TW201332551A - Method of treating gastrointestinal stromal tumors - Google Patents

Abstract

The present invention relates to a method of treating gastrointestinal stromal tumors (GIST), especially GIST, which is progressing after imatinib therapy or after imatinib and sunitinib therapy, using a combination comprising (a) a c-kit inhibitor and (b) a dual KIT inhibitor and FGFR inhibitor or FGFR inhibitor.

Description

Method for treating gastrointestinal stromal tumor

The present invention relates to a method of treating a gastrointestinal stromal tumor (GIST) of a human patient population using a combination comprising (a) a c-kit inhibitor and (b) a PI3K inhibitor or a FGFR inhibitor.

GIST is the most common gastrointestinal mesenchymal tumor. It is believed that these tumors are caused by interstitial cells of Cajal that constitute the intestinal muscle plexus found in the stomach and intestine. Primary GIST occurs most frequently in the stomach (50-60%), the small intestine (20-30%), and the large intestine (10%), and the remaining cases are involved in the esophagus, mesentery, omentum, and retroperitoneal cavity. Based on Swedish population-based morbidity, it has been estimated that approximately 5,000 new GIST cases are diagnosed each year in the United States. GIST occurs mainly in middle-aged and elderly people, with an average age of onset of approximately 60 years and no apparent gender preference.

GIST can present a variety of phenotypic characteristics, many of which are related to the patient's prognosis. Therefore, for the risk stratification of primary GIST, the consensus meeting focused on tumor size and mitotic index, which is associated with tumor recurrence. Currently, risk stratification based on pathological criteria preferably uses these terms as benign or malignant GIST. Patients with primary gastric GIST appear to perform slightly better than those with intestinal tumors. GIST tends to recur both locally and in the form of peritoneal and hepatic metastases, with lymph node metastasis being less frequent. Surgical resection is primarily relied upon for the treatment of primary GIST, but it is often difficult to cure the disease using cytotoxic chemotherapy. It has been found that positive staining can be performed on these tumors using immunohistochemical markers (CD117) previously used to stain for interstitial cells of Cajal, which facilitates the diagnosis of GIST. Used for The antibody in the immunohistochemical reaction recognizes the extracellular domain of the stem cell factor receptor (KIT). At present, KIT is the main diagnostic criteria for GIST, and other gastrointestinal KIT-positive mesenchymal tumors are almost impossible to be confused with GIST; notable exceptions include metastatic melanoma and malignant hemangioma. Approximately 95% of GISTs were positive for CD117 staining. In most of these cases, somatic mutations can be found in genes encoding KIT proteins (usually exons 11, 9 and 13). These mutations cause the receptor to gain function and become constitutively activated (regardless of the presence or absence of a ligand).

Therapies for patients with primary GIST rely primarily on surgical resection. However, surgery alone is usually incurable; a 5-year disease-specific survival rate is reported to be 54%. There is a recurrence rate of more than 50% within 2 years of resection of the primary GIST and a recurrence rate of nearly 90% after re-excision emphasizes the need for effective post-operative treatment.

Imatinib has been approved worldwide for the treatment of adult patients with KIT-positive (CD117) and unresectable and/or metastatic GIST, and by extending overall survival and progression-free survival (PFS) Increasing the 5-year survival rate significantly changes the prognosis of these patients. Imatinib doses ranging from 400 mg/day to 800 mg/day are used worldwide to treat patients with unresectable and/or metastatic KIT-positive GIST. In addition, 800 mg/day of imatinib significantly improved progression-free survival (PFS) in patients with advanced GIST with KIT exon 9 mutation compared to 400 mg/day.

Since imatinib has efficacy in treating patients with unresectable and/or metastatic GIST, a double-blind, randomized phase III study (ACOSOGZ9001) was performed to determine after complete resection compared to placebo. The use of 400 mg/day of imatinib for adjuvant treatment of GIST adult patients for 12 months improved recurrence-free survival (RFS). The results of this study indicate that treatment with imatinib significantly prolongs RFS. Based on these data, imatinib at a dose of 400 mg/day was approved worldwide to assist in the treatment of adult patients after removal of GIST. Now, using the results from SSGXVIII/AIO, a phase III multicenter, open-label, randomized study of 10 patients in GIST patients who are at high risk of postoperative disease and estimated to be at risk of disease recurrence Efficacy and safety of 400 mg imatinib administered once a month or 36 months. The study data confirmed that 36-month-old imatinib adjuvant therapy was well tolerated in GIST patients after surgical resection and was superior to 12-month therapy in prolonging RFS and overall survival.

Despite the efficacy of imatinib, it still does not meet the medical needs of the field of metastatic GIST therapy, with more than 50% of advanced GIST patients worsening after two years of first-line therapy with imatinib.

Nilotinib is a KIT inhibitor that inhibits the growth of GIST-T1 cells. However, in the presence of FGF2, the efficacy of nilotinib was significantly reduced. Dovtinib (4-amino-5-fluoro-3-[5-(4-methylhexahydropyrazin-1-yl)-1H-benzimidazol-2-yl]quinoline- 2(1H)-keto) is a dual KIT inhibitor and FGFR inhibitor that inhibits GIST-T1 cell growth to the same maximum inhibition in the presence or absence of added FGF2 (Figure 5).

Based on the discovery that the FGFR pathway can be a survival pathway in GIST, combining KIT inhibitors and dual KIT inhibitors and FGFR inhibitors targeting survival pathways in GIST can produce treatments comparable to those obtained by administering KIT inhibitors alone. The therapeutic effect should be better.

As shown herein, FGF2 growth factor and its receptor FGFR1 are overexpressed in primary GIST tissues, suggesting that the FGFR pathway can be a survival pathway activated in GIST. FGFR1, but not FGF2, is overexpressed in GIST cell lines. However, the FGFR signaling pathway is activated in the presence of exogenous FGF2 in the GIST cell line. In addition, GIST cell lines are less susceptible to treatment with KIT inhibitors in the presence of added FGF2. The combination of FGFR inhibitor and KIT inhibitor produces strong synergistic activity in GIST cells in the presence of FGF2 and significantly improves efficacy, which means that the combination of FGFR inhibitor and KIT inhibitor can improve the efficacy of current treatment strategies in GIST. .

More broadly, the present invention provides a method of treating GIST, preferably without any KIT mutations (including KIT mutations and KIT resistance mutations), by administering a therapeutically effective amount of a FGFR inhibitor to a patient in need thereof.

In addition, based on observations in the GIST cell line, it has now surprisingly been found that a combination of (a) c-kit inhibitor and (b) a combination of dual KIT inhibitors and FGFR inhibitors can be successfully used to treat a patient with imatinib. A patient with a worsening GIST after therapy.

It is further concluded that a combination of (a) c-kit inhibitor and (b) a dual KIT inhibitor and a FGFR inhibitor can be successfully treated to treat patients suffering from continuous therapy with imatinib and sunitinib. The patient of GIST.

Accordingly, the present invention provides a method of treating a GIST that is exacerbated after imatinib therapy or continuous imatinib and sunitinib therapy in a human patient, the package thereof A therapeutically effective amount of (a) a c-kit inhibitor and (b) a dual KIT inhibitor and a FGFR inhibitor or FGFR inhibitor are administered, for example, simultaneously or sequentially to the patient. More broadly, the present invention provides a method of treating a GIST in a human patient comprising, for example, co-administering a therapeutically effective amount of (a) a c-kit inhibitor and (b) dual KIT inhibition to the patient simultaneously or sequentially. And FGFR inhibitors or FGFR inhibitors.

In another aspect, the invention relates to a combination comprising (a) a c-kit inhibitor and (b) a dual KIT inhibitor and a FGFR inhibitor or a FGFR inhibitor for the manufacture of a GIST (especially in Imatin) The use of the agent for the deterioration of GIST after the first-line therapy.

Another aspect of the invention relates to the treatment of GIST (especially the GIST that worsens after imatinib therapy or the GIST that deteriorates after imatinib and sunitinib therapy) (a) c-kit inhibitors and b) a combination of a dual KIT inhibitor and a FGFR inhibitor or FGFR inhibitor.

As used herein, the expression "c-kit inhibitor" includes, but is not limited to, 4-(4-methylhexahydropyrazin-1-ylmethyl)-N-[4-methyl-3-(4-(pyridine). 3-yl)pyrimidin-2-ylamino)phenyl]-benzamide (imatinib), 4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino] - N - [5- (4- methyl--1H- imidazol-1-yl) -3- (trifluoromethyl) phenyl] benzoyl amine (nilotinib), masitinib ( Masitinib), sunitinib, sorafenib, regorafenib, motesanib, and their respective pharmaceutically acceptable salts.

In a preferred embodiment, the c-kit inhibitor used is imatinib. Imatinib is explicitly disclosed in the patent application US 5,521,184, the subject matter of which is cited This is incorporated into the present application. Imatinib can also be prepared according to the method disclosed in WO 03/066613. For the purposes of the present invention, imatinib is preferably applied in the form of its monomethanesulfonate. Imatinib monomethanesulfonate can be prepared according to the method disclosed in US 6,894,051. The invention likewise encompasses the corresponding polymorphs disclosed in US 6,894,051, such as crystalline variants.

In another preferred embodiment of the method described herein, the monomethanesulfonate of imatinib is orally administered in a dosage form as described in US 5,521,184, US 6,894,051 or US 2005-0267125. The mesylate salt of imatinib is sold under the trade name Glivec® (Gleevec®). Preferably, the daily oral dose of imatinib is 200-600 mg, specifically 400 mg/day, which is administered in a single dose or divided into multiple doses, for example twice daily.

In one embodiment of the invention, the c-kit inhibitor used is nilotinib. Nilotinib and its method of manufacture are disclosed in WO 04/005281, which is incorporated herein by reference. The pharmaceutically acceptable salts of nilotinib are especially disclosed in WO 2007/015871. For the purposes of the present invention, nilotinib is preferably applied in the form of its monohydrochloride monohydrate salt. WO 2007/015870 discloses certain polymorphs of nilotinib and its pharmaceutically acceptable salts useful in the present invention.

In the examples of the methods described herein, the monohydrochloride of nilotinib is orally administered in a dosage form as described in WO 2008/037716. The monohydrochloride salt of nilotinib is sold under the trade name Tasigna®. Preferably, the daily oral dose of imatinib is 200-1200 mg, for example 800 mg/day, which is administered or divided into multiple doses in a single dose, for example twice daily.

As used herein, the expression "FGFR inhibitor" includes, but is not limited to, (a) birvanib, indyneib, E-7080, ponatinib, SU-6668, and AZD-4547, (b) compounds disclosed in WO 2009/141386 and (c) compounds disclosed in WO 2006/000420 (including 3-(2,6-dichloro-3,5-dimethoxy-) Phenyl)-1-{6-[4-(4-ethyl-hexahydropyrazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea monophosphate , BGJ398). BGJ398 is a pan-FGFR kinase inhibitor that inhibits FGFR 1-3 (IC50 between 3 nM and 7 nM).

The dual KIT inhibitor and FGFR inhibitor of the pharmaceutical combination of the invention comprises at least one RTK inhibitor compound selected from the group consisting of a compound of formula I or a tautomer thereof, a compound of formula II or a tautomer thereof, A compound of the III or a tautomer thereof, a pharmaceutically acceptable salt of the compound, a pharmaceutically acceptable salt of the tautomer or a mixture thereof.

The dual KIT inhibitor and the FGFR inhibitor compound may be selected from the group consisting of a compound of formula I, a tautomer of a compound, a salt of a compound, a salt of a tautomer or a mixture thereof, wherein the compound of formula I has the formula: Wherein: R ¹ , R ² , R ³ and R ⁴ may be the same or different and are independently selected from the group consisting of H; Cl; Br; F; I; -OR ¹⁰ group; -NR ¹¹ R ¹² group; Or unsubstituted first alkyl, second alkyl or third alkyl; substituted or unsubstituted aryl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted heterocyclylalkyl group; R ⁵ , R ⁶ , R ⁷ and R ⁸ may be the same or different and independently selected from H; Cl; Br; F; I; -OR ¹³ group; -NR ¹⁴ R ¹⁵ group; -SR ¹¹ group; substituted or unsubstituted first alkyl group, second alkyl group or third alkyl group; substituted Or unsubstituted aryl; substituted or unsubstituted alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted heterocyclic; substituted or unsubstituted heterocycloalkane Substituted or unsubstituted alkoxyalkyl; substituted or unsubstituted aryloxyalkyl or substituted or unsubstituted heterocyclyloxyalkyl; R ¹⁰ and R ¹³ may be the same Or different, and independently selected from substituted Unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted alkoxy An alkyl group, a substituted or unsubstituted aryloxyalkyl group or a substituted or unsubstituted heterocyclic oxyalkyl group; R ¹¹ and R ¹⁴ may be the same or different and are independently selected from substituted Or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic; R ¹² and R ¹⁵ may be the same or different and are independently selected from substituted or unsubstituted Substituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heterocyclic; and R ¹⁶ is selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl A substituted or unsubstituted heterocyclic group.

The dual KIT inhibitor and the FGFR inhibitor compound may also be selected from a compound of formula II or a tautomer thereof, a pharmaceutically acceptable salt of the compound, a pharmaceutically acceptable salt of the tautomer or a mixture thereof, wherein The compound of formula II has the formula: Wherein: R ⁷ is a substituted or unsubstituted heterocyclic group. In some embodiments, R ⁷ is selected from substituted or unsubstituted heterocyclic groups of substituted or unsubstituted hexahydropyridyl, hexahydropyrazinyl or morpholinyl. In other embodiments, R ⁷ N- alkyl-based by-hexahydro-pyrazinyl, substituted or non-substituted. In other embodiments, R ⁷ N- alkyl-hexahydro-line by a substituted or unsubstituted pyrazinyl group, the N- alkyl and the alkyl group of the piperazine containing 1 to 4 carbon atoms.

The dual KIT inhibitor and the FGFR inhibitor compound may also be selected from a compound of formula III or a tautomer thereof, a pharmaceutically acceptable salt of the compound, a pharmaceutically acceptable salt of the tautomer or a mixture thereof, wherein The compound of formula III has the formula:

The compound of formula III includes 4-amino-5-fluoro-3-[5-(4-methylhexahydropyrazin-1-yl)-1H-benzoimidazol-2-yl]quinoline-2 (1H) -ketone (Compound A) and (4-Amino-5-fluoro-3-[6-(4-methylhexahydropyrazin-1-yl)-1H-benzimidazol-2-yl]quinoline- 2(1H)-keto) (Dovetinib).

In a preferred embodiment, the pharmaceutical combination of the invention comprises at least one compound of the formula I or a tautomer thereof, a compound of the formula II or a tautomer thereof, a compound of the formula III or a tautomer thereof, a pharmaceutically acceptable compound A salt, a pharmaceutically acceptable salt of a tautomer or a mixture thereof, which is a compound A.

In another preferred embodiment, the pharmaceutical combination of the invention comprises at least one compound of the formula I or a tautomer thereof, a compound of the formula II or a tautomer thereof, a compound of the formula III or a tautomer thereof, a pharmaceutical compound An acceptable salt, a pharmaceutically acceptable salt of a tautomer or a mixture thereof, which is dovetinib.

A dual KIT inhibitor of the formula I and a FGFR inhibitor compound or a tautomer thereof, a compound of the formula II or a tautomer thereof, a compound of the formula III or a tautomer thereof, a pharmaceutically acceptable salt of the compound, a tautomer The pharmaceutically acceptable salts of the constructs, or mixtures thereof, formulations thereof, and methods for their preparation are described, for example, in WO 2002/222598, WO 2003/087095, WO 2005/046589, WO 2006/127926, WO 2006/124413, Such cases are incorporated herein by reference in their entirety in WO 2007/064719, WO 2009/115562, and WO 2012/001074.

The compounds of the invention may be administered in free form or as a pharmaceutically acceptable salt.

As used herein, "pharmaceutically acceptable salts" include inorganic base salts, organic base salts, mineral acid salts, organic acid salts or basic or acidic amino acid salts. As the inorganic base salt, the present invention includes, for example, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as calcium and magnesium or aluminum; and an ammonia salt. As the organic base salt, the present invention includes, for example, a trimethylamine salt, a triethylamine salt, a pyridinium salt, a methylpyridine salt, an ethanolamine salt, a diethanolamine salt, and a triethanolamine salt. As the inorganic acid salt, the present invention includes, for example, a salt of hydrochloric acid, boroic acid, nitric acid, sulfuric acid, and phosphoric acid. As the organic acid salt, the present invention includes, for example, formate, acetate, trifluoroacetate, fumarate, oxalate, tartrate, maleate, lactate, citrate, succinate , malate, methanesulfonate, besylate and p-toluenesulfonate. As the basic amino acid salt, the present invention includes, for example, arginine, anisoamine, and aguanine. Acidic amino acids include, for example, aspartic acid and glutamic acid.

The monolactate of the compound of formula I is in various polymorphic forms (including, for example) The monohydrate form and the anhydrous form) are present. Polymorphs are produced if the same composition of matter (including its monohydrates and solvates) is crystallized in a different crystal lattice to produce different thermodynamic and physical properties characteristic of a particular crystalline form.

Other pharmaceutically acceptable salts of Compound A and dovetinib suitable for the present invention include the salts as disclosed in WO 2005/04658.

In one embodiment, dovastatin is administered to a suitable individual in a single dose or in divided doses daily at an effective dose ranging from about 0.001 mg/kg body weight/day to about 100 mg/kg body weight/day, preferably Administration is administered in a single dose or in divided doses from about 1 mg/kg/day to about 35 mg/kg/day. For a 70 kg human, this will total up from about 0.07 g/day to 2.45 g/day, preferably from about 0.05 g/day to about 1.0 g/day.

The following aspects of the invention are particularly important:

(1.) A method of treating a GIST in a human patient comprising administering to a human patient in need thereof a combination of effective doses against GIST (a) a c-kit inhibitor and (b) a KIT inhibitor or (b) a dual KIT Inhibitors and FGFR inhibitors or FGFR inhibitors or their respective pharmaceutically acceptable salts, in particular, wherein the c-kit inhibitor is selected from the group consisting of imatinib, nilotinib and massetinib or their respective pharmaceuticals Acceptable salt.

(2.) A method of treating GIST in a human patient comprising administering to a human patient in need thereof an effective dose against GIST, wherein the GIST deteriorates after imatinib therapy or after imatinib and sunitinib therapy.

(3.) A combination for the treatment of GIST comprising (a) a c-kit inhibitor and (b) a FGFR inhibitor or a respective pharmaceutically acceptable salt thereof.

For the purposes of the present invention, it comprises (a) a c-kit inhibitor and (b) a FGFR inhibitor. Preferably, the combination of the preparations is selected from the group consisting of (1) imatinib or a pharmaceutically acceptable salt thereof and Compound A or a pharmaceutically acceptable salt thereof, (2) imatinib or a pharmaceutically acceptable salt thereof and Vertinib or a pharmaceutically acceptable salt thereof.

The structure of the active agent identified by the generic or trade name may be obtained from the actual version of the standard overview "The Merck Index" or from a database (eg, an international patent (eg, IMS World Publications)). The corresponding content is hereby incorporated by reference.

Unless otherwise stated, c-KIT inhibitors, dual KIT inhibitors and FGFR inhibitors, and FGFR inhibitors are administered at a dose as specified in the product information for products containing the inhibitor for the proliferative disorder. , or (especially) if the product information is not available, it is used in the dose determined in the dose investigation study.

A suitable clinical study in a human patient (for example) is a non-randomized study of open-labeling performed in patients with GIST who have worsened after first-line therapy with imatinib. These studies demonstrate that treatment with the claimed method is particularly advantageous compared to a component of the treatment regimen alone. The beneficial effects on GIST can be directly determined by the results of such studies (e.g., RFS or no-deterioration survival-PFS) or by changes in the design design that are well known to those skilled in the art.

Instance

The following examples illustrate the above invention, however, it is not intended to limit the scope of the invention in any way. Others familiar with those skilled in the art The test model can also determine the beneficial effects of the claimed invention.

Example 1 - FGF receptor 1 (FGFR1) and FGF2 expressing cell lines and cultures in primary GIST

GIST882, GIST48 and GIST430 cell lines were obtained from Brigham and Women's Hospital, Boston, MA. GIST882 (Tuveson DA, Willis NA et al, Oncogene 2001; 20:5054-5058) was established from an untreated human GIST having a homozygous mis-sense mutation in KIT exon 13 encoding the K642E mutant KIT protein. GIST48 and GIST430 (Bauer S, Yu LK, Demetri GD, Fletcher JA. Cancer Res 2006; 66:9153-9161) were established from GISTs treated with imatinib for an initial clinical response. GIST48 has a primary homozygous exon 11 missense mutation (V560D) and a secondary heterozygous exon 17 misidentification mutation (D820A). GIST430 has a deletion in the primary heterozygous exon 11 reading frame and a secondary heterozygous exon 13 missense mutation (V654A). GIST-T1 was obtained from Kochi Medical School (Kochi). GIST-T1 was established by a metastatic human GIST having a 57 base heterozygous deletion in KIT exon 11 (Taguchi T, Sonobe H, Toyonaga S et al, Lab Invest 2002; 82: 663-665).

GIST882 cells were cultured in RPMI-1640 (ATCC Cat. No. 30-2001) supplemented with 15% FBS and 1% L-glutamic acid, supplemented with 15% FBS, 0.5% Mito+ (BD Bioscience Cat. No. 355006), GIST48 cells were cultured in 1% BPE (BD Bioscience/Fisher Cat. No. 354123) and 1% L-glutamic acid F10 (Gibco/Invitrogen catalog number 11550-043) supplemented with 15% FBS and 1% L-Bran Proline acid IMEM GIST430 cells were cultured in (Gibco/Invitrogen Cat. No. 12440-053), and GIST-T1 cells were cultured in DMEM supplemented with 10% FBS (Gibco/Invitrogen Cat. No. 11965).

Cell viability analysis

Imatinib and dovetinib were dissolved in DMSO to form a 10 mM stock solution, and then diluted with medium to produce a series of different concentrations (μM) (0, 0.02, 0.05, 0.16, 0.49, 1.48, 4.44, Working solutions for 13.3 and 40). 10,000 cells suspended in 80 μl of medium were seeded into each well of a 96-well cell-culture plate before treatment and allowed to grow for 24 hours. Add 10 μl of 60 μg/mL heparin (Sigma catalog number H3149) to each well, then add 10 μl of 50 μg/mL FGF2 (R&D Cat. No. 233-FB/CF) or medium to each well of the plates. . 10 μl of each of the above compound dilutions and 10 μl of the medium were added to each well until the final volume was 120 μl, so that all the paired combinations as well as the single agent were represented. After the addition of the compound, the cells were incubated at 37 ° C for 72 hr in a 5% CO ₂ incubator. Cell proliferation was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega Cat # G755B) and Victor 4 Plate Reader (Perkin Elmer). Coordination score values and CI ₇₀ calculated values were determined as described elsewhere (Lehar J, Krueger AS et al, Nat Biotechnol 2009; 27: 659-666).

Western blotting

Protein lysates were prepared from cell monolayers using RIPA buffer (Cell Signaling Technology Cat. No. 9806) according to the procedure set forth by the manufacturer. Detection of phosphoric acid-KIT (catalog number 3073S), total KIT (catalog) No. 3308), anti-system of phosphoric acid-AKT S473 (catalog number 4058), total AKT (catalog number 9272), phosphoric acid-ERK (catalog number 9101), total ERK (catalog number 9107) and phosphoric acid-FRS2 (catalog number 3864) Purchased from Cell Signaling Technology. The antibody to GAPDH (catalog number MAB374) was purchased from Millipore and anti-FRS2 (H-91) (catalog number sc-8318) was purchased from Santa Cruz. Binding antibodies were detected using a LI-COR Odyssey infrared imaging system.

result

The Novartis OncExpress database contains performance data for internal and public storage of 30,094 primary tumors (including 110 GIST samples) described by the Affymetrix Human Genome U133A or U133 Plus 2.0 array. Among the 41 tumor types included in this data set, FGF2 and its receptor FGFR1 also showed the highest average performance in GIST, except for known GIST-specific genes (such as KIT, ETV1 and PRKCQ) (Fig. 1). This indicates that the FGFR pathway can be a survival pathway in GIST. FGF2 was also found to be overexpressed at the protein level in primary GIST (Fig. 2). FGFR1, but not FGF2, is overexpressed in GIST cell lines. However, the FGFR signaling pathway is activated when multiple concentrations of exogenous FGF2 are added (Figure 3). KIT inhibition of imatinib and dovetinib was also measured by Western blots in the GIST cell line (Fig. 4).

GIST-T1 and GIST882 are sensitive to KIT inhibition achieved by nilotinib treatment (Figures 5 and 6, top). However, to show the two cell lines in the presence of added FGF2 less sensitive to inhibition of KIT and GI ₅₀ values more than 10 times the offset (FIGS. 5 and 6, upper panel), which indicates upon activation of FGFR signaling can act as a survival way. Therefore, combining KIT inhibitors with potent FGFR inhibitors should enhance growth inhibition in GIST cell lines.

In addition to dual KIT inhibitors and FGFR inhibitors, dovetinib is also an orally active, potent and selective FGFR inhibitor. When GIST-T1 and GIST882 were treated with dovetinib in the presence of added FGF2, maximal inhibition returned to a level comparable to that in the absence of FGF2, suggesting that dovetinib acts as a dual KIT and FGFR inhibitor. Both the KIT pathway and the FGFR pathway were inhibited (Figures 5 and 6, bottom panel). To determine the effect of a combination of a single agent and a combination of the FGFR inhibitor dovetinib and the KIT inhibitor imatinib (CGP057148B) on GIST cell growth inhibition, we compared each individual compound in a range of doses and in a paired combination. The cell proliferation reaction of the sky. As a single agent, imatinib effectively inhibited the growth of GIST-T1 and GIST882 in the absence of FGF2 (Fig. 7). In the presence of added FGF2, the two cell lines were less susceptible to imatinib treatment (Figure 7), which is similar to the results shown in Figures 5 and 6. Doverinib was effective in inhibiting GIST-T1 and GIST882, with or without the addition of FGF2, consistent with the findings shown in Figures 5 and 6 (Figure 7). The combination of dovetinib and the KIT inhibitor (imatinib) produced a weak combinatorial effect in GIST cells in the presence of FGF2, due to the fact that dovetinib is able to inhibit both the KIT pathway and the FGFR pathway. However, in the GIST-T1, GIST882, and GIST48, imatinib is a more potent KIT inhibitor than dovetinib (Figure 4), indicating that the combination of imatinib and dovetinib may still have clinical benefit. The combined effect is shown in Figure 7, which is determined by measuring the combination index (CI ₇₀ ) at 70% inhibition of the dose shift at 70% growth inhibition and determining the overall synergy observed throughout the dose matrix. The score value was determined (Lehar J, Krueger AS, al. Nat Biotechnol 2009; 27: 659-666).

The combination of imatinib and dovetini can demonstrate synergy in GIST cell lines even in the presence of FGF2 (Figure 7). Single agents and combinations of dovetinib and imatinib have been evaluated in the GIST882 (expressing K642E mutant KIT), GIST430 (expressing ex11del/V654A KIT) and GIST-T1 (expressing ex11del KIT) cell lines obtained from patients. The effect of the form. When the antiproliferative effects of imatinib and dovetinib were evaluated in combination, the observed growth inhibition was greater than the percent inhibition achieved by treatment with imatinib or dovetinib alone in the GIST882 and GIST430 cell lines. .

Coordination is based on the "weighted" synergy score S (where S 1 means little or no synergy, or S>1 means little synergy, and S>2 means significant synergy) or combination index CI (where CI=1 means dose additivity, CI< 0.5 indicates "true" synergy (2 x dose shift), CI < 0.3 indicates "useful" synergy (3 x displacement), and CI < 0.1 indicates "strong" synergy (10 x displacement) to quantify. A clear synergistic evaluation is indicated in bold form.

Example 2: Single dose of imatinib combined with dual KIT inhibitor and FGFR inhibitor dovetinib in patients with previously failed gastrointestinal stromal tumor (GIST) in imatinib and sunitinib therapy Investigating Phase Ib Study standard constrain:

Male or female patient 18 years old

2. WHO physical status (PS) is 0-2

3. Unremovable or metastatic GIST for diagnosis of histological confirmation

4. Available tissue specimens:

‧ Dose escalation group: The patient must have available archived tumor tissue that can be delivered during the course of the study.

‧ Dose escalation group: The patient must have available archived tumor tissue that can be delivered during the course of the study.

‧Dose Amplification Group: Patients must have available archived tumor tissue that can be delivered during the course of the study and must agree to a fresh pre-treatment biopsy.

5. After treatment with previous imatinib therapy failure, sunitinib was used to treat unresectable or metastatic GIST. Note the following specific criteria for the two test periods:

‧ Dose escalation group: The patient failed in the previous imatinib therapy and then failed in sunitinib therapy. Treatment failure can be attributed to disease progression after treatment (both imatinib and sunitinib) or intolerance to therapy (sunitinib).

‧Dose-amplified group: Patients must have documented disease progression after both imatinib and sunitinib. In addition, the patient’s previous therapy cannot be exceeded Twice (ie, treated with sunitinib after treatment with imatinib).

Figure 1: FGF2 and FGFR1 are highly expressed in primary GIST. The raw data (CEL file) of 30,094 primary tumor performance profiles was normalized by using the MAS5 algorithm with 150 as the target value.

Figure 2: FGF2 expression is substantially higher in KIT-positive primary gastrointestinal stromal tumors (GIST) than in other human primary tumor tissues. The GAPDH Western ink dot is displayed in the form of a loading control.

Figure 3: FGFR pathway is activated in GIST cell lines in the presence of various concentrations of added FGF2. FRS2 Tyr-phosphorylation was used as a readout for FGFR signaling activation and was measured by Western blots in the GIST cell line. The total FRS2 content is shown in an internal reference format.

Figure 4: KIT inhibition of imatinib and dovetinib as measured by Western blots in the GIST cell line.

Figure 5: GIST-T1 cells are less sensitive to nilotinib in the presence of added FGF2 than in the absence of added FGF2, and docetinib is compared to nilotinib in the presence of FGF2 In the case of the restoration of the maximum growth inhibition of GIST-T1.

Figure 6: GIST882 cells are less sensitive to nilotinib in the presence of added FGF2 than in the absence of added FGF2, and docetinib is compared to nilotinib in the presence of FGF2 Restore the maximum growth inhibition of GIST882.

Figure 7: Imatinib in the absence and presence of 20 ng/ml FGF2 And the combined effects of dovetinib in GIST-T1 (A) and GIST882. The left panel shows the percent inhibition of each single agent and combination treatment relative to DMSO treated cells. The concentration of imatinib (CGP057148B) increased from bottom to top along the left line, and the concentration of dovetinib increased from left to right along the bottom column. The middle graph shows the excessive suppression of each point in the left image. Excess inhibition was determined based on the Loewe synergistic model, which measures the expected growth effects relative to the additive effects of the two drugs. Positive numbers indicate synergy and negative numbers indicate antagonism. The graph on the right shows an equivalent line plot of the interaction between the two compounds. The line connecting the doses of imatinib and dovetinib represents the additive effect. The curves below and to the left of the line represent synergies.

Claims (9)

A pharmaceutical combination for treating GIST comprising (a) a c-kit inhibitor and (b) a dual KIT inhibitor and a FGFR inhibitor or FGFR inhibitor or a respective pharmaceutically acceptable salt thereof. The pharmaceutical combination of claim 1, wherein the c-kit inhibitor is selected from the group consisting of imatinib, nilotinib, and masitinib or a pharmaceutically acceptable salt thereof. The pharmaceutical combination according to claim 2, wherein the dual KIT inhibitor and the FGFR inhibitor are 4-amino-5-fluoro-3-[5-(4-methylhexahydropyrazin-1-yl)-1H- Benzimidazol-2-yl]quinolin-2(1H)-one or a pharmaceutically acceptable salt or tautomer thereof. Use of a combination of (a) a c-kit inhibitor and (b) a dual KIT inhibitor and a FGFR inhibitor or a FGFR inhibitor, or a respective pharmaceutically acceptable salt thereof, for the manufacture of a GIST for treating a human patient Pharmacy. The use of claim 4, wherein the c-kit inhibitor is selected from the group consisting of imatinib, nilotinib, and massetinib or a respective pharmaceutically acceptable salt thereof. The use according to any one of claims 4 or 5, wherein the dual KIT inhibitor and the FGFR inhibitor are 4-amino-5-fluoro-3-[5-(4-methylhexahydropyrazine-1- -1H-benzimidazol-2-yl]quinolin-2(1H)-one or a pharmaceutically acceptable salt or tautomer thereof. The use of any one of claims 4 or 5, wherein the GIST deteriorates after imatinib therapy. The use of any one of claims 4 or 5, wherein the GIST deteriorates after imatinib and sunitinib therapy. The use of claim 5, wherein imatinib is administered at a daily dose of between 300 mg and 600 mg.