WO2020162638A1

WO2020162638A1 - Composition for ameliorating malignant tumor diseases

Info

Publication number: WO2020162638A1
Application number: PCT/JP2020/005170
Authority: WO
Inventors: 升三西田; 正寛椿; 朋也武田; 元三田邉; 森川　敏生
Original assignee: 学校法人近畿大学
Priority date: 2019-02-08
Filing date: 2020-02-10
Publication date: 2020-08-13
Also published as: JPWO2020162638A1

Abstract

Compounds for ameliorating or treating malignant tumor diseases have been proposed, but many of those cannot be orally taken and have placed a burden on patients. Mangiferin has the effect of ameliorating malignant tumor diseases through oral intake, but the effect is small, and it has been realistically not easy to take mangiferin. Furthermore, a novel or improved form of an existing drug for inhibiting a kinase such as NIK has been constantly needed for development of a more effective medicine and the like for treating malignant tumor diseases. According to the present invention, formula (1), formula (2), formula (3), formula (4), formula (5), formula (6), and formula (7) are effective when being orally taken, and can exhibit the effect in small amounts compared to mangiferin.

Description

[Replenishment based on Rule 26 02.03.2020] Composition for improving malignant tumor disease

The present invention relates to a compound that inhibits an NF-κB-inducing kinase (also known as NIK-MAP3K14) useful for treating and/or preventing malignant tumors such as malignant lymphoma and multiple myeloma. The present invention also relates to a composition for preventing and/or treating malignant tumors such as malignant lymphoma and multiple myeloma using the compound, a pharmaceutical composition, and a processed food.

NF-κB (Nuclear factor kappa B) is a transcription factor that regulates the expression of various genes involved in immune response, cell proliferation, apoptosis and carcinogenesis. This NF-κB has five members: NF-κB p65 (p65), RelB, c-Rel, NF-κB1 (which is present in both precursor p105 and truncated p50) and NF-κB2 (which is Present in both precursor p100 and truncated p52). Mainly, NF-κB1 (truncated p50; NF-κBp50) and p65 form a heterodimer, and NF-κB2 (truncated p52; NF-κBp52) and RelB form a heterodimer.

In addition, activation of these NF-κB heterodimers is a signal transduction pathway that is strictly controlled by a series of events including phosphorylation reaction and proteolysis, and includes classical pathway (Canonical pathway) and non-classical pathway (Non-canonical pathway). It is classified into two routes (route).

NIK is a serine/threonine kinase that plays a role in both pathways, but is essential in non-classical signal transduction pathways. It phosphorylates IKKα to partially decompose NF-κBp100, resulting in NF-κBp52. To release.

NF-κBp52 translocates into the nucleus by forming a heterodimer with RelB and expresses the gene. Furthermore, in the classical pathway, IKKα, IKKβ and IKKγ complexes are activated to form a heterodimer of p65 and NF-κBp50, which translocates into the nucleus to regulate gene expression.

NIK is activated by ligands such as B cell activating factor (BAFF), CD40 ligand and tumor necrosis factor α (TNFα), and it is shown that NIK is important for activation of signal transduction pathway by these ligands. ing. Because of its important role, NIK expression is tightly regulated.

Under normal non-stimulation conditions, NIK is degraded by the interaction of TNF receptor-related factor (TRAF), which is a ubiquitin ligase, with NIK, and the amount of NIK protein in cells is small. It is believed that when the non-classical pathway is stimulated by the ligand, the activated receptor causes the TRAF-NIK complex to dissociate, thereby increasing NIK concentration. (Thu and Richmond, Cytokine Growth FR 2010, 21, 213-226: Non-Patent Document 1).

BAFF is produced and secreted from T cells, monocytes/macrophages, dendritic cells, etc., and is known to control B cell differentiation, activation, survival, etc. through three types of receptors on B cells. (Moore, et al., Science. 1999, 285, 260-263: Non-Patent Document 2).

As BAFF receptors, BAFF-R (BAFF-Receptor), TACI (Transmenbrane activator and calcium modulator and cyclophilin ligand interactor), and BMCA (Bcella permuta) are known.

BAFF-R and BMCA are mainly expressed in B cells, and TACI is expressed in B cells and activated T cells. The interaction between BAFF and BAFF-R activates the NIK-mediated nonclassical NF-κB signaling pathway.

It has been shown that the NF-κB pathway is constitutively activated in multiple myeloma (Annuziata, et al. Cancer Cell. 2007, 12, 115-130: Non-Patent Document 3 and Demchenko, et al. Blood 2010, 115, 3541-3552: Non-Patent Document 4). In addition, it has been shown that NIK gene amplification, TRAF gene deletion, and TRAF gene point mutations are observed in patients with multiple myeloma, and these increase NIK protein expression level and activate NIK constantly. It is a factor that activates the NF-κB pathway. It has also been shown that inhibition of NIK with shRNA (NIK shRNA) suppresses NF-κB activation and induces cell death in multiple myeloma cell lines (Annuziata, et al. Cancer Cell. 2007). 12, 115-130: Non-Patent Document 3).

It has also been shown that serum BAFF levels are elevated in patients with multiple myeloma, indicating that BAFF is secreted not only from monocytes and macrophages but also from multiple myeloma cells, and that BAFF It has been reported that autocrine enhances the growth of multiple myeloma cells (Sutherland, et al., Pharmacol. Ther. 2006, 112, 774-786: Non-Patent Document 5 and Novak, et al., Blood, 2004, 103, 689-694: Non-Patent Document 6).

It has been shown that a TRAF gene point mutation and an increase in NIK protein expression are also observed in Hodgkin's lymphoma patients, and it is also reported that NIK shRNA induces cell death (Ranuncolo, et al. , Blood. 2012, 120, 3756-3763: Non-Patent Document 7).

Furthermore, it has been shown that the cell mass of NIK protein is also increased in adult T cell leukemia cells, and that the NIK shRNA treatment suppresses the tumor growth of adult T cell leukemia in vivo (Saitoh, et al., Blood. 2008). , 111, 5118-5129: Non-Patent Document 8).

In addition, the API2-MALT1 fusion protein produced at the chromosomal translocation (t(11;18)(q21;q21)) in Mucosa associated lymphoid tissue (MALT) lymphoma caused protein cleavage at the 325th arginine position of NIK. It has been shown to induce constitutive activation of NIK when performed. This constitutive activation of NIK activates the non-classical NF-κB pathway, which is involved in cell adhesion and resistance to apoptosis (Rosebeck, et al., Science. 2011, 331, 468-472; Patent Document 9).

NIK is highly expressed in the cytoplasm by BAFF stimulation in diffuse large B-cell lymphoma (DLBCL) cells. The activation of NIK due to this high expression is an important signal transduction mechanism involved in lymphoma growth, and NIK shRNA suppresses NIK-induced NF-κB activation in vitro to suppress the growth of DLBCL cell lines. Is shown (Pham, et al. Blood. 2011, 117, 200-210: Non-Patent Document 10).

Moreover, BAFF expression was observed in B lymphoma cells collected from patients with chronic B lymphoma, and it has been shown that this BAFF reduces drug-induced apoptosis (Kern, et al., Blood. 2004, 103, 679-688: Non-Patent Document 11). It has also been reported that BAFF overexpressing mice induce the development of B lymphoma (Sutherland, et al., Pharmacol. Ther. 2006, 112, 774-786: Non-Patent Document 5).

Furthermore, BAFF expression was also found in serum and lymphoma cells of patients with MALT lymphoma, DLBCL, Hodgkin lymphoma and Burkitt lymphoma, which induces cell proliferation and apoptosis resistance of lymphomas, and BAFF high expression lymphoma patients. It has also been shown that the prognosis is poorer than that in low expression patients (He, et al., J. Immunol., 2004, 172, 3268-3279: Non-patent document 12, Novak, et al.,). Blood, 2004, 104, 2247-2253: Non-patent document 13 and Oki, et al, Haematologica. 2007, 92, 269-270: Non-patent document 14).

In T lymphoma collected from a patient with peripheral T lymphoma, NIK expression was higher than that in T cells collected from a healthy person, activating the downstream nonclassical NF-κB pathway, and NF-κB expression in the nucleus. Higher patients have been shown to have a poorer prognosis than lower patients. It has also been shown that treatment of NIK siRNA in peripheral T lymphoma cells can induce cell death (Odqvist, et, al., Clin. Cancer Res. 2013, 19, 2319-2330: Non-patent document 15).

The role of NIK in the growth of tumor cells is not limited to hematopoietic tumors, but it has been shown that NIK is highly expressed in certain pancreatic cancer cell lines, and that cell growth is suppressed by treatment with NIK siRNA (Nishina, et al., Biochem. Biophys. Res. Commun. 2009, 388, 96-101: Non-Patent Document 16).

It has also been shown that the BAFF concentration is higher in the serum of pancreatic cancer patients than in healthy subjects, and that BAFF concentration is correlated with tumor growth and progression of disease stage. Furthermore, it is also reported that BAFF-R is expressed in pancreatic cancer tissue, NF-κBp52 and RelB are highly expressed, and B lymphocytes existing around the pancreatic cancer tissue produce BAFF. (Koizumi, et al., PLoS One. 2013, 8, e71367: Non-Patent Document 17).

It has been reported that high expression of NIK induces constitutive activation of NF-κB in a Basal-like breast cancer cell line (Yamamoto, et al., Cancer Sci. 2011.101, 391-2397: non-patent reference). Reference 18).

In addition, in the case of malignant melanoma, analysis by tissue microarray analysis showed that NIK expression was significantly higher than that in benign tissue, and NIK shRNA suppressed tumor growth, induced apoptosis, and arrested cell cycle in vivo. It is recognized (Thu, et al., Oncogene. 2012, 31, 2580-2592: Non-Patent Document 19).

Furthermore, it has been shown that NF-κB is activated in non-small cell lung cancer tissues and cell lines, and it is also confirmed that treatment with NIK siRNA inhibits apoptosis induction and anchorage-independent cell proliferation (Saitoh. , Et al., Lung Cancer 2010, 70, 263-270: Non-Patent Document 20).

It has been shown that NIK is highly expressed also in liver cancer patients and liver cancer cell lines, and NIK siRNA treatment and miR-520e that reduces NIK expression suppress cell growth in vitro and tumor growth in vivo. (Zhang, et al., Oncogene, 2012, 31, 3607-3620: Non-Patent Document 21).

Helicobacter pylori is known to be deeply involved in the development of gastric cancer, and in patients infected with this bacterium, NIK is constantly activated at the site of gastric infection, and the non-classical NF-κB pathway is activated. It is shown. It has also been reported that gastric cancer cells introduced with a mutant that suppresses NIK activation suppress NF-κB activation by Helicobacter pylori (Feige, et al., Biochim. Biophys. Acta Mol. Cell Res. 2018, 1865, 545-550: Non-Patent Document 22 and Maeda, et al, Gastroenterology. 2000, 119, 97-108: Non-Patent Document 23).

Activation of the non-classical NF-κB pathway is associated with the development of colitis and colon cancer, and in mice lacking NLRP12, which negatively regulates NIK, non-classical NF-κB is mediated through NIK activation. The pathway has been shown to activate and induce colitis and cancer. In addition, it was reported that the expression of OLFM1 that negatively regulates NIK in colon cancer patients was higher in the non-cancerous part than in the cancerous part, and that cell growth and motility were suppressed by treating NIK siRNA in colonic cancer cells. (Allen, et al. Immunity 2012, 36, 742-754: Non-patent document 24 and Shi, et al., J. Pathol. 2016, 240, 352-365: Non-patent document 25)

NIK and RelB are highly expressed in head and neck tumor cells, and it has been shown that treatment of NIK and RelB siRNAs suppresses tumor cell motility and invasion (Das, et al., Mol. Carcinog. 2018). , In press: Non-Patent Document 26).

Overexpression of NIK and RelB has been observed in renal cancer patients, and it has been shown to reduce the 10-year survival rate compared to patients with low expression, and the state of NIK expression is a prognostic factor. Have been reported (Lua, et al., Urol. Int. 2018, 101, 190-196: Non-Patent Document 27).

It has been shown that NIK overexpression promotes tumorigenesis in glioma cells, and that non-classical NF-κB pathway activation by NIK activation enhances glioma cell motility and invasion (Cherry, et. al., Mol. Cancer. 2015, 14, 9: Non-Patent Document 28).

Compared to normal ovarian tissue, NIK mRNA expression was higher in ovarian cancer patient tissue, and the NIK/NF-κB p52 (non-classical) pathway was activated in ovarian cancer cells, and treatment with NIK shRNA resulted in scaffold dependence. It has been shown to suppress sexual and non-anchorage-dependent cell growth, and also to suppress tumor growth in vivo with NIK shRNA (Uno, et al., PLoS One. 2014, 9, e88347: non- (Patent Document 29)

In patients with endometrial cancer, it has been shown that the degree of differentiation of cancer tissues is low and that NIK activation is high as the disease progresses, and it has been reported that NIK activation suppresses apoptosis of cancer cells. (Zhou, et al., Int. J. Gynecol. Cancer. 2015, 25, 770-778: Non-Patent Document 30).

As described above, multiple myeloma, malignant lymphoma (MALT lymphoma, DLBCL, Burkitt lymphoma, Hodgkin lymphoma, adult T cell leukemia, peripheral T lymphoma, etc.), pancreatic cancer, breast cancer, malignant melanoma, lung cancer, liver cancer, gastric cancer, In malignant tumors such as colorectal cancer, head and neck tumor, glioma, renal cancer, ovarian cancer and endometrial cancer, BAFF overexpression and NIK overexpression occur, and NIK activation can activate NF-κB. It can be said that the causative disease is already scientific common sense.

Therefore, BAFF overexpression, NIK and non-classical NF can be used as drugs capable of inhibiting NIK activation and NIK activation associated with BAFF overexpression and suppressing the non-classical NF-κB signaling pathway. -Has a therapeutic effect on malignant tumors with over-activation of κB signaling.

Also, multiple myeloma is known to be accompanied by a characteristic symptom called CRAB (hypercalcemia, renal disorder, anemia, bone lesion).

For the development of CRAB, osteoclasts such as monoclonal immunoglobulin (M protein) expressed by multiple myeloma cells, immunoglobulin free light chain, interleukin 6 (IL-6) and Macrophage inflammatory protein 1α (MIP-1α) It has been reported that a cell activating factor is involved (Rajkumar, et al., Lancet Oncol. 2014, 15, e538-548: Non-Patent Document 31; Harmer, et al., Front. Endocrinol. 2019, 9, 9. 788: Non-patent document 32; Roussou, et al., Leukemia. 2009, 23, 2177-2181: Non-patent document 33).

M protein and immunoglobulin free light chain due to multiple myeloma are known to cause renal damage by depositing in the kidney, and also deposit in systemic organs, causing amyloidosis in various organs, resulting in neuropathy. , Causes various symptoms such as arrhythmia. In addition, it causes hyperviscosity syndrome in blood.

IL-6 secretion in multiple myeloma has been shown to enhance osteoclast differentiation and activate osteoclasts, which have been reported to lead to bone lesions (Harmer, et al., Front. Endocrinol. 2019, 9, 788: Non-Patent Document 32). MIP-1α secreted by multiple myeloma has also been shown to enhance osteoclast differentiation and activation, and to promote bone lesions (Roussou, et al., Leukemia. 2009, 23. , 2177-2181: Non-patent document 33; Tsubaki, et al., J. Cell. Biochem. 2010, 111, 1661-1672: Non-patent document 34). It has also been reported that hypercalcemia is caused by progression of bone lesions due to these factors (Lee, et al., Intern. Med. J. 2017, 47, 938-951: Non-patent document 35). That is, the osteoclast activating factor causes bone lesions, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms.

It has been reported that myeloma develops and expression of M protein is enhanced in NF-κB2 mutant mice (McCarhty, et al., BMC Cancer. 2012, 12, 203: Non-Patent Document 36). Furthermore, activation of the NF-κB pathway has also been reported to enhance the expression of osteoclast activating factors such as IL-6 and MIP-1α (Vrabel, et al., Blood Rev. 2019, 34. , 56-66: Non-Patent Document 37).

As described above, it is already common scientific knowledge that in multiple myeloma, activation of the NF-κB pathway causes M protein expression and bone lesions due to osteoclast activating factors.

Therefore, drugs capable of inhibiting NIK activation and NIK activation associated with NIK overexpression and suppressing the non-classical NF-κB signal transduction pathway include M protein and immunoglobulin free release due to multiple myeloma. Disorder due to increased chain expression, amyloidosis, bone lesion (bone destruction) due to hyperviscosity syndrome or increased expression of osteoclast activating factor, hypercalcemia associated with bone lesion, pathological fracture, spinal cord compression fracture, spinal cord compression It has therapeutic effects on spinal cord compression symptoms associated with fractures and neurological symptoms associated with spinal cord compression symptoms. That is, it has a therapeutic effect on CRB.

Patent Document 1 (Japanese Patent Publication No. 2016-531858) discloses 3-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-c]pyridine derivatives as NIK inhibitors. .. Here, it is shown that the compound is an inhibitor of NF-κB-inducible kinase also known as NIK-MAP3K14, and that the compound is a compound for use in the prevention or treatment of cancer. Is recognized as a right.

Note that, in Patent Document 1, the compound only cites EC50s against three types of cancer cells JJN-3, L-363, and LP-1 (all of which are multiple myeloma cell lines) as an example. From the above, it is recognized as a common general knowledge that NIK inhibitors have a wide therapeutic effect on malignant cancer cells.

Further, Patent Document 2 (Japanese Patent Laid-Open No. 07-082263) discloses that a xanthone compound having a structure of formula (10) has anticancer activity.

Note that (R ¹ to R ⁷ : H, —OH, C1-6 alkyl, C1-6 alkoxy, epoxypropoxy) and a benzophenone compound are represented.

Further, Patent Document 3 (JP-A-2017-031146) describes that mangiferin inhibits NIK and is effective for multiple myeloma and malignant melanoma. As described above, compounds having a xanthone skeleton have been found to have anticancer activity.

Special table 2016-531858 gazette JP-A-7-082263 JP, 2017-031146, A

As shown in Patent Document 1, a chemical substance is used as a drug that suppresses NIK. However, except for Mangiferin of Patent Document 3, the drug administration route is often intravenous administration, which imposes a heavy burden on the patient. Has become. Therefore, it is an object of the present invention to provide a therapeutic agent and an improved composition that inhibit NIK even if it is orally administered and improve malignant tumors such as malignant lymphoma and multiple myeloma.

Also, the mangiferin of Patent Document 3 is effective by oral administration, but the required amount of intake is large, and it could not be easily taken even by oral administration. Therefore, more effective or more active therapeutic agents and improved compositions have been sought.

Furthermore, new or improved forms of existing drugs that inhibit kinases such as NIK are always needed to develop more effective drugs etc. for treating malignant tumors.

The present inventors searched for a compound that inhibits NIK to solve the above problems, and found that the substance having a xanthone skeleton has an NIK inhibitory action including a substance having a structure that has not been found so far. It was confirmed that they actually induce cell death in malignant lymphoma and multiple myeloma.

Moreover, it was confirmed that the tumor growth of malignant lymphoma and multiple myeloma in vivo was significantly suppressed. Furthermore, it was confirmed that the expression of CD138, which is a malignancy marker for multiple myeloma, and the expression of CD20, which is a B cell marker, were increased, and the production of monoclonal immunoglobulin and immunoglobulin free light chain in multiple myeloma was confirmed. It was also confirmed that the production and the factor for inducing bone destruction are inhibited, and the present invention has been completed.

That is, the composition for improving malignant tumor disease according to the present invention has the following formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and (7). It is characterized in that it comprises a compound of formula.

Further, the composition for improving malignant tumor disease containing the above compound can be used as a composition for improving malignant tumor disease related to NIK overexpression and malignant tumor disease related to BAFF overexpression. It can also be used as a therapeutic drug for malignant tumor diseases.

Also, the above compounds can be used as a dedifferentiation inducer for plasma cells that have become malignant tumors into B cells. Furthermore, the above compound can be used as a remedy for a concomitant symptom called CRAB (hypercalcemia, renal disorder, anemia, bone lesion) characteristic of multiple myeloma.

The therapeutic agent for malignant tumors such as malignant lymphoma and multiple myeloma and the improving composition according to the present invention can improve malignant lymphoma and multiple myeloma. For example, cell death can be effectively induced in malignant tumor cells such as malignant lymphoma cells and multiple myeloma cells. It also does not affect normal cells at concentrations that induce cell death in malignant tumor cells.

Moreover, it is considered that the above-described malignant tumor enhances the development, proliferation and survival of malignant tumor cells by overexpression of NIK protein. Therefore, the composition for improving malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention is considered to have an improving effect not only on the malignant tumors shown in Examples but also on other malignant tumors. ..

Furthermore, it is possible to inhibit the production of monoclonal immunoglobulin, immunoglobulin free light chain production and bone destruction inducing factor production associated with multiple myeloma. Therefore, it is associated with renal disorders due to the increased expression of M protein and immunoglobulin free light chain associated with these production, amyloidosis, bone lesions (bone destruction) due to hyperviscosity syndrome and increased expression of osteoclast activating factor, and bone lesions. It is possible to suppress hypercalcemia, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms. That is, the expression of CRAB can be suppressed.

It is a figure which shows the synthetic|combination procedure of noraciriol. It is a graph which shows the result examined by the trypan blue dye method about the cell death induction effect by each chemical|medical agent using Raji cell. FIG. 6 is a graph showing the results of examining the cell death-inducing effect of each drug by the trypan blue dye method using CCRF-SB cells. It is a graph which shows the result examined by the trypan blue dye method about the cell death induction effect by each chemical|medical agent using Namalwa cell. It is a graph which shows the result examined by the trypan blue dye method about the cell death induction effect by each chemical|medical agent using Z138 cell. 7 is a graph showing the results of examining the cell death-inducing effect of each drug by the trypan blue dye method using SU-DHL-5 cells. It is a graph which shows the result examined by the trypan blue dye method about the cell death induction effect by each chemical|medical agent using L363 cell. FIG. 6 is a graph showing the results of examining the cell death-inducing effect of each drug by the trypan blue dye method using KMS-28BM cells. It is a graph which shows the result examined by the trypan blue dye method about the cell death induction effect by each chemical|medical agent using ARH77 cell. It is a graph which shows the result of having examined the cell death induction effect by each chemical|medical agent by the trypan blue dye method using IM9 cell. It is a graph which shows the result of having examined the cell death induction effect by each chemical|medical agent by the trypan blue dye method using RPMI1788 cell. 6 is a photograph showing the results of immunoblotting of the NIK inhibitory action of each drug using IM9 cells. 6 is a photograph showing the results of immunoblotting of activation kinetics of IKK, NF-κB p65, and NF-κB p52, which are downstream signals of NIK, using IM9 cells, depending on each drug. It is a panel which shows the result of having investigated CD138 expression suppression by each drug using L363 cell by Flow cytometry. It is a panel which shows the result of having investigated CD138 expression suppression by each drug using L363 cell by Flow cytometry. It is a panel which shows the result of having examined CD20 expression increase by each chemical|medical agent by Flow cytometry using L363 cell. It is a panel which shows the result of having examined CD20 expression increase by each chemical|medical agent by Flow cytometry using L363 cell. It is a graph which shows the result investigated by the enzyme-linked immunosorbent assay (ELISA) about the IgG secretion suppression by each drug using L363 cell. It is the result of having examined by the ELISA method the secretion suppression of the lambda chain of the immunoglobulin free light chain by each drug using L363 cells. Fig. 6 is a graph showing the results of an ELISA method using L363 cells to examine the inhibitory effect on bone destruction-related factors by each drug. It is a panel which shows the result of having examined CD138 expression suppression by Mangiferin, Mangiferin 8a, and noraciriol in KMS-28BM cell by Flow cytometry. It is a panel which shows the result of having examined CD20 expression increase by mangiferin, mangiferin 8a, and noraciriol using KMS-28BM cell by Flow cytometry. FIG. 9 is a graph showing the results of an ELISA method using KMS-28BM cells to examine IgG secretion inhibition by mangiferin, mangiferin 8a, and noraciriol. FIG. 9 is a graph showing the results of an ELISA method using KMS-28BM cells to suppress the secretion of the λ chain of the immunoglobulin free light chain by mangiferin, mangiferin 8a, and noraciriol. FIG. 7 is a graph showing the results of an examination by ELISA using KMS-28BM cells for the effect of suppressing bone destruction-related factors by mangiferin, mangiferin 8a, and noraciriol. 8 is a graph showing the time course of tumor growth when Raji cells were transplanted into NOD/ShiJic-scidJcl mice and noraciriol was orally administered. FIG. 26 is a photograph showing a tumor of an untreated mouse and a tumor of a mouse to which noractiliole was administered on day 27 in FIG. 26. FIG. 6 is a graph showing the time course of tumor growth when L363 cells were transplanted into NOD/ShiJic-scidJcl mice and noraciriol was orally administered. FIG. 29 is a photograph showing a tumor of an untreated mouse and a tumor of a mouse to which noractiliole was administered on day 8 in FIG. 28.

The composition for improving malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention will be described below. The following description is an exemplification of one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following embodiments can be modified without departing from the spirit of the present invention.

The therapeutic agent for malignant tumor such as malignant lymphoma and multiple myeloma according to the present invention has a xanthone skeleton (1), (2), (3), (4), (5), (6) ) And a substance represented by the formula (7) are contained as active ingredients. The formula (1) is 1,2',3',4',6-penta-O-propionyl mangiferin, and is hereinafter referred to as "mangiferin 8a". In addition, mangiferin is shown in Formula (8).

The formula (2) is 1,3,6,7-tetrahydroxyxanthone, which is hereinafter referred to as “norachiriol”. The formula (3) is 1,3,5,6-tetrahydroxyxanthone and is hereinafter referred to as "tetrahydroxyxanthone". The formula (4) is 1,3,6-trihydroxy-methoxyxanthone and is hereinafter referred to as “methoxyxanthone”. The formula (5) is 9-hydroxyxanthene, and is hereinafter referred to as "xanthydrol". The formula (6) is 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one. α-Mangostin”. The formula (7) is 1,3,6,7-tetrahydroxy-2,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one. Mangosteen” is called.

As can be seen from the formula (1), mangiferin 8a is obtained by ether-bonding a propionyl group to a part of the hydroxy groups of mangiferin (see the formula (8)). As shown in the Examples below, this compound exhibits superior inhibition of NIK and IKK activity than mangiferin and a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

Noraciliol (CAS number 3542-72-1) is a part of xanthone (CAS number: 90-47-1) to which a hydroxy group is attached, and has a xanthone skeleton like mangiferin 8a. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

Tetrahydroxyxanthone (CAS number 5084-31-1) is a part of xanthone to which a hydroxy group is attached and has a xanthone skeleton like mangiferin 8a. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

Methoxyxanthone (CAS number 41357-84-0) is a part of xanthone in which a hydroxy group and a part of the hydroxy group are ether-bonded with a methyl group, and has a xanthone skeleton like mangiferin 8a. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

Xanthohydrol (CAS number 96-46-0) has a hydroxy group at the position of the ketone of xanthone and has a xanthone skeleton. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

Α-Mangosteen (CAS number 6147-11-1) is a xanthone having a bismethylbutenyl group and a hydroxy group bonded to a part thereof and has a xanthone skeleton. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

γ-Mangostin (CAS No. 96-46-0) is a xanthone having a bismethylbutenyl group, a hydroxyl group and a methyl group bonded to this hydroxyl group, and having a xanthone skeleton. There is. And, like Mangiferin 8a, it exhibits more NIK and IKK activity inhibition than Mangiferin, and shows a high cell death-inducing effect on malignant tumors such as malignant lymphoma and multiple myeloma.

　These compounds are relatively low molecular weight compounds like mangiferin, they dissolve well in water and can be taken orally. The composition for improving malignant tumor disease according to the present invention contains at least one of these seven compounds as an active ingredient. It may also contain other pharmaceutically acceptable ingredients.

The composition for improving malignant tumor disease according to the present invention can be provided as a therapeutic agent for malignant tumor disease (a pharmaceutical composition containing an agent for inducing dedifferentiation into B cells and a CARB improving agent). The composition according to the present invention as a pharmaceutical composition can exert its effect by oral administration. Therefore, it can be provided as an internal preparation. For example, the powdery composition for improving malignant tumor disease may be provided in the form of a capsule, granule, powder, tablet or the like. In the case of oral preparations, additives such as binders, lubricants, disintegrating agents, coloring agents, flavoring agents, preservatives, antioxidants, stabilizers are added, and capsules, granules, powders and tablets are added. It can be produced by a conventional method.

Furthermore, the pharmaceutical composition according to the present invention may be formulated into an external preparation such as a solution, an ointment, a cream, a gelling agent, a patch, and an aerosol, and may be administered parenterally. When used as an external preparation, water, a lower alcohol, a solubilizing agent, a surfactant, an emulsion stabilizer, a gelling agent, an adhesive, and other necessary base components can be added. Further, additives such as a vasodilator, an adrenocortical hormone, a keratolytic agent, a moisturizer, a bactericide, an antioxidant, a cooling agent, a fragrance, and a pigment may be appropriately mixed.

Also, the composition for improving malignant tumor disease according to the present invention can be provided as a processed food. That is, the composition for improving malignant tumor disease according to the present invention has the same effect as that of the composition for improvement according to the present invention, even when ingested as a processed food.

As the processed food according to the present invention, for example, candy, gum, jelly, biscuits, cookies, rice crackers, bread, noodles, fish meat/meat paste products, tea, soft drinks, coffee drinks, milk drinks, whey drinks, lactic acid bacteria drinks , Yogurt, ice cream, pudding, etc., as well as general processed foods, including health foods, as well as health foods such as specified health foods and nutrition foods specified by the Ministry of Health, Labor and Welfare system. Further, processed foods include dietary supplements, feeds, food additives and the like.

The processed food according to the present invention can be prepared by adding the composition for improving malignant tumor disease to the raw material of these processed foods. It should be noted that, when adding noraciliol, α-mangostin and γ-mangostin to these processed foods, the use of a material containing noraciliol, α-mangostin and γ-mangostin as a raw material is excluded from the application. Hereinafter, the composition for improving malignant tumor disease according to the present invention will be described based on Examples.

<Example 1: Synthesis of Mangiferin 8a>
Mangiferin 8a was obtained in two steps by synthesizing 1,3,2',3',4',6,6',7-octa-O-propionyl mangiferin as a raw material and further synthesizing it. ..
(1) Synthesis of 1,3,2',3',4',6,6',7-octa-O-propionyl mangiferin

Mangiferin (2.1 g, 4.98 mmol), propionic anhydride (12.8 mL, 99.4 mmol) and dry pyridine (60 mL) were heated at 80°C for 5 hours. The reaction solution was poured into ice water (400 mL) and extracted with ethyl acetate. The ethyl acetate layer was washed successively with ice-cooled 10% sulfuric acid, saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (n-hexane/ethyl acetate=1/1), and 1,3,2′,3′,4′,6,6′,7-octa of the formula (9) was used. O-propionyl mangiferin (3.71 g, 86%) was obtained as a colorless solid.

¹ H-NMR (800 MHz, DMSO-d ₆ )δ: 0.70-1.26 (24H, m, COCH ₂ CH ₃ ), 1.92-2.89 (16H, m, COCH ₂ CH ₃ ), 3.83-3.91 (1H, m, H-6'a), 4.05-4.15 (2H, m, H-5' and H-6'b), 4.98-5.05 (1H, m, H-4'), 5.09-5.21 (1H, m, H-1'), 5.43-5.51 (2H, m, H-3' and H-2' ), 7.50-7.54 (1H, m, H-4), 7.68-7.71 (1H, m, H-5), 7.96-7.98 (1H, m, H-) 8).
¹³ C-NMR (200 MHz, DMSO-d ₆ ) δ:8.68/8.77/8.79/8.92/8.95/8.98/9.00/9.01/9.04/ 9.06/9.17/9.19/9.28 (COCH ₂ CH ₃ ), 26.6/26.7/26.9/26.8/26.93/26.97/27.00/ _{_{27.09 / 27.35 / 27.37 (COCH 2}} CH 3), 62.1 / 62.2 (C6 '), 68.20 / 68.23 (C4'), 69.6 / 70.1 ( C2'), 70.9/71.3 (C1'), 73.6/73.7 (C3'), 75.06/7.10 (C5'), 110.3/111.8 (C4) , 111.7/112.6 (C9a), 113.3/113.4 (C5), 118.9/119.0 (C2), 119.7/119.7 (C8a), 120.36/120 .40 (C8), 139.57/139.59 (C7'), 147.9 (C6), 149.1/150.9 (C1), 152.5/152.6 (C8b), 153.4. /154.8 (C4a), 156.6/156.8 (C3), 171.09/171.13/171.15/171.20/171.72/171.79/171.82/171.93 /172.37/172.44/172.78/172.87/173.16/1733.23/173.26 (COCH ₂ CH ₃ ), 173.56/173.60 (C9).
HRMS (FAB) m/z: [M+Na] ⁺ Calcd for C ₄₃ H ₅₀ O ₁₉ Na 893.844; Found 893.883.

(2) Synthesis of mangiferin 8a (1,2′,3′,4′,6-penta-O-propionyl mangiferin) 1,3,2′,3′,4′,6,6′,7-octa A mixed solution of -O-propionyl mangiferin (3.7 g, 4.25 mmol), ammonium acetate (3.8 g, 49.4 mmol), methanol (80 mL) and water (40 mL) was stirred at room temperature for 5.5 hours. After methanol was distilled off from the reaction solution by concentration under reduced pressure, the residue was diluted with ethyl acetate (100 mL). The mixture was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography [CHCl ₃ /CH ₃ OH (20:1)] and the obtained pale yellow solid (2.54 g) was dissolved in methanol (80 mL) and decolorized with activated carbon (1 ) Mangiferin 8a of the formula) (2.16 g, 73%) was obtained as a colorless solid.

¹ H-NMR (800 MHz, DMSO-d ₆ )δ::0.92-1.24 (15H,m,COCH ₂ CH ₃ ), 1.92-2.32 (10H,m,COCH ₂ CH ₃ ). , 3.85 (0.5H, dd-like, J=ca. 12.5, 1.5, H-6a'), 4.02-4.13 (2H, m, H-5', H-. 6a′, H-6b′), 4.21 (0.5H, dd, J=12.5, 4.7, H-6b′), 4.95 (0.5H, d, J=10.0) , H-1′), 5.00 (0.5H, dd, J=9.6, 9.6, H-1′), 5.01 (0.5H, dd, J=9.6, 9) .6, H-4′), 5.15 (0.5H, d, J=10.0, H-1′), 5.31 (0.5H, dd, J=9.6, 9.6) , H-3′), 5.39 (0.5H, dd, J=9.6, 9.6, H-3′), 5.48 (0.5H, dd, J=10.0, 9) .6, H-2'), 5.84 (0.5H, dd, J = 10.0, 9.6, H-2'), 6.71/6.73 (each 0.5H, s, H-4), 6.79/6.80 (each 0.5H, s, H-5), 7.278/7.279 (each 0.5H, s, H-8), 9.00-11 .5 (3H, br s, OH x 3).
¹³ C-NMR (200 MHz, DMSO-d ₆ )δ::8.84/8.89/9.00/9.05/9.10/9.14/9.15/9.21/9.23 (COCH ₂ CH ₃ ), 26.7/26.92/26.96/26.98/27.04/27.08/27.12/27.4 (COCH ₂ CH ₃ ), 62.1/62 .4 (C-6'), 68.2/68.4 (C-4'), 68.8/70.5 (C-2'), 71.0/71.1 (C-1') , 73.8/74.2 (C-3'), 74.7/75.3 (C-5'), 99.6/100.8 (C-4), 102.50/102.52 ( C-5), 106.6/107.7 (C-9a), 109.1 (C-8), 113.4 (C-2), 114.0/114.1 (C-8a), 143 .87/143.88 (C-7), 149.83/149.89 (C-6), 151.3 (C-1), 153.4 (C-8b), 157.6/157.7 (C-4a), 161.0/162.4 (C-3), 171.2/172.0/172.3/172.9/173.2/173.5/173.6 (COCH ₂ CH ₃ ) 172.7/172.8 (C-9).
HRMS (FAB) m/z: [M+Na] ⁺ Calcd for C ₃₄ H ₃₈ NaO ₁₆ 725.2058; Found. 725.2074.

<Example 2: Synthesis of noraciriol>
Noracilliol was synthesized according to the method of Non-Patent Document 38.
The synthesizing method shown in this example is briefly described as follows. That is, 2,4,5-trimethoxybenzoic acid (Compound II) is treated with thionyl chloride according to the method described in the literature (Non-Patent Document 38) to obtain 2,4,5-trimethoxybenzoic acid chloride (Compound III). It was Then, 2-hydroxy-2',4,4',5,6'-is obtained by Friedel-Crafts reaction between the obtained compound (Compound III) and 1,3,5-trimethoxybenzene (Compound IV). Pentamethoxybenzophenone (Compound V) was obtained. Further, this compound (V) is treated with tetrabutylammonium hydroxide to obtain 1,3,6,7-tetramethoxyxanthone (Compound VI), and then demethylation is carried out to obtain noratyliol (Compound I). Obtained in a yield of 39%.

The synthetic route of this example is described in detail below. Note that FIG. 1 is referred to.
(1) Method for producing 2,4,5-trimethoxybenzoic acid chloride (Compound III) 2,4,5-Trimethoxybenzoic acid (Compound II, 8.49 g, 0.040 mol) in an argon atmosphere at room temperature and thionyl chloride. (5 mL) was gradually added and dissolved, and then heated under reflux for 6 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give 2,4,5-trimethoxybenzoic acid chloride (Compound III, 8.30 g, 90%). The obtained compound (III) was immediately used for the next reaction.

(2) Method for producing 2-hydroxy-2',4,4',5,6'-pentamethoxybenzophenone (compound V) 2,4,5-trimethoxybenzoic acid chloride (compound obtained as described above III, 8.07 g, 0.035 mol), 1,3,5-trimethoxybenzene (compound IV, 6.48 g, 0.0385 mol) and anhydrous diethyl ether (500 mL) in a mixed suspension at room temperature under an argon atmosphere. After gradually adding aluminum chloride (16 g) at room temperature, the reaction mixture was stirred at room temperature for 48 hours. The solvent of the reaction solution was evaporated under reduced pressure, water was added to the residue, and the mixture was extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off with fold-fold filter paper, and the filtrate was evaporated under reduced pressure to give a crude product. The crude product thus obtained was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:1, v/v) to give 2-hydroxy-2′,4,4′,5,6′-pentamethoxy. Benzophenone (Compound V, 8.43 g, 69%) was obtained.

(3) Method for producing 1,3,6,7-tetramethoxyxanthone (Compound VI) 2-hydroxy-2′,4,4′,5,6′-pentamethoxybenzophenone (obtained as described above Compound V (6.97 g, 0.020 mol) was dissolved in a mixed solvent of pyridine (10 mL) and water (10 mL), 40% tetrabutylammonium hydroxide aqueous solution (5 mL) was added, and the mixture was heated under reflux for 6 hr. The obtained reaction mixture was poured into 5% hydrochloric acid and then extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off with fold-fold filter paper, and the filtrate was evaporated under reduced pressure to give a crude product. The obtained crude product was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:1, v/v) to give 1,3,6,7-tetramethoxyxanthone (Compound VI, 5.82 g, 92%).

(4) Method for producing noraciriol (Compound I) 1,3,6,7-tetramethoxyxanthone (Compound VI, 4.74 g, 0.015 mol) obtained as described above and pyridine hydrochloride (5.00 g) The mixture of 1) was heated and stirred at 200° C. for 6 hours. The obtained reaction mixture was allowed to cool to room temperature, poured into 5% hydrochloric acid, and extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off with fold-fold filter paper, and the filtrate was evaporated under reduced pressure to give a crude product. The obtained crude product was purified by silica gel column chromatography (chloroform:methanol=7:1, v/v) to obtain a noratyliol of the formula (2) (Compound I, 2.65 g, 68%).

<Example 3: Examination of cell death-inducing effect of each drug on Raji cells>
Raji cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. Raji cells were seeded on a 96-well plate, each concentration of drug was added, and cell viability was measured by the trypan blue dye method. The results are shown in Figure 2.

In the graph, the mangiferin 8a-administered group is "Mangeriferin 8a", the noraciriol-administered group is "Noratyryolol", the tetrahydroxanthone-administered group is "1,3,5,6-tetrahydroxyxanthone", and the methoxyxanthone-administered group is "1". , 3,6-trihydroxy-5-methoxyxanthone", xanthodol-administered group was "Xanthydolol", α-mangostin-administered group was "α-mangostin", and γ-mangostin-administered group was "γ-mangostin" (hereinafter the same). ..

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. Further, Table 1 shows the results of calculating IC50 (half-maximum inhibitory concentration: the same applies hereinafter). Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 4: Examination of cell death inducing effect of each drug on CCRF-SB cells>
CCRF-SB cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding CCRF-SB cells in 96-well plate, each concentration of drug was added, and cell viability was measured by trypan blue dye method. Results are shown in FIG.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 2 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 5: Examination of cell death inducing effect of each drug on Namalwa cells>
Namalwa cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. Namalwa cells were seeded on a 96-well plate, drugs at various concentrations were added, and cell viability was measured by the trypan blue dye method. The results are shown in Fig. 4.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 3 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 6: Examination of cell death inducing effect of each drug on Z138 cells>
Z138 cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. Z138 cells were seeded on a 96-well plate, each concentration of drug was added, and cell viability was measured by the trypan blue dye method. Results are shown in FIG.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 4 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 7: Examination of cell death-inducing effect of each drug on SU-DHL-5 cells>
SU-DHL-5 cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. SU-DHL-5 cells were seeded on a 96-well plate, drugs at various concentrations were added, and the cell viability was measured by the trypan blue dye method. Results are shown in FIG.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 5 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noraciliol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 8: Examination of cell death inducing effect of each drug on L363 cells>
L363 cells (multiple myeloma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding L363 cells in 96-well plate, each concentration of drug was added, and cell viability was measured by trypan blue dye method. The results are shown in Fig. 7.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 6 shows the results of calculating IC50 values. In the mangiferin 8a-administered group, the noratiriol-administered group, the tetrahydroxyxanthone-administered group, the methoxyxanthone-administered group, the xanthohydrol-administered group, and the γ-mangostin-administered group, it was observed that cell death was induced at a concentration lower than that of mangiferin.

<Example 9: Examination of cell death inducing effect of each drug on KMS-28BM cells>
KMS-28BM cells (multiple myeloma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding KMS-28BM cells on 96-well plate, each concentration of drug was added and cell viability was measured by trypan blue dye method. The results are shown in Fig. 8.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 7 shows the results of calculating IC50 values. In the mangiferin 8a-administered group, the noratiriol-administered group, the tetrahydroxyxanthone-administered group, the methoxyxanthone-administered group, the xanthohydrol-administered group, and the γ-mangostin-administered group, it was observed that cell death was induced at a concentration lower than that of mangiferin.

<Example 10: Examination of cell death inducing effect of each drug on ARH77 cells>
ARH77 cells (multiple myeloma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. ARH77 cells were seeded on a 96-well plate, each concentration of drug was added, and cell viability was measured by the trypan blue dye method. The results are shown in Fig. 9.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 8 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 11: Examination of cell death inducing effect of each drug on IM9 cells>
IM9 cells (multiple myeloma cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. IM9 cells were seeded on a 96-well plate, each concentration of drug was added, and the cell viability was measured by the trypan blue dye method. The results are shown in Fig. 10.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. A decrease in cell viability was confirmed in a concentration-dependent manner for all the drugs. In addition, Table 9 shows the results of calculating IC50 values. Inducing cell death at a lower concentration than mangiferin in the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group. Was recognized.

<Example 12: Examination of cell death inducing effect of each drug on RPMI1788 cells>
RPMI1788 cells (normal B lymphocyte cell line) were cultured under the conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. RPMI1788 cells were seeded on a 96-well plate, added with each concentration of drug, and the cell viability was measured by the trypan blue dye method. The results are shown in Fig. 11.

The horizontal axis shows the concentration of each drug and the vertical axis shows the cell viability (%). In addition, a control was prepared by adding only 0.5% DMSO in PBS used for dissolving the reagent. No decrease in cell viability was observed with any of the drugs. In addition, Table 10 shows the results of calculating IC50 values. IC50 values of all drugs were 100 μM or more.

Therefore, it was found that the composition for improving malignant tumor disease according to the present invention induces cell death in malignant tumor cells at a concentration that does not affect normal cells.

<Example 13: Examination of NIK activation inhibition by administration of each drug>
As a result of investigating the NIK inhibitory action by each drug administration using IM9 cells by immunoblotting, the NIK activation inhibitory action was recognized (FIG. 12).

IM9 cells were seeded in a 150 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 72 hours (Control). IM9 cells were seeded in a 150 cm ² flask and precultured for 24 hours, then 100 μM mangiferin, 10

μM Mangiferin

8a, 10 μM noractilol, 10 μM methoxyxanthone, 50 μM tetrahydroxyxanthone, 10 μM xanthohydrol, 50 μM α-mangostin and 50 μM γ-mangostin were added to final concentrations, and at 37° C. and 5% CO ₂ conditions. Cultured for 72 hours. Proteins were extracted from these cell suspensions with a cell lysate and used as samples. Note that 50 μM DMF, which is an NF-κB p65 nuclear translocation inhibitor, was used as a target.

Each sample was subjected to SDS-PAGE, transferred to a PVDF membrane, and examined for phosphorylation of NIK using an anti-phospho-NIK antibody and an anti-NIK antibody.

Figure 12 shows the results of immunoblotting. In the horizontal direction of the photograph, Control, 50 μM DMF, 100 μM mangiferin, 10

μM mangiferin

8a, 10 μM noraciriol, 10 μM methoxyxanthone, 50 μM tetrahydroxyxanthone, Control, 10 μM xanthohydrol, 50 μM α-mangosteen, and 50 μM γ-mangosteen were added. The cases are shown side by side.

▽The antibody type is shown in the vertical direction. Specifically, the case of an anti-phospho-NIK antibody (denoted as "Phospho-NIK") and the case of an anti-NIK antibody (denoted as "NIK") are shown. In the photograph of the anti-NIK antibody, no decrease in NIK band intensity was observed as compared with Control. On the other hand, in the case of the anti-phospho-NIK antibody, the immunoblotting result of the sample was diminished with mangiferin, mangiferin 8a, noraciliol, methoxyxanthone, tetrahydroxyxanthone, xanthohydrol, α-mangostin and γ-mangostin. NF-κB p65 nuclear translocation inhibitor, DMF, did not become thinner than Control.

<Example 14: Examination of NIK downstream signal by administration of each drug>
As a result of examining the activation kinetics of NIK downstream signals IKK, NF-κB p52 and NF-κB p65 by administration of each drug, inhibition of IKK activity and inhibition of nuclear translocation of NF-κB p52 and NF-κB p65 were examined. Yes (Fig. 13).

Each sample was prepared in the same manner as in Example 13, subjected to SDS-PAGE, transferred to a PVDF membrane, and used with anti-phospho-IKK antibody and anti-IKK antibody, anti-NF-κBp52 antibody, anti-NF-κBp65 antibody, and anti-Lamin antibody. The assay was carried out.

Figure 13 shows the results of immunoblotting. In the horizontal direction of the photograph, Control, 50 μM DMF, 100 μM mangiferin, 10

μM mangiferin

▽The antibody type is shown in the vertical direction. Specifically, anti-phospho-IKK antibody (described as “Phospho-IKK”), anti-IKK antibody (described as “IKK”), anti-NF-κBp52 antibody (described as “NF-κBp52nuclear”), anti-NF-κBp65. Antibodies (described as "NF-κBp65nuclear") and anti-Lamin antibodies (described as "Lamin").

Further, to separate the nucleus and the cytosol from the cells, the cytosolic fraction and the nuclear fraction were extracted using ProteoExtract (registered trademark) Subcellular Proteome Extraction Kit manufactured by Merck Ltd.

In the photograph of the anti-IKK antibody, no decrease in the intensity of the IKK band was observed as compared with Control. On the other hand, in the case of the anti-phospho-IKK antibody, the immunoblotting result of the sample was diminished with mangiferin, mangiferin 8a, noraciliol, methoxyxanthone, tetrahydroxyxanthone, xanthohydrol, α-mangostin and γ-mangostin. NF-κB p65 nuclear translocation inhibitor, DMF, did not become thinner than Control.

Next, refer to anti-NF-κBp52 antibody (described as “NF-κBp52nuclear”), anti-NF-κBp65 antibody (described as “NF-κBp65nuclear”), and anti-Lamin antibody (described as “Lamin”) against a substance in the cell nucleus. To do. Lamin is present regardless of all drugs, but nuclear NF-κBp52 is diminished by mangiferin, mangiferin 8a, noraciliol, methoxyxanthone, tetrahydroxyxanthone, xanthydrol, α-mangostin and γ-mangostin. Although it was confirmed, the increase in DMF was recognized as compared with Control. In addition, NF-κBp65 in the nucleus was decreased (shadows were reduced) with all drugs.

Lamin is a fibrous protein that maintains structure and regulates transcription in the cell nucleus. Therefore, it was shown that NF-κBp65 and NF-κBp52 in the nucleus do not exist or are very small even when they are present when each drug is added in the state where the nuclear substance is detected in all samples. ing.

<Example 15: CD138 expression suppression effect by administration of each drug in L363 cells>
As a result of investigating the CD138 expression-suppressing action by administration of each drug using L363 cells by Flow cytometry, the CD138 expression-suppressing action was confirmed.

L363 cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). L363 cells were seeded in a 75 cm ² flask and precultured for 4 hours, then 50 μM mangiferin, 1

μM Mangiferin

8a, 1 μM noractilol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to final concentrations, and the conditions were 37° C. and 5% CO ₂ . It was cultured for 10 days. After culturing for 10 days, the cells were stained with an anti-CD138 antibody that is a malignancy marker for multiple myeloma, and the expression of CD138 was measured using BD LSR Fortessa.

The results are shown in FIGS. 14 and 15. The horizontal axis represents the CD138 expression level, and the vertical axis represents the cell number. In addition, the solid line in the panel shows the Negative control that has not been treated with the anti-CD138 antibody and the drug, the dotted line shows the positive control that has not been treated with the drug and has been treated with the anti-CD138 antibody, and the broken line shows that that has been treated with the drug and the anti-CD138 antibody. ..

The CD138 expression was significantly increased in the Positive control as compared with the Negative control group that was not treated with the drug or the anti-CD138 antibody. Mangiferin administration group (FIG. 14(a)), mangiferin 8a administration group (FIG. 14(b)), noraciliol administration group (FIG. 14(c)), tetrahydroxyxanthone administration group, (FIG. 14(d)), methoxyxanthone. In the administration group, (FIG. 15(e)), the xanthohydrol administration group (FIG. 15(f)), the α-mangostin administration group (FIG. 15(g)), and the γ-mangostin administration group (FIG. 15(h)) Compared to Positive control, the expression level of CD138 was remarkably reduced, and was almost the same as Negative control. That is, it was found that mangiferin, mangiferin 8a, noractilol, tetrahydroxyxanthone, methoxyxanthone, xanthhydrol, α-mangostin, and γ-mangostin suppress the expression of CD138, which is a malignancy marker for multiple myeloma.

<Example 16: CD20 expression increasing effect by administration of each drug on L363 cells>
As a result of investigating the CD20 expression increasing action by each drug administration using L363 cells by Flow cytometry, the CD20 expression increasing action was confirmed.

μM Mangiferin

8a, 1 μM noractilol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to final concentrations, and the conditions were 37° C. and 5% CO ₂ . It was cultured for 10 days. After culturing for 10 days, the cells were stained with an anti-CD20 antibody that is a B cell marker, and the expression of CD20 was measured using BD LSR Fortessa. CCRF-SB cells were used as a CD20-positive control.

The results are shown in FIGS. 16 and 17. The horizontal axis represents the expression level of CD20, and the vertical axis represents the cell number. In addition, the solid line in the panel shows the Negative control that has not been treated with the anti-CD20 antibody and the drug, the dotted line shows the positive control that has not been treated with the drug and has been treated with the anti-CD20 antibody, and the broken line shows that that has been treated with the drug and the anti-CD20 antibody. .. The dashed-dotted line shows CCRF-SB cells treated with anti-CD20 antibody.

Compared to the Negative control group that was not treated with the drug or the anti-CD20 antibody, the CD20 expression was hardly increased in the Positive control, which was about the same as the Negative control. That is, it can be seen that L363 cells do not express CD20. On the other hand, in the CCRF-SB cells used as a CD20-positive control, the CD20 expression was significantly increased as compared with the L363 cells Negative control and Positive control. This indicates that CCRF-SB cells express CD20.

Furthermore, the mangiferin administration group (FIG. 16(a)), the mangiferin 8a administration group (FIG. 16(b)), the noratiliol administration group (FIG. 16(c)), the tetrahydroxyxanthone administration group, (FIG. 16(d)) methoxy. In the xanthone administration group (FIG. 17(e)), the xanthohydrol administration group (FIG. 17(f)), the α-mangostin administration group (FIG. 17(g)), and the γ-mangostin administration group (FIG. 17(h)). Compared with Positive control, the expression level of CD20 was remarkably increased.

That is, mangiferin, mangiferin 8a, noraciliol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, γ-mangostin suppress the anti-CD138 antibody, which is a malignancy marker for multiple myeloma, and are B cell markers. Increased CD20 expression. As a result, it was found that the above substances convert multiple myeloma cells into B cells.

In multiple myeloma, CD20 is not normally expressed, and the effect of the anti-CD20 antibody rituximab, which binds to CD20 and exerts an antitumor effect, is not observed. However, the increased expression of CD20 by the composition containing mangiferin 8a, noraciliol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, γ-mangostin according to the present invention enables rituximab to bind to cells. , Can exert an antitumor effect. That is, the antitumor effect of rituximab can be expected by converting multiple myeloma into B cell-like by the above substances.

The improvement of multiple myeloma using rituximab may be achieved by using a composition containing mangiferin 8a, noraciliol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, γ-mangostin and rituximab.

<Example 17: IgG secretion inhibitory effect by administration of each drug on L363 cells>
As a result of an enzyme-linked immunosorbent assay (ELISA) studying the inhibitory effect on IgG secretion by administration of each drug using L363 cells, an inhibitory effect on IgG secretion was confirmed.

μM Mangiferin

8a, 1 μM noractilol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to final concentrations, and the conditions were 37° C. and 5% CO ₂ . It was cultured for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of IgG secreted was measured by ELISA. For this measurement, a human anti-IgG antibody ELISA kit (Funakoshi) was used.

The results are shown in Fig. 18. The horizontal axis represents the sample group and the vertical axis represents the IgG secretion amount (ng/mL). In Control, IgG is 2861 ng/mL, whereas mangiferin administration group, mangiferin 8a administration group, noraciliol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangosteen administration group, γ In the mangosteen administration group, the doses were 140 ng/mL, 40 ng/mL, 59 ng/mL, 145 ng/mL, 67 ng/mL, 106 ng/mL, 24 ng/mL, and 61 ng/mL, respectively.

As described above, it was observed that the mangiferin administration group decreases IgG secretion, and the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthhydrol administration group, the α-mangosteen administration group. It was found that the γ-mangostin administration group markedly suppressed IgG secretion at a lower dose than the mangiferin administration group.

That is, the control group causes renal damage, amyloidosis, and hyperviscosity syndrome due to IgG production, and the mangiferin administration group, the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the xanthohydrol administration group, the α-mangosteen administration group It can be said that the group and the γ-mangostin administration group suppressed renal damage, amyloidosis, and hyperviscosity syndrome.

<Example 18: Inhibitory effect of λ chain of immunoglobulin free light chain by administration of each drug on L363 cells>
The inhibitory effect on the λ chain of the immunoglobulin free light chain by administration of each drug in L363 cells was examined by enzyme-linked immunosorbent assay (ELISA). As a result, the inhibitory effect on the λ chain of the immunoglobulin free light chain was suppressed. recognized.

μM Mangiferin

8a, 1 μM noractilol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to final concentrations, and the conditions were 37° C. and 5% CO ₂ . It was cultured for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of λ chain secreted immunoglobulin free light chain was measured by ELISA. This measurement was performed using a human anti-λ chain antibody ELISA kit (Funakoshi).

The results are shown in Fig. 19. The horizontal axis represents the sample group, and the vertical axis represents the amount of λ chain secretion (μg/L). In Control, the IgG is 142 μg/L, whereas the mangiferin administration group, the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangosteen administration group, the γ -In the mangosteen-administered group, the doses were 30 μg/L, 23 μg/L, 23 μg/L, 33 μg/L, 31 μg/L, 29 μg/L, 30 μg/L, 31 μg/L, respectively.

As described above, it was observed that the mangiferin administration group decreased the secretion of λ chain, and the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangosteen administration group were administered. It was found that the γ-chain secretion was significantly suppressed in the γ-mangostin administration group and the mangiferin administration group at a lower dose. That is, the control group causes renal damage, amyloidosis, and hyperviscosity syndrome due to the production of λ chain, and the mangiferin administration group, the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the xanthohydrol administration group, the α-mangosteen It can be said that the administration group and the γ-mangostin administration group suppressed renal damage, amyloidosis, and hyperviscosity syndrome.

<Example 19: Inhibitory effect of bone destruction-related factor secretion by administration of each drug on L363 cells>
As a result of Luminex's investigation of the inhibitory effect on the secretion of bone destruction-related factors by administration of each drug using L363 cells, the inhibitory effect on the secretion of bone destruction-related factors was confirmed.

μM Mangiferin

8a, 1 μM noractilol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to final concentrations, and the conditions were 37° C. and 5% CO ₂ . It was cultured for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of MIP-1α, which is a bone destruction-related factor, secreted was measured by Luminex. This measurement was performed using a Human Magnetic Luminex Assay (R&D).

The results are shown in Fig. 20. The horizontal axis represents the sample group, and the vertical axis represents the secreted amount of MIP-1α (pg/mL). In Control, the secreted amount of MIP-1α was 245 pg/mL. In addition, in the mangiferin administration group, the mangiferin 8a administration group, the noraciliol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthohydrol administration group, the α-mangostin administration group, and the γ-mangostin administration group, the amount of MIP-1α secretion was The values were 0 pg/mL, 0 pg/mL, 0 pg/mL, 0 pg/mL, 0.73 pg/mL, 0 pg/mL, 0 pg/mL, and 0 pg/mL, respectively.

As described above, the mangiferin administration group was found to reduce the secretion of MIP-1α, and the mangiferin 8a administration group, the noratiriol administration group, the tetrahydroxyxanthone administration group, the methoxyxanthone administration group, the xanthydrol administration group, α-mangosteen It was found that the administration group and the γ-mangostin administration group markedly suppressed MIP-1α secretion at a lower dose than the mangiferin administration group. That is, the control group has bone lesions (bone destruction) due to MIP-1α production, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms. Mangiferin-administered group, mangiferin-8a-administered group, noraciliol-administered group, tetrahydroxyxanthone-administered group, xanthydrol-administered group, α-mangosteen-administered group, γ-mangostin-administered group, bone lesion (bone destruction), bone lesion It can be said that the hypercalcemia, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms were suppressed.

<Example 20: CD138 expression suppression effect by administration of each drug on KMS-28BM cells>
As a result of investigating the CD138 expression inhibitory action by each drug administration using KMS-28BM cells by Flow cytometry, the CD138 expression inhibitory action was confirmed.

KMS-28BM cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). KMS-28BM cells were seeded in a 75 cm ² flask and precultured for 4 hours, 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM noratiliol were added to the final concentrations, and the mixture was cultured at 37° C. under 5% CO ₂ for 10 days. After culturing for 10 days, the cells were stained with an anti-CD138 antibody that is a malignancy marker for multiple myeloma, and the expression of CD138 was measured using BD LSR Fortessa.

The results are shown in Fig. 21. The horizontal axis represents the CD138 expression level, and the vertical axis represents the cell number. In addition, the solid line in the panel shows the Negative control that has not been treated with the anti-CD138 antibody and the drug, the dotted line shows the positive control that has not been treated with the drug and has been treated with the anti-CD138 antibody, and the broken line shows that that has been treated with the drug and the anti-CD138 antibody. .. The CD138 expression was significantly increased in the Positive control as compared with the Negative control group which was not treated with the drug or the anti-CD138 antibody.

In the mangiferin administration group (Fig. 21(a)), the mangiferin 8a administration group (Fig. 21(b)), and the noraciliol administration group (Fig. 21(c)), the CD138 expression level was remarkably reduced, and was almost the same as that of Positive control. It was about the same as Negative control. That is, it was found that mangiferin, mangiferin 8a, and noraciliol suppress the expression of CD138, which is a malignancy marker for multiple myeloma.

<Example 21: CD20 expression increasing effect by administration of each drug on KMS-28BM cells>
As a result of investigating the CD20 expression increasing action by administration of each drug using KMS-28BM cells by Flow cytometry, the CD20 expression increasing action was confirmed.

KMS-28BM cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). KMS-28BM cells were seeded in a 75 cm ² flask and precultured for 4 hours, 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM noratiliol were added to the final concentrations, and the mixture was cultured at 37° C. under 5% CO ₂ for 10 days. After culturing for 10 days, the cells were stained with an anti-CD20 antibody that is a B cell marker, and the expression of CD20 was measured using BD LSR Fortessa. CCRF-SB cells were used as a CD20-positive control.

The results are shown in Fig. 22. The horizontal axis represents the expression level of CD20, and the vertical axis represents the cell number. Also, the solid line in the panel shows the Negative control that has not been treated with the anti-CD20 antibody and the drug, the dotted line shows the positive control that has not been treated with the drug and has been treated with the anti-CD20 antibody, and the dashed line shows that that has been treated with the drug and the anti-CD20 antibody. .. The dashed-dotted line shows CCRF-SB cells treated with anti-CD20 antibody.

Compared to the Negative control group that was not treated with the drug or the anti-CD20 antibody, the CD20 expression was hardly increased in the Positive control, which was about the same as the Negative control. That is, it can be seen that KMS-28BM cells do not express CD20. On the other hand, in the CCRF-SB cells used as the CD20-positive control, the CD20 expression was significantly increased as compared with the KMS-28BM cells Negative control and Positive control. This indicates that CCRF-SB cells express CD20. Furthermore, in the mangiferin administration group (FIG. 22(a)), the mangiferin 8a administration group (FIG. 22(b)), and the noraciliol administration group (FIG. 22(c)), the CD20 expression level was remarkably increased as compared with Positive control. Was there.

That is, mangiferin, mangiferin 8a, and noraciliol suppressed the anti-CD138 antibody, which is a malignancy marker for multiple myeloma, and increased the expression of CD20, which is a B cell marker. As a result, it was found that the above substances convert multiple myeloma cells (KMS-28BM cells) into B cells.

<Example 22: IgG secretion inhibitory effect by administration of each drug on KMS-28BM cells>
The inhibitory effect on IgG secretion by administration of each drug using KMS-28BM cells was examined by enzyme-linked immunosorbent assay (ELISA), and the inhibitory effect on IgG secretion was confirmed.

KMS-28BM cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). KMS-28BM cells were seeded in a 75 cm ² flask and precultured for 4 hours, 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM noratiliol were added to the final concentrations, and the mixture was cultured at 37° C. under 5% CO ₂ for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of IgG secreted was measured by ELISA. For this measurement, a human anti-IgG antibody ELISA kit (Funakoshi) was used.

The results are shown in Fig. 23. The horizontal axis represents the sample group and the vertical axis represents the IgG secretion amount (ng/mL). IgG was 2018 ng/mL in Control, whereas 106 ng/mL, 40 ng/mL, and 0 ng/mL were respectively in the mangiferin administration group, the mangiferin 8a administration group, and the noratiriol administration group.

As described above, it was confirmed that the mangiferin-administered group decreased IgG secretion, and it was found that the mangiferin 8a-administered group and the noratiriol-administered group significantly suppressed IgG secretion at a lower dose than the mangiferin-administered group. That is, it can be said that the control group caused renal damage, amyloidosis, and hyperviscosity syndrome due to IgG production, and the mangiferin-administered group, mangiferin 8a-administered group, and noraciriol-administered group suppressed renal injury, amyloidosis, and hyperviscosity syndrome. ..

<Example 23: Inhibitory effect of λ chain of immunoglobulin free light chain by administration of each drug on KMS-28BM cells>
The inhibitory effect on the λ chain of the immunoglobulin free light chain by administration of each drug in KMS-28BM cells was examined by enzyme-linked immunosorbent assay (ELISA), and the inhibitory effect on the λ chain of the immunoglobulin free light chain was suppressed. The action was recognized.

KMS-28BM cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). KMS-28BM cells were seeded in a 75 cm ² flask and precultured for 4 hours, 1 µM mangiferin, 0.1 µM mangiferin 8a, and 0.05 µM noraciliol were added so that the final concentrations would be obtained, and the cells were cultured at 37°C under 5% CO ₂ for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of λ chain secreted immunoglobulin free light chain was measured by ELISA. This measurement was performed using a human anti-λ chain antibody ELISA kit (Funakoshi).

The results are shown in Fig. 24. The horizontal axis represents the sample group, and the vertical axis represents the amount of λ chain secretion (μg/L). In the Control, IgG was 16 μg/L, whereas in the mangiferin administration group, the mangiferin 8a administration group, and the noratiriol administration group, they were 0 μg/L, 0 μg/L, and 6.9 μg/L, respectively.

As described above, it was confirmed that the mangiferin-administered group decreased the secretion of the λ chain, and it was found that the mangiferin 8a-administered group and the noratiriol-administered group significantly suppressed the λ-chain secretion at a lower dose than the mangiferin-administered group. That is, the control group caused nephropathy, amyloidosis, hyperviscosity syndrome due to λ chain production, and the mangiferin administration group, the mangiferin 8a administration group, the noratiriol administration group suppressed the renal injury, amyloidosis, hyperviscosity syndrome. I can say.

<Example 24: Inhibitory effect of bone destruction-related factor secretion by administration of each drug on KMS-28BM cells>
As a result of Luminex's study on the inhibitory action on the secretion of bone destruction-related factors by administration of each drug using KMS-28BM cells, the inhibitory action on the secretion of bone destruction-related factors was confirmed.

KMS-28BM cells were seeded in a 75 cm ² flask and cultured under conditions of 37° C. and 5% CO ₂ for 10 days (Control). KMS-28BM cells were seeded in a 75 cm ² flask and precultured for 4 hours, 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM noratiliol were added to the final concentrations, and the mixture was cultured at 37° C. under 5% CO ₂ for 10 days. After culturing for 10 days, the culture supernatant was collected and the amount of IL-6, which is a bone destruction-related factor, secreted was measured by Luminex. This measurement was performed using a Human Magnetic Luminex Assay (R&D).

The results are shown in Fig. 25. The horizontal axis represents the sample group, and the vertical axis represents the amount of IL-6 secreted (pg/mL). In Control, the amount of IL-6 secreted was 153 pg/mL. Moreover, the IL-6 secretion amount in the mangiferin administration group, the mangiferin 8a administration group, and the noratiriol administration group was 36 pg/mL, 28 pg/mL, and 43 pg/mL, respectively.

As described above, it was confirmed that the mangiferin-administered group decreased IL-6 secretion, and that the mangiferin-8a-administered group and the noraciriol-administered group significantly suppressed IL-6 secretion at a lower dose than the mangiferin-administered group. It was That is, the control group had bone lesions (bone destruction) due to IL-6 production, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms. Mangiferin administration group, mangiferin 8a administration group, noraciliol administration group, bone lesions (bone destruction), hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, It can be said that the neurological symptoms associated with spinal cord compression symptoms were suppressed.

<Example 25: Tumor growth inhibitory effect of in vivo administration of noraciriol on Raji cells>
Raji cells were transplanted into NOD/ShiJic-scidJcl mice, and the tumor growth inhibitory effect when orally administered noraciriol was examined. As a result, a remarkable tumor growth inhibitory effect was observed.

Raji cells were transplanted to NOD/ShiJic-scidJcl mice, and when the mean tumor volume exceeded 100 mm ³ , the day 0 was treated with oral administration of noraciriol at 100 mg/kg to the mice for 27 consecutive days, and the tumor volume was measured. ..

　The group that has just been transplanted with Raji cells is called the target group, and the group that has been administered noraciriol is called the noraciriol-administered group. Each group consisted of 5 animals.

Results are shown in FIG. Referring to the figure, the horizontal axis represents the number of days elapsed (day) and the vertical axis represents the tumor volume (mm ³ ). White circles indicate a target group (displayed as "Control"). The black diamonds indicate the noractilol administration group (indicated as "100 mg/kg norathyriol"). In addition, “*” was shown for those that were significantly different (P<0.01) from the control group.

In the control group (open circle "-O-"), tumor growth was observed with the number of days elapsed, and it was 5790 mm ³ after 27 days. In the noraciliol-administered group (black diamond mark "-◆-"), remarkable tumor growth inhibition was observed.

That is, tumor growth was significantly suppressed in the noraciriol administration group compared to the control group.

Fig. 27 shows a photograph of the tumor 27 days after the start of administration. FIG. 27(a) shows a photograph of a tumor of one mouse in the control group, and FIG. 27(b) shows a photograph of one mouse in the noractilol administration group. In the target group of FIG. 27(a), a significant increase in tumor is observed. On the other hand, in the noraciliol-administered group in FIG. 27(b), a remarkable decrease in volume was observed as compared with the control group.

As mentioned above, it was found that the noractilol-administered group remarkably suppressed tumor growth.

<Example 26: Tumor growth inhibitory effect of in vivo administration of noraciriol on L363 cells>
When L363 cells were transplanted to NOD/ShiJic-scidJcl mice and noraciriol was orally administered, the tumor growth inhibitory effect was examined. As a result, a remarkable tumor growth inhibitory effect was observed.

L363 cells were transplanted into NOD/ShiJic-scidJcl mice, and when the mean tumor volume exceeded 100 mm ³ , the day 0 was treated with oral administration of noratiriol at 100 mg/kg to the mice for 8 days, and the tumor volume was measured. ..

The group that has just been transplanted with L363 cells is called the target group, and the group that has been administered noraciliol is called the noraciriol-administered group. Each group consisted of 5 animals.

The results are shown in Fig. 28. Referring to the figure, the horizontal axis represents the number of days elapsed and the vertical axis represents the tumor volume. White circles indicate a target group (displayed as "Control"). The black diamond marks indicate the noractilol administration group (indicated as "100 mg/kg norathyriol"). In addition, “*” was shown for those that were significantly different (P<0.01) from the control group.

In the control group (open circle "-O-"), tumor growth was observed with the lapse of days, and it became 745 mm ³ after 8 days. In the noraciliol-administered group (black diamond mark "-◆-"), remarkable tumor growth inhibition was observed.

FIG. 29 shows a photograph of the tumor 8 days after the start of administration. FIG. 29(a) shows a photograph of a tumor of one mouse in the control group, and FIG. 29(b) shows a photograph of one mouse in the noraciriol administration group. In the target group of FIG. 29(a), the tumor is remarkably increased. On the other hand, in the noraciliol-administered group in FIG. 29(b), a remarkable decrease in volume was observed as compared with the control group.

The composition for improving malignant tumor disease according to the present invention can be used as a pharmaceutical composition useful for malignant tumor disease caused by activation of NF-κB p52 and NF-κB p65 as a selective NIK inhibitor. .. Further, the composition for improving malignant tumor disease according to the present invention is applied to bone lesions (bone destruction) associated with multiple myeloma, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, and spinal cord compression fractures. It can be used as a useful pharmaceutical composition for spinal cord symptom associated therewith, neurological symptom associated with spinal cord compressive symptom, renal disorder, amyloidosis, hyperviscosity syndrome.

Claims

Malignant tumor containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton as an active ingredient A composition for improving a disease.
NIK excess containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton as an active ingredient. A composition for improving malignant tumor disease associated with expression.
BAFF excess containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton as an active ingredient A composition for improving malignant tumor disease associated with expression.
Malignant tumor containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton as an active ingredient Disease remedy.
A processed food containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton.
A multiple myeloma comprising at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton. B cell-like dedifferentiation inducer.
In multiple myeloma containing at least one compound of formula (1), formula (2), formula (3), formula (4), formula (5), formula (6) and formula (7) having a xanthone skeleton Accompanied CRB improver.