AU2017338564B2

AU2017338564B2 - Pharmaceutical combinations of histone deacetylase inhibitor and proteasome inhibitor or immunomodulatory drug for the treatment of hematological cancer

Info

Publication number: AU2017338564B2
Application number: AU2017338564A
Authority: AU
Inventors: Soo Jin Kim
Original assignee: Chong Kun Dang Corp
Current assignee: Chong Kun Dang Corp
Priority date: 2016-10-04
Filing date: 2017-09-29
Publication date: 2020-07-09
Anticipated expiration: 2037-09-29
Also published as: EP3522891A4; JP2019529562A; EP3522891A1; MX390025B; KR102002581B1; ZA201902340B; AU2017338564A1; RU2721409C1; WO2018066946A1; JP2021020953A; KR20180037507A; BR112019006633A2; CN109789138A; MX2019003907A; CA3038019A1

Abstract

The present invention relates to a pharmaceutical combination for treating a hematological cancer comprising the histone deacetylase (HDAC) inhibitor of the chemical formula 1, a proteasome inhibitor or an immunomodulatory drug and a steroidal anti-cancer agent together.The pharmaceutical combination of the present invention can be useful for treating a hematological cancer such as multiple myeloma, by reducing toxicity which is the problem of the conventional HDAC inhibitor and exhibiting an equivalent level of pharmaceutical effects due to a complex inhibitory mechanism against the cancer of the compound of chemical formula 1 and its pharmaceutically acceptable salt, the proteasome inhibitor or the immunomodulatory drug and the steroidal anti-cancer agent.

Description

Description Title of Invention: PHARMACEUTICAL COMBINATIONS OF HISTONE DEACETYLASE INHIBITOR AND PROTEASOME INHIBITOR OR IMMUNOMODULATORY DRUG FOR THE TREATMENT OF HEMATOLOGICAL CANCER Technical Field

[1] The present invention relates to a pharmaceutical combination for treating a hema tological cancer, more specifically, multiple myeloma, particularly, a hematological cancer which is resistant to conventional therapies, and more specifically, relates to a pharmaceutical combination for treating myeloma comprising a histone deacetylase (HDAC) inhibitor, a proteasome inhibitor or an immunomodulatory drug and a steroidal anti-cancer agent together. Background Art

[2] Myeloma of hematological cancers refers to a tumor consisting of cells of the type usually found in bone marrow. Furthermore, multiple myeloma (MM) means dis seminated malignant neoplasm of plasma cells characterized by secretion of multiple myeloid tumor lesions and M component (monoclonal immunoglobulin fragment), related to widespread osteolytic lesions that lead to bone pain, pathological fractures, hypercalcemia and normocytic normochromic anemia. Multiple myeloma cannot be treated by conventional, high-dose chemotherapies.

[3] On the other hand, acetylation status of nucleosomal histones plays an important role in the regulation of gene expression. Deacetylation of nucleosomal histones is fa cilitated by a group of enzymes known as histone deacetylases (HDACs) with 11 known isoforms. Histone deacetylation induces chromatin condensation leading to transcriptional repression, while acetylation allows approaching to transcriptional machinery which makes transcription easy by inducing localized relaxation within specific chromosomal regions. In tumor cells, selective inhibitors of HDAC enzymes lead to histone hyperacetylation. It is known to alter transcriptional regulation of a subset of genes, including many tumor suppressor genes involved in cell cycle control, cell division and apoptosis. In addition, HDAC inhibitors have been reported to inhibit tumor growth in vivo. Inhibition of tumor growth is accompanied by hyperacetylation of histone and tubulin and may include a number of mechanisms. HDAC inhibitors block cancer cell proliferation in both in vitro and in vivo. As described above, it has been known that HDAC inhibitors effectively inhibit cancer growth in multiple myeloma, but in case of drugs developed in the past, there is a problem that their use is limited due to severe toxicity.

[4] On the other hand, proteasome inhibitors and immunomodulatory drugs are

well known as the main agents for treating multiple myeloma.

[5] Meanwhile, immunomoduratory (IMiD) class of drugs exhibit significant

anti-myeloma characteristics in various multiple myeloma models and have been

shown to have significant clinical activity in patients with multiple myeloma.

Among these, bortezomib (also known as Velcade or PS-341) has been reported

to have activity in NSCLS cell lines as an FDA-approved small molecule

proteasome inhibitor in treatment of refractory multiple myeloma, where it was

found to induce concentration and time-dependent G2/M cell cycle arrest.

[6] Due to various problems of these therapies including toxicity, there is a strong

need for a combination and a method that provide alternatives for treating

hematological cancer.

[6A] Any discussion of documents, acts, materials, devices, articles or the like

which has been included in the present specification is solely for the purpose of

providing a context for the present invention. It is not to be taken as an

admission that any or all of these matters form part of the prior art base or were

common general knowledge in the field relevant to the present invention as it

existed before the priority date of each claim of this application.

[6B] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a

stated element, integer or step, or group of elements, integers or steps, but not

the exclusion of any other element, integer or step, or group of elements,

integers or steps.

Summary

[7] Under these circumstances, the present inventors have conducted intensive

studies on a more effective and less toxic hematological cancer drug, specifically

multiple myeloma. As a result, the present disclosure has been completed by

confirming that in case of combining the compound of chemical formula 1 as a

HDAC inhibitor and a proteasome inhibitor or an immunomodulatory drug and a

steroidal anti-cancer agent, it is possible to obtain synergistic anti-cancer effect

with reduced toxicity and comparable pharmaceutical effect to other HDAC

2a

inhibitors in multiple myeloma, which has been difficult to effectively treat due to various problems such as toxicity, etc. in the past.

[8] Thus, the purpose of the present disclosure, in one aspect, is to provide a combination for treating hematological cancer comprising the compound of the following chemical formula 1 as a histone deacetylase inhibitor, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti-cancer agent.

[8A] Accordingly, in one aspect, there is provided a combination when used for treating a hematological cancer, the combination comprising: a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti-cancer agent:

[chemical formula 1]

N |"

NN'OH H oA N ONN KNI wherein the compound of chemical formula 1 or its pharmaceutically acceptable salt, the proteasome inhibitor or the immunomodulatory drug, and the steroidal anti-cancer agent are administered simultaneously or sequentially.

[8B] In another aspect, there is provided a method for treating a hematological cancer in a subject, comprising administering to the subject a combination comprising a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti-cancer agent:

[chemical formula 1]

2b

WOH H 04-N N

[8C] In another aspect, there is provided a use of a combination comprising a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti-cancer agent, in the manufacture of a medicament for treating a hematological cancer:

[chemical formula 1]

N |" N 0 /-_ _OH H oAN N

L9] As one embodiment, the present invention relates to a pharmaceutical combination for treating a hematological cancer comprising,

[10] 1) a hydroxamic acid derivative represented by the following chemical formula 1, N-(7-(hydroxyamino)-7-oxoheptyl)-4-methyl-N-(4-(1-methyl-lH indazol-6-yl)phenyl)piperazine-1-carboxamide methanesulfonate or its pharmaceutically acceptable salt as the first active ingredient;

[11] 2) a proteasome inhibitor or an immunomodulatory drug as the second active ingredient; and

[12] 3) a steroidal anti-cancer agent as the third active ingredient. Preferably, the hema tological cancer is multiple myeloma.

[13] [Chemical formula 1]

[14]

N | NO H

OH 11 0 Advantageous Effects of Invention

[15] The improved efficacy of the combination according to the present invention can be demonstrated by measuring the therapeutic synergistic effect. The combination of the present invention exhibits a synergistic effect when the first active ingredient, which is a HDAC inhibitor, is administered in combination with the second active ingredient and the third active ingredient, and thus has excellent anti-cancer activity against a hematological cancer, particularly multiple myeloma. Brief Description of Drawings

[16] FIG. 1a is a graph of cancer growth of the control group and each experimental group in MM.Is xenograft mouse model.

[17] FIG. lb shows the tumor size change by individual of each experimental group on the last day of the experiment (day 57) in MM.Is xenograft mouse model.

[18] FIG. 2a is a graph of cancer growth of the control group and each experimental group in MM.Is xenograft mouse model.

[19] FIG. 2b shows the tumor size change by individual of each experimental group on the last day of the experiment (day 70) in MM.Is xenograft mouse model. Best Mode for Carrying out the Invention

[20] Hereinafter, the present invention will be described in more detail.

[21] The first active ingredient of the pharmaceutical combination according to the present invention is the compound of the above chemical formula 1, and this compound has activity as a histone deacetylase (HDAC). The description and preparation method of the compound of the above chemical formula 1 are described in detail in international patent publication No. WO 2010-110545, which is incorporated herein by reference. In the present invention, the compound of the above chemical formula 1 can be prepared by the preparation method disclosed in the above document. Generally, it is known that the compound of the above chemical formula 1 ad ministered to humans varies in dosage depending on various administration factors including individual specific factors of patients, but for example, it can be administered in 10 to 210 mg/M 2

.

[22] On the other hand, the second active ingredient of the pharmaceutical combination according to the present invention is a proteasome inhibitor or an immunomodulatory drug, and the proteasome inhibitor is an agent inhibiting activity of a proteasome (i.e. a cell complex degrading a protein) degrading a cell division regulating protein reversely, and it is based on the fact that the normal cells regenerate cell division ability even if the activity of the proteasome is temporarily inhibited, but various cancer cells enter the death process as soon as the proteasome activity is temporarily inhibited. The proteasome inhibitor that may be comprised in the pharmaceutical com bination of the present invention may be various classes of known proteasome in hibitors. For example, as a class of peptide boronate, there is bortezomib (INN, PS0341; Velcade) which is a compound approved in the United States for treating relapsed multiple myeloma. There is CEP-18770, as another peptide boronate. As other classes of proteasome inhibitors, there are peptide aldehyde (for example, MG132), peptide vinyl sulfone, peptide epoxyketone (for example, epoxomicin, carfilzomib), lactone inhibitor (for example, lactocystine, MLN 519, NPI-0052, salinosporamide A), a compound which produces dithiocarbamate complex with a metal (for example, disulfiram which is also used in treatment of chronic alcoholism), and specific an tioxidant (for example, epigallocatechin-3-gallate, catechin-3-galate, and sali nosporamide A. The proteasome inhibitor comprised in the pharmaceutical com bination of the present invention is preferably, bortezomib represented by the following chemical formula 2 or its pharmaceutically acceptable salt. The bortezomib can be administered intravenously or subcutaneously in a dose of 0.3-1.5 mg/m 2 , 1 to 2 times per week, depending on the administration schedule, but is not limited thereto.

[23] [Chemical formula 2]

[24]

N 0 NN N/N N 0

0

[25] Meanwhile, the immunomodulatory drug has effects such as expansion of immune cells and promotion of tumor cell death, etc. and specifically, a substance which regulates immunity by improving ADCC (antibody-dependent cell-mediated cyto toxicity). Specifically, the immunomodulatory drug comprised in the pharmaceutical combination of the present invention may be thalidomide lenalidomide, for malidomide, or a pharmaceutically acceptable salt, and preferably, lenalidomide rep resented by the following chemical formula 3 or its pharmaceutically acceptable salt. Lenalidomide is generally orally administered in a dose of 10 mg to 30 mg depending on the administration schedule, but is not limited thereto.

[26] [Chemical formula 3]

[27]

[28]

[29] The third active ingredient of the pharmaceutical combination according to the present invention is a steroidal anti-cancer agent, and for example, may be prednisone or dexamethasone, but is not limited thereto. Preferably, dexamethasone represented by the following chemical formula 4 that is, (8S, 9R, 10S, 11S, 13S, 14S, 16R, 17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11 ,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene- 3-on or isotope variant; or its pharmaceutically acceptable salt, hydrate, or solvate. In one em bodiment, dexamethasone is (8S, 9R, lOS, 11S, 13S, 14S, 16R, 17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11 ,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-on. In other em bodiment, dexamethasone is (8S, 9R, 10S, 11S, 13S, 14S, 16R,

17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11 ,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-3-on, wherein hydrogen atom is deuterated.

[30] [Chemical formula 4]

[31] 0

I H-m F H o A

[32] In the present invention, the pharmaceutically acceptable salt means a salt commonly used in the medical industry, and for example, there are inorganic ionic salts prepared with calcium, potassium, sodium and magnesium, etc., inorganic acid salts prepared with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid, etc., organic acid salts prepared with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc., amino acid salts prepared with methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, p-toluene sulfonic acid and naphthalene sulfonic acid, etc., and amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the kinds of salts in the present invention are not limited by these listed salts.

[33] In one specific embodiment, the first active ingredient, the second active ingredient, and the third active ingredient may be comprised per 100 parts by weight of the total pharmaceutical combination. When a proteasome inhibitor such as bortezomib is used as the second active ingredient, it is composed of 28.55 to 85.60 parts by weight of the first active ingredient, 1.28 to 8.63 parts by weight of the second active ingredient and 2.71 to 69.55 parts by weight of the third active ingredient. When an im munomodulatory drug such as lenalidomide is used as the second active ingredient, it is composed of 19.54 to 94.69 parts by weight of the first active ingredient, 2.46 to 52.63 parts by weight of the second active ingredient and 2.52 to 59.70 parts by weight of the third active ingredient.

[34] The pharmaceutical combination may comprise 3 kinds of separate formulations, and may consist of two or one formulation.

[35] The combination of the present invention may be administered orally or parenterally (For example, intravenously, subcutaneously, intraperitoneally or topically). In the present invention, the first active ingredient and the second active ingredient may be administered orally or parenterally, and preferably, may be administered parenterally. In addition, the third active ingredient may be parenterally or orally administered, preferably administered orally.

[36] In the combination of the present invention, the range of appropriate dose of the first active ingredient to the third active ingredient varies according to the body weight, age, sex, health condition of patient, diet, administration time, administration method, excretion rate, and severity of disease, etc. The daily dose of the first active ingredient is approximately 10 ~ 80 mg/kg, preferably 20 to 60 mg/kg. In addition, the daily dose of the second active ingredient of the present invention is approximately 0.1 - 5 mg/ kg, preferably 0.5 to 2 mg/kg.

[37] In the combination of the present invention, the appropriate administration period of the first active ingredient and the second active ingredient and the third active in gredient may be determined according to the dose. For example, in case that the second active ingredient of the present invention is a proteasome inhibitor, the first active in gredient may be administered once a day to once every two weeks, preferably once a week. In addition, the second active ingredient of the present invention may be ad ministered once a day to once a week, preferably twice a week. The third active in gredient of the present invention may be administered once a day to once a week, preferably 4 times a week. In addition, in case that the second active ingredient of the present invention is an immunomodulatory drug, the first active ingredient of the present invention may be administered once a day to once every two weeks, preferably once a week, and the second active ingredient may be administered once a day to once a week, preferably once a day, and the third active ingredient may be administered once a day to once a week, preferable once a week.

[38] The pharmaceutical combination of the present invention confirmed the combination effect of the first active ingredient, which is a HDAC inhibitor, the second active in gredient, which is a proteasome inhibitor or an immunomodulatory drug, and the third active ingredient, which is a steroidal anti-cancer agent, thereby exhibiting excellent anti-cancer activity.

[39] Therefore, the anti-cancer combination of the present invention can by usefully used as an anti-cancer treatment strategy, and it is preferable particularly for treating a hematological cancer. The hematological cancer may preferably be multiple myeloma.

[40] In the present invention, the first active ingredient, the second active ingredient, and the third active ingredient comprised in the combination of the present invention may be comprised in separate pharmaceutical composition respectively, and the pharma- ceutical composition may be prepared in a unit dose form or prepared by introducing in a multi-dose container by formulating it using a pharmaceutically acceptable carrier, according to a method which can be easily carried out by a person having ordinary skill in the art.

[41] The pharmaceutically acceptable carrier is conventionally used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc., but not limited thereto. The pharmaceutical combination of the present invention may comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. in addition to the above components. The ap propriate pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[42] As another embodiment, the present invention provides a method for treating a cancer comprising administering the pharmaceutical combination into an individual who needs it. In the present invention, the "individual" includes mammals, particularly humans. Mode for the Invention

[43] Hereinafter, the configuration and effect of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[44] <Example 1: Confirmation of anti-cancer effect of the combination of the present invention using bortezomib as the second active ingredient, in MM.Is xenograft model>

[45] 1-1)Experimental method

[46] a.Preparation of animal tumor model

[47] MM.Is which is a human multiple myeloma cell line was purchased from ATCC (USA). MM.Is cell line was maintained with RPMI1640 (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).

[48] After MM.Is cells which were mixed with matrigel were subcutaneously ad ministered into NOD.CB17-Prkdcscid/NCrHsd male mouse (2x10 7 cell/head) to organize groups to uniformly distribute the tumor size and then used in the experiment.

[49] b.Preparation of active ingredients

[50] The compound of the chemical formula 1 was prepared in the concentration of 4 mg/ ml by dissolving with physiological saline solution.

[51] Bortezomib was prepared in the concentration of 0.005 mg/ml by dissolving with physiological saline solution.

[52] Dexamethasone was prepared in the concentration of 0.05 mg/ml by diluting 5 mg/ mL original solution with physiological saline solution.

[53] The using solution was prepared on the day of use, and the remaining solution was discarded.

[54] c.Confirmation of anti-tumor activity

[55] The anti-cancer effect of the compound of the chemical formula 1 and bortezomib and dexamethasone was evaluated in MM.Is xenograft model. The experimental groups were divided to the following different administration groups.

[56] - control group : vehicle

[57] - Chemical formula 1 : the compound of the chemical formula 1 (40 mg/kg, once a week, injection)

[58] - Combination group of bortezomib and dexamethasone : bortezomib (0.05 mg/kg, twice a week, injection), dexamethasone (0.5 mg/kg, 4 times a week, injection)

[59] - Combination group of chemincal formula 1, bortezomib and dexamethasone : the compound of the chemical formula 1 (40 mg/kg, once a week, injection), bortezomib (0.05 mg/kg, twice a week, injection), dexamethasone (0.5 mg/kg, 4 times a week, injection)

[60] All experimental groups were administered intraperitoneally for 57 days. The toxicity was monitored by measuring mouse weight, and the tumor growth was measured by using caliper during the experiment. The tumor volume was calculated according to the following mathematical equation 1.

[61] [Mathematical equation 1]

[62] tumor volume = (length x width 2) / 2

[63] d.Statistical analysis

[64] For all the results of the present experiment, the comparison between experimental groups was performed by assuming normality of the data through parametric multiple comparison or nonparametric multiple comparison, and statistical analysis was performed by using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA), and the case where p value was below 0.05 was considered statistically significant.

[65] 1-2)Experimental result

[66] In order to evaluate anti-tumor effect by coadministration of the compound of the chemical formula 1 and bortezomib, dexamethasone, each substance was administered into xenograft mouse, and the weight and tumor growth were monitored for 57 days.

[67] In an aspect of tumor growth inhibiting effect, comparing the tumor sizes of group administering the compound of the chemical formula 1 and coadministration group of bortezomib and dexamethasone, and coadministration group of the compound of the chemical formula 1+bortezomib+dexamethasone to a group administering an excipient during administration period, the tumor growth was highly inhibited.

[68] In the coadministration group of bortezomib and dexamethasone, there was a slight decrease in the size, but the number of mice reaching a complete remission was only 1, and most of the mice had an effect of reducing the cancer less than 50%. While, in the coadministration group of the compound of the chemical formula 1+bortezomib+dexamethasone, the complete remission was observed in 3 mice, and most of the mice had an effect of reducing the cancer more than 50%. It is a very surprising effect that cancer reaches complete remission, and the increase of the rate of complete remission suggests that the combination of these drugs is very beneficial.

[69] These results suggest that the anti-cancer effect of 3-agent combination therapy of the compound of the chemical formula 1,bortezomib and dexamethasone is very ex ceptional.

[70] <Example 2:Confirmation of anti-cancer effect of the combination of the present invention using lenalidomide as the second active ingredient, in MM.Is xenograft model>

[71] 2-1)Experimental method

[72] a.Preparation of animal tumor model

[73] MM.Is which is a human multiple myeloma cell line was purchased from ATCC (USA). MM.Is cell line was maintained with RPMI1640 (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).

[74] After MM.Is cells which were mixed with matrigel were subcutaneously ad ministered into NOD.CB17-Prkdcscid/NCrHsd male mouse (2x10 7 cell/head) to organize groups to uniformly distribute the tumor size and then used in the experiment.

[75] b.Preparation of active ingredients

[76] The compound of the chemical formula 1 was prepared in the concentration of 6 mg/ ml by dissolving with physiological saline solution.

[77] Lenalidomide was prepared in the concentration of 1 mg/ml by dissolving with 1% HCl in PBS, and then titrating to a neutral pH.

[78] Dexamethasone was prepared in the concentration of 0.2 mg/ml by diluting 5 mg/ml dexamethasone with saline.

[79] The using solution was prepared on the day of use, and the remaining solution was discarded.

[80] c.Confirmation of anti-tumor activity

[81] The anti-cancer effect of the compound of the chemical formula 1 and lenalidomide and dexamethasone was evaluated in MM.Is xenograft model. The experimental groups were divided to the following different administration groups.

[82] - control group : vehicle

[83] - group administering only the compound of the chemical formula 1 : the compound of the chemical formula 1 (60 mg/kg, once a week, injection)

[84] - coadministration group of lenalidomide and dexamethasone : lenalidomide (10 mg/ kg, once a day, injection), dexamethasone (2 mg/kg, once a week, injection)

[85] - coadministration group: the compound of the chemical formula 1 (60 mg/kg, once a week, injection), lenalidomide (10 mg/kg, once a day, injection), dexamethasone (2 mg/kg, once a week, injection)

[86] All experimental groups were administered intraperitoneally for 70 days. The toxicity was monitored by measuring mouse weight, and the tumor growth was measured by using caliper during the experiment. The tumor volume was calculated according to the mathematical equation 1 in Example 1.

[87] d.Statistical analysis

[88] For all the results of the present experiment, the comparison between experimental groups was performed by assuming normality of the data through parametric multiple comparison or nonparametric multiple comparison, and statistical analysis was performed by using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA), and the case where p value was below 0.05 was considered statistically significant.

[89] 2-2)Experimental result

[90] In order to evaluate anti-tumor effect by coadministration of the compound of the chemical formula 1 and lenalidomide, dexamethasone, each substance was ad ministered into xenograft mouse, and the weight and tumor growth were monitored for 70 days.

[91] In an aspect of tumor growth inhibiting effect, comparing the tumor sizes of group administering the compound of the chemical formula 1 and coadministration group of lenalidomide and dexamethasone, and coadministration group of the compound of the chemical formula 1+ lenalidomide+dexamethasone to a group administering an excipient during administration period, the tumor growth was highly inhibited.

[92] In the coadministration group of lenalidomide and dexamethasone, there was a slight increase in the size of the first cancer, while in the coadministration group of the compound of the chemical formula 1+ lenalidomide+dexamethasone, most of the mice had an effect of reducing the cancer including complete remission.

[93] These results suggest that the anti-cancer effect of 3-agent coadministration therapy of the compound of the chemical formula 1 and lenalidomide, dexamethasone is very high.

Claims

CLAIMS:

[Claim 1]

A combination when used for treating a hematological cancer, the combination comprising: a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti-cancer agent:

[chemical formula 1]

NN aaN O N',OH H oA _N KN wherein the compound of chemical formula 1 or its pharmaceutically acceptable salt, the proteasome inhibitor or the immunomodulatory drug, and the steroidal anti-cancer agent are administered simultaneously or sequentially.
[Claim 2]

A method for treating a hematological cancer in a subject, comprising administering to the subject a combination comprising a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti cancer agent:

[chemical formula 1]

N |

NN'OH oAN H
[Claim 3]

Use of a combination comprising a compound of chemical formula 1 or its pharmaceutically acceptable salt, a proteasome inhibitor or an immunomodulatory drug, and a steroidal anti cancer agent, in the manufacture of a medicament for treating a hematological cancer:

[chemical formula 1]

N | N 0

4N N'OH OAN H N
[Claim 4]

The combination according to claim 1, the method according to claim 2, or the use according to claim 3, wherein the proteasome inhibitor is bortezomib of chemical formula 2 or its pharmaceutically acceptable salt:

[chemical formula 2]

N0 00
[Claim 5]

The combination according to claim 1, the method according to claim 2, or the use according to claim 3, wherein the immunomodulatory drug is lenalidomide of chemical formula 3 or its pharmaceutically acceptable salt:

[chemical formula 3] 0

0
[Claim 6]

The combination according to claim 1, the method according to claim 2, or the use according to claim 3, wherein the steroidal anti-cancer agent is dexamethasone of chemical formula 4 or its pharmaceutically acceptable salt:

[chemical formula 4]

O0

HO

F H
[Claim 7]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the pharmaceutically acceptable salt of the compound of chemical

formula 1 is methanesulfonate.
[Claim 8]

The combination, method, or use according to claim 5, wherein the lenalidomide is

lenalidomide free base.
[Claim 9]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the combination comprises a compound of chemical formula 1 or its

pharmaceutically acceptable salt, a proteasome inhibitor, and a steroidal anti-cancer agent;

and

wherein the compound of chemical formula 1 or its pharmaceutically acceptable salt, the

proteasome inhibitor, and the steroidal anti-cancer agent are comprised in an amount of

28.55~85.60 parts by weight of the compound of chemical formula 1 or its pharmaceutically

acceptable salt, 1.28-8.63 parts by weight of the proteasome inhibitor, and 2.71-69.55 parts

by weight of the steroidal anti-cancer agent, per 100 parts by weight of the total

pharmaceutical combination.
[Claim 10]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the combination comprises a compound of chemical formula 1 or its

pharmaceutically acceptable salt, an immunomodulatory drug, and a steroidal anti-cancer

agent;and

wherein the compound of chemical formula 1 or its pharmaceutically acceptable salt, the

immunomodulatory drug, and the steroidal anti-cancer agent are comprised in an amount of

19.54-94.69 parts by weight of the compound of chemical formula 1 or its pharmaceutically

acceptable salt, 2.46-52.63 parts by weight of the immunomodulatory drug, and 2.52~59.70 parts by weight of the steroidal anti-cancer agent, per 100 parts by weight of the total pharmaceutical combination.
[Claim 11]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the hematological cancer is multiple myeloma.
[Claim 12]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the compound of chemical formula 1 or its pharmaceutically acceptable

salt, the proteasome inhibitor or immunomodulatory drug, and the steroidal anti-cancer agent

are comprised in the form of separate formulations, respectively.
[Claim 13]

The combination according to claim 1, the method according to claim 2, or the use according

to claim 3, wherein the compound of chemical formula 1 or its pharmaceutically acceptable

salt, the proteasome inhibitor or immunomodulatory drug, and the steroidal anti-cancer agent

are combined and comprised in the form of a single formulation.
[Claim 14]

The combination, method or use according to claim 12, wherein the compound of chemical

formula 1 or its pharmaceutically acceptable salt, and the proteasome inhibitor or

immunomodulatory drug, are comprised as a formulation in a parenteral administration form,

and the steroidal anti-cancer agent is comprised as a formulation in an oral administration

form.
[Claim 15]

The method according to claim 2, or the use according to claim 3, wherein the compound of

chemical formula 1 or its pharmaceutically acceptable salt, the proteasome inhibitor or

immunomodulatory drug, and the steroidal anti-cancer agent are comprised in the form of separate formulations, respectively; and the formulation comprising the compound of chemical formula 1 or its pharmaceutically acceptable salt, the formulation comprising the proteasome inhibitor or immunomodulatory drug, and the formulation comprising the steroidal anti-cancer agent are in the form of a formulation that can be administered simultaneously or sequentially.
[Claim 16]

The method or use according to claim 15, wherein the compound of chemical formula 1 or its

pharmaceutically acceptable salt, and the proteasome inhibitor or immunomodulatory drug,

are comprised as a formulation in a parenteral administration form, and the steroidal anti

cancer agent is comprised as a formulation in an oral administration form.