JP7359363B2 - Molecular target combination tumor treatment/prevention drug - Google Patents

Description

TECHNICAL FIELD The present invention relates to molecular target combination therapy and prophylaxis in tumor treatment and prevention.

Malignant tumors such as cancer are one of the major causes of death caused by diseases. Although the effectiveness of tumor chemotherapy using chemotherapeutic agents is increasing, satisfactory clinical treatment results remain unsatisfactory. For example, 2 million people are newly diagnosed with tracheal, bronchial, and lung cancer and 1.7 million people die from it every year around the world (see Non-Patent Document 1), and the number of cases of advanced lung cancer is increasing significantly. As numbers increase, developing effective treatments is a challenge.

In recent years, molecules that target driver mutations in driver genes that directly play an important role in the development and progression of cancer, including epidermal growth factor receptor (EGFR), have been developed for advanced lung cancer. Targeted therapies have been attempted and have dramatically improved survival for patients with advanced lung cancer.

However, there is still no effective molecular target therapy for lung squamous cell carcinoma, and although immune checkpoint inhibitors have been indicated, cytotoxic anticancer drugs are still not available for chemotherapy for lung squamous cell carcinoma. It has become the mainstay. Clinical trials using FGFR inhibitors targeting fibroblast growth factor receptor (FGFR), which is the most common driver mutation in lung squamous cell carcinoma, are being conducted, but the response rate is only 7-19%. It has been reported that the cancer remains in the body (see Non-Patent Document 2), and the high efficacy that has been confirmed with molecular target drugs targeting other cancers has not been achieved at present. Therefore, the early development of new treatments for lung squamous cell carcinoma is desired.

Histone deacetylation is a molecular targeting drug that is being developed to combat and overcome malignant tumors and has been reported to have the activity of inhibiting cancer cell growth, inducing cell differentiation, and apoptosis. There are enzyme (HDAC) inhibitors. The present inventors found that HDAC inhibitors exhibit antitumor effects against malignant tumors with p53 mutations (see Non-Patent Documents 3 to 9), and by screening p21 ^WAF1/Cip1 -inducers, OBP-801/YM753 (spiruchostatin A) has also been discovered as a novel HDAC inhibitor (see Non-Patent Document 10, Patent Document 1, and Patent Document 2).

However, with the exception of cases in which SAHA/vorinostat, romidepsin/istodax, belinostat/bereodac have been approved for the treatment of cutaneous T-cell lymphoma, and panobinostat/farydac for the treatment of multiple myeloma, in the clinical treatment of malignant tumors such as cancer, There are no reports of HDAC inhibitors approved for use.

Although there are reports that HDAC inhibitors have shown antitumor effects in various non-small cell lung cancer cell lines, the results of clinical trials with single-agent HDAC inhibitors for non-small cell lung cancer are not satisfactory, and single-agent administration is not recommended. The response rate in tests remains at only 0-6% (Non-Patent Documents 11-13). Additionally, attempts to use existing anticancer drugs in combination with HDAC inhibitors for non-small cell lung cancer have been insufficient, with significantly more grade 3 or higher adverse events occurring in the combination group, and no prolongation of PFS observed in some cases. (Non-patent Documents 14-15), and there have been no reports yet that have confirmed safety and efficacy in clinical trials targeting lung cancer. There have been reports of the combined effects of HDAC inhibitors and several topoisomerase inhibitors, including the anthracycline anticancer drug amrubicin, on small cell lung cancer cell lines (Non-Patent Document 16); There are no reports targeting small cell lung cancer.

Despite these various attempts, it cannot be said that sufficient results have been achieved, and there is still a need to develop new therapeutic strategies to improve clinical outcomes in the treatment and prevention of malignant tumors such as cancer. There is a strong need for the development of new therapeutic agents based on this therapeutic strategy.

Patent No. 3554707 Japanese Patent Application Publication No. 2001-348340

Global Burden of Disease Cancer Collaboration, JAMA Oncol. 2018;4(11):1553-1568. Rebecca S. Heist, et al. J Thorac Oncol. 2012; 7(12): 1775-1780. Hirose T, Sowa Y, Takahashi S, et al. p53-independent induction of Gadd45 by histone deacetylase inhibitor: coordinate regulation by transcription factors Oct- 1 and NF-Y. Oncogene 2003;22:7762-73. Nakata S, Yoshida T, Horinaka M, et al. Histone deacetylase inhibitors upgrade death receptor 5/TRAIL-R2 and sensitize apoptosis induced by TRAIL/APO2-L in human malignant tumor cells. Oncogene 2004;23:6261-71. Yokota T, Matsuzaki Y, Miyazawa K, et al. Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter. Oncogene 2004;23:5340-9. Hitomi T, Matsuzaki Y, Yokota T, et al. p15(INK4b) in HDAC inhibitor-induced growth arrest. FEBS Lett 2003;554:347-50. Yokota T, Matsuzaki Y, Sakai T. Trichostatin A activates p18INK4c gene: differential activation and cooperation with p19INK4d gene. FEBS Lett 2004;574:171-5. Nakano K, Mizuno T, Sowa Y, et al. Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. J Biol Chem 1997;272:22199-206. Sowa Y, Orita T, Minamikawa S, et al. Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites. Biochem Biophys Res Commun 1997;241:142-50. Shindoh N, Mori M, Terada Y, et al. YM753, a novel histone deacetylase inhibitor, exhibits antitumor activity with selective, sustained accumulation of acety lated histones in tumors in the WiDr xenograft model. International journal of oncology. 2008; 32: 545-55. Wagner JM, et al. Clin Epigenetics. 2010 Dec;1(3-4):117-136. Traynor AM, et al. J Thorac Oncol. 2009 Apr;4(4):522-526. Gridelli C, et al. Crit Rev Oncol Hematol. 2008 Oct;68(1):29-36. Tambo Y, et al. Invest New Drugs. 2017 Apr;35(2):217-226. Ramalingam SS, et al. J Clin Oncol. 2010 Jan 1;28(1):56-62. Gray J, et al. Cancer Biol Ther. 2012 Jun; 13(8):614-622.

The problem to be solved by the present invention is to provide new therapeutic agents and therapeutic strategies that can treat and prevent malignant tumors, especially lung cancer.

The present inventor aimed to establish a new rational and highly effective treatment strategy for malignant tumors, especially lung cancer, and while proceeding with evaluation using new and existing molecular target drug compounds, the molecular target It was discovered that the use of OBP-801, a histone deacetylase (HDAC) inhibitor, and amrubicin, an anthracycline anticancer drug, exhibited significantly synergistic cancer treatment and prevention effects. Through the elucidation of its molecular mechanism, we have completed the invention of rational and highly effective new drugs for tumor treatment and prevention based on new therapeutic strategies.

The present invention enables the treatment and prevention of tumors, and in particular for the treatment and prevention of lung cancer. Its most important feature is that it is used in combination as an active ingredient.

The first means of the present invention for solving the above problems is a drug for tumor treatment, characterized in that it contains a histone deacetylase inhibitor and an anthracycline anticancer drug as active ingredients. A preventive pharmaceutical composition.

A second means of the present invention is a pharmaceutical composition for tumor treatment and prevention, which is characterized by containing a histone deacetylase inhibitor as an active ingredient, and is to be used in combination with an anthracycline anticancer drug.

A third means of the present invention is a pharmaceutical composition for tumor treatment and prevention, which is characterized in that it contains an anthracycline anticancer agent as an active ingredient, and is to be used in combination with a histone deacetylase inhibitor.

又は、次式（ＩＩ）：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩であることを特徴とする、本発明の第１の手段～第３の手段のいずれか１に記載の腫瘍治療用及び予防用医薬組成物である。 A fourth means of the present invention is that the histone deacetylase inhibitor is
The following formula (I):

Or the following formula (II):

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, an ethyl group, R4 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group, R5 to R8 each independently represents a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 represents a hydrogen atom, a methyl group, or a protective group; R10 and R11 independently represent a hydrogen atom, a methyl group, or a protective group.)
The pharmaceutical composition for tumor treatment and prevention according to any one of the first to third means of the present invention, which is a depsipeptide compound represented by or a pharmaceutically acceptable salt thereof. It is a thing.

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示される化合物であることを特徴とする、本発明の第４の手段に記載の腫瘍治療用及び予防用医薬組成物である。 A fifth means of the present invention is that the depsipeptide compound is
The following formula (III):

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
The pharmaceutical composition for tumor treatment and prevention according to the fourth aspect of the present invention is characterized by being a compound represented by:

A sixth means of the present invention is a pharmaceutical composition for tumor treatment and prevention according to the fifth means of the present invention, wherein R4 is an isopropyl group.

A seventh means of the present invention is a drug for tumor treatment and prevention according to any one of the first means to sixth means of the present invention, characterized in that the anthracycline anticancer drug is amrubicin. It is a composition.

An eighth means of the present invention is the pharmaceutical composition for tumor treatment and prevention according to any one of the first means to the seventh means of the present invention, wherein the tumor is lung cancer. It is.

A ninth means of the present invention is a method for producing a pharmaceutical composition for tumor treatment and prevention, which comprises a step of containing a histone deacetylase inhibitor and an anthracycline anticancer drug as active ingredients. It is.

A tenth aspect of the present invention is a pharmaceutical composition for tumor treatment and prevention for use in combination with an anthracycline anticancer drug, which comprises a step of containing a histone deacetylase inhibitor as an active ingredient. This is the manufacturing method.

An eleventh means of the present invention is a pharmaceutical composition for tumor treatment and prevention for use in combination with a histone deacetylase inhibitor, which comprises a step of containing an anthracycline anticancer drug as an active ingredient. This is the manufacturing method.

又は、次式（ＩＩ）：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩であることを特徴とする、本発明の第９の手段～第１１の手段のいずれか１に記載の腫瘍治療用及び予防用医薬組成物の製造方法である。 A twelfth means of the present invention is that the histone deacetylase inhibitor is
The following formula (I):

Or the following formula (II):

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, an ethyl group, R4 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group, R5 to R8 each independently represents a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 represents a hydrogen atom, a methyl group, or a protective group; R10 and R11 independently represent a hydrogen atom, a methyl group, or a protective group.)
The pharmaceutical composition for tumor treatment and prevention according to any one of the ninth to eleventh means of the present invention, which is a depsipeptide compound represented by or a pharmaceutically acceptable salt thereof. It is a method of manufacturing something.

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示される化合物であることを特徴とする、本発明の第１２の手段に記載の腫瘍治療用及び予防用医薬組成物の製造方法である。 A thirteenth means of the present invention is that the depsipeptide compound is
The following formula (III):

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
A method for producing a pharmaceutical composition for tumor treatment and prevention according to the twelfth means of the present invention, characterized in that the compound is a compound represented by:

A fourteenth means of the present invention is the pharmaceutical composition for tumor treatment and prevention according to the thirteenth means of the present invention, wherein R4 is an isopropyl group.

A fifteenth means of the present invention is a medicament for tumor treatment and prevention according to any one of the ninth means to the fourteenth means of the present invention, characterized in that the anthracycline anticancer drug is amrubicin. This is a method for producing a composition.

A sixteenth means of the present invention is the pharmaceutical composition for tumor treatment and prevention according to any one of the ninth means to the fifteenth means of the present invention, wherein the tumor is lung cancer. This is a manufacturing method.

A seventeenth means of the present invention is a pharmaceutical composition for tumor treatment and prevention, comprising a pharmaceutical composition containing a histone deacetylase inhibitor as an active ingredient and a pharmaceutical composition containing an anthracycline anticancer agent as an active ingredient. A kit of pharmaceutical compositions.

又は、次式（ＩＩ）：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩であることを特徴とする、本発明の第１７の手段に記載の腫瘍治療用及び予防用医薬組成物のキットである。 An eighteenth means of the present invention is that the histone deacetylase inhibitor is
The following formula (I):

Or the following formula (II):

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, an ethyl group, R4 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group, R5 to R8 each independently represents a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 represents a hydrogen atom, a methyl group, or a protective group; R10 and R11 independently represent a hydrogen atom, a methyl group, or a protective group.)
A kit of a pharmaceutical composition for tumor treatment and prevention according to the seventeenth means of the present invention, characterized in that it is a depsipeptide compound represented by or a pharmaceutically acceptable salt thereof.

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示される化合物であることを特徴とする、本発明の第１８の手段に記載の腫瘍治療用及び予防用医薬組成物のキットである。 A nineteenth aspect of the present invention provides that the depsipeptide compound is
The following formula (III):

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
A kit of a pharmaceutical composition for tumor treatment and prevention according to the eighteenth means of the present invention, characterized in that it is a compound represented by:

The 20th means of the present invention is a kit of the pharmaceutical composition for tumor treatment and prevention according to the 19th means of the present invention, wherein R4 is an isopropyl group.

A twenty-first means of the present invention is the tumor treatment and prevention drug according to any one of the seventeenth to twentieth means of the present invention, wherein the anthracycline anticancer drug is amrubicin. This is a composition kit.

A twenty-second means of the present invention is a kit of the pharmaceutical composition for tumor treatment and prevention according to the seventeenth means to the twenty-first means of the present invention, wherein the tumor is lung cancer. .

By using the pharmaceutical composition of the present invention in which a histone deacetylase inhibitor such as OBP-801 and an anthracycline anticancer drug are used in combination as active ingredients, synergistic apoptosis induction in tumor cells and tumor tissues and tumor A growth-inhibiting effect can be obtained, and tumor treatment and prevention can be achieved while avoiding side effects caused by the active ingredient. Furthermore, by combining a histone deacetylase inhibitor such as OBP-801 and an anthracycline anticancer drug, which each have different drug target molecules, it is possible to synergistically activate the caspase pathway, which cannot be achieved with a single drug. It has become possible to obtain a remarkable pharmacological effect of activating the mitochondrial apoptotic pathway by controlling the expression and activity of TXNIP and thioredoxin 2, and it is an effective drug that can be applied to a wide range of tumors, including tumors such as lung cancer, for which conventional therapies have low efficacy. It becomes possible to provide new clinically effective therapeutic and preventive strategies for tumors as therapeutic and preventive drugs.

This figure shows the in vitro cell proliferation inhibitory effect of histone deacetylase inhibitors and/or anthracycline anticancer agents on human lung squamous cell carcinoma cell lines. FIG. 1A shows the in vitro cell proliferation inhibitory effect of OBP-801 on Calu-1 cells. FIG. 1B shows the in vitro cytostatic effect of amrubicin on Calu-1 cells. Data represent mean ± SD. FIG. 1C shows the in vitro cell proliferation inhibitory effect on Calu-1 cells when OBP-801 and amrubicin were used in combination. FIG. 1D shows the in vitro cell proliferation inhibitory effect on H520 cells when OBP-801 and amrubicin were used in combination. FIG. 1E shows the in vitro cell proliferation inhibitory effect on A549 cells when OBP-801 and amrubicin were used in combination. Data represent mean ± SD. ** indicates P<0.01 (vs. control). FIG. 1F shows the combination index (CI) regarding the in vitro cell proliferation inhibitory effect on Calu-1 cells when OBP-801 and amrubicin were used together. The results of analysis of the proportion of Sub-G1 phase cells in Calu-1 cells are shown. FIG. 2A shows the analysis results of the percentage of Sub-G1 phase cells upon treatment of Calu-1 cells with OBP-801 and/or amrubicin for 72 hours. Data represent mean±SD, **indicates P<0.01. Figure 2B shows the sub-G1 phase induced by 72-hour treatment of Calu-1 cells with OBP-801, amrubicin, N-acetyl-L-cysteine (NAC), and/or the pancaspase inhibitor zVAD-fmk (Z-VAD). The analysis results of cell percentage are shown. FIG. 2C shows the analysis results of the percentage of Sub-G1 phase cells upon treatment of H520 cells with OBP-801, amrubicin, NAC, and/or Z-VAD for 72 hours. FIG. 2D shows the analysis results of the percentage of Sub-G1 phase cells upon treatment of A549 cells with OBP-801, amrubicin, NAC, and/or Z-VAD for 72 hours. Data represent mean±SD, **indicates P<0.01. FIG. 2E shows the results of Western blot analysis of Calu-1 cells treated with OBP-801 and/or amrubicin for 72 hours. The expression of Cleaved PARP (poly [ADP-ribose] polymerase) when used in combination is shown. It shows the in vitro cell growth suppressive effect of histone deacetylase inhibitors, anthracycline anticancer drugs, and/or kinase inhibitors on Calu-1 cells. FIG. 3A shows the in vitro cell proliferation inhibitory effects of OBP-801, amrubicin, and/or Selonsertib, an ASK1 (apoptosis signal-regulated kinase 1) inhibitor. FIG. 3B shows the in vitro cytostatic effects of OBP-801, amrubicin, and/or the p38 MAPK inhibitor SB203580. FIG. 3C shows the in vitro cytostatic effects of OBP-801, amrubicin, and/or the JNK inhibitor SP2600125. Data represent mean±SD, **indicates P<0.01. These are the results of Western blot analysis of Calu-1 cells treated with a histone deacetylase inhibitor and/or an anthracycline anticancer drug for 72 hours. FIG. 4A shows the results of Western blot analysis upon treatment with OBP-801 and/or amrubicin for 72 hours. TXNIP (thioredoxin-interacting protein) expression is shown. FIG. 4B shows the amount of intracellular reactive oxygen species (ROS) upon treatment of Calu-1 cells with OBP-801 and/or amrubicin for 48 hours. These are the results of Western blot analysis and the percentage of Sub-G1 phase cells upon treatment of Calu-1 cells with a histone deacetylase inhibitor and/or an anthracycline anticancer drug for 48 hours. FIG. 5A shows the results of Western blot analysis upon treatment with OBP-801 and/or amrubicin and siTrx2, an siRNA against thioredoxin 2, for 48 hours. Thioredoxin 2 expression is shown. FIG. 5B shows the analysis results of the percentage of Sub-G1 phase cells upon treatment with OBP-801 and/or amrubicin and siTrx2, an siRNA against thioredoxin 2, for 48 hours. Data represent mean±SD, **indicates P<0.01. The in vivo antitumor effects of histone deacetylase inhibitors and/or anthracycline anticancer agents are shown in an evaluation system using nude mice subcutaneously transplanted with human lung squamous cell carcinoma cell line H520. FIG. 6A shows the variation in tumor volume without or upon treatment with OBP-801 and/or amrubicin. Data represent mean±SD, **indicates P<0.01. FIG. 6B shows the results of a test that confirmed changes in the body weight of test mice when they were not treated or treated with OBP-801 and/or amrubicin. Data represent mean ± SD. This figure shows the tumor cell proliferation inhibitory effect on Calu-1 cells when treated in combination with OBP-801 or SAHA, a histone deacetylase inhibitor, and amrubicin, an anthracycline anticancer drug, for 72 hours. FIGS. 7A and 7B are the results of confirming the tumor cell growth suppressive effect of combined treatment of OBP-801 or SAHA and amrubicin on Calu-1 cells for 72 hours. A model of the mechanism of action when OBP-801 and amrubicin are used together is shown.

The present invention provides a pharmaceutical composition for tumor treatment and prevention, which is characterized by containing OBP-801, a histone deacetylase (HDAC) inhibitor, and an anthracycline anticancer drug as active ingredients. .

As used herein, the term "histone deacetylase inhibitor (HDAC inhibitor)" refers to a molecular targeting agent that targets and inhibits histone deacetylase (HDAC).

The HDAC inhibitor OBP-801 contained in the composition of the present invention can be obtained and used from Oncolys Biopharma Co., Ltd. (Tokyo, Japan).

HDAC inhibitors SAHA/vorinostat, romidepsin, panobinostat/LBH589, belinostat/PXD101, mocetinostat/MGCD0103, avexinostat/PCI-24781, entinostat/SNDX-275/MS-275, SB939, CS055/HBI- 8000, Resminostat/4SC-201/BYK408740, givinostat/ITF2357, quisinostat/JNJ-26481585, CHR-2845, CHR-3996, CUDC-101, AR-42, DAC-060, EVP-0334, M GCD-290, CXD- 101/AZD-9468, CG200745, arginine butyrate, 4SC-202, rocilinostat/ACY-1215 or SHP-141 may be used in the same manner as OBP-801 contained in the composition of the present invention.

または、次式（ＩＩ）：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩を使用してもよい。
中でも、次式（ＩＩＩ）：

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示される化合物であることがより好ましく、Ｒ４がイソプロピル基である化合物（ＯＢＰ－８０１）が特に好ましい。 Also, as a histone deacetylase inhibitor,
The following formula (I):

Or the following formula (II):

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, an ethyl group, R4 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group, R5 to R8 each independently represents a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 represents a hydrogen atom, a methyl group, or a protective group; R10 and R11 independently represent a hydrogen atom, a methyl group, or a protective group.)
A depsipeptide compound represented by or a pharmaceutically acceptable salt thereof may be used.
Among them, the following formula (III):

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
A compound represented by is more preferred, and a compound in which R4 is an isopropyl group (OBP-801) is particularly preferred.

In addition, as used herein, "anthracycline anticancer drug" refers to a drug that inhibits DNA topoisomerase II, intercalates between base pairs of DNA strands, inhibits DNA synthesis, replication, and transcription, and inhibits the proliferation of tumor cells. Refers to compounds that have anticancer activity.

Examples of anthracycline anticancer agents contained in the composition of the present invention include amrubicin, amrubicin hydrochloride, doxorubicin, doxorubicin hydrochloride, daunorubicin, daunorubicin hydrochloride, daunorubicin citrate, epirubicin, epirubicin hydrochloride, aclarubicin, aclarubicin hydrochloride, Use of zorubicin hydrochloride, idarubicin, idarubicin hydrochloride, pirarubicin, pirarubicin hydrochloride, valrubicin, pixantrone, pixantrone maleate, aldoxorubicin, aldoxorubicin hydrochloride, carubicin hydrochloride, esorubicin hydrochloride, berubicin hydrochloride, etc. I can do it. As the anthracycline anticancer agent contained in the composition of the present invention, preferably amrubicin or various salts of amrubicin such as amrubicin hydrochloride can be used.

Amrubicin and amrubicin hydrochloride, which are anthracycline anticancer agents contained in the composition of the present invention, can be obtained and used from ApexBio (Taiwan) or the like.

In the present invention, the term "tumor" includes malignant tumors such as epithelial malignant tumors such as cancer and non-epithelial malignant tumors such as sarcoma, as well as benign tumors. Preferably, the malignant tumor includes lung cancer, bronchial cancer, pharyngeal cancer, thyroid cancer, melanoma, breast cancer, bone tumor, bone and soft tissue tumor, osteosarcoma, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, and renal cell cancer. Includes cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, colon cancer, rectal cancer, skin cancer, mesothelioma, lymphoma, nervous system tumor, or neural tissue tumor. , more preferably lung cancer. Lung cancer includes non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC), more preferably includes non-small cell lung cancer, and even more preferably Non-small cell lung cancer is Squamous Non-Small Cell Lung Cancer (Sq NSCLC) or Non-Squamous Non-Small Cell Lung Cancer. er; Non- Sq NSCLC), and more preferably squamous Non-Small Cell Lung Cancer (Sq NSCLC).

In the present invention, "a pharmaceutical composition for tumor treatment and prevention characterized by containing a histone deacetylase inhibitor and an anthracycline anticancer drug as active ingredients" refers to a histone deacetylase inhibitor. It refers to a pharmaceutical composition in which an anticancer agent and an anthracycline anticancer agent are combined for simultaneous, separate, or sequential administration in the treatment or prevention of tumors.

The pharmaceutical composition of the present invention can be provided in the form of a combination drug containing both a histone deacetylase inhibitor and an anthracycline anticancer drug. Further, a pharmaceutical composition containing a histone deacetylase inhibitor and a pharmaceutical composition containing an anthracycline anticancer agent are provided separately, and these pharmaceutical compositions can be used simultaneously, separately, or sequentially. It's okay. Furthermore, it may be provided as a kit comprising a pharmaceutical composition containing a histone deacetylase inhibitor and a pharmaceutical composition containing an anthracycline anticancer agent.

Furthermore, according to the present invention, when a histone deacetylase inhibitor and an anthracycline anticancer drug are used together in tumor treatment and prevention, each is administered at a lower dosage than when either one is used alone. can be administered at

又は、次式（ＩＩ）：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩であるヒストン脱アセチル化酵素阻害剤、及び、アントラサイクリン系抗癌剤のみを有効成分として含有し、更に製剤化のための添加物を含有する医薬組成物である。
さらに好適には、次式（ＩＩＩ）：

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示される化合物又はその製薬学的に許容可能な塩であるヒストン脱アセチル化酵素阻害剤、及び、アントラサイクリン系抗癌剤のみを有効成分として含有し、更に製剤化のための添加物を含有する医薬組成物である。
特に好適には、前記式（ＩＩＩ）で示され、式中のＲ４がイソプロピル基である化合物（ＯＢＰ－８０１）又はその製薬学的に許容可能な塩であるヒストン脱アセチル化酵素阻害剤、及び、アントラサイクリン系抗癌剤のみを有効成分として含有し、更に製剤化のための添加物を含有する医薬組成物である。 The composition of the present invention containing a histone deacetylase inhibitor and an anthracycline anticancer agent contains the histone deacetylase inhibitor and an anthracycline anticancer agent as essential components, and optionally, It may contain additives for formulation.
Preferably, the pharmaceutical composition contains only a histone deacetylase inhibitor and an anthracycline anticancer agent as active ingredients, and further contains additives for formulation.
More preferably, the following formula (I):

Or the following formula (II):

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, an ethyl group, R4 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group, R5 to R8 each independently represents a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 represents a hydrogen atom, a methyl group, or a protective group; R10 and R11 independently represent a hydrogen atom, a methyl group, or a protective group.)
Contains only a histone deacetylase inhibitor, which is a depsipeptide compound represented by or a pharmaceutically acceptable salt thereof, and an anthracycline anticancer agent as active ingredients, and further contains additives for formulation. A pharmaceutical composition.
More preferably, the following formula (III):

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
A medicament containing only a histone deacetylase inhibitor, which is a compound represented by the above or a pharmaceutically acceptable salt thereof, and an anthracycline anticancer agent as active ingredients, and further contains additives for formulation. It is a composition.
Particularly preferred is a histone deacetylase inhibitor which is a compound (OBP-801) represented by the above formula (III), in which R4 is an isopropyl group, or a pharmaceutically acceptable salt thereof; , is a pharmaceutical composition containing only an anthracycline anticancer agent as an active ingredient, and further contains additives for formulation.

In the present invention, containing a histone deacetylase inhibitor and an anthracycline anticancer agent as active ingredients means that the main active ingredients include a histone deacetylase inhibitor and an anthracycline anticancer agent. However, the content of the histone deacetylase inhibitor and anthracycline anticancer drug is not limited.

In the present invention, the term "treatment" refers to killing a tumor or reducing the number of its cells, suppressing tumor growth, or treating a tumor by administering the pharmaceutical composition according to the present invention to a subject. This means that various symptoms caused by this condition are improved. Furthermore, in the present invention, the term "prevention" means prevention of an increase in the number of tumors that have been reduced by re-proliferation, and prevention of re-proliferation of a tumor whose growth has been suppressed.

The pharmaceutical composition for tumor treatment and prevention of the present invention can be produced through a step of containing a histone deacetylase inhibitor and an anthracycline anticancer agent as active ingredients. Furthermore, the pharmaceutical composition for tumor treatment and prevention to be used in combination with the anthracycline anticancer drug of the present invention can also be used in combination with a histone deacetylase inhibitor through the step of containing a histone deacetylase inhibitor as an active ingredient. A pharmaceutical composition for tumor treatment and prevention to be used in combination with an enzyme inhibitor can be manufactured by passing through a step of incorporating an anthracycline anticancer agent as an active ingredient. Furthermore, in addition to the above steps, a step of incorporating additives for formulation may be added, if desired.

In the pharmaceutical composition of the present invention, when the histone deacetylase inhibitor and the anthracycline anticancer agent are contained and provided in separate pharmaceutical compositions, the dosage forms of these pharmaceutical compositions are the same dosage form. or may be in a different dosage form. For example, both may be one of oral preparations, parenteral preparations, injections, infusions, and intravenous infusions and may be in different dosage forms, and both may be oral preparations, parenteral preparations, and injections. , a drip, or an intravenous drip, and may be in the same dosage form. Furthermore, the above pharmaceutical composition may be combined with one or more different formulations.

Pharmaceutical compositions of the invention can take the form of liquid compositions, such as bulk liquid solutions or suspensions, or solid compositions, such as bulk powders, tablets, etc. For example, prefilled and measured ampoules or syringes for liquid compositions, or pills, tablets, capsules, etc. for solid compositions. The amount of active ingredient in these preparations is determined so as to provide tumor therapeutic and/or preventive effects.

Examples of the liquid composition include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and suitable solubilizing agents, such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate 80 and HCO-50 may be used in combination as appropriate. Examples of oily liquids include sesame oil and soybean oil, which may be used in combination with benzyl benzoate and benzyl alcohol as solubilizing agents. It may also be suitably combined with buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants.

The solid composition may also include binders such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants. , such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavor, or any other ingredients of a similar nature. Compounds can be included.

The pharmaceutical compositions of the present invention can be administered orally or parenterally by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal. Examples of parenteral administration include injection administration, nasal administration, pulmonary administration, and transdermal administration. As an example of injection administration, the therapeutic antibodies of the present invention can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. Furthermore, the administration method and dosage may be appropriately selected depending on the age and symptoms of the patient.

Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention should not be construed as being limited by the descriptions.

We have demonstrated the remarkable therapeutic and preventive effects of the combination of histone deacetylase inhibitors and anthracycline anticancer drugs in tumor treatment and prevention. In this example, human lung squamous cell carcinoma cells were used as an example of a tumor to be treated by the present invention, and OBP-801 was used in combination with a histone deacetylase inhibitor as an example of a histone deacetylase inhibitor. Amrubicin hydrochloride was used as an example of an anthracycline anticancer drug.

<Materials and methods>
[1. Cell culture]
Calu-1 cells, a human lung squamous cell carcinoma cell line, and A549 cells, a human lung adenocarcinoma cell line, were cultured in a DMEM medium supplemented with 10% FBS and 4 mM glutamine at 37° C. under 5% CO ₂ conditions. H520 cells, a human lung squamous cell carcinoma cell line, were cultured in RPMI 1640 medium supplemented with 10% FBS and 2mM glutamine at 37°C and 5% _CO2 .

[2. reagent〕
OBP-801 was supplied by Oncolys Biopharma Co., Ltd. (Tokyo, Japan). Amrubicin hydrochloride was purchased from ApexBio (Taiwan). N-acetyl-L-cysteine (NAC) was purchased from Nacalai Tesque (Japan). Pan-caspase inhibitor zVAD-fmk was purchased from R&D Systems (Minneapolis, MN, USA). selonsertib, SB203580, SP2600125 was purchased from Selec Chemicals (Houston, TX, USA). siTrx2 was purchased from Salmon Fisher Scientific (Waltham, MA, USA).

[3. Cell viability test]
Cell viability was measured at a wavelength of 450 nm with a multiwell spectrophotometer Multiskan FC (Salmon Fisher Scientific, Waltham, MA, USA) using Cell Counting Kit-8 (Dojindo Laboratories, Japan). And it was decided.

[4. Analysis of apoptosis]
Cells were treated with or without OBP-801 and/or amrubicin hydrochloride at each concentration for 72 hours, and then collected. Cells were permeabilized with 0.1% Triton-X100 and nuclei were stained with propidium iodide (PI). The amount of DNA was measured using a FACSCalibur ^™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed using the Cell Quest ^™ software package (Becton-Dickinson).

[5. Combination coefficient〕
Combination coefficients (CI) were calculated using CalcuSyn software (Biosoft, UK) based on the method by Chou and Talalay. If CI<1, it is determined that there is a synergistic effect, ie, a larger than expected additive effect. When CI=1, it is determined that there is an additive effect, and when CI>1, it is determined that there is an antagonistic effect.

[6. Antitumor activity evaluation in xenograft model]
Female BALB/c nu/nu mice (5 weeks old) were purchased from Charles River Laboratory (Wilmington, MA, USA). H520 cells (4×10 ⁶ cells) were implanted by subcutaneous injection into the back of mice. The tumor volume was calculated using the formula: 1/2 x (length) x (width) ² . When the average tumor volume reached 40 ^mm3 (day 13), mice were randomly divided into four groups (5 animals/group) and treatment continued. Mice were administered dilution alone (control group) or OBP-801 (10 mg/kg) once a week (on days 15, 22, 29 and 36). In addition, dilution alone (control group) or amrubicin hydrochloride (AMR) (25 mg/kg) was administered only once (on day 14). OBP-801 was dissolved in 20% hydroxypropyl-β-cyclodextrin/saline and injected into the tail vein. Amrubicin hydrochloride was dissolved in physiological saline and injected into the tail vein. Mouse body weight and tumor volume were measured on days 13, 18, 22, 26, 30, 33, 37 and 40. All experiments and surgical procedures were performed in accordance with the guidelines established by the research institutional committee.

[7. Western blot analysis]
Cells were lysed in lysis solution (50mM Tris-HCl, 150mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 1mM dithiothreitol, and 0.5mM phenylmethylsulfonyl fluoride). Dissolved. The protein extract was loaded onto a polyacrylamide gel, subjected to electrophoresis, and transferred to a nitrocellulose membrane. Blots are blocked with blocking solution (5% skim milk/TBS-T) for 1 hour at room temperature and incubated with the appropriate primary antibody for 1 hour at room temperature. Signals were detected using Chemi-Lumi One L (Nacalai Tesque, Japan) or Immobilon Western (Millipore, Billerica, MA, USA).
The antibodies were rabbit polyclonal antibodies such as anti-Cleaved PARP antibody (#5625, Cell Signaling Technology, Danvers, MA, USA), anti-TXNIP antibody (ab188865, Abcam, Cambridge, UK), and anti-thioredoxin2 antibody (sc-133201, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin antibody (Sigma, St. Louis, MO, USA).

[8. Small interference (si) RNA transfection]
Knockdown of Thioredoxin2 was performed by gene introduction of small interfering RNA (siRNA) using Lipofectamine ^™ RNAiMAX (Invitrogen).

<Results>
[Test Example 1: In vitro cell proliferation inhibitory effect of OBP-801 and/or amrubicin on human lung squamous cell carcinoma cell line]

The effect of OBP-801 or amrubicin hydrochloride, an anthracycline anticancer drug, on tumor cell proliferation was investigated using Calu-1 cells, a human lung squamous cell carcinoma cell line.

OBP-801 exhibited a concentration-dependent cell proliferation inhibitory effect on human lung squamous cell carcinoma cells, and the concentration showing a 50% inhibitory effect was approximately 2.75 nM (FIG. 1A). In addition, amrubicin hydrochloride exhibited a concentration-dependent cell proliferation inhibitory effect on human lung squamous cell carcinoma cells, and the concentration showing a 60% inhibitory effect was about 2 μM (FIG. 1B).

[Example 2: Synergistic enhancement of in vitro cell proliferation inhibitory effect on human lung squamous cell carcinoma cell line by treating amrubicin hydrochloride, an anthracycline anticancer drug, in combination with OBP-801]

The growth inhibitory effect of combined treatment with OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, on tumor cells in tumor tissues was investigated in human lung squamous cell carcinoma cell lines Calu-1 cells, H520 cells, and human The study was conducted using A549 cells, a lung adenocarcinoma cell line.

For Calu-1 cells, single treatment with OBP-801 at a concentration of 2.75 nM or single treatment with amrubicin hydrochloride at a concentration of 2.5 μM only had an inhibitory effect of about 50-60%. Although not shown, it was confirmed that combined treatment with OBP-801 at a concentration of 2.75 nM and amrubicin hydrochloride at a concentration of 2.5 μM exhibited a significant cell growth inhibition effect of approximately 95% (FIG. 1C). The combination coefficient in this combination treatment was found to be less than 1.0, indicating a synergistic cell growth inhibition effect (FIG. 1F).

For H520 cells, single treatment with OBP-801 at a concentration of 4.5 nM or single treatment with amrubicin hydrochloride at a concentration of 4 μM only showed an inhibitory effect of about 50%; It was confirmed that when treated in combination with amrubicin hydrochloride at a concentration of 4.5 nM and 4 μM, a significant cell growth inhibition effect of approximately 85% was exhibited (FIG. 1D).

For A549 cells, single treatment with OBP-801 at a concentration of 2.5 nM or single treatment with amrubicin hydrochloride at a concentration of 400 nM only showed an inhibitory effect of about 50-70%; It was confirmed that when -801 was treated in combination with 2.5 nM of amrubicin hydrochloride and amrubicin hydrochloride at a concentration of 400 nM, a significant cell growth inhibition effect of approximately 90% was exhibited (FIG. 1D).

These results revealed that cancer cell proliferation was synergistically suppressed by treating cancer cells with OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug.

[Example 3: Synergistic apoptosis-inducing effect on tumor cells by combined treatment of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

The effects of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, on tumor cells were examined using Calu-1 cells. As a result, we interestingly found that when Calu-1 cells were treated with OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, for 72 hours, significant apoptosis induction occurred in Calu-1 cells ( Figure 2A).

From these results, it is clear that the combined treatment of OBP-801, which synergistically suppresses the proliferation of cancer cells, and amrubicin hydrochloride, an anthracycline anticancer drug, produces a remarkable apoptosis-inducing effect. It became.
[Example 4: Induction of caspase- and intracellular reactive oxygen species (ROS)-dependent apoptosis by combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

In order to examine whether the apoptosis that occurs in tumor cells caused by the combination of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, is caspase-dependent, Calu-1 cells, H520 cells, and A549 cells were used to investigate whether caspase inhibitor Analyzed using.

As a result, apoptosis induced by the combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, was suppressed by zVAD-fmk, a pan-caspase inhibitor (FIG. 2B, FIG. 2C, and FIG. 2D).

These results suggest that caspase-dependent apoptosis is induced in the combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, against tumors.

Furthermore, the apoptosis-inducing effect induced by the combination of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, was also suppressed by N-acetyl-L-cysteine (NAC) (Figure 2B, Figure 2C, Figure 2D). ).

From these results, it is understood that ROS-dependent apoptosis occurs when OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, are used in combination against tumors.

[Example 5: Increase in Cleaved PARP by combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

Since PARP is cleaved by caspases at the early stage of apoptosis, cleaved PARP increases after induction of apoptosis. Therefore, we confirmed whether Cleaved PARP increased by the combination of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, and examined whether apoptosis was induced.

As a result, it was confirmed that Cleaved PARP was increased by the combination of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug (FIG. 2E).

[Example 6: Contribution of apoptosis signal-regulated kinase 1 (ASK1), p38MAPK, and JNK to synergistic apoptosis induction of tumor cells by combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

The contribution of apoptosis signal-regulating kinase 1 (ASK1), p38MAPK, and JNK to the apoptosis-inducing effects of OBP-801 and the anthracycline anticancer drug amrubicin hydrochloride was examined using Calu-1 cells.

To examine the degree of contribution of apoptosis signal-regulated kinase 1 (ASK1), Selonsertib (GS-4997), an inhibitor of apoptosis signal-regulated kinase 1 (ASK1), was used. The p38 MAPK inhibitor SB203580 was used to examine the degree of contribution of p38 MAPK. The JNK inhibitor SP2600125 was used to examine the degree of contribution of JNK.

When Calu-1 cells are treated with 2.75 nM OBP-801 and 2 μM amrubicin hydrochloride, an anthracycline anticancer drug, for 72 hours, significant apoptosis induction occurs in Calu-1 cells, but ASK1 inhibitor Simultaneous treatment with Selonsertib partially reduced the extent of induced apoptosis (Figure 3A). On the other hand, when examining the contribution of the p38MAPK inhibitor SB203580 or JNK, simultaneous treatment with the JNK inhibitor SP2600125 did not result in any change in the degree of induced apoptosis (Figures 3B and 3C). .

These results demonstrate that the apoptosis signal-regulated kinase 1 (ASK1) pathway is involved in the synergistic induction of apoptosis in tumor cells by the combination of OBP-801 and the anthracycline anticancer drug amrubicin hydrochloride. It became.

[Example 7: Contribution of TXNIP (ThioredoXiN Interacting Protein) and reactive oxygen species (ROS) to induction of tumor cell apoptosis by combined use of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

Thioredoxin 2 (Trx2) is known to bind to and inhibit apoptosis signal-regulated kinase 1 (ASK1). When Trx2 is oxidized by reactive oxygen species (ROS) or TXNIP (ThioredoXiN Interacting Protein), it dissociates from ASK1, and as a result, ASK1 is activated and apoptosis is induced. Therefore, we analyzed the activation mechanism of apoptosis signal-regulated kinase 1 (ASK1), which has been shown to be involved in the induction of apoptosis in tumor cells, caused by the combination of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug. .

Calu-1 cells were treated with a combination of 2.75 nM OBP-801 and 2 μM amrubicin hydrochloride, an anthracycline anticancer drug, for 72 hours. As a result, it was revealed that the expression of TXNIP was significantly increased in treatment with OBP-801 alone and in combination (FIG. 4A). In addition, when Calu-1 cells were treated with 2.75 nM of OBP-801 and 2 μM of amrubicin hydrochloride, an anthracycline anticancer drug, for 48 hours, ROS increased when treated with amrubicin hydrochloride alone or in combination. This became clear (Fig. 4B).

[Example 8: Relationship between thioredoxin 2 (Trx2) and apoptosis in human lung squamous cell carcinoma cells]

The role of thioredoxin 2 (Trx2), which binds to and inhibits apoptosis signal-regulated kinase 1 (ASK1), in human lung squamous cell carcinoma cells was analyzed by suppressing its expression using siRNA.

By introducing siRNA (siTrx2 #1, #2, #3) specifically designed for thioredoxin 2 (Trx2) into Calu-1 cells, western blot analysis 48 hours after introduction showed that Trx2 expression was suppressed. This was confirmed (Fig. 5A). Furthermore, we found that apoptosis was induced 120 hours after introducing these siRNAs (FIG. 5B). Therefore, it has been revealed that apoptosis is induced in Calu-1 cells when Trx2 function is inhibited.

[Example 9: Effect of suppressing tumor growth and reducing tumor volume in mice by combined administration of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug]

Is it possible to inhibit the growth of tumor cells and tumor tissue within a host animal by co-administering OBP-801, a molecular target drug, and amrubicin hydrochloride, an anthracycline anticancer drug, in vivo? investigated. H520 cells, a human lung squamous cell carcinoma cell line, were used as tumor cells and were subcutaneously transplanted into nude mice to carry tumors, thereby obtaining a xenograft model. Then, they were divided into a control group, a group administered with OBP-801 alone, a group administered with amrubicin hydrochloride alone, which is an anthracycline anticancer drug, and a combination group with OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug. , each reagent was administered.

As a result, tumor volume was significantly suppressed in the group treated with the combined administration of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug (FIG. 6A). It was also confirmed that long-term combined administration of OBP-801 and amrubicin hydrochloride, an anthracycline anticancer drug, did not affect the animal's body weight (FIG. 6B).

From these results, in the treatment and prevention of tumors, the combined administration of OBP-801 and anthracycline anticancer drugs is very effective in that it can significantly reduce tumor growth. Even during long-term administration, it is effective without causing side effects such as weight loss, indicating that it is suitable for tumor treatment.

[Example 10: When used in combination with amrubicin, OBP-801 showed a stronger cell proliferation inhibitory effect than SAHA. ]

SAHA is the most clinically used HDAC inhibitor. The effects of OBP-801 and SAHA on tumor cell proliferation in combination with amrubicin were investigated. Calu-1 cells were used as tumor cells. OBP-801 or SAHA was used as the HDAC inhibitor. As shown in FIGS. 7A and 7B, treatment of Calu-1 cells with OBP-801 or SAHA alone showed approximately the same tumor cell proliferation inhibitory effect, but combined treatment with OBP-801 and amrubicin showed a more effective suppressive effect on tumor cell growth compared to the combined treatment of SAHA and amrubicin. These results indicate that OBP-801 exhibits a stronger effect than SAHA in combination with amrubicin and is more suitable for tumor treatment.

From the results of the above test examples and examples, it is clear that OBP-801 and amrubicin are effective against refractory cancers such as lung cancer, including lung squamous cell carcinoma, which cannot be treated with OBP-801 or amrubicin alone. The combined use of cyclin anticancer drugs, preferably OBP-801 and amrubicin, which have different drug target molecules, synergistically activates the caspase pathway and controls the mitochondrial apoptosis pathway by regulating the expression and activity of TXNIP and thioredoxin 2. It was revealed that activation can be expected to have a strong apoptosis-inducing effect (FIG. 8).

By using the pharmaceutical composition of the present invention in which a histone deacetylase inhibitor and an anthracycline anticancer drug are used in combination as active ingredients, a synergistic effect of inducing apoptosis and inhibiting tumor growth on tumor cells and tumor tissues can be obtained. This has made it possible to treat and prevent tumors while avoiding side effects caused by the active ingredients. Furthermore, by combining a histone deacetylase inhibitor and an anthracycline anticancer drug, which have different mechanisms of action, synergistic activation of the caspase pathway, TXNIP, and It has become possible to obtain the remarkable pharmacological effect of activating the mitochondrial apoptotic pathway by regulating the expression and activity of thioredoxin 2, and is an effective therapeutic and preventive drug that can be applied to a wide range of tumors, including those for which conventional therapy is less effective. , it has become possible to provide new clinically effective tumor treatment and prevention strategies.

Claims (7)

Formula (III):

(In the formula, R4 represents an isopropyl group.)
1. A pharmaceutical composition for the treatment and prevention of lung cancer , comprising a histone deacetylase inhibitor which is a depsipeptide compound represented by the formula or a pharmaceutically acceptable salt thereof, and amrubicin as active ingredients. Formula (III):

(In the formula, R4 represents an isopropyl group.)
A pharmaceutical composition for the treatment and prevention of lung cancer for use in combination with amrubicin , characterized in that it contains a histone deacetylase inhibitor, which is a depsipeptide compound represented by or a pharmaceutically acceptable salt thereof, as an active ingredient. thing. Formula (III), characterized in that it contains amrubicin as an active ingredient :

(In the formula, R4 represents an isopropyl group.)
A pharmaceutical composition for the treatment and prevention of lung cancer to be used in combination with a histone deacetylase inhibitor which is a depsipeptide compound represented by the formula or a pharmaceutically acceptable salt thereof . Formula (III):

(In the formula, R4 represents an isopropyl group.)
A medicament for treating and preventing lung cancer , which comprises a step of containing as active ingredients a histone deacetylase inhibitor which is a depsipeptide compound represented by the formula or a pharmaceutically acceptable salt thereof, and amrubicin . Method for producing the composition. Formula (III):

(In the formula, R4 represents an isopropyl group.)
A method for treating and preventing lung cancer for use in combination with amrubicin , comprising the step of containing a histone deacetylase inhibitor, which is a depsipeptide compound represented by the formula or a pharmaceutically acceptable salt thereof, as an active ingredient. A method for producing a pharmaceutical composition for use. Formula (III) , characterized by having a step of containing amrubicin as an active ingredient:

(In the formula, R4 represents an isopropyl group.)
A method for producing a pharmaceutical composition for the treatment and prevention of lung cancer for use in combination with a histone deacetylase inhibitor which is a depsipeptide compound or a pharmaceutically acceptable salt thereof . Formula (III):

(In the formula, R4 represents an isopropyl group.)
A pharmaceutical composition containing as an active ingredient a histone deacetylase inhibitor which is a depsipeptide compound or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing amrubicin as an active ingredient . Kits of therapeutic and prophylactic pharmaceutical compositions.

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