Use of adenosine diphosphate ribose as adjuvant therapy for radiation and / or anticancer therapy

Abstract

To provide an anticancer composition that improves the efficacy of anticancer therapy.SOLUTION: An anticancer composition comprising adenosine diphosphate ribose (ADP-ribose) or a pharmaceutically acceptable salt thereof is provided. The composition comprising ADP-ribose is used alone, in combination with a second anticancer drug, or in combination with radiation therapy.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to anti-cancer therapies using adenosine diphosphate ribose (ADP-ribose), and more particularly to anti-cancer therapies using ADP-ribose alone and in combination with an anti-cancer drug or radiation. [Background technology]

[0002] Radiation therapy is a cancer treatment that uses powerful energy beams, such as X-rays, to kill cancer cells. During radiation therapy, high-energy beams are emitted from a machine and aimed at precise locations in the body. Radiation therapy damages cells by destroying genetic material (such as DNA) that controls cell growth and division. The ultimate goal of radiation therapy is to target and destroy cancerous tissue while sparing normal, healthy tissue where possible. Advances in computer technology have led to the development of computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI) scans, allowing for precise tumor targeting to minimize damage to normal tissue. Intensity-modulated radiation therapy and proton beam therapy are performed by targeting photon and proton beams in various directions with minimal error to efficiently eliminate tumors and minimize damage to normal tissue. This method tends to significantly reduce side effects from radiation therapy. However, despite improvements in treatment technology, many patients experience cancer recurrence after treatment due to the inherent resistance of cancer cells to radiation.

[0003] Chemotherapy refers to any anti-cancer treatment that uses drugs to kill cancer cells. Chemotherapy can consist of a single drug or a combination of drugs and can be administered intravenously, intramuscularly, subcutaneously, injected into body cavities, or orally in tablet form. Chemotherapy differs from surgery and radiation therapy in that the cancer-fighting drugs spread through the bloodstream and circulate throughout the body, killing or eliminating the original cancer and distant cancer (metastatic) cells. More than half of all people diagnosed with cancer undergo chemotherapy. However, one of the obstacles to cancer chemotherapy treatment is the risk of side effects. Chemotherapy can affect all rapidly growing and dividing cells in the body, including new blood cells in the bone marrow and normal cells, including those in the mouth, stomach, skin, hair, and reproductive organs. Side effects occur when chemotherapy damages normal cells. The occurrence and severity of side effects vary depending on the type of drug and the response from the first treatment cycle to the next, but are particularly closely related to the drug dose. If side effects become severe, treatment should be discontinued immediately, and side effects tend to gradually improve after normal, healthy cells have recovered. Sometimes chemotherapy can induce long-term side effects that do not go away, and these may include damage to the heart, lungs, nerves, kidneys, or reproductive organs.

[0004] During the inevitable treatment process, patients with advanced cancer may experience initial treatment failure due to unexpected resistance. Even if treatments such as radiation therapy or anticancer therapy can be continued without resistance, the burden of various side effects, both major and minor, is considerable. In the case of radiation therapy, attempts have been made to maintain the anticancer effects induced by the treatment, such as suppressing cancer-specific genes induced in a hypoxic environment and regulating various molecular biological and genetic factors involved in DNA repair. However, known therapies for enhancing the efficacy of radiation therapy or overcoming resistance have been limited in their active use due to the possibility of exacerbating side effects caused by radiation therapy, such as chlorhexidine, gastrointestinal disorders, nausea, vomiting, and diarrhea, when used in combination with radiation therapy. Therefore, these therapies have not been clearly recognized as a means of fundamentally enhancing radiation therapy sensitivity or overcoming resistance. In the case of chemotherapy, reducing the administered dose is the most reliable method for reducing the side effects that must be tolerated compared to the efficacy of the treatment. However, due to the nature of the therapy, reducing the dose due to side effects can itself be considered a treatment failure. Therefore, the results of the methods known to date have not been satisfactory, and overcoming this is a medically unmet need that remains a rather difficult problem to solve.

[0005] Adenosine diphosphate ribose (ADP-ribose) is a biological substance derived from nicotinamide adenine dinucleotide (NAD) and is known to be involved in post-translational modification of the ADP-ribosylation unit molecule within cells. The use of exogenous NAD for malignant tumors or infectious diseases is partially disclosed in WO 99 / 12951, and the use of exogenous ADP-ribose for adenovirus-related diseases or conditions or mucositis is partially disclosed in WO 2017 / 143113 or WO 03 / 099297. However, these documents do not disclose specific anti-cancer or anti-cancer adjunctive uses of ADP-ribose. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to utilize adenosine diphosphate ribose (ADP-ribose) to improve the efficacy of anti-cancer therapies. [Means for solving the problem]

[0007] To achieve the above object, the present inventors have conducted extensive research and found that accumulation of ADP-ribose in cancer cells can exert anticancer effects by disrupting the homeostasis of ADP-ribose within the cancer cells, thereby significantly improving sensitivity to radiotherapy and / or chemotherapy. Accordingly, the present invention provides a method for using ADP-ribose in anticancer therapy, a method for combining ADP-ribose with radiotherapy and / or anticancer therapy, and an ADP-ribose formulation for anticancer therapy.

[0008] 1. Use of ADP-ribose for anti-cancer therapy

[0009] The present inventors have completed the present invention by specifically confirming the anti-cancer effect of ADP-ribose in various solid carcinomas. Accordingly, the present invention provides an anti-cancer composition comprising ADP-ribose or a pharmaceutically acceptable salt thereof; a method for treating cancer comprising administering a therapeutically effective amount of ADP-ribose or a pharmaceutically acceptable salt thereof to a patient; use of ADP-ribose or a pharmaceutically acceptable salt thereof for the prevention or treatment of cancer; or use of ADP-ribose or a pharmaceutically acceptable salt thereof for the manufacture of an anti-cancer agent.

[0010] ADP-ribose or a pharmaceutically acceptable salt thereof In the present invention, ADP-ribose or adenosine diphosphate ribose is a compound represented by the following Chemical Formula 1: [ka]

[0011] Methods for producing ADP-ribose are known in the art. In one embodiment, ADP-ribose can be synthesized by hydrolysis of nicotinamide adenine dinucleotide (NAD) in the presence of an alkaline base. ADP-ribose can also be isolated in the form of a monovalent or divalent salt of the corresponding metal ion of the base. Alternatively, ADP-ribose can be commercially available as a purified raw material (CAS Number: 68414-18-6).

[0012] In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, including salts of inorganic ions such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, and iron, and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid. In addition, salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, and acetylsalicylic acid, and salts of amino acids such as lysine, arginine, and guanidine, are also included. Also included are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium, which can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts referred to in the present invention are not limited to these listed salts. Preferably, the pharmaceutically acceptable salt of ADP-ribose in the present invention is not a lithium salt of ADP-ribose (including mono- and di-lithium salts).

[0013] In the present invention, ADP-ribose can be used in the form of a precursor drug of ADP-ribose, such as poly-ADP-ribose (Poly-ADPR). For example, the precursor drug of ADP-ribose can be formed by condensing one or more hydroxyl groups of the terminal ribose moiety of ADP-ribose represented by Chemical Formula 1 with a carboxylic acid, an amino acid, a fatty acid, or a combination thereof.

[0014] Anti-cancer effects of ADP-ribose The present inventors have found that the anti-cancer composition of the present invention exerts its anti-cancer effect by accumulating ADP-ribose in cancer cells and inducing disruption of the biochemical functions of cancer cells.

[0015] Specifically, the present inventors confirmed that when various solid cancer cell lines (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, and kidney cancer) were treated with ADP-ribose (at concentrations of several to several tens of μM), the ADP-ribose concentration in the cancer cells significantly increased (Example 1; Figure 1). In particular, the ADP-ribose concentration in cancer cells increased only transiently (for several hours) upon irradiation, which is used as an anti-cancer therapy, but the ADP-ribose concentration in cancer cells increased significantly and sustainedly upon exogenous ADP-ribose treatment compared to that upon irradiation (Example 7; Figure 17).

[0016] The present inventors predicted that artificial accumulation of ADP-ribose inside cancer cells would disrupt the biochemical homeostasis of ADP-ribose maintained in cancer cells, thereby exerting an effective anticancer effect. Specifically, we confirmed that ADP-ribose at low concentrations (several to several tens of μM) exhibits significant anticancer effects against various solid cancers (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer, and ovarian cancer) (Example 2; Figures 2 to 9; Example 4; Figures 12 to 14).

[0017] This confirmed that ADP-ribose or a pharmaceutically acceptable salt thereof can be useful for cancer treatment.

[0018] In the present invention, cancer, also known as malignant tumor or neoplasm, refers to a condition or disease associated with the regulation of cell death, which occurs when the normal balance of cell death is disrupted, resulting in excessive cell proliferation. Such abnormally proliferating cells may invade surrounding tissues and organs, forming tumors and destroying or deforming normal structures in the body.

[0019] Cancers that can be alleviated, ameliorated, or treated by the anticancer composition of the present invention include, but are not limited to, solid cancers whose proliferation, invasion, metastasis, etc. can be inhibited by disrupting intracellular ADP-ribose biochemistry. The solid cancers may be, but are not limited to, breast cancer, cervical cancer, glioma, brain cancer, melanoma, head and neck cancer, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gallbladder cancer, ovarian cancer, lymphoma, osteosarcoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, squamous cell carcinoma, thyroid cancer, parathyroid cancer, or urinary tract cancer. The cancer may be a cancer in which a specific gene is mutated. Preferably, the solid cancer is one or more types selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer and ovarian cancer.

[0020] In the present invention, cancer includes primary cancer and metastatic cancer. The anticancer composition of the present invention is effective in suppressing the penetration and metastatic ability of cancer cells. Specifically, the present inventors confirmed that ADP-ribose exhibits significant anticancer effects against metastatic cancers (pancreatic cancer, breast cancer) at low concentrations (several to several tens of μM) (Example 3; Figures 10 and 11). Therefore, the composition of the present invention can be useful in treating solid cancers, particularly metastatic solid cancers (e.g., metastatic pancreatic cancer, breast cancer).

[0021] Route of administration, dosage and administration The anti-cancer composition of the present invention can be administered via any common administration route as long as it can reach the target tissue. For example, depending on the purpose, it can be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and intrarectal administration. When administered orally, it can be administered into the oral cavity in an unformulated form, a form in which the active ingredient is coated or formulated to protect it from degradation in the stomach, or in the form of an oral patch. In addition, the anti-cancer composition of the present invention can be administered using any device that can deliver the active ingredient to the target cancer cells.

[0022] Specifically, the present inventors confirmed through animal experiments using mice that ADP-ribose exhibits anticancer effects against various solid cancers (pancreatic cancer, kidney cancer, and pancreatic cancer) when administered subcutaneously, intravenously, or orally (Example 4; Figures 12 to 14).

[0023] The anti-cancer composition of the present invention can be administered in a therapeutically effective amount, which means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The therapeutically effective amount level can be determined based on factors including the severity of the disease, drug activity, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, administration route, and excretion rate of the composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the composition of the present invention, and other factors well known in the medical field. The anti-cancer composition of the present invention can be administered once or several times a day.

[0024] The anti-cancer composition of the present invention can be administered alone or in combination with one or more anti-cancer therapies. When administered in combination, the one or more anti-cancer therapies can include chemotherapy, immunotherapy, radiation therapy, surgery, nanomedicine, or a combination thereof. The type and frequency of the anti-cancer therapies are not particularly limited as long as they are anti-cancer therapies that can enhance the anti-cancer effect of ADP-ribose. For example, the anti-cancer composition of the present invention can be used as adjuvant therapy before or after surgery, and can be administered simultaneously with, before, or after radiation therapy and / or anti-cancer therapy, as described below.

[0025] 2. Combination of radiation therapy and ADP-ribose for anti-cancer treatment The present inventors have found that the combined use of radiation therapy and ADP-ribose significantly synergizes the anti-cancer therapeutic effect. Therefore, the present invention provides an anti-cancer composition containing ADP-ribose or a pharmaceutically acceptable salt thereof for use in combination with radiation therapy; a method for treating cancer by administering a therapeutically effective amount of ADP-ribose or a pharmaceutically acceptable salt thereof to a patient in combination with radiation therapy; an anti-cancer adjuvant for radiation therapy containing ADP-ribose or a pharmaceutically acceptable salt thereof; a radiosensitizer containing ADP-ribose or a pharmaceutically acceptable salt thereof; or the use of ADP-ribose or a pharmaceutically acceptable salt thereof as an adjuvant for radiation therapy.

[0026] In the present invention, an anti-cancer adjuvant for radiotherapy means a preparation that can improve, enhance or increase the anti-cancer effect of radiotherapy when administered before or after radiotherapy.

[0027] Radiation therapy In the present invention, the term "radiation therapy" refers to the therapeutic procedure of irradiating cancer cells or tumor tissue with radiation for the purpose of killing the cancer cells. It is generally a standard treatment for controlling inoperable or inoperable tumors or tumor metastasis, and is based on the principle that radiation delivered to the target site causes the death of reproductive cells. In the present invention, anticancer radiation therapy may be, but is not limited to, ionizing radiation therapy, electromagnetic radiation therapy, brachytherapy, or external beam radiation therapy.

[0028] The composition comprising ADP-ribose or a pharmaceutically acceptable salt thereof according to the present invention exhibits a synergistic anticancer effect when used in combination with radiotherapy, and can therefore be useful as an anticancer adjuvant or a radiosensitizer for improving radiosensitivity to radiotherapy, and is not particularly limited in the type of cancer to which it can be applied. For example, the cancer may be solid cancer such as brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer, or ovarian cancer, but is not limited thereto.

[0029] In one embodiment, the solid tumor may be resistant to radiotherapy. Because the anti-cancer effect of radiotherapy is manifested by the formation of DNA breaks, resistance to radiotherapy is generally determined by the mechanisms capable of repairing DNA damage induced by radiotherapy. Many attempts have been made to inhibit DNA repair mechanisms as a way to synergize the anti-cancer efficacy of radiotherapy, and blocking repair of broken DNA strands can increase the sensitivity of radiotherapy. The two main forms of DNA damage are single-strand breaks (SSBs) and double-strand breaks (DSBs). Therefore, these can be described as two repair pathways targeting SSBs and DSBs. Base excision repair (BER) is one of the various pathways involved in repairing selected types of DNA SSBs.

[0030] PARP1 plays a key role in the repair of DNA single-stranded stubs (SSBs) through a process known as ADP-ribosylation. In the nucleus, PARP1 senses SSB DNA damage and recruits DNA repair complexes to SSB sites via ADP-ribosylation for repair. Excessive accumulation of poly-ADP-ribose, synthesized through ADP-ribosylation by PARP-1 activity, ultimately leads to cell death. To prevent this, cancer cells activate proteasomes, such as PARG and ARH3, to degrade poly-ADP-ribose and activate survival signaling. The present inventors have discovered that the accumulation of ADP-ribose and ADP-ribose polymers in cancer cells can act as a mediator that disrupts these biochemical survival mechanisms of cancer cells, potentially making them useful as an important anticancer adjuvant to overcome radiation therapy resistance.

[0031] In a specific example of the present invention, it was confirmed that when the ADP-ribose or a pharmaceutically acceptable salt thereof of the present invention was administered in combination with low-dose radiation in different cancer types, the response to radiation therapy was enhanced in all cancer types. Therefore, the ADP-ribose or a pharmaceutically acceptable salt thereof of the present invention can enhance the response to radiation therapy and can be very useful as an adjunct anticancer agent. Since it has excellent anticancer activity even when irradiated with a tolerable dose of radiation, it can minimize side effects that may occur due to radiation.

[0032] Specifically, the present inventors demonstrated that ADP-ribose treatment of various solid cancers can maintain intracellular ADP-ribose concentrations higher than those elevated by radiation (Example 7; Figure 17). The anti-cancer effect of radiation is expected through the killing of cancer cells by damaging their genetic material. To overcome this, cancer cells continuously repair DNA strands through ADP-ribosylation. However, as shown in Figure 17, sustaining the accumulation of ADP-ribose, which only temporarily increases due to the repair of DNA strands broken by radiation, can cause biochemical disruption in the DNA repair process in cancer cells, potentially enhancing the anti-cancer effects of radiation therapy. This may also enable the use of reduced doses of anti-cancer radiation to overcome radiation therapy resistance and reduce side effects.

[0033] Furthermore, the present inventors confirmed that the combined use of radiation therapy, ADP-ribose administration, and radiation therapy exhibited synergistic anticancer effects against various solid cancers (brain cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, and kidney cancer) (Example 8; Figure 18, Example 9; Figure 19).

[0034] Route of administration, dosage and administration Radiation therapy is generally defined in terms of radiation absorbed dose (Gy), time, and classification, and must be conservatively defined by the oncologist. The radiation dose a patient can tolerate is influenced by various considerations, the two most important being the location of the tumor relative to other critical structures or tissues in the body and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiation therapy may include, but is not limited to, a treatment schedule of approximately 1.8-2.0 Gy per day, 5 days per week, for a total dose of 10-80 Gy administered to the patient over a 1-6 week period, or a treatment schedule of low-dose radiation, such as 1-10 cGy or 5-40 cGy in multiple fractions.

[0035] The present inventors have confirmed that the combined use of a 2 Gy dose of radiation therapy and ADP-ribose administration in tumor mouse models exhibits synergistic anticancer effects against various solid cancers (Example 8; Figure 18, Example 9; Figure 19). However, the radiation absorbed dose of radiation therapy that can be used in combination with the anticancer composition of the present invention is not limited thereto, and a single dose can be 1 to 10 Gy, with a total dose of about 1 to 100 Gy, but is not limited thereto. For example, the total radiation dose irradiated to a patient can be about 1 cGy to about 100 Gy or about 1 cGy to about 50 Gy. In some embodiments, the individual is undergoing and / or can undergo radiation therapy. The radiation therapy can include one or more radiation treatments administered five days per week for one to ten weeks. As will be appreciated by those skilled in the art, radiation therapy can be administered over a specific period (e.g., 1-10 weeks) and can be administered to an individual intermittently, rather than continuously.

[0036] The administration route, dosage, and administration method of ADP-ribose in combination with radiation therapy are as described above, and it can be administered before or after direct radiation therapy. Furthermore, chemotherapy can be used in combination as described below.

[0037] 3. Combination of chemotherapy and ADP-ribose for anti-cancer treatment The present inventors have found that the combined use of chemotherapy and ADP-ribose significantly synergizes the effects of anti-cancer treatment. Therefore, the present invention provides: an anti-cancer adjuvant or sensitizer for chemotherapy containing ADP-ribose or a pharmaceutically acceptable salt thereof; an anti-cancer composition or combination containing (i) ADP-ribose or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer drug; a method for treating cancer comprising co-administering a therapeutically effective amount of ADP-ribose or a pharmaceutically acceptable salt thereof and a second anti-cancer drug; and the use of ADP-ribose or a pharmaceutically acceptable salt thereof as an anti-cancer adjuvant or sensitizer for chemotherapy for the prevention or treatment of cancer.

[0038] In the present invention, an anti-cancer adjuvant for chemotherapy means a preparation that can improve, enhance or increase the anti-cancer effect of an anti-cancer drug, and when used together with an anti-cancer drug, can improve, enhance or increase the anti-cancer effect of the anti-cancer drug.

[0039] Anticancer drugs that can be used in combination with ADP-ribose In the present invention, chemotherapy is a cancer treatment method using one or more chemotherapeutic agents as anticancer agents. In the present invention, the second anticancer agent that can be used in combination with ADP-ribose or its pharmaceutically acceptable salt can be selected appropriately for curing, controlling, or alleviating symptoms of cancer depending on the type and progression of cancer, and may be, for example, one or more selected from cytotoxic anticancer agents, targeted anticancer agents, immune anticancer agents, metabolic anticancer agents, or combinations thereof.

[0040] In the present invention, a cytotoxic anticancer drug is a drug that exerts an anticancer effect by attacking cancer cells, which indiscriminately divide at a rate faster than normal cells, and has the same meaning as commonly used in the technical field to which the present invention pertains. The cytotoxic anticancer drug includes alkylating agents, antimetabolites, and natural product anticancer drugs.

[0041] Examples of alkylating agents include, but are not limited to, nitrogen mustards (e.g., cyclophosphamide, chlormethine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, etc.), alkylsulfonates (e.g., busulfan, procarbazine, etc.), nitrosoureas (e.g., carmustine, lomustine, streptozocin, etc.), and platinum-based alkylating agents (e.g., cisplatin, carboplatin, dicycloplatin, eptaplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, etc.). Alkylating agents can induce cancer cell destruction by binding to DNA within cancer cells and damaging the DNA structure.

[0042] Examples of antimetabolites include, but are not limited to, pyrimidine derivatives (e.g., 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine, etc.), folate derivatives (e.g., methotrexate, pemetrexed, etc.), and purine derivatives (e.g., mercaptopurine, etc.). Antimetabolites can induce cancer cell death by suppressing metabolism necessary for DNA replication and cell survival.

[0043] Examples of the natural product anticancer drugs include, but are not limited to, topoisomerase inhibitors (camptothecin, epipodophyllotoxin, taxane-series drugs), antibiotics (e.g., dactinomycin, doxorubicin, daunorubicin, mitomycin, phleomycin, idarubicin, mitoxantrone HCl, etc.).

[0044] In the present invention, a targeted anticancer drug is an anticancer drug that induces cancer cell death by inhibiting a target protein (receptor or enzyme) involved in cancer growth, and has the same meaning as commonly used in the technical field to which the present invention pertains. The targeted anticancer drug includes small molecular weight compounds and monoclonal antibodies that inhibit target proteins (e.g., tyrosine kinases).

[0045] In the present invention, the targeted anticancer agent may be a receptor tyrosine kinase inhibitor that targets one or more targets selected from the group consisting of VEGF / VEGFR, EGFR, and HER2.

[0046] In one embodiment, the targeted anticancer drug that can be administered in combination with ADP-ribose is a VEGF / VEGFR inhibitor. In the present invention, the VEGF / VEGFR inhibitor may include, but is not limited to, small molecule compounds such as axitinib, cabozantinib, lapatinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, and vandetanib, as well as monoclonal antibodies such as bevacizumab, ramucirumab, and ranibizumab. In the present invention, cancers that can be treated by combined administration of ADP-ribose and a VEGF / VEGFR inhibitor are solid cancers, preferably brain cancer or liver cancer.

[0047] In one embodiment, the targeted anticancer drug that can be administered in combination with ADP-ribose is an EGFR inhibitor. In the present invention, EGFR inhibitors include, but are not limited to, small molecule compounds such as osimertinib, gefitinib, erlotinib, afatinib, brigatinib, icotinib, and vandetanib, as well as monoclonal antibodies such as cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab. In the present invention, cancers that can be treated by the combined administration of ADP-ribose and an EGFR inhibitor are solid cancers, preferably lung cancer.

[0048] In one embodiment, the targeted anticancer drug that can be administered in combination with ADP-ribose is a HER2 inhibitor. In the present invention, HER2 inhibitors include, but are not limited to, small molecule compounds such as lapatinib, neratinib, and afatinib, as well as monoclonal antibodies such as trastuzumab, pertuzumab, and margetuximab. In the present invention, cancers that can be treated by the combined administration of ADP-ribose and a HER2 inhibitor are solid cancers, preferably breast cancer.

[0049] In addition, the targeted anticancer drug of the present invention may also include Bcr-Abl targeted anticancer drugs such as imatinib, dasatinib, and nilotinib; Src targeted anticancer drugs such as bosutinib; JAK targeted anticancer drugs such as lestaurtinib, ruxolitinib, and pacritinib; MAP2 kinase targeted anticancer drugs such as cobimetinib, selumetinib, trametinib, and binimetinib; and MEL4-ALK targeted anticancer drugs such as ceritibin and crizotinib.

[0050] The second anticancer drug of the present invention may be a combination of one or more cytotoxic anticancer drugs and / or targeted anticancer drugs, which may be administered simultaneously or at different times. For example, the second anticancer drug may be nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, or ibritumomab. Tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimelacin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, bilephrine The anti-inflammatory drug may be one or more selected from the group consisting of norelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, lomustine, and carmustine, but is not limited thereto.

[0051] In the present invention, cancer immunotherapy is a cancer treatment method that activates the human immune system to fight cancer cells using an immunotherapy agent. In the present invention, the immunotherapy agent includes immune checkpoint inhibitors, immune cell therapy agents, anticancer vaccines, and antibody-drug conjugates, and an appropriate type can be selected for complete cure, control, or symptom relief of cancer depending on the type and progression of cancer.

[0052] In one embodiment, the immunological anticancer agent may be an immune checkpoint inhibitor, and may be one or more selected from the group consisting of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, CD28 antibody, KIR antibody, TCR antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, A2aR antibody, ICOS antibody, OX40 antibody, 4-1BB antibody, and GITR antibody. For example, the immune checkpoint inhibitor may be a PD-1 antibody such as nivolumab, pembrolizumab, cemiplimab, pidilizumab, or toripalimab; a PD-L1 antibody such as atezolizumab, avelumab, or durvalumab; or a CTLA-4 antibody such as ipilimumab or tremelimumab, or all of these.

[0053] In one embodiment, the immunosuppressant may be a cellular therapeutic agent, which may be a CAR-T therapeutic agent or a CAR-NK therapeutic agent, such as tisagenlecleucel or axicabtagene ciloleucel, but is not limited to these.

[0054] In the present invention, a metabolic anticancer drug refers to a drug that is involved in the growth and survival of cancer cells, such as by supplying nutrients to cancer cells, or that kills cancer cells by participating in various essential metabolic processes. Examples of metabolic anticancer drugs include, but are not limited to, IM-156, 3-bromopyruvate (3BP), NYH817100, WZB117, GNE-140, AZ93, AZD3965, CPI-613, MKT-077, CB-839, CB-1158, CPI-444, TVB-2640, NDI-010976, TCD-717, ADI-PEG20, epacadostat, indoximod, PX478, CPI-0610, RTA402, APO866, GMX1778, AG-221, and AG-120.

[0055] The ADP-ribose of the present invention can be administered in a form that exists independently in combination with a second anticancer drug, or can be administered after forming a physical / chemical bond with the second anticancer drug by any known method, depending on the purpose. For example, ADP-ribose can be used in a form that is directly bound to the second anticancer drug or linked to the second anticancer drug via a known linker. There are no particular limitations on the method of application as long as the ADP-ribose of the present invention acts together with the second anticancer drug to exhibit a synergistic anticancer effect.

[0056] Synergistic anticancer effects of ADP-ribose in combination with anticancer drugs The composition comprising ADP-ribose or a pharmaceutically acceptable salt thereof according to the present invention exhibits a synergistic anticancer effect when used in combination with chemotherapy, and can therefore be useful as an anticancer adjuvant or anticancer therapy sensitizer. In the anticancer adjuvant or chemotherapy sensitizer, the cancer may be any of the solid cancers described above. The solid cancer may be a solid cancer that exhibits resistance to radiation therapy.

[0057] The anticancer effects of existing anticancer drugs, such as the reduction of cancer cell viability, can be induced by ADP-ribose signaling. By inducing ADP-ribose to be actively utilized during the cell death process induced by existing anticancer drugs, a synergistic anticancer effect can be expected even when the existing anticancer drug is administered at a concentration lower than its required level. Specifically, the present inventors have confirmed that the combined administration of ADP-ribose and a second activator exhibits a synergistic anticancer effect against various solid cancers (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, and colon cancer) (Example 5; Figure 15; Example 6; Figure 16).

[0058] In one embodiment, the cytotoxic anticancer drug that can be administered in combination with ADP-ribose can be an antimetabolite anticancer drug, specifically a pyrimidine derivative, more specifically gemcitabine or 5-fluorouracil. In this case, the cancer that can be treated by the combination therapy is a solid cancer, specifically pancreatic cancer or colorectal cancer. The inventors have confirmed the synergistic anticancer effect of the combination of ADP-ribose and gemcitabine in pancreatic cancer (Example 5-3; Figure 15C; Example 6-3; Figure 16C), and the synergistic anticancer effect of the combination of ADP-ribose and 5-fluorouracil in colorectal cancer (Example 5-6; Figure 15F).

[0059] In one embodiment, the targeted anti-cancer drug that can be administered in combination with ADP-ribose may be a VEGF / VEGFR inhibitor, specifically an anti-VEGF monoclonal antibody. In this case, the treatable cancer may be a solid cancer, specifically brain cancer or liver cancer. The present inventors have confirmed the synergistic anti-cancer effect of combined administration of ADP-ribose and the anti-VEGF monoclonal antibody bevacizumab in brain cancer (Example 5-1; Figure 15A, Example 6-1; Figure 16A), and the synergistic anti-cancer effect of combined administration of ADP-ribose and sorafenib in liver cancer (Example 5-4; Figure 15D, Example 6-4; Figure 16D).

[0060] In one embodiment, the targeted anticancer drug that can be administered in combination with ADP-ribose may be an EGFR inhibitor, specifically a small molecule EGFR inhibitor. In this case, the treatable cancer may be a solid cancer, specifically lung cancer. The present inventors have confirmed the synergistic anticancer effect of the combined administration of ADP-ribose and the EGFR inhibitor osimertinib in lung cancer (Example 5-2; Figure 15B, Example 6-2; Figure 16B).

[0061] In one embodiment, the targeted anti-cancer drug that can be administered in combination with ADP-ribose may be a HER2 inhibitor, specifically an anti-HER2 monoclonal antibody. In this case, the treatable cancer may be a solid cancer, specifically breast cancer. The present inventors have confirmed the synergistic anti-cancer effect of the combined administration of ADP-ribose and the anti-HER2 monoclonal antibody trastuzumab in breast cancer (Example 5-5; Figure 15E).

[0062] Route of administration, dosage and administration In the anti-cancer composition or combination of the present invention, ADP-ribose or its pharmaceutically acceptable salt and the second anti-cancer drug may be administered simultaneously or at different times. For example, the composition or combination may be administered before or after the anti-cancer therapy using the second anti-cancer drug, in which case ADP-ribose or its pharmaceutically acceptable salt may be administered. Each therapy may be administered once or several times in a single cycle, but is not limited thereto.

[0063] In one embodiment, the administration schedule of a second anticancer agent and ADP-ribose, which was confirmed to have a synergistic anticancer effect in a tumor mouse model (FIG. 16), is shown in Table 1 below. The preferred patient administration doses of the anticancer composition of the present invention and the second anticancer agent can be calculated taking into account the optimal mouse administration dose specifically confirmed in the present invention, the dose-response relationship between mice and humans, and the NOAEL (No-observed-adverse-effect level), etc. [Table 1] The ADP-ribose or its pharmaceutically acceptable salt and the second anticancer drug according to the present invention can each be administered in amounts sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level of each can be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, administration route, and excretion rate of the composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the composition of the present invention used, and other factors well known in the medical field.

[0064] 4. ADP-ribose preparations for anti-cancer therapy The weight percentage of ADP-ribose or a pharmaceutically acceptable salt thereof contained in the anticancer composition described above is not particularly limited, but may be 0.0001 to 90 wt %, specifically 0.001 to 50 wt %, more specifically 0.01 to 20 wt %, based on the total weight of the final composition. The anti-cancer composition may be prepared in the form of a pharmaceutical composition or a food composition.

[0065] When the anti-cancer composition of the present invention is prepared in the form of a pharmaceutical composition, the pharmaceutical composition may further contain a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceuticals.

[0066] Specifically, the pharmaceutical composition of the present invention can be prepared into formulations for various administration routes by conventional methods.

[0067] In one embodiment, the pharmaceutical composition of the present invention may be prepared in the form of an oral administration formulation. Specifically, the composition may be prepared in the form of tablets (e.g., orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets), capsules (e.g., hard capsules, soft capsules), granules (e.g., sustained-release granules, enteric-coated granules, effervescent granules), powders, oral liquids (e.g., elixirs, suspensions, emulsions, lemonades, perfumes, decoctions and infusions, spirits, tinctures), syrups, oral jellies, teas, extracts (e.g., soft extracts, dry extracts), fluid extracts, or pills.

[0068] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a formulation for oral administration. Specifically, the composition may be prepared as an oral tablet (e.g., troche, sublingual tablet, buccal tablet, adhesive tablet, gum), oral liquid (e.g., gargle), oral spray, oral semisolid (e.g., oral cream, oral gel, oral ointment), or oral dissolving film.

[0069] In one embodiment, the pharmaceutical composition of the present invention may be prepared as an injection. In this case, the injection may be prepared in the form of an ampoule, a vial, a pre-filled syringe, a cartridge, etc., and more specifically, may be prepared as an infusion solution, a lyophilized injection, a powder injection, an implant, a sustained release injection, a dialysis or a flow-through preparation.

[0070] In one embodiment, the pharmaceutical composition of the present invention can be prepared as a dialysis or transfusion formulation. Specifically, the composition can be prepared as a dialysis agent (e.g., peritoneal dialysis agent, hemodialysis agent) or transfusion agent.

[0071] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a formulation for bronchial or pulmonary administration. Specifically, the composition may be prepared as an inhalant (e.g., inhalable powder, inhalable solution, or inhalable aerosol).

[0072] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a formulation for ocular administration. Specifically, the composition may be prepared as eye drops or eye ointment.

[0073] In one embodiment, the pharmaceutical composition of the present invention may be prepared as a preparation for nasal administration (e.g., nasal powder, nasal liquid), enema, rectal preparation (e.g., suppository, rectal semisolid, enema), vaginal preparation (e.g., vaginal tablet, vaginal suppository), or skin preparation (e.g., topical solid, topical liquid, aerosol, ointment, cream, gel, transdermal absorption preparation, cataplasm, patch, paste).

[0074] When the pharmaceutical composition is used for oral administration, it can be prepared in a sustained-release formulation by suitable encapsulation, enteric coating, compounding with a polymer, etc.

[0075] In one embodiment, the sustained release formulation may be prepared as a long-acting formulation.

[0076] In one embodiment, the long-acting formulation can be made by mixing the polymer and lipid in the appropriate ratio.

[0077] When the anti-cancer composition of the present invention is prepared in the form of a food composition, the food composition may contain additional ingredients commonly used in foods to improve the smell, taste, appearance, etc. For example, food additives may be added. The additives are selected depending on the type of food and used in appropriate amounts.

[0078] The food composition may be manufactured as a functional health food, which is the same term as food for special health use (FoSHU) and refers to a food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients. The functional health food may be manufactured in various forms, such as tablets, capsules, powders, granules, liquids, and pills, to achieve beneficial effects in treating cancer. [Effects of the Invention]

[0079] The composition comprising ADP-ribose or a pharmaceutically acceptable salt thereof according to the present invention not only exhibits effective anti-cancer therapeutic effects when used alone or in combination with radiotherapy and / or chemotherapy, but also does not exhibit toxicity to normal cells, and therefore can be usefully utilized in anti-cancer therapy. [Brief explanation of the drawings]

[0080] [Figure 1] FIG. 1 is a graph showing the results of confirming the accumulation of intracellular ADP-ribose after ADP-ribose treatment of various solid cancer cells (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, F: HCT116, G: Caki-1 cell line). [Figure 2] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on brain cancer (U-87MG) cells, confirming their growth inhibition and death. [Figure 3] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on lung cancer (H1975) cells, confirming their growth inhibition and death. [Figure 4] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on pancreatic cancer (AsPC-1) cells, confirming their growth inhibition and death. [Figure 5]This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on hepatoma (Hep G2) cells, confirming their growth inhibition and death. [Figure 6] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on breast cancer (MDA-MB-231) cells, confirming their growth inhibition and death. [Figure 7] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on colon cancer (HCT116) cells, confirming their growth inhibition and cell death. [Figure 8] This is a micrograph (A) and a graph (B) showing the effects of ADP-ribose treatment on kidney cancer (Caki-1) cells, confirming their growth inhibition and cell death. [Figure 9] This is a graph confirming the cancer cell killing effect after treating gastric cancer (SNU-1) or ovarian cancer (OVCAR-3) cells with ADP-ribose. [Figure 10] This is a micrograph (A) and a graph (B) showing that the penetration and metastatic ability of pancreatic cancer (AsPC-1) cells was reduced after treatment with ADP-ribose. [Figure 11] 1 shows a micrograph (A) and a graph (B) confirming the reduction in the penetration and metastatic ability of breast cancer (MDA-MB-231) cells after treatment with ADP-ribose. [Figure 12] 1A and 1B are graphs and photomicrographs showing the tumor volume reduction effect after subcutaneous (SC) injection of ADP-ribose in a xenograft animal model induced by a pancreatic cancer cell line. [Figure 13] 1A and 1B are graphs and photomicrographs showing the tumor volume reduction effect after intravenous (IV) injection of ADP-ribose in a xenograft animal model induced by a renal cancer cell line. [Figure 14] 1A and 1B are graphs and photomicrographs showing the tumor volume reduction effect after oral (PO) administration of ADP-ribose to a xenograft animal model induced by a pancreatic cancer cell line. [Figure 15]This graph shows the synergistic cell killing effect when various solid cancer cells (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, F: HCT116) were treated with low doses of anticancer drugs (A: bevacizumab, B: osimertinib, C: gemcitabine, D: sorafenib, E: Herceptin, F: 5-fluorouracil) in combination with ADP-ribose. [Figure 16] This is a graph showing the synergistic tumor volume reduction effect when low-dose anticancer drugs (A: bevacizumab, B: osimertinib, C: gemcitabine, D: sorafenib) and ADP-ribose were administered in combination to tumor-induced animals. [Figure 17] 1 is a graph showing the results of comparing the amount of intracellular ADP-ribose accumulation when solid cancer cells (A: U-87MG, B: Caki-1, C: AsPC-1, D: MDA-MB-231) were irradiated and when treated with ADP-ribose. [Figure 18] This graph confirms the synergistic cell killing effect when solid cancer cells (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, F: HCT116, G: Caki-1) are treated with a dose that shows radiation resistance in combination with ADP-ribose. [Figure 19] 1 is a graph showing the synergistic tumor volume reducing effect when low-dose radiation and ADP-ribose are administered in combination to tumor-induced animals. [Figure 20] These are photographs and graphs showing whether cytotoxicity occurs after ADP-ribose is administered to normal cells (A, B: human colon fibroblast, C, D: human hair dermal papilla cell). DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0082] material and method 1. ADP-ribose and Anticancer Drug Preparation ADP-ribose and the anticancer drugs bevacizumab, osimertinib, gemcitabine, sorafenib, Herceptin, and 5-fluorouracil were all ordered from Sigma (St. Louis, MO, USA). ADP-ribose and all anticancer drugs were stored frozen at −20°C until use.

[0083] 2. Cell Line Preparation Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), kidney cancer (Caki-1), gastric cancer (SNU-1), and ovarian cancer (OVCAR-3) cells were ordered from the American Type Culture Collection (ATCC, Manassas, VA, USA) and stored in liquid nitrogen until use in the study.

[0084] 3. ADP-ribose Quantitative Analysis 5×10 5Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), and kidney cancer (Caki-1) cells were cultured in their appropriate medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. After incubation, the medium was removed, the cells were washed with phosphate-buffered saline, and then added to RIPA buffer and incubated on ice for 20 minutes. The cells were then scraped, placed in a centrifuge tube, and centrifuged at 10,000 xg for 10 minutes at 4°C. The supernatant was collected, added with 1% SDS, boiled at 100°C for 5 minutes, and chilled on ice. The supernatant was centrifuged again at 10,000 g and 4°C for 10 minutes to obtain the final supernatant, and ADP-ribose was quantitatively analyzed and compared using an ELISA analysis kit (Cell Biolabs, Inc., San Diego, CA, USA).

[0085] 4. Verification of cancer cell death 3 x 10 cells in a 96-well plate 3 Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), kidney cancer (Caki-1), gastric cancer (SNU-1), and ovarian cancer (OVCAR-3) cells were cultured at 37°C and 5% CO2 for 24 hours and then treated with various concentrations of ADP-ribose. After an additional 24 hours of culture at 37°C and 5% CO2, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well. The absorbance was measured to determine the median lethality (LC50) and maximum lethality (maximum lethality) concentrations relative to untreated cells.

[0086] 5. Verification of inhibition of cancer cell metastasis and penetration ability The metastatic and invasive abilities of cancer cells were analyzed using transwells with 8.0 μm pore size polycarbonate membrane filters. The transwells were placed in a 24-well plate, and 3.5 × 10 pancreatic cancer (Aspc-1) and breast cancer (MDA-MB-231) cells were placed in each well. 5 Cells were seeded onto the top surface of the inserts at a density of 1000 μg / cm2 and divided into untreated and ADP-ribose-treated groups. They were then cultured for 24 hours at 37°C and 5% CO2. After culture, the transwells were rinsed gently with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and eosin to observe the percentage of cells that had migrated and infiltrated through the top surface of the inserts compared to the untreated group.

[0087] 6. Animal Preparation Five-week-old BALB / c nude mice were ordered from Charles River Laboratories (Wilmington, MA, USA). The animal facility was maintained in a specific-pathology-free environment at a temperature of 22–25°C with a 12-hour day / night cycle (lights on at 8:00 AM). Food and water were provided ad libitum. All animal studies were conducted in accordance with the regulations of the Animal Research Ethics Committee.

[0088] 7. Verification of anti-cancer efficacy of ADP-ribose by administration route and concentration using xenograft model Five-week-old BALB / c nude mice were inoculated with pancreatic cancer (AsPC-1) or renal cancer (caki-1) cells (1 × 10 7 ) were inoculated into the back of the flank of the mice, and the tumor volume was approximately 150 mm 3 The tumors were allowed to grow until the tumor volume reached 1000 μg / cm. Three concentrations of ADP-ribose were then administered subcutaneously, intravenously, and orally three times a week. Tumor size was measured every six days using a digital microscope, and the changes in tumor volume were compared between groups.

[0089] 8. Verification of cancer cell death by combined use of anti-cancer drugs 3 x 10 cells in a 96-well plate 3Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), and colon cancer (HCT116) cells were cultured at 37°C and 5% CO2 for 24 hours, and then treated with 15 mM bevacizumab for brain cancer (U-87MG), 1 nM osimertinib for lung cancer (H1975), 1 μM gemcitabine for pancreatic cancer (Aspc-1), 1 μM sorafenib for liver cancer (Hep G2), 2.5 μM Herceptin for breast cancer (MDA-MB-231), and 10 μM 5-fluorouracil for colon cancer (HCT116), either alone or in combination with ADP-ribose. After treatment, the cells were incubated for an additional 24 hours at 37°C and 5% CO2, and then 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After the reaction was completed, the reagent was removed, and 200 μl of dimethyl sulfoxide was added to each well. The absorbance was measured to compare the degree of cell death.

[0090] 9. Verification of anti-cancer efficacy by combining anti-cancer drugs using xenograft models Five-week-old BALB / c nude mice were inoculated with brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (AsPC-1), or liver cancer (Hep G2) cells (1 × 10 7 ) were inoculated into the back of the flank of each mouse, and the tumor volume was approximately 150 mm 3 At the time of tumor growth, the tumors were divided into control, anticancer drug monotherapy, and combination therapy groups. For the brain cancer (U-87MG) study, 25 mg / kg low-dose bevacizumab was administered intraperitoneally twice weekly. For the lung cancer (H1975) study, 1 mg / kg low-dose osimertinib was administered orally twice weekly. For the pancreatic cancer (AsPC-1) study, 50 mg / kg low-dose gemcitabine was administered intraperitoneally twice weekly. For the liver cancer (Hep G2) study, 10 mg / kg low-dose sorafenib was administered orally five times weekly, either alone or in combination with the aforementioned anticancer drugs and 10 mg / kg ADP-ribose (subcutaneously administered three times weekly). Tumor size was measured every six days using a digital microscope, and the results of tumor volume changes were compared between groups.

[0091] 10. Quantitative analysis of ADP-ribose in cancer cells using radiation therapy Brain cancer (U-87MG), kidney cancer (Caki-1), pancreatic cancer (AsPC-1), or breast cancer (MDA-MB-231) cells were cultured in 6-well plates and divided into untreated, irradiated, and ADP-ribose-treated groups. Irradiation was performed using an X-Rad 320 irradiator (Precision X-ray, North Branford, CT, USA) at 12.5 mA and a 2.0 mm Al X-ray beam filter at 300 kVp and a dose rate of 150 cGy / min, resulting in a total dose of 1, 2, or 5 Gy. Before and after treatment, the medium was removed at 4, 8, 16, and 24 hours. The cells were washed with phosphate-buffered saline (PBS), then added to RIPA buffer and placed on ice for 20 minutes. The cells were then scraped, placed in a centrifuge tube, and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was collected, and 1% SDS was added, followed by boiling at 100°C for 5 minutes and chilling on ice. The supernatant was then centrifuged again at 10,000 g and 4°C for 10 minutes to obtain the final supernatant, which was then quantitatively analyzed for ADP-ribose using an ELISA kit (Cell Biolabs, Inc., San Diego, CA, USA).

[0092] 11. Verification of cancer cell death by combined use of radiation 3 x 10 cells in a 96-well plate 3Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), and kidney cancer (Caki-1) cells were cultured for 24 hours at 37°C and 5% CO2, then treated with ADP-ribose or 2.5 Gy of radiation, or ADP-ribose and 2 Gy of radiation, and then cultured for another 24 hours at 37°C and 5% CO2. After incubation, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to compare cell death.

[0093] 12. Verification of anti-cancer efficacy in combination with radiation using xenograft models Five-week-old BALB / c nude mice were inoculated with brain cancer (U-87MG), pancreatic cancer (AsPC-1), breast cancer (MDA-MB-231), or kidney cancer (Caki-1) cells (1 × 10 7 ) were inoculated into the back of the flank of each mouse, and the tumor volume was approximately 150 mm 3 At the time of tumor growth, mice were divided into three groups: a control group, a 2 Gy dose of radiation alone, and a 2 Gy dose of radiation plus 10 mg / kg ADP-ribose (subcutaneously administered three times weekly). Mice were irradiated using an X-Rad 320 irradiator (Precision X-ray, North Branford, CT, USA). Mice were positioned at an appropriate distance from the radiation source to achieve the prescribed radiation dose. Lead shielding was used to protect other body parts and organs from radiation exposure beyond the tumorigenic site. Using an X-ray beam filter consisting of 12.5 mA and 2.0 mm Al, a total of 2 Gy of radiation was delivered at 300 kVp with a dose rate of 150 cGy / min, divided into two 1 Gy fractions (over two days). Tumor size was measured every six days using a digital microscope, and tumor volume changes were compared between groups.

[0094] 13. Analysis of statistical significance All graphs were expressed as mean ± standard deviation. Statistical significance was analyzed using Student's t-test analysis, depending on whether the data were normalized or not. Differences of P < 0.05 or greater were considered statistically significant. Statistical significance was analyzed using a program from Systat Software (Sigmastat ver. 3.5, Systat Software Inc., Chicago, IL, USA).

[0095] Example 1: Changes in ADP-ribose levels in cancer cells Example 1-1: Changes in ADP-ribose levels in U-87MG cells 5×10 5 U-87MG cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed, and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0096] As a result, it was confirmed that the amount of ADP-ribose significantly increased when brain cancer cells (U-87MG) were treated with ADP-ribose compared to the control group (P<0.001) (Figure 1A).

[0097] Example 1-2: Changes in ADP-ribose levels in H1975 cells 5×10 5 H1975 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed and treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0098] As a result, it was confirmed that the amount of ADP-ribose significantly increased (P<0.001) when lung cancer cells (H1975) were treated with ADP-ribose compared to the control group (Fig. 1B).

[0099] Example 1-3: Changes in ADP-ribose levels in AsPC-1 cells 5×10 5 AsPC-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed, and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0100] As a result, it was confirmed that the amount of ADP-ribose significantly increased when pancreatic cancer cells (AsPC-1) were treated with ADP-ribose compared to the control group (P<0.001) (Figure 1C).

[0101] Example 1-4: Changes in ADP-ribose levels in Hep G2 cells 5×10 5 Hep G2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed, and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0102] As a result, it was confirmed that the amount of ADP-ribose significantly increased (P<0.001) when ADP-ribose was treated in hepatoma cells (Hep G2) compared to the control group (Figure 1D).

[0103] Example 1-5: Changes in ADP-ribose levels in MDA-MB-231 cells 5×10 5 MDA-MB-231 cells were cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed and treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0104] As a result, it was confirmed that the amount of ADP-ribose significantly increased when breast cancer cells (MDA-MB-231) were treated with ADP-ribose compared to the control group (P<0.001) (Figure 1E).

[0105] Example 1-6: Changes in ADP-ribose levels in HCT116 cells 5×10 5 HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed and treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0106] As a result, it was confirmed that the amount of ADP-ribose significantly increased (P<0.001) when colon cancer cells (HCT116) were treated with ADP-ribose compared to the control group (Figure 1F).

[0107] Example 1-7: Changes in ADP-ribose levels in Caki-1 cells 5×10 5Caki-1 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium was removed, and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The supernatant was then used for ELISA analysis to detect ADP-ribose.

[0108] As a result, it was confirmed that the amount of ADP-ribose significantly increased (P<0.001) when kidney cancer cells (Caki-1) were treated with ADP-ribose compared to the control group (Figure 1G).

[0109] To summarize the above, Example 1 confirmed that intracellular ADP-ribose concentration can be significantly increased by exogenous ADP-ribose treatment in various solid cancer cells (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, and kidney cancer) (Figure 1).

[0110] Example 2: Changes in cancer cell viability depending on ADP-ribose concentration Example 2-1: Changes in viability of U-87MG cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3 U-87MG cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with ADP-ribose at final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After removing the reagent, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0111] As a result, in the control group without ADP-ribose treatment, rapid growth of brain cancer cells (U-87MG) was observed under a microscope, while in the experimental group with ADP-ribose treatment, growth of the brain cancer cells was inhibited and death was confirmed (Figure 2A). Quantitative measurements of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the brain cancer cells were killed at a concentration of 8 μM, and that almost all cells were killed at 32 μM (Figure 2B).

[0112] Example 2-2: Changes in viability of H1975 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3 H1975 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0113] As a result, in the control group without ADP-ribose treatment, rapid growth of lung cancer cells (H1975) was observed under a microscope, while in the experimental group with ADP-ribose treatment, growth of the lung cancer cells was inhibited and death was confirmed (Figure 3A). Quantitative measurements of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the lung cancer cells were killed at a concentration of 2 μM, and that almost all cells were killed at 32 μM (Figure 3B).

[0114] Example 2-3: Changes in viability of AsPC-1 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3AsPC-1 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0115] As a result, in the control group without ADP-ribose treatment, rapid growth of pancreatic cancer cells (AsPC-1) was observed under a microscope, while in the experimental group with ADP-ribose treatment, growth of the pancreatic cancer cells was inhibited and death was confirmed (Figure 4A). Quantitative measurements of the LD50 and LD60 values ​​for ADP-ribose treatment concentrations confirmed that approximately half of the pancreatic cancer cells were killed at a concentration of 2 μM, and that almost all cells were killed at 32 μM (Figure 4B).

[0116] Example 2-4: Changes in viability of Hep G2 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3 Hep G2 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0117] As a result, in the control group without ADP-ribose treatment, rapid growth of hepatoma cells (Hep G2) was observed under a microscope, while in the experimental group with ADP-ribose treatment, the growth of the hepatoma cells was inhibited and they died (Figure 5A). Quantitative measurements of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the hepatoma cells were killed at a concentration of 4 μM, and all cells were killed at 32 μM (Figure 5B).

[0118] Example 2-5: Changes in viability of MDA-MB-231 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3 MDA-MB-231 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0119] As a result, in the control group without ADP-ribose treatment, rapid growth of breast cancer cells (MDA-MB-231) was observed under a microscope, while in the experimental group with ADP-ribose treatment, growth of the breast cancer cells was inhibited and death was confirmed (Figure 6A). Quantitative measurements of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the breast cancer cells were killed at a concentration of 16 μM, and that almost all cells were killed at 32 μM (Figure 6B).

[0120] Example 2-6: Changes in viability of HCT116 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3HCT116 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0121] As a result, in the control group without ADP-ribose treatment, colon cancer cells (HCT116) were observed under a microscope to be growing rapidly, while in the experimental group with ADP-ribose treatment, the growth of colon cancer cells was inhibited and they died (Figure 7A). Quantitative measurements of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the colon cancer cells were killed at a concentration of 8 μM, and that almost all of the cells were killed at 32 μM (Figure 7B).

[0122] Example 2-7: Changes in viability of Caki-1 cells with increasing ADP-ribose concentration 3 x 10 cells in a 96-well plate 3 Caki-1 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM ADP-ribose. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0123] As a result, rapid growth of renal cancer cells (Caki-1) was observed under a microscope in the control group without ADP-ribose treatment, while in the experimental group with ADP-ribose treatment, growth of the renal cancer cells was inhibited and death was confirmed (Figure 8A). Quantitative measurement of the LD50 and LD60 for different ADP-ribose concentrations confirmed that approximately half of the renal cancer cells were killed at a concentration of 16 μM, and that almost all cells were killed at 32 μM (Figure 8B).

[0124] Example 2-8: Changes in viability of SNU-1 or OVCAR-3 cells by ADP-ribose treatment 3 x 10 cells in a 96-well plate 3 SNU-1 or OVCAR-3 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 32 μM ADP-ribose and cultured for another 24 hours at 37°C and 5% CO2. After incubation, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0125] As a result, it was confirmed that gastric cancer cells (SNU-1) or ovarian cancer cells (OVCAR-3) were significantly killed when treated with ADP-ribose compared to the control group (Figure 9).

[0126] To summarize the above, through the cell experiments in Example 2, it was confirmed that ADP-ribose has anticancer effects against various solid cancer cells (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer, and ovarian cancer) (Figures 2 to 9).

[0127] Example 3: ADP-ribose alters cancer cell migration and penetration ability Inhibition of cancer cell growth and induction of cancer cell death may also affect the metastatic and invasive abilities of cancer cells. As shown in Example 2, the effects of ADP-ribose treatment on cancer cell growth inhibition and induction of cancer cell death were clearly observed, and it is expected that ADP-ribose treatment will induce a decrease in the metastatic and invasive abilities of cancer cells.

[0128] Example 3-1: Changes in translocation and permeability of AsPC-1 cells by ADP-ribose The assay was performed using transwells with 8.0 μm pore size polycarbonate membrane filters. The transwells were placed in 24-well plates, and AsPC-1 cells were added to each well at 3.5 × 10 cells per well. 5 Cells were seeded onto the top surface of the insert at a density of 1000 μg / cm2 and cultured at 37°C with 5% CO2 for 24 hours. After culture, the transwells were rinsed gently with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and eosin reagent for observation.

[0129] As a result, while the penetration and migration of pancreatic cancer cells (AsPC-1) from the top to the bottom of the transwell was easily observed in the control group, the penetration and migration of pancreatic cancer cells was significantly inhibited in the experimental group treated with 16 μM ADP-ribose (Figure 10A). Quantitative measurement of the inhibitory effect of ADP-ribose on pancreatic cancer cells on migration and migration confirmed a significant (P<0.001) decrease in migration and migration ability compared to the control group (Figure 10B).

[0130] Example 3-2: Changes in translocation and permeability of MDA-MB-231 cells by ADP-ribose The assay was performed using transwells with 8.0 μm pore size polycarbonate membrane filters. The transwells were placed in 24-well plates, and 3.5 × 10 MDA-MB-231 cells were added to each well. 5Cells were seeded onto the top surface of the insert at a density of 1000 μg / cm2 and cultured at 37°C with 5% CO2 for 24 hours. After culture, the transwells were rinsed gently with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and eosin reagent for observation.

[0131] As a result, while the penetration and migration of breast cancer cells (MDA-MB-231) from the top to the bottom of the transwell was easily observed in the control group, the penetration and migration of breast cancer cells was significantly inhibited in the experimental group treated with 32 μM ADP-ribose (Figure 11A). Quantitative measurement of the inhibitory effect of ADP-ribose on breast cancer cell migration and migration confirmed a significant (P<0.001) decrease in migration and migration ability compared to the control group (Figure 11B).

[0132] In summary, Example 3 confirmed that ADP-ribose has excellent therapeutic effects against metastatic cancer by inhibiting the metastasis and penetration ability of solid cancer (pancreatic cancer, breast cancer) cells (Figures 10 and 11).

[0133] Example 4: Comparison and verification of anti-cancer efficacy depending on the concentration and administration route of ADP-ribose using an animal model Example 4-1: Changes in tumor volume in animal models injected subcutaneously with various concentrations of ADP-ribose Five-week-old BALB / c nude mice were inoculated with pancreatic cancer (AsPC-1) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into four groups: a control group and a subcutaneous injection of 2, 20, or 40 mg / kg ADP-ribose. Tumors with a volume of approximately 150 mm 3 When tumors grew to 100% WT, various concentrations of ADP-ribose were injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the results of changes in tumor volume were compared between groups.

[0134] The volume of the injected pancreatic cancer tumor was measured over time, and the final volume of the control group tumor after the final administration was approximately 2155.8 mm 3 The final tumor volumes of the groups injected subcutaneously with 2, 20, and 40 mg / kg ADP-ribose were 1015.8 mm, respectively.3 , 496.6mm 3 , 336.2mm 3 All treatment groups showed statistically significant (P<0.001) reductions in tumor volume compared to the control group. Specifically, tumor volume in the group subcutaneously injected with 20 mg / kg ADP-ribose was significantly (P<0.001) reduced compared to the group subcutaneously injected with 2 mg / kg ADP-ribose. The group subcutaneously injected with 40 mg / kg ADP-ribose had the smallest tumor volume, demonstrating a statistically significant (P<0.001) reduction compared to the group subcutaneously injected with 2 mg / kg ADP-ribose (Figure 12A and B).

[0135] Example 4-2: Changes in tumor volume in animal models after intravenous injection of various concentrations of ADP-ribose Five-week-old BALB / c nude mice were inoculated with renal cancer (caki-1) cells (1 × 10 7 Mice were inoculated into the hind flank and divided into four groups: a control group and those receiving intravenous injections of 2, 10, and 20 mg / kg ADP-ribose. Tumors with a volume of approximately 150 mm 3 When tumors grew to 100% thyroid cancer, various concentrations of ADP-ribose were intravenously injected three times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0136] The volume of the injected kidney tumor was measured over time, and the final volume of the control group tumor after the final administration was approximately 2074.9 mm 3 The final tumor volume was 706.7 mm in the groups receiving intravenous injections of 2, 10, and 20 mg / kg ADP-ribose. 3 , 331.7mm 3 , 227.2mm 3All injection groups showed statistically significant (P<0.001) reductions in tumor volume compared to the control group. Specifically, tumor volume was significantly (P<0.001) reduced in the group injected intravenously with 10 mg / kg ADP-ribose compared to the group injected intravenously with 2 mg / kg ADP-ribose, and tumor volume was smallest in the group injected intravenously with 20 mg / kg ADP-ribose, showing a statistically significant (P<0.001) reduction compared to the groups injected intravenously with 2 mg / kg and 10 mg / kg ADP-ribose (Figure 13A and B).

[0137] Example 4-3: Changes in tumor volume in animal models after oral administration of various concentrations of ADP-ribose Five-week-old BALB / c nude mice were inoculated with pancreatic cancer (AsPC-1) cells (1 × 10 7 Mice were inoculated into the hind flank and divided into four groups: a control group and groups receiving oral administration of 10, 40, and 80 mg / kg ADP-ribose. Tumors with a volume of approximately 150 mm 3 When tumors grew to 100 mg / kg, various concentrations of ADP-ribose were administered orally five times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0138] The volume of the injected pancreatic cancer was measured over time, and the final volume of the control tumor after the final administration was approximately 3194.3 mm 3 The final tumor volume was 1798.6 mm in the groups orally administered 10, 40, and 80 mg / kg ADP-ribose. 3 , 1175.9mm 3 , 1056.9mm 3 All oral administration groups showed statistically significant (P<0.001) reductions in tumor volume compared to the control group. Compared with the group receiving oral administration of 10 mg / kg ADP-ribose, the groups receiving oral injections of 40 and 80 mg / kg ADP-ribose showed significantly (P<0.001) reductions in tumor volume (Figure 14A and B).

[0139] In summary, following the results of cell experiments in Examples 2 and 3, the animal experiment in Example 4 reconfirmed that ADP-ribose has anticancer effects against various solid cancers (FIGS. 12 to 14).

[0140] Example 5: Changes in cancer cell viability by combined treatment with ADP-ribose and low-concentration anticancer drugs Example 5-1: Changes in viability of U-87MG cells treated with ADP-ribose and low-concentration bevacizumab 3 x 10 cells in a 96-well plate 3 Brain cancer (U-87MG) cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 1.6 μM ADP-ribose and / or 15 mM bevacizumab. After further culture at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0141] As a result, when brain cancer cells were treated with bevacizumab alone, approximately 68% of all cancer cells survived, but when treated in combination with ADP-ribose, the survival rate of the cancer cells was significantly reduced to approximately 18% (P<0.001) (Figure 15A).

[0142] Example 5-2: Changes in viability of H1975 cells treated with ADP-ribose and low-concentration osimertinib 3 x 10 cells in a 96-well plate 3 After culturing 1000 lung cancer (H1975) cells at 37°C and 5% CO2 for 24 hours, they were treated with 1.6 μM ADP-ribose and 1 nM osimertinib, either individually or in combination, and then cultured for another 24 hours at 37°C and 5% CO2. After incubation, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After removing the reagent, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0143] As a result, when lung cancer cells were treated with osimertinib alone, approximately 50% of all cancer cells survived, but when treated in combination with ADP-ribose, the survival rate of cancer cells was significantly reduced to approximately 0.9% (P<0.001) (Figure 15B).

[0144] Example 5-3: Changes in viability of AsPC-1 cells after combined treatment with ADP-ribose and low-concentration gemcitabine 3 x 10 cells in a 96-well plate 3 Pancreatic cancer (AsPC-1) cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 1.6 μM ADP-ribose and 1 μM gemcitabine, either individually or in combination, and then cultured for another 24 hours at 37°C and 5% CO2. After incubation, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0145] As a result, when pancreatic cancer cells were treated with gemcitabine alone, approximately 54% of all cancer cells survived, but when treated in combination with ADP-ribose, the survival rate of the cancer cells was significantly reduced to approximately 1% (P<0.001) (Figure 15C).

[0146] Example 5-4: Changes in viability of Hep G2 cells treated with ADP-ribose and low-concentration sorafenib 3 x 10 cells in a 96-well plate 3 Hepatocellular carcinoma (Hep G2) cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 1.6 μM ADP-ribose and / or 1 μM sorafenib. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0147] As a result, when liver cancer cells were treated with 1 μM sorafenib alone, approximately 49% of all cancer cells survived, but when treated in combination with ADP-ribose, the survival rate of cancer cells was significantly reduced to approximately 0.9% (P<0.001) (Figure 15D).

[0148] Example 5-5: Changes in viability of MDA-MB-231 cells treated with ADP-ribose and low-concentration Herceptin 3 x 10 cells in a 96-well plate 3 Breast cancer (MDA-MB-231) cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 3.2 μM ADP-ribose and 2.5 μM Herceptin, either individually or in combination, and then cultured for another 24 hours at 37°C and 5% CO2. After that, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0149] As a result, when breast cancer cells were treated with 2.5 μM Herceptin alone, a whopping 81% of all cancer cells survived due to Herceptin resistance, but when treated in combination with ADP-ribose, the survival rate of the cancer cells was significantly reduced to approximately 20% (P<0.001) (Figure 15E).

[0150] Example 5-6: Changes in viability of HCT116 cells treated with ADP-ribose and low-concentration 5-fluorouracil 3 x 10 cells in a 96-well plate 3 Colon cancer (HCT116) cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 1.6 μM / 100 μl ADP-ribose and / or 10 μM 5-fluorouracil (5-FU). After further incubation at 37°C and 5% CO2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0151] As a result, when colon cancer cells were treated with 10 μM 5-fluorouracil alone, approximately 51% of all cancer cells survived, but when treated in combination with ADP-ribose, the survival rate of cancer cells was significantly reduced to approximately 12% (P<0.001) (Figure 15F).

[0152] To summarize the above, Example 5 shows that ADP-ribose exhibits a synergistic anticancer effect against solid cancers when administered in combination with existing anticancer drugs, and that ADP-ribose can be used not only as an anticancer drug for solid cancers but also as an adjuvant to existing anticancer drugs (Figure 15).

[0153] Example 6: Changes in tumor volume in animal models administered ADP-ribose and low-concentration anticancer drugs in combination Example 6-1: Changes in tumor volume in brain cancer animal models administered ADP-ribose and low-concentration bevacizumab in combination Five-week-old BALB / c nude mice were inoculated with brain cancer (U-87MG) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a single intraperitoneal injection of Avastin (active ingredient: bevacizumab) at 25 mg / kg, and a combination of Avastin and 10 mg / kg ADP-ribose. 3 When tumors grew to 100 mg / kg, Avastin was administered intraperitoneally twice a week, and 10 mg / kg ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the results of tumor volume change were compared between groups.

[0154] The volume of the brain cancer was measured over time and compared. The final volume of the control tumor after the final administration was approximately 2527.1 mm 3 The final tumor volume in the Avastin-treated group was approximately 1598.9 mm 3 The final tumor volume in the group receiving Avastin and ADP-ribose was approximately 334.5 mm. 3 The tumor volume in the combined administration group was significantly reduced by approximately 87% (P<0.001) compared to the control group, and was also significantly reduced by approximately 79% (P<0.001) compared to the group treated with Avastin alone (Figure 16A).

[0155] Example 6-2: Changes in tumor volume in a lung cancer animal model administered ADP-ribose and low-concentration osimertinib in combination Five-week-old BALB / c nude mice were inoculated with lung cancer (H1975) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, oral administration of osimertinib at 1 mg / kg, and oral administration of osimertinib plus 10 mg / kg ADP-ribose. 3 When tumors reached 1000 mg / kg, osimertinib was administered orally twice weekly, and 10 mg / kg ADP-ribose was injected subcutaneously three times weekly. Tumor size was measured using a digital microscope, and the results of tumor volume change were compared between groups.

[0156] The volume of lung cancer was measured over time and compared. The final volume of the control tumor after the final administration was approximately 2265.5 mm 3 The final tumor volume in the osimertinib-treated group was approximately 1881.9 mm 3 The final tumor volume in the osimertinib and ADP-ribose co-administration group was approximately 424.7 mm 3 The tumor volume in the combination group was significantly reduced by approximately 81% (P<0.001) compared to the control group, and was significantly reduced by approximately 77% (P<0.001) compared to the group treated with osimertinib alone (Figure 16B).

[0157] Example 6-3: Changes in tumor volume in pancreatic cancer animal models administered ADP-ribose and low-concentration gemcitabine in combination Five-week-old BALB / c nude mice were inoculated with pancreatic cancer (AsPC-1) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a single intraperitoneal injection of gemcitabine at 50 mg / kg, and a combination of gemcitabine and 10 mg / kg ADP-ribose. Tumors with a volume of approximately 150 mm 3 When tumors grew to 1000 mg / kg, gemcitabine was administered intraperitoneally twice weekly, and 10 mg / kg ADP-ribose was injected subcutaneously three times weekly. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0158] The volume of the pancreatic cancer was measured over time and compared. The final volume of the control tumor after the final administration was approximately 2174.3 mm 3 The final tumor volume in the gemcitabine-administered group was approximately 1815.3 mm 3 The final tumor volume in the group receiving gemcitabine and ADP-ribose was approximately 444.3 mm 3 The tumor volume in the combination group was significantly reduced by approximately 80% (P<0.001) compared to the control group, and was significantly reduced by approximately 76% (P<0.001) compared to the group treated with gemcitabine alone (Figure 16C).

[0159] Example 6-4: Changes in tumor volume in a liver cancer animal model administered ADP-ribose and low-dose sorafenib in combination Five-week-old BALB / c nude mice were inoculated with hepatoma (Hep G2) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, oral administration of 10 mg / kg sorafenib alone, and oral administration of sorafenib plus 10 mg / kg ADP-ribose. 3 When tumors reached the tumor size, sorafenib was administered orally five times a week, and 10 mg / kg ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the results of tumor volume change were compared between groups.

[0160] The volume of liver cancer was measured over time and compared. The final volume of the control tumor after the final administration was approximately 2322.4 mm 3 The final tumor volume in the sorafenib-treated group was approximately 1849.8 mm 3 The final tumor volume in the sorafenib and ADP-ribose co-administration group was approximately 546 mm 3 The tumor volume in the combination group was significantly reduced by approximately 76% (P<0.001) compared to the control group, and was also significantly reduced by approximately 70% (P<0.001) compared to the group treated with sorafenib alone (Figure 16D).

[0161] To summarize the above, the cell experiment in Example 5 and the animal experiment in Example 6 showed that ADP-ribose showed a synergistic anticancer effect against solid cancers even when administered in combination with existing anticancer drugs at low concentrations, demonstrating that ADP-ribose can be used not only as an anticancer drug for solid cancers but also as an adjuvant to existing anticancer drugs (Figure 16).

[0162] Example 7: Changes in ADP-ribose in cancer cells due to radiation and ADP-ribose treatment Example 7-1: Changes in ADP-ribose over time in U-87MG cells treated with various doses of radiation and ADP-ribose U-87MG cells were divided into five groups: untreated (control), treated with 1, 2, or 5 Gy of radiation, or treated with ADP-ribose. They were cultured in 6-well plates for 0, 4, 8, 16, and 24 hours, respectively. The medium was then removed and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS) to prepare supernatants. The supernatants were then used in ELISA assays to detect ADP-ribose.

[0163] Figure 17A is a graph showing the percentage increase in intracellular ADP-ribose in U-87MG cells treated with increasing radiation doses of 1, 2, and 5 Gy and 8 μM ADP-ribose compared to the control group. In the groups treated with increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose peaked at 4 hours, then began to decrease at 8 hours, restoring to a level similar to that of the untreated group by 24 hours. However, the ADP-ribose-treated group maintained a significantly (P<0.001) increased amount of intracellular ADP-ribose compared to the control group even after 24 hours (Figure 17A).

[0164] Example 7-2: Changes in ADP-ribose over time in Caki-1 cells treated with various doses of radiation and ADP-ribose Caki-1 cells were divided into five groups: untreated (control), treated with 1, 2, or 5 Gy of radiation, or treated with ADP-ribose. They were cultured in 6-well plates for 0, 4, 8, 16, and 24 hours, respectively. The medium was then removed and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS) to prepare supernatants. The supernatants were then used in ELISA assays to detect ADP-ribose.

[0165] Figure 17B is a graph showing the percentage increase in intracellular ADP-ribose in Caki-1 cells treated with increasing radiation doses of 1, 2, and 5 Gy and 16 μM ADP-ribose compared to the control group. In the groups treated with increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose peaked at 4 hours and was restored to approximately the same level as the untreated group by 8 hours. However, the ADP-ribose-treated group maintained a significantly (P<0.001) increased amount of intracellular ADP-ribose compared to the control group even after 24 hours (Figure 17B).

[0166] Example 7-3: Changes in ADP-ribose over time in AsPC-1 cells treated with various doses of radiation and ADP-ribose AsPC-1 cells were divided into five groups: untreated (control), treated with 1, 2, or 5 Gy of radiation, or treated with ADP-ribose. They were cultured in 6-well plates for 0, 4, 8, 16, and 24 hours, respectively. The medium was then removed and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS) to prepare supernatants. The supernatants were then used in ELISA assays to detect ADP-ribose.

[0167] Figure 17C is a graph showing the percentage increase in intracellular ADP-ribose in AsPC-1 cells treated with increasing radiation doses of 1, 2, and 5 Gy and 2 μM ADP-ribose compared to the control group. In the groups treated with increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose peaked at 4 hours and then recovered to almost the same level as the untreated group by 16 hours. However, the ADP-ribose-treated group maintained a significantly (P<0.001) increase in intracellular ADP-ribose compared to the control group even after 24 hours (Figure 17C).

[0168] Example 7-4: Changes in ADP-ribose over time in MDA-MB-231 cells treated with various doses of radiation and ADP-ribose MDA-MB-231 cells were divided into five groups: untreated (control), treated with 1, 2, or 5 Gy of radiation, or treated with ADP-ribose. They were cultured in 6-well plates for 0, 4, 8, 16, and 24 hours, respectively. The medium was then removed and the cells were treated with RIPA buffer and 1% sodium dodecyl sulfate (SDS) to prepare supernatants. The supernatants were then used in ELISA assays to detect ADP-ribose.

[0169] Figure 17D is a graph showing the percentage increase in intracellular ADP-ribose in MDA-MB-231 cells treated with increasing radiation doses of 1, 2, and 5 Gy and 16 μM ADP-ribose compared to the control group. In the groups treated with increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose peaked at 4 hours and then recovered to almost the same level as the untreated group by 16 hours. However, the ADP-ribose-treated group maintained a significantly (P<0.001) increase in intracellular ADP-ribose compared to the control group even after 24 hours (Figure 17D).

[0170] To summarize the above, Example 7 demonstrated that exogenous ADP-ribose treatment in various solid cancer cells can maintain intracellular ADP-ribose levels higher than those elevated by radiation (Figure 17). The anti-cancer effect of radiation is expected through the killing of cancer cells by damaging their genetic material. To overcome this, cancer cells continually repair DNA strands through a process known as adenosine diphosphate ribosylation. However, as shown in Figure 17, if ADP-ribose, which only temporarily increases due to the repair process caused by radiation, is continuously accumulated, a synergistic anti-cancer effect can be expected with radiation therapy through disruption of biochemical processes. Furthermore, the combined use of ADP-ribose may enable the use of reduced doses of anti-cancer radiation to overcome radiation therapy resistance and reduce side effects.

[0171] Example 8: Changes in cancer cell viability by combined treatment with ADP-ribose and radiation Example 8-1: Changes in survival rate of U-87MG cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 U-87MG cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 8 μM ADP-ribose and / or 2.5 Gy of radiation. After further culture at 37°C and 5% CO2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0172] As a result, when U-87MG cells were treated with 2 Gy of radiation alone, approximately 88% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with a 2 Gy dose of radiation, the survival rate of the cancer cells was significantly reduced to approximately 12.3% (P<0.001) (Figure 18A).

[0173] Example 8-2: Changes in survival rate of H1975 cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 H1975 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 2 μM ADP-ribose and / or 2.5 Gy of radiation. After further culture at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0174] As a result, when H1975 cells were treated with 2 Gy of radiation alone, approximately 75% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with 2 Gy of radiation, the survival rate of the cancer cells was significantly reduced to approximately 8% (P<0.001) (Figure 18B).

[0175] Example 8-3: Changes in survival rate of AsPC-1 cells following combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 AsPC-1 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 2 μM ADP-ribose and / or 2.5 Gy of radiation. After further culture at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0176] As a result, when AsPC-1 cells were treated with 2 Gy of radiation alone, approximately 87.3% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with 2 Gy of radiation, the survival rate of the cancer cells was significantly reduced to approximately 8% (P<0.001) (Figure 18C).

[0177] Example 8-4: Changes in survival rate of Hep G2 cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 Hep G2 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 4 μM ADP-ribose and / or 2.5 Gy of radiation. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0178] As a result, when Hep G2 cells were treated with 2 Gy of radiation alone, approximately 82.3% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with a 2 Gy dose of radiation, the survival rate of the cancer cells was significantly reduced to approximately 7.3% (P<0.001) (Figure 18D).

[0179] Example 8-5: Changes in survival rate of MDA-MB-231 cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 MDA-MB-231 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 16 μM ADP-ribose and / or 2.5 Gy of radiation. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0180] As a result, when MDA-MB-231 cells were treated with 2 Gy of radiation alone, approximately 90.6% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with 2 Gy of radiation, the survival rate of the cancer cells was significantly reduced to approximately 11.3% (P<0.001) (Figure 18E).

[0181] Example 8-6: Changes in survival rate of HCT116 cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3 HCT116 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 16 μM ADP-ribose and / or 2.5 Gy of radiation. After further culture at 37°C and 5% CO2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0182] As a result, when HCT116 cells were treated with 2 Gy of radiation alone, approximately 90.3% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with 2 Gy of radiation, the survival rate of the cancer cells was significantly reduced to approximately 14% (P<0.001) (Figure 18F).

[0183] Example 8-7: Changes in survival rate of Caki-1 cells after combined treatment with ADP-ribose and a resistant dose of radiation 3 x 10 cells in a 96-well plate 3Caki-1 cells were cultured at 37°C and 5% CO2 for 24 hours, then treated with 16 μM ADP-ribose and / or 2.5 Gy of radiation. After further incubation at 37°C and 5% CO2 for 24 hours, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and incubated for 1 hour. After the reagent was removed, 200 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0184] As a result, when Caki-1 cells were treated with 2 Gy of radiation alone, approximately 94.7% of the cancer cells showed resistance and survived compared to the untreated group (control). However, when treated in combination with ADP-ribose, even with 2 Gy of radiation, the survival rate of the cancer cells was significantly reduced to approximately 22.7% (P<0.001) (Figure 18G).

[0185] To summarize the above, Example 8 demonstrates that ADP-ribose, when administered in combination with radiation, exhibits a synergistic anticancer effect against solid cancers, and that ADP-ribose can be used not only as an anticancer agent for solid cancers but also as an adjuvant for radiation therapy (Figure 18).

[0186] Example 9: Changes in tumor volume in animal models treated with ADP-ribose and a tolerable dose of radiation Example 9-1: Changes in tumor volume in brain cancer animal models treated with ADP-ribose and a tolerable dose of radiation Five-week-old BALB / c nude mice were inoculated with brain cancer (U-87MG) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a group receiving 2 Gy of radiation alone, and a group receiving 2 Gy of radiation plus 10 mg / kg of ADP-ribose. 3 When tumors reached the tumor size, they were given two 2-Gy doses of 1-Gy radiation and 10 mg / kg of ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0187] Figure 19A shows the results of measuring and comparing the volume of brain cancer over time. The final volume of the control tumor after the final administration was approximately 2545.2 mm 3 The final tumor volume in the group receiving 2 Gy of radiation alone was approximately 1472.1 mm 3 The final tumor volume in the group receiving 2 Gy of radiation and ADP-ribose was approximately 725.2 mm 3 The tumor volume in the combined administration group was significantly reduced by approximately 71.5% (P<0.001) compared to the control group, and was also significantly reduced by approximately 50.7% (P<0.001) compared to the group that received 2 Gy of radiation alone (Figure 19A).

[0188] Example 9-2: Changes in tumor volume in pancreatic cancer animal models treated with ADP-ribose and a resistant dose of radiation Five-week-old BALB / c nude mice were inoculated with pancreatic cancer (AsPC-1) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a group receiving 2 Gy of radiation alone, and a group receiving 2 Gy of radiation plus 10 mg / kg of ADP-ribose. 3 When tumors reached the tumor size, they were given two 2-Gy doses of 1-Gy radiation and 10 mg / kg of ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0189] Figure 19B shows the results of measuring and comparing the volume of pancreatic cancer over time. The final volume of the control tumor after the final administration was approximately 2384.3 mm 3 The final tumor volume in the group receiving 2 Gy of radiation alone was approximately 1725.5 mm 3 The final tumor volume in the group receiving 2 Gy of radiation and ADP-ribose was approximately 736.2 mm. 3The tumor volume in the combined administration group was significantly reduced by approximately 69.1% (P<0.001) compared to the control group, and was also significantly reduced by approximately 57.3% (P<0.001) compared to the group that received 2 Gy of radiation alone (Figure 19B).

[0190] Example 9-3: Changes in tumor volume in breast cancer animal models treated with ADP-ribose and a tolerable dose of radiation Five-week-old BALB / c nude mice were inoculated with breast cancer (MDA-MB-231) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a group receiving 2 Gy of radiation alone, and a group receiving 2 Gy of radiation plus 20 mg / kg of ADP-ribose. 3 When tumors reached the tumor size, they were given two 2Gy doses of 1Gy each, and 20mg / kg of ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0191] Figure 19C shows the results of measuring and comparing the volume of breast cancer over time. The final volume of the control tumor after the final administration was approximately 2336.1 mm 3 The final tumor volume in the group receiving 2 Gy of radiation alone was approximately 1459.6 mm 3 The final tumor volume in the group receiving 2 Gy of radiation and ADP-ribose was approximately 640.4 mm. 3 The tumor volume in the combined administration group was significantly reduced by approximately 72.6% (P<0.001) compared to the control group, and was also significantly reduced by approximately 56.1% (P<0.001) compared to the group that received 2 Gy of radiation alone.

[0192] Example 9-4: Changes in tumor volume in a renal cancer animal model treated with ADP-ribose and a tolerable dose of radiation Five-week-old BALB / c nude mice were inoculated with renal cancer (Caki-1) cells (1 × 10 7 Mice were inoculated into the back of the flank and divided into three groups: a control group, a group receiving 2 Gy of radiation alone, and a group receiving 2 Gy of radiation plus 20 mg / kg of ADP-ribose.3 When tumors reached the tumor size, they were given two 2Gy doses of 1Gy each, and 20mg / kg of ADP-ribose was injected subcutaneously three times a week. Tumor size was measured using a digital microscope, and the changes in tumor volume were compared between groups.

[0193] Figure 19D shows the results of measuring and comparing the volume of kidney cancer over time. The final volume of the control tumor after the final administration was approximately 2620.5 mm 3 The final tumor volume in the group receiving 2 Gy of radiation alone was approximately 2454.5 mm 3 The final tumor volume in the group receiving 2 Gy of radiation and ADP-ribose was approximately 786.4 mm. 3 The tumor volume in the combined administration group was significantly reduced by approximately 70% (P<0.001) compared to the control group, and was also significantly reduced by approximately 68% (P<0.001) compared to the group that received 2 Gy of radiation alone (Figure 19D).

[0194] In summary, the cell experiments in Example 8 and the animal experiments in Example 9 demonstrated that ADP-ribose, when administered in combination with radiation, exhibited a synergistic anticancer effect against solid cancers, demonstrating that ADP-ribose can be used not only as an anticancer agent for solid cancers but also as an adjuvant for radiation therapy (Figure 19).

[0195] Example 10: Confirmation of toxicity when ADP-ribose is treated in normal cells It is important for anticancer drugs to be non-toxic to normal cells and to be toxic specifically to cancer cells. Therefore, we evaluated whether ADP-ribose is toxic to normal cells in the present invention.

[0196] Human colon fibroblast (CCD-18Co) and human dermal papilla cell (HDPC) cells were used as normal cells. First, 5 × 10 3Each normal cell line was cultured in DMEM medium at 37°C and 5% CO2 for 24 hours. Then, cells were divided into four groups: untreated and treated with ADP-ribose (25, 50, and 100 μl). After 24, 48, and 72 hours of drug treatment, cell viability was compared using the MTT assay.

[0197] As a result, it was confirmed that no toxicity was observed in any of the normal cells (Figure 20A and B: CCD-18Co, Figure 20C and D: HDPC) upon treatment with ADP-ribose. Therefore, together with the experimental results of the above examples, this example demonstrates that ADP-ribose acts specifically on cancer cells and can be useful as an anticancer agent.

Claims

1. An anti-cancer composition comprising ADP-ribose or a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, wherein the cancer is one or more solid cancers selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer and ovarian cancer.

3. The composition of claim 1 , wherein the cancer is a metastatic cancer.

4. The composition according to claim 1, which induces ADP-ribose accumulation in cancer cells, thereby causing disruption of cancer cell biochemical functions.

5. An anti-cancer adjuvant for radiotherapy, comprising ADP-ribose or a pharmaceutically acceptable salt thereof.

6. The anticancer adjuvant according to claim 5, which improves radiation sensitivity.

7. An anti-cancer composition comprising (i) ADP-ribose or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent.

8. The composition of claim 7, wherein the second anticancer agent is one or more selected from the group consisting of cytotoxic anticancer agents, targeted anticancer agents, and immune anticancer agents.

9. 9. The composition of claim 8, wherein the second anticancer agent is a cytotoxic anticancer agent.

10. 10. The composition of claim 9, wherein the cytotoxic anticancer drug is an alkylating agent and the ADP-ribose or a pharmaceutically acceptable salt thereof enhances sensitivity to the alkylating agent.

11. The composition of claim 8 , wherein the second anticancer agent is a targeted anticancer agent.

12. The composition according to claim 11, wherein the targeted anticancer drug is a targeted anticancer drug that targets one or more targets selected from the group consisting of VEGF / VEGFR, EGFR, and HER2, and ADP-ribose or a pharmaceutically acceptable salt thereof enhances sensitivity to the targeted anticancer drug.

13. The composition for treating brain cancer or liver cancer according to claim 12, wherein the targeted anticancer drug is a VEGF / VEGFR inhibitor.

14. The composition for treating lung cancer according to claim 12, wherein the targeted anticancer drug is an EGFR inhibitor.

15. The composition for treating breast cancer according to claim 12, wherein the targeted anticancer drug is a HER2 inhibitor.

16. The composition of claim 8 , wherein the second anti-cancer agent is an immunological anti-cancer agent.

17. An anti-cancer adjuvant to chemotherapy comprising ADP-ribose or a pharmaceutically acceptable salt thereof.

18. The anticancer adjuvant according to claim 17, wherein the anticancer drug therapy and the anticancer adjuvant are administered simultaneously or at different times.

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