# Patent

## Title
Use of tyrosine kinase inhibitors for treating allergic diseases

## Abstract

The present invention relates to a method for treating allergic diseases such as asthma, comprising administering a tyrosine kinase inhibitor to a human in need of such treatment, more particularly a non-toxic, selective and potent c-kit inhibitor. Preferably, said inhibitor is unable to promote death of IL-3 dependent cells cultured in presence of IL-3.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This Application is a 371 of PCT/IB02/03297, filed Jun. 28, 2002, which claims benefit of U.S. Provisional Application No. 60/301,408, filed Jun. 29, 2001.


BACKGROUND OF THE INVENTION
The present invention relates to a method for treating allergic diseases such as asthma, comprising administering a tyrosine kinase inhibitor to a human in need of such treatment, more particularly a non-toxic, selective and potent c-kit inhibitor. Preferably, said inhibitor is unable to promote death of IL-3 dependent cells cultured in presence of IL-3.
Recent surveys show that human are more and more afflicted in modern societies with allergic disorders such as allergic sinusitis, allergic rhinitis and asthma. For example, in the USA alone, it is estimated that more than 87 million people are coping with some form of allergic diseases. The financial burden of the treatments rises to a total of several billion dollars and is due to the recurrence of these diseases.
Different treatments are available to alleviate the symptoms associated with allergic diseases. For example, regarding severe allergic diseases such as asthma, histamine H.sub.1-receptor antagonists have been proposed together with antagonists of leukotriene receptors (U.S. Pat. No. 5,420,143), but anti-histamine compounds have been found to be less effective and do not provide a solution to the recurrence of asthma. Similar strategies have been proposed in U.S. Pat. No. 6,221,880 using 5-lipoxygenase inhibitors, but again this treatment only reduces inflammation symptoms associated with allergic diseases and cannot be considered as a cure on the long run.
In response to this problem, the suppression of allergic disorders by treatment with interleukin-2 (IL-2) has been proposed in U.S. Pat. No. 5,989,546, but the induction of death by apoptosis of a subpopulation of T lymphocytes has many side effects limiting such therapy to the most severe forms of allergic diseases.
Therefore, there is a need for alternative treatments of these diseases that would be more effective on the long term and which would be well tolerated especially in respect to repeated administration.
Among these allergic diseases, we can cite allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis and insect bite skin inflammation, but bronchial asthma is the most prevalent and recurrent disease severely afflicting the human population.
Asthma is characterized by airflow obstruction, bronchial hyperresponsiveness and airway inflammation. Airway inflammation is the major factor in the development and perpetuation of asthma. In allergic asthma, which is the most frequent, especially in children, and better studied form of the disease, allergens are thought to initiate the inflammatory process by inducing a T-lymphocyte mediated response (TH2) that results in the production of allergen-specific IgE. IgE bind to its high-affinity receptor FceR1 on pulmonary mast cells triggering a type 1 (IgE-mediated) immediate allergic response. Mast cell activation induces diverse effector responses, such as secretion of allergic mediators, proteases, chemokines such as MCP-1 and RANTES (reviewed in Marshall et al, Allergy Asthma Proc. 2000 September-October; 21(5):309-13), leukotrienes, prostaglandins, neurotrophins (reviewed in Carr et al, Curr Opin Pulm Med. 2001 January; 7(1):1-7), induction of cytokine gene transcription (IL-4, IL-5, IL-6, IL-13, TNFa and GM-CSF) (Bradding et al, Am J Respir Cell Mol Biol 10, 471-80 (1994). These mediators contribute to creating the asthmatic phenotype by their effects on endothelial cells, smooth muscle cells and fibroblasts and on extracellular matrix, and by recruiting other inflammatory cells (Martin et al, J Clin Invest 91, 1176-82 (1993); Bischoff et al, J Exp Med 175, 245-5 (1992), and reviewed in Galli and Costa, Allergy 50, 851-62 (1995); Galli, Curr Opin Hematol. 2000 January; 7(1):32-9; Bingham et al, Mast-cell responses in the development of asthma. J Allergy Clin Immunol. 2000 February; 105(2 Pt 2):S527-34; Busse et al, Asthma. N. Engl. J. Med. 2001 February 1;344(5):350-62).
Newly developed therapeutic approaches to asthma have suggested a role of mast cells in asthma. One is a humanized anti-IgE monoclonal antibody that is now in phase III of clinical trials (reviewed in Fick et al, Curr Opin Pulm Med. January 1999;5(1):76-80; Chang T W, Nat Biotechnol. 2000 February; 18(2):157-62; Barnes P J Int Arch Allergy Immunol. 2000 November; 123(3):196-204). The rationale of the anti-IgE therapy is to specifically target IgE with the result of inactivating free IgE and halting further IgE production. In addition, since IgE levels are a major regulator of the level of expression of IgE receptor FceRI, one aim of this therapy is to decrease FceRI expression on mast cells and basophils, and, as a consequence, to decrease the capacity of these cells to be activated. These trials have shown that the anti-IgE therapy is capable of improving some of the parameters of asthma, for example corticosteroid usage. Nevertheless, antibody based therapy is not suitable to repeated treatment of the most recurrent forms of allergic diseases.
The capacity of the anti-IgE therapy to decrease FceRI expression has been demonstrated on basophils. The decrease in FceRI expression on basophils is associated with a decrease in the capacity of basophils to secrete mediators upon activation. Even though the effect of the anti-IgE therapy on pulmonary mast cells has not been studied because these cells are difficult to harvest.
In addition, compositions comprising tryptase inhibitors for treating mast-cell mediated conditions are proposed in U.S. Pat. No. 5,656,660, but decreasing the activity of free tryptase released by activated mast cells is not sufficient to block chain reactions caused by the others mast cells released factors.
Mast cells (MC) are tissue elements derived from a particular subset of hematopoietic stem cells that express CD34, c-kit and CD13 antigens (Kirshenbaum et al, Blood. 94: 2333-2342, 1999 and Ishizaka et al, Curr Opin Immunol. 5:937-43, 1993). Immature MC progenitors circulate in the bloodstream and differentiate in tissues. These differentiation and proliferation processes are under the influence of cytokines, one of utmost importance being Stem Cell Factor (SCF), also termed Kit ligand (KL), Steel factor (SL) or Mast Cell Growth Factor (MCGF). SCF-receptor is encoded by the protooncogene c-kit, that belongs to type III receptor tyrosine kinase subfamily (Boissan and Arock, J Leukoc Biol. 67: 135-48, 2000). This receptor is also expressed on others hematopoietic or non hematopoietic cells. Ligation of c-kit receptor by SCF induces its dimerization followed by its transphosphorylation, leading to the recruitement and activation of various intracytoplasmic substrates. These activated substrates induce multiple intracellular signaling pathways responsible for cell proliferation and activation (Boissan and Arock, 2000). Mast cells are characterized by their heterogeneity, not only regarding tissue location and structure but also at the functional and histochemical levels (Aldenborg and Enerback., Histochem. J. 26:587-96, 1994; Bradding et al. J Immunol. 155:297-307, 1995; Irani et al, J Immunol. 147:247-53, 1991; Miller et al, Curr Opin Immunol. 1:637-42, 1989 and Welle et al, J Leukoc Biol. 61:233-45, 1997).
In connection with the invention, evidence of focal and complete degranulation of mast cells was frequently observed. Besides, mast cells produce a large variety of mediators categorized here into three groups: preformed granule-associated mediators (histamine, proteoglycans, and neutral proteases), lipid-derived mediators (prostaglandins, thromboxanes and leucotrienes), and various cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, TNF-a, GM-CSF, MIP-1a, MIP-1b and IFN-g). Then, liberation by activated mast cells of mediators (TNF-a, histamine, leucotrienes, prostaglandines etc . . . ) is proposed here to induce severe allergic diseases.
More recently, an approach directed towards mast cells has been developed and tested in a mouse model of asthma. This approach targets SCF and is based on the critical role of c-kit and its ligand, SCF, on mast cell growth, differentiation and activation. Intranasal administration of antisense oligonucleotides to SCF was shown to suppress various signs of lung inflammation, such as IL-4 production and eosinophil infiltration in a classical OVA-induced model of asthma (Finotto et al, J. Allergy Clin. Immunol. February 2001;107(2):279-86). However, the efficacy of the antisens technology as far as clinical uses is concerned has not been actually demonstrated and the cost for producing such active nucleic acid molecules is not commensurate with a global marketing.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A. depicts the production of PGE1 in mice following treatment with 4-(4-methylpiperazine-1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide, or vehicle alone, and subsequently challenged with antigen.
FIG. 1B. depicts the body weight of mice following treatment with 4-(4-methylpiperazine-1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide, or vehicle alone, and subsequently challenged with antigen.
FIG. 2. depicts superficial pyodermatitis and generalized Malassezia dermatitis, presentations of severe atopic dermatitis, in a dog prior to treatment with 4-(4-methylpiperazine-1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide.
FIG. 3. depicts the resolution of the superficial pyodermatitis and generalized Malassezia dermatitis following treatment with 4-(4-methylpiperazine- 1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide.
FIG. 4. depicts total cell number in lavage fluid from mice following pre-treatment with saline, vehicle alone, or 4-(4-methylpiperazine-1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide, and subsequent challenge with methacholine.
FIG. 5. depicts the enhanced pause value (Penh), an indicator of airway resistance, in mice following pre-treatment with saline, vehicle alone, or 4-(4-methylpiperazine-1-ylmethyl) -N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phenyl]-benzamide, and Subsequent challenge with methacholine.



In contrast, the present invention propose to use c-kit specific kinase inhibitors to inhibit mast cell proliferation, survival and activation. A new route for treating allergic diseases is provided, which consists of destroying mast cells playing a role in the pathogenesis of these disorders. It has been found that tyrosine kinase inhibitors and more particularly c-kit inhibitors are especially suited to reach this goal.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for treating allergic diseases comprising administering a tyrosine kinase inhibitor to a mammal in need of such treatment.
Tyrosine kinase inhibitors are selected for example from bis monocyclic, bicyclic or heterocyclic aryl compounds (WO 92/20642), vinylene-azaindole derivatives (WO 94/14808) and 1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992), Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), seleoindoles and selenides (WO 94/03427), tricyclic polyhydroxylic compounds (WO 92/21660) and benzylphosphonic acid compounds (WO 91/15495), pyrimidine derivatives (U.S. Pat. No. 5,521,184 and WO 99/03854), indolinone derivatives and pyrrol-substituted indolinones (U.S. Pat. No. 5,792,783, EP 934 931, U.S. Pat. No. 5,834,504, U.S. Pat. No. 5,883,116, U.S. Pat. No. 5,883,113, U.S. Pat. No. 5,886,020, WO 96/40116 and WO 00/38519), as well as bis monocyclic, bicyclic aryl and heteroaryl compounds (EP 584 222, U.S. Pat. No. 5,656,643 and WO 92/20642), quinazoline derivatives (EP 602 851, EP 520 722, U.S. Pat. No. 3,772,295 and U.S. Pat. No. 4,343,940) and aryl and heteroaryl quinazoline (U.S. Pat. No. 5,721,237, U.S. Pat. No. 5,714,493, U.S. Pat. No. 5,710,158 and WO 95/15758).
Preferably, said tyrosine kinase inhibitors are unable to promote death of IL-3 dependent cells cultured in presence of IL-3.
In another embodiment, the invention is directed to a method for treating allergic diseases comprising administering a c-kit inhibitor to a mammal in need of such treatment.
Preferably, said c-kit inhibitor is a non-toxic, selective and potent c-kit inhibitor. Such inhibitors can be selected from the group consisting of indolinones, pyrimidine derivatives, pyrrolopyrimidine derivatives, quinazoline derivatives, quinoxaline derivatives, pyrazoles derivatives, bis monocyclic, bicyclic or heterocyclic aryl compounds, vinylene-azaindole derivatives and pyridyl-quinolones derivatives, styryl compounds, styryl-substituted pyridyl compounds, seleoindoles, selenides, tricyclic polyhydroxylic compounds and benzylphosphonic acid compounds.
Among preferred compounds, it is of interest to focus on pyrimidine derivatives such as N-phenyl-2-pyrimidine-amine derivatives (U.S. Pat. No. 5,521,184 and WO 99/03854), indolinone derivatives and pyrrol-substituted indolinones (U.S. Pat. No. 5,792,783, EP 934 931, U.S. Pat. No. 5,834,504), U.S. Pat. No. 5,883,116, U.S. Pat. No. 5,883,113, U.S. Pat. No. 5,886,020, WO 96/40116 and WO 00/38519), as well as bis monocyclic, bicyclic aryl and heteroaryl compounds (EP 584 222, U.S. Pat. No. 5,656,643 and WO 92/20642), quinazoline derivatives (EP 602 851, EP 520 722, U.S. Pat. No. 3,772,295 and U.S. Pat. No. 4,343,940), 4-amino-substituted quinazolines (U.S. Pat. No. 3,470,182), 4-thienyl-2-(1H)-quinazolones, 6,7-dialkoxyquinazolines (U.S. Pat. No. 3,800,039), aryl and heteroaryl quinazoline (U.S. Pat. No. 5,721,237, US 5,714,493, U.S. Pat. No. 5,710,158 and WO 95/15758), 4-anilinoquinazoline compounds (U.S. Pat. No. 4,464,375), and 4-thienyl-2-(1H)-quinazolones (U.S. Pat. No. 3,551,427).
So, preferably, the invention relates to a method for treating allergic diseases comprising administering a non toxic, potent and selective c-kit inhibitor. Such inhibitor can be selected from pyrimidine derivatives, more particularly N-phenyl-2-pyrimidine-amine derivatives of formula I:




wherein the R1, R2, R3, R13 to R17 groups have the meanings depicted in EP 564 409 B1, incorporated herein in the description.

Preferably, the N-phenyl-2-pyrimidine-amine derivative is selected from the compounds corresponding to formula II:




    Wherein R1, R2 and R3 are independently chosen from H, F, Cl, Br, I, a C1-C5 alkyl or a cyclic or heterocyclic group, especially a pyridyl group;
    R4, R5 and R6 are independently chosen from H, F, Cl, Br, I, a C1-C5 alkyl, especially a methyl group;
    and R7 is a phenyl group bearing at least one substituent, which in turn possesses at least one basic site, such as an amino function.


Preferably, R7 is the following group:




Among these compounds, the preferred are defined as follows:

    R1 is a heterocyclic group, especially a pyridyl group,
    R2 and R3 are H,
    R4 is a C1-C3 alkyl, especially a methyl group,
    R5 and R6 are H,
    and R7 is a phenyl group bearing at least one substituent, which in turn possesses at least one basic site, such as an amino function, for example the group:






Therefore, in a preferred embodiment, the invention relates to a method for treating allergic diseases comprising the administration of an effective amount of the compound known in the art as CGP57148B:

    4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-yl-)pyrimidine-2Yl)amino-)]phényl]-benzamide






The preparation of this compound is described in example 21 of EP 564 409 and the β-form, which is particularly useful is described in WO 99/03854.
Alternatively, the c-kit inhibitor can be selected from:

    
    
        indolinone derivatives, more particularly pyrrol-substituted indolinones,
        monocyclic, bicyclic aryl and heteroaryl compounds, quinazoline derivatives,
        and quinaxolines, such as 2-phényl-quinaxoline derivatives, for example 2-phenyl-6,7-dimethoxy quinaxoline.
    
    


In a preferred aspect, the invention contemplated the method mentioned above, wherein said c-kit inhibitor is unable to promote death of IL-3 dependent cells cultured in presence of IL-3.
Among allergic diseases, asthma, allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis and insect bite skin inflammation and blood sucking parasitic infestation are embraced by the invention. Preferably, the method depicted above is practiced in human but also in animals in respect to insect bites, blood sucking parasitic infestation, especially the infestation of pets (cats and dogs) by fleas, which has been a continued problem in the art.
In a further embodiment, c-kit inhibitors as mentioned above are inhibitors of activated c-kit. In frame with the invention, the expression “activated c-kit” means a constitutively activated-mutant c-kit including at least one mutation selected from point mutations, deletions, insertions, but also modifications and alterations of the natural c-kit sequence (SEQ ID No. 1). Such mutations, deletions, insertions, modifications and alterations can occur in the transphosphorylase domain, in the juxtamembrane domain as well as in any domain directly or indirectly responsible for c-kit activity. The expression “activated c-kit” also means herein SCF-activated c-kit. Preferred and optimal SCF concentrations for activating c-kit are comprised between 5.10−7 M and 5.10−6 M, preferably around 2.10−6 M. In a preferred embodiment, the activated-mutant c-kit in step a) has at least one mutation proximal to Y823, more particularly between amino acids 800 to 850 of SEQ ID No 1 involved in c-kit autophosphorylation, notably the D816V, D816Y, D816F and D820G mutants. In another preferred embodiment, the activated-mutant c-kit in step a) has a deletion in the juxtamembrane domain of c-kit. Such a deletion is for example between codon 573 and 579 called c-kit d(573-579). The point mutation V559G proximal to the juxtamembrane domain c-kit is also of interest.
In this regard, the invention contemplates a method for treating allergic diseases comprising administering to a mammal in need of such treatment a compound that is a selective, potent and non toxic inhibitor of activated c-kit obtainable by a screening method which comprises:

    a) bringing into contact (i) activated c-kit and (ii) at least one compound to be tested; under conditions allowing the components (i) and (ii) to form a complex,
    b) selecting compounds that inhibit activated c-kit,
    c) testing and selecting a subset of compounds identified in step b), which are unable to promote death of IL-3 dependent cells cultured in presence of IL-3.


This screening method can further comprise the step consisting of testing and selecting a subset of compounds identified in step b) that are inhibitors of mutant activated c-kit (for example in the transphosphorylase domain), which are also capable of inhibiting SCF-activated c-kit wild.
Alternatively, in step a) activated c-kit is SCF-activated c-kit wild.
A best mode for practicing this method consists of testing putative inhibitors at a concentration above 10 μM in step a). Relevant concentrations are for example 10, 15, 20, 25, 30, 35 or 40 μM.
In step c), IL-3 is preferably present in the culture media of IL-3 dependent cells at a concentration comprised between 0.5 and 10 ng/ml, preferably between 1 to 5 ng/ml.
Examples of IL-3 dependent cells include but are not limited to:

    
    
        cell lines naturally expressing and depending on c-kit for growth and survival. Among such cells, human mast cell lines can be established using the following procedures: normal human mast cells can be infected by retroviral vectors containing sequences coding for a mutant c-kit comprising the c-kit signal peptide and a TAG sequence allowing to differentiate mutant c-kits from c-kit wild expressed in hematopoetic cells by means of antibodies.
    
    


This technique is advantageous because it does not induce cellular mortality and the genetic transfer is stable and gives satisfactory yields (around 20%). Pure normal human mast cells can be routinely obtained by culturing precursor cells originating from blood obtained from human umbilical vein. In this regard, heparinated blood from umbilical vein is centrifuged on a Ficoll gradient so as to isolate mononucleated cells from other blood components. CD34+ precursor cells are then purified from the isolated cells mentioned above using the immunomagnetic selection system MACS (Miltenyi biotech). CD34+ cells are then cultured at 37° C. in 5% CO2 atmosphere at a concentration of 105 cells per ml in the medium MCCM (α-MEM supplemented with L-glutamine, penicillin, streptomycin, 5 10−5 M β-mercaptoethanol, 20% veal foetal serum, 1% bovine albumin serum and 100 ng/ml recombinant human SCF. The medium is changed every 5 to 7 days. The percentage of mast cells present in the culture is assessed each week, using May-Grünwal Giemsa or Toluidine blue coloration. Anti-tryptase antibodies can also be used to detect mast cells in culture. After 10 weeks of culture, a pure cellular population of mast cells (>98%) is obtained.
It is possible using standard procedures to prepare vectors expressing c-kit for transfecting the cell lines established as mentioned above. The cDNA of human c-kit has been described in Yarden et al., (1987) EMBO J.6 (11), 3341-3351. The coding part of c-kit (3000 bp) can be amplified by PCR and cloned, using the following oligonucleotides:










5′AAGAAGAGATGGTACCTCGAGGGGTGACCC3′
(SEQ ID No 2)
sens



 


5′CTGCTTCGCGGCCGCGTTAACTCTTCTCAACCA3′
(SEQ ID No 3)
antisens






The PCR products, digested with Not1 and Xho1, has been inserted using T4 ligase in the pFlag-CMV vector (SIGMA), which vector is digested with Not1 and Xho1 and dephosphorylated using CIP (Biolabs). The pFlag-CMV-c-kit is used to transform bacterial clone XL1-blue. The transformation of clones is verified using the following primers:










5′AGCTCGTTTAGTGAACCGTC3′
(SEQ ID No 4)
sens,



 


5′GTCAGACAAAATGATGCAAC3′
(SEQ ID No 5)
antisens.






Directed mutagenesis is performed using relevant cassettes is performed with routine and common procedure known in the art.
The vector Migr-1 (ABC) can be used as a basis for constructing retroviral vectors used for transfecting mature mast cells. This vector is advantageous because it contains the sequence coding for GFP at the 3′ and of an IRES. These features allow to select cells infected by the retrovirus using direct analysis with a fluorocytometer. As mentioned above, the N-terminal sequence of c-kit c-DNA can be modified so as to introduce a Flag sequence that will be useful to discriminating heterogeneous from endogenous c-kit.
Other IL-3 dependent cell lines that can be used include but are not limited to:

    
    
        BaF3 mouse cells expressing wild-type or mutated form of c-kit (in the juxtamembrane and in the catalytic sites) are described in Kitayama et al, (1996), Blood 88, 995-1004 and Tsujimura et al, (1999), Blood 93, 1319-1329.
        IC-2 mouse cells expressing either c-kitWT or c-kitD814Y are presented in Piao et al, (1996), Proc. Natl. Acad. Sci. USA 93, 14665-14669.
    
    


IL-3 independent cell lines are:

    
    
        HMC-1, a factor-independent cell line derived from a patient with mast cell leukemia, expresses a juxtamembrane mutant c-kit polypeptide that has constitutive kinase activity (Furitsu T et al, J Clin Invest. 1993;92:1736-1744; Butterfield et al, Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res. 1988;12:345-355 and Nagata et al, Proc Natl Acad Sci USA. 1995;92:10560-10564).
        P815 cell line (mastocytoma naturally expressing c-kit mutation at the 814 position) has been described in Tsujimura et al, (1994), Blood 83, 2619-2626.
    
    


The extent to which component (ii) inhibits activated c-kit can be measured in vitro or in vivo. In case it is measured in vivo, cell lines expressing an activated-mutant c-kit, which has at least one mutation proximal to Y823, more particularly between amino acids 800 to 850 of SEQ ID No1 involved in c-kit autophosphorylation, notably the D816V, D816Y, D816F and D820G mutants, are preferred.
Example of cell lines expressing an activated-mutant c-kit are as mentioned.
In another preferred embodiment, the method further comprises the step consisting of testing and selecting compounds capable of inhibiting c-kit wild at concentration below 1 μM. This can be measured in vitro or in vivo.
Therefore, compounds are identified and selected according to the method described above are potent, selective and non-toxic c-kit wild inhibitors.
Alternatively, the screening method as defined above can be practiced in vitro. In this regard, the inhibition of mutant-activated c-kit and/or c-kit wild can be measured using standard biochemical techniques such as immunoprecipitation and western blot. Preferably, the amount of c-kit phosphorylation is measured.
In a still further embodiment, the invention contemplates a method for treating allergic diseases as depicted above wherein the screening comprises:

    a) performing a proliferation assay with cells expressing a mutant c-kit (for example in the transphosphorylase domain), which mutant is a permanent activated c-kit, with a plurality of test compounds to identify a subset of candidate compounds targeting activated c-kit, each having an IC50<10 μM, by measuring the extent of cell death,
    b) performing a proliferation assay with cells expressing c-kit wild said subset of candidate compounds identified in step (a), said cells being IL-3 dependent cells cultured in presence of IL-3, to identify a subset of candidate compounds targeting specifically c-kit,
    c) performing a proliferation assay with cells expressing c-kit, with the subset of compounds identified in step b) and selecting a subset of-candidate compounds targeting c-kit wild, each having an IC50<10 μM, preferably an IC50<1 μM, by measuring the extent of cell death.


Here, the extent of cell death can be measured by 3H thymidine incorporation, the trypan blue exclusion method or flow cytometry with propidium iodide. These are common techniques routinely practiced in the art.
The method according to the invention includes preventing, delaying the onset and/or treating allergic diseases in human, dogs and cats.
Therefore, the invention embraces the use of the compounds defined above to manufacture a medicament for treating allergic diseases such as asthma, allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis, insect bite skin inflammation and blood sucking parasitic infestation.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
More particularly, the invention relates to a pharmaceutical composition intended for administration with aerosolized formulation to target areas of a patient's respiratory tract, intranasal or topical administration.
Devices and methodologies for delivering aerosolized bursts of a formulation of a drug is disclosed in U.S. Pat. No. 5,906,202. Formulations are preferably solutions, e.g. aqueous solutions, ethanoic solutions, aqueous/ethanoic solutions, saline solutions, colloidal suspensions and microcrystalline suspensions. For example aerosolized particles comprise the active ingredient mentioned above and a carrier, (e.g., a pharmaceutically active respiratory drug and carrier) which are formed upon forcing the formulation through a nozzle which nozzle is preferably in the form of a flexible porous membrane. The particles have a size which is sufficiently small such that when the particles are formed they remain suspended in the air for a sufficient amount of time such that the patient can inhale the particles into the patient's lungs.
The invention encompasses systems described in U.S. Pat. No. 5,556,611:

    
    
        liquid gas systems (a liquefied gas is used as propellent gas (e.g. low-boiling FCHC or propane, butane) in a pressure container,
        suspension aerosol (the active substance particles are suspended in solid form in the liquid propellent phase),
        pressurized gas system (a compressed gas such as nitrogen carbon dioxide, dinitrogen monoxide, air is used.
    
    


Thus, according to the invention the pharmaceutical preparation is made in that the active substance is dissolved or dispersed in a suitable nontoxic medium and said solution or dispersion atomized to an aerosol, i.e. distributed extremely finely in a carrier gas. This is technically possible for example in the form of aerosol propellent gas packs, pump aerosols or other devices known per se for liquid misting and solid atomizing which in particular permit an exact individual dosage.
Therefore, the invention is also directed to aerosol devices containing a formulation as depicted above, preferably with metered dose valves.
Regarding intranasal administration, pharmaceutically acceptable carriers for administering tyrosine kinase or c-kit inhibitors to the nasal mucosal surfaces will be readily appreciated by the ordinary artisan. Such carriers are disclosed, simply by way of example, by Remington's Pharmaceutical Sciences” 16th edition, 1980, Ed. By Arthur Osol, the disclosure of which is incorporated herein by reference.
The selection of appropriate carriers depends upon the particular type of administration that is contemplated. For administration via the upper respiratory tract. The composition can be formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's, Id. at page 1445). Of course, the ordinary artisan can readily determine a suitable saline content and pH for an innocuous aqueous carrier for nasal and/or upper respiratory administration.
Common intranasal carriers include nasal gels, creams, pastes or ointments with a viscosity of, e.g., from about 10 to about 3000 cps, or from about 2500 to 6500 cps, or greater, may also be used to provide a more sustained contact with the nasal mucosal surfaces. Such carrier viscous formulations may be based upon, simply by way of example, alkylcelluloses and/or other biocompatible carriers of high viscosity well known to the art (see e.g., Remington's, cited supra. A preferred alkylcellulose is, e.g., methylcellulose in a concentration ranging from about 5 to about 1000 or more mg per 100 ml of carrier. A more preferred concentration of methyl cellulose is, simply by way of example, from about 25 to about mg per 100 ml of carrier.
Other ingredients, such as art known preservatives, colorants, lubricating or viscous mineral or vegetable oils, perfumes, natural or synthetic plant extracts such as aromatic oils, and humectants and viscosity enhancers such as, e.g., glycerol, can also be included to provide additional viscosity, moisture retention and a pleasant texture and odor for the formulation. For nasal administration of solutions or suspensions according to the invention, various devices are available in the art for the generation of drops, droplets and sprays.
A premeasured unit dosage dispenser including a dropper or spray device containing a solution or suspension for delivery as drops or as a spray is prepared containing one or more doses of the drug to be administered and is another object of the invention. The invention also includes a kit containing one or more unit dehydrated doses of tyrosine kinase or c-kit inhibitors, together with any required salts and/or buffer agents, preservatives, colorants and the like, ready for preparation of a solution or suspension by the addition of a suitable amount of water.
Therefore, the invention relates to a nasal dropper or a nasal spray device comprising a tyrosine kinase inhibitor, more particularly a c-kit inhibitors depicted above.
The compositions according to the invention may also be presented in all forms normally used for topical application, in particular in the form of a gel, paste, ointment, cream, lotion, liquid suspension aqueous, aqueous-alcoholic or, oily solutions, or dispersions of the lotion or serum type, or anhydrous or lipophilic gels, or emulsions of liquid or semi-solid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous phase or vice versa, or of suspensions or emulsions of soft, semi-solid consistency of the cream or gel type, or alternatively of microemulsions, of microcapsules, of microparticles or of vesicular dispersions to the ionic and/or nonionic type. These compositions are prepared according to standard methods.
The composition according to the invention comprises any ingredient commonly used in dermatology and cosmetic. It may comprise at least one ingredient selected from hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, emollients, viscosity enhancing polymers, humectants, surfactants, preservatives, antioxidants, solvents, and fillers, antioxidants, solvents, perfumes, fillers, screening agents, bactericides, odor absorbers and coloring matter.
As oils which can be used in the invention, mineral oils (liquid paraffin), vegetable oils (liquid fraction of shea butter, sunflower oil), animal oils, synthetic oils, silicone oils (cyclomethicone) and fluorinated oils may be mentioned. Fatty alcohols, fatty acids (stearic acid) and waxes (paraffin, carnauba, beeswax) may also be used as fatty substances.
As emulsifiers which can be used in the invention, glycerol stearate, polysorbate 60 and the PEG-6/PEG-32/glycol stearate mixture are contemplated.
As hydrophilic gelling agents, carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylate/alkylacrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, clays and natural gums may be mentioned, and as lipophilic gelling agents, modified clays such as bentones, metal salts of fatty acids such as aluminum stearates and hydrophobic silica, or alternatively ethylcellulose and polyethylene may be mentioned.
As hydrophilic active agents, proteins or protein hydrolysates, amino acids, polyols, urea, allantoin, sugars and sugar derivatives, vitamins, starch and plant extracts, in particular those of Aloe vera may be used.
As lipophilic active, agents, retinol (vitamin A) and its derivatives, tocopherol (vitamin E) and its derivatives, essential fatty acids, ceramides and essential oils may be used. These agents add extra moisturizing or skin softening features when utilized.
In addition, a surfactant can be included in the composition so as to provide deeper penetration of the ingredients and of the tyrosine kinase inhibitor.
Among the contemplated ingredients, the invention embraces penetration enhancing agents selected for example from the group consisting of mineral oil, water, ethanol, triacetin, glycerin and propylene glycol; cohesion agents selected for example from the group consisting of polyisobutylene, polyvinyl acetate and polyvinyl alcohol, and thickening agents.
Chemical methods of enhancing topical absorption of drugs are well known in the art. For example, compounds with penetration enhancing properties include sodium lauryl sulfate (Dugard, P. H. and Sheuplein, R. J., “Effects of Ionic Surfactants on the Permeability of Human Epidermis: An Electrometric Study,” J. Ivest. Dermatol., V.60, pp. 263-69, 1973), lauryl amine oxide (Johnson et. al., U.S. Pat. No. 4,411,893), azone (Rajadhyaksha, U.S. Pat. Nos. 4,405,616 and 3,989,816) and decylmethyl sulfoxide (Sekura, D. L. and Scala, J., “The Percutaneous Absorption of Alkylmethyl Sulfides,” Pharmacology of the Skin, Advances In Biolocy of Skin, (Appleton-Century Craft) V. 12, pp. 257-69, 1972). It has been observed that increasing the polarity of the head group in amphoteric molecules increases their penetration-enhancing properties but at the expense of increasing their skin irritating properties (Cooper, E. R. and Berner, B., “Interaction of Surfactants with Epidermal Tissues: Physiochemical Aspects,” Surfactant Science Series, V. 16, Reiger, M. M. ed. (Marcel Dekker, Inc.) pp. 195-210, 1987).
A second class of chemical enhancers are generally referred to as co-solvents. These materials are absorbed topically relatively easily, and, by a variety of mechanisms, achieve permeation enhancement for some drugs. Ethanol (Gale et. al., U.S. Pat. No. 4,615,699 and Campbell et. al., U.S. Pat. Nos. 4,460,372 and 4,379,454), dimethyl sulfoxide (U.S. Pat. Nos. 3,740,420 and 3,743,727, and U.S. Pat. No. 4,575,515), and glycerine derivatives (U.S. Pat. No. 4,322,433) are a few examples of compounds which have shown an ability to enhance the absorption of various compounds.
Topical composition referred herein are particularly relevant for treating skin allergic disorders such as urticaria, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis, insect bite skin inflammation and blood sucking parasitic infestation especially in dogs and cats.
Pharmaceutical compositions suitable for use in the invention include compositions wherein c-kit inhibitors are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therpeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. As mentioned above, a tyrosine kinase inhibitor and more particularly a c-kit inhibitor according to the invention is unable to promote death of IL-3 dependent cells cultured in presence of IL-3.
As mentioned above, the invention also contemplates a composition suitable for oral administration comprising a tyrosine kinase inhibitor, more particularly a c-kit inhibitor for the treatment of skin allergic disorders such as urticaria, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis, insect bite skin inflammation and blood sucking parasitic infestation especially in dogs and cats.
In another embodiment, the composition according to the invention is suitable for intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or rectal administration and comprises a tyrosine kinase inhibitor, more particularly a c-kit inhibitor for the treatment of skin allergic disorders such as urticaria, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis, insect bite skin inflammation and blood sucking parasitic infestation especially in dogs and cats.
Utility of the invention will further ensue from the detailed description below.
EXAMPLE 1
Use of 4-(4-méthylpipérazine- 1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide for treating Anaphylaxis.
Anaphylaxis is a life-threatening rapid allergic reaction that affects millions of people. It can be caused by a variety of allergens such as food, medications, insect venom, and latex. As of today, it is treated with epinephrine but side effects are commonly observed.
Passive Cutaneous Anaphylaxis:
The experimental group has been treated with the compound for one week prior antigen challenge. Each animal received ip injection of 1 mg/day in 2 doses of 0.5 mg, and control group received vehicle at equal volume. A second group received 2× the amount of the first experiment. Basically, each animal received 2 mg per day in 2 doses of 1 mg via ip injection. They were treated every day for one week prior antigen challenge. The p value is 0.043, and n is 5 for treated ground and 4 for vehicle treated group.
On day 8, anesthetize mice with avertin. Inject the right ears with 20 ng/20 μL of IgE and 20 uL of PBS to the left ears intradermally.
After 24 hours, 100 μL of 1% Evans Blue containing 100 μg of DNP-albumin are injected in tail vein of mice. Mice are sacrificed after 90 minutes of tail vein injection. The ears are cut off as close to the base of ear and incubated in 1 ml of formamide at 54 C for 48 hours for quantitative analysis of formamide extracts at 610 nm.
Reagents: Anti-DNP IgE (monoclonal anti-Dinitrophenyl)
Human-Dinitrophenyl Albumin (DNP-Albumin)
1% Evans Blue (in PBS)
The results are presented in FIG. 1A (PGE1 is protaglandine 1, which is released during anaphylaxis. FIG. 1B shows that at 2 mg/day the animals are in good health.
EXAMPLE 2
Treatment of Atopic Dermatitis in Dogs
DeMora F et al, Skin mast cell releasability in dogs with atopic dermatitis, Inflamm Res 1996 August;45(8):424-7 have found that the total histamine content found per isolated skin mast cell was higher in the allergic dogs than in nonatopic animals. This correlates with our observation that mast cells number increases in the derm of dogs afflicted with atopic dermatitis.
Garcia G and DeMora F, Effect of H1-antihistamines on histamine release from dispersed canine cutaneous mast cells, Am J Vet Res March 1997;58(3):293-7 tried H1-antihistamines as an alternative to glucocorticoid therapy. Using an in vitro method, they showed that loratadine is the only antihistamine that has potent inhibition of histamine release from dog cutaneous mast cells without a substantial prodegranulating effect.
While loratadine could provide a solution for short term treatment, we propose in frame with the present invention to use tyrosine kinase inhibitors to deplete mast cells that are involved in atopic dermatitis.
In this regards, we have tested 4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide on a 3 year old dog afflicted with severe atopic dermatitis presenting superficial pyodermatitis and generalized Malassezia dermatitis.
4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide was administered at day 0 at 3 mg/kg/d together with cefalexine (30 mg/kg/d) and ketoconazole (10 mg/kg/j). Doses of the c-kit inhibitor can be increased up to 20 mg/kg/d since a very good tolerance was observed. Results are presented in FIG. 2 (before treatment) and FIG. 3 (after treatment).
EXAMPLE 3
Treatment of Asthma with 4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide.
4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide was tested in asthmatic mice provoked by immunization and administration of ovalbumin.
Ovalbumin Protocol:


    Day 0: Inject mice intraperitoneally with the compound 4-(4-méthylpipérazine-1-ylméthyl)-N-[4-méthyl-3-[(4-pyridine-3-ylpyrimidine-2yl)amino]phényl]-benzamide or vehicle in 2 doses of 0.5 mg/100 μl (1 mg/day).
    Day 21: Harvest day. Give mice one last dose of the compound or saline before methacholine challenge. Measure respiratory rate for 2 minutes after aerosolization with methacholine. Draw blood for IgE analysis. Lavage lungs with saline for cell counts and cytokine analysis. Fix lungs in 10% formalin for histology.


Note: PenH is a dimensionless value that is used to calculate airway function. And is further explicated in Am J Respir Crit Care Med Vol. 156, pp. 766-775, 1997.
Results are presented in FIGS. 4 and 5.

## Claims

1. A method for treating asthma, passive cutaneous anaphylaxis or atopic dermatitis, comprising administering an effective amount of the following compound:




4-(4-methylpiperazine-1-ylmethyl)-N-[4-methyl-3-[(4-pyridine-3-ylpyrimidine-2-yl)amino]phenyl]-benzamide, or a pharmaceutically acceptable salt thereof, to a human, dog or cat in need of such treatment.

2. The method according to claim 1 for treating asthma.