Combination therapy of ADP-ribose binding peptide and ADP-ribose for cancer prevention or treatment

Abstract

The present invention relates to an ADP (adenosine diphosphate)-ribose binding peptide; and an anticancer use using ADP-ribose. The pharmaceutical composition of the present invention accumulates ADP-ribose in cancer cells, disrupts the cell balance, causes cancer cells to die, and has an excellent anticancer effect especially in cancers resistant to existing PRAP inhibitors.

Description

Technical Field

[0001] The present invention relates to the use of ADP (adenosine diphosphate)-ribose binding peptides and ADP-ribose for anti-cancer applications.

Background Art

[0002] Poly(ADP-ribose) polymerase (PARP) contains ADP-ribosyltransferases that promote the ligation of ADP-ribose, and a total of 18 PARP family members have been identified. The typical roles of PARP are to perform essential functions in the processes of cell survival such as DNA damage repair, chromatin remodeling, transcription, and cell death signaling. Among the diverse PARP family members, PARP1 was first described as the major enzyme responsible for the repair mechanism in DNA base damage and single strand breaks (SSBs) caused by harmful stimuli inside and outside the cell. PARP1 recruits the base excision repair (BER) scaffold protein XRCC1 (X-ray cross-complementing group 1) to the damage site and is involved in the repair of DNA base damage. It is also known to play a central role in the repair of DNA double strand breaks (DSBs) via homologous recombination (HR) and non-homologous end joining (NHEJ). When PARP family members such as PARP1, PARP2, PARP3, PARP4, and PARP5 (tankyrases 1 and 2) function, inducing poly(ADP-ribosylation) (PARylation) and synthesizing poly(ADP-ribose) (PAR) chains is one of the important biochemical actions. Most of the PAR synthesis induced by the PARP family is utilized in the response to DNA damage, and through the binding action to the surfaces of numerous intracellular proteins, in addition to DNA repair, it also regulates transcription, cell death, chromatin remodeling, antioxidant, inflammation, metabolic regulation, cell cycle regulation, differentiation, proteasomal degradation, RNA processing, and tumor suppressors. That is, the induction of PARylation by PARP is involved in the response to cell stress, and more importantly, it is emphasized that PARP is an enzyme that regulates diverse and sophisticated cell physiological processes.Under such a background, recently, in the field of cancer treatment, attention has been focused on the inhibition of PARP, and various PARP inhibitors have been developed and utilized clinically.

[0003] In December 2014, the European Medicines Agency (EMA) and the US FDA approved Olaparib as a single-agent therapy for patients with germline BRCA mutation advanced ovarian cancer who had previously received three or more chemotherapy treatments. Additionally, Rucaparib was rapidly approved by the US FDA on December 19, 2016, as a treatment for previously treated BRCA-mutated ovarian cancer and received formal approval in April 2018. Niraparib was approved by the US FDA in March 2017 for epithelial ovarian cancer, fallopian tube cancer, and primary peritoneal cancer, and Talazoparib, an inhibitor of PARP1 and PARP2, was approved by the US FDA in 2018 for breast cancer with germline BRCA mutations. PARP inhibitors such as those described above were developed based on synthetic lethality intervention technology and have been recognized as clinically important cancer therapeutics as described above. However, recently, the resistance of PARP inhibitors and their limitations in clinical application have been taken up as unmet medical needs. When treating cancer using PARP inhibitors, the reasons for unmet medical needs are very diverse, but the problem of resistance during cancer treatment is well known. In the process of synthetic lethality intervention using PARP inhibitors, cancer cells with the characteristics of microsatellite stable / microsatellite instable-low (MSS / MSI-L) develop resistance to PARP inhibitors. In the case of cancer cells with a deficiency in the P53 binding protein 1 (TP53BP1), resistance occurs due to secondary mutations that restore homologous recombination function. It is not easy to overcome the resistance to PARP inhibitors caused by such factors.The tolerance condition can create a tumor microenvironment in which the expression of PAR can increase due to the reactivation of PARP. When the increased PAR accumulates excessively while playing an important role in overcoming stress and survival of cancer cells as described above, it may trigger a unique cell death mechanism called Parthanatos. Therefore, the expression level of poly(ADP-ribose) glycohydrolase (PARG) is very dynamically regulated in cancer cells. Thus, if a method for continuously maintaining PAR synthesized in cancer cells becomes possible, it can induce the death of cancer cells via Parthanatos, and can be utilized for the treatment of cancers with various indications, and can also solve the problem of unmet medical needs due to PARP inhibitor resistance.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The inventor has confirmed that when ADP (adenosine diphosphate)-ribose binding peptide and ADP-ribose are used in combination, they have excellent anti-cancer effects not only in general cancer cells but also in cancer cells resistant to PARP inhibitors, and thus completed the present invention.

Means for Solving the Problems

[0005] One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising a) an ADP (adenosine diphosphate)-ribose binding peptide or a pharmaceutically acceptable salt thereof, and b) ADP-ribose, a precursor thereof or a pharmaceutically acceptable salt thereof.

[0006] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer in PARP (Poly(ADP-ribose)polymerase) inhibitor-resistant cancer.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which contains an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient, and the ADP-ribose binding peptide is derivatized to have sustained release properties.

[0008] Another object of the present invention is to provide a method for preventing or treating cancer, which includes the step of administering the pharmaceutical composition to an individual in need thereof.

[0009] Another object of the present invention is to provide the use of the pharmaceutical composition for preventing or treating cancer.

Effects of the Invention

[0010] The pharmaceutical composition of the present invention accumulates ADP-ribose in cancer cells, disrupts the cell balance, and causes cancer cells to die. It has an excellent anti-cancer effect, especially in cancers resistant to existing PRAP inhibitors.

Brief Description of the Drawings

[0011]

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Best Mode for Carrying Out the Invention

[0012] Specifically, it is as follows. Note that each explanation and embodiment disclosed in the present invention can be applied to other explanations and embodiments respectively. That is, all combinations of all elements disclosed in the present invention belong to the scope of the present invention. Also, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

[0013] One object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising a) an ADP (adenosine diphosphate)-ribose binding peptide or a pharmaceutically acceptable salt thereof, and b) ADP-ribose, a precursor thereof or a pharmaceutically acceptable salt thereof.

[0014] Another aspect of the present invention is a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition to an individual in need thereof.

[0015] Another aspect of the present invention is the use of the pharmaceutical composition for preventing or treating cancer.

[0016] The term "ADP-ribose binding peptide" in the present invention means all peptides having an activity of binding to ADP-ribose or an ADP-ribose polymer and suppressing its degradation. The peptides in the present invention include not only those simply formed by peptide bonds between the amino acids constituting them, but also all forms of peptide analogs, derivatives, etc. having a partially modified form in order to improve characteristics such as stability and efficacy from the perspective of protein pharmaceuticals.

[0017] In one aspect, the "ADP-ribose binding peptide" in the present invention may be composed of any amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14. The peptides of SEQ ID NOs: 1 to 14 are all derived from the WWE domain. The WWE domain is a globular domain conserved in a number of proteins such as deltex, Trip12, and poly-ADP-ribose polymerase homolog, and is named after the name of the most conserved residue within the domain (L. Aravind, TRENDS in Biochemical Sciences, 2001, 26(5):273). In some proteins having the WWE domain, it is known that an ADP-ribose binding motif exists within the domain. It has been reported that the WWE domain of intracellular enzymes mainly binds to ADP-ribose and induces degradation. That is, it is considered that the binding of a specific enzyme to PAR or ADPR via the WWE domain is of great significance for the survival of cancer cells (PNAS August 23, 2011 108(34)14103-14108).

[0018] As described above, although reports on the functions of the WWE domain in proteins were already known, when a peptide in which a part of the WWE domain was isolated was produced and treated with cells as in the present invention, conversely, ADP-ribose degradation was suppressed, ADP-ribose was accumulated in the cells, and as a result, the fact that it has an excellent anticancer effect has never been reported at all.

[0019] The ADP-ribose binding peptide not only includes peptides consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 14, but also any sequences to which one or more amino acids are added, substituted or deleted from these sequences, provided that they fall within the equivalent scope. For example, a peptide having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 14 of the present invention, and having corresponding efficacy to a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 14, that is, a peptide showing ADP-ribose binding activity and anticancer activity, even if it has an amino acid sequence in which some sequences are added, substituted or deleted from the amino acid sequences of SEQ ID NOs: 1 to 14, it is obvious that it is included within the scope of the present invention.

[0020] Also, if it has an activity corresponding to a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 14, the addition of meaningless sequences before and after the amino acid sequence, or naturally occurring mutations, or their silent mutations can also be included within the scope of the present invention.

[0021] As used herein, the term "homology" in the present invention refers to the degree of identity with a given amino acid sequence or nucleotide sequence, which can be expressed as a percentage. In this specification, the homologous sequence having the same or similar activity as the given amino acid sequence or nucleotide sequence is represented as "% homology". For example, it can be confirmed by using standard software for calculating parameters such as score, identity, and similarity, specifically BLAST 2.0, or by comparing sequences through Southern hybridization experiments under defined stringent conditions. The appropriate hybridization conditions defined are within the scope of the art and can be determined by methods known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0022] In addition, the peptides of the present invention can further include modifications such as chemical derivatization in one or more amino acids constituting the peptide in order to improve desired properties. Despite such modifications, it is obvious to those skilled in the art that any of them fall within the scope of the present invention as long as they have anti-cancer activity equivalent to that of the peptides of the present invention. The said derivatization can include acetylation, hydroxylation, methylation, amidation, pegylation; or addition of fatty acids, carbohydrates, lipid components, cofactors, etc., but is not limited thereto.

[0023] As a specific embodiment of the present invention, the peptide can be additionally used in a form fused with a cell-penetrating peptide to enhance cell permeability. That is, the ADP-ribose binding peptide can further include a cell-penetrating peptide at the N-terminus, C-terminus, or both termini. At this time, a linker can be further included between the ADP-ribose binding peptide and the cell-penetrating peptide, which can be appropriately carried out by those skilled in the art.

[0024] The term "cell-penetrating peptide" in the present invention refers to a peptide having the property of promoting the intracellular uptake / absorption of various substances such as nanoparticles, compounds, DNA, and proteins. Specifically, the cell-penetrating peptide may be a peptide derived from TAT, buforin, maurocalcine, penetratin, poly-arginine, Antennapedia, Transportan, VP22, Hph-1, poly-arginine, R11 (R9), Pep-1, HP4, LAH4, Vetofusin-1, a signal sequence-based peptide, or an amphipathic peptide, but is not limited thereto, and can be appropriately selected by those skilled in the art as long as it can promote the intracellular movement of the ADP-ribose binding peptide of the present invention. As a specific embodiment, the ADP-ribose binding peptide containing a cell-penetrating peptide may be a peptide consisting of any amino acid sequence among SEQ ID NOs: 15 to 29, but is not limited thereto.

[0025] Furthermore, those skilled in the art can appropriately modify and use the ADP-ribose binding peptide of the present invention in order to apply it according to the type of cell membrane permeable peptide used. That is, the ADP-ribose binding peptide of the present invention is not limited to only the amino acid sequence presented in the present invention even when it is used by fusing it with a cell membrane permeable peptide. The amino acid sequence can be added / substituted / removed in a form appropriate for the application of the cell membrane permeable peptide within a range obvious to those skilled in the art, that is, within an equivalent range.

[0026] In addition, the ADP-ribose binding peptide of the present invention can be used together with reagents known in the art that can deliver proteins to cells or enhance delivery efficiency in order to enhance cell membrane permeability. The reagents include, for example, not only commercially available reagents such as Chariot (trademark) (Active motif, Cat.30025), Xflect (trademark) (Takara, Cat.631324), Pierce (trademark) (ThermoFisher Scientific, Cat.89850), ProteoJuice (trademark) (Merck, Cat.71281), PULSinTM (Poylplus transfection), but also all other non-commercial reagents, and are not particularly limited to the above examples as long as the ADP-ribose binding peptide of the present invention can be delivered to cells.

[0027] As a specific embodiment of the present invention, the ADP-ribose binding peptide can further contain a cancer-specific peptide at the N-terminus, C-terminus or both termini. By adding a peptide sequence specific to the target cancer according to the desired purpose, the anti-cancer effect can be further improved in vivo. The term "cancer-specific peptide" in the present invention refers to a peptide that can specifically recognize a protein expressed in cancer cells. The protein expressed in the cancer cells may be a cancer-specific receptor such as Integrin αvβ3, EGFR, HER, SSTR2, GnRH-R, Bn receptor, VIP receptor, NTSR1, CCK2R, MC1R, hY1R, etc., but is not limited thereto. For example, when targeting HER2-positive cancer, a KCCYSL sequence that recognizes HER2 expression can be added as the cancer-specific peptide. When targeting EGFR-positive cancer, a YHWYGYTPQNVI sequence that recognizes EGFR expression can be added. When targeting NTSR1-positive cancer, an RRPYIL sequence that recognizes the expression of NTSR1 can be added, but is not particularly limited thereto.

[0028] The term "ADP-ribose" in the present invention is a compound represented by the following formula 1.

[0029]

Chemical formula

[0030] Methods for producing ADP-ribose are known in the technical field to which the present invention pertains. In one embodiment, ADP-ribose can be synthesized by hydrolysis of nicotinamide adenine dinucleotide (NAD + ) in the presence of an alkaline base. Also, ADP-ribose can be isolated in the form of a monovalent or divalent salt of the corresponding metal ion of the base. Further, ADP-ribose is commercially available as a purified raw material (CAS registration number: 68414-18-6).

[0031] The term "precursor of ADP-ribose" in the present invention refers to a substance that can be converted into ADP-ribose in vivo, for example, NAD + , cADPR, poly-ADPR, nicotinic acid, nicotinamide, nicotinamide riboside, but is not limited thereto.

[0032] The term "NAD + " in the present invention is used as a concept including all forms of isolated NAD + and NADH reduced by receiving electrons, and is a compound represented by the following formula 2.

[0033]

Chemical formula

[0034] NAD + is the most abundant coenzyme in vivo involved in oxidation-reduction reactions, acts as a coenzyme for many dehydrogenases, and serves as a hydrogen acceptor for oxidation-reduction reactions such as glycolysis and fermentation to oxidize the hydrogen of various substrates. NAD + is a precursor synthesized from ADP-ribose by PARP in cells, and can generate various forms of ADP-ribose through a pathway as shown in the following reaction formula 1.

[0035]

Chemical formula

[0036] The pharmaceutically acceptable salts in the present invention mean salts commonly used in the pharmaceutical industry. For example, there are salts of inorganic ions such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., and salts of inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, etc. In addition, there are 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, orotate acid, acetylsalicylic acid, etc., and amino acid salts such as lysine, arginine, guanidine, etc. There are also salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, benzethonium, etc., which can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts meant in the present invention are not limited by these listed salts. Preferably, the pharmaceutically acceptable salt of ADP-ribose in the present invention is not the lithium salt of ADP-ribose (including mono- and di-lithium salts).

[0037] The term "cancer" in the present invention refers to a disease related to the regulation of cell death, and when the balance of normal cell death is disrupted, it refers to a disease caused by excessive cell proliferation. In the present invention, the cancer includes both malignant tumors and benign tumors. For example, it may be brain cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, or skin cancer, but the types of cancer in the present invention are not limited by the above examples. The composition for preventing or treating cancer in the present invention has a therapeutic effect on any cancer in which cell death can occur due to the accumulation of ADP-ribose in cells.

[0038] As an example, the cancer may be a solid cancer such as kidney cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, or colorectal cancer, but is not limited thereto.

[0039] As one aspect of the present invention, the cancer may be a PARP (Poly(ADP-ribose)polymerase)-inhibitor-resistant cancer.

[0040] The term "PARP inhibitor" in the present invention refers to an anti-cancer therapeutic agent capable of treating cancer by suppressing PARP, which is a major enzyme for DNA single-strand breaks that performs essential functions in cell survival processes such as DNA damage repair, chromatin remodeling, transcription, and cell death signaling. As an example, PARP inhibitors may be olaparib, rucaparib, niraparib, iniparib, talazoparib, niraparib, veliparib, fluzoparib, Simmiparib, venadaparib, atamparib, pamiparib, stenoparib, 3-aminobenzamide, ME0328, PJ34, AG-14361, INO-1001, UPF-1069, AZD-2461, AZD-5305, AZD-9574, CEP9722, XAV-939, M2912, DR2313, RBN012759, GeA-69, BYK204165, MN64, RK-287107, 4-hydroxyquinazoline, NMS-P118, picolinamide, NVP-TNKS656, NU1025, WIKI4, G007-LK, 3-aminobenzamide, berberine chloride hydrate, HI-TOPK-032, 4’,5,7-trimethoxyflavone, BGP-15 2HCl or A-966492, but are not limited thereto.

[0041] The term "PARP-inhibitor-resistant cancer" in the present invention refers to a cancer in which resistance to the above-described PARP inhibitors has occurred.

[0042] As used herein, the term "treatment" refers to intervening to alter the natural processes of an individual or cell having a disease, which can be done during or to prevent the progression of a pathological condition. The intended therapeutic effects include preventing the onset or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, reducing or temporarily alleviating the disease state, ameliorating or improving the prognosis. In particular, in the present invention, it includes any act of improving the course of cancer by administration of the pharmaceutical composition of the present invention. Also, the term "prevention" refers to any act of suppressing or delaying the onset of cancer by administration of the composition.

[0043] The ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof, ADP-ribose, its precursor or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition can be contained in a content of 0.0001 to 90% by weight, specifically 0.001 to 50% by weight, more specifically 0.01 to 20% by weight, based on the total weight of the final composition, although not limited thereto.

[0044] The pharmaceutical composition can be further prepared by including suitable carriers, excipients or diluents commonly used in the manufacture of drugs. Specifically, the pharmaceutical composition of the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral patches, etc., topical agents, topical patches, suppositories and sterile injections by ordinary methods.

[0045] When the composition is used for oral administration, it can be manufactured as a sustained-release formulation through appropriate encapsulation, enteric coating, blending of polymers, etc. Further, when the composition is used as an injection, various approaches can be considered during formulation in view of the properties of the ADP-ribose binding peptide. For example, as described above, the peptide can be modified by PEGylation, binding to fatty acids, etc., or the drug release rate can be adjusted by binding to a functional moiety through an ester linker, peptide linker, etc., or the serum residence time can be adjusted. Further, it can be blended with nanovehicles such as liposomes, polymersomes, micelles, niosomes, dendrimers, or hydrogels, polymers, etc. to enhance the stability of the drug or deliver the drug to a desired site. However, in the examples as described above, the scope of the present invention is not limited.

[0046] In one embodiment, the sustained-release formulation can be manufactured as a long acting formulation.

[0047] In one embodiment, as the long acting formulation, a polymer and a lipid can be mixed at an appropriate ratio and can further contain methylcellulose, albumin, etc., but is not limited thereto.

[0048] The pharmaceutical composition of the present invention can be administered to an individual having or at risk of developing cancer. The term "individual" in the present invention means all animals including humans.

[0049] The pharmaceutical composition of the present invention can be administered to a subject individual in a pharmaceutically effective amount. The term "administration" in the present invention refers to introducing the pharmaceutical composition of the present invention into a subject individual by any appropriate method, and the administration route can be through various oral or parenteral routes as long as it can reach the target tissue. Examples of the administration route include, but are not limited to, oral, intramuscular, intravenous, arterial, subcutaneous, intraperitoneal, pulmonary, and nasal.

[0050] The term "pharmaceutically effective amount" in the present invention means an amount sufficient to prevent and / or treat cancer with a reasonable benefit / risk ratio applicable to medical use. Appropriate dosage and frequency of administration can be selected by methods known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention actually administered can be appropriately determined by various factors such as the type of symptoms to be treated, the route of administration, gender, health status, diet, age, weight of the individual, and the severity of the disease.

[0051] The pharmaceutical composition of the present invention may further contain a third anti-cancer agent.

[0052] The term "third anti-cancer agent" in the present invention means that an appropriate type can be selected for the complete cure, regulation, and symptom relief of cancer depending on the type and progression of the cancer, and for example, it may be a cytotoxic anti-cancer agent, a targeted anti-cancer agent, an immune anti-cancer agent, a metabolic anti-cancer agent, a synthetic lethality anti-cancer agent, or a combination thereof.

[0053] The cytotoxic anti-cancer agent in the present invention is a drug that attacks cancer cells that divide indiscriminately at a faster rate than normal cells and exhibits an anti-cancer effect, and its meaning is the same as that commonly used in the technical field to which the present invention belongs. The cytotoxic anti-cancer agent includes alkylating agents, antimetabolites, and natural anti-cancer agents.

[0054] The alkylating agents include, but are not limited to, nitrogen mustards (such as cyclophosphamide, chloromethine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, etc.), alkyl sulfonates (such as busulfan, procarbazine, etc.), nitrosoureas (such as carmustine, lomustine, streptozocin, etc.), platinum-based alkylating agents (such as cisplatin, carboplatin, dicycloplatin, eptaplatin, lobaplatin, miloplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, etc.). Alkylating agents can bind to DNA within cancer cells and cause damage to the DNA structure, thereby inducing the destruction of cancer cells.

[0055] The antimetabolites include, but are not limited to, pyrimidine derivatives (such as 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine, etc.), folic acid derivatives (such as methotrexate, pemetrexed, etc.), purine derivatives (such as mercaptopurine, etc.). Antimetabolic drugs can induce cancer cell death by suppressing the metabolism necessary for DNA replication and cell survival.

[0056] The natural product anticancer agents include, but are not limited to, topoisomerase inhibitors (camptothecin, epipodophyllotoxin, taxane drugs), antibiotics (such as dactinomycin, doxorubicin, daunorubicin, mitomycin, phleomycin, idarubicin, mitoxantrone HCl, etc.).

[0057] The target anticancer agent in the present invention is an anticancer agent that induces the death of cancer cells by controlling target proteins (receptors or enzymes) involved in cancer growth, and its meaning is the same as that commonly used in the technical field to which the present invention belongs. The target anticancer agent includes small molecule compounds that inhibit target proteins (such as tyrosine kinase) and monoclonal antibodies.

[0058] The target anti-cancer agent in the present invention can be a receptor tyrosine kinase inhibitor that targets one or more targets selected from the group consisting of VEGF / VEGFR, EGFR, and HER2.

[0059] In one embodiment, the target anti-cancer agent that can be used as the third anti-cancer agent is a VEGF / VEGFR inhibitor. In the present invention, VEGF / VEGFR inhibitors include small molecule compounds such as axitinib, cabozantinib, lapatinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, vandetanib, and monoclonal antibodies such as bevacizumab, ramucirumab, ranibizumab, but are not limited thereto.

[0060] In one embodiment, the target anti-cancer agent that can be used as the third anti-cancer agent is an EGFR inhibitor. In the present invention, EGFR inhibitors include small molecule compounds such as osimertinib, gefitinib, erlotinib, afatinib, brigatinib, icotinib, vandetanib, and monoclonal antibodies such as cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, but are not limited thereto.

[0061] In one embodiment, the target anti-cancer agent that can be used as the third anti-cancer agent is a HER2 inhibitor. In the present invention, HER2 inhibitors include monoclonal antibodies such as trastuzumab, pertuzumab, and margetuximab, together with small molecule compounds such as lapatinib, neratinib, and afatinib, but are not limited thereto.

[0062] In addition, the target anti-cancer agents in the present invention can also include Bcr-Abl target anti-cancer agents such as imatinib, dasatinib, and nilotinib; Src target anti-cancer agents such as bosutinib; JAK target anti-cancer agents such as lestaurtinib, ruxolitinib, and pacritinib; MAP2 Kinase target anti-cancer agents such as cobimetinib, selumetinib, trametinib, and binimetinib; MEL4-ALK target anti-cancer agents such as ceritibin and crizotinib, etc.

[0063] Cancer immunotherapy in the present invention is a cancer treatment method that activates the human immune system through immune anti-cancer agents to fight cancer cells. The immune anti-cancer agents in the present invention include immune checkpoint inhibitors, immune cell therapy agents, anti-cancer vaccines, and antibody-drug conjugates, and appropriate types can be selected for the complete cure, regulation, and symptom relief of cancer according to the type and progression of cancer.

[0064] In one embodiment, the immune anti-cancer agent may be an immune checkpoint inhibitor, and may be one or more selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a CD28 antibody, a KIR antibody, a TCR antibody, a LAG-3 antibody, a TIM-3 antibody, a TIGIT antibody, an A2aR antibody, an ICOS antibody, an OX40 antibody, a 4-1BB antibody, and a GITR antibody. For example, the immune checkpoint inhibitor may be a PD-1 antibody such as nivolumab, pembrolizumab, cemiplimab, pidilizumab, toripalimab; a PD-L1 antibody such as atezolizumab, avelumab, duralumab; a CTLA-4 antibody such as ipilimumab, tremelimumab, or all of these.

[0065] In one embodiment, the immune anti-cancer agent may be a cell therapy agent, and may be a CAR-T therapy agent or a CAR-NK therapy agent such as tisagenlecleucel, axicabtagene ciloleucel, but is not limited thereto.

[0066] In the present invention, a metabolic anti-cancer agent refers to a drug that is involved in the growth and survival of cancer cells, such as supplying nutrients to cancer cells, or is involved in multiple essential metabolic actions, and kills cancer cells. The metabolic anti-cancer agent may be, for example, IM-156, 3-bromopyruvic acid (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 or AG-120, etc., but is not limited thereto.

[0067] In the present invention, a synthetic lethality anti-cancer agent refers to an anti-cancer agent that uses synthetic lethality, where cells can survive when only one of two genes (or two gene products) has a mutation, but cells will die when both genes have mutations. As an example, the synthetic lethality intervention anti-cancer agent may be olaparib, niraparib, talazoparib, veliparib, a PRMT5 inhibitor (e.g., TNG908, MRTX1719, AMG193, PRT811, GSK3326595, PF-06939999, JNJ-64619178, PRT543, PRT811, JBI-778, TNG462, AGX323, AT101, AT201), a PRMT1 inhibitor (e.g., GSK3368715, SKL27969), a Mat2A inhibitor (e.g., AG-270, IDE397, ISM020), a PKMYT1 inhibitor (e.g., RP-6306), a PARG inhibitor (e.g., IDE161), etc., but is not limited thereto.

[0068] The third anti-cancer agent of the present invention may be a combination of one or more cytotoxic anti-cancer agents, target anti-cancer agents and / or synthetic lethal anti-cancer agents, and these can be administered simultaneously or at different times. Further, the third anti-cancer agent may be dichloroacetic acid, tamoxifen, osimertinib, olaparib, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinate, amsacrine, alemtuzumab, procarbazine, alprostadil, formium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, lomustine, and carmustine, and may be one or more selected from the group consisting of these, but is not limited thereto.

[0069] Since the ADP-ribose and ADP-ribose binding peptide of the present invention have excellent anti-cancer activity, they can be produced not only in the form of a pharmaceutical composition but also in the form of a functional food composition.

[0070] When the composition of the present invention is manufactured in the form of a food composition, the food composition can contain additional ingredients that are commonly used in foods and can improve odor, taste, visual appearance, etc. For example, food additives can be added. The additives are selected according to the type of food and used in appropriate amounts.

[0071] The food composition can be manufactured as a functional food, where the functional food is the same term as food for special health use (FoSHU), and means a medicine or a food with high medical effects that is processed so that its biological regulatory function is efficiently shown in addition to nutrient supply. The functional food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects for improving cancer.

[0072] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, which contains an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient, and the ADP-ribose binding peptide is derivatized to have sustained release properties.

[0073] The ADP-ribose binding peptide and the derivatization are as described above.

[0074] The derivatization of the ADP-ribose binding peptide may be, for example, but not limited to, one or more amino acids constituting it being pegylated or a fatty acid being bound to one or more amino acids, showing sustained release properties. The derivatization may directly modify the amino acids or may modify them using additional elements such as a linker.

[0075] Specifically, the PEG used for the pegylation may be, for example, but not limited to, miniPEG2, dPEG4, dPEG6, dPEG8, dPEG12 or dPEG24, etc.

[0076] Specifically, the fatty acid may be, but is not limited to, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, melissic acid, corynomycolic acid, etc.

[0077] In a specific embodiment of the present invention, miniPEG2, a cell-penetrating peptide, an ADP-ribose binding peptide, and a fatty acid are combined to produce an ADP-ribose binding peptide derivative. In particular, the peptide of SEQ ID NO: 29 (GRKKRRQRRRPQFLYVADLENMVQYRRNEHGRRRKIKR) is pegylated and a derivative produced by binding a fatty acid is named a "sustained-release derivative". The specific structure of the sustained-release derivative of the present invention is as shown in Formula 3 below.

[0078]

Chemical formula

[0079] In another aspect of the present invention, in the case of a derivative produced by pegylating the peptides of SEQ ID NOs: 1 to 14 and binding a fatty acid and an additional cell-penetrating peptide, it is possible to confirm an effect equivalent to that of the "sustained-release derivative" using the peptide of SEQ ID NO: 29.

[0080] Embodiments of the present invention can be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Further, the embodiments of the present invention are provided to more fully explain the present invention to those with average knowledge in the art. Furthermore, throughout the specification, when a component is described as "including", it does not exclude other components and may further include other components unless otherwise stated to the contrary.

Example

[0081] Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the following examples and experimental examples are merely for illustrating the present invention, and the scope of the present invention is not limited thereto.

[0082] Examples 1 to 29. Production of ADP (adenosine diphosphate)-ribose binding peptides The ADP-ribose binding peptides of Examples 1 to 14 were synthesized from WWE domains present in various types of proteins and used in experiments. Specific information on the peptides used is shown in Table 1 below.

[0083]

Table 1

[0084] In addition, cell-penetrating peptides were bound to the N-terminus of the peptides of Examples 1 to 14 to produce the peptides of Examples 15 to 28. The peptide sequences of Examples 15 to 28 are shown in Table 2 below.

[0085]

Table 2

[0086] In addition, a cell membrane-permeable peptide (GRKKRRQRRRPQ) was conjugated to the peptide N-terminus of Example 2 to produce the peptide of Example 29. The peptide sequence of Example 29 is shown in Table 3 below.

[0087]

Table 3

[0088] The peptides of Examples 1 to 15 and 17 to 29 produced above were processed, and the change in cancer cell viability was measured. In Aspc-1 cells (left side of Fig. 1) and MDA-MB-231 cells (right side of Fig. 1), the anti-cancer effect of the peptide alone of all examples could be confirmed, and the GI50 (Half maximal growth inhibition concentration) of each example was confirmed.

[0089] Example 30. Production of a sustained-release derivative using the peptide of Example 29 Peptron was commissioned to conjugate the peptide of Example 29 with PEG and bind stearic acid to synthesize a derivative of the ADP-ribose binding peptide, which was named "sustained-release derivative", and mass spectrometry (SHIMADZU LCMS-2020 System) and HPLC analysis (SHIMADZU Prominence HPLC System) were performed.

[0090] In the case of HPLC analysis, the column used was Shiseido capcell pak C18, 120 angstroms (4.6 x 50 mm), 5 μM, and the column temperature was maintained at room temperature. The flow rate was 1.0 mL / min, and detection was performed at 220 nm. Mobile phase A was 0.1% TFA (Trifluoroacetic acid) in water, and mobile phase B was 0.1% TFA in acetonitrile. The gradient was 35% B for 2 minutes, 30% B for 10 minutes, 10% B for 1 minute, and 77% B for 4 minutes.

[0091] The results are shown in Table 4, and the LC-MS [M+H]+ value was confirmed to be 5363.43.

[0092]

Table 4

[0093] Experimental Example 1. Changes in cancer cell viability by combined treatment with the peptide of the example and ADP-ribose Experimental Example 1-1. Changes in viability by combined treatment with the peptide of the example and ADP-ribose in Caki-1 cells 2.5×10 3 renal cancer (Caki-1) cells were cultured in a 96-well plate at 37°C and 5% CO 2 for 24 hours under the conditions, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination.

[0094] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with 25 μM concentration of the peptide of Example 1 alone 4) Groups treated with 10 μM concentration of the CPP-peptide of Example 15 alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 1 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 15 (Combination 2) Thereafter, the cells were cultured again for 96 hours at 37°C and 5% CO 2 Under the conditions, 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 then the absorbance was measured to confirm the cell viability.

[0095] As a result, in the peptide-untreated group, Caki-1 cells grew rapidly, whereas a decrease in survival rate was confirmed in all of the peptide and ADPR treatment groups of the examples of the present invention. In particular, in the group treated with the combination of the peptide of the example and ADPR, the growth of Caki-1 cells was significantly suppressed, and a morphology of cell death was observed (Figure 2).

[0096] Experimental Example 1-2. Change in survival rate by combined treatment with the peptide of the example and ADP-ribose in HCC1937 cells 2.5Х10 3 breast cancer (HCC1937) cells were cultured at 37 °C and 5% CO 2 for 24 hours under the conditions, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination.

[0097] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with 25 μM concentration of the peptide of Example 2 alone 4) Groups treated with 10 μM concentration of the CPP-peptide of Example 16 alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 2 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 16 (Combination 2) Then, the cells were cultured again for 96 hours at 37 °C and 5% CO 2 under the conditions, 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 then the absorbance was measured to confirm the cell survival rate.

[0098] As a result, in the peptide-untreated group, HCC1937 cells grew rapidly, while it was confirmed that the survival rate decreased in all of the peptide and ADPR treatment groups of the examples of the present invention. In particular, in the group treated with the combination of the peptide of the example and ADPR, the growth of HCC1937 cells was significantly suppressed, and a dying morphology could be observed (Figure 3).

[0099] Experimental Example 1-3. Changes in survival rate due to combined treatment with the peptide of the example and ADP-ribose in AsPC-1 cells 2.5×10 3 pancreatic cancer (AsPC-1) cells were cultured at 37°C and 5% CO 2 for 24 hours under the conditions of, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination.

[0100] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with 25 μM concentration of the peptide of Example 3 alone 4) Groups treated with 10 μM concentration of the CPP-peptide of Example 17 alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 3 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 17 (Combination 2) Then, they were cultured again for 96 hours at 37°C and 5% CO 2 under the conditions of, 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 then the absorbance was measured to confirm the cell survival rate.

[0101] As a result, in the peptide-untreated group, AsPC-1 cells grew rapidly, while in all of the peptide and ADPR treatment groups of the examples of the present invention, it was confirmed that the survival rate decreased. In particular, in the group treated with the combination of the peptide of the example and ADPR, the growth of AsPC-1 cells was significantly suppressed, and a dying morphology could be observed (Figure 4).

[0102] Experimental Example 1-4. Changes in survival rate by combined treatment of the peptide of the example and ADP-ribose in HepG2 cells 2.5×10 3 liver cancer (HepG2) cells were cultured at 37°C and 5% CO 2 for 24 hours under the conditions, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination.

[0103] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with 25 μM concentration of the peptide of Example 4 alone 4) Groups treated with 10 μM concentration of the CPP-peptide of Example 18 alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 4 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 18 (Combination 2) Then, they were cultured again for 96 hours at 37°C and 5% CO 2 under the conditions, 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 then the absorbance was measured to confirm the cell survival rate.

[0104] As a result, in the peptide-untreated group, HepG2 cells grew rapidly, whereas in all of the peptide and ADPR treatment groups of the examples of the present invention, it was confirmed that the survival rate decreased. In particular, in the group treated with the combination of the peptide of the example and ADPR, the growth of HepG2 cells was significantly suppressed, and a morphology of cell death was observed (Figure 5).

[0105] Experimental Example 1-5. Change in survival rate by combined treatment of the peptide of the example and ADP-ribose in H1975 cells 2.5×10 3 lung cancer (H1975) cells were cultured at 37°C and 5% CO 2 for 24 hours under the conditions, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination.

[0106] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with 25 μM concentration of the peptide of Example 5 alone 4) Groups treated with 10 μM concentration of the CPP-peptide of Example 19 alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 5 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 19 (Combination 2) Then, they were cultured again for 96 hours at 37°C and 5% CO 2 under the conditions, 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 then the absorbance was measured to confirm the cell survival rate.

[0107] As a result, in the peptide-untreated group, H1975 cells grew rapidly, while in all of the peptide and ADPR treatment groups of the examples of the present invention, it was confirmed that the survival rate decreased. In particular, in the group treated with the combination of the peptide of the example and ADPR, the growth of H1975 cells was significantly suppressed, and a morphology of cell death was observed (Figure 6).

[0108] Experimental Example 1-6. Changes in survival rate due to combined treatment with the peptide of the example and ADP-ribose in HCT116 cells 2.5×10 3 colorectal cancer (HCT116) cells were cultured at 37°C and 5% CO 2 for 24 hours under the conditions of, and then divided into the following groups, and the GI50 concentration of the peptide of each example was treated alone or in combination with cell lines.

[0109] 1) Untreated group 2) Group treated with 1 mM concentration of ADPR alone 3) Group treated with the peptide of Example 6 at a concentration of 25 μM alone 4) Groups treated with the CPP-peptide of Example 20 at a concentration of 10 μM alone 5) Group treated with a combination of 1 mM concentration of ADPR and 25 μM concentration of the peptide of Example 6 (Combination 1) 6) Group treated with a combination of 1 mM concentration of ADPR and 10 μM concentration of the CPP-peptide of Example 20 (Combination 2) Then, they were cultured again for 96 hours at 37°C and 5% CO 2 under the conditions of, 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 then the absorbance was measured to confirm the cell survival rate.

[0110] As a result, while the HCT116 cells grew rapidly in the peptide-untreated group, it was confirmed that the survival rate decreased in all of the peptide and ADPR treatment groups of the examples of the present invention. In particular, in the group treated with the peptide of the example in combination with ADPR, the growth of HCT116 cells was significantly suppressed, and a morphology of cell death was observable (Figure 7).

[0111] Experimental Example 2. Confirmation of normal cell toxicity when the peptide of the example and ADP-ribose are used in combination 2.5 x 10 3 human-derived normal CCD-18-Co or MRC5 cells were cultured at 37°C and 5% CO 2 for 24 hours under the conditions, and then treated with the peptides of Examples 1 to 15 and 17 to 29 in combination with ADP-ribose.

[0112] Then, they were cultured again for 96 hours at 37°C and 5% CO 2 conditions, 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 the reaction was completed, the MTT reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell survival rate.

[0113] As a result, it was confirmed that when the peptides of each example were used in combination with ADPR as compared with the peptide-untreated group, about 90% or more of the normal CCD-18-Co cells survived. Therefore, it was confirmed that when the peptides of each example were used in combination with ADPR, almost no toxicity was shown to normal cells (Figure 8).

[0114] Experimental Example 3. Comparison and verification of the anti-cancer efficacy of the peptide of the example and ADP-ribose in combination using an animal model Experimental Example 3-1. Change in tumor volume by the combined treatment of the peptide of the example and ADP-ribose in AsPC-1 cells Pancreatic cancer (AsPC-1) cells (1 × 10 7(The cells were) inoculated behind the flanks of the mice, and the administration groups were classified as follows. The GI50 concentrations of the peptides in each example were treated alone or in combination.

[0115] 1) Untreated group 2) Group administered ADP-ribose alone 3) Groups treated with the peptides of Examples 1-6 alone 4) Groups treated with ADPR and the peptides of Examples 1-6 in combination (Combination 1-6) When the tumor volume grew to about 75 mm 3 ADP-ribose and the peptides of Examples 1-6 were administered alone or in combination. Here, ADP-ribose was used at a concentration of 10 mg / kg, and the peptides of Examples 1-6 were used at a concentration of 20 mg / kg, and dosed subcutaneously twice a week for 4 weeks.

[0116] Thereafter, the tumor size was measured using digital calipers, and the results of changes in tumor volume were compared by group.

[0117] As a result, in all dosing groups, the tumor volume was significantly reduced compared to the control group. In particular, it was confirmed that the combined administration group had a significantly greater reduction in tumor volume than the single administration group (Figs. 9 and 10).

[0118] Experimental Example 3-2. Change in tumor volume by combined treatment of the CPP-peptide of Example and ADP-ribose in HCC1937 cells 5-week-old BALB / c nude mice were inoculated with breast cancer (HCC1937) cells (1 × 10 7 (The cells were) inoculated behind the flanks of the mice, and the administration groups were classified as follows. The GI50 concentrations of the CPP-peptides in each example were treated alone or in combination.

[0119] 1) Untreated group 2) Group administered ADP-ribose alone 3) Groups treated with the CPP-peptides of Examples 15-20 alone 4) Group (Combination A - F) treated with ADPR and CPP - peptides of Examples 15 - 20 in combination respectively When the tumor volume grew to about 75 mm 3 ADP - ribose, CPP - peptides of Examples 15 - 20 were administered alone or in combination. Here, ADP - ribose was used at a concentration of 10 mg / kg, and CPP - peptides of Examples 15 - 20 were used at a concentration of 10 mg / kg, and dosed subcutaneously twice a week for 4 weeks.

[0120] Thereafter, the tumor size was measured using digital calipers, and the results of changes in tumor volume were compared by group.

[0121] As a result, in all dosing groups, the tumor volume was significantly reduced compared to the control group. In particular, it was confirmed that the combined - administration group had a significantly greater reduction in tumor volume than the single - administration group (Figures 11 and 12).

[0122] Experimental Example 4. Confirmation of anti - cancer synergistic effect by combined treatment of peptides and ADP - ribose of the examples on PARP - resistant cell lines Next, an attempt was made to confirm the combined treatment effect of the peptides of the examples and ADP - ribose in PARP - inhibitor - resistant cancer cell lines.

[0123] Specifically, 2.5Х10 3 PARP - inhibitor - resistant breast cancer (HCC1937) cells were cultured in a 96 - well plate at 37°C and 5% CO 2 for 24 hours under the conditions.

[0124] Thereafter, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0125] 1) Untreated group 2) Group treated with 2 μM concentration of PARP inhibitor (Olaparib) 3) Group treated with 1 mM concentration of ADPR alone 4) Groups treated with 25 μM concentration of the peptides of Examples 1 - 6 alone respectively 5) Groups treated with CPP - peptides of Examples 15 - 20 at a concentration of 10 μM alone 6) Groups treated with a combination of 1 mM ADPR and peptides of Examples 1 - 6 at a concentration of 25 μM (Combination 1) 6) Groups treated with a combination of 1 mM ADPR and CPP - peptides of Examples 15 - 20 at a concentration of 10 μM (Combination 2) Thereafter, they were cultured again for 96 hours under the conditions of 37 °C and 5% CO 2 Then, 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 then the absorbance was measured to confirm the cell viability.

[0126] As a result, when HCC1937 cells were treated with olaparib, a PARP inhibitor, alone, about 80% of the cancer cells survived. However, in all cases where the peptides of the examples or ADP - ribose were treated, it was confirmed that the cell viability decreased significantly. In particular, in all cases where the peptides of the examples and ADP - ribose were treated in combination, it was confirmed that the viability of cancer cells decreased significantly compared to the single treatment (Figure 13).

[0127] Experimental Example 5. Changes in cancer cell viability by combined treatment of the peptides of the examples and NAD + The present inventors focused on the possibility that NAD which is a precursor of ADP - ribose, could replace ADP - ribose, and confirmed the anti - cancer effect when NAD + was administered alone and in combination with the ADP - ribose - binding peptides of the examples. + Specifically, 2.5×10

[0128] ovarian cancer (OVCAR - 3) cells were cultured in a 96 - well plate at 37 °C and 5% CO 3 for 24 hours, and then divided into the following groups, and the GI50 concentration of the peptides of each example was treated alone or in combination. 2 After culturing for 24 hours under the conditions of 37 °C and 5% CO

[0129] 1) Untreated group 2) Group treated with NAD at a concentration of 1 mM + alone 3) Group treated with the peptide of Example 2 at a concentration of 25 μM alone 4) Group treated with the CPP - peptide of Example 16 at a concentration of 10 μM alone 5) Group treated with a combination of NAD at a concentration of 1 mM + and the peptide of Example 2 at a concentration of 25 μM (Combination 1) 6) Group treated with a combination of NAD at a concentration of 1 mM + and the CPP - peptide of Example 16 at a concentration of 10 μM (Combination 2) Then, they were cultured again for 96 hours under the conditions of 37°C and 5% CO 2 2. 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 then the absorbance was measured to confirm the cell viability.

[0130] As a result, in the peptide - untreated group, OVCAR - 3 cells grew rapidly, and it was confirmed that there was no significant difference from the control group even when treated with NAD alone. However, when the peptide of the example of the present invention was treated in combination with NAD + it was confirmed that the viability of cancer cells was significantly decreased compared to the case of treating the peptide of the example alone, showing an excellent anti - cancer effect (Figure 14). +

[0131] Experimental Example 6. Changes in the viability of cancer cells by combined treatment of the peptide of the example, ADP - ribose, NAD + and various anticancer agents 2.5x10 was added to a 96 - well plate 3 ​Individual human-derived pancreatic cancer (Aspc-1), renal cancer (Caki-1), breast cancer (HCC1937), liver cancer (HepG2), lung cancer (H1975), colorectal cancer (HCT116), ovarian cancer (OVCAR-3), brain cancer (U87) cells were each cultured at 37°C, 5% CO 2 for 24 hours under the conditions, and then divided into the following groups, and the GI20 / IC20 concentrations of each preparation were treated alone or in combination.

[0132] 1) Untreated group 2) Groups in which the peptides of Examples 1 to 14 at a concentration of 25 μM and Examples 15, 17 to 29 at a concentration of 10 μM were each treated alone with each cancer cell 3) Group treated alone with 2 mM concentration of ADPR 4) 2 mM concentration of NAD + treated alone group 5) Groups treated alone with various anticancer agents (2 μM concentration of Cisplatin, 1 μM concentration of Docetaxel, 3 mM concentration of Dichloroacetate, 2 μM concentration of Doxorubicin, 2 μM concentration of Sorafenib, 10 nM concentration of Osimertinib, 0.2 μM concentration of Trastuzumab, 0.2 nM concentration of Bevacizumab, 0.2 μM concentration of Tamoxifen, or 1 μM concentration of Olaparib) 6) Groups treated in combination with 2 mM concentration of ADPR and the peptides of the examples (Examples 1 to 14 at 25 μM, Examples 15, 17 to 29 at 10 μM) 7) 2 mM concentration of ADPR, 2 mM concentration of NAD + and the peptides of the examples (Examples 1 to 14 at 25 μM, Examples 15, 17 to 29 at 10 μM) were each treated in combination 8) Groups treated in combination with 2 mM concentration of ADPR, the peptides of the examples (Examples 1 to 14 at 25 μM, Examples 15, 17 to 29 at 10 μM), and various anticancer agents (the same concentration as in the group 5) Then, at 37°C, 5% CO 2Under the conditions of , the cells were cultured again for 96 hours. 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, the MTT reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability. As a result, in all cancer cells used in the experiment, compared with the case where the peptides of Examples 1 to 15 and 17 to 29, ADPR, NAD + and various anticancer agents were treated alone, when each of the peptides of the above examples was treated in combination with ADPR, about 40% of the cancer cell viability was shown. When the peptides of the above examples were treated in combination with ADPR and NAD + in combination, about 20% of the cancer cell viability was shown. In addition, when the peptides of Examples 1 to 15 and 17 to 29 were treated in combination with ADPR and various anticancer agents, the cancer cell viability was significantly reduced compared with all other groups, showing about 10% viability. Therefore, it was confirmed that when the peptides of the produced examples were treated in combination with ADPR and various anticancer agents, excellent anticancer effects were shown (Figs. 15 to 28).

[0133] Experimental Example 7. Changes in cancer cell viability when the sustained-release derivative produced using the peptide of Example 29 was treated alone or in combination 2.5×10 3 human-derived pancreatic cancer (Aspc-1), renal cancer (Caki-1), breast cancer (HCC1937), liver cancer (HepG2), lung cancer (H1975), colorectal cancer (HCT116), ovarian cancer (OVCAR-3), and brain cancer (U87) cells were each cultured at 37 °C and 5% CO 2 for 24 hours under the conditions of, and then the sustained-release derivative was treated alone with the cell line. In addition, the cells were divided into the following groups, and the GI20 concentration of each formulation was treated alone or in combination with MDA-MB-231 cancer cells.

[0134] 1) Untreated group 2) Group treated with 5 μM concentration of the sustained-release derivative alone 3) Group treated with 2 mM concentration of ADPR alone 4) 2 mM concentration of NAD+ Group treated with [the agent] alone 5) Groups treated with various anticancer agents alone (cisplatin at a concentration of 2 μM, docetaxel at a concentration of 1 μM, dichloroacetate at a concentration of 3 mM, doxorubicin at a concentration of 2 μM, sorafenib at a concentration of 2 μM, osimertinib at a concentration of 10 nM, trastuzumab at a concentration of 0.2 μM, bevacizumab at a concentration of 0.2 nM, tamoxifen at a concentration of 0.2 μM, or olaparib at a concentration of 1 μM) 6) Group treated with a combination of 2 mM ADPR and a sustained-release derivative at a concentration of 5 μM 7) 2 mM ADPR, 2 mM NAD + and a group treated with a combination of a sustained-release derivative at a concentration of 5 μM 8) Group treated with a combination of 2 mM ADPR, a sustained-release derivative at a concentration of 5 μM, and various anticancer agents (same concentrations as in 5) above) Then, they were cultured again for 96 hours under the conditions of 37 °C and 5% CO 2 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 then the absorbance was measured to confirm the cell viability.

[0135] As a result, when treated with the sustained-release derivative alone, a survival rate of less than 10% was confirmed in most cancer cell lines (left side of Figure 29). In the case of the triple-negative breast cancer cell line MDA-MB-231, drug resistance was shown at a survival rate of around 80% when treated with the sustained-release derivative, ADPR, NAD + or various anticancer agents alone. However, when the sustained-release derivative was combined with ADPR, cytotoxicity was shown. When NAD + or various anticancer agents were further added and the three agents were used in combination, it was confirmed that significantly excellent cytotoxicity was shown (right side of Figure 29).

[0136] Experimental Example 8. Confirmation of normal cell toxicity during single or combined treatment with the sustained-release derivative produced using the peptide of Example 29 2.5 x 10 3 normal human-derived MRC5 cells were cultured in a 96-well plate at 37°C and 5% CO 2 for 24 hours, and then treated with the sustained-release derivative and ADPR alone or in combination.

[0137] After that, they were cultured again for 96 hours at 37°C and 5% CO 2 conditions, 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 the reaction was completed, the MTT reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0138] As a result, when comparing the sustained-release derivative with the untreated group, it was confirmed that when the sustained-release derivative was treated alone or in combination with ADPR, about 90% or more of the normal MRC5 cells survived. Therefore, it was confirmed that the sustained-release derivative showed almost no toxicity in normal cells regardless of single or combined treatment (Figure 30).

[0139] Experimental Example 9. Comparison and verification of the anti-cancer efficacy of the sustained-release derivative alone or in combination with an immunotherapeutic agent using an animal model Six-week-old C57BL / 6-hPD1 (PD-1 humanized mouse) male mice were inoculated with colorectal cancer (MC38) cells (1 × 10 6 cells / 100 μl) at the right dorsal forelimb site of the mice, and the administration groups were classified as follows.

[0140] 1) Untreated group 2) Group injected intravenously with the sustained-release derivative at the GI50 concentration alone 3) Group injected subcutaneously with the sustained-release derivative at the GI50 concentration alone 4) Group injected intraperitoneally with the immunotherapeutic agent nivolumab at the GI50 concentration alone 5) Group treated with a sustained-release derivative of the GI50 concentration (intravenous injection) and nivolumab (intraperitoneal injection) in combination (Combination 1) 6) Group treated with a sustained-release derivative of the GI50 concentration (subcutaneous injection) and nivolumab (intraperitoneal injection) in combination (Combination 2) When the tumor volume grew to about 100 mm 3 The sustained-release derivative and nivolumab were administered alone or in combination. Here, the sustained-release derivative was used by mixing at a ratio of 3:1 with albumin at a concentration of 5 mg / kg, and nivolumab was used at a concentration of 5 mg / kg, and each was administered twice a week for 4 weeks.

[0141] Thereafter, a digital caliper was used to measure the tumor size, and the results of changes in tumor volume were compared by group.

[0142] As a result, in all dosing groups, the tumor volume was significantly reduced compared to the control group. In particular, it was confirmed that the combined administration group had a significantly greater reduction in tumor volume than the single administration group (Figure 31). Also, in all dosing groups, no change in body weight was shown, so it was confirmed that there was no toxicity (Figure 32).

[0143] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it should be understood that the above-described embodiments are exemplary in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims and all equivalent concepts, rather than the above detailed description.

Claims

1. a) an ADP (adenosine diphosphate)-ribose binding peptide or a pharma- ceutically acceptable salt thereof; and b) A pharmaceutical composition for preventing or treating cancer, comprising ADP-ribose, a precursor thereof or a pharma- ceutically acceptable salt thereof.

2. The ADP-ribose binding peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14. The pharmaceutical composition for preventing or treating cancer according to claim 1.

3. The precursor of ADP-ribose is NAD ＋ (nicotinamide adenine dinucleotide), cADPR, poly-ADPR, nicotinic acid, nicotinamide or nicotinamide riboside; The pharmaceutical composition for preventing or treating cancer according to claim 1.

4. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the peptide further comprises a cell-penetrating peptide at the N-terminus, C-terminus or both termini.

5. The pharmaceutical composition for preventing or treating cancer according to claim 4, wherein the cell membrane permeable peptide is at least one selected from the group consisting of TAT, buforin, maurocalcinde, penetratin, poly-arginine-derived peptide, antennapedia, transportan, VP22, Hph-1, poly-arginine, R11 (R9), Pep-1, HP4, LAH4, Vetofusin-1, signal sequence-based peptides, and amphipathic peptides.

6. The pharmaceutical composition for preventing or treating cancer according to claim 4, wherein the peptide consists of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 15 to 29.

7. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein one or more amino acids constituting the peptide are acetylated, hydroxylated, methylated, amidated or pegylated.

8. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the peptide has a fatty acid, carbohydrate, lipid moiety or cofactor bound to one or more amino acids constituting the peptide.

9. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is one or more solid cancers selected from the group consisting of brain cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, colon cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

10. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a PARP (Poly(ADP-ribose) polymerase) inhibitor-resistant cancer.

11. The pharmaceutical composition for preventing or treating cancer according to claim 1 , wherein the pharmaceutical composition is formulated as a sustained release formulation.

12. The pharmaceutical composition for preventing or treating cancer according to claim 1 , further comprising a third anticancer drug.

13. The pharmaceutical composition for preventing or treating cancer according to claim 12, wherein the third anti-cancer agent is a cytotoxic anti-cancer agent, a targeted anti-cancer agent, an immune anti-cancer agent, a metabolic anti-cancer agent, a synthetic lethality anti-cancer agent, or a combination thereof.

14. A pharmaceutical composition for preventing or treating cancer, comprising an ADP-ribose binding peptide or a pharma- ceutical acceptable salt thereof as an active ingredient, the ADP-ribose binding peptide being derivatized so as to have sustained release properties.

15. The pharmaceutical composition for preventing or treating cancer according to claim 14, wherein the derivatization of the ADP-ribose binding peptide is such that one or more of the amino acids constituting the ADP-ribose binding peptide are pegylated or one or more of the amino acids are bound to a fatty acid.

16. The pharmaceutical composition for preventing or treating cancer according to claim 14, wherein the derivatized ADP-ribose binding peptide is represented by the following formula 3: 【Chemistry 1】

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