Combined medicine

Abstract

The present invention relates to a drug, a combination agent, a pharmaceutical composition, a preparation, a treatment or prevention method, a drug for inhibiting drug resistance, a method for screening patients, and the like for ALK fusion gene-positive cancer, which has positive expression of FGFR1 and positive expression of FGF2, and which is obtained by combining a compound having ALK inhibitory activity with an FGFR inhibitor. The present invention also relates to a drug, a combination agent, a pharmaceutical composition, a preparation, a treatment or prevention method, or a drug for inhibiting drug resistance, a method for screening patients, and the like.

Description

Technical Field

[0001] The present invention relates to a drug, a combination agent, a pharmaceutical composition, or a preparation which is useful for the treatment or prevention of ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, and which is a combination of an ALK inhibitor and a FGFR kinase inhibitor, as well as a method and a product for treating or preventing the target cancer, and a method for screening patients with ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive. Background Art

[0002] Anaplastic lymphoma kinase (ALK) is a receptor-type tyrosine kinase proto-oncogene identified as a fusion gene in 1994 (Non-Patent Document 1).

[0003] So far, it has been reported that the result of ALK gene abnormality (translocation) leads to the generation of abnormal kinase, thereby participating in canceration (Science. 1994 Mar 4; 263 (5151): 1281-4.: Non-patent document 1). For example, it has been reported that in lung cancer, ALK binds to the microtubule-associated protein EML4 through chromosomal translocation, thereby generating EML4-ALK with active tyrosine kinase activity and acquiring canceration ability (Cancer Res. 2008 Jul 1; 68 (13): 4971-6.: Non-patent document 2). It has been reported that compounds having ALK kinase inhibitory activity are useful for cancers having this gene abnormality (Non-patent document 2).

[0004] Alectinib (generic name: alectinib hydrochloride) has been approved in Japan and overseas as an ALK inhibitor.

[0005] Fibroblast growth factor receptor (FGFR) is a receptor tyrosine kinase. So far, it has been reported that gene abnormalities (mutations, translocations) result in the generation of abnormal kinases, thereby participating in canceration (Wiley Interdiscip Rev Dev Biol. May-Jun 2015; 4(3): 215-66.: Non-patent document 4). FGFR inhibitors are known to inhibit the proliferation of cancer cells by inhibiting the phosphorylation of FGFR kinases (Nat Med. 2018 May; 24(4): 512-517: Non-patent document 5).

[0006] On the other hand, it is known that although these inhibitors are effective immediately after the start of treatment, cancer cells develop drug resistance and their effects weaken due to long-term administration (Cell 2010.141(1):69-8: Non-patent document 3).

[0007] As a mechanism of drug resistance of cancer cells caused by other drugs, it has been reported that FGF2 and FGFR1 are involved in drug resistance to the EGFR inhibitor (gefitinib) (Oncogenesis. 2013 Mar; 2(3): e39: Non-Patent Document 6).

[0008] For cancer cells that have become resistant to inhibitor treatment, the combination of different inhibitors has been investigated to inhibit cell proliferation. For example, the combined effect of ceritinib and an FGFR inhibitor (infigratinib) has been reported for resistant strains established by exposing ceritinib, an ALK inhibitor, to ALK lung cancer cell lines from ALK lung cancer patients (Non-Patent Document 5).

[0009] However, in the mechanism of resistance to EGFR inhibitors, the involvement of FGFR is not the main one (Annals of Oncology 29 (Supplement 1): i10-i19, 2018: non-patent document 7). For the mechanism of resistance caused by ALK inhibitors, changes in the three-dimensional structure of ALK fusion protein such as G1202R mutation and I1171N mutation are considered to be the main resistance mechanism (Frontiers in Oncology. 2021 October; Vol. 11, 1-13: non-patent document 8, Journal of Clinical Oncology 37, no. 16 (June 01, 2019) 1370-1379: non-patent document 9).

[0010] Prior art literature

[0011] Non-patent literature

[0012] Non-patent literature 1: Science. 1994 Mar 4; 263(5151): 1281-4.

[0013] Non-patent document 2: Cancer Res. 2008 Jul 1; 68(13): 4971-6.

[0014] Non-patent document 3: Cell 2010.141(1):69-8

[0015] Non-patent literature 4: Wiley Interdiscip Rev Dev Biol. May-Jun 2015; 4(3): 215-66.

[0016] Non-patent literature 5: Nat Med. 2018 May; 24(4): 512-517

[0017] Non-patent literature 6: Oncogenesis. 2013 Mar; 2(3): e39

[0018] Non-patent document 7: Annals of Oncology 29 (Supplement 1): i10-i19, 2018 Non-patent document 8: Frontiers in Oncology. 2021 October; Vol. 11, 1-13

[0019] Non-patent literature 9: Journal of Clinical Oncology 37, no. 16 (June 01, 2019) 1370-1379 Summary of the invention

[0020] Under the above circumstances, it is desired to provide a therapeutic agent that is effective against drug-resistant ALK fusion gene-positive cancer and / or that can suppress the drug resistance of ALK fusion gene-positive cancer.

[0021] The present invention focuses on the previously unknown association between ALK fusion gene-positive cancer and two oncogenes (FGFR1 and FGF2), and provides a new treatment based on the combined administration of ALK inhibitors and FGFR kinase inhibitors for ALK fusion gene-positive cancers having this combination of oncogenes.

[0022] That is, the present invention identifies genes involved in the drug resistance mechanism of ALK fusion gene-positive cancer cells caused by ALK inhibitors, and provides a therapeutic agent with excellent proliferation inhibition effect for ALK fusion gene-positive cancers that are FGFR1-positive and FGF2-positive by combining a compound with ALK inhibitory activity with an FGFR inhibitor.

[0023] Another aspect of the present invention provides an inhibitor for drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive before drug resistance by using a compound having ALK inhibitory activity in combination with an FGFR inhibitor.

[0024] That is, the present invention relates to the following inventions.

[0025] <1> A drug for treating or preventing ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising a combination of an ALK inhibitor and a FGFR inhibitor.

[0026] <1-a1> The drug according to <1>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0027] <1-a2> The drug according to <1>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0028] <1-b1> The drug according to any one of <1> to <1-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0029] <1-b2> The drug according to any one of <1> to <1-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0030] <1-c1> The drug according to any one of <1> to <1-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0031] <1-c2> The medicament according to any one of <1> to <1-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0032] <1-d> The medicament according to any one of <1> to <1-b2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0033] <1-e> The medicament according to any one of <1> to <1-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0034] <1-f> The drug according to any one of <1> to <1-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0035] <2> A drug for inhibiting drug resistance in ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising a combination of an ALK inhibitor and a FGFR inhibitor.

[0036] <2-a1> The drug according to <2>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0037] <2-a2> The drug according to <2>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0038] <2-b1> The drug according to any one of <2> to <2-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0039] <2-b2> The drug according to any one of <2> to <2-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0040] <2-c1> The drug according to any one of <2> to <2-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0041] <2-c2> The medicament according to any one of <2> to <2-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0042] <2-d> The medicament according to any one of <2> to <2-b2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0043] <2-e> The medicament according to any one of <2> to <2-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0044] <2-f> The drug according to any one of <2> to <2-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0045] <3> A therapeutic or preventive agent for ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising an ALK inhibitor as an active ingredient and used in combination with a FGFR inhibitor.

[0046] <3-a1> The drug according to <3>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0047] <3-a2> The drug according to <3>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0048] <3-b1> The drug according to any one of <3> to <3-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0049] <3-b2> The drug according to any one of <3> to <3-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0050] <3-c1> The drug according to any one of <3> to <3-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0051] <3-c2> The medicament according to any one of <3> to <3-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0052] <3-d> The medicament according to any one of <3> to <3-b2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0053] <3-e> The medicament according to any one of <3> to <3-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0054] <3-f> The drug according to any one of <3> to <3-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0055] <4> A drug for inhibiting drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, comprising an ALK inhibitor as an active ingredient and used in combination with a FGFR inhibitor.

[0056] <4-a1> The drug according to <4>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0057] <4-a2> The drug according to <4>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0058] <4-b1> The drug according to any one of <4> to <4-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0059] <4-b2> The drug according to any one of <4> to <4-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0060] <4-c1> The drug according to any one of <4> to <4-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0061] <4-c2> The medicament according to any one of <4> to <4-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0062] <4-d> The medicament according to any one of <4> to <4-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0063] <4-e> The medicament according to any one of <4> to <4-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0064] <4-f> The drug according to any one of <4> to <4-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0065] <5> A drug for treating or preventing ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising a FGFR inhibitor as an active ingredient and used in combination with an ALK inhibitor.

[0066] <5-a1> The drug according to <5>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0067] <5-a2> The drug according to <5>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0068] <5-b1> The drug according to any one of <5> to <5-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0069] <5-b2> The drug according to any one of <5> to <5-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0070] <5-c1> The drug according to any one of <5> to <5-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0071] <5-c2> The medicament according to any one of <5> to <5-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0072] <5-d> The medicament according to any one of <5> to <5-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0073] <5-e> The medicament according to any one of <5> to <5-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0074] <5-f> The drug according to any one of <5> to <5-e>, wherein the FGFR inhibitor and the ALK inhibitor are administered simultaneously.

[0075] <6> A drug for inhibiting drug resistance in ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, comprising a FGFR inhibitor as an active ingredient and used in combination with an ALK inhibitor.

[0076] <6-a1> The drug according to <6>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0077] <6-a2> The drug according to <6>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0078] <6-b1> The drug according to any one of <6> to <6-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0079] <6-b2> The drug according to any one of <6> to <6-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0080] <6-c1> The drug according to any one of <6> to <6-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0081] <6-c2> The medicament according to any one of <6> to <6-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0082] <6-d> The medicament according to any one of <6> to <6-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0083] <6-e> The medicament according to any one of <6> to <6-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0084] <6-f> The drug according to any one of <6> to <6-e>, wherein the FGFR inhibitor and the ALK inhibitor are administered simultaneously.

[0085] <7> A method for treating or preventing ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, comprising administering to a subject an effective amount of a compound having ALK inhibitory activity and an effective amount of a FGFR inhibitor in combination.

[0086] <7-a1> The drug according to <7>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0087] <7-a2> The method according to <7>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib or a salt thereof.

[0088] <7-b1> The method according to any one of <7> to <7-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0089] <7-b2> The method according to any one of <7> to <7-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0090] <7-c1> The method according to any one of <7> to <7-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0091] <7-c2> The method according to any one of <7> to <7-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0092] <7-d> The method according to any one of <7> to <7-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0093] <7-e> The method according to any one of <7> to <7-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0094] <7-f> The method according to any one of <7> to <7-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0095] <8> A method for inhibiting drug resistance in ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising administering to a subject an effective amount of a compound having ALK inhibitory activity and an effective amount of a FGFR inhibitor in combination.

[0096] <8-a1> The drug according to <8>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0097] <8-a2> The method according to <8>, wherein the ALK inhibitor is a compound selected from alectinib and lorlatinib, or a salt thereof.

[0098] <8-b1> The method according to any one of <8> to <8-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0099] <8-b2> The method according to any one of <8> to <8-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0100] <8-c1> The method according to any one of <8> to <8-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0101] <8-c2> The method according to any one of <8> to <8-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0102] <8-d> The method according to any one of <8> to <8-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0103] <8-e> The method according to any one of <8> to <8-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0104] <8-f> The method according to any one of <8> to <8-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0105] <9> A product comprising (1) a preparation containing a compound having ALK inhibitory activity, (2) a container, and (3) an instruction sheet or label indicating that the compound having ALK inhibitory activity is administered to a subject in combination with at least one FGFR inhibitor for the purpose of treating ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive.

[0106] <9-a1> The drug according to <9>, wherein the ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

[0107] <9-a2> The product according to <9>, wherein the ALK inhibitor is a compound or a salt thereof selected from alectinib and lorlatinib.

[0108] <9-b1> The product according to any one of <9> to <9-a2>, wherein the FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdafitinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

[0109] <9-b2> The product according to any one of <9> to <9-a2>, wherein the FGFR inhibitor is AZD4547 or infigratinib.

[0110] <9-c1> The product according to any one of <9> to <9-b2>, wherein the ALK fusion gene-positive cancer is a cancer selected from non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer and lung cancer.

[0111] <9-c2> The product according to any one of <9> to <9-b2>, wherein the ALK fusion gene-positive cancer is non-small cell lung cancer.

[0112] <9-d> The product according to any one of <9> to <9-c2>, wherein the ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

[0113] <9-e> The product according to any one of <9> to <9-d>, wherein the ALK fusion gene-positive cancer is ALK-sensitive cancer.

[0114] <9-f> The product according to any one of <9> to <9-e>, wherein the ALK inhibitor and the FGFR inhibitor are administered simultaneously.

[0115] <10> A method for screening patients with ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising: obtaining tumor tissue from a patient diagnosed with ALK fusion gene-positive cancer, and measuring the expression levels of FGFR1 and FGF2 in the tumor tissue sample.

[0116] <10-a> The method according to <10>, wherein the expression level is the expression level of a protein.

[0117] <10-b> The method according to <10>, wherein the expression level is the expression level of mRNA.

[0118] <10-c> The method according to any one of <10> to <10-c>, wherein the sample is a fresh frozen, fixed frozen, or formalin fixed paraffin embedded (FFPE) sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 : Graphs showing the cell proliferation inhibitory effects of each inhibitory drug in (A) untreated NCI-H2228 cells (parental cells), (B) DTP cells, and (C) repopulating cells.

[0120] Figure 2 : (A) Western blot showing the detection of ALK or FGFR1 signaling molecules in untreated NCI-H2228 (parental cells) and DTP cells. (B) A graph showing the change in the amount of phosphorylated FGFR1 caused by BGJ398 treatment in DTP cells.

[0121] Figure 3 : Western blot showing the temporal changes of (A) ALK or FGFR1 signaling molecules in untreated NCI-H2228 (parental cells) induced by alectinib treatment. (B) is a graph showing the temporal changes of the amount of phosphorylated FGFR1.

[0122] Figure 4 : These are photographs comparing the morphological changes of cells in untreated NCI-H2228 (parental cells) and DTP cells.

[0123] Figure 5-1 : (Fig. 5A) is a protein blot showing the expression of FGFR1 or FGF2 in FGFR1 or FGF2 knockout cells using siRNA. (Fig. 5B) is a diagram showing the cell proliferation inhibitory effect of alectinib treatment in FGFR1 or FGF2 knockout cells using the CRISPR / Cas9 system. (Fig. 5C) is a protein blot showing the expression of FGFR1 or FGF2 in FGFR1 or FGF2 knockout cells using the CRISPR / Cas9 system. (Fig. 5D) is a diagram showing the cell proliferation inhibitory effect of alectinib treatment in FGFR1 or FGF2 knockout cells using the CRISPR / Cas9 system.

[0124] Figure 5-2 : (Fig. 5E) is a graph showing the cell proliferation inhibitory effect of lorlatinib treatment in FGFR1 or FGF2 knockout cells using the CRISPR / Cas9 system. (Fig. 5F) is a graph showing the induced apoptosis induction effect of alectinib treatment in FGFR1 or FGF2 knockout cells (Fig. 5G) is a protein blot showing changes in FGFR1 downstream signals caused by control and alectinib treatment in FGFR1 or FGF2 knockout cells using the CRISPR / Cas9 system.

[0125] Figure 6: Western blot comparing the amount of FGFR1 and FGF2 proteins in SNU2535 parental cells and NCI-H2228 parental cells.

[0126] Figure 7 : (A) is a graph showing the cell proliferation inhibitory effect of alectinib treatment in SNU-2535 cells overexpressing FGFR1 and FGF2. (B) is a Western blot showing changes in ALK and FGFR1 signaling molecules caused by alectinib treatment.

[0127] Figure 8 : It is a figure which shows the cell proliferation inhibitory effect of single use and combined use of alectinib and BGJ398 in alectinib-resistant NCI-H2228 cells.

[0128] Fig. 9 : is a Western blot showing the detection of ALK and FGFR1 signaling molecules in NCI-H2228 parental cells and alectinib-resistant cells.

[0129] Fig.10 : is a Western blot showing the detection of ALK and FGFR1 signaling molecules in NCI-H2228 parental cells and alectinib-resistant cells treated with alectinib and BGJ398 alone or in combination.

[0130] Fig.11 : (A) is a graph showing the cell proliferation inhibitory effect of the combined treatment of alectinib and a FGFR inhibitor in NCI-H2228 and (C) SNU-2535 cells. (B) is a graph showing the cell proliferation inhibitory effect of the combined treatment of lorlatinib and a FGFR inhibitor in NCI-H2228 and (D) SNU-2535 cells.

[0131] Fig.12 : is a Western blot showing the detection of ALK and FGFR1 signaling molecules in NCI-H2228 and SNU-2535 cells treated with alectinib and BGJ398 alone or in combination.

[0132] Fig.13 : (A) is a graph showing apoptosis induction by combined treatment of alectinib and a FGFR inhibitor in NCI-H2228 and (B) SNU-2535 cells.

[0133] Fig.14 : (A) is a graph showing the tumor growth inhibition effect of alectinib and GJ398 alone or in combination in the NCI-H2228 tumor transplanted mouse model, (B) is a Western blot showing the detection of ALK and FGFR1 signaling molecules. (C) is a photograph showing tumor size. (D) is a graph showing tumor weight.

[0134] Fig.15 : A graph showing changes in body weight during single or combined administration of alectinib and BGJ398 in the NCI-H2228 tumor transplanted mouse model.

[0135] Fig.16 : A graph showing the cell proliferation inhibitory effect of long-term single or combined treatment with alectinib and BGJ398 on NCI-H2228 cells.

[0136] Fig.17 : is a graph showing the mRNA expression level of FGFR1 and the risk ratio.

[0137] Fig.18 : is a graph showing the mRNA expression level of FGF2 and the risk ratio.

[0138] Fig.19 : This is a graph in which the mRNA expression levels of FGFR1 and FGF2 were plotted. DETAILED DESCRIPTION

[0139] The present invention relates to a drug, combination agent, pharmaceutical composition, preparation, and product for treating or preventing ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, which is composed of a compound having ALK inhibitory activity and an FGFR inhibitor. In addition, the present invention also relates to a method for treating or preventing ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, or a drug for inhibiting drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, or a method for inhibiting drug resistance.

[0140] ALK inhibitors

[0141] The ALK inhibitor used in the present invention refers to a substance that can directly or indirectly neutralize, block, inhibit, reduce or hinder the activity of ALK. As an example of the activity of the aforementioned ALK, tyrosine kinase activity can be cited. The aforementioned substances include: low molecular weight compounds, antibodies, antisense substances, transcription inhibitors, low molecular weight interfering RNA (siRNA), etc.

[0142] As examples of the aforementioned low molecular weight compounds, the following have been approved to date: the first generation of crizotinib (compound name: 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine), the second generation of alectinib (compound name: 9-ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)-piperidin-1-yl]-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile monohydrochloride), and the second generation of ceritinib (compound name: 5-chloro-N2-(2-isopropoxy-5-methyl-4-piperidin-4-yl-phenyl)-N4-[2-(propane -2-sulfonyl)-phenyl]-pyrimidine-2,4-diamine), and the third-generation lorlatinib (compound name: (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-4,8-methanepyrazolo[4,3-h][2,5,11]benzoxadiazepinecyclotetradecine (cyclotetradecine)-3-carbonitrile), and brigatinib (compound name: {2-[(5-chloro-2-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl)amino]phenyl}dimethyl-λ 5 -phosphone) as the fifth agent.

[0143] Alectinib is widely used as the first choice drug. These compounds or their salts can be produced by the methods described in WO2010 / 143664, WO2004 / 076412, WO2006 / 021881, WO2008 / 073687, etc. These compounds or their salts also include hydrates, various pharmaceutically acceptable solvates, polymorphs, etc.

[0144] The administration route of the ALK inhibitor used in the present invention is suitably oral or non-oral, preferably oral administration. As a dosage form for oral administration, for example, it can be appropriately selected from any dosage form such as liquid preparations, powders, granules, tablets, enteric solvents and capsules. ALK inhibitors with such a dosage form can be formulated by methods known to those skilled in the art. For example, with a pharmaceutically acceptable carrier or medium, specifically, with sterile water, saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, medium, preservative, adhesive, etc., after mixing in a generally recognized clinically required unit dosage form, it is formulated by freeze drying, tableting and other formulation operations.

[0145] ALK inhibitors can also be used non-orally in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. The amount of active ingredients in these preparations can be appropriately selected to be able to administer the indicated range of dosage. The sterile composition for injection can be prepared in accordance with the usual formulation practice using a medium such as distilled water for injection. As an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride can be exemplified, and can be used in combination with appropriate cosolvents, such as alcohol, specifically ethanol, polyols, such as propylene glycol, polyethylene glycol, nonionic surfactants, such as polysorbate 80 (TM), HCO-50. As an oily liquid, sesame oil and soybean oil can be exemplified, and can be used in combination with benzyl benzoate and benzyl alcohol as cosolvents. In addition, it can be appropriately combined with a buffer, such as a phosphate buffer, a sodium acetate buffer, an analgesic, such as procaine hydrochloride, a stabilizer, such as benzyl alcohol, phenol, and an antioxidant.

[0146] As the dosage of the ALK inhibitor of the present invention, for example, the dosage can be selected in the range of 0.0001 mg to 1000 mg / 1 kg body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.001 mg to 100000 mg / body per patient. However, the dosage of the ALK inhibitor of the present invention is not limited to these dosages.

[0147] More specific dosages of the aforementioned alectinib, its salt, or hydrate thereof include: twice a day, 20 mg, 40 mg, 60 mg, 80 mg, 120 mg, 160 mg, 220 mg, 240 mg, 300 mg, 460 mg, 600 mg, 760 mg or 900 mg per dose in terms of free form.

[0148] The administration period of the ALK inhibitor of the present invention is appropriately determined according to the degree of symptoms or side effects, and can be administered until the cancer is treated or the desired therapeutic effect is achieved. Specifically, it can be administered continuously for 7 days to 3 years, or there is a method of setting a 1 to 14 day drug-free period during the administration period, and administering the drug in 1 to 36 cycles with 2 days to 3 months as one cycle. Preferably, it is administered in 3 to 24 cycles with 14 to 30 days as one cycle.

[0149] It is known that FGFR (Fibroblast growth factor receptor) gene abnormalities activate downstream STAT3 pathway, MAPK pathway, or PI3K-AKT pathway, etc. These activations promote the proliferation, survival, and migration of cancer cells, thus contributing to canceration.

[0150] Therefore, FGFR inhibitors are known to be useful for inhibiting the proliferation of cancer cells.

[0151] There are four types of FGFR receptors: FGFR1, FGFR2, FGFR3, and FGFR4. Genetic abnormalities of FGFR1, FGFR2, and FGFR3 have been reported in various tumors.

[0152] There are 23 FGF (Fibroblast growth factor) family members, which activate FGFR signaling by binding to FGFR. FGF2 binds to all FGFRs.

[0153] The FGFR inhibitor used in the present invention refers to a substance that can directly neutralize, block, inhibit, reduce or hinder the activity of FGFR tyrosine kinase. The aforementioned substances include: low molecular weight compounds, antibodies, antisense, transcription inhibitors, low molecular weight interfering RNA (siRNA, sgRNA), etc.

[0154] For example, non-selective FGFR inhibitors include sorafenib (chemical name: 4-[2-(4-chloro-2-(5-nitro-2-fluorophenyl)phenyl)-1-piperazinyl]-N-(2-pyridyl)benzamide), dobatinib (chemical name: 4-[(4-(4-hydroxy-3-(trifluoromethyl)phenyl)piperazin-1-yl)carbonyl]-2-(3,5-dimethoxyphenyl)benzonitrile), sibutinib (chemical name: N-(4-(4-{2- (4-(5-fluoro-2-fluorophenyl)piperazin-1-yl}pyrimidin-4-yl)phenyl)-2-(2-methoxyethoxy)benzamide), dabrafenib (chemical name: 4-[2-(4-(5-fluoro-2-fluorophenyl)phenyl)-1-piperazinyl]-N-(2-pyridinyl)benzamide), octotinib (chemical name: N-[4-(4-{2-(4-(5-fluoro-2-fluorophenyl)piperazin-1-yl}pyrimidin-4-yl)phenyl]-2-(2-methoxyethoxy)benzamide In addition, as FGFR1-3 selective oral tyrosine kinase inhibitors, infigratinib (BGJ398, chemical name: N'-(2,6-dichloro-3,5-dimethoxyphenyl)-N-[6-[[4-(4-ethyl-1-piperazinyl)phenyl]amino]-4-pyrimidinyl]-N-methyl-urea), delazentinib (ARQ087, chemical name: (6R)-6-(2-fluorophenyl)- 5,6-dihydro-N-[3-[2-[(2-methoxyethyl)amino]ethyl]phenyl]-benzo[h]quinazolin-2-amine), peraminerb (INCB054828, chemical name: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-1,3,4,7-tetrahydro-8-(4-morpholinomethyl)-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one), erdafitinib (chemical name: N 1 -(3,5-dimethoxyphenyl)-N 2 -(1-Methylethyl)-N 1-[3-(1-methyl-1H-pyrazol-4-yl)-6-quinoxalinyl]-1,2-ethylenediamine), Ponatinib (chemical name: 3-(2-imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-benzamide), Debio1347 (chemical name: [5-amino-1-(2-methyl-1H-benzimidazol-6-yl)-1H-pyrazol-4-yl]-1H-indol-2-yl-methanone), Fobatinib (TAS-120, chemical name: 1-[(3S)-3-[4-amino-3-[2-(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-pyrrole [alkyl]), tasurgratinib (Tasurgratinib, E7090, chemical name: 5-((2-(4-(1-(2-hydroxyethyl)piperidin-4-yl)benzamide)pyridin-4-yl)oxy)-6-(2-methoxyethoxy)-N-methyl-1H-indole-1-carboxamide), AZD4547 (chemical name: N-(5-(3,5-dimethoxyphenethyl)-1H-pyrazol-3-yl)-4-((3S, 5R)-3,5-dimethylpiperazin-1-yl)benzamide), PD173074 (chemical name: 1-(tert-butyl)-3-(2-((4-(diethylamino)butyl)amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)urea) and other compounds.

[0155] Preferred is infigratinib, pemitinib, erdafitinib, AZD4547 or a salt thereof.

[0156] These compounds or their salts are manufactured and sold in the form of pharmaceutical preparations, or can be obtained as research reagents, or can be manufactured by conventional methods. These compounds or their salts also include hydrates, various pharmaceutically acceptable solvates, polymorphs, and the like.

[0157] FGFR inhibitors are formulated according to common methods (e.g., Remington's Pharmaceutical Science, latest edition, Mack Publishing Company, Easton, USA), and pharmaceutically acceptable carriers and additives may be included together. For example, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, adhesives, disintegrants, lubricants, fluidity promoters, flavoring agents, etc. may be cited, but are not limited to these, and other commonly used carriers may be appropriately used. Specifically, trehalose, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, polysorbate, etc. may be appropriately cited.

[0158] In the present invention, the administration method of the FGFR inhibitor can be implemented by any of oral and parenteral administration. Particularly preferred is an administration method based on oral administration. As the administration method involved, specific examples can be appropriately exemplified: administration by liquid preparations, powders, granules, tablets, enteric preparations and capsules, etc. As an example of injection administration, for example, systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., for the treatment of the present invention. In addition, the administration method can be appropriately selected according to the age and symptoms of the patient. As a dosage, for example, in the case of oral preparations, the dosage can be selected in the range of 0.1 mg / kg to 100 mg / kg, preferably 0.1 mg / kg to 5 mg / kg per administration. For example, each patient can select the dosage in the range of 9 mg to 150 mg. However, the dosage of the FGFR inhibitor used in the present invention is not limited to these dosages.

[0159] Depending on the type and severity of the disease, for example, the preferred daily dose of Infigratinib is 75 mg to 125 mg, but it is not limited and can be reduced in stages according to the symptoms and degree of side effects. The frequency of administration is usually once a day, and the prescribed period (for example, 3 weeks) is considered a cycle. A prescribed drug-free period (for example, 1 week) is set and the cycle is repeated. The number of cycles varies according to the type and severity of the disease. The treatment is measured according to methods known in the art and maintained until the target cancer is treated or the desired therapeutic effect is achieved. In one example, Infigratinib is 125 mg once a day for 3 consecutive weeks. If side effects are found, the dose is reduced to 100 mg once and reduced to 75 mg twice. However, other dosage regimens may also be useful.

[0160] The FGFR inhibitor can be administered during the period when the target cancer is treated or the desired therapeutic effect is achieved, with the above-mentioned 3-week administration and 1-week drug-free period constituting one cycle. Specifically, the FGFR inhibitor can be administered for 1 to 36 cycles.

[0161] For example, erdafitinib is administered once a day at an initial dose of 8 mg, and the dose is increased to 9 mg while monitoring the serum phosphate level.

[0162] Drugs, combinations, pharmaceutical compositions, and treatment and prevention methods

[0163] One embodiment of the present invention is a drug for treating or preventing ALK fusion gene-positive cancer (hereinafter referred to as target cancer) that is FGFR1-positive and FGF2-positive, comprising a combination of a compound having ALK inhibitory activity and a FGFR inhibitor.

[0164] The "drug for treating or preventing a target cancer, which is a combination of a compound having ALK inhibitory activity and a FGFR inhibitor" in the above scheme means a drug that is combined for the purpose of administering a compound having ALK inhibitory activity and a FGFR inhibitor simultaneously, separately, or sequentially in the treatment or prevention of the target cancer. The drug of the present invention can also be provided in the form of a compound containing both a compound having ALK inhibitory activity and a FGFR inhibitor. In addition, a preparation containing a compound having ALK inhibitory activity and a preparation containing a FGFR inhibitor can be provided separately, and these preparations can be used simultaneously or sequentially.

[0165] It should be noted that in the present application, "combination" specifically includes the use with other drugs recorded in the usage and dosage of the pharmaceutical preparation. Specifically, it includes: the use of the compound with ALK inhibitory activity is recorded in the instructions of the pharmaceutical preparation, and the use of the compound with ALK inhibitory activity is recorded in the instructions of the pharmaceutical preparation.

[0166] In the present invention, simultaneous administration means that the compound having ALK inhibitory activity and the FGFR inhibitor are administered at the same time, and can be administered as a compound, or as a mixture prepared at the time of administration, or different forms of preparations can be administered at the same time. In the case of simultaneous administration, the administration can be carried out by separate routes or by the same route, and the dosage forms can be the same or different.

[0167] In the present invention, when a compound having ALK inhibitory activity and a FGFR inhibitor are administered separately, the order of administration of the compound having ALK inhibitory activity and the FGFR inhibitor may be administration of the compound having ALK inhibitory activity after administration of the FGFR inhibitor, or administration of the compound having ALK inhibitory activity and the FGFR inhibitor at the same time. In addition, the FGFR inhibitor may be administered after administration of the compound having ALK inhibitory activity.

[0168] Preferably, the compound having ALK inhibitory activity is administered simultaneously with the FGFR inhibitor.

[0169] Specifically, the instructions for use of the pharmaceutical preparation of the compound having ALK inhibitory activity describe the combined use with the FGFR inhibitor, and when used in combination, the compound having ALK inhibitory activity is administered in accordance with the prescribed usage and dosage.

[0170] Alternatively, the instructions for use of the FGFR inhibitor pharmaceutical preparation may state that the compound has ALK inhibitory activity is used in combination, and when used in combination, the compound is administered according to the prescribed usage and dosage of the compound has ALK inhibitory activity.

[0171] In the above-mentioned drugs, when the compound having ALK inhibitory activity and the FGFR inhibitor are provided in respective preparations, the dosage forms of these preparations may be the same dosage form or different dosage forms. For example, both parties may be different dosage forms selected from oral preparations, non-oral preparations, injections, drips, and intravenous drips, or both parties may be the same dosage form selected from oral preparations, non-oral preparations, injections, drips, and intravenous drips. Preferably, the dosage forms of both parties are oral preparations. In addition, more than one different preparation may be further combined in the above-mentioned drugs.

[0172] It should be noted that in the present invention, the pharmaceutical composition comprises the compound having ALK inhibitory activity and / or FGFR inhibitor for treatment and / or prevention of the present invention, and may further comprise a pharmaceutically acceptable carrier.

[0173] In the present invention, the term "pharmaceutically acceptable carrier" means one or more suitable solid or liquid excipient diluents or encapsulating materials as exemplified above, which are suitable for administration to mammals.

[0174] In the present invention, "combination agent" means a form in which a compound having ALK inhibitory activity and a compound being an FGFR inhibitor can be used separately for the same object, or a form in which these compounds are formulated separately without mixing and combined. For example, it includes a drug preparation of a compound having ALK inhibitory activity and a drug preparation of an FGFR inhibitor, which are prepared as a single dose in one package so that the drug preparation can be taken simultaneously.

[0175] In the present invention, the "pharmaceutical preparation" composed of a compound having ALK inhibitory activity and a FGFR inhibitor includes: preparations obtained by separately formulating these active ingredients, or solid preparations such as tablets, capsules, granules, powders, pills, etc. contained in the same preparation, aqueous and non-aqueous oral solutions and suspensions, and liquid preparations such as non-oral solutions filled in containers suitable for subdividing into individual dosage amounts, lyophilized preparations that can be dissolved and used when used, or preparations obtained by combining any of these dosage forms. Each unit dosage form of the pharmaceutical preparation contains 150 mg to 800 mg, preferably 150 mg to 400 mg, and particularly preferably 150 mg to 300 mg of alectinib or its salt in terms of free body. The preparations obtained by separately formulating alectinib or its salt specifically include 150 mg capsule preparations, 150 mg, 300 mg, 600 mg tablets, etc.

[0176] In another viewpoint, the present invention provides: a drug containing a compound having ALK inhibitory activity as an active ingredient, which is used in combination with an FGFR inhibitor to treat or prevent a target cancer. "A drug containing a compound having ALK inhibitory activity as an active ingredient, which is used in combination with an FGFR inhibitor to treat or prevent a target cancer" means a drug containing a compound having ALK inhibitory activity as an active ingredient, which is used to treat or prevent a target cancer on the condition that it is used in combination with an FGFR inhibitor. When the drug of the present invention containing a compound having ALK inhibitory activity as an active ingredient is used in combination with an FGFR inhibitor, it can be administered simultaneously with the FGFR inhibitor, or it can be administered before or after the administration of the FGFR inhibitor. When a compound having ALK inhibitory activity is administered before or after the administration of the FGFR inhibitor, the administration period can be optimized by measuring the residual concentration of the FGFR inhibitor in the subject. The concentration of the sample collected from the subject can be determined based on an immunoassay such as ELISA described later, which is well known to those skilled in the art.

[0177] In another viewpoint, the present invention provides: a drug containing a FGFR inhibitor as an active ingredient for use in combination with a compound having ALK inhibitory activity to treat or prevent a target cancer. The "drug containing a FGFR inhibitor as an active ingredient for use in combination with a compound having ALK inhibitory activity to treat or prevent a target cancer" in the present invention means a drug containing a FGFR inhibitor as an active ingredient for use in combination with a compound having ALK inhibitory activity to treat or prevent a target cancer. When a drug containing a FGFR inhibitor as an active ingredient is used in combination with a compound having ALK inhibitory activity, it can be administered simultaneously with the compound having ALK inhibitory activity, or before or after the administration of the compound having ALK inhibitory activity. When a FGFR inhibitor is administered before or after the administration of a compound having ALK inhibitory activity, the administration period can be optimized by measuring the residual concentration of the compound having ALK inhibitory activity in the subject. The sample collected from the subject can be separated using various separation devices such as chromatographs, and the concentration can be determined based on an analytical method known to those skilled in the art.

[0178] The above invention means that a compound having ALK inhibitory activity and a FGFR inhibitor are administered or used together (hereinafter referred to as "administration"), and is not to be construed as limiting the order of administration or the interval between administrations, etc. In addition, the present invention can be used in the form of a product consisting of a compound having ALK inhibitory activity and a FGFR inhibitor. Furthermore, in the present invention, when a compound having ALK inhibitory activity is used in combination with a FGFR inhibitor, each can be administered in a smaller dosage as needed compared to the dosage of either party used alone.

[0179] Cancer Type

[0180] The drug of the present invention can be used for the prevention or treatment of diseases such as leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid leukemia, chronic lymphoid leukemia, etc.), malignant lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), brain tumors, neuroblastomas, gliomas, thyroid cancer, myelodysplastic syndrome, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, liver cancer, gallbladder cancer, skin cancer, malignant melanoma, kidney cancer, renal pelvis and ureter cancer, bladder cancer, uterine cancer, testicular cancer, prostate cancer, etc. Further, the compound of the present invention can also be used for the prevention or treatment of the infiltration / metastasis of solid cancer.

[0181] In particular, the drug of the present invention can be used for the prevention or treatment of ALK fusion gene-positive cancers. Examples of ALK fusion gene-positive cancers include, but are not limited to, non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer, and lung cancer.

[0182] In the present invention, "ALK fusion gene positive" means the presence of a fusion gene of ALK and other genes. Examples of the aforementioned other fusion genes include: EML4 (echinoderm microtubule-associated protein like protein 4), GenBank TM Accession number NM_019063), TFG (TRK-fused gene, GeneBank TM Accession No. NM_006070), etc., but is not limited to these.

[0183] Methods for identifying ALK fusion gene-positive cancers are described in WO2008 / 127248, Japanese Patent Application Laid-Open No. 2008-295444, "ALK Gene Testing Guidelines" (Biomarker Committee of the Japanese Lung Cancer Society), etc. As a kit for detecting this fusion gene, the Vysis LSI ALK Break Apart Rearrangement Probe Kit is available. TM (Abbott), Histofine ALK iAEP TM Kit (Nichirei Corporation).

[0184] "ALK sensitivity" means sensitivity to ALK inhibitors. Specifically, it means that when a patient with ALK fusion gene-positive cancer or ALK fusion gene-positive cancer cells is given an ALK inhibitor once, a certain degree of shrinking effect or growth inhibition effect is shown when compared with a control such as a solvent, or when compared with before administration of the ALK inhibitor.

[0185] "FGFR1 expression positive" or "FGF2 expression positive" means that the level of messenger RNA (mRNA) encoding FGFR1 protein or FGF2 protein is higher than that of normal cells. It should be noted that the normal cells used for comparison are cells of the same tissue type, especially phenotype as the tumor, or cells obtained from the tissue type that produces the tumor.

[0186] Whether a cell is "FGFR1 expression-positive" or "FGF2 expression-positive" can be determined using a known method for determining mRNA levels or protein levels.

[0187] Methods suitable for determining gene expression levels at the mRNA level are not limited, and include qPCR, RT-PCR, RNA protection analysis, Northern blot hybridization, RNA dot blot, in situ hybridization, microarray technology, methods using tags (including serial analysis of gene expression (SAGE) such as LongSAGE and SuperSAGE), microarrays, fluorescence in situ hybridization (FISH) (including Flow-FISH, qFiSH and double fusion FISH (D-FISH) and other variations) and the like for determining standard assays for mRNA expression levels.

[0188] Specifically, it can be determined by, for example, the following method.

[0189] The expression levels of FGFR1 and FGF2 mRNA in tumor tissue samples were quantified by RNA in situ hybridization using FGFR1 or FGF2 probes. In situ hybridization is well known in the art, for example, as described by Wang et al. in J Mol Diagn. 2012 Jan; 14(1): 22-9.

[0190] In situ hybridization is usually performed on cells or tissue sections fixed on slides, using both direct and indirect methods.

[0191] In the direct method, a detectable molecule (e.g., a fluorophore, i.e., fluorescence in situ hybridization or FISH) is directly bound to a nucleic acid probe so that the probe-target hybrid can be observed under a microscope immediately after the hybridization reaction. In this method, it is extremely important that the probe-reporter combination can withstand very stringent hybridization and washing conditions. It should be noted that it is important that the reporter molecule does not interfere with the hybridization reaction. Methods that meet these criteria include, for example, the terminal fluorescent dye labeling method of RNA probes developed by Bauman et al. (1980, 1984) and the direct enzyme labeling method of nucleic acids described by Renz and Kurz (1984). Radioactive labels can also be used. In the indirect method, it is necessary to contain a detectable molecule into which the probe is chemically or enzymatically introduced, for example, detection can be achieved by using a biotin-streptavidin system or the like. ISH probes for detecting FGFR1 or FGF2 mRNA expression are designed, for example, according to Jin and Lloyd (J Clin Lab Anal. 1997; 11(1): 2-9.). The sequence used in the design of the probe of the present invention is a sequence with GenBank sequence accession number NM_023110.2 (FGFR1). It should be noted that the poly A tail provided by the above GenBank accession number is not used for probe design. In addition, the method for formalin fixation, paraffin embedding tissue specimens or frozen specimens can use known methods. In bright field microscopy, conventional color pigments or fluorescent pigments for multiple analysis can be used (Levsky and Singer, J Cell Sci. 2003 Jul 15; 116 (Pt14): 2833-8).

[0192] Preferably, the expression levels of FGFR1 and FGF2 mRNA in tumor tissue samples can be measured by RNAscope (registered trademark) (Advanced Cell Diagnostics, Inc., 3960 Point Eden Way, Hayward, CA 94545, USA) using RNA in situ hybridization using ACD (Advanced Cell Diagnostics, Inc., 3960 Point Eden Way, Hayward, CA 94545, USA) technology. RNAscope is a commercially available fully automated multiplex assay that is a highly sensitive and specific assay that can detect up to 4 different RNAs per slide simultaneously and 1 RNA in fresh frozen, fixed frozen, and FFPE tissue samples (Journal Pathology, 2021.254(4): p405-417).

[0193] In addition, judging "FGFR1 expression positive" or "FGF2 expression positive" by protein level is not limited, and includes conventional methods for determining protein expression levels, such as using an antibody having the ability to specifically bind to a protein encoded by the aforementioned gene (or a fragment thereof containing an antigenic determinant), and then quantifying the resulting antibody-antigen complex. For example, the protein level of FGFR1 or FGF2 can be quantified by using a standard assay for determining protein expression levels, such as Western blot or protein transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical methods and immunohistochemical methods, methods based on biochips or microarrays using proteins containing specific antibodies, or assays based on colloidal precipitation in the form of dipsticks, etc.

[0194] The antibodies used in these assays can be, for example, polyclonal serum, hybridoma supernatant or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, double-chain antibodies, three-chain antibodies, four-chain antibodies and humanized antibodies. At the same time, the antibody can be labeled or unlabeled. As illustrative but non-exclusive examples of labels that can be used, radioisotopes, enzymes, fluorophores, chemiluminescent reagents, substrates or cofactors of enzymes, inhibitors of enzymes, particles, colorants, etc. can be cited. There are a wide range of well-known assays that can be used in the present invention, which use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These methods include: protein blotting or protein transfer, ELISA, RIA, competitive EIA, DAS-ELISA, immunocytochemistry and immunohistochemistry, methods based on biochips or microarrays of proteins containing specific antibodies, or assays based on colloidal precipitation in the form of dipsticks, etc. Other methods for detecting and quantifying target protein levels include: affinity chromatography, binding ligand assays, etc.

[0195] Specifically, when using the Western blotting method, the FGFR1 or FGF2 protein separated by electrophoresis can be transferred to a membrane, and the presence of the protein can be detected using an antibody against the protein.

[0196] When using Western blotting, "FGFR1 expression positive" means cells in which the expression of FGFR1 protein is detected more strongly than the control (SNU-2535 cells) in Western blotting. "FGF2 expression positive" means cells in which the expression of FGF2 protein is detected more strongly than the control (SNU-2535 cells) in Western blotting.

[0197] In addition, the quantitative of the protein level of FGFR1 or FGF2 based on immunostaining can comprise the tissue microarray (TMA) through assembling object sample by construction, and utilize immunohistochemical method to determine the expression level of related protein to carry out.In order to maintain the reproducibility of method, immunostaining intensity can be evaluated with more than 2 people, and use the same and clear cut-off value benchmark to score.Inconsistent can be solved by re-evaluating simultaneously.For example, considering the specific cut-off value of expression in tumor cell and each marker, the result of immunostaining can be recorded as expression negative (0) vs expression positive and low expression (1+) vs moderate expression (2+) and high expression (3+).As general benchmark, select cut-off value to make reproducibility easy and translate biological event as far as possible. Alternatively, the intensity of immunostaining can be evaluated by using imaging techniques and automated methods such as those disclosed in Rojo, MG et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

[0198] In another embodiment of the present invention, a method is provided for enhancing the therapeutic effect of a compound having ALK inhibitory activity when a cancer patient is treated with a compound having ALK inhibitory activity by using an FGFR inhibitor. Here, enhancement of the therapeutic effect means any of the following: the efficacy of the treatment is increased compared to when a single dose is administered; the amount of the compound having ALK inhibitory activity administered for treatment is reduced compared to when a single dose is administered; a therapeutic effect is shown for more severe cases, such as cases where the previous treatment is ineffective or worsening; due to the disappearance of cancer, the treatment period using a compound having ALK inhibitory activity is shortened compared to the expected treatment period when the drug is administered alone, or the treatment period predetermined before the start of the treatment using a combined administration; or due to the inhibition of disease progression, the treatment period including a compound having ALK inhibitory activity and / or other maintenance therapy is extended. In addition, in another embodiment of the present invention, a method for extending progression-free survival (PFS) in a subject is provided, which comprises administering an effective amount of a compound having ALK inhibitory activity and an effective amount of a FGFR inhibitor in combination. In another embodiment of the present invention, a method for using a compound having ALK inhibitory activity or a FGFR inhibitor in the manufacture of a pharmaceutical composition for treating or preventing a target cancer, the composition comprising a compound having ALK inhibitory activity and a FGFR inhibitor as active ingredients is provided.

[0199] It should be noted that, based on the insights of the present invention, it is also confirmed that, similar to compounds with ALK inhibitory activity, by using EGFR inhibitors, HER2 inhibitors or BRAF inhibitors in combination with FGFR inhibitors, the effects of these inhibitors on ALK fusion gene-positive cancers, especially ALK fusion gene-positive cancers with positive FGFR1 expression and positive FGF2 expression, can be enhanced. As EGFR inhibitors, epirubicinitib, gefitinib, afatinib, erlotinib, dasatinib, osimertinib, etc. can be cited. As HER2 inhibitors, neratinib, trastuzumab, lapatinib, pertuzumab, adavopatib, alectinib, panitumumab, etc. can be cited. As BRAF inhibitors, dabrafenib, trametinib, vemurafenib, cobimetinib, encorafenib, ubekinumab, etc. can be cited.

[0200] Preferred combinations of EGFR inhibitors and FGFR inhibitors include: epirubicinib / infigratinib, epirubicinib / pemitinib, epirubicinib / erdatinib, epirubicinib / AZD4547, epirubicinib / tasugatinib, gefitinib / infigratinib, gefitinib / pemitinib, gefitinib / erdatinib, gefitinib / AZD4547, gefitinib / tasugatinib, afatinib / infigratinib, afatinib / pemitinib, afatinib / erdatinib, afatinib / AZD 4547, afatinib / tasugatinib, erlotinib / infigratinib, erlotinib / pemitinib, erlotinib / erdatinib, erlotinib / AZD4547, erlotinib / tasugatinib, dasatinib / infigratinib, dasatinib / pemitinib, dasatinib / erdatinib, dasatinib / AZD4547, dasatinib / tasugatinib, osimertinib / infigratinib, osimertinib / pemitinib, osimertinib / erdatinib, osimertinib / AZD4547, osimertinib / tasugatinib. Particularly preferred are erlotinib / infigratinib, erlotinib / AZD4547, osimertinib / infigratinib, and osimertinib / AZD4547.

[0201] Preferred combinations of a HER2 inhibitor and a FGFR inhibitor include: neratinib / infigratinib, neratinib / pemitinib, neratinib / erdafitinib, neratinib / AZD4547, neratinib / tasugatinib, trastuzumab / infigratinib, trastuzumab / pemitinib, trastuzumab / erdafitinib, trastuzumab / AZD4547, trastuzumab / tasugatinib, lapatinib / infigratinib, lapatinib / pemitinib, lapatinib / erdafitinib, lapatinib / AZD4547, lapatinib / tasugatinib, pertuzumab / infigratinib, pertuzumab / Pemetinib, Pertuzumab / Erdafitinib, Pertuzumab / AZD4547, Pertuzumab / Tasugatinib, Adavopatibin / Infigratinib, Adavopatibin / Pemetinib, Adavopatibin / Erdafitinib, Adavopatibin / AZD4547, Adavopatibin / Tasugatinib, Alectinib / Infigratinib, Alectinib / Pemetinib, Alectinib / Erdafitinib, Alectinib / AZD4547, Alectinib / Tasugatinib, Panitumumab / Infigratinib, Panitumumab / Pemetinib, Panitumumab / Erdafitinib, Panitumumab / AZD4547, Panitumumab / Tasugatinib. Particularly preferred examples include neratinib / infigratinib, neratinib / AZD4547, lapatinib / infigratinib, and lapatinib / AZD4547.

[0202] Preferred combinations of BRAF inhibitors and FGFR inhibitors include dabrafenib / infigratinib, dabrafenib / pemetinib, dabrafenib / erdafitinib, dabrafenib / AZD4547, dabrafenib / tasugatinib, trametinib / infigratinib, trametinib / pemetinib, trametinib / erdafitinib, trametinib / AZD4547, trametinib / tasugatinib, vemurafenib / infigratinib, vemurafenib / pemetinib, vemurafenib / erdafitinib, vemurafenib / AZD4547, Vemurafenib / tasugatinib, cobimetinib / infigratinib, cobimetinib / pemetinib, cobimetinib / erdafitinib, cobimetinib / AZD4547, cobimetinib / tasugatinib, encorafenib / infigratinib, encorafenib / pemetinib, encorafenib / erdafitinib, encorafenib / AZD4547, encorafenib / tasugatinib, ubetumab / infigratinib, ubetumab / pemetinib, ubetumab / erdafitinib, ubetumab / AZD4547, ubetumab / tasugatinib. Particularly preferred are dabrafenib / infigratinib, dabrafenib / AZD4547, trametinib / infigratinib, and trametinib / AZD4547.

[0203] In the present invention, containing a compound having ALK inhibitory activity and / or a FGFR inhibitor as an effective ingredient means containing a compound having ALK inhibitory activity and / or a FGFR inhibitor as a main active ingredient, and does not limit the content rate of the compound having ALK inhibitory activity and / or the FGFR inhibitor. In addition, the term "treatment" means any of the following situations: by administering the drug of the present invention to the subject, cancer cells die or their cell number is reduced; the proliferation of cancer cells is inhibited; the metastasis of cancer cells is inhibited; the progression-free survival (PFS) is prolonged compared to the single treatment; various symptoms caused by the subject cancer are improved. In addition, the term "prevention" means any of the following situations: preventing an increase in the number of reduced cancer cells caused by their re-proliferation; preventing the re-proliferation of cancer cells whose proliferation is inhibited.

[0204] In the present invention, "effective amount" means the daily dosage of each inhibitor when a compound having ALK inhibitory activity and a FGFR inhibitor are administered in combination. The dosage in the present invention may be the same as the dosage when each inhibitor is used alone, or a dosage lower than the dosage when used alone. Alternatively, the effective amount in the present invention may be: one is the same as the dosage when used alone, and the other is a dosage lower than the dosage when used alone.

[0205] In addition, other schemes of the present invention include: a drug for inhibiting the drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, which is a combination of an ALK inhibitor and a FGFR inhibitor; a drug for inhibiting the drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, which contains an ALK inhibitor as an active ingredient and is used in combination with a FGFR inhibitor; a drug for inhibiting the drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, which contains an FGFR inhibitor as an active ingredient and is used in combination with an ALK inhibitor; and a method for inhibiting the drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, which comprises administering to a subject a combination of an effective amount of a compound having ALK inhibitory activity and an effective amount of a FGFR inhibitor.

[0206] The drug or method for inhibiting drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive in the present invention refers to a drug or method that can inhibit drug resistance by combining ALK inhibitors and FGFR inhibitors for ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive and has not received previous treatment with ALK inhibitors, etc. It should be noted that "inhibiting drug resistance" means that in a subject treated with a combination of ALK inhibitors and FGFR inhibitors, the sensitivity to ALK inhibitors or the cell proliferation inhibitory effect in a known in vitro test (e.g., Anticancer Drugs. 2022 Feb 1; 33(2): 124-131, etc.) using cultured cells of tumor cells obtained from the same subject at any time before and after the start of treatment is compared, and the sensitivity or cell proliferation inhibitory effect of tumor cells at any time after the start of treatment is the same as or not significantly reduced compared to the start of treatment.

[0207] In the present invention, "progression-free survival (PFS)" refers to the time from treatment (or randomization) to initial disease progression or death. In one embodiment of the present invention, PFS can be evaluated based on the treatment effect determination criteria (RECIST) for solid tumors. In one embodiment of the present invention, PFS can be evaluated based on the CA-125 level as a determinant of progression.

[0208] In the present invention, as a specific example of "prolonging progression-free survival", it can be cited that the combined use of a compound having ALK inhibitory activity and a FGFR inhibitor prolongs PFS compared to the PFS when a single dose of the compound having ALK inhibitory activity or the FGFR inhibitor is administered at the same dose as that used in combination.

[0209] In the present invention, "subject" is not limited, and means a human or non-human mammal as the subject of administration of the drug of the present invention or a mammal that needs the administration of the drug of the present invention, such as a cow, horse, dog, sheep or cat. The preferred subject is human. The subject includes patients (including humans and non-human mammals). Specifically, it refers to patients with various cancers such as leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid leukemia, chronic lymphoid leukemia, etc.), malignant lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), brain tumors, neuroblastomas, gliomas, thyroid cancer, myelodysplastic syndrome, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, liver cancer, gallbladder cancer, skin cancer, malignant melanoma, kidney cancer, renal pelvis and ureteral cancer, bladder cancer, uterine cancer, testicular cancer, prostate cancer, tumors transferred by the tumor, or a human or non-human mammal with a high probability of illness.

[0210] product

[0211] As another embodiment of the present invention, a product is provided. In one embodiment, the product is provided for the treatment or prevention of a target cancer in a subject, comprising: (a) (1) a preparation containing a FGFR inhibitor, (2) a container, and (3) an instruction or label indicating that the aforementioned FGFR inhibitor is administered to the subject in combination with at least one compound having ALK inhibitory activity for the treatment of the target cancer in the subject; or (b) (1) a preparation containing a compound having ALK inhibitory activity, (2) a container, and (3) an instruction or label indicating that the aforementioned compound having ALK inhibitory activity is administered to the subject in combination with at least one FGFR inhibitor for the treatment of the target cancer in the subject.

[0212] The product comprises: a container comprising a formulation comprising a FGFR inhibitor or a compound having ALK inhibitory activity.

[0213] The product may also include: a label or instructions on or included with the container.

[0214] In the present invention, "instructions" refers to documents usually included in the commercial packaging of the preparation, which contain information about indications, usage, dosage, administration, contraindications and / or warnings related to the use of the preparation. "Labels" refers to sheet-like items including the product name, dosage, dosage form, indications, etc. of the preparation containing the FGFR inhibitor or the compound having ALK inhibitory activity, and refers to those directly attached to the container.

[0215] The label or instruction sheet indicates the indication for the preparation, i.e., the treatment of a target cancer, etc. In one embodiment, the label or instruction sheet indicates that the preparation can be used to treat a target cancer, etc. In addition, the label or instruction sheet may also indicate that the preparation can be used to treat other disorders.

[0216] Suitable containers include, for example, PTP, bottles, vials, syringes, blister packs, etc. The container may be formed of various materials such as glass or plastic. These containers may also be packaged in a paper outer box printed with the above-mentioned label or the like.

[0217] As a further embodiment of the present invention, there is also included a method for screening a patient having ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive.

[0218] The method comprises: obtaining a tumor tissue of a patient diagnosed with ALK fusion gene-positive cancer, and measuring the expression levels of FGFR1 and FGF2 in the aforementioned tumor tissue sample. As the expression levels of FGFR1 and FGF2, the expression level of the protein or the expression level of mRNA can be used. Specifically, the aforementioned method for determining "FGFR1 expression positive" or "FGF2 expression positive" can be used.

[0219] Tumor tissue obtained from patients diagnosed with ALK fusion gene-positive cancer can be prepared as fresh frozen, fixed frozen, formalin fixed paraffin embedded (FFPE) specimens for use in this method.

[0220] [Example]

[0221] The contents of all patents and references explicitly cited in this specification are fully incorporated into this specification by reference.

[0222] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not to be construed as being limited to the description.

[0223] The drugs, reagents, and cells used in the Examples are as follows.

[0224] Alectinib was synthesized by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Infigratinib (BGJ398) was obtained from LC Laboratories (Woburn, Massachusetts, USA). AZD4547 was obtained from ChemScene (Monmouth Junction, NJ, USA). Lorlatinib was obtained from Selleck Chemicals (Houston, TX, USA). PPP was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). All agents were dissolved in dimethyl sulfoxide (DMSO: Sigma-Aldrich, St. Louis, MO, USA) for in vitro assays and in a 6% (w / v) solution of Captisol (ChemScene) for in vivo assays. DMSO and Captisol were used as solvent controls in each assay. Recombinant human FGF1, FGF2, FGF7, and FGF12 proteins were obtained from R&D Systems (Minneapolis, MN, USA) and dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich). Recombinant human FGF9 and FGF18 proteins were obtained from Abcam (Cambridge, England) and dissolved in distilled water. Recombinant human FGF11 protein was obtained from LifeSpan Biosciences (Seattle, WA, USA) and dissolved in Tris buffer (Horizon Discovery in Cambridge, England).

[0225] The tissues, suppliers and culture media of the human cancer cell lines used in the examples are as follows. The cells were cultured at 37°C under 5% CO2.

[0226] [Table 1]

[0227] Cell lines organize Oncogenic driver mutations supplier Culture medium NCI-H2228 NSCLC <![CDATA[EML4-ALK 1 ]]> ATCC 10% FBS-RPMI1640 SNU-2535 NSCLC <![CDATA[EML4-ALK,G1269A 2 ]]> KCLB 10% FBS-RPM11640

[0228] The in vitro data were statistically analyzed using Student's t test or Dunnett's test. The in vivo data were statistically analyzed using the Wilcoxon rank sum test based on the Holm-Bonferroni method. The threshold for significance was set at P < 0.05.

[0229] It should be noted that the [numbers] in the examples correspond to the numbers of the references described below.

[0230] Example 1

[0231] NCI-H2228 cells (parental cells) were treated with alectinib for 13 days to produce DTP cells that are transiently resistant to alectinib. It should be noted that DTP cells (drug tolerant persister cells) refer to cells that acquire drug resistance without accompanying gene mutations through a certain period of drug exposure. The cells have the reversibility of showing drug sensitivity again by terminating drug exposure.

[0232] First, a screening test for the proliferation inhibition effect on parental cells and DTP cells was performed using an anticancer drug library (TargetMol) of 3114 compounds containing compounds that inhibit various signals. As a result, nine compounds were found that did not affect the cell proliferation of parental cells but strongly inhibited the cell proliferation of DTP cells (Table 2). Next, a cell proliferation inhibition test was performed on these nine compounds, and the IC values ​​of DTP cells were calculated. 50 As a result, among the 9 compounds, the FGFR1 inhibitor erdafitinib showed the strongest cell proliferation inhibitory effect on DTP cells compared with the parent strain, indicating that the FGFR1 signal of DTP cells was most strongly activated, contributing to cell proliferation (Table 3).

[0233] [Table 2]

[0234]

[0235] [Table 3]

[0236] ND: Not detected, as the IC of the parental cell could not be calculated 50 .

[0237] Next, the proliferation inhibitory effect in DTP cells was evaluated according to a known method (Anticancer Drugs. 2022 Feb 1; 33(2): 124-131) using an FGFR inhibitor (BGJ398) [3] that inhibits FGFR1, FGFR2, and FGFR3 and inhibits FGFR4 38 times less than other FGFRs, or an IGF1R inhibitor (picropodophyllotoxin, PPP) [4].

[0238] The result is as follows ( Figure 1 , Table 4).

[0239] [Table 4]

[0240] A

[0241]

[0242] As a result, NCI-H2228 cells showed sensitivity to ALK inhibitors (alectinib and lorlatinib) but not to FGFR inhibitors (BGJ398), but DTP cells showed no sensitivity to ALK inhibitors but showed sensitivity to FGFR inhibitors.

[0243] Western blotting

[0244] Cells were seeded in six-well plates, and the agents were added the next day and cultured for a certain period of time. The same amount of protein lysate was loaded for each Western blot using a protein analysis system based on Sally Sue or Jess capillary electrophoresis (ProteinSimple, Santa Clara, CA, USA). Antibodies used were antibodies against ALK, phospho-ALK, FGFR1, FGFR2, FGFR3, FGFR4, ERK, phospho-ERK, AKT, phospho-AKT, EGFR, phospho-EGFR, MET, β-actin, phospho-MET, HER2, CD44, BIM, VIM, CDH1, S6, pS6, and STAT3 (Cell Signaling Technology, Danvers, MA, USA), Cleaved PARP, phospho-HER2, and FGF2, FGF12 (Abcam).

[0245] As a result, the cell proliferation inhibition effect based on PPP treatment in parental cells and DTP cells was roughly the same ( Figure 1 A, B). In addition, in the parental cells, phosphorylation of ALK and phosphorylation inhibition of STAT3, AKT, ERK, and S6 of ALK or FGFR1 signaling molecules were observed by alectinib treatment, while phosphorylation inhibition of STAT3, AKT, ERK, and S6 was not observed in cells with DTP ( Figure 2 A).

[0246] In the parental cells, no phosphorylation inhibition of STAT3, AKT, ERK, and ALK was observed after BGJ398 treatment, but in DTP cells, phosphorylation inhibition of FGFR1 and ERK was observed ( Figure 2 A, B).

[0247] Furthermore, the repopulated cells prepared by culturing DTP cells in alectinib-free medium for 37 days showed alectinib sensitivity recovery and BGJ398 sensitivity loss compared to DTP cells ( Figure 1 C, Table 2).

[0248] The sensitivity of the repopulating cells to these agents was the same as that of the parental cells, indicating that the reversible biological characteristics of the DTP cells were the same as those of other cell DTP cells reported by Mikubo et al. [1].

[0249] In order to investigate the mechanism of the acquisition of FGFR1 signaling dependence in DTP cells, the time-dependent changes in ALK and FGFR1 signaling molecules caused by alectinib treatment were analyzed. Among the 23 FGF family members, 11 FGF ligands, including FGF-1, 2, 3, 4, 5, 6, 10, 19, 20, 21, and 22, are known to bind specifically to FGFR1. In addition, due to the activation of FGFR1, signaling molecules downstream of FGFR1, such as AKT and ERK, are activated [2,5]. The mRNA expression levels of these 11 FGFs in NCI-H2228 parental cells were analyzed, and the results showed that only the mRNA of FGF2 was expressed at more than one transcript per million (TPM) (Table 5).

[0250] [Table 5] B

[0251] Therefore, the expression of FGFR1 and FGF2 was studied. The phosphorylation of FGFR1 and the amount of FGF2 protein increased after 13 days of alectinib treatment ( Figure 3 A, B), but the amount of FGFR1 protein remained basically unchanged ( Figure 3 A).

[0252] Furthermore, although ALK phosphorylation was completely inhibited by alectinib treatment, the phosphorylation of STAT3, AKT, and ERK increased after 13 days of alectinib treatment ( Figure 3 A). DTP cells are known to exhibit morphological changes, stemness, or epithelial-mesenchymal transition (EMT) including increased vimentin (VIM) and decreased cadherin 1 (CDH1) [1,6], and even alectinib-treated DTP cells showed morphological changes ( Figure 4 ), an increase in stem cell markers involving CD44 and CD133 proteins, and EMT accompanied by an increase in VIM protein and a decrease in CDH1 protein ( Figure 3 A).

[0253] Example 2

[0254] To verify whether FGFR1 signaling activation caused by increased FGF2 protein expression contributes to the cell proliferation inhibitory effect caused by ALK inhibitor treatment, gene-specific knockdown of FGFR1 and FGF2 was performed using NCI-H2228 cells and low-molecular-weight interfering RNA (siRNA).

[0255] (1) Knockdown of FGFR1 or FGF2 using siRNA

[0256] Using NEPA21 electroporation (Nepa Gene, Japan), siRNA targeting FGFR1, FGF2 or control (Horizon Discovery) was introduced into cells, and the next day, the prescribed concentration of the drug was added and cultured for 6 days. The knockdown of FGFR1 or FGF2 was confirmed by Western blotting after 2 days of siRNA introduction, and the results confirmed the disappearance of FGFR1 and FGF2 (Figure 5A).

[0257] FGFR1 or FGF2 knockout cells showed increased sensitivity to alectinib compared with control cells. 40 47.0 nM and 14.7 nM for FGFR1 knockout cells, 41.5 nM and 14.9 nM for FGF2 knockout cells, and >1000 nM and 246.4 nM for control cells ( FIG. 5B ).

[0258] (2) Establishment of FGFR1 and FGF2 knockout cells using CRISPR / Cas9 system

[0259] Using NEPA21 electroporation, crRNA and Cas9 protein (NipponGene) targeting FGFR1 or FGF2 (Fasmac) were introduced into the cells, and single clones of each clonal cell were isolated by Smart Aliquotor (NT Science, Aichi, Japan), with 2 clones for each clone, and the disappearance of FGFR1 or FGF2 protein in FGFR1 or FGF2 knockout cells was confirmed by the aforementioned western blot analysis ( Figure 5C ).

[0260] Increased sensitivity to alectinib in FGFR1 or FGF2 knockout cells compared with parental cells. 50 For FGFR1 knockout cells, it was 5.4 nM and 7.3 nM, for FGF2 knockout cells, it was 5.6 nM and 6.1 nM, and for parental cells, it was 219.6 nM (Figure 5D). In addition, for the sensitivity caused by treatment with lorlatinib, which is another ALK inhibitor, the IC 50 For FGFR1 knockout cells, it was 0.7 nM, 0.7 nM, for FGF2 knockout cells, it was 0.7 nM, 0.9 nM, and for parental cells, it was >1000 nM (Figure 5E).

[0261] The FGFR1 or FGF2 knockout cells obtained above were inoculated in 384-well plates, and the agents were added at the indicated concentrations the next day and cultured for the indicated time. According to the manufacturer's protocol, the Caspase-Glo 3 / 7 assay (Promega) was used to measure the activity of caspase 3 and 7 induced by signal transduction apoptosis in cells. The activity was corrected according to the cell viability determined as described above, and then the relative caspase 3 and 7 activity of the agent relative to the medium was calculated. Each point represents the mean + standard deviation of 3 experiments. As a result, it was observed that in each FGFR1 or FGF2 knockout cell, the induced apoptosis induction caused by alectinib treatment was significantly increased compared with the parental cells ( Figure 7 B) and enhanced ERK inhibition (Figure 5F, 5G).

[0262] These insights suggest that cell survival relative to ALK inhibitors is activated through FGFR1 signaling in FGFR1-high-expressing and FGF2-high-expressing ALK fusion gene-positive NSCLC cells, and that both FGFR1 and FGF2 protein molecules are important in escaping cell death due to cell proliferation inhibition caused by ALK inhibitor treatment in ALK fusion gene-positive NSCLC cells.

[0263] Example 3

[0264] In SNU2535 parental cells, the expression levels of FGFR1 and FGF2 proteins were decreased compared with those in NCI-H2228 parental cells ( Figure 6 ).

[0265] To further confirm that both FGFR1 and FGF2 are essential for cell survival under ALK inhibitor treatment, we overexpressed FGFR1 and FGF2 in SNU2535 cells, a non-small cell lung cancer (NSCLC) cell line positive for ALK fusion gene. Figure 7 B).

[0266] The human coding sequence (CDS) of FGFR1, FGF2 or control was synthesized and cloned downstream of the EF1A promoter in the lentiviral vector using VectorBuilder (Chicago, Illinois, USA), and SNU-2535 cells were infected with the lentiviral vector. One day later, the culture medium was cultured in a medium containing puromycin for 4 days, and FGFR1 and FGF2 overexpressing cells were selected and clones were isolated using Smart Aliquotor. FGFR1 or FGF2 protein expression was confirmed by Western blot analysis as described above.

[0267] Compared with control cells or FGF2-overexpressing cells, alectinib-treated cells showed reduced inhibitory effects on cell proliferation and ERK phosphorylation in FGFR1- and FGF2-overexpressing cells ( Figure 7 A, B).

[0268] These results suggest that in FGFR1- and FGF2-high-expressing ALK fusion gene-positive NSCLC cells, FGFR1 signaling is activated through the expression of both FGFR1 and FGF2 proteins, thereby avoiding the cell proliferation inhibition caused by ALK inhibitor treatment.

[0269] Example 4

[0270] Using alectinib-resistant NCI-H2228 cells, we investigated whether FGFR1 signaling was activated even after alectinib resistance.

[0271] NCI-H2228 cells were exposed to 1000 nM alectinib for 348 days and cultured in alectinib-free medium for more than 60 days to establish drug-resistant cells.

[0272] The above cells were seeded in 384-well plates, treated with ALK inhibitors (alectinib or lorlatinib (Selleck Chemicals LLC)) and FGFR inhibitors (infigratinib (BGJ) (LC laboratories)) for 8 days, and cell proliferation assays were performed according to the description of Example 1.

[0273] As a result, the IC of alectinib 50 The value was >1000 nM in the resistant cells, whereas it was 219.6 nM in the parental cells. 50 The value was 4.6 times that of the parental cells (Table 6, Figure 8 ).

[0274] [Table 6]

[0275] A

[0276]

[0277] In the resistant cells, there was no ALK resistance mutation and increased copy number of FGFR1 and FGF2 genes, but an increase in FGFR1 and FGF2 protein was observed (Table 7, Fig. 9 ).

[0278] [Table 7]

[0279]

[0280] In addition, in drug-resistant cells, combined treatment with BGJ398 and alectinib strongly inhibited cell proliferation and reduced ERK phosphorylation. 50 The value was >1000 nM when alectinib was used alone, whereas it was 38.8 nM when used in combination (Table 8, Fig.10 ).

[0281] [Table 8]

[0282]

[0283] The above results suggest that in drug-resistant cells, FGFR1 signaling may be activated due to the increased expression of FGFR1 and FGF2 proteins.

[0284] Example 5

[0285] The combined effects of ALK inhibitors and FGFR inhibitors were studied in NCI-H2228 cells with high FGFR1 and FGF2 expression and SNU-2535 cells with low FGFR1 and FGF2 expression and ALK fusion gene.

[0286] The results are shown in Fig.11 , 12 It should be noted that Fig.11 In the figure, the horizontal axis and the vertical axis respectively show the concentration of each drug and the cell survival rate.

[0287] NCI-H2228 cells showed no sensitivity to FGFR inhibitors alone (BGJ398 and AZD4547) (IC 50 Value>1000nM), but inhibition of cell proliferation was observed by the combination of ALK inhibitor and FGFR inhibitor ( Fig.11 A, B). Furthermore, in NCI-H2228 cells, the combined use of alectinib and BGJ398 showed an inhibitory effect on the phosphorylation of AKT and ERK compared with each single agent ( Fig.12 ).

[0288] On the other hand, in SNU-2535 cells, no effect of the combination of ALK inhibitor and FGFR inhibitor was observed ( Fig.11 C,D).

[0289] Therefore, it was shown that in ALK fusion gene-positive NSCLC cells with high FGFR1 expression and high FGF2 expression, FGF2 binds to FGFR1 and causes FGFR1 signaling activation, but by using ALK inhibitors together with FGFR inhibitors, both ALK and FGFR1 signals are inhibited, thereby observing a strong proliferation inhibitory effect.

[0290] Example 6

[0291] In NCI-H2228 cells, alectinib treatment-induced apoptosis induction was enhanced by co-administration with FGFR inhibitors ( Fig.13 A). On the other hand, in SNU-2535 cells, no effect of combined use of FGFR inhibitors was observed ( Fig.13 B).

[0292] Example 7

[0293] To evaluate the in vivo efficacy of the combined treatment of a FGFR inhibitor and an ALK inhibitor, nu / nu nude mice bearing subcutaneous xenografts of NCI-H2228 cells were used to administer BGJ398 and alectinib alone or in combination.

[0294] Male 4- to 5-week-old BALB / c-nu / nu mice (CAnN Cg-Foxn1<nu> / CrlCrlj nu / nu) were purchased from Charles River Laboratories Japan (Yokohama, Japan).

[0295] Each mouse was inoculated subcutaneously with 5 × 10 6 Tumor volume and body weight were measured twice a week, and tumor volume was estimated as follows.

[0296] Tumor volume = ab 2 / 2 (a and b represent the length and width of the tumor respectively)

[0297] After tumor establishment, mice were randomly assigned to a solvent administration group, a drug administration group, or a combined administration group in which each drug was administered at the same dose / same schedule as a single dose, and the drugs were orally administered for 11 consecutive days.

[0298] No tumor growth inhibition was observed in NCI-H2228 tumors when BGJ398 was used alone, but a significantly stronger tumor growth inhibition effect was observed when BGJ398 was used in combination with alectinib compared with alectinib alone ( Fig.14 A, C, D) and decreased ERK phosphorylation ( Fig.14 B).

[0299] Furthermore, the combined use was well tolerated, with no weight loss observed during treatment ( Fig.15 ).

[0300] In addition, the effects of long-term exposure of BGJ398 and alectinib alone or in combination were evaluated in NCI-H2228 cells in vitro. The treatment period of the drugs is as described in Table 9.

[0301] [Table 9]

[0302]

[0303] like Fig.16 As shown, a strong cell proliferation inhibitory effect was observed in the 5-week combined use group (treatment No. 7) compared with the groups treated with each drug alternately (treatment No. 4 and 6). In addition, compared with the group treated with alectinib for 4 weeks after only 1 week of combined use (treatment No. 5), the cell proliferation inhibitory effect of treatment No. 7 was roughly the same until 5 weeks, but the proliferation inhibitory effect continued after switching to drug-free medium from the 6th week. Since treatment No. 5 was switched to drug-free medium from the 6th week, cell re-growth was observed ( Fig.16 ).

[0304] These results suggest that in order to strongly inhibit the cell growth of ALK fusion gene-positive NSCLC cells that highly express FGFR1 and FGF2, it is useful to continue the combined use of an ALK inhibitor and a FGFR inhibitor for as long as possible.

[0305] Example 8

[0306] Using samples from an open-label randomized phase III trial (J-ALEX clinical trial: JO28928), the mRNA expression levels of FGFR1 and FGF2 were measured and their correlation with the hazard ratio was analyzed. The open-label randomized phase III trial compared the efficacy and safety of crizotinib and alectinib in patients with ALK fusion gene-positive progressive and recurrent non-small cell lung cancer.

[0307] Tumor samples collected before the start of treatment with alectinib (n = 38) and crizotinib (n = 31) were used to measure the expression of FGFR1 or FGF2 mRNA, and various cutoff values ​​(subgroups) were set in each expression level to calculate the estimated hazard ratio (HR) of alectinib and crizotinib. The adjusted hazard ratio (HR) and 95% confidence interval of PFS were estimated using a multivariate Cox proportional hazard model. In each subgroup, drugs considered to be associated with disease progression (PD) or death, ECOGPerformance Status (PS) at baseline, treatment at the start of treatment, disease status, and brain metastases at baseline caused by IRF were set as covariates for adjusted analysis (Table 10).

[0308] [Table 10]

[0309] A

[0310] Covariate parameters variable FGFR1 or FGF2 mRNA High / Low Treatment options Alectinib / crizotinib ECOGPS 0 / 1 Processing line 1 / 2 Disease status Stage IIB or IV / recurrence after surgery Brain metastases at baseline due to IRF whether

[0311] The distribution of PFS relative to specific cutoff values ​​was estimated using the Kaplan-Meier method. These analyses were performed using RStudio version 1.0153 and R version 3.6.0. The number of patients and PFS events in each subgroup are shown in Table 11.

[0312] [Table 11]

[0313] C

[0314]

[0315] It should be explained that Fig.17 , 18 In the figure, the horizontal axis and the vertical axis show the respective mRNA expression levels and risk ratios.

[0316] As a result, it was observed that the hazard ratios for both FGFR1 and FGF2 mRNA expression levels tended to increase as the mRNA levels increased ( Fig.17 , 18 ).

[0317] In particular, in the cases with high FGF2 mRNA expression corresponding to subgroups 18 to 26, the logHR value was 1.0 or more, and the hazard ratio was the highest ( Fig.18 In addition, the expression of FGFR1 mRNA was relatively higher in patients with high FGF2 mRNA expression compared with patients with low FGF2 mRNA expression ( Fig.19 ).

[0318] As described above, even in clinical trials, the effectiveness of ALK inhibitors in one treatment was short for ALK fusion gene-positive NSCLC patients with high FGFR1 mRNA expression and high FGF2 mRNA expression, which is consistent with the results of the above non-clinical trials.

[0319] Investigation

[0320] Molecular targeted therapy is now the standard of care, but despite dramatic effects observed for many cancers with activated oncogenes, these treatments rarely provide complete cures. In ALK fusion gene-positive NSCLC, complete responses are observed in less than 5% of patients given ALK inhibitors [7]. In nonclinical trials, DTP cells based on treatment with molecular targeted agents have been identified, but the mechanism of induction of DTP is not yet fully understood [1].

[0321] The present application shows that DTP cells treated with alectinib based on FGFR1 high-expressing and FGF2 high-expressing cells undergo cell proliferation dependent on FGFR signals. Further, the cell proliferation inhibitory effect of alectinib and FGFR inhibitors in combination was not observed in FGFR1 low-expressing and FGF2 low-expressing cells, but was observed in FGFR1 high-expressing and FGF2 high-expressing cells.

[0322] In addition, the data obtained through clinical trials were analyzed in this application, and the results showed that the majority of patients with FGF2 high expression and ALK mutation, which is equivalent to about 20% of the total, had relatively higher FGFR1 expression levels, and among these FGF2 high expression patients, the PFS of ALK inhibitor treatment was shorter than that of patients with FGF2 low expression.

[0323] The above shows that the present application further clarifies that in ALK fusion gene-positive cancer, high expression of both FGFR1 and FGF2 is involved in the acquisition of resistance to ALK inhibitors. Therefore, by performing a combined therapy of ALK inhibitors and FGFR inhibitors before the acquisition of resistance rather than after the acquisition of resistance, that is, in actual clinical practice, in one treatment, a powerful treatment that can improve the prognosis of lung cancer patients with ALK fusion gene-positive cancer can be achieved without causing the acquisition of resistance caused by ALK inhibitors.

[0324] (References)

[0325] 1. Mikubo, M., et al., Mechanism of Drug Tolerant Persister Cancer Cells: The Landscape and Clinical Implication for Therapy. J Thorac Oncol, 2021.16(11):p.1798-1809.

[0326] 2. Ornitz, DM and N. Itoh, The Fibroblast Growth Factor signaling pathway. Wiley Interdiscip Rev Dev Biol, 2015.4(3):p.215-66.

[0327] 3. Guagnano, V., et al., FGFR Genetic Alterations Predict for Sensitivity to NVP-BGJ398, a Selective Pan-FGFR Inhibitor. Cancer Discovery, 2012.2(12):p.1118-1133.

[0328] 4.Menu,E.,et al.,Inhibiting the IGF-1receptor tyrosine kinase withthe cyclolignan PPP:an in vitro and in vivo study in the 5T33MM mousemodel.Blood,2006.107(2):p.655-60.

[0329] 5.Tiong,K.H.,L.Y.Mah,and C.-O.Leong,Functional roles of fibroblastgrowth factor receptors(FGFRs)signaling in human cancers.Apoptosis:aninternationaljournal on programmed cell death,2013.18(12):p.1447-1468.

[0330] 6.Hangauer,M.J.,et al.,Drug-tolerant persister cancer cells arevulnerable to GPX4 inhibition.Nature,2017.551(7679):p.247-250.

[0331] 30.Takezawa,K.,et al.,Role of ERK-BIM and STAT3-Survivin SignalingPathways in ALK Inhibitor-Induced Apoptosis in EML4-ALK-Positive LungCancer.Clinical Cancer Research,2011.17(8):p.2140-2148.

[0332] 7. Camidge, DR, et al., Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non-Small Cell Lung Cancer in the Global Phase III ALEXStudy. Journal of Thoracic Oncology, 2019.14(7):p.1233-1243.

[0333] Industrial Applicability

[0334] The combined drug of the present invention can be used for the prevention or treatment of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive. In addition, the combined drug of the present invention can be used as an inhibitor of drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive. In addition, the present invention can provide a method for screening patients with ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive.

Claims

1. A drug for treating or preventing ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising a combination of an ALK inhibitor and a FGFR inhibitor.

2. A drug for inhibiting drug resistance in ALK fusion gene-positive cancers that are both FGFR1-positive and FGF2-positive, comprising a combination of an ALK inhibitor and a FGFR inhibitor.

3. A drug for treating or preventing ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising an ALK inhibitor as an active ingredient and used in combination with a FGFR inhibitor.

4. A drug for inhibiting drug resistance of ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising an ALK inhibitor as an active ingredient and used in combination with a FGFR inhibitor.

5. A drug for treating or preventing ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising a FGFR inhibitor as an active ingredient and used in combination with an ALK inhibitor.

6. A drug for inhibiting drug resistance of ALK fusion gene-positive cancer that is FGFR1-positive and FGF2-positive, comprising a FGFR inhibitor as an active ingredient and used in combination with an ALK inhibitor.

7. The drug according to any one of claims 1 to 6, wherein The ALK inhibitor is a compound or a salt thereof selected from crizotinib, alectinib, ceritinib, lorlatinib and brigatinib.

8. The drug according to any one of claims 1 to 6, wherein The FGFR inhibitor is a compound or a salt thereof selected from infigratinib, delazentinib, pemitinib, erdatinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074.

9. The drug according to any one of claims 1 to 6, wherein The ALK fusion gene-positive cancer is a cancer selected from the group consisting of non-Hodgkin's lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, renal cancer and lung cancer.

10. The drug according to any one of claims 1 to 6, wherein The ALK fusion gene-positive cancer has not received prior treatment with an ALK inhibitor.

11. The drug according to any one of claims 1 to 6, wherein The ALK fusion gene-positive cancer is an ALK-sensitive cancer.

12. The drug according to any one of claims 1 to 6, wherein The ALK inhibitor is administered concurrently with the FGFR inhibitor.

13. A method for screening a patient with ALK fusion gene-positive cancer that is both FGFR1-positive and FGF2-positive, comprising: Tumor tissues of patients diagnosed with ALK fusion gene-positive cancer are obtained, and the expression levels of FGFR1 and FGF2 in the tumor tissue samples are measured.

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