Pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating NK-T cell lymphoma comprising a phosphodiesterase 5 inhibitor, a composition for auxiliary anticancer treatment and a health functional food, which inhibits the reactivation of Epstein barr virus (EBV) caused by histone deacetylase (HDAC) inhibitors. Using the pharmaceutical composition for preventing, improving or treating NK-T cell lymphoma of the present invention, the side effects of HDAC inhibitors can be reduced by inhibiting the reactivation of Epstein barr virus caused by HDAC inhibitors used as NK-T cell lymphoma anticancer drugs, thereby significantly improving the anticancer effect. Therefore, based on the above effects, the present invention can be widely used in the field of medicine.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating natural killer T cell (NK-T) lymphoma, a composition for auxiliary anti-cancer treatment and a health functional food, comprising a phosphodiesterase 5 inhibitor, and in particular to a pharmaceutical composition for inhibiting the reactivation of Epstein barr virus (EBV) caused by a histone deacetylase (HDAC) inhibitor. Background Art

[0002] Epstein-Barr virus (EBV) belongs to the herpes virus. It is known that more than 90% of normal people will be infected, and for most people it only causes asymptomatic latent infection. However, for people whose immune system is long-term weakened due to bone marrow transplantation or organ transplantation, EBV is the cause of tumors such as lymphoproliferative disease, Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease. Lymphocytic diseases associated with EBV include Hodgkin's lymphoma, nasal type extranodal NK-T cell lymphoma, and post-transplant lymphoproliferative disease (PTLD). Lymphoproliferative diseases are non-Hodgkin's lymphomas. After solid organ transplantation or hematopoietic stem cell transplantation, EBV lurking in the donor and continued use of immunosuppressants can cause benign diseases such as polyclonal B lymphoproliferative diseases that cause lymphocytes or plasma cells to proliferate, as well as malignant diseases such as malignant lymphomas. Its clinical manifestations vary, and it is a serious systemic complication that occurs in 1-15% of organ transplant patients. Recently, studies have found that some gastric cancers express EBV genes, and it is known that in Japan and South Korea, the proportion of gastric cancer accounts for 5-10% of all gastric cancers. A research team from Kagoshima University investigated the distribution of EBV-positive gastric cancer in seven countries including Japan, and the results showed that the odds ratio of EBV-related gastric cancer in Russia (1.9), Colombia (2.5), Chile (2.7) and other countries showed a higher ratio than that in Japan (1.0).

[0003] Existing treatments include reducing immunosuppressants, using antiviral drugs, anticancer chemotherapy, administering rituximab antibodies, etc. However, due to differences in treatment response rates, no effective treatment has been developed so far. Therefore, there is an urgent need to develop new therapies to treat and cure EBV-related tumors.

[0004] Regarding adoptive immunotherapy using EBV antigen-specific T cells for EBV infection, Dr. Brenner's research group conducted research by inducing EBV antigen-specific CTL in vitro to treat EBV infection diseases or EBV-related lymphomas, laryngeal cancer and other tumors after transplantation of hematopoietic blasts. In South Korea, the research group of the inventor successfully used T cells of inactivated antigen-specific cells for the first time to perform adoptive immunotherapy on EBV-positive lymphoma patients and acute leukemia patients. Although adoptive immunotherapy using antigen-specific T cells cultured in vitro has the advantage of overcoming the limitations of antibody therapeutic drugs, it is limited in general clinical application and industrialization due to the need to accumulate high-affinity antigen-specific T cell in vitro culture technology, which can overcome the immune tolerance state of tumor-related antigens and can identify and remove tumor cells, as well as the high cost of antigen-specific T cell production.

[0005] Recently, research on targeted immunotherapy using gene-engineered T cells has been actively conducted for adoptive immunotherapy for cancer patients, which can overcome the limitations of antibody therapeutic drugs, therapeutic cellular tumor vaccines, and adoptive cell immunotherapy. In particular, the strategy of using tumor antigen-specific TCR (T cell antigen receptor) genes is expected to develop into a universal targeted immunotherapy at the off-the-shelf stage that can overcome the limitations of tailor-made therapies. However, in order to obtain antigen-specific TCR genes, there are difficulties in obtaining tumor antigen-specific T cell clones and TCR gene clones, and the research conditions in Korea are not perfect, so limited clinical research is currently being conducted by foreign researchers.

[0006] On the other hand, histones are proteins that wrap around DNA (deoxyribonucleic acid) chains as genetic information in the cell nucleus. They condense DNA and increase or regulate gene expression through acetylation and methylation. If this acetylation and methylation are reduced and the regulation fails, cancer may be caused. Histone deacetylase, or HDAC (Histonedeacetylase), plays a role in regulating the degree of acetylation of histones and inhibiting DNA expression. If HDAC is abnormally overexpressed, it may cause cancer. Therefore, overexpression of HDAC may induce cancer. In order to inhibit this mechanism, HDAC inhibitors are used as anticancer drugs. However, HDAC inhibitors can induce the reactivation of Epstein-Barr virus (EBV) in lymphoma patients, and there is currently no further treatment for EBV reactivation. Therefore, there is an urgent need to develop a therapeutic drug that can prevent EBV reactivation in lymphoma. Summary of the invention

[0007] Technical issues

[0008] Therefore, the present inventors studied substances capable of inhibiting the reactivation of Epstein-Barr virus caused by HDAC inhibitors and confirmed that phosphodiesterase 5 inhibitors can inhibit the reactivation of Epstein-Barr virus caused by HDAC inhibitors, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK (natural killer) cell leukemia, comprising a phosphodiesterase 5 inhibitor.

[0010] Furthermore, another object of the present invention is to provide a method for treating NK-T cell lymphoma or NK cell leukemia, comprising the step of administering a phosphodiesterase 5 inhibitor to an individual.

[0011] Another object of the present invention is to provide a composition for adjuvant anticancer treatment of NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor.

[0012] Another object of the present invention is to provide a health functional food for preventing or improving NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor.

[0013] Technical Solution

[0014] In order to achieve the above-mentioned object, the present invention provides a pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor, wherein the pharmaceutical composition is used to inhibit the reactivation of Epstein barr virus (EBV) caused by a histone deacetylase (HDAC) inhibitor.

[0015] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor and an HDAC inhibitor.

[0016] In order to achieve the above-mentioned further object, the present invention provides a method for treating NK-T cell lymphoma or NK cell leukemia, comprising the step of administering a phosphodiesterase 5 inhibitor to an individual, characterized in that the reactivation of Epstein barr virus (EBV) caused by a histone deacetylase (HDAC) inhibitor is inhibited.

[0017] Furthermore, the present invention provides a method for treating NK-T cell lymphoma or NK cell leukemia, comprising the step of administering a phosphodiesterase 5 inhibitor and an HDAC inhibitor to an individual.

[0018] In order to achieve the above another object, the present invention provides a composition for adjuvant anticancer treatment of NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor, wherein the above NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0019] In order to achieve the above-mentioned another object, the present invention provides a health functional food for preventing or improving NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor (phosphodiesterase 5inhibitor), wherein the above-mentioned NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0020] Furthermore, the present invention provides a health functional food for preventing or improving NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor and an HDAC inhibitor, wherein the NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0021] Effects of the Invention

[0022] The use of the phosphodiesterase 5 inhibitor of the present invention can inhibit EBV reactivation caused by HDAC inhibitors used as anticancer drugs for NK-T cell lymphoma or NK cell leukemia, reduce the side effects of HDAC inhibitors, and thus significantly improve the anticancer effect. Therefore, based on the above effects, the present invention can be widely used in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1a The graph shows the expression of BZLF1 at different treatment times when SNK6 cells were treated with romidepsin or sildenafil alone or simultaneously.

[0024] Figure 1b The graph shows the expression of BZLF1 at different treatment times when NK92MI cells were treated with romidepsin or sildenafil alone or simultaneously.

[0025] Figure 2a The graph shows the expression of BZLF1 in response to different treatment concentrations of sildenafil when SNK6 cells were treated with romidepsin or sildenafil alone or simultaneously.

[0026] Figure 2b The graph shows the expression of BZLF1 in NK92MI cells when romidepsin or sildenafil was treated alone or simultaneously with sildenafil, and the expression of BZLF1 was confirmed according to different treatment concentrations of sildenafil.

[0027] Figure 3a The figure shows whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (Daudi) other than NK-T cell lymphoma or NK cell leukemia cell lines, as confirmed by BZLF1 mRNA (messenger RNA) expression.

[0028] Figure 3b This figure confirms whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (Daudi) other than NK-T cell lymphoma or NK cell leukemia cell lines, as determined by BRLF1 mRNA expression.

[0029] Figure 3c The figure shows whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (Raji) other than NK-T cell lymphoma or NK cell leukemia cell lines, as confirmed by BZLF1 mRNA expression.

[0030] Figure 3dThe figure shows whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (Raji) other than NK-T cell lymphoma or NK cell leukemia cell lines, as confirmed by BRLF1 mRNA expression.

[0031] Figure 3e The figure shows whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (1A2) other than NK-T cell lymphoma or NK cell leukemia cell lines, as confirmed by BZLF1 mRNA expression.

[0032] Figure 3f The figure shows whether EBV reactivation caused by romidepsin occurs in EBV-positive cell lines (1A2) other than NK-T cell lymphoma or NK cell leukemia cell lines, as confirmed by BRLF1 mRNA expression.

[0033] Figure 4a The figure confirms whether sildenafil also has an inhibitory effect on EBV reactivation caused by romidepsin in EBV-positive cell lines (Daudi) other than NK-T cell lymphoma or NK cell leukemia cell lines, based on EBV copy number.

[0034] Figure 4b The figure confirms whether sildenafil also has an inhibitory effect on EBV reactivation caused by romidepsin in EBV-positive cell lines (Daudi) other than NK-T cell lymphoma or NK cell leukemia cell lines, as measured by BZLF1 mRNA expression.

[0035] Figure 4c The figure confirms whether sildenafil also has an inhibitory effect on EBV reactivation caused by romidepsin in EBV-positive cell lines (Daudi) other than NK-T cell lymphoma or NK cell leukemia cell lines, as measured by BRLF1 mRNA expression.

[0036] Figure 5a The graph shows whether sildenafil inhibits EBV reactivation caused by HDAC inhibitors in SNK6 cells, as determined by EBV copy number.

[0037] Figure 5b The figure confirms whether sildenafil inhibits EBV reactivation caused by HDAC inhibitor in SNK6 cells through the mRNA expression of BZLF1.

[0038] Figure 5cThe figure confirms whether sildenafil inhibits EBV reactivation caused by HDAC inhibitor in SNK6 cells through BRLF1 mRNA expression.

[0039] Figure 5d This figure confirms that the tumor cell survival rate is reduced due to the inhibition of EBV reactivation by sildenafil in SNK6 cells.

[0040] Figure 6a The figure confirms whether sildenafil inhibits EBV reactivation caused by HDAC inhibitor in NK92MI cells through the mRNA expression of BRLF5.

[0041] Figure 6b The figure confirms whether sildenafil inhibits EBV reactivation caused by HDAC inhibitor in NK92MI cells through the mRNA expression of BZLF1.

[0042] Figure 6c The figure confirms whether sildenafil inhibits EBV reactivation caused by HDAC inhibitor in NK92MI cells through BRLF1 mRNA expression.

[0043] Figure 6d This figure confirms that the tumor cell survival rate is reduced due to the inhibition of EBV reactivation by sildenafil in SNK6 cells.

[0044] Figure 7a The figure confirms whether the PDE5 inhibitor vardenafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor vardenafil alone or simultaneously with romidepsin through the mRNA expression of BZLF1.

[0045] Figure 7b The figure confirms whether the PDE5 inhibitor vardenafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor vardenafil alone or simultaneously with romidepsin through BRLF1 mRNA expression.

[0046] Figure 7c The figure confirms whether the PDE5 inhibitor udenafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor udenafil alone or simultaneously with romidepsin through BZLF1 mRNA expression.

[0047] Figure 7dThe figure is a diagram for confirming whether the PDE5 inhibitor udenafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor udenafil alone or simultaneously with romidepsin through BRLF1 mRNA expression.

[0048] Figure 7e The figure confirms whether the PDE5 inhibitor tadalafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor tadalafil alone or simultaneously with romidepsin through the mRNA expression of BZLF1.

[0049] Figure 7f The figure confirms whether the PDE5 inhibitor tadalafil has an inhibitory effect on EBV reactivation caused by romidepsin when SNK6 cells are treated with the PDE5 inhibitor tadalafil alone or simultaneously with romidepsin through BRLF1 mRNA expression. DETAILED DESCRIPTION

[0050] Hereinafter, the present invention will be described in detail.

[0051] The present invention provides a pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor. The pharmaceutical composition is used to inhibit the reactivation of Epstein-Barr virus (EBV) caused by a histone deacetylase (HDAC) inhibitor.

[0052] In the present invention, the above-mentioned NK-T cell lymphoma or NK cell leukemia can be Epstein-Barr virus-positive NK-T cell lymphoma or NK cell leukemia, preferably, it can be Epstein-Barr virus-positive NK-T cell lymphoma or NK cell leukemia reactivated by EBV caused by HDAC inhibitor.

[0053] In the present invention, the above-mentioned phosphodiesterase 5 inhibitor (PDE5 inhibitor) is known to be a drug that inhibits the decomposition of cGMP-specific phosphodiesterase type 5 in the vascular smooth muscle cells that supply blood to the corpus cavernosum of the penis. Therefore, it is used to treat erectile dysfunction, and PDE5 is also known to exist in the arterial blood vessel wall, so it can effectively treat pulmonary hypertension. However, the inhibitory effect on the side effects of HDAC inhibitors as in the present invention has not been published.

[0054] In the present invention, the phosphodiesterase 5 inhibitor may be at least one selected from the group consisting of sildenafil, mirodenafil, vardenafil, tadalafil, udenafil, dasantafil and avanafil, preferably, sildenafil, vardenafil, tadalafil or udenafil, but is not limited thereto.

[0055] In the present invention, the HDAC inhibitor may have an anticancer effect, but may also induce EBV reactivation when administered to patients with NK-T cell lymphoma or NK cell leukemia, thereby inhibiting the anticancer effect. The phosphodiesterase 5 inhibitor of the present invention may inhibit EBV reactivation caused by the HDAC inhibitor.

[0056] In the present invention, the HDAC inhibitor may be at least one selected from the group consisting of Romidepsin, Vorinostat, Panobinostat, Belinostat, Entinostat, Mocetinostat, Givinostat, Pracinostat, Quisinostat, Abexinostat, chr-3996 and AR-42.

[0057] In the present invention, the pharmaceutical composition can be used to inhibit the reactivation of Epstein barr virus (EBV) caused by HDAC inhibitors.

[0058] The term "prevention" used in the present invention refers to any action of inhibiting the reactivation of the above-mentioned Epstein-Barr virus or delaying its progression by administering the composition of the present invention.

[0059] The term "treatment" used in the present invention refers to inhibiting the reactivation of Epstein barr virus (EBV) by administering the composition of the present invention, and refers to all actions that improve or ameliorate diseases caused by the above viruses, such as lymphoproliferative disease, Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), Hodgkin's disease, etc., and refers to attempts to obtain useful results or ideal results including clinical results. Useful or ideal clinical results may be detectable or undetectable, and may include relieving or improving at least one symptom or state, reducing the scope of the disease, stabilizing the disease state, inhibiting the occurrence of the disease, inhibiting the spread of the disease, delaying or delaying the progression of the disease, delaying or delaying the onset of the disease, improving or alleviating and (partially or completely) reducing the disease state, but are not limited thereto. In addition, "treatment" may refer to extending the patient's survival beyond what would be expected without treatment. In addition, "treatment" may refer to inhibiting the progression of the disease, temporarily delaying the progression of the disease, and more preferably, is related to permanently stopping the progression of the disease. Preferably, in the present invention, it may refer to enhancing the anti-cancer effect of HDAC inhibitors by inhibiting the side effects of HDAC inhibitors, thereby improving the survival of patients.

[0060] The pharmaceutical composition of the present invention can be made into the dosage forms such as oral dosage forms such as powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods. The carriers, excipients and diluents that can be included in the above-mentioned pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil. When making the preparation, the diluents or excipients such as conventional fillers, extenders, adhesives, wetting agents, disintegrants, surfactants are used for preparation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which are prepared by adding at least one excipient to the extract or compound of the present invention, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talcum powder are also used in addition to simple excipients. Oral liquid preparations include suspensions, internal liquids, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, a variety of excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspensions, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, injectable esters such as ethyl oleate, etc. can be used. As the matrix of the suppository, synthetic fatty acid esters (witepsol), polyethylene glycol, tween 61, cocoa butter, lauric acid glyceride, glyceryl gelatin, etc. can be used.

[0061] The dosage of the pharmaceutical composition of the present invention may vary according to the age, sex, weight and specific disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration and the judgment of the prescriber. A person of ordinary skill in the art can determine the dosage based on these factors, and the dosage range is generally 0.01 mg / kg / day to about 2000 mg / kg / day. A more preferred dosage is 0.1 mg / kg / day to 1000 mg / kg / day. Administration may be once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0062] The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. Any route of administration is contemplated, such as oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine epidural, or intracerebral injection.

[0063] In the present invention, the pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia may include, in addition to the active ingredients, any known compound or natural extract that has been verified to have safety and anti-cancer activity, so as to enhance and strengthen the anti-cancer effect.

[0064] The pharmaceutical composition of the present invention can inhibit EBV reactivation caused by HDAC inhibitors, thereby effectively preventing or treating NK-T cell lymphoma or NK cell leukemia.

[0065] To this end, the present invention provides a method for treating NK-T cell lymphoma or NK cell leukemia, comprising the step of administering a phosphodiesterase 5 inhibitor to an individual, characterized in that the reactivation of Epstein barr virus (EBV) caused by an HDAC (Histone deacetylase) inhibitor is inhibited.

[0066] The term "individual" used in the present invention refers to an individual suffering from cancer, preferably, refers to a mammal including humans, such as horses, sheep, pigs, goats, dogs, etc., who suffers from a cancer for which HDAC inhibitors have a therapeutic effect, and when the above-mentioned phosphodiesterase 5 inhibitor is administered according to the present invention, EBV reactivation caused by the above-mentioned HDAC inhibitor is inhibited, thereby enhancing the therapeutic effect thereon, but preferably refers to humans.

[0067] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor and an HDAC inhibitor.

[0068] The pharmaceutical composition of the present invention can inhibit EBV reactivation and effectively exert the anti-cancer effect of HDAC inhibitors, thereby effectively preventing or treating NK-T cell lymphoma or NK cell leukemia.

[0069] In addition, the pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and methods using biological response modifiers to prevent or treat NK-T cell lymphoma or NK cell leukemia.

[0070] Furthermore, the present invention provides a method for treating NK-T cell lymphoma or NK cell leukemia, comprising the step of administering a phosphodiesterase 5 inhibitor and an HDAC inhibitor to an individual.

[0071] In the above method for treating NK-T cell lymphoma or NK cell leukemia, the "individual" is as described above.

[0072] Furthermore, the present invention provides a composition for adjuvant anticancer treatment of NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor, wherein the NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0073] The Epstein-Barr virus-positive NK-T cell lymphoma or NK cell leukemia may be caused by EBV reactivation caused by an HDAC inhibitor, and the phosphodiesterase 5 inhibitor is characterized in that it enhances the anticancer therapeutic effect of the HDAC inhibitor by inhibiting the EBV reactivation.

[0074] The term "composition for auxiliary anti-cancer treatment" used in this specification refers to a composition that, when used in combination with anti-cancer drug therapy, synergistically enhances the anti-cancer treatment effect by preventing the side effects of the above anti-cancer drugs.

[0075] Therefore, the above-mentioned adjuvant anti-cancer therapy composition can be used to assist in cancer treatment using HDAC inhibitors or other anti-cancer drugs, and can be used as an anti-cancer adjuvant and administered simultaneously or sequentially with the anti-cancer drug.

[0076] In addition, the present invention provides a health functional food for preventing or improving NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor, wherein the NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0077] Furthermore, the present invention provides a health functional food for preventing or improving NK-T cell lymphoma or NK cell leukemia, comprising a phosphodiesterase 5 inhibitor and an HDAC inhibitor, wherein the NK-T cell lymphoma or NK cell leukemia is Epstein-Barr virus positive.

[0078] The above-mentioned Epstein-Barr virus-positive NK-T cell lymphoma or NK cell leukemia may be caused by EBV reactivation caused by HDAC inhibitors, and the above-mentioned phosphodiesterase 5 inhibitor is characterized in that it prevents the onset or improves the symptoms of diseases caused by the above-mentioned viruses, such as lymphoproliferative disease, Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), Hodgkin's disease, etc., by inhibiting the above-mentioned EBV reactivation.

[0079] The health functional food of the present invention includes all forms of functional food, nutritional supplement, health food and food additives, etc. The above-mentioned health functional food can be prepared into various forms according to conventional methods known in the art.

[0080] For example, as a health food, the health functional food of the present invention can be made into granules, capsules and powders for ingestion, or made into tea, juice and beverages for drinking. In addition, the health functional food of the present invention can be mixed with a known substance or active ingredient known to have an improvement or therapeutic effect on NK-T cell lymphoma or NK cell leukemia to form a composition. In particular, the above-mentioned known substance can be a substance that causes the reactivation of Epstein-Barr virus, but is not limited thereto.

[0081] Furthermore, as functional foods, the health functional foods of the present invention can be added to beverages (including alcoholic beverages), fruits and processed foods thereof (such as canned fruits, bottled foods, jams, marmalade, etc.), fish, meat and processed foods thereof (such as ham, sausage tortillas, etc.), breads and noodles (such as udon noodles, buckwheat, instant noodles, pasta, macaroni, etc.), fruit juices, various beverages, cookies, maltose, dairy products (such as butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, steamed foods, frozen foods, various seasonings (such as miso, soy sauce, sauces, etc.), etc.

[0082] The preferred content of the health functional food of the present invention can be, for example, 0.01 weight percent to 80 weight percent in the final prepared food, but is not limited thereto. Preferably, it can be 0.01 weight percent to 50 weight percent of the final prepared food.

[0083] Furthermore, in order to use the health functional food of the present invention as a food additive, it can be prepared in the form of a powder or a concentrated liquid.

[0084] To avoid complexity of this specification, repeated contents will be omitted, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which the present invention belongs.

[0085] Hereinafter, the present invention will be described in detail by way of examples, but the following examples are merely examples of the present invention, and the content of the present invention is not limited to the following examples.

[0086] Example 1. Confirmation of the inhibitory effect of sildenafil on EBV reactivation caused by HDAC inhibitors

[0087] NK-T cell lymphoma cell line SNK6 (source: Seoul National University) and NK cell leukemia cell line NK92MI (source: Seoul National University) were treated with 10nM romidepsin as an HDAC inhibitor and 100nM sildenafil as a PDE5 inhibitor for 24, 48, and 72 hours, and the cells were collected every hour. Then, RNA (ribonucleic acid) was extracted using TRIzol reagent (Ambion, Life technologies), and cDNA (complementary DNA) was synthesized using a reverse transcription kit (OmniscriptRT kit, Qiagen), and the expression of BZLF1, an mRNA expressed during EBV reactivation, was confirmed by real-time fluorescent quantitative polymerase chain reaction (real rimePCR). The group treated only with DMSO (dimethyl sulfoxide) was used as the control group, and the results of SNK6 cells were Figure 1a The results for NK92MI cells are shown in Figure 1b The primer sequences used in real-time fluorescence quantitative PCR are shown in FIG. 1 .

[0088] Table 1:

[0089]

[0090]

[0091] like Figure 1a and Figure 1b As shown, it was confirmed that when treated with romidepsin alone, the expression of BZLF1 in SNK6 cells and NK92MI cells increased compared with the control group, thereby confirming that EBV (epstein-Barr virus) was reactivated. On the contrary, when treated with romidepsin and sildenafil for 72 hours, the expression of BZLF1 was significantly reduced compared with the group treated with romidepsin alone, thereby confirming that sildenafil inhibited the EBV reactivation increased by romidepsin.

[0092] Example 2. Confirmation of the inhibitory effect of different concentrations of sildenafil on EBV reactivation caused by HDAC inhibitors

[0093] SNK6 and NK92MI cells were treated with sildenafil alone at a concentration of 10 nM, 100 nM or 1000 nM and simultaneously treated with 10 nM romidepsin as experimental groups, and the mRNA expression of BZLF1 was measured using the same method as in Example 1. The treatment time was 72 hours, and the results of SNK6 cells were Figure 2a The results for NK92MI cells are shown in Figure 2b Shown in.

[0094] like Figure 2a and Figure 2b As shown, it was confirmed that the combination treatment with sildenafil had an inhibitory effect on EBV reactivation caused by romidepsin.

[0095] Example 3. Validation of the inhibitory effect of sildenafil on EBV reactivation caused by HDAC inhibitors in NK-T cell lymphoma or NK cell leukemia

[0096] 3.1 Screening of non-NK-T cell lymphoma and non-NK cell leukemia cell lines with EBV reactivation induced by romidepsin

[0097] EBV reactivation caused by romidepsin and the effect of sildenafil were confirmed by collecting EBV-positive cell lines other than SNK6 and NK92MI. As EBV-positive cell lines, Daudi cell line (ATCC, CCL-213) and Raji cell line (ATCC, CCL-86) as Burkitt's lymphoma cells, and 1A2 cell line (ATCC, CRL-8119) as lymphoma cells were treated with 1nM or 10nM romidepsin for 72 hours to confirm the mRNA expression of BZLF1 and BRLF1, genes whose expression increases during EBV reactivation. The mRNA expression results of BZLF1 in the above-mentioned Daudi cell line were as follows: Figure 3a As shown in Figure 2, the mRNA expression results of BRLF1 were Figure 3b In addition, the mRNA expression results of BZLF1 in the above Raji cell line are shown in Figure 3c The results of BRLF1 mRNA expression were shown in Figure 3d In addition, the results of BZLF1 mRNA expression in the above 1A2 cell line are shown in Figure 3e The results of BRLF1 mRNA expression were shown in Figure 3f Shown in.

[0098] like Figures 3a to 3f As shown, it was confirmed that the expression of BZLF1 and BRLF1 in the Daudi cell line was increased by romidepsin treatment. The expression of BZLF1 and BRLF1 in the Raji cell line did not increase, and only the expression of BRLF1 increased in the 1A2 cell line. Therefore, it was confirmed that romidepsin caused EBV reactivation only in the Daudi cell line.

[0099] 3.2 Validation of the specificity of sildenafil in NK-T cell lymphoma or NK cell leukemia cells for the inhibitory effect on EBV reactivation induced by HDAC inhibitors

[0100] An experiment was conducted to confirm whether EBV reactivation caused by romidepsin is inhibited when Daudi cell lines are treated with sildenafil and romidepsin at the same time. The Daudi cell lines have been confirmed to cause EBV reactivation by romidepsin by Example 3.1. The Daudi cell lines were treated with 10nM romidepsin and 100nM sildenafil for 72 hours respectively and the group treated simultaneously were used as experimental groups, and the group treated only with DMSO was used as a control group. In order to measure the EBV copy number secreted to the extracellular space due to EBV reactivation, DNA was extracted from the culture medium, and the EBV copy number was measured by using real-time fluorescence quantitative PCR (real rime PCR) to measure the Ct value, and the EBV copy number was measured by comparing it with the baseline EBV number, and then Figure 4a The mRNA expression of BZLF1 was measured by the same method as in Example 3.1, and Figure 4b As shown, BRLF1 mRNA was measured and Figure 4c Shown in.

[0101] like Figures 4a to 4c As shown, no significant difference was confirmed between the group treated with romidepsin alone and the group treated with romidepsin and sildenafil simultaneously in the Daudi cell line. Therefore, it was confirmed that sildenafil did not show an inhibitory effect on EBV reactivation in the case of non-NK-T cell lymphoma or NK cell leukemia cells.

[0102] Example 4. Confirmation of EBV inhibitory effect of sildenafil

[0103] SNK6 and NK92MI cell lines were treated with 10 nM of romidepsin for 72 hours, and the inhibition of EBV reactivation by sildenafil (100 nM, 72 hours) was confirmed by measuring the EBV copy number, BALF5, BZLF1 and BRLF1 expression. In addition, cell growth was measured by counting living cells by trypan blue staining. BALF5 is a gene that has the function of generating DNA from the virus produced during EBV reactivation. DNA was extracted from the culture medium using QIAamp DNA mini kit (Qiagen) and real-time quantitative PCR was performed to measure BALF5 mRNA. The BALF5 primer sequences used in real-time quantitative PCR are shown in Table 2. The EBV copy number measurement results in SNK6 cells confirmed as above are shown in Table 2. Figure 5a As shown in Figure 2, BZLF1 mRNA expression was Figure 5b As shown in Figure 2, BRLF1 mRNA expression was Figure 5c As shown in Figure 5dIn addition, the mRNA expression of BRLF5 in NK92MI cells was Figure 6a As shown in Figure 2, BZLF1 mRNA expression was Figure 6b As shown in Figure 2, BRLF1 mRNA expression was Figure 6c As shown in Figure 6d Shown in.

[0104] Table 2:

[0105] Primer name Primer sequence (5'->3') Serial Number BALF5-Forward Primer CGT CTC ATT CCC AAG TGT TTC 7 BALF5-reverse primer GCC CTT TCC ATC CTC GTC 8

[0106] like Figure 5a to Figure 5d As shown, by comparing the romidepsin alone-administered group and the romidepsin and sildenafil-administered group of the SNK6 cell line, it was confirmed that the EBV copy number and the expression of BZLF1 and BRLF1 were reduced in the romidepsin and sildenafil-administered group, thereby confirming that EBV reactivation was inhibited, and it was confirmed that the survival ability of the NK-T cell lymphoma cell line SNK6 cell line was reduced in the romidepsin and sildenafil-administered group.

[0107] And, if Figures 6a to 6d As shown, by comparing the romidepsin alone administration group and the romidepsin and sildenafil administration group of the NK92MI cell line, it was confirmed that the expression of BALF5, BZLF1 and BRLF1 was reduced in the romidepsin and sildenafil administration group, thereby confirming that EBV reactivation was inhibited, and the trypan blue staining results confirmed that the survival ability of the NK-T cell lymphoma cell line NK92MI cell line was reduced in the romidepsin and sildenafil administration group.

[0108] Based on the above results, it was confirmed that sildenafil can effectively inhibit the oncogenic virus EBV.

[0109] Example 5. Confirmation of the inhibitory effect of PDE5 inhibitors other than sildenafil on EBV

[0110] In addition, the inhibitory effects of PDE5 inhibitors other than sildenafil on EBV were confirmed.

[0111] The mRNA expression of BZLF1 and BRLF1 was measured by real-time fluorescence quantitative PCR (qPCR) using the same method as in Example 3.1 to confirm the inhibition of EBV reactivation by vardenafil, udenafil or tadalafil as PDE5 inhibitors on SNK6 cell lines treated with 10 nM romidepsin. At this time, vardenafil was treated with 100 nM for 72 hours, udenafil was treated with 100 nM for 48 hours, and tadalafil was treated with 1000 nM for 24 hours. The mRNA expression of BZLF1 in the vardenafil-administered experimental group was Figure 7a As shown in Figure 2, BRLF1 mRNA expression was Figure 7bIn addition, the mRNA expression of BZLF1 in the Udenafil administration experimental group was Figure 7c As shown in Figure 2, BRLF1 mRNA expression was Figure 7d In addition, the mRNA expression of BZLF1 in the tadalafil administration experimental group was Figure 7e As shown in Figure 2, BRLF1 mRNA expression was Figure 7f Shown in.

[0112] Finally, similar to the experimental group treated with romidepsin and sildenafil in the above example, the experimental groups treated with vardenafil, udenafil or tadalafil and romidepsin also showed a decrease in the mRNA expression of BZLF1 and BRLF1, thus confirming that EBV reactivation was suppressed.

[0113] In summary, when NK-T cell lymphoma cell lines and NK cell leukemia cell lines were treated simultaneously with HDAC inhibitors and phosphodiesterase 5 inhibitors, the reactivation of Epstein barr virus (EBV) caused by HDAC inhibitors was suppressed, thus confirming that it can reduce the side effects of HDAC inhibitors and significantly enhance the anti-cancer effect.

[0114] Hereinafter, the preparation examples of the pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia comprising a phosphodiesterase 5 inhibitor of the present invention will be described, but it is not intended to limit the present invention, but is only for the purpose of specifically describing the present invention.

[0115] Preparation Example 1. Preparation of powder

[0116] Phosphodiesterase 5 inhibitor of the present invention 10mg

[0117] Lactose 1g

[0118] The powder is prepared by mixing the above ingredients and filling in a sealed bag.

[0119] Preparation Example 2. Preparation of tablets

[0120] Phosphodiesterase 5 inhibitor of the present invention 10mg

[0121] Corn starch 100mg

[0122] Lactose 100mg

[0123] Magnesium stearate 2mg

[0124] After the above ingredients are mixed, tablets are prepared by tableting according to a conventional tablet preparation method.

[0125] Preparation Example 3. Preparation of capsules

[0126] Phosphodiesterase 5 inhibitor of the present invention 10mg

[0127] Corn starch 100mg

[0128] Lactose 100mg

[0129] Magnesium stearate 2mg

[0130] After the above ingredients are mixed, capsules are prepared by filling into gelatin capsules according to a conventional capsule preparation method.

[0131] Preparation Example 4. Preparation of pills

[0132] Phosphodiesterase 5 inhibitor of the present invention 10mg

[0133] Lactose 1.5g

[0134] Glycerin 1g

[0135] Xylitol 0.5g

[0136] After mixing the above ingredients, prepare 4 g of each pill according to the conventional method.

[0137] Preparation Example 5. Preparation of granules

[0138] Phosphodiesterase 5 inhibitor of the present invention 10mg

[0139] Soybean extract 50mg

[0140] Glucose 200mg

[0141] Starch 600mg

[0142] After the above components were mixed, 100 mg of 30% ethanol was added, and the mixture was dried at 60°C, formed into granules, and then packed in bags.

[0143] Above, specific parts of the present invention are described in detail. It is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention should be defined by the attached invention claims and their equivalents. Sequence Listing <110> Samsung Life Public Welfare Foundation <120> Pharmaceutical composition for preventing or treating NK-T cell lymphoma or NK cell leukemia <130> 1-25 <150> KR 20200030208 <151> 2020-03-11 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <400> 1 aatgccgggc caagtttaag caac 24 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <400> 2 ttgggcacat ctgcttcaac agga 24 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 tggcttggaa gactttctga ggct 24 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 aatctccaca ctcccggctg taaa 24 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 ggcatggact gtggtcatga g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tgcaccacca actgcttagc 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 cgtctcattc ccaagtgttt c 21 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 gccctttcca tcctcgtc 18

Claims

1. Use of a phosphodiesterase 5 inhibitor selected from the group consisting of sildenafil, vardenafil, tadalafil and udenafil in the preparation of a medicament for preventing or treating NK-T cell lymphoma, characterized in that: The phosphodiesterase 5 inhibitor is used to inhibit the reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin; The NK-T cell lymphoma is Epstein-Barr virus positive NK-T cell lymphoma.

2. The use according to claim 1, characterized in that The NK-T cell lymphoma is caused by reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin.

3. Use of a combination of a phosphodiesterase 5 inhibitor selected from the group consisting of sildenafil, vardenafil, tadalafil and udenafil and a histone deacetylase inhibitor romidepsin in the preparation of a medicament for preventing or treating NK-T cell lymphoma, characterized in that: The combination is useful for inhibiting the reactivation of Epstein-Barr virus induced by the histone deacetylase inhibitor romidepsin.

4. The use according to claim 3, characterized in that The NK-T cell lymphoma is Epstein-Barr virus positive.

5. The use according to claim 3, characterized in that: The NK-T cell lymphoma is caused by reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin.

6. Use of sildenafil as a phosphodiesterase 5 inhibitor in the preparation of a medicament for preventing or treating NK cell leukemia, characterized in that: The sildenafil is used to inhibit the reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin; The NK cell leukemia is Epstein-Barr virus positive NK cell leukemia.

7. The use according to claim 6, characterized in that The NK cell leukemia is caused by reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin.

8. Use of a combination of sildenafil as a phosphodiesterase 5 inhibitor and romidepsin as a histone deacetylase inhibitor in the preparation of a medicament for preventing or treating NK cell leukemia, characterized in that: The combination is useful for inhibiting the reactivation of Epstein-Barr virus induced by the histone deacetylase inhibitor romidepsin.

9. The use according to claim 8, characterized in that The NK cell leukemia is Epstein-Barr virus positive NK cell leukemia.

10. The use according to claim 8, characterized in that The NK cell leukemia is caused by reactivation of Epstein-Barr virus caused by the histone deacetylase inhibitor romidepsin.

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