO92/20791 , which are herein incorporated by reference), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No. WO91 /10741 , which are herein incorporated by reference). In one embodiment, the first and/or second agent is an antibody fragment. Various fragments of antibodies have been defined, including Fab, (Fab')₂, Fv, dsFV and single- chain Fv (scFv) which have specific antigen binding. These antibody fragments are defined as follows: (1 ) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain or equivalents by genetic engineering; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalents by genetic engineering; (4) F(Ab')₂, a dimer of two Fab' fragments held together by disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; dsFV, which is the variable region of the light chain and the variable region of the heavy chain linked by disulfide bonds and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Single chain antibodies may also be referred to as single chain variable fragments (scFv). Methods of making these fragments are routine in the art.

Reference is made to the numbering scheme from Kabat, E. A., et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991 ). In these compendiums, Kabat lists many amino acid sequences for antibodies for each subclass, and lists the most commonly occurring amino acid for each residue position in that subclass. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. CDR and FR residues are also determined according to a structural definition (as in Chothia and Lesk, J. MoI. Biol. 196:901 -917 (1987). Where these two methods result in slightly different identifications of a CDR, the structural definition is preferred, but the residues identified by the sequence definition method are considered important FR residues for determination of which framework residues to import into a consensus sequence.

Nucleic Acids In one embodiment, an agent of the invention, e.g. the first agent and/or the second agent may independently be a nucleic acid, for example, an interfering RNA (RNAi) e.g. an siRNA. In one embodiment, the first agent is an RNAi against a STIM1 protein.

The term "short interfering RNA" or "siRNA" as used herein refers to a double stranded nucleic acid molecule capable of RNA interference "RNAi", see for example Bass, 2001 , Nature, 41 1 ,428-429; Elbashir et al., 2001 , Nature, 41 1 ,494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zemicka-Goetz et al., International PCT Publication No. WO01 /36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; MeIIo and Fire, International PCT Publication No. WO01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and nonnucleotides.

In one embodiment, the first agent is an RNAi molecule which inhibits a STIM1 protein in a mast cell. In one embodiment, the first agent is an RNAi molecule which comprises the sequence; GCAUGGAAGGCAUCAGAAGUGUAUA (SEQ ID. No 1 ). In an alternative embodiment, the first agent is an RNAi molecule which has a nucleic acid sequence which has approximately 75%, 80% or greater identity with the sequence of SEQ ID. No.1 . In one embodiment, the first agent is an RNAi molecule which has approximately 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96% 97%, 98% or 99% identity to SEQ ID. No. 1 .

In one embodiment, the second agent is an siRNA molecule or a mixture thereof. In one embodiment, the second agent is an siRNA molecule or mixture thereof which interfere with a cysteinyl leukotriene type I receptor. In one embodiment, the second agent comprises one or more RNAi molecule selected from SEQ ID No .2, SEQ ID No .3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No. 7, SEQ ID No 8 and SEQ ID No 9.

In one embodiment, the siRNA molecule is a double stranded molecule comprising (a) a sense strand comprising the nucleic acid sequence of SEQ ID. No. 2 and an anti-sense strand comprising the nucleic acid sequence of SEQ ID. No. 3; (b) a sense strand comprising the nucleic acid sequence of SEQ ID. No. 4 and an anti-sense strand comprising the nucleic acid sequence of SEQ ID. No. 5; (c) a sense strand comprising the nucleic acid sequence of SEQ ID. No. 6 and an anti-sense strand comprising the nucleic acid sequence of SEQ ID. No. 7; (d) a sense strand comprising the nucleic acid sequence of SEQ ID. No. 8 and an anti-sense strand comprising the nucleic acid sequence of SEQ ID. No. 9; or (e) a combination of one or more of (a), (b), (c) and (d).

In one embodiment, the second agent is an siRNA molecule or mixture of siRNA molecules which have approximately 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96% 97%, 98% or 99% identity to SEQ ID. No. 2, 3, 4, 5, 6, 7, 8 and/or 9.

In one embodiment, the second agent is an siRNA or a mixture of siRNA molecules generated against the 5-lipoxyenase enzyme. Such siRNA molecules are commercially available from e.g. Dharmacon Inc. Chicago, IL.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the

Needleman et al. (1970) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In one embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

In one embodiment, an agent of the invention, e.g. the first and/ or second agent is an isolated nucleic acid molecule. With regards to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and/or 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

In a further aspect the invention provides an expression vector comprising a nucleic acid as described above and associated regulatory sequences necessary for expression of a protein or polypeptide in a host cell. Such regulatory sequences include promoters, termination sequences and enhancers, for example.

Aptamers A further class of agents of the invention e.g. the first and/or second agents are aptamers. Aptamers have been defined as artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and re-amplification.

RNA aptamers are nucleic acid molecules with affinities for specific target molecules. They have been likened to antibodies because of their ligand binding properties. They may be considered as useful agents for a variety of reasons. Specifically, they are soluble in a wide variety of solution conditions and concentrations, and their binding specificities are largely undisturbed by reagents such as detergents and other mild denaturants. Moreover, they are relatively cheap to isolate and produce. They may also readily be modified to generate species with improved properties. Extensive studies show that nucleic acids are largely non-toxic and non-immunogenic and aptamers have already found clinical application. Furthermore, it is known how to modulate the activities of aptamers in biological samples by the production of inactive dsRNA molecules in the presence of complementary RNA single strands (Rusconi et al., 2002).

It is known from the prior art how to isolate aptamers from degenerate sequence pools by repeated cycles of binding, sieving and amplification. Such methods are described in US 5,475,096, US 5,270,163 and EP0533 38 and typically are referred to as SELEX (Systematic Evolution of Ligands by EX-ponential Enrichment). The basic SELEX system has been modified for example by using Photo-SELEX where aptamers contain photo-reactive groups capable of binding and/or photo cross-linking to and/or photo- activating or inactivating a target molecule. Other modifications include Chimeric- SELEX, Blended-SELEX, Counter-SELEX, Solution-SELEX, Chemi-SELEX, Tissue- SELEX and Transcription-free SELEX which describes a method for ligating random fragments of RNA bound to a DNA template to form the oligonucleotide library. However, these methods even though producing enriched ligand-binding nucleic acid molecules, still produce unstable products. In order to overcome the problem of stability it is known to create enantiomeric "spiegelmers" (WO 01/92566). The process involves initially creating a chemical mirror image of the target, then selecting aptamers to this mirror image and finally creating a chemical mirror image of the SELEX selected aptamer. By selecting natural RNAs, based on D-ribose sugar units, against the non- natural enantiomer of the eventual target molecule, for example a peptide made of D- amino acids, a spiegelmer directed against the natural L-amino acid target can be created. Once tight binding aptamers to the non-natural enantiomer target are isolated and sequenced, the Laws of Molecular Symmetry mean that RNAs synthesised chemically based on L-ribose sugars will bind the natural target, that is to say the mirror image of the selection target. This process is conveniently referred to as reflection- selection or mirror selection and the L-ribose species produced are significantly more stable in biological environments because they are less susceptible to normal enzymatic cleavage, i.e. they are nuclease resistant.

In one embodiment, the first agent is selected from an aptamer which binds to the STIM- 1 protein and an aptamer that binds to the CRACM1 protein. In one embodiment, the second agent is selected from an aptamer which binds to a leukotriene receptor e.g. a cysteine leukotriene type I receptor.

Proteins

In an embodiment, an agent of the invention, e.g. the first agent and/or the second agent is a protein. As used herein, the term "protein" refers to a polymer molecule comprising a plurality of amino acid residues linked via the peptide linkage, as will be appreciated by one skilled in the art. Peptides and polypeptides are encompassed with the term "protein".

In one embodiment, an agent, whether it be a first agent or a second agent as described above, is an isolated protein, peptide, antibody, antibody fragment or fusion protein. An

"isolated" or "purified" protein or biologically active fragment thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of the protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.

Methods of Screening

In one aspect of the present invention, there is provided a method of identifying a candidate agent for use in treating a disorder associated with mast cell activation in combination with a second agent which is an inhibitor of mast cell activation by leukotrienes, wherein the method comprises screening for a candidate first agent which inhibits a component of a CRAC channel pathway in mast cells, and detecting the effect of the candidate first agent on mast cell activation. In one embodiment, the step of detecting comprises measuring leukotriene secretion from a mast cell. In one embodiment, the leukotriene is LTC₄. Leukotriene secretion can be measured e.g. by enzyme immunoassay such as those supplied by Cayman Chemicals, Ann Arbor, Ml. In one embodiment, the method is carried out in vitro. In one embodiment, the method, prior to detection, comprises contacting a candidate first agent or plurality of candidate first agents with a cell which expresses the components of the CRAC channel pathway and measuring leukotriene secretion from the cell. In one embodiment, the cell is a mast cell, optionally a human mast cell.

In one embodiment, the candidate first agent is a CRACM1 antagonist. In one embodiment, the candidate agent is a STIM1 antagonist.

In one embodiment, the method further comprises screening for a candidate second agent which is an inhibitor of activation of mast cells by a cysteinyl leukotriene. In one embodiment, the method further comprises contacting the cell with a candidate second agent or plurality of candidate second agents. In one embodiment the candidate second agent is a cysteinyl leukotriene type I receptor antagonist. The method may further comprise detecting the effect on cell activation of the combined contacting by a candidate first agent and candidate second agent. The step of detecting may comprise administering an agent which is known to activate a mast cell and detecting the effect of the candidate first agent, and optionally the candidate second agent, on mast cell activation in response to the known agent. In one embodiment, the candidate first agent reduces or inhibits leukotriene secretion from the cell as compared to administration of the known agent alone. In one embodiment, the contacting of the candidate first agent and the candidate second agent together has a greater effect on inhibition of mast cell activation than the combined additive effect of sole administration of the candidate first agent and the sole administration of the candidate second agent. Such an effect may be measured in for example leukotriene secretion and/or Ca²⁺ influx.

In one embodiment, the known agent is thapsigargin or a cysteinyl leukotriene. In embodiments of the invention, the step of detecting further comprises detecting levels of other inflammatory mediators released by the cell e.g. histamine and cytokines.

In one embodiment, the activation of the cell is monitored using e.g. patch clamping. The patch clamping technique is in common use to monitor the flow of ions across a membrane (Neher E (1992) "Nobel lecture, Ion channels for communication between and within cells" Neuron. 8(4):605-12). A variant of the classical patch clamp that can be adapted to the present invention is the planar patch clamp, which uses a planar array of PDMS electrodes that mimic a classical glass electrode (Klemic et al. (2002), Biosensors and Bioelectronics 597-6040. Patch clamp devices are commercially available e.g. from Axon Instruments.

Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. High throughput methods of screening can be useful in identifying candidate first agents and second agents. Modulation of the CRAC channel activity and therefore mast cell activation can be detected, thereby identifying one or more agents that inhibit a component of the CRAC channel pathway.

Any available compound library can be screened in such a high throughput format. Many libraries of compounds are commercially available e.g. from the Sigma Chemical Company (Saint Louis, MO) and many can be custom synthesised.

Products and Kits

In one aspect of the present invention, there is provided a pharmaceutical product (i.e. for pharmaceutical use) comprising a first agent and a second agent as disclosed herein. The product may comprise the first and second agent as a single dosage form. Alternatively, the product may comprise the first agent and the second agent as separate dosage forms.

In a further aspect of the invention, there is provided a kit comprising a product comprising a first agent and a second agent as described herein.

Also included in the present invention, are products and kits comprising at least two agents which each inhibit a different component involved in the leukotriene synthesis and activation of mast cells.

Methods

The present invention includes a method of treating a disorder associated with mast cell activation comprising administering a therapeutically effective amount of a first agent as described herein. In embodiments of the present invention, the first agent is an inhibitor of a component of a CRAC channel in mast cells. In one embodiment, the method further comprises administering a second agent as defined herein in combination with the first agent. The combination may be for simultaneous, sequential or separate adminstration.

In one aspect of the present invention, there is provided use of a first agent for the manufacture of a medicament for the treatment of a disorder which is associated with mast cell activation, wherein the first agent is an inhibitor of a component of a CRAC channel in mast cells. In one embodiment, the first agent is for administration with a second agent which is an inhibitor of mast cell activation by a leukotriene. In one embodiment, the medicament is for the treatment of a disorder which is caused by or exacerbated by a local activation of mast cells.

The term "local activation of mast cells" as used herein is taken to mean non-systemic activation of mast cells. For example, "local activation of mast cells" may include mast cell activation within a particular tissue or portion of a tissue, for example in response to an agonist e.g. an allergen. As described above, it is believed that activation of mast cells in a tissue leads to activation of further mast cells in the local area (e.g. the same tissue) as a result of the positive feedback system following leukotriene release. The activation of further mast cells via this positive feedback may be exacerbated in tissues which have a poor blood supply, thereby preventing or reducing removal of leukotrienes from the tissue. Thus, in one embodiment, the disorder involves mast cell activation in a tissue or area of a subject's body which has a poor or reduced blood supply. Examples of local activation of mast cells include e.g. activation of mast cells in nasal polyp tissue and nasal mucosa.

Also included in the present invention, are method which comprises administering at least two agents which each inhibit a different component involved in the leukotriene synthesis and activation of mast cells, wherein the agents are administered simultaneously, sequentially or separately.

A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In one embodiment the patient is a mammal, and is preferably human. The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease. The term "treatment" may also include alleviating symptoms of a disorder.

In one embodiment, the method is for treating or alleviating the symptoms of mast cell related disorder e.g. an allergic disorder. As used herein, the term "allergic disorder" means a disease, condition or disorder associated with an allergic response against normally innocuous substances. These substances may be found in the environment e.g. indoor air pollutants or they may be non-environmental e.g. those causing dermatological or food allergies. Allergens can enter the body through a number of routes, including by inhalation, ingestion, contact with the skin or injection (including by insect sting). For the purpose of this invention, allergic disorders include any hypersensitivity which results from at least mast cell activation and that occurs upon re- exposure to the sensitizing allergen, which in turn causes the release of inflammatory mediators. Disorders which may be treated by the present invention include without limitation, allergic rhinitis (e.g., hay fever), sinusitis, rhinosinusitis, otitis media, nasal pruritus, insect sting reactions, latex reactions, urticaria, atopic dermatitis, eczema and asthma. The present invention also includes alleviating symptoms of such allergic disorders e.g. sneezing, nasal congestion, coughing, rhinorrhea, nasal pruritus, shortness of breath and chest tightness.

In an alternative embodiment, the disorder is an inflammatory disorder. In one embodiment, the disorder is asthma. As used herein, the term "asthma" means a pulmonary disease, disorder or condition characterized by reversible airway obstruction, airway inflammation, and increased airway responsiveness to a variety of stimuli. These paragraphs provide basis for claims specific to any one of the listed diseases, i.e. each recited disease or disorder may be included in a claim directed solely to a product, method or use relating to that, and only that, disease or disorder. In one embodiment, the method comprises the combined use of the first agent and the second agent to treat a disorder which is caused by mast cell activation. "Combined use" and "combination" in the context of the invention means the simultaneous, sequential or separate administration of the first agent as defined herein on the one hand and of the second agent as defined herein on the other hand.

As discussed herein, the use of a combination of the first agent and the second agent may be more effective than the effects of the administration of the two agents as monotherapies. In one embodiment, the combination of the first agent and the second agent may permit the use of lower dosages of one or more of the therapies and/or less frequent administration of the therapies to a subject with a mast cell related disorder. The ability to utilize lower dosages of a therapy and/or to administer the therapy less frequently may reduce the toxicity associated with the administration of the therapy to a subject without reducing the efficacy of the therapy in the prevention or treatment of a disorder. Also, the combination of the first agent and second agent may avoid or reduce adverse or unwanted side effects associated with the use of either agent alone.

In one embodiment, the combination may include a dosage of the first agent which is at least 10% less than a therapeutically effective amount of the first agent if used as a monotherapy i.e. when the agent is administered alone. In one embodiment, the combination comprises the first agent in a dosage at least 15% less than a therapeutically effective amount of the first agent if used as a monotherapy, e.g. the dosage of the first agent is reduced by at least 20%, 25%, 30%, 35%, 40%, 45% or 50%, 55%, 60% or more as compared to a therapeutically effective dosage of the first agent as a monotherapy i.e. when the agent is administered alone.

In one embodiment, the combination may include a dosage of the second agent which is at least 10% less than a therapeutically effective amount of the second agent if used as a monotherapy i.e. when the agent is administered alone. In one embodiment, the combination comprises the second agent in a dosage at least 15% less than a therapeutically effective amount of the second agent if used as a monotherapy, e.g. the dosage of the second agent is reduced by at least 20%, 25%, 30%, 35%, 40%, 45% or 50%, 55%, 60% or more as compared to a therapeutically effective dosage of the second agent as a monotherapy i.e. when the agent is administered alone. In one embodiment, the combination is for treating a subject who fails to respond to either the first agent or the second agent when said agent is administered alone.

In one embodiment, the first agent and the second agent are administered by separate routes.

In one embodiment, the first agent and the second agent may be administered simultaneously. Simultaneous administration includes for example (1 ) the simultaneous uptake of two separate dosage forms containing the first agent in one of the dosage forms and the second agent in the other dosage form or (2) pharmaceutical compositions containing both active ingredients in one single dosage form (fixed unit dose form).

In one embodiment, the at least two agents which each inhibit a different component involved in the leukotriene synthesis and activation of mast cells may be administered simultaneously. Simultaneous administration includes for example (1 ) the simultaneous uptake of two separate dosage forms or (2) pharmaceutical compositions containing both active ingredients in one single dosage form (fixed unit dose form).

"Combined use" and "combination" in the context of the invention also includes a pharmaceutical product comprising both the first agent and the second agent, or at least two second agents, as discrete separate dosage forms, in separate containers or e. g. in blisters containing both types of drugs in discrete solid dosage units, e.g. in a form in which the dosage units which have to be taken together or which have to be taken within one day are grouped together in a manner which is convenient for the patient. Said pharmaceutical product itself or as a part of a kit may contain instructions for the simultaneous, sequential or separate administration of the discrete separate dosage units, to a patient in need thereof.

Sequential administration in the context of the invention means the administration of the first agent as defined herein on the one hand and of the second agent as defined herein on the other hand in separate dosage forms within less than 12 hours, e.g. within less than one hour, or e.g. within 5 minutes or less.

Sequential administration in the context of some aspects of the invention means the administration of at least one agent which inhibits a component involved in the leukotriene synthesis and activation of mast cells, and of at least one agent which inhibits a different component involved in the leukotriene synthesis and activation of mast cells in separate dosage forms within less than 12 hours, e.g. within less than one hour, or e.g. within 5 minutes or less.

In one embodiment, the first agent and the second agent may be administered separately. Separate administration within the context of the invention means the administration of the first agent on the one hand and of the second agent on the other hand in separate dosage forms within 12 hours or more.

The pharmaceutical composition of the present invention may be prepared by mixing the first agent with the second agent. In this embodiment, the first agent and the second agent can a) in a first step be mixed and subsequently processed with pharmaceutically acceptable auxiliaries and/or excipients and finally pressed to tablets or caplets or b) in a first step separately be processed with pharmaceutically acceptable auxiliaries and/or excipients to give granules or pellets containing each only one of the two active ingredients; the pellets or granules for their part then can be mixed in an appropriate ratio and either be pressed, optionally with further pharmaceutically acceptable auxiliaries and/or excipients, to give for example, tablets or caplets, or can be filled in more or less loose form in capsules.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The one or more agents of the invention, e.g., the first agent and/or second agent, may be administered to the patient via a number of routes, which may depend on the nature of the disorder to be treated.

In one embodiment, one or more agents of the invention, e.g. the first agent and/or the second agent may be administered via inhalation in the same product or as separate products. Methods of administering pharmaceuticals to the lung by inhalation are well- known to those skilled in the art. The design of suitable inhaler devices is described, for example in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985, p. 181 -182, incorporated herein by reference.

In one embodiment, one or more agents as described herein are for oral administration. In one embodiment, the first agent is for oral administration. The second agent may be also be for oral administration, e.g. in a product which comprises both the first agent and the second agent. Alternatively, the first agent is comprised within a first product and the second agent is comprised within a second product.

Suitable oral administration forms include solid dosage forms. Solid dosage forms for oral administration include capsules, tablets (also called pills), powders and granules. In such solid dosage forms, the active compound(s) is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules and tablets, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.

Suitably, the oral formulations may contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkylamine oxides; bile acid and salts thereof (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface active agents, such as sodium laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts.

The agents of the invention, e.g. the first and/or second agent may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

In one embodiment, the one or more agents of the invention, e.g. the first agent and/or the second agents of the invention are for administration in liquid dosage form. The agents may be administered as a single product or as separate products. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active agents, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.

Also included in the present invention is a pharmaceutical formulation comprising a first agent and, in some embodiments, a second agent as described herein. In embodiments, the formulation is a composition comprising the agent(s) and a pharmaceutically acceptable diluent, carrier or excipient. Such formulations may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The formulations may also include antioxidants and/or preservatives. As antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid. Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.

The product of the disclosure may be presented as solids in finely divided solid form, for example they may be micronised. Powders or finely divided solids may be encapsulated.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the agent(s) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration (referred to herein as a "therapeutically effective amount"). The selected dosage level will depend upon the activity of the particular agent, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the agent(s) at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

In one embodiment, the dosage levels of a second agent in a combined preparation of the first and second agents may be less than the dosage level of the second agent as currently prescribed. For example, in some embodiments, the second agent is montelukast sodium. Montelukast sodium is currently supplied in 10mg tablets for adults, (it is also available in other dosage forms). In one embodiment of the present invention, the montelukast may be administered in a dosage form which is less than 10mg e.g. 1 mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg or 9mg when for adult use.

The methods of the present invention may comprise administering the first agent, optionally together with the second agent at least once a day, e.g. every four hours. In one embodiment, the methods comprise administering the agent(s) at the onset of mast cell activation e.g. when a patient starts to experience symptoms e.g. sneezing or tightness of breath. Alternatively, the agent(s) may be for prophylaxis administration and are taken before symptoms are experienced.

Examples

Materials and Methods

Cell Culture and Transfection- Rat basophilic leukemia (RBL-1 ) cells (a mast cell line) and HEK293 were bought from ATCC. RBL-1 cells were cultured (37 C, 5% CO₂) in Dulbecco's modified Eagle medium with 10% fetal bovine serum, 2 mM L-glutamine and penicillin-streptomycin, as previously described (Bakowski, D., Glitsch, M. D., and Parekh, A. B. (2001 ) Journal of Physiology (Lond.) 532, 55-71 ). Cell transfection was as described in Moreau, B., Straube, S., Fisher, R. J., Jr., P. J. W., and B., P. A. (2005) Journal of Biological Chemistry 280, 8776-8783. Briefly, HEK293 cells were cultured in RPMI with 10% fetal bovine serum, 2 mM L-glutamine and penicillin-streptomycin. HEK293 cells were cotransfected with cDNA encoding human cysteinyl leukotriene receptor type I (Origene) using the lipofectamine method. RBL-1 cells were transfected with RNAi having the sequence of SEQ ID. No. 1 , against STIM1 together with enhanced GFP using the nucleofection method (Amaxa). Alternatively, RBL-1 cells were transfected with RNAi against the cysteinyl leukotriene type I receptor purchased from Dharmacon together with enhanced GFP using the nucleofection method (Amaxa). Cells were passaged onto glass coverslips and used 24-48 h after plating for patch clamp or Ca²⁺ imaging experiments.

Peritoneal mast cells were isolated from female, Sprague-Dawley rats, weighing approximately 30Og. The animals were sacrificed according to 'Schedule 1 ', (carbon dioxide overdose and neck dislocation). Immediately, 100-15OmI of sterile HEPES buffer (in mM: NaCI 150, KCL 5.6, HEPES 10, NaOH 1 .5, MgCI₂ 1 , CaCI₂ 2, glucose 10 1 g/l BSA, pH 7.4) was injected into the peritoneal cavity. The abdomen was massaged for 2 minutes and then the buffer removed. This was centrifuged at 20Og for 10 minutes. The pellet was resuspended in DMEM, triturated and plated onto glass coverslips and used within 6 hours of isolation. Human mast cells were obtained from nasal tissue with full patient consent. Nasal tissue was minced with fine scissors, trypsinized, mechanically agitated, centrifuged twice at 1000g and then resuspended in DMEM. Cells were used within 3-6 hours of isolation. Ic_RAc recordings- Patch-clamp experiments were conducted in the tight-seal whole-cell configuration at room temperature (20-25 ⁰C) as previously described (Bakowski, D., Glitsch, M. D., and Parekh, A. B. (2001 ) Journal of Physiology (Lond.) 532, 55-71 ). Sylgard-coated, fire-polished pipettes had d.c. resistances of 4.2-5.5MΩ when filled with standard internal solution that contained (in mM): 145 mM cesium glutamate, 8 mM NaCI, 1 mM MgCI₂ 1 , 2mM Mg-ATP, , 10 mM HEPES, pH 7.2 with CsOH. For some experiments, this was supplemented with 0.1 mM Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N',-tetraacetic acid (EGTA) and 30 μM lnsP₃.

In the experiments shown in Figures 2B and C (see example 3), the pipette solution contained reduced EGTA (from 10 to 0.1 mM). In the experiment shown in Figure 7C, 1 OmM EGTA and 4.6mM CaCI₂ was added to the standard solution such that free Ca²⁺ was clamped at ~140nM. A correction of +10 mV was applied for the subsequent liquid junction potential that arose from this glutamate-based internal solution. Extracellular solution contained (in mM): 145 mM NaCI , 2.8mM KCI, 1 OmM CaCI₂, 2mM MgCI₂, 1 OmM CsCI, 1 OmM D-glucose, 1 OmM HEPES, pH 7.4 with NaOH. I_CR_AC was measured by applying voltage ramps (-100 to +100 mV in 50 msec) at 0.5 Hz from a holding potential of 0 mV as previously described (Gilabert and Parekh, (2000) EMBO Journal 19 (23), 6401 -6407 and Parekh et al (1997) Ce// 89, 973-981 ). Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitised at 100 μs. Currents were normalised by dividing the amplitudes (measured from the voltage ramps at - 80 mV) by the cell capacitance. Capacitative currents were compensated before each ramp by using the automatic compensation of the EPC 9 -2 amplifier. All leak currents were subtracted by averaging the ramp currents obtained just before I_CR_AC had started to develop, and then subtracting this from all subsequent currents.

Ca²⁺ imaging-Ca²⁺ imaging experiments were carried out at room temperature using the IMAGO CCD camera-based system from TILL Photonics, as described previously (Moreau, B. et al, (2005) Journal of Biological Chemistry 280, 8776-8783). Cells were alternately excited at 356 and 380 nm (20 msec exposures) and images were acquired every 2-3 seconds. Images were analysed offline using IGOR Pro for Windows. Cells were loaded with Fura 2-AM (1 μM) for 40 minutes at room temperature in the dark and then washed three times in standard external solution of composition (in mM) NaCI 145, KCI 2.8, CaCI₂ 2, MgCI₂ 2, D-glucose 10, HEPES 10, pH 7.4 with NaOH. Cells were left for 15 minutes to allow further desertification. Ca²⁺-free solution had the following composition (in mM) NaCI 145, KCI 2.8, MgCI₂ 2, D-glucose 10, HEPES 10, EGTA 0.1 , pH 7.4 with NaOH). The rate of Ba²⁺ influx was obtained by measuring the initial slope of the fluorescence rise following readmission of Ba²⁺ to cells with depleted stores. Ca²⁺ signals are plotted as ΔR, which denotes the change in 356/380 nm ratio.

/.TCrFollowing stimulation of attached cells with thapsigargin, the supernatant was collected and LTC₄ levels were measured by enzyme immunoassay (Cayman Chemicals, Ann Arbor, Ml) as described previously (Chang, W. C, and Parekh, A. B. (2004) Journal of Biological Chemistry 279, 29994-29999). In brief, 50 μl of supernatant, leukotriene C₄ acetylcholinetransferase and leukotriene C₄ antiserum were added to each enzyme immunoassay well. Following incubation for 18 hr at room temperature, the wells were emptied and rinsed five times with enzyme immunoassay washing buffer. 200 μl of Ellman's reagent (prepared fresh) was added to each well and the plate was placed on an orbital shaker for 1 .5 hr in the dark. Plate absorbance was measured at a wavelength of 405 nm.

Histology

The human nasal polyps surgically removed with full patient consent were fixed in 4% paraformalydehyde for 12 hours. The polyps were washed in 1 OmM PBS then transferred to a cryoprotectant (20% sucrose in PBS) until the polyps sank. 5 micron sections were cut using a Reichert-Jung Cryocut 1800 and mounted on gelatine-coated glass slides and air dried. The sections were immersed in 0.5% acetic acid for 2 minutes and then transferred to 0.5% toluidine blue dissolved in 0.7N HCI (pH 0.5) for 15 minutes. The sections were rinsed with distilled water, blotted bry and immersed in xylene for 2 minutes, then mounted in DPX. They were viewed on a Leica DMRB, and captured with a Nikon DXM 1200 digital camera.

Data are presented as the mean±sem. Statistical significance was determined using a student t test. ^* denotes p<0.05 and ^** p<0.01 .

Example 1 : Leukotrienes as paracrine signals.

To test whether leukotrienes can operate as paracrine signals, cysteinyl leukotriene release from one group of RBL-1 cells was stimulated, the supernatant collected and then applied to a second group of RBL-1 cells pre-loaded with a Ca²⁺ -sensitive fluorescent dye, fura 2 since a Ca²⁺ rise is a key trigger for mast cell activation. A schematic representation of the protocol for collecting the supernatant is shown in Figure 1 1 . Cells in the first group were stimulated by application of the sarco-endoplastic reticulum Ca²⁺ ATPase inhibitor thapsigargin in Ca²⁺ -free solution for 4 minutes. Thapsigargin depletes the intracellular Ca²⁺ stores and thus opens CRAC channels in the plasma membrane (Parekh A. B and Putney J. W. (2005) Physiological Reviews 85, 757-814), leading to robust secretion of LTC₄. As the cells were bathed in Ca²⁺ -free solution, no Ca²⁺ influx occurred. Thereafter, the cells were washed extensively in Ca²⁺ - free solution (without thapsigargin) and the supernatant was collected and stored on ice (control supernatant). Cells were then exposed to Ca²⁺ -containing external solution an after five minutes the supernatant was collected (stimulated supernatant).

As shown in Figures 1 A and 1 B, application of supernatant from stimulated cells evoked a Ca²⁺ rise in fura 2-loaded RBL-1 cells (averaged data from 103 cells) (Figure 1 A, open circles) whereas supernatant from resting (i.e. non-stimulated) control supernatant (to which CaCI₂ had been added to bring free Ca²⁺ back to 2mM) did not evoke a Ca²⁺ rise in fura 2-loaded RBL-1 cells within 300 seconds of recording (Figure 1 B, filled circles), demonstrating that this solution contained insufficient levels of thapsigargin or paracrine signal to illicit a response (n=73 cells) (Figure 1 B, filled circles). The effects of active supernatant were blocked by the cysteinyl leukotriene receptor antagonist SR-2640 (1 - 10 μM) (n=103 cells) (Figure 1 A) and also by the structurally distinct leukotriene receptor blocker montelukast (10OnM) (n=145 cells) as well as the 5-lipoxynenase inhibitor zileuton (10μM) (n=91 cells) (Figure 1 B).

Figure 1 C shows that blocking the generation of cysteinyl leukotrienes by pre-treatment of those cells used as the source of active supernatant by knocking down expression of the 5-lipoxygenase enzyme using an RNAi approach or zileuton (1 μM and applied 10 minutes prior to stimulation with thapsigargin) resulted in an ineffective supernatant, as seen by lack of Ca²⁺ response in fura 2-loaded cells.

Example 2: Effect of store-operated Ca²⁺ influx on neighbouring cells To gain insight into the spatial profile of leukotriene-dependent paracrine signalling, CRAC channel activation was restricted to just one cell by dialysing it with lnsP₃ in a weak intracellular Ca²⁺ buffer via a patch pipette to evoke store-operated Ca²⁺ influx whilst recording Ca²⁺ signals in adjacent cells. Figure 1 D shows a brightfield image of this recording condition. Activation of I_CR_AC resulted in a robust cytoplasmic Ca²⁺ rise in the patched cell, and this triggered a cytoplasmic Ca²⁺ rise in adjacent cells (Figure 1 E). Pre-treatment with montelukast had no effect on the cytoplasmic Ca²⁺ rise in the patched cell but suppressed the Ca²⁺ rise in adjacent ones (Figure 1 F). Hence leukotriene secretion from one cell activates nearby cells, demonstrating a role as a local paracrine signal.

Example 3

HEK293 cells do not express cysteinyl leukotriene type I receptors and therefore unable to respond to supernatant (see Figure U). These cells were therefore transfected with the gene encoding the cysteinyl leukotriene type I receptor and supernatant subsequently applied. Robust Ca²⁺ signals were now generated (Figure 2A (n=14 cells)), which were similar in pattern to those seen in the mast cells (compare with Figures 1 A and B). The responses to supernatant in the transfected cells were suppressed by pre-treatment of the cells with montelukast (Figure 2A). If the main component of supernatant is a cysteinyl leukotriene (or combination of cysteinyl leukotrienes), acute application of leukotrienes should evoke Ca²⁺ signals in resting mast cells and that the signals should mimic supernatant. As shown in Figure 2B, this prediction is met.

Application of 160 nM LTC₄ evoked oscillatory cytoplasmic Ca²⁺ signals that were similar in pattern to supernatant. Similar responses were observed when LTD₄ was applied instead (Figure 2B). The leukotrienes were reconstituted in ethanol but the final solvent control (0.1 % ethanol) was ineffective (Figure 2B). Cysteinyl leukotriene responses were dose-dependent in that application of 80 nM LTC₄ evoked a small Ca²⁺ signal (Figure

2C) and 0.1 nM failed to trigger a detectable response. Responses to LTC₄ were fully suppressed by montelukast (Figure 2C). Finally, following desensitization of the ATP response, LTC₄ was still able to evoke a prominent cytoplasmic Ca²⁺ rise (Figure 2D), ruling out an action of P2Y receptors.

Example 4

To see whether leukotrienes functioned as paracrine signals in primary mast cells, RBL- derived supernatant was applied onto fura 2-loaded acutely isolated rat peritoneal mast cells. A clear and sustained Ca²⁺ rise occurred (Figure 3A) showing supernatant releases Ca²⁺ from intra-cellular stores and trigger store-operated Ca²⁺ entry (n=79 cells) which was suppressed by either pre-treatment with SR-2640 or montelukast (Figure 3A (n=43, SR-2640 n=65 montelukast)). Stimulation with thapsigargin resulted in secretion of LTC₄ from these cells (data not shown). Hence primary peritoneal mast cells generate LTC₄ and express functional cysteinyl leukotriene type I receptors, consistent with the idea that leukotrienes are effective paracrine signals.

Example 5

Experiments were designed to see whether modest stimulation of RBL cells resulted in secretion of sufficient levels of leukotriene to activate Ca²⁺ signals in resting cells. Cells were stimulated in the presence of different external Ca²⁺ concentrations to vary the extent of Ca²⁺ entry. Graded Ca²⁺ influx occurs when cells are stimulated in the presence of 0.25-2 mM external Ca²⁺ In the presence of 0.25 mM Ca²⁺, the rate of rise and the extent of the cytoplasmic Ca²⁺ signal is -30% and 50% of those seen in 2 mM Ca²⁺ (Chang et al, 2008) Journal of Biological Chemistry, 283, 4622-4631 ). CRAC channels were stimulated in the presence of 0.25 mM, 0.5 mM or 2 mM external Ca²⁺, the supernatant collected, Ca²⁺ added to the supernatant make the final Ca²⁺ concentration 2 mM and then applied to resting cells loaded with fura 2.

As shown in Figure 4A-C, larger responses were observed when cells were stimulated in the presence of higher Ca²⁺ concentrations but even the modest Ca²⁺ influx that occurred in 0.25 mM Ca²⁺ still evoked significant leukotriene secretion. Importantly, montelukast suppressed the responses regardless of stimulus intensity. Hence paracrine signalling by cysteinyl leukotrienes is maintained following modest levels of Ca²⁺ influx.

Allergic responses like type I immediate hypersensitivity have two kinetically distinct phases: an initial component that develops rapidly after mast cell stimulation and lasts for 10-20 minutes and this is followed by a delayed phase that can last for a few hours. To see whether cysteinly leukotrienes can contribute to both phases, cells were stimulated with thapsigargin for different periods of time, the supernatant collected and then its ability to trigger Ca²⁺ responses in resting cells was tested. Results are summarised in Figures 4D-G.

Stimulation for up to 25 minutes resulted in supernatant that elicited robust Ca²⁺ signals in fura 2-loaded resting cells (Figure 4D-F). Responses were also obtained after 40-60 minutes stimulation, although now there was more variability in the time course of the response on a cell to cell basis (Figure 4G). Cells deteriorated after ~ an hour in thapsigargin and it was therefore not possible to test longer times. Over the entire time range tested, the ability of supernatant to evoke a Ca²⁺ signal in fura 2-loaded cells was fully prevented by pre-treating the latter with montelukast (one example shown in Figure 4F). The results establish that cysteinyl leukotrienes function as powerful paracrine signals for several tens of minutes and thus can contribute both to early and the initial stages of the delayed phase of type I immediate hypersensitivity.

Example 6

The signalling pathway through which activation of cysteinyl leukotriene type I receptors triggers Ca²⁺ signals in mast cells was investigated. Fura 2-loaded cells were preheated with the phospholipase C inhibitor U73122 and active supernatant was subsequently applied. The Ca²⁺ signal was suppressed in response to active supernatant (Figure 3B with U73122), whereas the inactive analogue U73433 did not (data not shown). Hence the cysteinyl leukotriene receptor couples to the phospholipase C pathway, generating lnsP₃ Consistent with this, stimulation with active supernatant in Ca²⁺ -free solution resulted in Ca²⁺ release from intracellular stores (Figure 3D). These stores were lnsP₃-sensitive because pre-treatment with thapsigargin, which depletes the lnsP₃-mobilizable store, prevented supernatant from evoking a response (Figure 3B). Using ionomycin to assess the amount of stores intracellular Ca²⁺, it was found that supernatant (applied in Ca²⁺ -free external solution) reduced store content by 36.4±2.5% (Figure 3C (n=77 for control and 86 for supernatant)). Ionomycin (5mM) was also applied in Ca²⁺ -free solution. In accordance with this, supernatant triggered store- operated Ba²⁺ influx (Figure 3D) (n= 45 cells) and this was significantly less than that seen following more substantial store deletion with thapsigargin (Figure 3E) (approximately half).

In accordance with a loss in store Ca²⁺ content, both supernatant and LTC₄ triggered store-operated Ba²⁺ influx (Figures 6A). Low concentrations of Gd³⁺ in the mM range are widely used to block CRAC channels selectively. Ba²⁺ entry in response to LTC₄ was suppressed by a low concentration of Gd³⁺ (1 mM; Figure 6A and B), suggesting LTC₄ was able to activate CRAC channels. To test this directly, whole cell patch clamp experiments were carried out to record the CRAC current. Initial experiments using Ca²⁺ as the charge carrier failed to reveal a store-operated current to LTC₄. This is not unexpected since the agonist only partially depletes the stores and Ba²⁺ influx to LTC₄ was significantly less than that evoked by thapsigargin (51 .4±4%). The size of I_CR_AC was amplified by measuring the current in divalent-free external solution where Na⁺ is the charge carrier.

Under these conditions, application of 160 nM LTC₄ resulted in the development of I_CR_AC (Figure 6C). The current-voltage relationship revealed the key characteristics of I_CR_AC, namely inward rectification and a positive reversal potential ~ +60 mV, very close to the predicted Na⁺ equilibrium potential. The amplitude of the current was -3.1 ±0.4 pA/pF, which was significantly smaller than the size of I_CR_AC evoked by thapsigargin (-10.3±-0.2 pA/pF), consistent with the more modest store depletion by LTC₄. The current declined in the continuous presence of LTC₄ but could be rescued partially by subsequent application of thapsigargin (Figure 6C). The current-voltage relationship for the thapsigargin-evoked I_CR_AC (labelled 2 in Figure 6D) was identical to that elicited by LTC₄ (labelled 1 in Figure 6D). Following full activation of I_CR_AC by thapsigargin (Figure 6E), application of LTC₄ failed to generate a current, suggesting both stimuli converge on the same pool of channels.

Example 7: Method to determine whether ATP might contribute to Ca²⁺ rise evoked by supernatant It has been reported previously that ATP is released from RBL cells and evokes, via P2Y receptors, Ca²⁺ signals in adjacent cells (Osipchuk, Y. and Cahalan, M. D (1992) Nature 359, 241 -244). Since cysteinyl leukotriene receptors can also respond to pyrimidinergic ligands (Mellor, E. A et al (2001 ) P.N.A.S USA 98, 7964-7969), experiments were designed to assess whether ATP might contribute to the Ca²⁺ response evoked by supernatant. The RBL-1 cells were pre-treated with 10μ SR-2640 (a concentration that suppressed the effects of the supernatant) and 10OmM (a maximal concentration) of ATP was applied to RBL-1 cells and the Ca²⁺ signal measured.

As shown in Figure 1 G, acute application of 10OmM ATP to RBL-1 cells evoked a Ca²⁺ signal that was unaffected by pre-treating cells with 1 OmM SR-3640 (a concentration that suppressed the effects of supernatant (see Figure 1 A)) (n=54 for control and 61 for SR-2640). As shown in Figure 1 H, the Ca²⁺ rise to application of 50 mM ATP, a concentration that is just over threshold, was unaffected by SR-2640 (n=43 for control and 30 for SR-2640). Similar results were observed with montelukast (data not shown). Example 8: Examining whether supernatant results in cross-desensitization between pyrimidinerqic receptors and cysteinyl receptor.

The effects of pyrimidinergic agonists acting on cysteinyl receptors are suppressed by leukotriene receptor antagonists (Mellor at al, PNAS USA, 98, 7964-7969). Pyrimidinergic agonists and leukotrienes show cross-desensitization when acting on cysteinyl leukotriene receptors and it was examined whether this was the case with supernatant. 300μM ATP evoked no further Ca²⁺ rise when applied after 100 μM ATP

(Figure 1 1), showing desensitization of the ATP response. However, subsequent application of supernatant to the same cells evoked a clear Ca²⁺ rise (Figure 1 1) (n=53 cells), demonstrating a lack of cross desensitization.

In addition, HEK293 cells were used as a bioassay to see whether RBL cells supernatant contained enough ATP to trigger Ca²⁺ signals. HEK293 cells express P2Y but not cysteinyl leukotriene receptors. Application of supernatant to fura-2 loaded HEK293 cells failed to evoke a Ca²⁺ rise, although the cells subsequently responded to ATP (Figure U). Since P3Y receptors on RBL-1 and HEK293 cells have similar EC₅₀ ¹S for ATP (70μM and 40μM respectively, data not shown) and HEK293 cells do not respond to RBL cell-derived supernatant then clearly insufficient ATP is in the supernatant to activate the RBL cells.

Example 9: CRAC channel activity

Store-operated Ca²⁺ influx is critically dependent on the ER resident protein STIM1 , which functions as the sensor linking store depletion to the opening of plasmalemmal CRAC channels. RNAi knockdown of STIM1 resulted in a substantial reduction of I_CR_AC (Figure 5A-C) and the associated store-operated Ca²⁺ signal (Figure 5D). The pattern of the Ca²⁺ signal to supernatant was also changed by knocking down STIM1 (Figure 5E- H). The number of Ca²⁺ oscillations measured over a 400 second period fell (Figure 5E versus 5F, aggregate data is summarised in Figure 5G) and the interval between the first and second Ca²⁺ oscillation rose significantly in the STI M 1 -depleted cells (Figure 5H).

Example 10: Positive feedback of LTC₄ production

RBL-1 cells were stimulated with thapsigargin and LTC₄ secretion was measured in the absence and then presence of SR-3640 or montelukast. If positive feedback occurred, then one would predict the leukotriene receptor antagonists to reduce LTC₄ secretion. Figures 8A and B show that both antagonists dose-dependently reduced LTC₄ secretion. SR-2640 and montelukast do not block store-operated Ca²⁺ influx ruling out an inhibitory action on the CRAC channels themselves or the subsequent cytoplasmic Ca²⁺ rise. These results suggest therefore is that LTC₄ secretion is a positive feedback cycle, ensuring regenerative release. Because thapsigargin should fully deplete the stores and thus activates CRAC channels maximally, leukotrienes are unlikely to enhance further LTC₄ production by mobilising intracellular Ca²⁺. Since protein kinase C increases LTC₄ secretion, it is possible that cysteinyl leukotriene receptor activation enhances thapsigargin-driven LTC₄ through a similar pathway. An alternative explanation is that cysteinyl leukotriene antagonists block the enzyme 5-lipoxygenase, in addition to inhibiting the cysteinyl leukotriene receptor.

Inspection of the montelukast-inhibition curve in Figure 8B reveals a biphasic curve, with -20% block at 100 nM and -70% block at 10μM. Both concentrations are well below that reported for block of the 5-lipoxygenase (Ranures, R et al, Biochemical and Biophysical Communications, 324, 815-821 ). Nevertheless, positive feedback in other ways was considered. A critical step in LTC₄ production is migration of Ca²⁺ -dependent protein kinase Ca isozyme to the plasma membrane. Application of active supernatant to resting cells resulted in modest but clear translocation of protein kinase Ca to the plasma membrane (Figures 8C and D), consistent with the small Ca²⁺ rise that it triggered. Hence supernatant is able to activate the two critical early steps in cysteinyl leukotriene production (a Ca²⁺ rise and protein kinase Ca translocation), supporting the notion of a positive feedback step.

Example 1 1 : Pattern of Ca²⁺ signal

Knocking down STIM1 reduces CRAC channel activity and alters the pattern of Ca²⁺ signal evoked by supernatant. Figure 5A shows a comparison of the time course of Ic_RAc development of a control cell (filled circles) and one treated with RNAi and STIM1 (open circles).

Figure 5B, I-V curves from panel A, taken once the currents had peaked, are depicted. Figure 5C shows the results of aggregate data from 6 control cells and 7 cells treated with RNAi to STIM1 , indicating a reduction in l_CRAcwhen STIM1 is knocked down.

Figure 5D shows that store-operated Ca²⁺ influx, measured with fura 2, is substantially reduced in cells exposed to RNAi directed against STIM1 (n=86 for control and 77 for STIM1 knock down). Cells were treated with thapsigargin in Ca²⁺ -free solution to 10 minutes and then 2mM external Ca²⁺ readmitted. Only the Ca²⁺ influx signal is shown.

Figure 5E shows the series of Ca²⁺ spikes in a control cell in response to active supernatant, whereas Figure 5F shows that the number of Ca²⁺ spikes was reduced in cells treated with RNAi to STIM1 . Figure 5G shows the comparison of the number of spikes on a 400 second time frame for control cells and those in which STIM1 had been knocked down. The number of control spikes is taken as 100%. Figure 5H indicated the time interval between the first and second Ca²⁺ spike following application of supernatant in control cells and those treated with RNAi to STI M1 .

Example 12: RNAi knock down of the cvsteinyl leukotriene type I receptor. A mixture of siRNA molecules were used to knock out the cysteinyl leukotriene type I receptor. The siRNA molecules were obtained using SmartPool from Dharmacon.

Strand 1 :

Sense: UAUCAUAUUCAACGAAGCUAUU Antisense: 5'-P UAGCUCGUUGAAUAUGAUAUU

Strand 2:

Sense: GUACAUUGCCUCUCCGUGUUU

Antisense: 5'-P ACACGGAGAGGCAAUGUACUU

Strand 3:

Sense: GUGGGUUUCUUUGGCAAUAUU Antisense: 5'-P UAUUGCCAAAGAAACCCACUU

Strand 4: Sense: ACCUAUGCCUUAUAUGUUAUU

Antisense: 5'-P UAACAUAUAAGGCAUAGGUUU

As shown in Figure 9A-B, the ability of supernatant to elicit a Ca2+ response is suppressed by RNAi knockdown of the cysteinyl leukotriene type 1 receptor. The fraction of cells responding to supernatant was dramatically reduced in the RNAi-treated cells. The control was a nonsense siRNA. Example 13: Clinical Relevance

LTC₄ secretion was measured in surgically removed nasal tissue from patients with either severe allergic rhinitis or nasal polyposis. In both cases, stimulation with thapsigargin (2mM for 4 minutes) evoked a significant increase in LTC₄ secretion (Figures 8E and F), which was slightly but consistently larger in nasal polyp tissue (each experiment was carried out in triplicate). LTC₄ production in human polyps following thapsigargin exposure was suppressed by removing external Ca²⁺ or by inhibiting protein kinase C with GO-6983 (1 mM 15 minutes pre-treatment) (Figure 8G), implicating major role for Ca²⁺ influx followed by protein kinase C activation (Figure 8G), which suggests that the pathway linking CRAC channel activation to LTC₄ secretion in human mast cells is identical to that in RBL-1 cells. This experiment was carried out in triplicate. LTC₄ secretion following CRAC channel activation in human tissue was significantly reduced by SR-2640, consistent with a positive feedback cascade in maintaining leukotriene production (data not shown). Application of supernatant from RBL-1 cells evoked robust sustained Ca²⁺ signals in fura 2-loaded human mast cells (Figure 8H) from a patient with nasal polyposis and these were suppressed by blocking cysteinyl leukotriene type I receptors with pre-treatment of either montelukast or SR-2640 (Figure 8H). Similar results were obtained in samples from 3 patients (14-23 cells).

Example 14: Determining whether paracrine signalling between mast cells involving cvsteinyl leukotrienes operate in vivo. If such signalling occurs in vivo then three conditions need to be satisfied. First, mast cells in vivo should be close enough for paracrine signalling to take place. Second, the diffusion coefficient for LTC₄ should be sufficiently high for it to couple cells over the distances encountered in vivo and third, cysteinyl leukotriene receptors should have high affinity for ligand so that dilution in the interstitial fluid does not reduce the concentration to levels too low to elicit a response. Experiments were carried out to determine whether these conditions were satisfied. The results are summarised in Figure 10.

Slices cut through acutely isolated human nasal polyps were stained with the aniline dye toluidine blue to identify mast cells (arrows). Sections from two different patients are shown in Figures 1 OA and B and reveal that mast cells could often be located within 20-

50 mm of each other, a distance short enough to match that seen in experiments conducted on cultured mast cells. To estimate the rate of diffusion of LTC₄, I_CR_AC was activated in one mast cell and measured the spread of the cytoplasmic Ca²⁺ signal through the population (Figure 10C).

The patched cell was dialysed with lnsP₃ but clamped at +50 mV. Although CRAC channels activate, the driving force for Ca²⁺ influx at this potential is very small. Upon hyperpolarisation to -80 mV, robust Ca²⁺ influx occurred which drove the release of LTC₄. This resulted in an intercellular Ca²⁺ wave spreading through the cell population (Figure 10D). By measuring the delay before Ca²⁺ started to rise in the non-patched cells as a function of their distance from the patched cell, a latency of ~ 50 seconds was calculated before cells 50-60 mm away responded. The diffusion coefficient of LTC₄ was estimated by plotting the measured diffusion coefficients from the literature against the corresponding molecular weights (Figure 10E). With a molecular weight of 626, the interpolated diffusion coefficient is ~ 400 μm²/s. This figure is an approximation but nonetheless a time of -0.9 seconds for LTC₄ was calculated to diffuse a distance of 50 μm.

Although this is considerably faster than the spread of the intercellular Ca²⁺ wave in Figure 10D, it is not unexpected because the latter reflects not only the diffusion of LTC₄ but also the synthesis and secretion of the leukotriene from the patched cell as well as subsequent lnsP₃ production and diffusion to the stores in the target cell. To estimate receptor affinity, different concentrations of LTC₄ were applied and the ability of mast cells to respond was measured (Figure 10F-H). Even concentrations as low as 200 pM were able to activate Ca²⁺ signals in all cells. 2 pM LTC₄ triggered a Ca²⁺ signal in almost 25% of the cells, revealing that the cysteinyl leukotriene type I receptor has high affinity for its ligand.

Example 15: Montelukast blocks LTC^responses with an IC^of around 7nM

RBL mast cells were loaded with fura 2 to measure cytoplasmic Ca²⁺ concentration. 160 nM LTC₄ was applied to the cells in the presence of different concentrations of montelukast. As shown in Figure 12, montelukast blocks responses to 160 nM LTC₄ in a concentration dependent manner. A substantial reduction in the cell response to LTC₄ was seen using 1 OnM montelukast. The IC₅₀ of montelukast was shown to be approximately 7nM. Example 16: Effect of combination of blockade of CRAC channel and cysLTI receptors. La³⁺ is an effective inhibitor of CRAC channels. The application of 1 μM La³⁺ resulted in approximately 50% blockade of CRAC channels (data not shown). Supernatant from mast cells treated with 1 μM La³⁺ was applied to a second population of mast cell and was able to evoke responses in a second pool of fura 2-loaded mast cells (see Figure 13a). Application of low concentrations of montelukast to the second population of mast cells resulted in a blockade in the response of the mast cells to the supernatant (Figure 13b and 13c). Figure 13c shows that the response of the mast cells to the supernatant was almost completely abolished by application of 7.5nM montelukast. Controls are shown in Figures 13 d to g.

The results indicate that a combination of partial CRAC channel blockade and partial cysLTI receptor blockade results in abolition of paracrine signalling and mast cell response.

Example 17: Effect of combination of blockade of 5-lipoxqenase and cvsLTI receptors. Cysteinyl leukotriene release from one group of RBL-1 cells was stimulated, the supernatant collected and then applied to a second group of RBL-1 cells pre-loaded with a Ca²⁺ -sensitive fluorescent dye, fura 2. Figure 14 shows activation of the second groups of cells in the presence of differing concentrations of montelukast (control, Figure 14A to C) and varying concentrations of montelukast and zileuton, a 5-lipoxgenase inhibitor (Figure 14D-F). The results showed that 1 OnM montelukast substantially reduced but did not abolish mast cell response to the active supernatant (Figure 14C) whilst a combination of 50OnM zileuton and 1 OnM montelukast almost completely abolished mast cell response. This is also shown in Figure 14G. This shows that partial blockade of an event downstream of CRAC channel activation i.e. synthesis of leukotrienes, by the 5-lipoxgenase inhibitor zileuton in combination with cysLTI receptor inhibitor montelukast results in almost complete abolition of mast cell response to cysteine leukotrienes in active supernatant.

Discussion

The present invention demonstrates that cysteinyl leukotrienes are a dominant component of mast cell activation, as indicated by the following; first, pre-treatment of fura 2-loaded cells with the cysteinyl leukotriene receptor antagonist SR-2640 (1 -10 μM) abolished the Ca²⁺ signal in response to challenge with active supernatant (Figure 1 A). Similar inhibition was observed with the structurally distinct and clinically prescribed blockers montelukast (Figure 1 B; 100 nM), pranalukast and zafirlukast (data not shown), antagonists specific for the cysteinyl leukotriene type I receptor. Second, blocking generation of leukotrienes by pre-treatment of those cells used as the source of active supernatant with the 5-lipoxygenase blockers NDGA or zileuton (both at 10 μM) prior to stimulation with thapsigargin resulted in an ineffective supernatant, as seen by lack of Ca²⁺ response in fura 2-loaded cells. Collectively, these results identify cysteinyl leukotrienes as major components of the active supernatant and establish a role for cysteinyl leukotrienes but not ATP as paracrine signals in mast cells.

The results described above have directly shown that cysteinyl leukotrienes are effective local paracrine signals in primary rat and human mast cells. Moreover, since leukotriene secretion is driven by CRAC channel activation and the secreted leukotrienes in turn stimulate CRAC channels, these findings suggest a regenerative positive feedback cascade that might underlie sustained inflammatory response and chronic mast cell activation characteristic of allergic rhinitis/nasal polyposis and other mast cell related disorders. Furthermore, results described herein show that partial blockade of both the CRAC channels and the cysLT receptor activation can lead to near total abolition of paracrine signalling and therefore mast cell response to leukotrienes. These results indicate that combination therapies which target both the CRAC channel pathway and the cysLT receptor pathways may have utility in the treatment of mast cell relates disorders, particularly with the effect that reduced dosages of the active ingredients may be required.

Claims

1 . An agent which is an inhibitor of a component of a Calcium Release Activated Calcium (CRAC) channel pathway in mast cells for use in treating a disorder associated with activation of mast cells, wherein said agent is a first agent and is for use in combination with a second agent which is an inhibitor of activation of mast cells by a cysteinyl leukotriene.

2. An agent according to claim 1 , wherein the CRAC channel pathway comprises a CRACM1 protein and a STIM1 protein, and wherein the first agent is a direct or indirect inhibitor of the CRACM1 protein.

3. An agent according to claim 1 , wherein the CRAC channel pathway comprises a CRACM1 protein and a STIM1 protein, and wherein the first agent is a direct or indirect inhibitor of the STIM1 protein.

4. An agent according to claim 2, wherein the first agent and the second agent are independently selected from an antibody, a small molecule, a protein, a peptide and a nucleic acid.

5. An agent according to claim 4, wherein the first agent and/or the second agent is a nucleic acid sequence, and further wherein the first and/or second agent is an siRNA.

6. An agent according to claim 5, wherein the first agent is an siRNA comprising the sequence of GCAUGGAAGGCAUCAGAAGUGUAUA (SEQ ID. No 1 ) and optionally the second agent is an siRNA molecule or mixture of siRNA molecules selected from; SEQ ID. No. 2, SEQ ID. No. 3, SEQ ID. No. 4, SEQ ID. No. 5, SEQ ID. No. 6, SEQ ID. No. 7, SEQ ID. No. 8 and SEQ ID. No. 9; and further optionally wherein the second agent is a mixture of siRNA comprising one or more of the following duplexes (a) SEQ ID. No. 2 and SEQ ID. No. 3, (b) SEQ ID. No. 4 and SEQ ID. No. 5, (c) SEQ ID. No. 6 and SEQ ID. No. 7 and (d) SEQ ID. No. 8 and SEQ ID. No. 9.

7. An agent according to any preceding claim, wherein the cysteinyl leukotriene is selected from leukotriene C4 (LTC₄) and leukotriene D4 (LTD₄).

8. An agent according to any preceding claim, which is for simultaneous, separate or sequential use with the second agent.

9. An agent according to any preceding claim, wherein the second agent is a cysteinyl leukotriene type I receptor antagonist.

10. An agent according to any of claims 1 to 8, wherein the second agent is an inhibitor of LTC₄ production by mast cells.

1 1 . An agent according to claim 10, wherein the second agent is an inhibitor of 5- lipoxygenase.

12. An agent according to any preceding claim, wherein the disorder is a disorder associated with or caused by local activation of mast cells.

13. An agent according to claim 12, wherein the disorder affects a tissue with a reduced or poor blood supply.

14. An agent according to claim 12, wherein the disorder is an allergic disorder.

15. An agent according to claim12 or claim 13, wherein the disorder is selected from allergic rhinitis, nasal polyposis and asthma.

16. An agent according to claim 15, wherein the disorder is allergic rhinitis.

17. A pharmaceutical composition comprising a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene.

18. A pharmaceutical product comprising a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene.

19. A kit comprising a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene.

20. A combination of a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene for use in treating a disorder associated with mast cell activation.

21 . A combination according to claim 20, wherein the first agent is for use at a dosage which is reduced as compared to the dosage used if the first agent is for administration without the second agent, wherein optionally the first agent is for use at a dosage which is at least 10% less, and optionally at least 20%, and further optionally at least 30% than the dosage when if for administration alone.

22. A combination according to claim 20 or claim 21 , wherein the second agent is for use at a dosage which is reduced as compared to the dosage used if the second agent is for administration without the first agent, wherein optionally the second agent is for use at a dosage which is at least 10% less, and optionally at least 20%, and further optionally at least 30% than the dosage if for administration alone.

23. A composition, product, kit or combination according to any of claims 17 to 22, wherein the first agent is as claimed in any of claims 1 to 6 and wherein the second agent is selected from a cysteinyl leukotriene type I receptor antagonist and an inhibitor of 5-lipoxygenase.

24. A composition, product, combination or kit according to claim 23, wherein the second agent is selected from an antibody, a small molecule, a protein, a peptide and a nucleic acid.

25. A composition, product, combination or kit according to claim 24, wherein the second agent is selected from montelukast, zafirlukast, pranlukast and zileuton and pharmaceutically acceptable salts thereof.

26. A method of reducing the probability of or treating a disorder associated with mast cell activation comprising; administering a therapeutically effective amount of a first agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell and a second agent which is an inhibitor of activation of mast cells by leukotriene to a subject in need thereof.

27. A method according to claim 26, wherein the second agent is an inhibitor of activation of mast cells by leukotriene C4 (LTC₄), wherein said first agent is administered simultaneously, separately or sequentially to said second agent.

28. A method according to claim 26 or claim 27, for treating a disorder which is caused by local activation of mast cells.

29. A method according to claim 26 or claim 27, wherein the disorder is selected from asthma, allergic rhinitis and nasal polyposis.

30. A method according to any of claims 26 to 29, wherein the first agent and the second agent are independently selected from the group consisting of a protein, an antibody, an antibody fragment, a peptide, a small molecule and a nucleic acid.

31 . A method according to any of claims 26 to 30, wherein the first agent is a direct or indirect inhibitor of a CRACM1 channel protein.

32. A method according to any of claims 26 to 30, wherein the first agent is a direct or indirect inhibitor of a STIM1 protein.

33. A method according to any of claims 26 to 32, wherein the second agent is a direct or indirect inhibitor of a cysteinyl leukotriene type I receptor protein.

34. A method according to claim 33, wherein the second agent is a cysteinyl leukotriene type I receptor antagonist.

35. A method according to claim 33, wherein the second agent is selected from montelukast, zafirlukast and pranlukast and pharmaceutically salts thereof.

36. A method according to any of claims 26 to 32, wherein the second agent is a 5- lipooxygenase inhibitor.

37. A method according to claim 36, wherein the second agent is zileuton or a pharmaceutically acceptable salt thereof.

38. A method according to any of claims 26 to 37, wherein the subject is a human.

39. Use of an agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell for the manufacture of a medicament for the treatment of a disorder which is associated with mast cell activation, wherein the agent is a first agent and is for sequential, simultaneous or separate administration with a second agent which is an inhibitor of activation of mast cells by leukotrienes.

40. Use according to claim 38 or claim 39, wherein the disorder is caused by or associated with local activation of mast cells and is optionally selected from allergic rhinitis, asthma, and nasal polyposis.

41 . Use according to any of claims 38 to 40, wherein the medicament is for a human patient.

42. Use according to any of claims 38 to 41 , wherein the second agent is a cysteinyl leukotriene type I receptor antagonist.

43. Use of an inhibitor of activation of mast cells by leukotrienes for the manufacture of a medicament for the treatment of a disorder associated with mast cell activation, wherein said inhibitor is for sequential, simultaneous or separate administration with an agent which is an inhibitor of a component of a CRAC channel pathway in a mast cell.

44. A combination of at least two agents, wherein each of said agents targeting a different component involved in a pathway for leukotriene synthesis by, and activation of mast cells, wherein optionally said combination is for use in treating a mast cell related disorder.

45. A pharmaceutical product comprising at least two pharmaceutically acceptable agents, each of which targets a different component involved in the pathways involved in a pathway for leukotriene synthesis by, and activation of mast cells, wherein optionally said product is for use in treating a mast cell related disorder.

46. A combination of claim 44 or a product of claim 45 which comprises an agent which is a 5-lipoxgenase inhibitor.

47. A combination of claim 44 or 46 or a product of claim 45 or claim 46, which comprises an agent which is a cysteinyl leukotriene type I receptor antagonist.

48. A combination of claim 44, 46 or 47 or a product of claims 45 to 47, wherein the leukotriene is LTC₄.

49. A method of identifying a candidate agent for use in treating a disorder associated with mast cell activation in combination with a second agent which is an inhibitor of mast cell activation by leukotrienes, wherein the method comprises; (a) screening for a candidate first agent which inhibits a component of a CRAC channel pathway in mast cells, and detecting the effect of the candidate first agent on mast cell activation.

50. The method according to claim 49, wherein the step of detecting comprises measuring leukotriene secretion from a mast cell.