WO2004016270A1 - USE OF Itk INHIBITORS FOR THE TREATMENT OF MAST CELL-DRIVEN OR BASOPHIL-DRIVEN DISEASES - Google Patents

Abstract

The present invention relates to the treatment of mast cell-driven or basophil-driven conditions or diseases by inhibiting the activity of Inducible T cell kinase (Itk). Particular conditions or diseases that may be treated include asthma, rhinitis, COPD.

Description

Field of the invention Use of Itk inhibitors for the treatment of mast cell-driven or basophil-driven diseases

The present invention relates to the treatment of mast cell- or basophil-driven conditions or diseases by inhibiting the activity of Inducible T cell kinase (hereinafter referred to as Itk).

Background to the invention

Mast cells and basophils

Mast cells and basophils each have a complex role in acquired and innate immunity. These roles include both effector cell and, potentially, immunoregulatory activities. Mast cells and basophils display a number of similarities but they also differ in many aspects of natural history and function, as explained below.

Both mast cells and basophils are derived from CD34⁺ hematopoietic progenitor cells, such as those present in adult bone marrow. Basophils (like other granulocytes) typically mature in the bone marrow and then circulate in the peripheral blood, from where they can then be recruited into the tissues. However mature mast cells typically do not circulate in the blood but complete their differentiation in vascularized tissues (Galli et al. 1999). Under physiological conditions, basophils have a short life-span of a few days. IL-3 can promote the production and survival of human basophils in vitro and can induce basophilia in vivo (Galli 2000; Valent et al. 1989; Lantz et al. 1998). Unlike basophils, mast cells can be very long-lived and mast cells that are apparently mature can proliferate under certain conditions (Galli et al. 1999; Schwartz and Huff 1998).

Both mast cells and basophils express the αβγ2 form of the high-affinity receptor for IgE (the FcεRI receptor) on their surface.

Both cell types can be activated to secrete diverse preformed mediators, lipid mediators (synthesized de novo), and cytokines, after cross-linking of FcεRI-bound IgE with bivalent or multivalent antigen. Upon appropriate stimulation (for example, via the FcεRI receptor), mast cells and basophils can secrete mediators that are either preformed and granule- associated (for example, histamine, proteoglycans and neutral proteases) or are synthesized de novo (for example, leukotriene C₄ [LTC₄], platelet activating factor [PAF] and [in mast cells only] prostaglandin D₂ [PGD₂]). Furthermore, mouse or human mast cells represent potential sources of many cytokines with effects in inflammation, immunity, haematopoiesis, tissue remodelling and diverse other biological processes (for example, IL-1 , IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-16, TNF-α, bFGF, VPF/VEGF, TGF-β and several C-C chemokines, including MIP-1 α and MCP-1 ). By contrast, the spectrum of basophil-derived cytokines appears to be more limited but includes IL-4 and IL-13 (Brunner et al. 1993; MacGlashan et al. 1994).

Mast cells and basophils are not only important effector cells in acute IgE-associated allergic reactions but may also contribute significantly to the expression of aspects of acquired immune responses that develop over hours (for example, late-phase reactions [LPRs]) or days to weeks (for example, chronic allergic inflammation) (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000). Mast cells and basophils may also mediate immunoregulatory functions, both through their ability to produce certain cytokines and by other mechanisms (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000; Mecheri and David 1997).

Studies in reconstituted mast cells, mast-cell-deficient mice and correlative analyses in humans indicate that mast cells can contribute to leukocyte infiltration, tissue remodelling and long-term functional changes in the context of IgE-associated acquired immunity. Basophils can be prominent in the leukocytic infiltrates elicited at sites of IgE-associated late-phase responses in the skin, nose and airways of humans, and may contribute to the expression of acquired immunity to certain nematodes or to the feeding of ectoparasites such as ticks.

Beyond influencing immune responses via the secretion of cytokines, in vitro evidence has suggested that mast cells and/or basophils can function as antigen-presenting cells (Mecheri and David 1997) and represent sources of co-stimulatory activity.

Animal studies have demonstrated that mast cells are essential for virtually all of the augmented vascular permeability and tissue swelling that are associated with IgE- dependent passive cutaneous anaphylaxis reactions (Galli et al. 1999). Several findings indicate that mast cells are critical for the immediate phase of IgE-associated 'Type I' reactions also in humans (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000; Schwartz 1994). However, in many allergic individuals, the immediate reaction to cutaneous antigenic challenge is followed 4-8 hours later by persistent swelling and leukocyte infiltration (i.e. the late phase response) (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000). Many of the clinically significant consequences of IgE-dependent reactions, both in the respiratory tract and in the skin, are now thought to reflect the actions of the leukocytes recruited to these sites during LPRs rather than the direct effects of the mediators released by mast cells at early time points after antigen challenge (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000).

Several lines of evidence, derived from both clinical and animal studies, indicate that mast cell activation can contribute to the leukocyte infiltration associated with late phase responses. The leukocytes recruited to sites of LPRs can include basophils, eosinophils, neutrophils, lymphocytes and monocytes/macrophages. All of these cells may influence the reactions by providing additional proinflammatory mediators and cytokines, with the relative importance of the various potential effector cells varying depending on the individual circumstances (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000).

The release of cytokines and other mediators from mast cells and basophils has the potential to influence many aspects of the pathophysiology at sites of allergic diseases, including some of the chronic changes (for example, airway hyper-reactivity, connective tissue changes and enhanced mucus production) that are associated with these disorders (Galli et al. 1999; Schwartz and Huff 1998; Metcalfe et al. 1997; Galli 2000; Gordon and Galli 1994). Studies with adoptively reconstituted mast-cell-deficient mice having genetically manipulated mast cell lines have shown that mast cells can contribute significantly to the development of at least three of the long-term features of asthma in mouse models of the disorder: allergen-induced bronchial hyper-reactivity to cholinergic stimulation (Kobayashi et al. 2000; Williams and Galli 2000), eosinophil infiltration, and increased numbers of proliferating cells in the airway epithelium (Williams and Galli 2000). Furthermore, IL-4 which can be released from either T cells or basophils recruited to sites of allergic inflammation, as well as from at least some mast cells, may contribute to the local enhancement of mast cell effector function and proliferation (Bischoff et al. 1999; Lorentz et al. 2000). It has been demonstrated that not only mast cells but also basophils accumulate in nasal epithelium after allergen challenge (Klein 2000). In addition to the Th2 cells, mast cells also play an important role in initiating and maintaining the allergic response in asthma and allergic rhinitis (Holgate 2000). Exposure to allergens trigger a range of cellular events in the mast cell including release of pre- formed granule-held mediators such as histamine and tryptase, synthesis and release of newly formed mediators such as PGD2 and LTC4, and the stimulation of a wide range of pro-inflammatory cytokines and chemokines such as TNFα, IL-4, IL-5, IL-6, IL-8, IL-13 and GM-CSF (Bingham and Austen, 2000). Thus the mast cell cannot only initiate an immediate reaction but can also initiate and co-ordinate the later inflammatory response, This process has also been shown to occur in seasonal allergic rhinitis (Bentley et al 2000).

In summary, a variety of mediators and cells contribute to the allergic response. IgE and mast cells have been implicated both in the acute and late phases of an allergic response. The T cell, and in particular the Th2 cell, is thought to orchestrate the allergic responses through the production of cytokines such as IL-4, IL-5, IL-9 and IL-13. Together these Th2-derived cytokines will drive the production of allergen-specific IgE (IL-4), the IL-5 together with IL-3 regulates the eosinophil component, and the IL-3 and IL-4 will also be involved in the differentiation of basophils and mast cells. Recent data implicate IL-13, and potentially IL-9, to be key players in regulating the hyper-reactivity and mucus production seen in asthmatic patients.

At the effector end of the allergic reaction is the acute-phase classical hypersensitivity reaction mediated by cross-linking of allergen specific IgE bound to the FcεRI on mast cells and basophils. This induces an immediate release of granule-associated mediators such as histamine and leukotrienes, followed by subsequent release of chemokines and cytokines involved in the recruitment of cells responsible for the late phase reactions. The late phase reaction is characterized by the influx of eosinophils and activated Th2 cells, and is manifested by oedema and mucus production. The late phase also triggers the influx of other inflammatory cells such as monocytes and neutrophils, which contribute to a sustained general inflammation.

Preventing activation of mast cells and/or basophils has for a long time been considered an attractive strategy for treatment of allergic symptoms. Such activation may occur either by IgE-dependent or IgE-independent stimuli that leads to the secretion of several mediators including histamine, heparin, pro-inflammatory cytokines and proteolytic enzymes. A variety of different mechanisms and targets have been suggested as suitable to intervene in the FcεRI mediated mast cell / basophil activation and degranulation process (Oliver ef a/2000). These include, among others, inhibition of the two kinases Syk and Lyn located proximally to the FcεRI receptor. Lyn mediates the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the β and γ subunits of the tetrameric (αβγ₂) FcεRI. The dually phosphorylated γ subunit ITAM is needed for the recruitment and activation of Syk. Syk, when activated, is responsible for the downstream propagation of the signal (Jouvin 1994). Data from Syk knock-out mice shows that mast cells from these mice fail to degranulate (Costello era/, 1996). However, Syk is also expressed in platelets and studies of Syk-/- mice showed that this enzyme is critical for effective blood coagulation and vascular integrity (Law et al, 1999) and hence may not provide an attractive drug target. Studies of Lyn-/- mice showed accumulation of plasma cells in spleen as they age, resulting in elevated levels of serum IgM and glomerulonephritis due to the presence of immune complexes containing auto-reactive antibodies (Wang et al, 1996). Therefore, inhibiting Lyn for therapeutic purposes may present a significant challenge.

Mast cell stabilizers are currently being used to reduce the release of histamine and other inflammatory mediators by stabilizing mast cells, the mechanism of action has yet to be determined (Spector 1999).

The use of recombinant anti-lgE antibodies blocking the binding of circulating IgE to the FcεRI has been reported to prevent mast cell / basophil degranulation (MacGlashan et al, 1997). Reduced serum IgE levels and basophil FceRI expression levels accompany the symptomatic improvements in allergic rhinitis and asthmatic symptoms observed.

Itk

Itk (jnducible T cell kinase) is a 72 kDa kinase expressed in the cytoplasm of T cells, NK cells, mast cells and basophils, but not in other immune cells or outside the haematopoietic system. Itk is also known as lnterleukin-2-inducible T cell kinase, Emt (expressed mainly in T cells) or Tsk (J_cell specific tyrosine kinase). Itk is a member of the Tec-family of cytosolic protein tyrosine kinases. In mammalians, this family also includes Btk (Bruton's tyrosine kinase), Tec, Bmx, and Txk. The Tec-family kinases regulate various immune cell functions that integrate signals given by the other cytosolic tyrosine kinases as well as by serine/threonine kinases, lipid kinases, and small G proteins (Yang et al 2000).

Tec-family kinases have the following general structure: a N-terminal pleckstrin-homology (PH) domain, a Tec-homology domain that includes a Btk motif and one or two proline-rich (PR) motifs, a SH3 domain, a SH2 domain and a c-terminal catalytic (SH1) domain. These kinases are expressed almost exclusively in hematopoietic tissues. Tec and Bmx have also been detected in endothelial cells. The cellular distribution differs for each Tec-family member. For example, Itk is expressed by T cells, NK cells, mast cells and basophils, whereas Btk is expressed by all hematopoietic cells except T cells. Thus hematopoietic cells may express one or several Tec-family kinases. For example T cells express Itk, Tec and Txk, and mast cells express Btk, Itk and Tec.

The gene for Itk (the itk gene) is located on human chromosome 5q in a cluster of genes associated with a Th2 type immune response (for example, IL-4 and IL-5). No disease association has been described for the itk gene. In contrast, the gene for Btk has been very thoroughly studied due to its association with X-linked agammaglobulinemia (XLA). XLA patients are virtually devoid of mature B cells and their Ig levels are strongly reduced. The B cell deficiency is due to a defective expansion of the pre-B cell pool. Btk-/- mice, and x/^'d mice carrying a mutation which results in the substitution of Arg with Cys at residue 28 in the Btk protein, have about half the number of B cells as compared to normal mice (Smith et al 2001 ). Mast cell development is normal in Btk-/- mice, but bone- marrow derived mast cells (BMMCs) exhibit a reduction in antigen-induced histamine release.

In T and B cells, signalling through T cell receptors and B cell receptors leads to activation of Itk and Btk, respectively. Downstream of Itk and Btk a number of different messengers are engaged, including scaffolding proteins (SLP-76, LAT, SLP-65), Src kinases, MAP kinases, and PI3-K. These events are followed by PLC-γ activation that leads to IP3 generation and sustained Ca^z+ flux, and subsequently activation of transcription factors. PLC-γ1 has been suggested as a direct substrate for Itk (Miller and Berg 2002). In T cells, Itk (and Tec) may also mediate signalling through the CD28 co-receptor. Published studies, mainly involving Btk-/- animals and their mast cells, have strongly suggested that Btk is the Tec-family kinase mediating signalling from the FcεRI. No other Tec family kinase has yet been shown to have a critical role in the signalling pathways downstrem of FcεRI. .

Signalling from Tec-family kinases can also be regulated by PH domain-mediated plasma membrane localization, and by Src-family-mediated phosphorylation of critical tyrosine residues.

In T cells, it is known that Itk is involved in the signalling cascade downstream of the T cell receptor (TcR), and is also associated with the co-stimulatory molecule CD28. Most of current knowledge on the role of Itk in T cell signalling has been generated in Itk-deficient knockout mice (Itk-/-). Such animals have a decreased number of mature thymocytes and a reduced response to anti-CD3 stimulation, but no general T cell defect, as demonstrated by their intact response to PMA/ionomycin stimulation (Liao and Liftman 1995). From studies using Itk-/- mice, it has been proposed that Itk is required for Th2 but not Th1 cell development. This was demonstrated in the N. brasiliensis and L. major infection models where the Itk-/- animals are protected in the Leishmania model indicating an intact Th1 response, whereas they are susceptible to infection with N. Brasiliensis that requires an intact Th2 response for resolution of the infection (Fowell et al 1999). Thus, in T cells Itk apparently modulates the TcR signal in a way that will allow Th1 but not Th2 differentiation. However, the functional role of Itk in mast cell development and function has not been previously identified or described.

Itk inhibitors and their uses

The international patent application published as WO 02/50071 discloses particular thiazolyl compounds that are inhibitors of the Tec-family kinases and may be used as immunosuppressive, anti-inflammatory, anti-allergic and anti-cancer agents. The compounds are described as inhibitors of Itk and other Tec-family kinases including Btk, Txk, Tec and Bmx.

The international patent application published as WO 01/25220 discloses particular triazine compounds that are inhibitors of a wide range of phosphoryl transferases or kinases, including the Tec-family kinases (Bmx, Btk, Itk, Tec and Txk). The compounds are described as useful for treating a wide range of diseases.

The international patent application published as WO 01/66107 describes treatment of conditions associated with cytokine production by administering Tec-family kinase inhibitors, preferably inhibitors of Btk, Tec, Bmx or Txk and most preferably of Btk.

The international patent application published as WO 02/34899 describes methods of screening for compounds that modulate the activity of truncated Tec-family kinase polypeptides, and therapeutic uses of the compounds identified.

Description of the invention

We have identified the critical role of Itk in regulating important mast cell and basophil functions. The activity of mast cells or basophils may be blocked through inhibition of Itk. Thus Itk inhibitors may be used as pharmaceutical agents for the treatment or prevention of mast cell-driven or basophil-driven conditions or diseases.

In particular we have identified Itk as a target for inhibiting several key events in both acute and late phase allergic reactions common to allergic rhinitis and asthma.

We have demonstrated that inhibiting Itk activity in primary human mast cells and/or basophils leads to reduced levels of histamine release after stimulation through FcεRI. Itk inhibitors are able to inhibit histamine release in αlgE stimulated primary human basophils and in human lung mast cells. This effect is not observed when using Btk selective inhibitors. Thus Itk is the critical, rate-limiting enzyme that controls signalling from the FcεRI and drives the downstream responses.

Our findings indicate that an Itk inhibitor should reduce both the acute and late phase responses of an allergic reaction, as seen in both rhinitis and asthma and other conditions or diseases. Thus an Itk inhibitor may be used as a pharmaceutical agent to treat either the acute phase or the late phase responses of an allergic reaction. It is also possible to treat both the acute phase and the late phase responses with a single pharmaceutical agent (the Itk inhibitor), avoiding the need for polytherapy . Thus a single drug (Itk inhibitor) may replace the use of two separate drugs (such as an anti-histamine drug plus a glucocorticosteroid drug).

In a first aspect of the invention, we provide a method of treating or preventing a mast cell- driven or a basophil-driven condition or disease which comprises administering a therapeutically effective amount of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof to a human or mammal suffering from or susceptible to the condition or disease.

Preferably we provide a method of treating or preventing a mast cell-driven condition or disease.

In a further aspect of the invention, we provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of a mast cell-driven or a basophil-driven condition or disease. The Itk inhibitor (or its salt or ester) is used to inhibit mast cell responses and/or basophil responses, including activation and degranulation.

Preferably we provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of a mast cell-driven condition or disease.

In particular we provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of a mast cell-driven or a basophil-driven condition or disease.

Preferably we provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of a mast cell-driven condition or disease.

The treatment or prevention of a condition or disease covers all forms of medical therapy, including prophylactic, diagnostic and therapeutic regimens carried out in vivo or ex vivo on humans or other mammals. Prevention of a condition or disease includes reducing the risk of that condition or disease. Humans or mammals susceptible to a condition or disease are those at risk of suffering from the condition or disease. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the condition or disease in question. Persons at risk of developing a particular condition or disease generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition or disease.

Mast cell-driven and basophil-driven conditions or diseases include allergic, inflammatory, autoimmune, proliferative and hyper-proliferative diseases. Particular types and specific examples of conditions and disease are discussed below.

Allergic, inflammatory or auto-immune conditions or diseases of the respiratory tract include: reversible obstructive airway diseases such as asthma (for example, bronchial, allergic, intrinsic asthma, extrinsic and chronic asthma), and associated manifestations of the disease (late responses, hyper-responsiveness); farmer's lung and related diseases; fibrosis; idiopathic interstitial pneumonia; chronic obstructive airway disease (also known as chronic obstructive pulmonary disease or COPD); bronchoiectasis; cystic fibrosis; eosinophilic pneumonias; adult respiratory distress syndrome (ARDS); emphysema; and alveolitis (for example cryptogenic fibrosing alveolitis).

Allergic, inflammatory or auto-immune conditions or diseases in the nose include all conditions or diseases characterised by inflammation of the nasal mucosal membrane such as acute rhinitis, allergic rhinitis, chronic rhinitis including caseosa, hypertrophic rhinitis, rhinitis purulenta, and rhinitis siccia, rhinitis medicamentosa, membranous rhinitis including croupous, fibrinous and pseudomembranous rhinitis, scrofulous rhinitis, seasonal rhinitis including rhinitis nervosa (hay fever) and vasomotor rhinitis. Of particular interest are allergic rhinitis and seasonal rhinitis including rhinitis nervosa (hay fever). Suitable conditions or diseases in the nose also include nasal polyps and allergic manifestations of nasopharynx.

Allergic, inflammatory or auto-immune conditions or diseases of the eye include conjunctivitis (allergic, acute, vernal, of hay fever, chronic) and inflammation disorders of the eyelids, cornea, uveal tract and retina. Allergic, inflammatory and auto-immune conditions or diseases of the gastrointestinal tract include food allergy and food intolerance, ulcerative colitis, Crohn^'s disease, irritable bowel disease, gastric ulcers, and food related allergic diseases which have symptomatic manifestations remote from the gastrointestinal tract (for example migraine, rhinitis and eczema).

Allergic, inflammatory or auto-immune conditions or diseases of the skin include psoriasis, atopical dermatitis, contact dermatitis/dermatitis herpetiformis, erythema nodosum, urticaria, cutaneous eosinophilias, acne, Alopecia areata, eosinophilic fasciitis dermatomyositis, photoallergic sensitivity and periodontal disease.

Allergic, inflammatory or auto-immune conditions or diseases of the joints and connective tissue include osteoarthritis, systemic lupus erythematosus, vasculitis, Wegener^'s granulomatosis, polyarthritis nodosa, bursitis, tendonitis, gout, Behcet's syndrome, ankylosing sponditis, Reiter^'s syndrome and psoriatic arthritis.

Allergic, inflammatory, and auto-immune conditions or diseases of the circulatory system include artheroma, reperfusion injury (such as angioplasty), myocardial infarction, thrombosis, and vascular and tissue damage caused by ischaemia or injury.

Allergic, inflammatory, and auto-immune conditions or diseases of the central nervous system (CNS) include Parkinsons ^'s disease, Alzheimers and other dementias, stroke and subarachnoid heamorrage.

Inflammatory conditions or diseases of the liver include hepatitis, cirrhosis and glomerulonenephritis.

Allergic, inflammatory, and auto-immune conditions or diseases of the bladder and uro- genital tract include cystitis.

In a preferred aspect of the invention, we provide a method for treating or preventing a reversible obstructive airway disease (especially asthma) which comprises administering a therapeutically effective amount of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof to a human or mammal suffering from or susceptible to the disease. We further provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of a reversible obstructive airway disease (especially asthma). We also provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of a reversible obstructive airway disease (especially asthma).

In another preferred aspect of the invention, we provide a method for treating or preventing rhinitis which comprises administering a therapeutically effective amount of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof to a human or mammal suffering from or susceptible to rhinitis. We further provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of rhinitis. We also provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of rhinitis. (In this context, the term rhinitis includes all the various types of rhinitis discussed above. Of particular interest are allergic rhinitis and seasonal rhinitis including rhinitis nervosa or hay fever).

In a further preferred aspect of the invention, we provide a method for treating or preventing chronic obstructive pulmonary disease which comprises administering a therapeutically effective amount of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof to a human or mammal suffering from or susceptible to chronic obstructive airway disease. We further provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of chronic obstructive pulmonary disease. We also provide the use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of chronic obstructive pulmonary disease.

An Itk inhibitor for use in the invention is any compound having Itk inhibitory activity. The Itk inhibitor is a direct inhibitor of the Itk enzyme. The level of Itk inhibitory activity must be sufficient for the Itk inhibitor to possess useful therapeutic properties. When tested in the in vitro assay described in the Examples, the Itk inhibitor preferably gives an IC₅₀ value for inhibition of Itk activity of less than 5 μM (most preferably less than 1 μM).

The Itk inhibitor may be a compound of any chemical structure that possesses the necessary Itk inhibitory activity. For example, suitable Itk inhibitors include the compounds described in the Examples. The Itk inhibitor may be a racemate or may exist in enantiomeric or diastereoisomeric forms or mixtures thereof.

Preferably the Itk inhibitor is a selective Itk inhibitor (that is, an inhibitor that is more active against Itk than it is against Btk). A selective Itk inhibitor for use in the invention is any compound which has Itk inhibitory activity that is significantly greater than its Btk inhibitory activity, preferably selectivity is over 50-fold against Btk (ie the Itk inhibitor gives an IC₅₀ value for inhibition of Btk activity in vitro which is more than 50-fold greater than its IC₅₀ value for inhibition of Itk activity in vitro). Most preferably selectivity of the Itk inhibitor is also over 10-fold (preferably over 20-fold) against Syk and/or over 10-fold (preferably over 50-fold) against Lyn. (The kinases Syk and Lyn are believed to have a regulatory role in the signalling pathway directly downstream of the FcεRI receptor).

Preferably the selective Itk inhibitor gives an IC₅₀ value for inhibition of Itk activity of less than 5 μM (most preferably less than 1 μM) when tested in the assay described in the Examples.

For the above mentioned therapeutic indications, the dose of the Itk inhibitor to be administered will depend on the compound employed, the disease being treated, the mode of administration, the age, weight and sex of the patient. Such factors may be determined by the attending physician. However, in general, satisfactory results are obtained when the compounds are administered to a human at a daily dosage of between 0.1 mg/kg to 100 mg/kg (measured as the active ingredient).

The Itk inhibitor may be used on its own, or in the form of appropriate pharmaceutical formulations comprising the Itk inhibitor in combination with a pharmaceutically acceptable diluent, adjuvant or carrier. Particularly preferred are compositions not containing material capable of causing an adverse reaction, for example, an allergic reaction. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988. The Itk inhibitors may be administered topically, for example, to the lungs and/or the airways, in the form of solutions, suspensions, HFA aerosols or dry powder formulations, for example, formulations in the inhaler device known as the Turbuhaler^® ; or systemically, for example, by oral administration in the form of tablets, pills, capsules, syrups, powders or granules; or by parenteral administration, for example, in the form of sterile parenteral solutions or suspensions; or by rectal administration, for example, in the form of suppositories.

Dry powder formulations and pressurized HFA aerosols of the Itk inhibitor may be administered by oral or nasal inhalation. For inhalation the compound is desirably finely divided. The finely divided compound preferably has a mass median diameter of less than 10 μm, and may be suspended in a propellant mixture with the assistance of a dispersant, such as a C₈-C₂₀ fatty acid or salt thereof, (for example, oleic acid), a bile salt, a phospholipid, an alkyl saccharide, a perfluorinated or polyethoxylated surfactant, or other pharmaceutically acceptable dispersant.

The Itk inhibitor may also be administered by means of a dry powder inhaler. The inhaler may be a single or a multi dose inhaler, and may be a breath actuated dry powder inhaler.

One possibility is to mix the finely divided Itk inhibitor with a carrier substance, for example, a mono-, di- or polysaccharide, a sugar alcohol, or an other polyol. Suitable carriers are sugars, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; and starch. Alternatively the finely divided Itk inhibitor may be coated by another substance. The powder mixture may also be dispensed into hard gelatine capsules, each containing the desired dose of the active compound.

Another possibility is to process the finely divided powder into spheres which break up during the inhalation procedure. This spheronized powder may be filled into the drug reservoir of a multidose inhaler, for example, that known as the Turbuhaler^® in which a dosing unit meters the desired dose which is then inhaled by the patient. With this system the active Itk inhibitor, with or without a carrier substance, is delivered to the patient.

For oral administration the Itk inhibitor may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone, and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain for example, gum arabic, gelatine, talcum, titanium dioxide, and the like. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the Itk inhibitor may be admixed with for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above mentioned excipients for tablets. Also liquid or semisolid formulations of the drug may be filled into hard gelatine capsules.

Liquid preparations for oral application may be in the form of syrups or suspensions, for example solutions containing the Itk inhibitor, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, saccharine and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

The Itk inhibitor may also be administered in conjunction with other compounds used for the treatment of the above conditions or diseases.

In a further aspect of the invention, we provide methods for identifying and/or developing Itk inhibitors as useful pharmaceutical agents.

To identify Itk inhibitors that may be useful pharmaceutical agents, it is advantageous to screen for and select compounds that inhibit Itk activity in mast cells or basophils using appropriate in vitro or in vivo assays. In particular, it is advantageous to screen for and select compounds that have the potential to inhibit Itk activity in these cells in vivo (for example, because they are targeted to these cells).

It may also be advantageous to develop Itk inhibitors by improving their ability to inhibit Itk activity in mast cells or basophils (for example, by developing compound features or other mechanisms that help to target the inhibitors to these cells). Appropriate assays that may be useful in identifying and/or developing Itk inhibitors (particularly selective Itk inhibitors) include one or more assays selected from those described in the Examples: Itk LANCE TRF assay, Btk LANCE TRF assay, Lyn LANCE TRF assay, Syk assay, Basophil Histamine Release assay (algE induced), Basophil Leukotriene C4 (LTC4) Release assay (algE induced), Basophil lnterleukin-4 (IL-4)

Release assay (algE induced), Lung Mast Cell Histamine Release assay (algE induced). One or more of the following assays may be particularly useful in identifying and/or developing Itk inhibitors that act in basophils or mast cells: Basophil Histamine Release assay (algE induced), Basophil Leukotriene C4 (LTC4) Release assay (algE induced), Basophil lnterleukin-4 (IL-4) Release assay (algE induced), Lung Mast Cell Histamine Release assay (algE induced).

The present invention will now be described with reference to the following non-limiting Examples.

EXPERIMENTAL DETAILS FOR THE EXAMPLES

Preparation of compounds

General methods

All reactions were performed in dried glassware in an argon atmosphere at room temperature, unless otherwise noted. All reagents and solvents were used as received. Merck Silica gel 60 (0.040-0.063 mm) was used for preparative silica gel chromatography. A Kromasil KR-100-5-C18 column (250 x 20 mm, Akzo Nobel) and mixtures of acetonitrile/water at a flow rate of 10 ml/min was used for preparative HPLC. Reactions were monitored at 254 nm by analytical HPLC, using a Kromasil C-18 column (150 x 4.6 mm) and a gradient (containing 0.1% trifluoroacetic acid) of 5 to 100% of acetonitrile in water at a flow rate of 1 ml/min. Evaporations of solvents were performed under reduced pressure using a rotary evaporator at a maximum temperature of 60°C. Products were dried under reduced pressure at 40 °C.

¹H NMR spectra were recorded on a Varian lnova-400 or Unity-500+ instrument. The central solvent peak of chloroform-d (δ_H 7.27 ppm), dimethylsulfoxide-Oe (D_H 2.50 ppm) or methanol-d₄ (δ_H 3.35 ppm) were used as internal references. Low resolution mass spectra obtained on a Hewlett Packard 1100 LC-MS system equipped with a APCI ionisation chamber. Preparation of Compound A5 5-Bromo-3-(4-morpholin-4-ylphenyl)-2-phenyl-1 H-pyrrolo[2,3-Jb]pyridine

2-(4-morpholin-4-ylphenyl)-1 -phenylethanone (150 mg, 0.53 mmol) and (5-bromo-pyridin- 2-yl)-hydrazine (100 mg, 0.53 mmol) in benzene (40 mL) containing acetic acid (0.4 mL) was heated at reflux temperature for 20 h. Water was continuously distilled off using a Dean-Starch trap. Crude, impure title compound was crystallized from the reaction mixture at 8 °C. This material was heated in an inert atmosphere at 230°C for 7 min and then partitioned between toluene and water. The toluene phase was washed with water and brine and then evaporated. The residue was chromatographed (SiO₂; EtOAc-heptane 1 :3) to give the title compound (67 mg, 29 %).

¹H NMR (400 MHz, DMSO- ₆): 512.21 (1 H, s); 8.24 (1 H, d); 7.88 (1 H, d); 7.27-7.44 (7H, m); 6.90 (2H, d); 3.70 (4H, dd); 3.14 (4H,dd). APCI-MS m/z: 434.1/436.1 [MH+]

Preparation of Compound A 14

5-Bromo-3-[4-(2-morpholin-4-ylethoxy)phenyl]-2-phenyl-1 r -pyrrolo[2,3-J ]pyridine trifluoroacetate A mixture of 4-(5-bromo-2-phenyl-1 /-/-pyrrolo[2,3-£>]pyridin-3-yl)phenol (100 mg, 0.27 mmol), NaH (60% susp. in mineral oil, 45 mg, 1.25 mmol) and DMF (2 ml) was heated at 60°C for 30 min. Mixture of 4-(2-chloroethyl)morpholine hydrochloride (53 mg, 0.28 mmol), NaH (60% susp. in mineral oil, 15 mg, 0.42 mmol) and DMF (500 μl) was added and the reaction mixture was further stirred at 60°C for 75 min. Water (1 ml) and AcOH (200 μl, 3.5 mmol) were added and product was purified with preparative HPLC (RP-18, acetonitrile/water/trifluoroacetic acid gradient from 10:90:0.1 to 95:5:0.1 ) to give the title compound (59 mg, 36%).

Η NMR (400 MHz, CD₃CN): .58.31 (1 H, d); 8.00 (1 H, d); 7.28-7.54 (7H, m); 7.01-7.12 (2H, m); 4.36-4.50 (2H, m); 3.84-4.26 (2H, m); 3.52-3.68 (4H, m); 3.20-3.45 (4H, m). APCI-MS m/z: 478.0/480.0 [MH+]. General method for preparation of 2-arylbenzothiazole-6-carboxamides A mixture of 4-amino-3-mercaptobenzoic acid (5 mmol) and an aromatic aldehyde 5.1 mmol) were dissolved in acetic acid (5 ml) and nitrobenzene (15 ml). The solution was heated at 185°C for 15 min. After cooling the reaction mixture was made alkaline by the addition of 1 M NaOH and extracted with EtOAc. Following acidification with HCI (cone.), the water phase was extracted with EtOAc. Drying and evaporation of the solvent gave the 2-arylbenzothiazole-6-carboxylic acid.

A mixture of 2-arylbenzothiazole-6-carboxylic acid (1 mmol), HATU (1.2 mmol) and N,N- diisopropylethylamine (5 mmol) were dissolved in 20 ml of DMF. After stirring for 5 min at rt, the amine (2 mmol) was added and the mixture stirred at rt overnight. CH₂CI (50 ml) was added and the solution was washed with 1 M NaHCO₃ and water. Drying and evaporation of the solvent yielded the crude title product which was purified by column chromatography (SiO₂; MeOH-CH₂CI₂, 5:95).

Preparation of Compound B3

2-(4-chloro-2-hydroxyphenyl)-N-(2-pyrrolidin-1-ylethyl)-1,3-benzothiazole-6- carboxamide

Compound B3 was synthesized following the protocol for the preparation of 2- arylbenzothiazole-6-carboxamides. From 2-(4-chloro-2-hydroxyphenyl)benzothiazole-6- carboxylic acid (0.059 g, 0.19 mmol) and 2-pyrrolidin-1 -ylethanamine (0.050 ml, 0.40 mmol), the title compound was isolated (0.018 g, 23%).

APCI-MS m/z: 402.1 [MH+].

General procedure for the reduction of 2-arylbenzothiazole-6-(amine-1 -ylcarbonyl) into 2- arylbenzothiazole-6-(amine- 1 -ylmethyl)

The 2-arylbenzothiazole-6-(amine-1 -ylcarbonyl) (0.2 mmol) was dissolved in 5 ml of THF. After cooling at 0°C, a 1 M solution of LiAIH₄ in THF (0.4 mmol) was added dropwise. The ice-bath was removed and the reaction mixture stirred at rt until completion (LC-MS).

Water (0.5 ml) was added followed by CH₂CI₂ (30 ml). After washing with brine, drying and evaporation, the crude title product was purified by column chromatography (MeOH- CH₂CI₂, 5:95). Compound B1 was synthesized following this general procedure (and the general procedures for the synthesis of the corresponding amides). The starting amides were synthesized according to the methods outlined above.

Preparation of Compound B1

5-chloro-2-[6-(pyrrolidin-1-ylmethyl)-1 ,3-benzothiazol-2-yl]phenol

From 2-(4-chloro-2-hydroxyphenyl)benzothiazole-6-(pyrrolidin-1 -ylcarbonyl) (0.065 g, 0.18 mmol), the title compound was isolated (0.028 g, 45%).

¹H NMR (400 MHz, CD₂CI₂): 612.72 (1 H, brs); 8.03 (1 H, s); 7.97 (1 H, d); 7.68 (1 H, d); 7.57 (1 H, d); 7.13 (1 H, d); 6.99 (1 H, dd); 3.87 (2H, s); 2.74-2.60 (4H, m); 1.91-1.82 (4H, m). APCI-MS m/z: 345.1 [MH+]

Preparation of Compound C1 6-Phenyl-1 r/-pyrazolo[4,3-c]cinnolin-3-ol

A solution of 6-bromo-1 H-pyrazolo[4,3-c]cinnolin-3-ol (20 mg, 0.075 mmol), phenyl boronic acid (27 mg, 0.22 mmol, 3.0 equiv.) and {1 ,1 '-Bis(diphenylphosphino)ferrocene}- dichloropalladium(ll) (3.1 mg, 5 mol%) in a mixture of dioxaπe (400 μL), EtOH (40 μL) and aquous 2M sodium carbonate was heated in a sealed vial to 90°C for 3 hours. After cooling, EtOAc (5mL) was added and the resulting suspension was filtered through a pad of celite. The solid was washed with acetonitrile and 50% aquous acetonitrile. After evaporation of solvents the remaining crude was dissolved in 50% aqueous acetonitrile, acidified by 2-3 drops of trifluoroacetic acid and purified by reversed phase HPLC. After freeze-drying, the title product was isolated as its trifluoroacetic acid salt as a red powder.

¹H NMR (400 MHz, DMSO-ofe DaO); .58.06 (1 H, brd); 7.80-7.43 (7H, m).

¹H NMR (400 MHz, D SO-αfc NaOH):. .58.14 (1 H, dd); 7.62 (2H, dd); 7.48-7.35 (3H, m);

7.35-7.31 (2H, m). APCI-MS m/z: 263.1 [MH+j.

Preparation of Compound C7 6-Pyridin-3-yl-1 H-pyrazolo[4,3-c]cinnolin-3-amine

A solution of 6-bromo-1H-pyrazolo[4,3-c]cinnolin-3-amine (20 mg, 0.075 mmol), and 3- pyridyl boronic acid (28 mg, 0.22 mmol, 3.0 equiv.) and {1 ,1 '- Bis(diphenylphosphino)ferrocene}-dichloropalladium(ll) (3.1 mg, 5 mol%) in a mixture of dioxane (400 μL), EtOH (40 μL) and aquous 2M sodium carbonate was heated in a sealed vial to 90°C for 3 hours After cooling, EtOAc (5mL) was added and the resulting suspension was filtered through a pad of celite The solid was washed with acetonitrile and 50% aqueous acetonitrile After evaporation of solvents the remaining crude was dissolved in 50% aqueous acetonitrile, acidified by 2-3 drops of trifluoroacetic acid and purified by reversed phase HPLC After freeze-drying, the title product was isolated as its trifluoroacetic acid salt as a red powder

¹H NMR (400 MHz, DMSO-cfe): 5 13.12 (1 H, brs), 8.92 (1 H, d), 8.64 (1 H, dd), 8.34 (1 H, dd), 8.16 (1 H, dt), 7.98 (1 H, t), 7.92 (1 H, d), 7.55 (1 H, dd), 6.21 (2H, brs). APCI-MS m/z: 263.1 [MH+]

Preparation of Compound D5 6-Bromo-2-(4-isopropylphenyl)-3H-imidazo[4,5-b]pyridine trifluoroacetate A mixture of 5-bromo-2,3-dιamιnopyrιdιne (0 060 g, 0.32 mmol) and 4- isopropylbenzaldehyde (0.044 g, 0 30 mmol) and ιron(lll)chlorιde hexahydrate (0.48 g, 1.8 mmol) in DMF (200 ml) was heated to 120°C with continuous air bubbling through the solution for 4-16 h until the starting materials has been consumed. The reaction mixture was poured onto ice/water, filtrated, washed with water, EtOH, MeOH and dried. Recrystailized twice from DMF (250 ml resp.150 ml), the crystalls were washed with MeOH, diethyl ether and dried to afford the title compound.

¹H NMR (400 MHz, DMSO-δ₆): 58.40 (1 H, brd); 8.25 (1 H, brs); 8.15 (2H, brd); 7.46 (2H, brd); 2.98 (1 H, m); 1.25 (6H, d). APCI-MS m/z: 316.0/318.0 [MH+]

Preparation of Compound D6 6-Bromo-2-(4-isopropoxyphenyl)-3H-imidazo[4,5-b]pyridine trifluoroacetate

A mixture of 5-bromo-2,3-dιamιnopyπdιne (0 060 g, 0.32 mmol) and 4- isopropoxybenzaldehyde (0 049 g, 0 30 mmol) and ιron(lll)chloπde hexahydrate (0.48 g,

1.8 mmol) in DMF (200 ml) was heated to 120°C with continuous air bubbling through the solution for 4-16 h until the starting materials has been consumed.

The reaction mixture was poured into icewater, filtrated, washed with water, EtOH, MeOH and dried Recrystailized twice from DMF (250 ml resp 150 ml), filtered, washed with MeOH, diethyl ether and dried to afford the title compound Η NMR (400 MHz, DMSO- ₆): .58.36 (1 H, brs); 8.20 (1 H, brs); 8.15 (2H, brd); 7.10 (2H, brd); 4.75 (1 H, m); 1.31 (6H, d). APCI-MS m/z: 332.0/334.0 [MH+]

Assays

Itk LANCE TRF assay

The Itk kinase assay utilized recombinant human Itk kinase domain fused with GST (Glutathione S-Transferase). The protein was expressed in High five insect cells, purified in one step on an affinity chromatography glutathione column and stored in 50 mM Tris/HCI (pH 7.6), 150 mM NaCI, 5% (w/v) mannitol, 1 mM DTT, 30% glycerol at -70°C. The kinase substrate used in the assay was a biotinylated peptide derived from the Src- optimal substrate (Nair et al, J. Med. Chem., 38: 4276, 1995; biotin- AEEEIYGEFEAKKKK). The assay additions were as follows: Test compounds (or controls; 1 μl in 100% DMSO) were added to black 96-well flat-bottomed plates (Greiner 655076) followed by 20 μl Itk in assay buffer and the reaction was started by adding 20 μl ATP and peptide substrate in assay buffer. The assay buffer constitution during phosphorylation was: 50 mM HEPES (pH 6.8), 10 mM MgCI₂, 0.015% Brij 35, 1 mM DTT, 10% glycerol, 160 ng/well Itk, 2 μM peptide substrate and 50 μM ATP. The assay was stopped after 50 minutes (RT) by adding 150 μl ice-cold Stop solution (50 mM Tris/HCI, pH 7.5, 10 mM EDTA, 0.9% NaCI and 0.1 % BSA) together with LANCE reagents (2 nM PT66-Eu³⁺, Wallac AD0069 and 5 μg/mL Streptavidin-APC, Wallac AD0059. Both concentrations were final in stopped assay solution). The plates were measured on a Wallac 1420 Victor 2 instrument with TRF settings after 1 h incubation, and the ratio (665 signal/615 signal)^*10000 was used to calculate the inhibition values. IC50 values were determined using XLfit.

Btk LANCE TRF assay The Btk kinase assay utilized recombinant human full length Btk fused with GST

(Glutathione S-Transferase). The protein was expressed in baculo virus transfected Sf9 insect cells, purified in one step on an affinity chromatography glutathione-sepharose column and stored in 10 mM T s-HCI (pH 7.4), 150 mM NaCI, 1 mM DTT and 30 % glycerol and stored at -70°C. The kinase substrate used in the assay was Btk217-229 (biotm-KKVVALYDYMPMN) comprising the Btk autophosphorylation site. The assay additions were as follows Test compounds (or controls, 1 μl in 100% DMSO) were added to black 96-well flat-bottomed plates (Greiner 655076) followed by 20 μl Btk and substrate in assay buffer and the reaction was started by adding 20 μl ATP. The assay buffer constitution during phosphorylation was 50 μM Tns/HCI (pH 7 4), 7.5 mM MnCI₂, 0.002% Brij 35, 10 mM DTT, 5% glycerol, 40 ng/well Btk, 2 μM peptide substrate and 20 μM ATP. The assay was stopped after 60 minutes (RT) by adding 120 μl ice-cold Stop solution (50 mM Tris/HCI, pH 7.5, 10 mM EDTA, 0 9% NaCI and 0.1% BSA) together with LANCE reagents (1 nM PT66-Eu³⁺, Wallac AD0069 and 10 nM Streptavidin-APC, Wallac AD0059. Both concentrations were final in stopped assay solution). The plates were measured on a Wallac 1420 Victor 2 instrument with TRF settings after 1h incubation, and the ratio (665 sιgnal/615 sιgnal)^*10000 was used to calculate the inhibition values IC50 values were determined using XLfit

Lyn LANCE TRF assay

The Lyn kinase assay utilized recombinant N-terminal 6Hιs-tagged human Lyn The protein was expressed in baculo virus infected Sf9 insect cells and used as a crude cell lysate, stored at -70°C. The autophosphorylation site Lyn 363-369 (biotin-EDNEYTA) from human p56 Lyn was used as kinase substrate in the asay. The assay additions were as follows Test compounds (or controls, 1 μl in 100% DMSO) were added to black 96- well flat-bottomed plates (Greiner 655076) followed by 20 μl Lyn in assay buffer and the reaction was started by adding 20 μl ATP/substrate The assay buffer constitution during phosphorylation was 90 mM Hepes (pH 7 4), 1.5 mM MnCI₂, 0.09% Triton X-100, 80 μM DTT, 80 μM Na₃VO₄, Lyn lysate diluted 1/20000, 7 μM peptide substrate and 7.5 μM ATP. The assay was stopped after 100 minutes (RT) by adding 20 μl ice-cold Stop solution (100 mM Hepes, pH 7 4, 0 1 % Triton X-100 and 10 mM EDTA) together with LANCE reagents (2 nM PT66-Eu³⁺, Wallac AD0069 and 1 16 nM Phycolink Streptavidin-APC, Prozyme PJ25S Both concentrations were final in stopped assay solution). The plates were measured on a Wallac 1420 Victor 2 instrument with TRF settings after 1 h incubation, and the ratio (665 sιgnal/615 sιgnal)^*10000 was used to calculate the inhibition values IC50 values were determined using XLfit Syk assay

Syk kinase was cloned in baculovirus and expressed as a full-length protein in Sf9 cells (AZAP). A crude enzyme extract was used as enzyme source. As substrate SLP76(107- 120) (Biotin-6-aminohexanoate-Ser-Phe-Glu-Glu-Asp-Asp-Tyr-Glu-Ser-Pro-Asn-Asp-Asp- s Gln-OH) synthesised in house was used. The reaction was performed in flat bottom 384- well solid black plates (Costar) in a final volume of 20 μl, containing 50 mM Hepes pH 7.4, 0.05% Triton X-100, 0.2 mM DTT, 1 μM ATP, 3 mM MnCI₂, 0.2 mM peptide substrate and a 5000-fold dilution of SYK insect cell extract. After 30 min incubation at room temperature the reaction was terminated by addition of 15 μl EDTA to a final concentration of 20 mM diluted in detection buffer (50 mM Hepes pH 7.4, 0.17% BSA, 0.033% Tx-100, 250 mM NaCI). For detection 15 μl of PT66-acceptor and SA-donor beads in detection buffer were added to a final concentration of 15 μg/ml. The plates were sealed and incubated in the dark for 2-3 hours at room temperature under constant agitation before measurement in the AlphaQuest™ AD microplate analyser.

Basophil Histamine Release assay:

Basophils were obtained from human blood by density centrifugation on polymorphprep (AXIS-SHIELD). Mononuclear cells were collected from the top layer and washed in THG^" buffer (0.8% NaCI, 0.24% HEPES, 0.1% D-glucose anhydrous, 0.1% Gelatine Type B, 0.1 % 1.8M NaH₂PO₄ • 2 H₂O, 0.1 % 2.7M KCI/ 1 L H₂O). Basophils were purified by negative selection using MACS Basophil Isolation Kit (Miltenyi Biotech). Briefly, cells were labelled with magnetic beads in cold MACS buffer (PBS, 2 mM EDTA, and 0.5% BSA) and separated using the AutoMACS separation instrument. The cells were subsequently washed in RPMI 1640 supplemented with 25 mM HEPES (GIBCO-BRL) and 5% heat inactivated Fetal Calf Serum (GIBCO-BRL), and incubated with 5 μg IgE (Serotec

PHP008) for 60 minutes at 37°C. The cells were washed in THG^" buffer and resuspended in THG⁺⁺ buffer (THG^", supplemented with 1.8 mM CaCI₂ • 2 H₂O and 1 mM MgCI ^■ 6 H₂O). The basophils (1 .5 x 10⁴ /well) were added to round bottomed 96-well polypropylene plates (Costar 3365) in THG⁺⁺ buffer and stimulated through FcεRI cross linking by addition of 10 ng anti-lgE/well (The Binding Site). Plates were sealed with plate sealers (Labsystem) and incubated at 37°C in a water bath for 25 minutes. The release was stopped by centrifugation at 4°C and supernatants collected.

Histamine content was measured in the supernatants by addition of 20 μl HMT/³H- SAM [1 ml Histamine Methyl Transferase (HMT), purified from male rat kidneys (for description see below) and 1 Mbq S-Adenosyl-L-[methyl-³H] methionine (Amersham Pharmacia Biotech) in 5 ml THG⁺⁺ buffer] to 100 μl sample (volume adjusted with THG⁺⁺ buffer). For total histamine release 1.5 x 10⁴ boiled basophils were used. Samples were incubated at 37°C for 60 minutes and the reaction stopped by addition of 50 μl NaOH (10 mM) followed by 400 μl toluene/isoamyl alcohol (4:1 ). Tubes were capped and vigorously shaken for 40 sec. to extract methyl histamine and subsequently centrifuged to separate the phases. To measure ³H-methyl histamine content in the top (organic) phase, samples were transferred to LumaPlates (Packard), dried and read in 1450 MicroBeta instrument (Wallac).

HMT - Histamine Methyl Transferase preparation: HMT was obtained from male rat kidneys. Briefly, the membrane was peeled off and kidneys (80 g) homogenized with 10 volumes of 0.25 M sucrose.The homogenate was centrifuged at 15000xg for 90 minutes at 0°C. The supernatant was collected and saturated to 45% with ammonium sulphate (277 mg/ ml) whilst stirring in the cold and subsequently centrifuged at 10000g for 20 min. The supernatant was collected, adjusted to 70% saturation with ammonium sulphate (171 mg/ ml) and centrifuged at 10000g for 15 min. Supernatants were discarded and pellets dissolved in 0.1 M sodium phosphate buffer pH 7.4, and dialysed against 2 x 4 L of cold 1 mM sodium phosphate buffer pH 7.4.

Basophil Leukotriene C4 (LTC4) Release assay

Human basophils (purified according to the protocol described for basophil histamine release assay above) were resuspended to a density of 600,000/ ml in THG^" buffer. Cells (50 μl) were added to 96 well plates containing 25 μl compound (4x final concentration i.e. 4 x 10^"9 to 4 x 10^"6M). Release was started by the addition of 10 μl of 10 times concentrated anti-human IgE to give a final dilution of 1 :10000 (Sigma code I-0632) at 37°C for 25 minutes . Cells were spun at 300xg for 5 minutes at 4°C and 50 μl supernatant sampled for the measurement of LTC4.

LTC4 was measured by a commercial ELISA kit ( BioTRAK™ Leukotriene C4/D4/E4 Enzyme immunoassay system RPN224). Assays were carried out as per kit instructions Briefly, 50 μl supernatant from basophil release experiments were sampled into the biotrak ELISA plate, 50 μl of anti-serum was added and incubated for 2 hours at room temperature. Subsequently 50 μl of peroxidase conjugate was then added and incubated for a further hour at room temperature. Wells were then emptied and washed four times with 300 μl wash buffer. Substrate (150 μl) was then added to each well and incubated for 30 minutes on a plate shaker at room temperature. Sulphuric acid (100 μl) was then added and the OD determined at 450 nM LTC4 released was calculated by comparison to a standard curve set up as in the kit, but using release buffer (THG). Briefly, serial dilutions of LTC4 were made in THG and 50 μl of these transferred to the Biotrak plate to give standards of 0, 0.75, 1.5, 3,6, 12, 24, and 48 pg of LTC4/well.

Basophil lnterleukιn-4 (IL-4) Release assay

Human basophils (purified according to the protocol described for basophil histamine release assay above) were resuspended to a density of 2x10⁶/ ml in HEPES-buffered RPMI 1640 with 10 % FCS and 2 mM L-glutamine. Cells (50 μl) were added to 96 well plates containing 25 μl compound (4x final concentration i.e. 4 x 10^"9 to 4 x 10^'6M). Release was started by the addition of 10 μl of 10 times concentrated anti-human IgE to give a final dilution of 1.10,000 (Sigma code I-0632) at 37°C for 4 hours. Cells were spun at 300xg for 5 minutes at 4°C and 50 μl supernatant sampled for the measurement of IL-4. IL-4 was measured by a commercial ELISA kit ( BioTRAK™, lnterleukin-4 [(h)IL-4] Human, Elisa System, code RPN2753). Assays were carried out as per kit instructions. Briefly, 50 μl of biotinylated antibody reagent was added to the BioTRAK™ plate. Supernatant from basophil release experiments were sampled into the plate. This was then incubated at room temperature for two hours at room temperature. Wells were then emptied and washed three times with 300 μl wash buffer. Streptavidin-HRP conjugate (100μL) was then added and incubated at room temperature for 30 minutes. Wells were then emptied and washed three times with wash buffer. Substrate (100 μl) was then added to each well and incubated for 30 minutes in the dark at room temperature. Stop solution (100 μl 0.18 M sulphuric acid) was then added and the OD determined at 450nM. IL-4 released was calculated by comparison to a standard curve set up as in the kit, but using release buffer (RPMI 1640). Briefly, serial dilutions of IL-4 were made and 50 μl of these transferred to the Biotrak plate to give standards of 0, 0.08, 0.2, 0.51 , 1.28, 3.2, 8, and 20 pg of IL-4/well. Lung Mast Cell Histamine Release assay

Human lung tissue was obtained from from lung resection patients The tissue (usually 5- 20 g) was chopped on arrival with scissors into strips of approximately 5 mm by 20 mm, and stored overnight in 50ml RPMI 1640 with 5% FCS and Penicillin streptomycin solution (Gibco, 10,000 μg /mL of Streptomycin sulphate and 10,000 Units/mL of penicillin G ) 1 ml/100 ml RPMI The following day the tissue was finely chopped using a mechanical tissue chopper (Mcllwain tissue chopper™) into small cubes (~1 mm³) and washed through nylon gauze with 1 L THG (Tyrodes-HEPES-gelatin pH 7.4). The washed tissue was then suspended in 10X volume (w/v) of digestion buffer (THG containing 1 mM MgCI₂, 5 mg/ml bovine serum albumin, 0 01 mg/ml DNase 1 type IV (Sigma), and 1 mg/ml collagenase type 1 A (Sigma)) and incubated for 30 minutes at 37°C Tissue was further disrupted by passing through a 50 ml syringe (with tip removed) 10 times, and incubated for a further 30 minutes in the digestion buffer The digestion buffer was then diluted 1 :1 with THG Isolated cells were harvested by passing through 120 μm nylon gauze and centrifugation at 300xg for 5 minutes at 20°C The cells were washed 3 x in THG After the final wash the cells were resuspended in 50 ml RPMI-1640 containing 5% FCS 1 % glutamine and 1 % Penicillin /streptomycin and stored overnight at 4°C Mast cells were purified by positive magnetic staining using a PAN mouse lgG1 CELLection™ kit (Dynal). Beads (300 μl) were incubated overnight at 4°C with 50 μl of anti-human CD117 (Pharmingen) The beads were then washed 4 X in THG and collected using a Dynal magnet The prepared cells were then centrifuged at 300g for 5 minutes at 4°C and resuspended in 2 mL of THG and pre-treated Dynal beads added (10 beads/mast cell). The cells and beads were mixed and left for 1 hour on a roller mixer at 4°C At the end of this incubation the cells were diluted with a further 8 ml of THG and cells attached to beads were isolated using the Dynal magnet, and washed 5 x in THG (10 mis) . The beads were then removed from the cells by incubation with 200 μl THG containing 20 μl of releasing buffer (supplied with Dynal CELLection™ kit) for 15 minutes at 37°C. Cells were aspirated in a pipette 5 times and detached beads removed using the Dynal magnet Purified mast cells were collected in the supernatant The beads were washed in THG (200 ul) a further 3 times to maximise recovery of mast cells The supernatants were pooled and centrifuged at 300xg 5 minutes at room temperature. The cells were then washed in 10 ml THG and resuspended into 1 ml RPMI-1640 with 5%FCS. Mast cells were counted in a haemocytometer (20 μl cells + 180 μl Kimura's stain). Pure mast cells (1 ml in RPMI 1640 containing 5% FCS) were sensitised by incubating with 5 μg/ml human IgE (Serotec) for 1 hour at 37°C. Cells were then diluted to 10 ml with THG and centrifuged, 300xg for 5 minutes at room temperature. The pellet was washed in 10 ml THG^" and cells recovered by centrifugation, 300xg for 5 minutes at room temperature. The pellet was resuspended in 1 ml THG⁺⁺. 10 μl of the cells were then taken into 90 μl Kimura's stain and counted for purity and number in a haemocytometer. Mast cells were then resupended to a density of 300,000 mast cells/ml in THG⁺⁺ for use in release experiments.

Stock compounds at 4 mM were made up in DMSO and serially diluted in DMSO to 4000X final concentration. Each of these serial dilutions was then diluted 1000X into THG⁺⁺. These solutions were then used at A final volume in release wells (25 μl compound + 50 μl cells, 15 μl buffer and 10 μl anti-lgE (Sigma)).

50 μl cells were added to wells of a 96 well plate (15,000 cells/well) and compound or vehicle added (25 μl). The cells were pre-warmed to 37°C and left for 3 minutes with compound before activation with 10 μl of 10x concentrated anti-human IgE (1/3,000 final anti-lgE (sigma I-0632)) in THG⁺⁺ buffer. Release was allowed to continue for 20 minutes at 37°C. Plates were spun at 200xg for 5 minutes at 4°C and 75 μl supernatant sampled into 1 ml deep well plates for analysis of histamine.

Total histamine was determined by freeze-thawing 50 μl of mast cells with 150 μl distilled water and microwaving for 30 seconds. 25 μl of this is sampled for Histamine analysis. Histamine content was determined according to the same protocol as for the basophil histamine release assay described above.

EXAMPLE 1

The structurally diverse compounds A5, A14, B1 , B3, C1 , C7, D5 and D6 were tested for their ability to inhibit histamine release from primary human basophils isolated from blood obtained from healthy volunteers. The basophils were stimulated with anti-lgE. The compounds were also profiled to determine their capability to inhibit the catalytic activity of the protein tyrosine kinases Btk, Syk, and Lyn which are all present in mast cells and important in the signalling downstream of the FcεRI receptor.

Results are shown in the table below.

Results show that inhibition of anti-lgE induced histamine release was dependent on Itk inhibition.

Compound C7 is a selective Btk inhibitor which does not have the preferred Itk inhibitory activity (Itk IC₅₀ is not less than 5 μM). Compound C7 does not inhibit histamine release. Each of the other compounds is a good Itk inhibitor (Itk IC₅₀ less than 5 μM) and each inhibits histamine release. Compounds A5, B1 , B3, D5 and D6 are selective Itk inhibitors (Btk IC₅₀ >50 μM compared to Itk IC₅₀ <5 μM). Compounds A5, A14, B1 , B3, D5 and D6 are more than 10-fold selective over the kinases Syk and Lyn. Compound C1 is a non- selective Itk inhibitor which is more active against Btk. EXAMPLE 2

Two of the compounds tested in Example 1 were tested for their ability to inhibit IL-4 and/or LTC4 release from anti-lgE stimulated human primary basophils. Results are shown in the table below. Note that the IL-4 release was measured in the presence of 10% serum, which reduces the effect of the compound due to compound binding to the serum proteins.

Compounds A5 and C1 inhibited LTC4 release. Compound A5 also inhibited IL-4 release. Compound C1 was not tested in the IL-4 assay.

EXAMPLE 3

Two of the compounds tested in Example 1 were tested for their ability to inhibit histamine release from human lung mast cells prepared from human lung tissue. These compounds were found to inhibit histamine release from primary human lung mast cells (results shown in the table below).

REFERENCES

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Claims

What we claim is

1 ) A method of treating or preventing a mast cell-driven or a basophil-driven condition or disease which comprises administering a therapeutically effective amount of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof to a human or mammal suffering from or susceptible to the condition or disease, wherein the Itk inhibitor gives an IC₅o value for inhibition of Itk activity in vitro of less than 5 μM

2) A method as claimed in claim 1 for treating or preventing a mast cell-driven condition or disease wherein the Itk inhibitor gives an IC₅₀ value for inhibition of Btk activity in vitro which is more than 50-fold greater than its IC₅₀ value for inhibition of Itk activity in vitro

3) A method as claimed in either claim 1 or claim 2 for treating or preventing a reversible obstructive airway disease

4) A method as claimed in claim 3 for treating or preventing asthma.

5) A method as claimed in either claim 1 or claim 2 for treating or preventing rhinitis

6) A method as claimed in either claim 1 or claim 2 for treating or preventing chronic obstructive pulmonary disease

7) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof for the treatment or prevention of a mast cell-driven or a basophil-driven condition or disease, wherein the Itk inhibitor gives an IC₅₀ value for inhibition of Itk activity in vitro of less than 5 μM

8) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in claim 7 wherein the Itk inhibitor gives an IC₅₀ value for inhibition of Btk activity in vitro which is more than 50-fold greater than its IC₅₀ value for inhibition of Itk activity in vitro 9) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 7 or claim 8 for the treatment or prevention of a reversible obstructive airway disease

10) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in claim 9 for the treatment or prevention of asthma

11 ) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 7 or claim 8 for the treatment or prevention of rhinitis.

12) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 7 or claim 8 for the treatment or prevention of chronic obstructive pulmonary disease

13) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the treatment or prevention of a mast cell-driven or a basophil-driven condition or disease, wherein the Itk inhibitor gives an IC₅₀ value for inhibition of Itk activity in vitro of less than 5 μM

14) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in claim 13 wherein the Itk inhibitor gives an IC₅₀ value for inhibition of Btk activity in vitro which is more than 50-fold greater than its IC₅₀ value for inhibition of Itk activity in vitro

15) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 13 or claim 14 in the manufacture of a medicament for the treatment or prevention of a reversible obstructive airway disease

16) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in claim 15 in the manufacture of a medicament for the treatment or prevention of asthma

17) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 13 or claim 14 in the manufacture of a medicament for the treatment or prevention of rhinitis 8) Use of an Itk inhibitor or a pharmaceutically acceptable salt or ester thereof as claimed in either claim 13 or claim 14 in the manufacture of a medicament for the treatment or prevention of chronic obstructive pulmonary disease