NO325454B1 - Use of N- {5- [4- (4-methyl-piperazino-methyl) -benzoylamido] -2-methylphenyl} -4- (3-pyridyl) -2-pyrimidine-amine. - Google Patents

Description

The present invention relates to the use of N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-rmethylphenyl}-4-(3-pyridyl)-2-pyrimidine-arnine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of mastocytosis.

Heinrich et al. reports in "BLOOD", August 2000, Volume 96(3), Pages 925-932, results obtained with N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4 -(3-pyridyl)-2-pyrimidine amine mesylate (STI571) in studies of a mast cell leukemia cell line and mainly in a human myeloid leukemia cell line, but does not describe the use of STI571 in mastocytosis. The present invention relates to the treatment of mastocytosis in human patients as well as dogs using STI571.

Salt-forming groups in the compound used in the invention are groups or radicals which have basic or acidic properties. Compounds which have at least one basic group or at least one basic radical, e.g. a free amino group, a pyrazinyl radical or a pyridyl radical, can form acid addition salts, e.g. with inorganic acids, such as hydrochloric acid, sulfuric acid or phosphoric acid, or with suitable organic carboxyl - or sulfonic acids, e.g. aliphatic mono- or dicarboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids such such as benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids such as mandelic acid or cinnamic acid, heteroaromatic carboxylic acids such as nicotinic acid or isonicotinic acid, aliphatic sulphonic acids such as methane-, ethane- or 2-hydroxyethanesulphonic acid, or aromatic sulphonic acids, for example benzene-, p-toluene- or naphthalene-2-sulphonic acid. When several basic groups are present, mono- or polyacid addition salts can be formed.

For isolation or purification purposes, it is also possible to use pharmaceutically unacceptable salts. However, only pharmaceutically acceptable, non-toxic salts are used for therapeutic purposes, and these salts are therefore preferred.

The term "mastocytosis" used herein refers to cystemic mastocytosis, e.g. mastocytoma, and especially mast cell neoplasms in dogs.

Mast cells play an important role as the primary effector cells in the allergic disorders mentioned here. Antigen-specific IgE-mediated degranulation of mast cells leads to the subsequent release of chemical mediators and a majority of cytokines as well as to leukotriene synthesis. Mast cells are also involved in the pathogenesis of multiple sclerosis.

Mast cell neoplasms occur in both humans and animals. In dogs, mast cell neoplasms are called mastocytomas, and the disease is common, representing 7 - 21% of tumors in dogs. A distinction must be made between human mastocytosis, which is usually transient or painless, and canine mast cell neoplasia, which is unpredictable and often aggressive and metastatic. For example, isolated mastocytomas in humans never metastasize; in contrast, 50% of canine mastocytomas are malignant, as determined by Hottendorf & Nielsen (1969) after reviewing 46 published reports of tumors in 938 dogs.

Cancer in the pet population is a spontaneous disease. Motivated by extending their animals' quality of life, pet owners often seek specialized care and treatment from veterinary oncologists at private veterinary hospitals and veterinary teaching hospitals throughout the country. Therapeutic modalities for veterinary cancer patients are similar to those for humans, including surgery, chemotherapy, radiation therapy, and biotherapy. It is estimated that there are 42 million dogs and about 20 million cats in the United States. Rough calculations of cancer incidence show that approximately 4 million new cancer diagnoses are made in dogs and a similar number in cats each year.

Cutaneous mast cell tumors in dogs are a common problem. Most mast cell tumors are benign and can be cured with simple resection, but if they recur or metastasize to distant sites, therapeutic options are limited. Treatment options for recurrent lesions may include external beam radiation therapy. For distant metastases or disseminated disease, the use of Lomustine® and vinblastine containing chemotherapy protocols has been shown to have a certain beneficial effect. Sites of metastases for mast cell tumors include skin, regional lymph nodes, spleen, liver, and bone marrow.

The involvement of the KIT receptor in the pathogenesis of mastocytosis is suggested through the observation that several mutations resulting in constitutive activation of KIT have been detected in a number of mast cell lines. A point mutation in human c-KIT, which causes replacement of Asp816 with Val in the phosphotransferase domain and receptor autoactivation, for example, occurs in a long-term human mast cell leukemia line (HMC-1) and the corresponding codon in two rodent mast cell lines. This activating mutation has also been identified in situ in some cases of human mastocytosis. Two other activating mutations have been found in the intracellular juxtamembrane region of KIT, i.e. the Val560Gly substitution in the human HMC-1 mast cell line and a seven-amino deletion (Thr573-His579) in a rodent mast cell line designated FAM3.

Using established test models and especially the test models described herein, it can be shown that the compound used according to the invention or in each case a pharmaceutically acceptable salt thereof, results in an effective prevention or, preferably, treatment of mastocytosis. The pharmacological activity can, for example, be shown in a clinical study or in the test procedure substantially as described below.

Part A

Example 1

This example demonstrates the in vitro effects of the compound of the invention on the SCF-dependent development of cultured human mast cell growth developed from CD34<+> cord blood cells using the culture system described by Kinoshita T, Sawai N, et al in Blood 1999, 94, 496- 508. More than 90% of the isolated cells were CD34 positive according to the flow cytometric analysis.

Reagents and antibodies

The compound according to the invention is solubilized in PBS at a concentration of 10"<2>M and stored at -80°C. AII-trans retinoic acid (Sigma) is dissolved in ethanol at a concentration of 10"<2>M and stored in light-protected small bottles at -80°C. Purified mAb for tryptase (MAB 1222) can be obtained from Chemicon International Inc, CA. For the flow cytometric analysis, mAbs for CD34 (8G12, FITC) and CD1 lb (Leul5, PE) are obtained from Becton Dickinson Immunocytometry Systems (Mountain View, CA), and mAb for CD41 (SZ22, FITC) from Immunotech S.A. (Marseilles, France). mAb for glycophorin A (GPA, JC159, FITC) can be obtained from Dako (Glostrup, Denmark). For Western blotting and immunoprecipitation, mAbs for c-kit (NU-c-kit) and phosphotyrosine (4G10) can be obtained from Nichirei and Upstate Biotechnology, Inc (Lake Placid, NY), respectively.

Suspension cultures

Serum-depleted liquid cultures are performed in 24-well culture plates (#3047; Becton Dickinson) 20,000 CD34<+> cells are cultured in each well containing 2 ml of a medium supplemented with 1% BSA, 300 ug/ml fully iron-saturated human transferrin (approximately 98% pure, Sigma), 16 µg/ml soy lecithin (Sigma), 9.6 µg/ml cholesterol (Nakalai Chemicals Ltd, Japan) and 20 ng/ml SCF, 10 ng/ml GM-CSF, 2 U/ml EPO, 10 ng/ml TPO, different concentrations of a compound according to the invention, alone or in combination. To investigate the effects of a compound according to the invention on the SCF-dependent development of mast cells, 10-week cultured cells developed with 20 ng/ml SCF from CD34<+> cord blood cells are used as target cells. 5 to 10 x 10<4>10-week cultured cells are incubated for 2 weeks in 24-well culture plates containing 20 ng/ml SCF with or without various concentrations of a compound according to the invention. The plates are incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2 and 90% N2. If the culture is continued for up to 4 weeks, half of the culture medium is replaced every 2 weeks with fresh medium containing the factor(s). The number of viable cells is determined by the trypan blue exclusion test using a hemocytometer.

Clonal cell cultures

The mast cell colony assay was performed in 35-mm Lux suspension culture dishes (#171099; Nunc, IL). The culture consisted of 10-week cultured cells (4,000 cells/ml) developed with 10 ng/ml SCF, α-medium, 0.9% methylcellulose (Shinetsu Chemical, Japan), 1% BSA, 300 µg/ml fully iron-saturated human transferrin , 16 ug/ml soy lecithin, 9.6 ug/ml cholesterol and 100 ng/ml SCF with or without 10"<6> M of a compound according to the invention. Dishes are incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. After 4 weeks, aggregates consisting of 30 cells or more are marked as mast cell colonies, and those consisting of 10 to 29 cells as mast cell clusters. 30 individual colonies and clusters are removed and stained with anti- tryptase mAb or mouse IgGl using an alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique Almost all constituent cells are positive for tryptase.

Cytochemical and immunological stainings

The cultured cells are spread on slides using a Cytospin II. Cytochemical reaction with peroxidase (POX) is carried out by the conventional method. Reaction with mAb against tryptase is detected using the APAAP method (Dako APAAP Kit System, Dako Corp. CA), as described by F. Ma, K. Koike, et al. in Bro. J. Haematol. 1988, 100, 427-35.

Immunoprecipitation and Western blotting

Immunoprecipitation and Western blotting are performed, as described by T. Kamijo, K. Koike et al. in Leuk. Res. 1997, 21, 1097-106.

Flow cytometric analysis

For analysis of surface markers on the cultured cells, 1-2 x 10<5> cells are collected in plastic tubes and incubated with appropriately diluted FITC or PE mAb, as described by Kinoshita T, Sawai N, et al in Blood 1999,94,496-505 . The cells are washed twice after which their surface markers are analyzed with FACScan flow cytomers. Viable cells are separated according to their forward light scattering properties and lateral scattering properties. The proportion of positive cells is determined by comparison with cells stained with FITC- or PE-conjugated mouse isotype-matched Ig.

Detection of cellular apoptosis

The analysis of cellular apoptosis is performed by a flow cytometric assay using propidium iodide (PI, Sigma) according to the procedure described by N. Sawai, K. Koike, et al in Stem Cells. 1999, 17, 45-53. To reduce cells undergoing apoptosis, necrosis or already dead, a Percoll gradient centrifugation can be used. 10 week cultured cells are layered on 27% percoll (Sigma) in α-medium and 54% percoll in PBS. After centrifugation, the cells are collected from the interface of the two different concentrations of percoll solution, washed with PBS and treated with 1 ml of Ortho PermeaFix™ for 40 min at room temperature. The cells are then incubated with DNase-free RNase (Sigma) for 15 min at 37°C and stained with PI for 15 min. The DNA content of 2 x 10<4> cells is monitored with a flow cytometer. Said 10-week cultured cells (2 x 10<6>) exposed to SCF or SCF and the compound used in the invention are lysed for 10 min on ice in 100 µl hypotonic lysis buffer (10 mm Tris (pH 7.5), 10 mM EDTA, pH 8.0, 0.5% Triton X-100). After centrifugation for 10 min at 14,000 g, the supernatant is transferred to a new test tube and treated with 0.2 mg/ml RNase A (Sigma) and 0.2 mg/ml Proteinase K (Sigma). DNA is precipitated with 120 µl isopropanol and 20 µl 5 M NaCl overnight at -20°C. After centrifugation at 14,000 g, the pellets are dried, dissolved in 20 ul Tris-EDTA, and then the samples are analyzed by gel electrophoresis in 2% agarose and ethidium bromide staining.

Analysis of histamine, tryptase and cytokine levels

Histamine concentrations in cell lysates obtained by treating the cultured cells with 0.5% Nonidet P-40 and in supernatant are measured with the Histamine Radioimmunoassay (RIA) kit (hnmunotech) as described in Kinoshita T, Sawai N, et al in Blood 1999, 94, 496-508.

Statistical analysis

All experiments should be performed at least three times. To determine the significance of the difference between two independent groups, the unpaired t-test can be used, or the Mann-Whitney-U test when the data are not normally distributed.

Results as obtained for N-{5-[4-(4-methylpiperazinomethyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidinamine monomesylate (STI571B)

Addition of STI571B at 10'<6> M or higher to the culture with SCF almost completely inhibits progeny generation.

In SCF plus STI571B, the number of viable cells is unchanged at day 2 and then decreases in a time-dependent manner. On day 14, the total cell count reaches an insignificant level. Consistent with the results obtained with 10-week cultured mast cells, STI571B at IO"<6> M significantly inhibits the cell production of CD34<+> cells under stimulation with SCF. Furthermore, STI571B induces a programmed death of the cultured mast cells developed under stimulation with SCF . The percentage of sub-diploid nuclei increases with the culture period. This observation can be confirmed by DNA ladder formation in cells exposed to SCF plus STI571B on gel electrophoresis. STI571B exerts its inhibitory effects at the early phase of mast cell development as well as late emerging mast cells.

Part B: Mastocytosis

Example 2: Methods

Reagents: Novartis Pharma; Basel, Switzerland provided SALT I for use in these experiments. Fresh 10 mM stock solutions of the inhibitor were prepared before each experiment by dissolving compound in 1 ml of phosphate-buffered saline (PBS; Gibco-BRL).

Antibodies: A polyclonal rabbit anti-KIT antibody (c-kit Ab-1) was used at a dilution of 1:500 (c-kit Ab-1; Oncogene, Cambridge, MA). The anti-phosphotyrosine antibody (PY20) was used at a dilution of 1:1000 (PY20 Transduction Laboratories; Lexington, NY). Peroxidase-conjugated goat anti-mouse antibody was used at a dilution of 1:5000 and goat anti-rabbit antibody at a dilution of 1:10,000 (Pierce; Rockford, IL).

Cell lines: BR and C2 canine mastocytoma cell lines were obtained from Dr George Caughey (University of California at San Francisco, San Francisco, CA). Both cell lines were maintained in DMEM supplemented with 2% bovine calf, 1 mM L-glutamine, 12.5 mM HEPES (pH 7.5), 0.25 mg/ml histidine, 1% penicillin-streptomycin, and 1% fungizone. The BR and C2 cells were derived from canine mast cell tumors and were originally established in long-term culture after initial passage in immunodeficient mice (DeVinney R et al, Am J Respir Cell Mol Biol 1990; 3(5):413-420; Lazarus SC et al , Am J Physiol 1986;

251 (6 Pt 1):C935-C944). The BR cell line has a point mutation (T1752C) that results in a leucine-to-proline substitution at amino acid 575 (juxtamembrane domain). The C2 cell line has an internal tandem duplication (ITD) in the KIT juxtamembrane region. The translation of this ITD results in reduplication of amino acid residues at the 3' end of exon 11 (London CA et al, Exp Hematol 1999; 27(4):689-697; Ma Yet al, J Invest Dermatol 1999; 112(2): 165-170).

Proliferation assays: Cells were added to 96-well plates at a density of 40,000 cells/well in normal culture media and varying concentrations of SALT I. Proliferation was measured at 48-72 hours using an XTT-based assay (Roche Molecular Biochemicals; Indianapolis, IN). (Heinrich MC et al, Blood 2000; 96(3):925-932.

Protein lysates: BR and C2 cells were washed x 2 in PBS and then allowed to rest in Optimem (Gibco-BRL) at 37°C for about 18 hours. Cells were then incubated for 90 min in the presence of various concentrations of SALT I. After this incubation, cells were pelleted and lysed using 100-250 µl of protein lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.25 % deoxycholate, with the addition of the inhibitors aprotinin, leupeptin, pepstatin, PMSF, and sodium orthovanadate [Sigma]). Western immunoblot analysis was performed as described previously (Hoatlin ME et al, Blood 1998; 91(4):1418-1425; Heinrich MC et al, Blood 2000; 96(3):925-932).

Example 3: The compound used according to the invention inhibits constitutively activated KIT kinase associated with canine mast cell tumors.

To test the effectiveness of the compound in inhibiting the kinase activity of mutant forms of canine kit, two cell lines (BR and C2) expressing two different constitutively activated kit isoforms were used. The Kit mutations in these cell lines are both located in the juxtamembrane domain and are homologous to mutations seen in gastrointestinal stromal tumors (GISTs) (Lux ML et al, Am J. Pathol 2000; 156(3):791-795; Rubin BP et al, Cancer Res 2001;61(22):8118-8121). Lysates prepared from BR or C2 cells were probed with an anti-P-Tyr antibody and kit receptor activation was determined by measuring autophosphorylation. As reported previously, kit autophosphorylation in these cells was observed in the absence of SLF (Ma Yet al, J Invest Dermatol 1999; 112(2):165-170; Ma Y et al, Journal of Investigative Dermatology 2000; 114(2): 392-394). Inhibition of kit autophosphorylation using the compound was dose-dependent with complete inhibition observed using 10 and 1.0 mM doses. Almost complete inhibition was shown using a dose of 0.1 µm. Limited autophosphorylation of c-kit was achieved using 0.001-0.01 µm doses of the compound. The compound thus not only inhibits the autophosphorylation of the mutated c-kit receptor in these cells, but is also a more potent inhibitor of this mutated receptor than it is of the c-kit wild-type receptor (IC50 100-200 nM) (Heinrich MC et al , Blood 2000;96(3):925-932). To determine whether the compound modulated kit protein expression, the membrane was removed and reprobed with an anti-c-kit antibody. There was no change in expression of c-kit protein in compound-treated cells. The compound therefore reduces autophosphorylation of canine mutant kit polypeptide by inhibition of kit kinase activity rather than a downregulation of kit protein expression.

Example 4: The compound used according to the invention inhibits the proliferation of cell lines in canine mast cell tumors

To test the biological effect of inhibiting the kinase activity of a mutant c-kit receptor, BR or C2 cells were cultured for 48-72 hours in the presence of

different concentrations of the compound. At inhibitor concentrations of 0.1-10 nM, proliferation was reduced by 90-95% compared to cells treated only with media. Partial inhibition of proliferation was noted at doses of 0.001-0.01 (im of the compound. The decreased proliferation observed at doses of 0.01-10

Hm inhibitor was statistically significant (p<0.001). The compound therefore inhibits proliferation of BR and C2 cells with the same dose response range seen for inhibition of receptor autophosphorylation. Morphological observations of inhibitor-treated cells showed changes consistent with apoptosis (data not shown).

c

Tables 1 and 2: BR or C2 cells were spread on 96-well plates at a concentration of 40,000 cells/well and cultured in normal growth media and varying concentrations of the compound. Cell proliferation was measured at 72 hours using an XTT-based assay system. Each compound concentration was analyzed in triplicate. Results are expressed as a percentage of maximal proliferation (cells only, no compound 1) ± 1 standard deviation. Representative results from one of six independent experiments are shown.

Example 5: Example of a prospective series of companion dogs with measurable cutaneous mast cell tumors.

The study patients are companion dogs with measurable and histologically confirmed mast cell tumors. Cases are limited to those with measurable lesions amenable to biopsy.

Eligible criteria are:

- histologically confirmed measurable cutaneous mast cell tumors

- cases will require serial biopsy at 2 mm Keyes punch before and during therapy

- histological stage of development (TJ-intermediate or III-poorly differentiated)

- functional status 0 or 1 (modified Karnofsky - Table 3)

informed owner's consent

(a) Exclusion criteria are:

- concurrent cytotoxic chemotherapy

- prednisone and non-steroidal anti-inflammatory drugs cannot be initiated within 30 days of the study; if prednisone or non-steroidal anti-inflammatory drugs have been administered for more than 30 days they can be continued

- abnormal serum bile acid test (liver function)

Pretreatment evaluation of all cases includes physical examination, complete blood count, "buffy" coat, serum biochemistry, urinalysis, serum bile acids (fasting and postprandial), documentation of regional lymph node size, abdominal radiographs, and abdominal ultrasound. The treatment regimen is 25 mg/kg PO QD x 60 days of SALT I.

Treatment is continued in all cases for 60 days unless disease progression is noted. In cases where a partial or complete response is experienced, ongoing therapy for a further 60 days can be considered. Cases with successful complete therapy are eligible for repeat study participation.

The efficacy of the compound used is determined against measurable cutaneous mast cell tumors using clinical endpoints. Biological endpoints can be obtained from serial biopsies collected from cutaneous tumors and from blood samples available throughout the course of treatment.

Clinical endpoints include measurable tumor response rate, objective response to measurable tumor, and time to progression of measurable tumor. All harmful side effects will be recorded.

"Objective tumor responses", as defined below, are observed during treatment with the compound used according to the invention and indicate efficacy of the treatment regimen.

In particular, complete responses and partial responses to treatment with the compound can be observed. Furthermore, it can be observed that more animals that achieve treatment show stable disease, while less treated animals show progressive disease. It can also be observed that fewer animals that achieve treatment show disease relapse compared to non-treated animals. Time to progression, duration of remission, and survival may be increased in animals treated with the compound.

"Complete response (CR)" is defined as the disappearance of all clinical signs of cancer and any signs related to the cancer.

"Partial response (PR)" is defined as a 50% or more reduction in the sum of the measurements of representative lesions, without an increase in the size of any lesions or the appearance of any new lesions.

"Stable disease (SD)" is defined as no response or a response less than that defined for partial response or progressive disease without the appearance of any new lesions or worsening of clinical signs.

"Progressive disease (PD)" is defined as an unequivocal increase of at least 50% in the size of any measurable lesion or the appearance of new lesions.

"Relapse (R)" is defined as the appearance of new lesions or the reappearance of old lesions in dogs that have had a complete response; in dogs that had only a partial response, relapse was defined as at least a 50% increase in the sum of measurements for representative lesions, compared to measurements obtained at the time of maximum response.

"Time to progression (TTP)" is reported from day 0 in the protocol. TTP will be defined as the number of days from the start of therapy (from day 0) to relapse (R).

"Duration of remission" is defined as the number of days from the objective response (PR or CR) to relapse.

"Survival" is defined as the number of days from the start of treatment with the compound to death. Cause of death will be noted, but may include disease progression, toxicity, and others.

Particularly advantageous results are shown by the compound used according to the invention, which is CGP 57148B {N-{5-[4-(4-methylpiperazino-methyl)-benzoylaminod]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidinamine monomesylate}.

It is highly preferred that the compound used according to the invention is used in the form of its monomesylate salt.

According to the special findings in connection with the invention, the compound used according to the present invention can also be used in a method for the treatment of warm-blooded animals, including humans, where a therapeutically effective dose of the compound is administered to such a warm-blooded animal, preferably a human or a dog , very preferably a human, who suffers from one of the diseases mentioned herein.

Application may also be in a method for administration to a dog having canine mast cell neoplasms, and this method comprises administering a pharmaceutically effective amount of the compound or a pharmaceutically acceptable salt thereof to said dog once a day for a period that preferably exceeds 1 month, 2 months or even 3 months. The invention relates in particular to such a method where a daily dose of about 20-200 mg, preferably 80-160 mg, especially 125 mg, of SALT I is administered.

The application preferably takes place in a method where a daily dose of the monomethanesulfonate salt of N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine -amine is administered to a dog, and wherein said daily dose includes an amount of said monomethanesulfonate salt sufficient to maintain plasma levels of at least 0.2 µm.

When used according to the invention, a pharmaceutical preparation is obtained for the treatment of mastocytosis. Pharmaceutical compositions for the treatment of mastocytosis comprise an effective amount of the compound used together with pharmaceutically acceptable carriers suitable for topical, enteral, eg oral or rectal, or parenteral administration, and may be inorganic or organic, solid or liquid. For oral administration, tablets or gelatin capsules are used in particular, including the compound used together with diluents and/or lubricants, e.g.

silicic acid, talc, stearic acid or salts thereof, and/or polyethylene glycol, but also lotions, gels or creams. Tablets may include binders, starches, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, disintegrants and/or effervescent mixtures, or adsorbents, colourants, flavorings and sweeteners. The compound can also be used in the form of parenterally administrable preparations or in the form of infusion solutions. Topical administration is, for example, to the skin. A further form of topical administration is to the eye, for example for the treatment of vernal conjunctivitis. Pharmaceutical preparations can be sterilized and/or can include excipients, e.g. preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizing agents, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical preparations are prepared in a manner known per se, e.g. by means of conventional mixing, granulating, confectioning, dissolving or lyophilizing processes, and include from about 1 to 100%, especially from about 1 to about 20 %, active ingredient(s).

The compound used according to the invention can, for example, be formulated as described in examples 4 and 6 in WO 99/03854.

The dosage range of the compound to be used depends on factors known to those skilled in the art, including the species of warm-blooded animal, body weight and age, route of administration. Unless otherwise stated herein, the compound used according to the invention is preferably administered from one to four times per day or immediately when a reaction is observed. Furthermore, N-{5-[4-(4-methylpiperazinomethyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidinamine (STI57IB) is preferably administered to a warm-blooded animal, especially a human in a dosage in the range of about 10 to 750 mg/day, preferably 30 to 600 mg/day, more preferably 30 to 300 mg/day.

In dogs, depending on the species, age, individual condition, method of administration and the relevant clinical picture, effective doses, e.g. daily doses of about 20 - 200 mg, preferably 80-160 mg, especially 125 mg, are administered to warm-blooded animals on approx. 5 kg body weight. For adult dogs of approximately 5 kg with unresectable and/or metastatic malignant canine mast cell neoplasms, a starting dose of 125 mg daily can be recommended. For dogs with an inadequate response after a determination of response to therapy at 125 mg daily, dose escalation can be safely considered and dogs can be treated as long as they benefit from treatment and in the absence of limited toxicities. Doses can be titrated to thereby achieve plasma levels of at least 0.2 µM (micromolar), preferably 0.5 µM, more preferably at least 1 µM. Achieving and/or maintaining a plasma level of about 1 uM is particularly preferred.

Claims (3)

1. Use of N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amine or a pharmaceutically acceptable salt thereof for the preparation of a drug for the treatment of mastocytosis. 2. Use according to claim 1, where the compound is used in the form of its monomesylate salt. 3. Use according to claim 1 of N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amine or a pharmaceutically acceptable salt thereof for the production of a drug for the treatment of mast cell neoplasms in dogs.