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

Abstract

The present invention relates to an anti-cancer use of an adenosine diphosphate (ADP)-ribose binding peptide using an adenosine diphosphate (ADP)-ribose binding peptide and an ADP-ribose binding peptide using an ADP-ribose binding peptide. The pharmaceutical composition of the present invention destroys cell balance and causes apoptosis of cancer cells by accumulating ADP-ribose in cancer cells, and particularly has an excellent anti-cancer effect even in cancers tolerant to existing PRAP inhibitors.

Description

Technical Field

[0001] The present invention relates to an anti-cancer use of adenosine diphosphate (ADP)-ribose binding peptide and ADP-ribose. Background Art

[0002] It has been confirmed that poly(ADP-Ribose) polymerase (PARP) includes ADP-ribosyl transferases that promote ADP-ribose attachment, and there are 18 PARP family members. The representative role of PARP is to perform essential functions in cell survival, such as DNA damage repair and chromatin remodeling, transcription, and apoptosis signals. Among the various PARP family members, PARP1 was first reported as a key enzyme that performs repair mechanisms based on DNA base damage or in single strand breaks (SSBs) based on harmful stimuli inside and outside the cell. PARP1 participates in the repair of DNA base damage by recruiting X-ray cross-complementing group 1 (XRCC1), a base excision repair (BER) scaffold protein, to the damage site. In addition, it is known that it plays an important role in the repair of double strand breaks (DSBs) of DNA by homologous recombination (HR) and non-homologous end joining (NHEJ). Among the PARPs of the PARP1, PARP2, PARP3, PARP4 and PARP5 (tankyrases 1 and 2) family, the synthesis of poly(ADP-ribose) (PAR) chains by inducing poly(ADP-ribosylation) (PARylation) is one of the important biochemical actions. Most of the PAR synthesis induced by the PARP family is used in response to DNA damage, and in addition to DNA repair, it regulates transcription, apoptosis, chromatin remodeling, anti-oxidation, inflammation, metabolic regulation, cell cycle regulation, differentiation, proteasome degradation, RNA processing and tumor suppressor factors by binding to the surface of many intracellular proteins. That is, PARP poly (ADP-ribosylation) induction is involved in the response to cellular stress, and more importantly, it is an enzyme that regulates diverse and delicate cellular physiological processes. Based on this background, in recent years, the inhibition of PARP has received attention in the field of cancer treatment, and a variety of PARP inhibitors have been developed and used clinically.

[0003] In December 2014, the European Medicines Agency (EMA) and the U.S. FDA approved Olaparib as a single-agent therapy for patients with progressive ovarian cancer with a germline BRCA mutation who have received more than three chemotherapy regimens. In addition, Rucaparib was approved by the U.S. FDA on December 19, 2016 as a treatment for ovarian cancer with a previous BRCA mutation, and was approved by the U.S. FDA on December 19, 2016, and formally approved in April 2018. Niraparib was approved by the U.S. 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 U.S. FDA in 2018 for breast cancer with a germline BRCA mutation. The PARP inhibitors described above are developed based on the synthetic lethality intervention technology, and as described above, are certified as clinically important cancer therapeutics, but recently the tolerance of PARP inhibitors or the limitations of clinical application have been raised as unmet medical needs. It is well known that there are many reasons for the use of PARP inhibitors and the generation of unmet medical needs in the treatment of cancer, but the most representative one is the problem of tolerance in the treatment of cancer. In the process of synthetic lethality intervention using PARP inhibitors, cancer cells with the characteristics of microsatellite stability (MSS) / microsatellite instable-low (MSI-L) develop resistance to PARP inhibitors. For cancer cells with defects in P53 gene binding protein (TP53 binding protein 1, TP53BP1), resistance is generated based on secondary mutations that restore homologous recombination function. Because the resistance to PARP inhibitors generated by these reasons is difficult to overcome. Tolerance conditions can create a tumor microenvironment in which PAR expression can be increased due to the reactivation of PARP, and the increased PAR plays an important role in the stress overcoming and survival of cancer cells described above. At the same time, when excessively accumulated, it can cause a unique apoptotic mechanism called Parthanatos (a new form of cell death). Therefore, based on poly (ADP-ribose) hydrolase (poly (ADP-ribose) glycohydrolase, PARG) in cancer cells, its expression level is very dynamically regulated.Therefore, if a method for continuously maintaining PAR synthesized in cancer cells can be developed, apoptosis of cancer cells can be induced by Parthanatos, which can be used to treat cancers of various indications and can also solve the problem of unmet medical needs due to resistance to PARP inhibitors. Summary of the invention

[0004] Technical Problems to be Solved by the Invention

[0005] The present inventors have confirmed that the combined use of ADP-ribose and an ADP-ribose-binding peptide has an excellent anticancer effect not only on conventional cancer cells but also on cancer cells resistant to PARP inhibitors, thereby completing the present invention.

[0006] Technical Solution

[0007] An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising: a) an adenosine diphosphate (ADP)-ribose binding peptide or a pharmaceutically acceptable salt thereof; and b) ADP-ribose, a precursor thereof or a pharmaceutically acceptable salt thereof.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, wherein the cancer is a poly (ADP-ribose) polymerase (PARP) inhibitor-resistant cancer.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the ADP-ribose binding peptide is a peptide derivatized in a sustained-release manner.

[0010] Another object of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition to an individual in need of the pharmaceutical composition.

[0011] Another object of the present invention is to provide a use of the pharmaceutical composition in preventing or treating cancer.

[0012] Beneficial Effects

[0013] The pharmaceutical composition of the present invention destroys the cell balance by accumulating ADP-ribose in cancer cells and causes apoptosis of cancer cells, and in particular, has excellent anti-cancer effects in cancers that are resistant to existing PRAP inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the results of confirming the survival rate and the GI50 concentration of each sequence peptide after treating pancreatic cancer (Aspc-1) cells and breast cancer (MDA-MB-231) cells with peptides of SEQ ID NO: 1 to SEQ ID NO: 15 and SEQ ID NO: 17 to SEQ ID NO: 29 individually. The left graph shows the results of Aspc-1 cells, and the right graph shows the results of MDA-MB-231 cells. **p<0.001vs untreated control group.

[0015] Figure 2 The results show the results of confirming the cell survival rate after treating renal cancer (Caki-1) cells with ADP-ribose and peptides of SEQ ID NO: 1 or SEQ ID NO: 15 at various GI50 concentrations alone or in combination. The upper figure shows a cell photograph, and the lower graph shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination treatment (Combination) 1 group.

[0016] Figure 3 The results show the results of confirming the cell survival rate after treating breast cancer (HCC1937) cells with ADP-ribose and peptides of SEQ ID NO: 2 or SEQ ID NO: 16 at various GI50 concentrations alone or in combination. The upper figure shows a cell photograph, and the lower graph shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination 1 group.

[0017] Figure 4 The results of confirming the cell survival rate after treating pancreatic cancer (AsPC-1) cells with ADP-ribose and peptides of SEQ ID NO: 3 or SEQ ID NO: 17 at various GI50 concentrations alone or in combination are shown. The upper figure shows a cell photograph, and the lower graph shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination 1 group.

[0018] Figure 5The results show the results of confirming the cell survival rate after treating liver cancer (HepG2) cells with ADP-ribose and peptides of SEQ ID NO: 4 or SEQ ID NO: 18 at various GI50 concentrations alone or in combination. The upper figure shows a cell photograph, and the lower graph shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination treatment (Combination) 1 group.

[0019] Figure 6 The results of confirming the cell survival rate after treating lung cancer (H1975) cells with ADP-ribose and peptides of SEQ ID NO: 5 or SEQ ID NO: 19 at various GI50 concentrations alone or in combination are shown. The upper figure shows a cell photograph, and the lower figure shows the cell survival rate. The upper figure shows a cell photograph, and the lower figure shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination treatment (Combination) 1 group.

[0020] Figure 7 The results show the results of confirming the cell survival rate after treating colorectal cancer (HCT116) cells with ADP-ribose and peptides of SEQ ID NO: 6 or SEQ ID NO: 20 at various GI50 concentrations alone or in combination. The upper figure shows a cell photograph, and the lower graph shows the cell survival rate. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination 1 group.

[0021] Figure 8 The results show the results of confirming the cell survival rate after treating human normal colon (CCD-18-Co) fibroblasts with ADP-ribose alone or in combination with peptides of SEQ ID NO: 1 to SEQ ID NO: 15 and SEQ ID NO: 17 to SEQ ID NO: 29. The lower graph shows a cell photograph, and the upper graph shows the cell survival rate.

[0022] Fig. 9 The figure shows the changes in tumor volume over time after subcutaneous treatment of ADP-ribose and peptides of SEQ ID NO: 1 to SEQ ID NO: 6 at various GI50 concentrations, either alone or in combination, in a pancreatic cancer (AsPC-1) transplanted animal model. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group.

[0023] Fig.10 It is a graph showing the tumor volume on the last day of the experiment after subcutaneous treatment of ADP-ribose and peptides of SEQ ID NO: 1 to SEQ ID NO: 6 at various GI50 concentrations, either alone or in combination, in a pancreatic cancer (AsPC-1) transplanted animal model. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group.

[0024] Fig.11 The figure shows the tumor volume changes over time after ADP-ribose and peptides of SEQ ID NO: 15 to SEQ ID NO: 20 at various GI50 concentrations were treated subcutaneously in breast cancer (HCC1937) transplanted animal models alone or in combination. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group.

[0025] Fig.12 It is a graph showing the tumor volume on the last day of the experiment after oral administration of peptides of SEQ ID NO: 15 to SEQ ID NO: 20 alone or in combination with ADP-ribose and various GI50 concentrations to breast cancer (HCC1937) transplanted animal models. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group.

[0026] Fig.13 The figure shows the cell survival rate of PARP inhibitor-resistant breast cancer (HCC1937) cells treated with olaparib alone, ADP-ribose alone or in combination with peptides of SEQ ID NO: 1 to SEQ ID NO: 6 or peptides of SEQ ID NO: 15 to SEQ ID NO: 20 at various GI50 concentrations. *p<0.001 vs. untreated control group and olaparib-treated group. **p<0.001 vs. untreated control group and single-treatment group. ***p<0.001 vs. untreated control group, single-treatment group and combination 1 group.

[0027] Fig.14 It is shown that treatment with NAD alone or in combination with ovarian cancer (OVCAR-3) cells +The results of confirming cell viability after adding peptides of SEQ ID NO: 2 or SEQ ID NO: 16 at different GI50 concentrations were shown in the figure on the left, and cell viability was shown in the graph on the right. *p<0.001 vs. untreated control group. **p<0.001 vs. untreated control group and single treatment group. ***p<0.001 vs. untreated control group, single treatment group, and combination treatment group 1.

[0028] Fig.15 The present invention shows that the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 1, and the right graph shows the results of using the peptide of SEQ ID NO: 2. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0029] Fig.16 The present invention shows that the peptide of SEQ ID NO: 3 or SEQ ID NO: 4 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 3, and the right graph shows the results of using the peptide of SEQ ID NO: 4. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0030] Fig.17 The present invention shows that the peptide of SEQ ID NO: 5 or SEQ ID NO: 6 alone or in combination with ADP-ribose, NAD +The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 5, and the right graph shows the results of using the peptide of SEQ ID NO: 6. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0031] Fig.18 The present invention shows that the peptide of SEQ ID NO: 7 or SEQ ID NO: 8 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 7, and the right graph shows the results of using the peptide of SEQ ID NO: 8. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0032] Fig.19 The results show that the peptide of SEQ ID NO: 9 or SEQ ID NO: 10 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 9, and the right graph shows the results of using the peptide of SEQ ID NO: 10. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0033] Fig. 20 The present invention shows that the peptide of SEQ ID NO: 11 or SEQ ID NO: 12 alone or in combination with ADP-ribose, NAD +The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 11, and the right graph shows the results of using the peptide of SEQ ID NO: 12. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0034] Fig.21 The present invention shows that the peptide of SEQ ID NO: 13 or SEQ ID NO: 14 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 13, and the right graph shows the results of using the peptide of SEQ ID NO: 14. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0035] Fig. 22 The present invention shows that the peptide of SEQ ID NO: 15 or SEQ ID NO: 17 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 15, and the right graph shows the results of using the peptide of SEQ ID NO: 17. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0036] Fig.23 The present invention shows that the peptide of SEQ ID NO: 18 or SEQ ID NO: 19 alone or in combination with ADP-ribose, NAD +The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 18, and the right graph shows the results of using the peptide of SEQ ID NO: 19. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0037] Fig.24 The present invention shows that the peptide of SEQ ID NO: 20 or SEQ ID NO: 21 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 20, and the right graph shows the results of using the peptide of SEQ ID NO: 21. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0038] Fig.25 The present invention shows that the peptide of SEQ ID NO: 22 or SEQ ID NO: 23 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 22, and the right graph shows the results of using the peptide of SEQ ID NO: 23. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0039] Fig.26 The present invention shows that the peptide of SEQ ID NO: 24 or SEQ ID NO: 25 alone or in combination with ADP-ribose, NAD +The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 24, and the right graph shows the results of using the peptide of SEQ ID NO: 25. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0040] Fig. 27 The present invention shows that the peptide of SEQ ID NO: 26 or SEQ ID NO: 27 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 26, and the right graph shows the results of using the peptide of SEQ ID NO: 27. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0041] Fig.28 The present invention shows that the peptide of SEQ ID NO: 28 or SEQ ID NO: 29 alone or in combination with ADP-ribose, NAD + The results of confirming cell viability after combined treatment with various anticancer agents. The left graph shows the results of using the peptide of SEQ ID NO: 28, and the right graph shows the results of using the peptide of SEQ ID NO: 29. *p<0.05 vs. untreated control group. **p<0.001 vs. anticancer agent combination group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0042] Fig.29 The results show that the sustained-release derivative (prepared by PEGylating the peptide of SEQ ID NO: 29 and then binding fatty acids) alone or in combination with ADP-ribose, NAD +The results of confirming cell survival rate after combined treatment with various anticancer agents. The left graph shows the results of single treatment with sustained-release derivatives, and the right graph shows the results of combined treatment. *p<0.05 vs. untreated control group. **p<0.001 vs. combined anticancer agent group and groups other than ADP-ribose and NAD + Other groups except the combination group. ##p<0.001vs other groups except the anticancer agent combination group. $$p<0.001vs other groups.

[0043] Fig.30 The results show the results of examining the cell viability of human normal lung (MRC5) fibroblasts treated with ADP-ribose and a sustained-release derivative alone or in combination. The upper graph shows a cell photograph, and the lower graph shows the cell viability.

[0044] Fig.31 The figure shows the tumor volume changes over time after the immunotherapy agent nivolumab and sustained-release derivatives were treated intravenously, subcutaneously or intraperitoneally at the concentration of GI50 in the colorectal cancer (MC38) transplanted animal model. **p<0.001 vs. untreated control group. ##p<0.001 vs. untreated control group and single treatment group.

[0045] Fig.32 The figure shows the changes in body weight over time after the immunotherapeutic agent nivolumab and its sustained-release derivative were administered alone or in combination to the colorectal cancer (MC38) transplanted animal model intravenously, subcutaneously, or intraperitoneally. DETAILED DESCRIPTION

[0046] The specific description is as follows. On the other hand, each description and embodiment disclosed in the present invention can also be applied to different descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention cannot be limited to the specific description described below.

[0047] In one aspect of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, the pharmaceutical composition comprising: a) an adenosine diphosphate (ADP)-ribose binding peptide or a pharmaceutically acceptable salt thereof; and b) ADP-ribose, a precursor thereof or a pharmaceutically acceptable salt thereof.

[0048] Another aspect of the present invention is a method for preventing or treating cancer, the method comprising the step of administering the pharmaceutical composition to an individual in need of the pharmaceutical composition.

[0049] Another aspect of the present invention is use of the pharmaceutical composition in preventing or treating cancer.

[0050] In the present invention, the term "ADP-ribose binding peptide" refers to all peptides having the activity of binding to ADP-ribose or ADP-ribose polymers to inhibit degradation. In the present invention, the peptide is not only formed by the peptide bonds between the amino acids constituting it, but also includes all forms of modified forms (analogs and derivatives, etc.) having partial modifications in order to improve the properties (stability and efficacy, etc.) from the perspective of protein pharmaceuticals.

[0051] As one aspect, in the present invention, the "ADP-ribose binding peptide" may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14. The peptides of SEQ ID NO: 1 to SEQ ID NO: 14 are all derived from the WWE domain. The WWE domain is a globular domain conserved in many proteins such as deltex, Trip12, poly-ADP-ribose polymerase homologs, and is named after the most conserved residue in the domain (L. Aravind, TRENDS in Biochemical Sciences, 2001, 26 (5): 273). It is well known that in some proteins having a WWE domain, an ADP-ribose binding motif exists in the domain. It is reported that the WWE domain of intracellular enzymes mainly binds to ADP-ribose and induces its decomposition. That is, specific enzymes bind to PAR or ADPR via the WWE domain mainly for the purpose of cancer cell survival (PNAS August 23, 2011 108(34)14103-14108).

[0052] On the other hand, although the functions of the WWE domain in the protein as described above have been reported, it has never been reported that, as shown in the present invention, when a part of the WWE domain is prepared as an isolated peptide and used for cell treatment, the decomposition of ADP-ribose is inhibited and intracellular ADP-ribose is accumulated, ultimately having an excellent anti-cancer effect.

[0053] The ADP-ribose binding peptides include not only peptides consisting of any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14, but also peptides having one or more amino acid sequences added, substituted or deleted from these sequences, as long as they fall within the equivalent scope. For example, it is obvious that as long as the peptides have at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with the peptides having any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14 of the present invention and exhibit the same efficacy (i.e., ADP-ribose binding activity and anticancer activity) as the peptides consisting of any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14, even if they have an amino acid sequence in which a part of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 14 is added, substituted or deleted, they are also included in the scope of the present invention.

[0054] In addition, as long as it has an activity corresponding to a peptide consisting of any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14, the addition of meaningless sequences before and after the amino acid sequence or naturally occurring mutations, or silent mutations thereof, are also included in the scope of the present invention.

[0055] In the present invention, the term "homology" may refer to the degree of consistency with a given amino acid sequence or base sequence, and may be expressed as a percentage. In this specification, a homology sequence having the same or similar activity as a given amino acid sequence or base sequence is expressed as "% homology". For example, standard software (specifically, BLAST 2.0) for calculating parameters (parameters) (scores, identity, and similarity, etc.) may be used, or, under defined stringent conditions, sequences may be compared by gel electrophoresis (Southern) hybridization experiments, so that confirmation may be performed, and the defined appropriate hybridization conditions may be within the technical scope, and may be determined by methods well 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; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0056] In addition, in order to further improve the desired properties, the peptide of the present invention may include chemical derivatization and other modifications in one or more amino acids constituting the peptide. Even if such modifications are performed, as long as they have equivalent anticancer activity to the peptide of the present invention, they are within the scope of the present invention, which is obvious to those skilled in the art. The derivatization may be acetylation, hydroxylation, methylation, amidation, pegylation; or may also include fatty acids, carbohydrates, lipid components, cofactors, etc., but are not limited thereto.

[0057] As a specific aspect of the present invention, in order to improve cell permeability, the peptide can also be used in the form of a fusion with a cell-penetrating peptide. That is, the ADP-ribose binding peptide can also include a cell-penetrating peptide at the N-terminus, the C-terminus or both ends. At this time, a linker can also be included between the ADP-ribose binding peptide and the cell-penetrating peptide, and it can be appropriately performed by a person skilled in the art.

[0058] In the present invention, the term "cell-penetrating peptide" 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 can be TAT, buforin, maurocalcine, penetratin, poly-arginine derived peptide, antennapedia, transporter, VP22, Hph-1, poly-arginine, R11 (R9), Pep-1, HP4, LAH4, Vetofusin-1, signal sequence-based peptides or amphipathic peptides, 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 including the cell-penetrating peptide may be a peptide consisting of any one of the amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 29, but is not limited thereto.

[0059] Furthermore, in order to be applicable according to the type of cell-penetrating peptide used, those skilled in the art can appropriately modify and use the ADP-ribose binding peptide of the present invention. That is, the ADP-ribose binding peptide of the present invention is not limited to the amino acid sequence proposed in the present invention even when it is fused with a cell-penetrating peptide and used, and can be used in a suitable form suitable for the cell-penetrating peptide by adding / substituting / deleting the amino acid sequence within a range that is obvious to those skilled in the art (i.e., an equivalent range).

[0060] In addition, in order to improve cell permeability, the ADP-ribose binding peptides of the present invention can be used together with agents known in the art that can deliver proteins to cells or increase delivery efficiency. For example, the agents include not only Chariot TM (Active motif, Cat.30025), Xflect TM (Takara, Cat. 631324), Pierce TM (ThermoFisher Scientific, Cat. 89850), ProteoJuice TM (Merck, Cat. 71281), PULSin TM(Poylplus transfection company), and other commercially available reagents, and other non-commercial reagents are also included, and are not particularly limited to the examples as long as the ADP-ribose binding peptide of the present invention can be delivered to cells.

[0061] As a specific aspect of the present invention, the ADP-ribose binding peptide may also include cancer-specific peptides at the N-terminus, C-terminus or both ends. According to the desired purpose, the anti-cancer effect in vivo can be further improved by adding a peptide sequence specific to the targeted cancer. In the present invention, the term "cancer-specific peptide" refers to a peptide that can specifically recognize a protein expressed in a cancer cell. The protein expressed in the cancer cell may be a cancer-specific receptor, such as integrin αvβ3, EGFR, HER, SSTR2, GnRH-R, Bn receptor, VIP receptor, NTSR1, CCK2R, MC1R and hY1R, but is not limited thereto. For example, when HER2-positive cancer is targeted, the cancer-specific peptide may be added with a KCCYSL sequence for identifying HER2 expression, when EGFR-positive cancer is targeted, a YHWYGYTPQNVI sequence for identifying EGFR expression may be added, and when NTSR1-positive cancer is targeted, a RRPYIL sequence for identifying NTSR1 expression may be added, but is not particularly limited thereto.

[0062] In the present invention, the term "ADP-ribose" is a compound represented by the following Chemical Formula 1.

[0063] [Chemical formula 1]

[0064]

[0065] The preparation method of ADP-ribose is well known in the art to which the present invention belongs. In one embodiment, under the condition of having an alkaline base, ADP-ribose can be prepared by nicotinamide adenine dinucleotide (NAD + ). In addition, ADP-ribose can be isolated as a monovalent or divalent salt of its corresponding alkaline metal ion. Alternatively, ADP-ribose can be obtained as a purified raw material through commercial channels (CAS No.: 68414-18-6).

[0066] In the present invention, the term "precursor of ADP-ribose" refers to a substance that can be converted into ADP-ribose in vivo, for example, NAD + , cADPR, poly-ADPR, nicotinic acid, nicotinamide and nicotinamide riboside, but are not limited thereto.

[0067] In the present invention, the term "NAD +"Used as including isolated NAD + And the concept of all forms of NADH reduced by receiving electrons, and it is a compound represented by the following Chemical Formula 2.

[0068] [Chemical formula 2]

[0069]

[0070] NAD + NAD is the most abundant coenzyme in the body that participates in redox reactions. It functions as a coenzyme for many dehydrogenases and as a hydrogen acceptor in redox reactions such as glycolysis and fermentation, oxidizing hydrogen in various substrates. + It is a precursor substance for ADP-ribose synthesized by PARP in cells, and various forms of ADP-ribose can be generated through the pathway shown in the following reaction formula 1.

[0071] [Reaction 1]

[0072]

[0073] In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, for example, inorganic ion salts including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc and iron, and inorganic acid salts such as hydrochloric acid, phosphoric acid and sulfuric acid, in addition, organic acid salts 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, acetylsalicylic acid, and amino acid salts such as lysine, arginine and guanidine. In addition, organic ion salts such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium and benzethonium chloride that can be used in pharmaceutical reactions, purification and separation processes are provided, however, the types of salts referred to in the present invention are not limited to the listed salts. Preferably, in the present invention, the pharmaceutically acceptable salt of ADP-ribose is not a lithium salt of ADP-ribose (including monolithium salt and dilithium salt).

[0074] In the present invention, the term "cancer" is a disease related to apoptosis regulation, and refers to a disease caused by excessive cell proliferation when the normal apoptosis balance is broken. In the present invention, the cancer includes both malignant tumors and benign tumors, for example, 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 tumors, 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 of the present invention are not limited to the examples. The composition for preventing or treating cancer of the present invention has a therapeutic effect on all cancers in which apoptosis may occur due to the accumulation of ADP-ribose in cells.

[0075] 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.

[0076] As one aspect of the present invention, the cancer may be a poly (ADP-ribose) polymerase (PARP) inhibitor-resistant cancer.

[0077] In the present invention, the term "PARP inhibitor" refers to an anticancer therapeutic agent for treating cancer by inhibiting PARP, which is a key enzyme for single-strand breaks of DNA that performs required functions in cell survival processes such as DNA damage repair, chromatin remodeling, transcription, and apoptosis signaling. As an example, the PARP inhibitor can 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, CEP 9722, 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.

[0078] In the present invention, the term "PARP inhibitor-resistant cancer" refers to a cancer that has developed resistance to the PARP inhibitors described above.

[0079] In the present invention, the term "treatment" refers to the process of intervening in order to change the natural course of the diseased individual or cell, which can be performed during the development of the pathological condition or for the prevention thereof. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological results of the disease, preventing metastasis, reducing the rate of disease progression, alleviating the disease state or temporarily alleviating, improving or improving prognosis. In particular, in the present invention, all actions to improve the cancer process by administering the pharmaceutical composition of the present invention are included. In addition, the term "prevention" refers to all actions to inhibit or delay the onset of cancer by administering the composition.

[0080] The ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof, ADP-ribose, a precursor substance thereof or a pharmaceutically acceptable salt thereof included in the pharmaceutical composition is not limited thereto, however, based on the total weight of the final composition, each may contain 0.0001 wt % to 90 wt %, specifically 0.001 wt % to 50 wt %, more specifically 0.01 wt % to 20 wt %.

[0081] The pharmaceutical composition may further include suitable carriers, excipients or diluents commonly used in drug preparation. Specifically, according to conventional methods, the pharmaceutical composition of the present invention can be prepared and used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral patches and other oral dosage forms, external preparations, external patches, suppositories and sterile injections.

[0082] When the composition is used for oral administration, it can be prepared into a sustained-release preparation by appropriate encapsulation, enteric coating and polymer formulation. In addition, when the composition is used as an injection, taking into account the characteristics of the ADP-ribose binding peptide, a variety of permeation-enhancing methods can be considered during formulation. For example, as described above, the peptide can be modified by pegylation, binding with fatty acids, etc., or by combining with functional parts through ester joints, peptide joints, etc. to adjust the drug release rate or adjust the serum residual time. In addition, the drug stability can be increased or the drug can be delivered to the desired site by formulation with nanocarriers (such as liposomes, polymer vesicles, micelles, vesicles, dendrimers) or hydrogels and polymers. However, the scope of the present invention is not limited to the above examples.

[0083] In one embodiment, the sustained-release preparation can be prepared as a long-acting preparation.

[0084] In one embodiment, in the long-acting preparation, the polymer and the lipid may be mixed in an appropriate ratio, and may further include methylcellulose and albumin, etc., but are not limited thereto.

[0085] The pharmaceutical composition of the present invention can be administered to individuals who have developed or are at risk of developing cancer. In the present invention, the term "individual" refers to all animals including humans.

[0086] The pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective dose. In the present invention, the term "administration" refers to introducing the pharmaceutical composition of the present invention into a subject by any appropriate method, and as long as it can reach the target tissue, the administration route can be administered by various routes such as oral or parenteral. Examples of administration routes include, but are not limited to, oral, intramuscular, intravenous, arterial, subcutaneous, abdominal, pulmonary and nasal.

[0087] In the present invention, the term "pharmaceutically effective dose" refers to a sufficient amount to prevent and / or treat cancer at a reasonable benefit / risk ratio that is suitable for medical use. The appropriate dosage and frequency of administration can be selected according to methods known in the art, and the amount of the pharmaceutical composition of the present invention actually administered and the frequency of administration can be appropriately determined by various factors such as the type of symptoms to be treated, the route of administration, gender, health status, diet, personal age, weight and severity of the disease.

[0088] The pharmaceutical composition of the present invention may further comprise a third anticancer agent.

[0089] In the present invention, the term "third anticancer agent" can be selected according to the type and progression of cancer to cure, regulate and alleviate symptoms of cancer. For example, it can be a cytotoxic anticancer agent, a targeted anticancer agent, an immune anticancer agent, a metabolic anticancer agent, a synthetic lethality anticancer agent or a combination thereof.

[0090] In the present invention, cytotoxic anticancer agents are drugs that exhibit anticancer effects by attacking cancer cells that divide more rapidly and indiscriminately than normal cells, and their meanings are the same as those commonly used in the technical field to which the present invention belongs. The cytotoxic anticancer agents include alkylating agents, antimetabolites, and natural anticancer agents.

[0091] The alkylating agent may include, but is not limited to, nitrogen mustards (e.g., cyclophosphamide, chlormethine, uramustine, melphalan, chlorambucil, ifosfamide, and bendamustine, etc.), alkyl sulfonates (e.g., busulfan and procarbazine, etc.), nitrosoureas (e.g., carmustine, lomustine, and streptozotocin, etc.), platinum alkylating agents (e.g., cisplatin, carboplatin, dicycloplatin, epplatin, loboplatin, miplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, etc.). Alkylating agents can damage the DNA structure by binding to the DNA in cancer cells, thereby inducing the destruction of cancer cells.

[0092] The antimetabolite may include, but is not limited to, pyrimidine derivatives (e.g., 5-fluorouracil, capecitabine, cytarabine, gemcitabine, and fludarabine, etc.), folic acid derivatives (e.g., methotrexate and pemetrexed, etc.), purine derivatives (e.g., mercaptopurine, etc.). Antimetabolites can induce apoptosis of cancer cells by inhibiting the metabolism required for DNA replication and cell survival.

[0093] The natural anticancer agents may include, but are not limited to, topoisomerase inhibitors (camptothecin, epipodophyllotoxin and taxanes) and antibiotics (eg, actinomycin, doxorubicin, daunorubicin, mitomycin, phleomycin, idarubicin, mitoxantrone hydrochloride, etc.).

[0094] In the present invention, targeted anticancer agents are anticancer agents that induce apoptosis of cancer cells by inhibiting target proteins (receptors or enzymes) involved in cancer growth, and their meanings are the same as those commonly used in the technical field to which the present invention belongs. The targeted anticancer agents include low molecular weight compounds and monoclonal antibodies that inhibit target proteins (tyrosine kinases, etc.).

[0095] In the present invention, the targeted anticancer agent may be a receptor tyrosine kinase inhibitor targeting one or more targets selected from the group consisting of VEGF / VEGFR, EGFR and HER2.

[0096] In one embodiment, the targeted anticancer agent that can be used as the third anticancer agent is a VEGF / VEGFR inhibitor. In the present invention, VEGF / VEGFR inhibitors may include, but are not limited to, low molecular weight compounds (such as axitinib, cabozantinib, lapatinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib and vandetanib); and monoclonal antibodies (such as bevacizumab, ramucirumab and ranibizumab).

[0097] In one embodiment, the targeted anticancer agent that can be used as the third anticancer agent is an EGFR inhibitor. In the present invention, the EGFR inhibitor may include, but is not limited to, low molecular weight compounds (such as osimertinib, gefitinib, erlotinib, afatinib, brigatinib, icotinib and vandetanib); and monoclonal antibodies (such as cetuximab, panitumumab, zalutumumab, nimotuzumab and matuzumab).

[0098] In one embodiment, the targeted anticancer agent that can be used as the third anticancer agent is a HER2 inhibitor. In the present invention, the HER2 inhibitor may include but is not limited to low molecular weight compounds (such as lapatinib, neratinib and afatinib); and monoclonal antibodies (such as trastuzumab, pertuzumab and margetuximab).

[0099] In addition, the targeted anticancer agents of the present invention may also include Bcr-Abl targeted anticancer agents (such as imatinib, dasatinib, nilotinib); Src targeted anticancer agents (such as bosutinib); JAK targeted anticancer agents (such as lestaurtinib, ruxolitinib, pacritinib); MAP2 kinase (Kinase) targeted anticancer agents (such as cobimetinib, selumetinib, trametinib, binimetinib); MEL4-ALK targeted anticancer agents (such as ceritibin and crizotinib), etc.

[0100] In the present invention, cancer immunotherapy is a cancer treatment method that activates the human immune system and fights cancer cells through immune anticancer agents. In the present invention, immune anticancer agents may include immune checkpoint inhibitors, immune cell therapy agents, anticancer vaccines and antibody-drug conjugates, and appropriate types may be selected according to the type and progression of cancer for the purpose of curing, regulating and alleviating symptoms of cancer.

[0101] In one embodiment, the immune anticancer agent can be an immune checkpoint inhibitor, which can be selected from one or more of 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 can be a PD-1 antibody (such as nivolumab, pembrolizumab, cemiplimab, pidilizumab and toripalimab); a PD-L1 antibody (such as atezolizumab, avelumab and duralumab); and a CTLA-4 antibody (such as ipilimumab and tremelimumab) or all of them.

[0102] In one embodiment, the immune anticancer agent may be a cell therapy agent, and may be a CAR-T therapy agent (such as tisagenlecleucel and axicabtagene ciloleucel) or a CAR-NK therapy agent, but is not limited thereto.

[0103] In the present invention, metabolic anticancer agents refer to drugs that kill cancer cells by participating in the growth and survival of cancer cells or participating in multiple necessary metabolic actions such as supplying nutrition to cancer cells. For example, the metabolic anticancer agent can be 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, but are not limited thereto.

[0104] In the present invention, synthetic lethality anticancer agents refer to those that allow cells to survive only when one of two genes (or two gene products) is mutated, and cause cell apoptosis when both genes are mutated. As an example, the synthetic lethal intervention anticancer agent can be olaparib, niraparib, talazoparib, veliparib, PRMT5 inhibitors (e.g., TNG908, MRTX1719, AMG 193, PRT811, GSK3326595, PF-06939999, JNJ-64619178, PRT543, PRT811, JBI-778, TNG462, AGX323, AT101 and AT201), PRMT1 inhibitors (e.g., GSK3368715 and SKL27969), Mat2A inhibitors (e.g., AG-270, IDE397 and ISM020), PKMYT1 inhibitors (e.g., RP-6306) and PARG inhibitors (e.g., IDE161), etc., but are not limited thereto.

[0105] The third anticancer agent of the present invention may be a combination of more than one cytotoxic anticancer agent, targeted anticancer agent and / or synthetic lethal anticancer agent, and they may be administered simultaneously or at different times. In addition, the third anticancer agent can be selected from dichloroacetic acid, tamoxifen, osimertinib, olaparib, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, mistletoe, asparaginase, retinoic acid, hydroxyurea, dasatinib, estramustine, gemtuzumab, ibritumomab tiuxetan, epplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, docetaxel, pemetrexed, tegafur, capecitabine, gemelacil, oteracil, azacitidine, methotrexate, uracil, aza One or more of the group consisting of cytosine, 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, actinomycin, pirarubicin, aclarubicin, peplomycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, hexamethylmelamine, dacarbazine, thiotepa, nimustine, chlorambucil, dibromodulan, lomustine and carmustine, but not limited thereto.

[0106] The ADP-ribose and ADP-ribose binding peptides of the present invention have excellent anticancer activity, and therefore can be prepared not only in the form of a pharmaceutical composition but also in the form of a functional food composition.

[0107] When the composition of the present invention is prepared in the form of a food composition, the food composition may contain additional ingredients commonly used in food to improve smell, taste, vision, etc. For example, food additives may be added. The additives may be selected according to the type of food and used in an appropriate amount.

[0108] The food composition can be prepared into health functional food, wherein health functional food is the same term as food for special health use (FoSHU), which refers to food with high medical and therapeutic effects that is processed in a way that effectively regulates the body in addition to providing nutrition. In order to achieve the beneficial effect of improving cancer, the health functional food can be prepared in various forms, such as tablets, capsules, powders, granules, liquids, pills, etc.

[0109] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an effective ingredient, wherein the ADP-ribose binding peptide is derivatized to have sustained release.

[0110] ADP-ribose binding peptides and derivatization are described above.

[0111] For example, the derivatization of the ADP-ribose binding peptide can be performed by pegylation of one or more amino acids constituting the peptide, or by binding fatty acids to one or more amino acids, so as to exhibit sustained release, but is not limited thereto. The derivatization can be performed by directly modifying amino acids or by using additional elements such as linkers.

[0112] Specifically, the PEG used for the PEGylation may be miniPEG2, dPEG4, dPEG6, dPEG8, dPEG12 or dPEG24, etc., but is not limited thereto.

[0113] Specifically, the fatty acid can be butyric acid, caprylic 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 or corynomycolic acid, etc., but is not limited thereto.

[0114] In one embodiment of the present invention, an ADP-ribose binding peptide derivative is prepared by combining miniPEG2, a cell-penetrating peptide, an ADP-ribose binding peptide and a fatty acid. In particular, the derivative prepared by PEGylating the peptide of SEQ ID NO: 29 (GRKKRRQRRRPQFLYVADLENMVQYRRNEHGRRRKIKR) and combining it with a fatty acid is named "sustained-release derivative". The specific structure of the sustained-release derivative of the present invention is shown in the following chemical formula 3.

[0115] [Chemical formula 3]

[0116]

[0117] As another aspect of the present invention, in the case of derivatives prepared by pegylation of the peptides of SEQ ID NO: 1 to SEQ ID NO: 14 and combining fatty acids and additional cell-penetrating peptides, the same level of effect as that of the "sustained-release derivatives" using the peptide of SEQ ID NO: 29 can also be confirmed.

[0118] The embodiments of the present invention may be modified into a variety of different forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art to which the present invention belongs. Furthermore, throughout the specification, unless otherwise specifically stated to the contrary, "comprising" a certain component means that other components may also be included, rather than excluding other components.

[0119] Embodiments of the invention

[0120] Hereinafter, the present invention will be described in detail by way of examples and experimental examples. However, the following examples and experimental examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0121] Examples 1 to 29. Preparation of adenosine diphosphate (ADP)-ribose binding peptides

[0122] The ADP-ribose binding peptides of Examples 1 to 14 were synthesized from wwE domains present in various proteins and used in the experiments. Specific information of the peptides used is shown in Table 1 below.

[0123] Table 1

[0124] Example Sequence information (SEQ ID NO.) Derivatized Protein 1 RRYTVQFTTMVQVNEETGNRRPVM(SEQ ID NO: 1) HUWE1 2 FLYVADLENMVQYRRNEHGRRRKIKR(SEQ ID NO: 2) RNF146 3 RVYTIDFNSMQQINEDTGTARAIQR(SEQ ID NO: 3) TRIP12 4 VPYIIDLQSMHQFRQDTGTMRPVRR(sEQ ID NO: 4) DTX1 5 FSYKIDFAEMKQMNLTTGKQRLIKR(SEQ ID NO: 5) PARP11 6 GRYDVHLGERMRYAVYWDELASEVRR (SEQ ID NO: 6) DDHD2 7 APYIIDLQSMNQFRQDTGTLRPVRR (SEQ ID NO: 7) DTX4 8 APYIIDLPSWTQFRQDTGTMRAVRR(SEQ ID NO: 8) DTX2 9 FCYLIYFNSMSQMNRQTRRRRRLRR(SEQ ID NO: 9) PARP7 10 FSYVIDFNTMGQINRQTQRQRRVRR(SEQ ID NO: 10) PARP12 11 YNYTVNYTTHTQTNKTSSFCRSVRR (SEQ ID NO: 11) PIK3C2G 12 WIWYWKNESGTWIQYGEEKDKRKN(SEQ ID NO: 12) ZC3HAV1 13 RHYTVNLNTYTATDTKGHSLSVQR (SEQ ID NO: 13) PARP14 14 TAYEASVCDYLEQQVARGN (SEQ ID NO: 14) HNRNPA2B1

[0125] In addition, the peptides of Examples 15 to 28 were prepared by binding a cell-penetrating peptide to the N-terminus of the peptides of Examples 1 to 14. The sequences of the peptides of Examples 15 to 28 are shown in Table 2 below.

[0126] Table 2

[0127] Example Sequence information (sEQ ID NO.) 15 GRKKRRQRRRPQRRYTVQFTT3VQVNEETGNRRPVM(SEQ ID NO: 15) 16 RAGLQFPVGRLLRRLLRFLYVADLENMVQVRRNEHGRRKIKR (SEQ ID NO: 16) 17 GRKKRRQRRRPQRVYTIDFNSMQQINEDTGTARAIQR(sEQ ID NO: 17) 18 GRKKRRQRRRPQVPYIIDLQSMHQFRQDTGTMRPVRR(SEQ ID NO: 18) 19 GRKKRRQRRRPQFSYKIDFAEMKQMNLTTGKQRLIKR(SEQ ID NO: 19) 20 GRKKRRQRRRPQGRYDVHLGERMRYAVYWDELASEVRR(SEQ ID NO: 20) 21 GRKKRRQRRRPQAPYIIDLQSMNQFRQDTGTLRPVRR (SEQ ID NO: 21) 22 GRKKRRQRRRPQAPYIIDLPSWTQFRQDTGTMRAVRR (SEQ ID NO: 22) 23 GRKKRRQRRRPQFCYLIYFNSMSQMNRQTRRRRRLRR(SEQ ID NO: 23) 24 GRKKRRQRRRPQFSYVIDFNTMGQINRQTQRQRRVRR(SEQ ID NO: 24) 25 GRKKRRQRRRPQYNYTVNYTTHTQTNKTSSFCRSVRR(SEQ ID NO: 25) 26 GRKKRRQRRRPQWIWYWKNESGTWIQYGEEKDKRKN(SEQ ID NO: 26) 27 GRKKRRQRRRPQRHYTVNLNTYTATDTKGHSLSVQR(SEQ ID NO: 27) 28 GRKKRRQRRRPQTAYEASVCDYLEQQVARGN(SEQ ID NO: 28)

[0128] In addition, the peptide of Example 29 was prepared by combining a cell-penetrating peptide (GRKKRRQRRRPQ) at the N-terminus of the peptide of Example 2. The sequence of the peptide of Example 29 is shown in Table 3 below.

[0129] Table 3

[0130] Example Sequence information (SEQ ID No) 29 GRKKRRQRRRPQFLVVADLENMVQYRRNEHGRRRKIKR(SEQ ID NO: 29)

[0131] As described above, the changes in cancer cell survival rate were measured by treating the peptides prepared in Examples 1 to 15 and Examples 17 to 29. Figure 1 Left) and MDA-MB-231 cells ( Figure 1 In the figure (right), the anticancer effects of the peptides of all Examples alone were confirmed, and the half-maximal growth inhibition concentration (GI50) of each Example was confirmed.

[0132] Example 30. Preparation of sustained-release derivatives using the peptide of Example 29

[0133] Peptron was commissioned to name the derivative of the ADP-ribose binding peptide synthesized by pegylation of the peptide of Example 29 and conjugation with stearic acid as "sustained-release derivative", and mass analysis (SHIMADZU LCMS-2020 System) and HPLC analysis (SHIMADZU Prominence HPLC System) were performed.

[0134] For HPLC analysis, the chromatographic column used was Shiseido capcell pak C18, 120 angstroms (4.6×50 mm), 5 μm, and the column temperature was kept at room temperature. The flow rate was 1.0 mL / min, and the detection was 220 nm. Mobile phase A was water containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile containing 0.1% TFA. The gradient was set to 35% B for 2 minutes, 30% B for 10 minutes, 10% B for 1 minute, and 77% B for 4 minutes.

[0135] The results are shown in Table 4, and LC-MS [M+H] + The value was confirmed to be 5363.43.

[0136] Table 4

[0137]

[0138] Experimental Example 1. Changes in the survival rate of cancer cells treated with the peptide and ADP-ribose according to the example

[0139] Experimental Example 1-1. Changes in survival rate of Caki-1 cells treated with the peptides of the examples and ADP-ribose

[0140] At 37°C and 5% CO2, 2.5 × 10 3 After culturing renal cancer (Caki-1) cells in a 96-well plate for 24 hours, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0141] 1) Group without any treatment;

[0142] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0143] 3) a group treated with the peptide of Example 1 alone at a concentration of 25 μM;

[0144] 4) groups treated with the CPP-peptide of Example 15 at a concentration of 10 μM;

[0145] 5) a group treated with a combination of 1 mM ADPR and 25 μM peptide of Example 1 (combination treatment 1);

[0146] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 15 at a concentration of 10 μM (combination treatment 2).

[0147] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0148] The results showed that in the group without peptide treatment, Caki-1 cells grew rapidly, while in the group treated with peptide of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with peptide of the present invention and ADPR, it was observed that the growth of Caki-1 cells was significantly inhibited and in the form of apoptosis ( Figure 2 ).

[0149] Experimental Example 1-2. Changes in survival rate of HCC1937 cells treated with the peptides of the examples and ADP-ribose

[0150] At 37°C and 5% CO2, 2.5 × 10 3 After culturing breast cancer cells (HCC1937) in 96-well plates for 24 hours, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0151] 1) Group without any treatment;

[0152] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0153] 3) a group treated with the peptide of Example 2 alone at a concentration of 25 μM;

[0154] 4) groups treated with the CPP-peptide of Example 16 at a concentration of 10 μM;

[0155] 5) a group treated with a combination of 1 mM ADPR and 25 μM peptide of Example 2 (combination treatment 1);

[0156] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 16 at a concentration of 10 μM (combination treatment 2).

[0157] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0158] The results showed that in the group without peptide treatment, HCC1937 cells grew rapidly, while in the group treated with peptides of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with peptides of the present invention and ADPR, it was observed that the growth of HCC1937 cells was significantly inhibited and in the form of apoptosis ( Figure 3 ).

[0159] Experimental Example 1-3. Changes in survival rate of AsPC-1 cells treated with peptides and ADP-ribose according to the examples

[0160] At 37°C and 5% CO2, 2.5 × 10 3 After culturing pancreatic cancer (AsPC-1) cells in 96-well plates for 24 hours, they were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0161] 1) Group without any treatment;

[0162] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0163] 3) a group treated with the peptide of Example 3 alone at a concentration of 25 μM;

[0164] 4) groups treated with the CPP-peptide of Example 17 at a concentration of 10 μM;

[0165] 5) a group treated with a combination of 1 mM ADPR and 25 μM peptide of Example 3 (combination treatment 1);

[0166] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 17 at a concentration of 10 μM (combination treatment 2).

[0167] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0168] The results showed that in the group without peptide treatment, AsPC-1 cells grew rapidly, while in the group treated with the peptide of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with the peptide of the present invention and ADPR, it was observed that the growth of AsPC-1 cells was significantly inhibited and in the form of apoptosis ( Figure 4 ).

[0169] Experimental Example 1-4. Changes in survival rate of HepG2 cells treated with the peptide of the example and ADP-ribose

[0170] At 37°C and 5% CO2, 2.5 × 10 3 After culturing each hepatoma (HepG2) cell in a 96-well plate for 24 hours, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0171] 1) Group without any treatment;

[0172] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0173] 3) a group treated with the peptide of Example 4 alone at a concentration of 25 μM;

[0174] 4) groups treated with the CPP-peptide of Example 18 at a concentration of 10 μM;

[0175] 5) a group treated with a combination of 1 mM ADPR and 25 μM peptide of Example 4 (combination treatment 1);

[0176] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 18 at a concentration of 10 μM (combination treatment 2).

[0177] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0178] The results showed that in the group without peptide treatment, HepG2 cells grew rapidly, while in the group treated with the peptide of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with the peptide of the present invention and ADPR, it was observed that the growth of HepG2 cells was significantly inhibited and in the form of apoptosis ( Figure 5 ).

[0179] Experimental Example 1-5. Changes in survival rate of H1975 cells treated with the peptides of the examples and ADP-ribose

[0180] At 37°C and 5% CO2, 2.5 × 10 3 After culturing lung cancer (H1975) cells in a 96-well plate for 24 hours, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0181] 1) Group without any treatment;

[0182] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0183] 3) a group treated with the peptide of Example 5 alone at a concentration of 25 μM;

[0184] 4) groups treated with the CPP-peptide of Example 19 at a concentration of 10 μM;

[0185] 5) a group treated with a combination of 1 mM ADPR and 25 μM peptide of Example 5 (combination treatment 1);

[0186] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 19 at a concentration of 10 μM (combination treatment 2).

[0187] The cells were cultured again for 96 hours at 37°C and 5% CO2, and 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 reagent that had completed the reaction was removed, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell survival rate.

[0188] The results showed that in the group without peptide treatment, H1975 cells grew rapidly, while in the group treated with peptide of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with peptide of the present invention and ADPR, it was observed that the growth of H1975 cells was significantly inhibited and in the form of apoptosis ( Figure 6 ).

[0189] Experimental Example 1-6. Changes in survival rate in HCT116 cells by combined treatment with the peptide of the example and ADP-ribose

[0190] At 37°C and 5% CO2, 2.5 × 10 3 After culturing in a 96-well plate for 24 hours, colorectal cancer (HCT116) cells were divided into the following groups, and the peptides of each example were administered to the cell lines alone or in combination at the GI50 concentrations.

[0191] 1) Group without any treatment;

[0192] 2) a group treated with ADPR alone at a concentration of 1 mM;

[0193] 3) a group treated with the peptide of Example 6 alone at a concentration of 25 μM;

[0194] 4) groups treated with the CPP-peptide of Example 20 at a concentration of 10 μM;

[0195] 5) a group treated with a combination of ADPR at a concentration of 1 mM and the peptide of Example 6 at a concentration of 25 μM (combination treatment 1);

[0196] 6) A group treated with a combination of ADPR at a concentration of 1 mM and the CPP-peptide of Example 20 at a concentration of 10 μM (combination treatment 2).

[0197] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0198] The results showed that in the group without peptide treatment, HCT116 cells grew rapidly, while in the group treated with peptides of the present invention and ADPR, the survival rate was reduced. In particular, in the group treated with peptides of the present invention and ADPR, it was observed that the growth of HCT116 cells was significantly inhibited and in the form of apoptosis ( Figure 7 ).

[0199] Experimental Example 2: Confirmation of normal cytotoxicity when the peptide of the example is treated with ADP-ribose in combination

[0200] At 37°C and 5% CO2, 2.5 × 10 3 After human normal CCD-18-Co or MRC5 cells were cultured in a 96-well plate for 24 hours, the peptides of Examples 1 to 15 and 17 to 29 and ADP-ribose were treated in combination.

[0201] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 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.

[0202] The results showed that when the peptides of each example were used in combination with ADPR, the survival rate of normal CCD-18-Co cells was about 90% or more, compared with the group without peptide treatment. Therefore, when the peptides of each example and ADPR were used in combination, it was confirmed that there was almost no toxicity to normal cells ( Figure 8 ).

[0203] Experimental Example 3. Comparison and verification of anticancer efficacy of combined treatment with peptides and ADP-ribose according to the examples using animal models

[0204] Experimental Example 3-1. Changes in tumor volume in AsPC-1 cells treated with a combination of the peptides of the examples and ADP-ribose

[0205] Pancreatic cancer (AsPC-1) cells (1×10 7 The mice were inoculated with 100 mg / kg of glutathione (mg / kg) at the posterior flank of the mice, and the mice were divided into the following administration groups, and the peptides of each example were treated alone or in combination with the GI50 concentration.

[0206] 1) Group without any treatment;

[0207] 2) ADP-ribose alone-administered group;

[0208] 3) groups of peptides of Examples 1 to 6 were treated separately;

[0209] 4) Groups treated with ADPR and the peptides of Examples 1 to 6 in combination (combination treatments 1 to 6), respectively.

[0210] When the tumor volume grows to about 75 mm 3 When ADP-ribose and the peptides of Examples 1 to 6 were administered alone or in combination. ADP-ribose was used at a concentration of 10 mg / kg, and the peptides of Examples 1 to 6 were used at a concentration of 20 mg / kg, and were administered subcutaneously twice a week for a total of 4 weeks.

[0211] Next, digital calipers were used to measure tumor size, and the results of changes in tumor volume were compared among the groups.

[0212] The results showed that the tumor volume was significantly reduced in all the administration groups compared with the control group. In particular, it was confirmed that the tumor volume of the combined administration group was significantly reduced compared with the single administration group ( Fig. 9 and Fig.10 ).

[0213] Experimental Example 3-2. Changes in tumor volume in HCC1937 cells based on combined treatment with CPP-peptide and ADP-ribose in Example

[0214] 5-week-old BALB / c nude mice were inoculated with breast cancer (HCC1937) cells (1×10 7 The mice were inoculated with the following GI50 concentrations of the CPP-peptides of each example alone or in combination.

[0215] 1) Group without any treatment;

[0216] 2) ADP-ribose alone-administered group;

[0217] 3) groups of CPP-peptides of Examples 15 to 20 were treated separately;

[0218] 4) Groups treated with ADPR and the CPP-peptides of Examples 15 to 20 in combination (combination treatments A to F), respectively.

[0219] When the tumor volume grows to about 75 mm 3 When ADP-ribose and the CPP-peptides of Examples 15 to 20 were administered alone or in combination. ADP-ribose was used at a concentration of 10 mg / kg, and the CPP-peptides of Examples 15 to 20 were used at a concentration of 10 mg / kg, and were administered subcutaneously twice a week for a total of 4 weeks.

[0220] Next, digital calipers were used to measure tumor size, and the results of changes in tumor volume were compared among the groups.

[0221] The results showed that the tumor volume was significantly reduced in all the administration groups compared with the control group. In particular, it was confirmed that the tumor volume of the combined administration group was significantly reduced compared with the single administration group ( Fig.11 and Fig.12 ).

[0222] Experimental Example 4. Confirmation of the synergistic anticancer effect of the combined treatment with the peptide of the example and ADP-ribose in PARP-resistant cell lines Next, the combined treatment effect of the peptide of the example and ADP-ribose was confirmed in PARP inhibitor-resistant cancer cell lines.

[0223] Specifically, 2.5×10 3 PARP inhibitor-resistant breast cancer (HCC1937) cells were cultured in 96-well plates for 24 h.

[0224] Next, the cells were divided into the following groups, and the cells were treated with the GI50 concentration of the peptides of each example alone or in combination.

[0225] 1) Group without any treatment;

[0226] 2) A group treated with 2 μM of PARP inhibitor (Olaparib);

[0227] 3) a group treated with ADPR alone at a concentration of 1 mM;

[0228] 4) groups treated with peptides from Examples 1 to 6 at a concentration of 25 μM, respectively;

[0229] 5) groups treated with the CPP-peptides of Examples 15 to 20 at a concentration of 10 μM, respectively;

[0230] 6) A group treated with ADPR at a concentration of 1 mM and the peptides of Examples 1 to 6 at a concentration of 25 μM (combination treatment 1);

[0231] 6) A group in which ADPR at a concentration of 1 mM and the CPP-peptides of Examples 15 to 20 at a concentration of 10 μM were treated in combination (combination treatment 2).

[0232] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0233] The results showed that when HCC1937 cells were treated with olaparib as a PARP inhibitor alone, about 80% of the cancer cells survived, while in all cases where the peptide of the example or ADP-ribose was treated, the cell survival rate was significantly reduced. In particular, in all cases where the peptide of the example and ADP-ribose were treated in combination, it was confirmed that the survival rate of cancer cells was significantly reduced compared to single treatment ( Fig.13 ).

[0234] Experimental Example 5. Peptides and NAD based on the examples + Changes in cancer cell survival rate after combined treatment

[0235] The present inventors have focused on NAD, a precursor of ADP-ribose. + The possibility of replacing ADP-ribose confirmed that NAD + Anticancer effects when administered alone or in combination with the ADP-ribose binding peptides of the Examples.

[0236] Specifically, 2.5×10 3 After culturing ovarian cancer (OVCAR-3) cells in a 96-well plate for 24 hours, the cells were divided into the following groups and treated with the GI50 concentration of the peptides of each example alone or in combination.

[0237] 1) Group without any treatment;

[0238] 2) Treat with 1 mM NAD alone + Group;

[0239] 3) a group treated with the peptide of Example 2 alone at a concentration of 25 μM;

[0240] 4) a group treated with the CPP-peptide of Example 16 alone at a concentration of 10 μM;

[0241] 5) Combined treatment with 1 mM NAD + and a group containing the peptide of Example 2 at a concentration of 25 μM (combination treatment 1);

[0242] 6) Combined treatment with 1 mM NAD + and the CPP-peptide of Example 16 at a concentration of 10 μM (combination treatment 2).

[0243] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0244] The results showed that in the group without peptide treatment, OVCAR-3 cells grew rapidly, and only NAD treatment alone + However, when the peptides of the present invention and NAD + When the peptide of the example was treated alone, it was confirmed that the survival rate of cancer cells was significantly reduced and an excellent anticancer effect was shown ( Fig.14 ).

[0245] Experimental Example 6. Changes in the survival rate of cancer cells treated with the peptide of the example, ADP-ribose, NAD+, and various anticancer agents

[0246] At 37°C and 5% CO2, 2.5 × 10 3 Human pancreatic cancer (Aspc-1), kidney 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 cultured in 96-well plates for 24 hours and then divided into the following groups and treated with the GI20 / IC20 concentrations of each agent alone or in combination.

[0247] 1) Group without any treatment;

[0248] 2) Each cancer cell was treated with the peptides of Examples 1 to 14 at a concentration of 25 μM, and the peptides of Examples 15 and 17 to 29 at a concentration of 10 μM.

[0249] 3) a group treated with 2 mM ADPR alone;

[0250] 4) Treat with 2 mM NAD alone + Group;

[0251] 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);

[0252] 6) A group in which ADPR at a concentration of 2 mM and the peptides of the examples (25 μM for Examples 1 to 14, and 10 μM for Examples 15 and 17 to 29) were co-treated;

[0253] 7) Combined treatment with 2 mM ADPR and 2 mM NAD + and the peptides of the Examples (25 μM for Examples 1 to 14; 10 μM for Examples 15 and 17 to 29);

[0254] 8) Groups treated with 2 mM ADPR and the peptides of the examples (25 μM as in Examples 1 to 14; 10 μM in Examples 15, 17 to 29; and various anticancer agents (same concentrations as in Group 5 above)).

[0255] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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.

[0256] The results showed that, in all cancer cells used in the experiment, the peptides, ADPR, and NAD + Compared with the cases of various anticancer agents, the combined treatment of the peptide of the embodiment and ADPR showed a survival rate of about 40% of cancer cells, and the combined treatment of the peptide of the embodiment, ADPR and NAD + In the case of combined treatment of peptides, ADPR and various anticancer agents of Examples 1 to 15 and Examples 17 to 29, the survival rate of cancer cells was reduced and showed a survival rate of about 10% compared with all other groups. Therefore, it was confirmed that when the peptides, ADPR and various anticancer agents of the prepared examples were combined, excellent anticancer effects were shown ( Figures 15 to 28 ).

[0257] Experimental Example 7. Changes in the survival rate of cancer cells when the sustained-release derivatives prepared using the peptides of Example 29 were treated alone or in combination

[0258] At 37°C and 5% CO2, 2.5 × 10 3 Human pancreatic cancer (Aspc-1), kidney 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 cultured in 96-well plates for 24 hours, and the sustained-release derivatives were treated alone to the cell lines. In addition, they were divided into the following groups, and MDA-MB-231 cancer cells were treated alone or in combination with the GI20 concentration of each preparation.

[0259] 1) Group without any treatment;

[0260] 2) a group treated with the sustained-release derivative alone at a concentration of 5 μM;

[0261] 3) a group treated with 2 mM ADPR alone;

[0262] 4) Treat with 2 mM NAD alone + Group;

[0263] 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);

[0264] 6) a group treated with a combined treatment of 2 mM ADPR and 5 μM sustained-release derivatives;

[0265] 7) Combined treatment with 2 mM ADPR and 2 mM NAD + and a group with a sustained-release derivative at a concentration of 5 μM;

[0266] 8) A group treated with a combination of 2 mM ADPR, 5 μM sustained-release derivatives, and various anticancer agents (the concentrations were the same as in 5)).

[0267] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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 reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell survival rate.

[0268] The results showed that when the sustained-release derivatives were treated alone, a survival rate of less than 10% was confirmed in most cancer cell lines ( Fig.29 On the other hand, for MDA-MB-231, a triple-negative breast cancer cell line, when the sustained-release derivative, ADPR, and NAD + When the cells were exposed to various anticancer drugs, the survival rate was 80%, indicating drug resistance. However, when the sustained-release derivative and ADPR were used in combination, cytotoxicity was observed. + When various anticancer agents were used as a combination of three drugs, it was confirmed that they showed significantly excellent cytotoxicity ( Fig.29 on the right side of the screen).

[0269] Experimental Example 8. Confirmation of normal cytotoxicity when the sustained-release derivatives prepared using the peptides of Example 29 are treated alone or in combination

[0270] At 37°C and 5% CO2, 2.5 × 10 3 Human normal MRC5 cells were cultured in 96-well plates for 24 hours and then treated with slow-release derivatives and ADPR alone or in combination.

[0271] Next, the cells were cultured for 96 hours at 37°C and 5% CO2, and 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.

[0272] The results showed that when the sustained-release derivatives were treated alone or in combination with ADPR, the survival of normal MRC5 cells was confirmed to be about 90% or more, compared with the group not treated with the sustained-release derivatives. Therefore, it was confirmed that the sustained-release derivatives showed almost no toxicity to normal cells, regardless of the treatment alone or in combination ( Fig.30 ).

[0273] Experimental Example 9. Comparison and verification of the anticancer efficacy of sustained-release derivatives alone or in combination with immunotherapeutic agents using animal models. Six-week-old C57BL / 6-hPD1 (PD-1 humanized mice) male mice were inoculated with colorectal cancer (MC38) cells (1×10 6 / 100 μL) were inoculated into the forelimb area of ​​the right back of mice, and the mice were divided into the following administration groups.

[0274] 1) Group without any treatment;

[0275] 2) a group that received intravenous injection of sustained-release derivatives at GI50 concentration alone;

[0276] 3) a group that received a subcutaneous injection of the sustained-release derivative at GI50 concentration alone;

[0277] 4) A group that received intraperitoneal injection of nivolumab, an immunotherapeutic agent, at a GI50 concentration alone;

[0278] 5) the group treated with combined GI50 concentration of sustained-release derivative (intravenous injection) and nivolumab (intraperitoneal injection) (combination treatment 1);

[0279] 6) The group treated with combined treatment of the sustained-release derivative (subcutaneous injection) and nivolumab (intraperitoneal injection) at GI50 concentration (combination treatment 2).

[0280] When the tumor volume grows to about 100 mm 3The sustained-release derivative and nivolumab were administered alone or in combination. The sustained-release derivative was mixed with albumin at a concentration of 5 mg / kg at a ratio of 3:1, and nivolumab was used at a concentration of 5 mg / kg, and they were administered twice a week for a total of 4 weeks.

[0281] Next, digital calipers were used to measure tumor size, and the results of changes in tumor volume were compared among the groups.

[0282] The results showed that the tumor volume was significantly reduced in all the administration groups compared with the control group. In particular, it was confirmed that the tumor volume of the combined administration group was significantly reduced compared with the single administration group ( Fig.31 In addition, no changes in body weight were observed in any of the administration groups, thus confirming that there was no toxicity ( Fig.32 ).

[0283] From the above description, without changing the technical idea or essential features of the present invention, a person skilled in the art to which the present invention belongs can understand that the present invention can be implemented in other specific forms. In this regard, it should be understood that the above-described embodiments are only illustrative and not restrictive in all aspects. The scope of the present invention should be interpreted as all changes and deformations derived from the meaning, scope and equivalent concepts of the patent claims described later are included within the scope of the present invention relative to the above detailed description.

Claims

1. A pharmaceutical composition for preventing or treating cancer, wherein: Include: a) adenosine diphosphate (ADP)-ribose binding peptide or a pharmaceutically acceptable salt thereof; and b) ADP-ribose, a precursor thereof or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein: The ADP-ribose binding peptide consists of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

14.

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

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

5. The pharmaceutical composition for preventing or treating cancer according to claim 4, wherein: The cell-penetrating peptide is selected from the group consisting of TAT, buforin, maurocalcinde, penetratin, poly-arginine-derived peptides, 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 amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO:

29.

7. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein: The peptide is obtained by acetylation, hydroxylation, methylation, amidation or pegylation of one or more amino acids constituting the peptide.

8. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein: The peptide is formed by binding a fatty acid, a carbohydrate, a lipid component or a cofactor to one or more amino acids constituting the peptide.

9. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein: The cancer is selected from one or more solid cancers in the group consisting of brain cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, colon cancer, liver cancer, stomach cancer, pancreatic cancer, bile duct 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 poly (ADP-ribose) polymerase (PARP) 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 preparation.

12. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein: The composition also includes a third anticancer agent.

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

14. A pharmaceutical composition for preventing or treating cancer, wherein: The active ingredient comprises an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof, wherein the ADP-ribose binding peptide is a peptide derivatized so as to have a sustained release property.

15. The pharmaceutical composition for preventing or treating cancer according to claim 14, wherein: The ADP-ribose binding peptide is derivatized by pegylation of one or more amino acids constituting the peptide, or by binding a fatty acid to one or more amino acids.

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 chemical formula 3: [Chemical formula 3]

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