Composition containing suramin

Abstract

To provide a composition containing suramin.SOLUTION: A pharmaceutical composition contains suramin and is used to treat cancer, where the cancer is one containing a cancer cell with RAD52-mediated DNA repair pathway being enhanced. The pharmaceutical composition may be a preparation for oral administration that contains suramin and is used to treat cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions comprising suramin. The present invention also relates to RAD52 inhibitors or RAD52 expression suppressants comprising suramin. The present invention further relates to inhibitors of the RAD52-mediated DNA repair pathway comprising suramin. The present invention further relates to pharmaceutical compositions comprising suramin for use in treating cancer. The present invention further relates to pharmaceutical compositions comprising suramin, which are orally administered formulations for use in treating cancer. [Background technology]

[0002] According to Non-Patent Literature 1, cell lines with a C-terminal domain deficiency of RECQL4 (RECQL4ΔC) have improved erroneous single-strand annealing (SSA) activity and decreased alternative end joining (Alt-EJ) activity, suggesting that RECQL4 may regulate the DNA repair pathway in double-strand breaks. Furthermore, cancer cells with RECQL4ΔC increase the focus of RPA2 / RAD52, a factor involved in SSA, after cancer treatments such as ionizing radiation and cisplatin treatment, and accumulate RPA2 / RAD52 at DNA damage sites. Furthermore, using in vitro and in vivo xenograft models, we have shown that knockdown of RAD52 and RAD52 inhibitors, specifically 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), epigallocatechin (EGC), and RAD52 phenylalanine 79 aptamer, suppress the proliferation of colorectal cancer cell lines (HCT116 cells) possessing RECQL4ΔC.

[0003] According to Patent Document 1, it has been shown that epigallocatechin (EGC) and AICAR, which are RAD52 inhibitors, may inhibit SSA activity in RECQL4-deficient cells. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2019-19082 [Non-patent literature]

[0005] [Non-Patent Document 1] Kohzaki M. et al., International Journal of Cancer, 146(11): 3098-3113, 2020 Brief Description of the Invention

[0006] The present invention provides compositions comprising suramin. The present invention also provides RAD52 inhibitors or RAD52 expression suppressants comprising suramin. The present invention further provides inhibitors of the RAD52-mediated DNA repair (e.g., single-strand annealing (SSA)) pathway comprising suramin. The present invention further provides pharmaceutical compositions comprising suramin for use in treating cancer. The present invention further provides pharmaceutical compositions comprising suramin, which are orally administered formulations for use in treating cancer.

[0007] The inventors have found that suramin inhibits RAD52. The inventors have also found that suramin suppresses RAD52 expression. The inventors have further found that suramin inhibits RAD52-mediated DNA repair. The inventors have found that suramin inhibits RAD52-mediated DNA repair and exerts an antitumor effect against cancer cells.

[0008] The inventors have further found that RAD52 inhibitors such as suramin exert antitumor effects against RECQL4 wild-type cancer cells and RECQL4-deficient cancer cells. The inventors have also found that RAD52 inhibitors such as suramin exert antitumor effects in homologous recombination repair (HRR)-deficient cells (e.g., BRCA1-deficient cells). Furthermore, the inventors have found that RAD52 inhibitor treatment enhances its antitumor effect when combined with platinum-based drugs or PARP inhibitors. This combined effect was observed in RECQL4 wild-type and BRCA1 wild-type cells, as well as in RECQL4-deficient and BRCA1-deficient cells.

[0009] The present invention is based on the above findings. According to the present invention, for example, the following inventions are provided. (1) A pharmaceutical composition containing suramin for use in treating cancer. (2) A pharmaceutical composition according to (1) above, wherein the cancer is a cancer that includes cancer cells that have enhanced the RAD52-mediated DNA repair pathway. (3) The pharmaceutical composition according to (2) above, wherein the cancer cells with enhanced RAD52-mediated DNA repair pathway are cancers having a RECQL4-decreasing mutation. (4) A pharmaceutical composition according to (2) above, wherein the cancer is a cancer that includes cancer cells having homologous recombination repair (HRR) deficiency. (5) A pharmaceutical composition according to any of (1) to (4) above, which is an oral administration preparation. (6) The pharmaceutical composition according to any one of (1) to (5) above, wherein the cancer cells are cells that have DNA damage to their genomic DNA. (7) A pharmaceutical composition according to any one of (1) to (6) above, which is used in combination with radiotherapy or chemotherapy with a platinum-based agent. (8) Suramin, an inhibitor of the RAD52-mediated DNA repair pathway. (9) Prepare a compound selected from the group consisting of analogs, related compounds, varieties, and derivatives of suramin, The process involves bringing cancer cells with enhanced RAD52-mediated DNA repair pathways into contact with the compound, Selecting compounds that suppress the proliferation of cancer cells with enhanced RAD52-mediated DNA repair pathways. A method for selecting compounds that include [the specified element]. (10) A pharmaceutical composition comprising suramin for use in treating cancer, which is formulated as an oral preparation. (11) A pharmaceutical composition for use in inhibiting the acquisition of resistance to platinum formulations and / or PARP inhibitor treatment, comprising a RAD52 inhibitor. (12) A pharmaceutical composition for use in treating cancer in patients who have undergone platinum-based and / or PARP inhibitor treatment, which is a pharmaceutical composition to be used in combination with the treatment that has been undergone, and which comprises a RAD52 inhibitor. (13) The pharmaceutical composition according to (11) or (12) above, wherein the RAD52 inhibitor is suramin. (14) A pharmaceutical composition according to any of (1) to (7) and (10) to (12) above, which is used in combination with a PARP inhibitor. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the SSA inhibitory effect of suramin in step 6 of the primary assay system of Example 1. [Figure 1B] Figure 1B shows the inhibitory effect of suramin on the number of RAD52 foci in the secondary assay system of Example 1. [Figure 2] Figure 2 shows the effect of suramin on colony formation in human colorectal cancer cell line HCT116 and human breast cancer cell line MCF7, which have wild-type RECQL4 (WT) or RECQL4 loss-of-function (RECQL4). In the figure, the survival rate is shown as the ratio of the number of colonies formed to the number of colonies in untreated control cells. [Figure 3A] Figure 3A shows the effect of intraperitoneal administration of suramin on tumor growth in a xenograft model obtained by subcutaneous transplantation of MCF7 cells. [Figure 3B]Figure 3B shows the effect of oral administration of slamine on tumor growth in a xenograft model obtained by subcutaneous transplantation of MCF7 cells. [Figure 4] Figure 4 shows the effect of oral administration of slamine on tumor growth in a xenograft model obtained by subcutaneous transplantation of MCF7 cells. [Figure 5] Figure 5 shows the effect of oral administration of slamine on tumor growth in an orthotopic xenograft model obtained by transplanting MCF7 cells into the mammary fat pad. [Figure 6] Figure 6 shows the effect of oral administration of slamine on tumor growth in an orthotopic xenograft model obtained by transplanting MCF7 cells into the mammary fat pad. [Figure 7A] Figure 7A shows the effect of slamine at concentrations in the μM order on the colony formation of MCF7 cells. In the figure, the ratio of the colony number of untreated control cells to the colony formation number is shown as the survival rate. [Figure 7B] Figure 7B shows the effect of slamine at concentrations in the nM order on the colony formation of MCF7 cells. In the figure, the ratio of the colony number of untreated control cells to the colony formation number is shown as the survival rate. [Figure 7C] Figure 7C shows the effect of slamine at concentrations in the pM order on the colony formation of MCF7 cells. In the figure, the ratio of the colony number of untreated control cells to the colony formation number is shown as the survival rate. [Figure 7D] Figure 7D shows the effect of slamine at concentrations in the fM order on the colony formation of MCF7 cells. In the figure, the ratio of the colony number of untreated control cells to the colony formation number is shown as the survival rate. [Figure 8] Figure 8 shows that slamine suppresses the expression of RAD52 in cells. [Figure 9A] Figure 9A shows an experimental scheme for confirming the effect of a RAD52 inhibitor on a cancer-bearing model based on C57BL / 6 mice with a normal immune system. [Figure 9B] Figure 9B shows the effect of RAD52 inhibitors on a cancer-carrying model based on C57BL / 6 mice with a normal immune system. [Figure 9C] Figure 9C shows the results of comparing the effects of RAD52 inhibitors on a cancer-carrying model based on C57BL / 6 mice with a normal immune system, as well as the effects on negative controls and polyI:C. [Figure 10] Figure 10 shows the changes in DNA repair activity induced by Cyclin E expression induction. [Figure 11] Figure 11 shows an example of a RECQL4 dysfunction mutation. [Figure 12] Figure 12 illustrates a method for quantitatively evaluating the activity of DNA pathways by homologous recombination (HR), alternative end joining (Alt-EJ), single-strand annealing (SSA), break-induced replication (BIR), and classic non-homologous end join repair (c-NHEJ). [Figure 13] Figure 13 shows the combined effect of olaparib, a PARP inhibitor, and a RAD52 inhibitor against cancer cell lines. [Figure 14] Figure 14 shows the antitumor effects of RAD52 inhibitors on BRCA1-deficient cancer cells, which are HRR-deficient cancer cells, and on cancer cells in which BRCA1 expression was restored to BRCA1-deficient cancer cells. [Figure 15] Figure 15 shows the antitumor effect of combining the platinum-based drug cisplatin with a RAD52 inhibitor. [Figure 16] Figure 16 shows the antitumor effect of combining a RAD52 inhibitor with a platinum-based drug (cisplatin) in BRCA1-deficient cancer cells, which are HRR-deficient cancer cells. [Figure 17] Figure 17 shows the antitumor effect of combining a RAD52 inhibitor with a higher concentration of platinum-based drug (cisplatin) in cancer cells in which BRCA1 expression was restored from BRCA1-deficient HRR-deficient cancer cells. [Figure 18]Figure 18 shows the antitumor effects of the combined use of a RAD52 inhibitor and a PARP inhibitor (olaparib) in BRCA1-deficient cancer cells (HRR-deficient cancer cells) and cancer cells in which BRCA1 expression was restored to BRCA1-deficient cancer cells. [Figure 19] Figure 19 shows the antitumor effects of combining a RAD52 inhibitor with a higher concentration of a PARP inhibitor (olaparib) in BRCA1-deficient cancer cells (HRR-deficient cancer cells) and cancer cells in which BRCA1 expression was restored to BRCA1-deficient cancer cells. [Figure 20] Figure 20 shows the antitumor effect of combining a RAD52 inhibitor with a platinum-based drug (cisplatin) in BRCA1-deficient cancer cells, which are HRR-deficient cancer cells. [Figure 21] Figure 21 shows the antitumor effect of combining a RAD52 inhibitor (EGC or D-103) with a platinum-based drug (cisplatin) in BRCA1-deficient cancer cells, which are HRR-deficient cancer cells. [Figure 22] Figure 22 shows the antitumor effect of the combination of a RAD52 inhibitor (suramin) and a platinum-based drug (cisplatin) in BRCA1-deficient cancer cells, which are HRR-deficient cancer cells. Specific description of the invention

[0011] In this specification, the subject may be a mammal (e.g., mouse, rat, rabbit, hamster, monkey, dog, cat, cow, horse, sheep, goat, llama, and donkey), such as a human.

[0012] In this specification, "RECQL4" is a member of the RecQ helicase family and is required for maintaining genomic stability. As shown in Figure 11, RECQL4 has, from the N-terminus, an Sld2-like domain, a nuclear localization signal (NLS), a helicase domain, and an Hrq1 domain in that order. It is known that deletion of the C-terminal region of RECQL4 (the region containing the helicase domain) induces Rosmund-Thomson syndrome (RTS). RECQL4 mutations are also associated with Barre-Gerold syndrome (BGS) and Lapaderino syndrome. RECQL4 is involved in several DNA repair mechanisms, interacts with PARP-1, regulates BER, and repairs single-strand breaks (SSBs). According to the inventors' previous report (Kohzaki M. et al., International Journal of Cancer, DOI: 10.1002 / ijc.32670), cell lines with a C-terminal domain deficiency of RECQL4 (RECQL4ΔC) have improved erroneous single-strand annealing (SSA) activity and decreased alternative end joining (Alt-EJ) activity, suggesting that RECQL4 may regulate the DNA repair pathway in double-strand breaks. Furthermore, cancer cells with RECQL4ΔC increase RPA2 / RAD52 focus after cancer treatments such as ionizing radiation irradiation and platinum-based drug treatment (e.g., cisplatin treatment). Furthermore, in vitro and in vivo xenograft models have shown that knockdown of RAD52 or RAD52 inhibitors, specifically 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), epigallocatechin (EGC), and RAD52 phenylalanine 79 aptamer, suppress the proliferation of colorectal cancer cell lines (HCT116 cells) possessing RECQL4ΔC. Human RECQL4 may be RECQL4 having the amino acid sequence registered under GenBank registry number: AAH13277.2, or human RECQL4 having an amino acid sequence corresponding to that sequence. The C-terminal domain may be the region of amino acids 490-1208 in the amino acid sequence registered under GenBank registry number: AAH13277.2.In non-human cells, orthologs of human RECQL4 exist in the genome and are expressed.

[0013] In this specification, "RECQL4ΔC cells" means cells that express RECQL4ΔC. RECQL4ΔC includes at least the N-terminal Sld2-like domain of RECQL4 (i.e., the region of RECQL4 having amino acid sequences corresponding to amino acid sequences 1-490 in the amino acid sequence registered with GenBank registry number: AAH13277.2). RECQL4ΔC may include, for example, the Sld2-like domain. RECQL4ΔC may include, for example, the Sld2-like domain and a nuclear localization signal (NLS). RECQL4ΔC may consist of, for example, the Sld2-like domain and a nuclear localization signal (NLS). RECQL4ΔC may, for example, have a deletion in part or all of the C-terminal region (the region of RECQL4 having amino acid sequences corresponding to amino acid sequences 490-1208 in the amino acid sequence registered with GenBank registry number: AAH13277.2). Cells expressing RECQL4ΔC may be cells in which the gene encoding endogenous RECQL4 present in the genome has been modified to become RECQL4ΔC. Modifications can be made, for example, by introducing frameshift mutations or nonsense mutations, or by introducing stop codons. Furthermore, while not particularly limited, gene modification can be appropriately carried out using methods well known to those skilled in the art, such as genome editing technology.

[0014] In this specification, "RECQL4-deficient cells" means cells in which the function and / or structure of the endogenous RECQL4 helicase is lost or inhibited. In RECQL4-deficient cells, the endogenous RECQL4 has reduced or lost helicase activity due to genetic modification. Examples of RECQL4-deficient cells include cells lacking the helicase domain of RECQL4, and cells lacking both the helicase domain and the Hrq1 domain, such as cells possessing RECQL4ΔC. RECQL4-deficient cells may possess endogenous RECQL4 with an Sld2-like domain.

[0015] In this specification, "RAD52" is a protein involved in DNA repair. Human RAD52 may be RAD52 having the amino acid sequence registered with GenBank registry number: AAA85793.1 or human RAD52 having the amino acid sequence corresponding to that amino acid sequence. Orthologs of RAD52 exist in the animal species from which the cell type used is derived. RECQL4 suppresses the RAD52-mediated single-strand annealing pathway, and therefore, it is known that deficient mutations in RECQL4 enhance the RAD52-mediated single-strand annealing pathway (SSA) (Int J Cancer. 2020, 146(11): 3098-3113). Homologous recombination (HR), error-prone single-strand annealing (SSA), and break-induced replication (BIR) are among the DNA damage repair pathways.

[0016] In this specification, “treatment” means therapeutic treatment and preventive treatment.

[0017] In this specification, "platinum preparation" and "platinum preparation" are synonymous and mutually interchangeable, and refer to anticancer agents having antitumor activity, such as cisplatin, carboplatin, and oxaliplatin.

[0018] In this specification, "ionizing radiation therapy" refers to radiotherapy administered to a target malignant tumor for the purpose of treating that malignant tumor. Ionizing radiation therapy may be performed by irradiation with X-rays, gamma rays, or particle beams. Ionizing radiation therapy induces double-strand breaks in the DNA within the irradiated cells.

[0019] In this specification, "suramin" refers to the compound shown in the following chemical formula (I). Suramin is used as a treatment for African sleeping sickness (African trypanosomiasis). Suramin is used against Trypanosoma bruseyi rhodesiensis. Trypanosoma brucei rhodesienseIt is considered effective against infection. Suramin has been developed as an anticancer agent due to its antitumor effect (Stain, Cancer Res, 1993, 53: 2239-2248), but high doses of suramin need to be administered by intravenous infusion, which has resulted in serious side effects such as nausea, hypotension, liver dysfunction, and kidney damage. Suramin may be administered in the form of its pharmaceutically acceptable salts (especially sodium salts). [ka]

[0020] According to the present invention, suramin can inhibit RAD52-mediated DNA repair even at fM-order concentrations, thereby suppressing cell proliferation or causing cell death. Suramin is known to be poorly absorbed into the body when administered orally (Odgen et al, Pharm Res, 2004). However, fM-order blood or tumor tissue concentrations can be achieved by oral administration or by other methods of administration.

[0021] The present invention provides an inhibitor of RAD52 expression, comprising suramin. The present invention also provides an inhibitor of RAD52-mediated DNA repair pathways (e.g., single-strand annealing (SSA) and / or break-induced replication (BIR)), comprising suramin. According to the present invention, these inhibitors can be used in vivo, i.e., they can be pharmaceutical compositions. Such pharmaceutical compositions can be administered to a subject. In one preferred embodiment, the subject has RAD52-positive cells. In one preferred embodiment, the subject has immunity (e.g., T-cell immunity). Cells having immunity (e.g., T-cell immunity) do not have a RECQL4-downfunction mutation. In one preferred embodiment, the subject has cancer cells having a RECQL4-downfunction mutation. A subject having cancer cells with a RECQL4-downfunction mutation may be an immune subject (e.g., T-cell immunity). Cancer cells may be cells in which one or more DNA repair pathways selected from the group consisting of SSA, BIR, and HR are activated. Cancer cells may be cells that have activated SSA. Cancer cells may be cells that have activated BIR. Cancer cells may be cells that have activated HR or cells that have inactivated HR. In one embodiment, cancer cells may be cancer cells having a RECQL4-reducing mutation and in which one or more DNA repair pathways selected from the group consisting of SSA, BIR, and HR are activated. In one embodiment, cancer cells may be cancer cells having a RECQL4-reducing mutation and in which SSA is activated. In one embodiment, cancer cells may be cancer cells having a RECQL4-reducing mutation and in which BIR is activated. In one embodiment, cancer cells may be cancer cells having a RECQL4-reducing mutation and in which HR is activated or cells that have inactivated HR. Examples of cells with inactivated HR include BRCA1 and / or BRCA2-reducing or loss-of-function mutant cells. In one preferred embodiment, the subject has cells with DNA damage. DNA damage may, for example, be a double-strand break in genomic DNA. The double-strand break may preferably have a cleavage end. In some embodiments, the subject may be a subject with cancer.Cancer may include cancers in which the RAD52-mediated DNA repair pathway has been activated. Activation of the RAD52-mediated DNA repair pathway can, in some embodiments, be induced or enhanced by a loss-of-function mutation in RECQL4. Therefore, in some embodiments, cancers in which the RAD52-mediated DNA repair pathway has been activated may include cancer cells having a loss-of-function mutation in RECQL4. In some preferred embodiments, the loss-of-function mutation in RECQL4 may be a loss-of-function mutation. Furthermore, activation of the RAD52-mediated DNA repair pathway can be induced or enhanced by DNA damage. In addition, the RAD52-mediated DNA repair pathway can be induced or enhanced by increased expression or activity of oncogenes such as Cyclin E.

[0022] The present invention also provides inhibitors of the RAD52-mediated DNA repair pathway, such as SSA, which include suramin.

[0023] The present invention provides a pharmaceutical composition comprising suramin for use in treating cancer in subjects having cancer. In one preferred embodiment, the subjects have RAD52-positive cancer cells. In one preferred embodiment, the subjects have cancer cells in which RAD52-mediated DNA repair, such as single-strand annealing pathway (SSA), has been activated. In one preferred embodiment, the subjects have cancer cells having DNA damage. Activation of RAD52-mediated DNA repair, such as SSA, is mediated by RAD52 in one embodiment. Activation of RAD52-mediated DNA repair, such as SSA, can be induced or enhanced by a downfunction mutation in RECQL4 in one embodiment. Therefore, in one embodiment, activation of RAD52-mediated DNA repair, such as SSA, can be RAD52-mediated DNA repair, such as RAD52-mediated SSA, induced or enhanced by a downfunction mutation in RECQL4. Cancer in which RAD52-mediated DNA repair, such as RAD52-mediated SSA, has been activated may include cancer cells having a downfunction mutation in RECQL4. In one preferred embodiment, a RECQL4-reducing mutation may be a loss-of-function mutation. Furthermore, activation of RAD52-mediated DNA repair, such as RAD52-mediated SSA, may be enhanced by DNA damage. DNA damage may be, for example, a double-strand break. In one embodiment, the double-strand break is repaired by DNA ligase III. In another embodiment, the double-strand break is repaired by annealing of DNA breaks with sticky ends.

[0024] Cancer cells with activated RAD52-mediated DNA repair, such as SSA, may be cancer cells in which RAD52-mediated DNA repair, such as SSA, is activated due to DNA damage. Cancer cells with activated RAD52-mediated DNA repair, such as SSA, may also be cancer cells in which RAD52-mediated DNA repair, such as SSA, is activated due to a RECQL4-reducing mutation. Cells with activated RAD52-mediated DNA repair, such as SSA, may be cancer cells that have DNA damage and a RECQL4-reducing mutation, and have activated RAD52-mediated DNA repair, such as SSA. Cancer cells with activated RAD52-mediated DNA repair, such as SSA, may be cancer cells with enhanced RAD52 expression and / or function, for example. RAD52-mediated DNA repair is preferably SSA.

[0025] A RECQL4-reducing mutation is a genomic mutation that can either reduce or eliminate the function of RECQL4. A RECQL4-reducing mutation means that RECQL4 functions less than wild-type RECQL4 (e.g., RECQL4 with the amino acid sequence registered under GenBank registry number: AAH13277.2). The function of RECQL4 may be, for example, the ability to suppress the activation of RAD52-mediated DNA repair pathways such as the SSA pathway. A RECQL4-loss mutation means a mutant that has little to no function. Such mutants may result, for example, from the deletion of part or all of the coding region of RECQL4. Whether RECQL4 has a RECQL4-reducing mutation can be determined by comparing its function with that of the wild-type RECQL4 (e.g., RECQL4 with the amino acid sequence registered under GenBank registry number: AAH13277.2).

[0026] The activity of RAD52-mediated DNA repair pathways such as the SSA pathway is, for example, Cells (for example, cells with a RECQL4-deficient mutation, e.g., RECQL4-deficient cells) To introduce a reporter system to evaluate the repair of double-strand breaks in damaged cells, Here, the reporter system has a marker gene, which is divided into a first part and a second part. The first part is operably linked to a regulatory sequence, and a DNA sequence is interposed between the first and second parts, so that at least the second part is not transcribed or translated. The interposed DNA sequence has a cleavage site for a restriction enzyme (e.g., I-SceI) with sequence specificity of 16 bases or more. Cleavage by this restriction enzyme induces RAD52-mediated DNA repair, such as the SSA pathway, within the cell. When the first and second parts, which were divided by this RAD52-mediated DNA repair, are relinked in-frame, the in-frame relinked first and second parts are expressed, thereby enabling the detection of the marker gene containing the first and second parts. To introduce the aforementioned restriction enzyme with sequence specificity of 16 or more bases into the above-mentioned cells (for example, cells with a functionally impaired RECQL4 mutation, for example, RECQL4-deficient cells), DNA double-strand breaks are induced, thereby inducing RAD52-mediated DNA repair such as the SSA pathway. This can be measured by methods or test systems that include the following. Higher expression of the marker gene indicates higher activity of RAD52-mediated DNA repair, such as the SSA pathway, and lower expression indicates lower activity of RAD52-mediated DNA repair, such as the SSA pathway. By comparing the expression of the marker gene in the test cells with that of control cells (e.g., cells expressing wild-type RECQL4), it is possible to evaluate whether the test cells enhance or suppress RAD52-mediated DNA repair inhibition, such as SSA.

[0027] In this invention, the suppression of RAD52-mediated DNA repair activity, such as that of SSA, can be confirmed by the above-described test system. The above-described test system can be used to confirm whether the suppression of RAD52-mediated DNA repair activity, such as that of SSA, can be more strongly compared to a positive control (e.g., in the presence of suramin) or a negative control (e.g., in the absence of suramin).

[0028] In some aspects of the present invention, cancer can be solid tumors and hematological cancers. Solid tumors can be highly malignant tumors, particularly malignant tumors. The cancers to be treated are not particularly limited, but include hematological cancers such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, myelodysplastic syndromes, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, anaplastic carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, and liver cancer. This includes solid cancers such as visceral cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, bladder cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue. Other examples include sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreaticblastoma, pleuropneumonblastoma, and retinoblastoma.

[0029] In some aspects of the present invention, cancer has DNA damage. In some aspects of the present invention, cancer may be cells with impaired DNA repair pathways. Such cancers have a reduced ability to repair DNA damage and may have DNA damage. For example, in some aspects of the present invention, cancer may be cancer cells having functional RECQL4. In some aspects of the present invention, cancer may be cancer cells having impaired RECQL4, or cancer cells lacking functional RECQL4. In some aspects of the present invention, cancer may be cancer cells having a functional HR repair pathway. In some aspects of the present invention, cancer may be cancer cells having impaired HR repair pathways, or cancer cells lacking functional HR repair pathways. In some aspects of the present invention, cancer may be cells with impaired HR repair pathways or cells lacking functional HR repair pathways due to BRCA1 mutations and / or BRCA2 mutations. Such cancers have a reduced ability to repair DNA damage and may have DNA damage. In addition to or in addition to the above, DNA damage can be caused by various chemotherapy or radiotherapy. Therefore, in some aspects, cancer has experienced chemotherapy or radiotherapy. Examples of chemotherapy include platinum-based treatments and DNA damaging agents (alkylating agents such as mitomycin C and melphalan; platinum-based drugs such as cisplatin and oxaliplatin; DNA methylase inhibitors such as 5-aza-2'-cytidine; topoisomerase inhibitors such as camptothecin and etoposide; and DNA-protein crosslinking agents, interstrand / intrastrand crosslinking agents, and double-strand cleavage agents). Examples of radiotherapy include X-ray, electron beam, and gamma-ray therapy. Radiation destroys cancer cells by damaging the DNA within the cancer cells. According to the present invention, RAD52 inhibitors have a therapeutic effect against the above-mentioned cancers. According to the present invention, RAD52 inhibitors can inhibit the acquisition of cancer resistance to the above-mentioned chemotherapy or radiotherapy. According to the present invention, RAD52 inhibitors can also treat cancers that have become resistant to the above-mentioned chemotherapy or radiotherapy. In some aspects of the present invention, these cancers may be any of the cancers exemplified above.

[0030] Cell death can be induced in cells where DNA double-strand breaks are not repaired. Therefore, inhibiting the repair of DNA double-strand breaks can induce cell death in cells. Inhibiting the repair of DNA double-strand breaks in cancer cells can induce cell death in cancer cells and may be beneficial in cancer treatment. In this respect, RAD52 inhibitors may be advantageous in cancer treatment. According to the present invention, the RAD52 inhibitor is preferably suramin, although it is not particularly limited.

[0031] According to the present invention, a pharmaceutical composition containing suramin for use in treating cancer in a subject having cancer can be used in combination with cancer therapies selected from the group consisting of chemotherapy with other anticancer agents, radiotherapy, and surgery. In one preferred embodiment, a pharmaceutical composition containing suramin for use in treating cancer in a subject having cancer can be used in combination with chemotherapy. In a more preferred embodiment, the chemotherapy may be chemotherapy using a platinum-based agent. In another more preferred embodiment, the chemotherapy may be chemotherapy using an anthracycline anticancer agent. In yet another more preferred embodiment, the chemotherapy may be chemotherapy using a PARP inhibitor. In one other preferred embodiment, the chemotherapy may be chemotherapy using an alkylating agent (e.g., ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, ranimustine, etc.). In one preferred embodiment, chemotherapy may be a topoisomerase inhibitor (e.g., irinotecan, camptothecin, topotecin, nogitecan, hycamtin, etoposide, anthracycline anticancer agents, etc.). In one preferred embodiment, a pharmaceutical composition containing suramin for use in treating cancer in a subject with cancer may be used in combination with radiotherapy. When used in combination with cancer therapies such as chemotherapy and radiotherapy, the pharmaceutical composition of the present invention may be administered before, concurrently with, or during an overlapping period with, said cancer therapy, or after said cancer therapy. In one embodiment, examples of platinum preparations include platinum chelates of platinum and a chelating agent, such as cisplatin, carboplatin, oxaliplatin, miriplatin, and nedaplatin. In one embodiment, examples of anthracycline anticancer agents include doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, acralubicin, amrubicin, and mitoxatron. By combining platinum-based drugs with RAD52 inhibitors, the impact of developing resistance to platinum-based drugs in cancer treatment can be reduced compared to platinum-based monotherapy, and cancer treatment outcomes can be improved.In some embodiments, PARP inhibitors include, for example, olaparib, rucaparib, niraparib, veliparib, and talazoparib. In some embodiments, cancers treated with PARP inhibitors may exhibit enhanced BIR and SSA. Such cancers can be preferably treated with RAD52 inhibitors. Furthermore, combining PARP inhibitor treatment with RAD52 inhibitors can reduce the impact of developing resistance to PARP inhibitors in cancer treatment compared to PARP inhibitor monotherapy, and thus improve cancer treatment outcomes. Examples of radiotherapy include therapies that treat cancer by irradiating it with medical radiation such as electron beams, proton beams, heavy ion beams, alpha rays, beta rays, X-rays, and gamma rays. In some preferred embodiments, radiotherapy induces RAD52-mediated DNA repair, such as the SSA pathway, in cancer cells. RAD52-mediated DNA repair, such as the SSA pathway, can be induced by double-strand breaks in genomic DNA. For example, X-rays or gamma rays can efficiently induce double-strand breaks in genomic DNA. Chemotherapy or radiotherapy can damage genomic DNA and activate RAD52-mediated DNA repair pathways such as the SSA pathway, and can therefore be preferably used in combination with the administration of the pharmaceutical composition of the present invention to a subject.

[0032] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable excipients, additives, and / or auxiliaries such as bulking agents, wetting agents, surfactants, disintegrants, binders, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring and odor-correcting agents, analgesics, stabilizers, lubricants, and colorants.

[0033] The pharmaceutical compositions of the present invention can be administered by various routes of administration. In one preferred embodiment, the pharmaceutical compositions of the present invention are administered orally, for example. In this embodiment, the pharmaceutical compositions of the present invention are formulated for oral administration, i.e., they may be oral formulations. In another preferred embodiment, the pharmaceutical compositions of the present invention are administered parenterally, for example. In this embodiment, the pharmaceutical compositions of the present invention are formulated for parenteral administration, i.e., they may be parenteral formulations (e.g., infusion formulations or injection formulations). Parenteral administration includes, for example, intratumoral administration, intravenous administration, and intraperitoneal administration. The intratumoral suramin concentration after administration of these pharmaceutical compositions may be or can be in the range of fM to nM. Parenteral formulations, such as injection formulations, may be formulations in which suramin is dissolved, or may be kits comprising a composition containing suramin in lyophilized form and water (e.g., water for injection). Here, the composition containing suramin in lyophilized form may further contain pharmaceutically acceptable excipients, additives, and / or auxiliaries.

[0034] In the case of orally administered formulations, the pharmaceutical composition of the present invention is not particularly limited, but can be administered in doses ranging from 0.01 mg / kg body weight to 100 mg / kg body weight. In one preferred embodiment, it can be administered in doses ranging from 0.3 mg / kg body weight to 30 mg / kg body weight. In one preferred embodiment, it can be administered in doses ranging from 0.5 mg / kg body weight to 20 mg / kg body weight, or from 1 mg / kg body weight to 15 mg / kg body weight. In a more preferred embodiment, it may be 0.01 mg / kg body weight to 0.1 mg / kg body weight. Administration can be carried out once to several times a day (e.g., once, twice, or three times a day), once to several times a week (e.g., once, twice, three, four, five, or six times a week), or once to several times a month (e.g., once, twice, or three times a month). Oral administration of 1 mg / kg of suramin to mice is thought to be equivalent to oral administration of 0.081 mg / kg of suramin to humans (FASEB J, 2008, 22, 659-61). Considering that the dose of suramin used to treat African sleeping sickness is 20 mg / kg administered intravenously on days 1, 3, 7, 14, and 21, and given the low absorption of suramin when administered orally, it can be understood that suramin inhibits RAD52 at very low concentrations. Suramin can be administered to cancer patients to achieve trough levels of, for example, 1 fM to 1 μM (e.g., 1 pM to 1 nM, 1 pM to 100 nM, 1 pM to 10 pM, 100 fM to 100 pM, or 100 fM to 10 pM). Normally, when a drug is administered, its blood concentration increases and then decreases due to metabolism and excretion. When a drug is administered repeatedly, its blood concentration fluctuates, eventually settling within a certain range. This state is called the steady state. The highest blood concentration of the drug in the steady state is called the peak value, and the lowest value is called the trough value.

[0035] The pharmaceutical composition of the present invention can be administered at doses below the dose at which one or more adverse reactions selected from the group consisting of nausea, hypotension, and renal impairment occur. Suramin can cause serious adverse reactions such as renal impairment, hepatic impairment, coagulation disorders, and polyneuropathy. When the relationship between the blood concentration of suramin and adverse reactions was examined, renal impairment may occur at concentrations of 300 μM (428.7 μg / mL) or higher, coagulation disorders do not occur at 250 μg / mL, and polyneuropathy may occur at 350 μg / mL or higher. Furthermore, adverse reactions are less likely to occur with suramin at blood concentrations of 300 μg / mL or lower. Therefore, in the present invention, suramin can be administered to a blood concentration of 300 μg / mL or less, 250 μg / mL or less, 200 μg / mL or less, 150 μg / mL or less, 100 μg / mL or less, 50 μg / mL or less, 10 μg / mL or less, 5 μg / mL or less, 1 μg / mL or less, or 100 ng / mL or less.

[0036] The present invention provides for the use of suramin in the manufacture of a pharmaceutical for use in treating cancer. The pharmaceutical is administered to a subject having cancer, and the preferred cancers are as described above. The present invention provides suramin for use in treating cancer. Suramin is administered to subjects with cancer, and preferred cancers are as described above. The present invention provides a method for treating cancer in a subject having cancer, comprising administering to the subject a therapeutically effective amount of suramin or a pharmaceutical composition containing suramin. The suramin or pharmaceutical composition containing suramin is administered to the subject having cancer, and the preferred cancers are as described above. In one aspect of the present invention, the target patients for administration of suramin may be patients who have received chemotherapy with DNA-damaging anticancer agents (for example, anthracycline drugs (e.g., doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, acralubicin, amrubicin, bleomycin, and mitoxatron, etc.), platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin, miriplatin, and nedaplatin, etc.) and PARP inhibitors (e.g., olaparib, rucaparib, niraparib, beriparib, and talazoparib, etc.), patients undergoing such chemotherapy, or patients scheduled to undergo such chemotherapy. In one aspect of the present invention, suramin may be administered to patients who have received, are undergoing, or are scheduled to receive radiation therapy with medical radiation such as electron beams, proton beams, heavy ion beams, alpha rays, beta rays, X-rays, and gamma rays. Thus, in one aspect of the present invention, suramin can be used in combination with chemotherapy using DNA-damaging anticancer agents or radiation therapy using medical radiation. In a preferred embodiment of the present invention, the subjects to whom suramin is administered may be subjects having cancer having cancer cells that have activated the RAD52-mediated DNA repair pathway. Activation of the RAD52-mediated DNA repair pathway may occur, for example, by DNA damage. DNA damage may be, for example, a double-strand break. DNA damage may be induced by DNA-damaging anticancer agents (e.g., the above-mentioned anticancer agents) and / or radiotherapy (e.g., the above-mentioned radiotherapy). In one preferred embodiment of the present invention, the target population for RAD52 inhibitor (preferably suramin) is a patient having cancer with functional RECQL4. In one preferred embodiment of the present invention, the target population for RAD52 inhibitor (preferably suramin) is a patient having a decrease in the function of RECQL4. In patients having a decrease in the function of RECQL4, the RAD52 inhibitor (preferably suramin) may exhibit a higher antitumor effect than in patients without such a mutation. In one preferred embodiment of the present invention, the target population for RAD52 inhibitor (preferably suramin) may be a patient having cancer with functional BRCA1 and / or BRCA2 (particularly functional BRCA1 and functional BRCA2). In one preferred embodiment of the present invention, the target population for RAD52 inhibitor (preferably suramin) is a patient having a decrease in the function of BRCA1 and / or BRCA2. In subjects with BRCA1 and / or BRCA2 downfunction mutations, RAD52 inhibitors (preferably suramin) may exhibit a higher antitumor effect than in subjects without such mutations. In one aspect of the present invention, subjects receiving suramin may be those who have received cancer therapy selected from the group consisting of chemotherapy with other anticancer agents, radiotherapy, and surgery. In one aspect of the present invention, subjects may be those who are scheduled to receive cancer therapy selected from the group consisting of chemotherapy with other anticancer agents, radiotherapy, and surgery. In one aspect of the present invention, subjects receiving suramin may be those who have a RECQL4 downfunction mutation, and who have received, are receiving, or are scheduled to receive cancer therapy selected from the group consisting of chemotherapy with other anticancer agents, radiotherapy, and surgery.

[0037] The present invention provides a method for selecting a compound. In this embodiment, the method of the present invention includes designing, synthesizing, and / or providing a compound selected from the group consisting of analogs, related compounds, varieties, and derivatives of suramin. The method of the present invention may include contacting the compound with cancer cells that have enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathways). The method of the present invention may include selecting a compound that inhibits the proliferation of cancer cells that have enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathways). The compound thus obtained may be an anticancer agent or a candidate thereof. In some embodiments, the method may further include contacting the compound with cancer cells that do not have enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathways). The present invention allows for the selection of compounds as RAD52-mediated DNA repair pathway (e.g., RAD52-mediated single-strand annealing pathway)-specific compounds, by including the selection of compounds that suppress the proliferation of cancer cells with enhanced RAD52-mediated DNA repair pathway (e.g., RAD52-mediated single-strand annealing pathway) compared to cancer cells that do not enhance the RAD52-mediated DNA repair pathway (e.g., RAD52-mediated single-strand annealing pathway). Cancer cells with enhanced RAD52-mediated DNA repair pathway (e.g., RAD52-mediated single-strand annealing pathway) may be cancer cells with a dysfunctional RECQL4 mutation, while cancer cells without enhanced RAD52-mediated DNA repair pathway (e.g., RAD52-mediated single-strand annealing pathway) may be cancer cells with wild-type or wild-type-equivalent mutant RECQL4. The cells may be, for example, primary cells or cell lines. Methods for establishing cell lines from primary somatic cells are well known to those skilled in the art.

[0038] The present invention provides compounds selected from the group consisting of analogs, related compounds, varieties, and derivatives of suramin. Contacting cancer cells with enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathway) with the compound, Selecting compounds that suppress the proliferation of cancer cells with enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathway) and This provides a method for selecting compounds that include [the specified compound]. The present invention provides a composition comprising a compound, particularly a compound selected from the group consisting of analogs, related compounds, varieties, and derivatives of suramin. Contacting cancer cells with enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathway) with the compound, Select a composition containing a compound that suppresses the proliferation of cancer cells with enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathway). A method for selecting a composition containing the above compounds is provided. The composition may further contain pharmaceutically acceptable excipients in addition to the above compounds. The composition may be formulated for administration to a subject. The composition may also be formulated for oral administration to a subject. Cancer cells with enhanced RAD52-mediated DNA repair pathways (e.g., RAD52-mediated single-strand annealing pathway) may be, for example, cancer cells with DNA damage or cancer cells with RECQL4-deficient mutations.

[0039] Analogues, related compounds, varieties, and derivatives of suramine can be obtained, for example, by modifying suramine, substituting groups of suramine, or removing groups of suramine. A derivative of suramine may, for example, be a fragment of suramine. A derivative of suramine may, for example, be a substituted suramine or a fragment thereof.

[0040] Substituents include halogens, hydroxyls, optionally substituted lower alkyls, optionally substituted cycloalkyls, optionally substituted cycloalkyl lower alkyls, optionally substituted lower alkenyls, optionally substituted lower alkoxys, optionally substituted lower alkenyloxys, optionally substituted aryls, optionally substituted aryl lower alkyls, optionally substituted aryloxys, optionally substituted heterocyclic groups, optionally substituted heterocyclic lower alkyl groups, optionally substituted heterocyclic groups, oxys, hydroxyls, optionally substituted aminos, optionally substituted lower alkylcarbonyls, optionally substituted cycloalkylcarbonyls, optionally substituted cycloalkyl lower alkylcarbonyls, optionally substituted lower Lower alkoxycarbonyl, optionally substituted arylcarbonyl, optionally substituted aryl lower alkylcarbonyl, optionally substituted aryloxycarbonyl, optionally substituted heterocyclic carbonyl, optionally substituted heterocyclic lower alkylcarbonyl, optionally substituted heterocyclic oxycarbonyl, optionally substituted aminocarbonyl, optionally substituted phosphate residue, aryl substituted with optionally substituted phosphate residue, aralkyl substituted with optionally substituted phosphate residue, hydroxy substituted with optionally substituted phosphate residue, amino substituted with optionally substituted phosphate residue, or lower alkyl substituted with optionally substituted phosphate residue [the lower alkyl is CO, O, S, SO, SO2, NR] a (R aThe lower alkyl group is hydrogen, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkoxy, optionally substituted aryl, optionally substituted aryl lower alkyl, optionally substituted aryloxy, optionally substituted heterocyclic group, optionally substituted heterocyclic lower alkyl, optionally substituted heterocyclic group oxy, hydroxy, optionally substituted amino, optionally substituted phosphate residue, aryl substituted with optionally substituted phosphate residue, aralkyl substituted with optionally substituted phosphate residue, hydroxy substituted with optionally substituted phosphate residue, amino substituted with optionally substituted phosphate residue, or a lower alkyl group substituted with optionally substituted phosphate residue (where the lower alkyl group is CO, O, S, SO, SO2, NR b (R b The substituent may be hydrogen or a lower alkyl group, a heteroatom group selected from the group consisting of -N= and =N-, O or CH2, a heteroatom group selected from the group consisting of -N= and =N-, and the substituent may have one or more carbons replaced by heteroatoms selected from O, S, and N. Here, lower means having 1 to 6 carbon atoms, preferably 1 to 4, for example, 1, 2, 3, or 4. The substitution in the substituent may be made by a second substituent.

[0041] The second substituent may be a halogen, hydroxy, an optionally substituted lower alkyl, an optionally substituted cycloalkyl, an optionally substituted cycloalkyl lower alkyl, an optionally substituted lower alkenyl, an optionally substituted lower alkoxy, an optionally substituted lower alkenyloxy, an optionally substituted aryl, an optionally substituted aryl lower alkyl, an optionally substituted aryloxy, an optionally substituted heterocyclic group, an optionally substituted heterocyclic lower alkyl, an optionally substituted heterocyclic group oxy, hydroxy, an optionally substituted amino, an optionally substituted lower alkylcarbonyl, an optionally substituted cycloalkyl carbonyl, an optionally substituted cycloalkyl lower alkylcarbonyl, or an optionally substituted Lower alkoxycarbonyl, optionally substituted arylcarbonyl, optionally substituted aryl lower alkylcarbonyl, optionally substituted aryloxycarbonyl, optionally substituted heterocyclic carbonyl, optionally substituted heterocyclic lower alkylcarbonyl, optionally substituted heterocyclic oxycarbonyl, optionally substituted aminocarbonyl, optionally substituted phosphate residue, aryl substituted with optionally substituted phosphate residue, aralkyl substituted with optionally substituted phosphate residue, hydroxy substituted with optionally substituted phosphate residue, amino substituted with optionally substituted phosphate residue, or lower alkyl substituted with optionally substituted phosphate residue [the lower alkyl is CO, O, S, SO, SO2, NR] a (R aThe lower alkyl group is hydrogen, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl lower alkyl, optionally substituted lower alkenyl, optionally substituted lower alkoxy, optionally substituted aryl, optionally substituted aryl lower alkyl, optionally substituted aryloxy, optionally substituted heterocyclic group, optionally substituted heterocyclic lower alkyl, optionally substituted heterocyclic group oxy, hydroxy, optionally substituted amino, optionally substituted phosphate residue, aryl substituted with optionally substituted phosphate residue, aralkyl substituted with optionally substituted phosphate residue, hydroxy substituted with optionally substituted phosphate residue, amino substituted with optionally substituted phosphate residue, or a lower alkyl group substituted with optionally substituted phosphate residue (where the lower alkyl group is CO, O, S, SO, SO2, NR b (R b The substituent may be hydrogen or a lower alkyl group, a heteroatom group selected from the group consisting of -N= and =N-, O or CH2, a heteroatom group selected from the group consisting of -N= and =N-, and the substituent may have one or more carbons replaced by heteroatoms selected from O, S, and N. In the above, lower means having 1 to 6 carbon atoms, preferably 1 to 4, for example, 1, 2, 3, or 4. Also, may be substituted means either unsubstituted or substituted. The second substituent may be an unsubstituted group.

[0042] The present invention provides a method for testing whether a test compound is equivalent to or more strongly inhibits the RAD52-mediated DNA repair pathway (e.g., the RAD52-mediated single-chain annealing pathway) than a reference compound. In this embodiment, the method may include contacting the test compound with cells in which the RAD52-mediated DNA repair pathway (e.g., the RAD52-mediated single-chain annealing pathway) is activated, and determining whether the test compound inhibits the RAD52-mediated DNA repair pathway (e.g., the RAD52-mediated single-chain annealing pathway) as strongly as or more strongly than the reference compound in cells that have been contacted with the reference compound. In one preferred embodiment, the reference compound is suramin. In one preferred embodiment, the reference compound may be epigallocatechin (EGC). In one preferred embodiment, the reference compound may be any RAD52-mediated DNA repair pathway inhibitor (e.g., a RAD52-mediated single-chain annealing pathway inhibitor). These compounds may preferably be further tested by a secondary assay system. For example, a secondary assay system may include a method that further evaluates whether RAD52 activity is suppressed in cells treated with the test compound. Enhancement and suppression of RAD52 activity can be assessed by measuring whether RAD52 forms foci in the nucleus, or by measuring the number of foci formed. When RAD52 activity is enhanced, it forms foci in the nucleus, and the number of foci increases. Conversely, suppression of RAD52 activity can be assessed by a decrease in the number of foci formed. [Examples]

[0043] Example 1: Construction of a compound assay system targeting the DNA repair pathway by RAD52 As previously shown by Kohzaki M. et al., International Journal of Cancer, DOI: 10.1002 / ijc.32670, human RECQL4-deficient cells (e.g., human colorectal cancer cell line HCT116, human lung cancer cell line A549, and human breast cancer cell line MCF7, all lacking RECQL4ΔC) are sensitive to DNA damage-inducing treatments such as ionizing radiation therapy and platinum-based treatments (e.g., cisplatin therapy), but have been found to activate the RAD52-mediated DNA repair pathway (particularly SSA) that acts after such treatment. Human RECQL4-deficient colorectal cancer cell line HCT116 cells activated the RAD52-mediated DNA repair pathway while suppressing the activation of alternative non-homologous end joining (Alt-EJ). In other words, human RECQL4 deficiency is thought to selectively activate the SSA pathway within the repair mechanism in HCT116 cells.

[0044] In this example, an assay system was constructed to test compounds that can suppress the RAD52-mediated DNA repair pathway using HCT116 cells lacking human RECQL4. As shown in the example below, RAD52 inhibition exerted an antitumor effect by suppressing the DNA repair pathway in human RECQL4-deficient cells at low concentrations. The cells used in the example below, as shown in Figure 11, are cell lines in which the C-terminal region of RECQL4 after the established helicase domain was deleted by introducing a stop codon and a blasticidin S resistance gene near amino acid number 490 of endogenous RECQL4, i.e., the start site of the helicase domain, and selecting genetically modified cells through drug selection.

[0045] Primary assay system Step 1: The hprtSAGFP vector (Addgene_41594, Addgene) was transfected into RECQL4-deficient colorectal cancer HCT116 cells, selected with puromycin (2 - 4 μg / ml), and cells stably incorporating the vector were established. In RECQL4-deficient cells, RAD52 expression is upregulated. As shown in Figure 10, the hprtSAGFP vector is used as a substrate for the DNA repair pathway by RAD52. In the vector, the green fluorescent protein GFP is divided into two parts by a drug selection marker. A cleavage site for the restriction enzyme I-SceI was introduced into the gene encoding the above-divided GFP. By forcibly expressing I-SceI in HCT116 cells, a double-strand break is added to the gene encoding the divided GFP. Then, the divided GFP gene is religated by the DNA repair pathway by RAD52 in the cells, and the activity intensity of the DNA repair pathway by RAD52 can be measured by the expression of GFP. In the said vector, the GFP gene is driven by the CAG promoter.

[0046] Step 2: 0.5×10 5 ~2×10 5 of the above cells were seeded into a 24-well plate containing 300 μl of medium.

[0047] Step 3: The next day, 0.4 μg each of the pCBASceI vector (Addgene_26477, Addgene), which is an expression vector for the restriction enzyme I-SceI, or the empty control vector pCAGGS was transfected into the seeded cells using Lipofectamine LTX Reagent with PLUS Reagent (15338100, ThermoFisher Scientific), or Amaxa nucleofector (program D-032) with Kit V (VCA-1003, Lonza, Basel, Switzerland). As shown in Figure 2, this treatment resulted in a 2-3 fold increase in the number of GFP-positive cells after DNA cleavage in RECQL4-deficient cells compared to wild-type cells. Therefore, this assay system was considered suitable for testing the DNA repair pathway inhibitory activity of RAD52. Accordingly, as step 3, cells were treated with 10 μM of the test compound several hours before or immediately after the transfection.

[0048] Step 4: After 36-48 hours, SSA activity was quantified by flow cytometry based on the percentage of GFP-positive cells, and it was investigated whether the test compound had an inhibitory effect on the RAD52-mediated DNA repair pathway, which is activated after DNA double-strand breaks. Screening was performed independently twice, and the mean and standard deviation of RAD52-mediated DNA repair pathway activity were obtained.

[0049] Step 5: When an inhibitor candidate was obtained, the pmaxGFP (VDC-1040, Lonza) vector was transfected in Step 3 above, and the test compound was treated with 10 μM. After 24-48 hours, the analysis was performed by flow cytometry to confirm that it did not inhibit the transfection itself.

[0050] Step 6: To further confirm the concentration-dependent inhibitory effect of the test compound, the concentration was varied to 10, 5, and 2.5 μM, and the results were analyzed independently at least twice to confirm the concentration-dependent inhibitory effect and reproducibility.

[0051] Secondary assay system For test compounds evaluated as having RAD52-mediated DNA repair pathway inhibitory activity in the above assay system, molecular evidence that the suppression of the DNA repair pathway is mediated by RAD52 may be obtained by confirming the intracellular localization of RAD52 in cells treated with the test compound. The specific method is as follows. If information on the inducibility of RAD52 is to be obtained, evaluation can be performed using a secondary assay system in addition to the primary assay system.

[0052] Step 1: RECQL4-deficient colorectal cancer HCT116 cells 1 × 10⁶ 5 ~4×10 5 The cells were seeded into a 12-well plate containing 1 ml of culture medium. Cover slips were placed in the 12 wells beforehand, and the cells were attached to them.

[0053] Step 2: Two to three days later, the test compound was added at a concentration of 10 μM six hours before cisplatin treatment. Subsequently, the HCT116 cells were treated with 3 μg / ml cisplatin for 16 hours, and then the treated cells were fixed by treating them with phosphate-buffered saline (PBS) containing 2% sucrose and 3% paraformaldehyde at room temperature for 15 minutes.

[0054] Step 3: After treating with PBS containing 0.2% Triton-X for 2 minutes, the samples were blocked by treating with PBS containing 1% BSA at room temperature for 15 minutes.

[0055] Step 4: As the primary antibody, mouse RAD52 antibody (1:200 dilution; sc-365341) or rabbit RAD52 antibody (1:500 dilution; sc-8350) was treated at room temperature for 1 hour.

[0056] Step 5: After washing three times with PBST (PBS and 0.05% Tween-20), the cells were treated with either goat anti-mouse Alexa Fluor 488 (1:2,000; A-11001), a goat anti-mouse IgG antibody, or goat anti-rabbit Alexa Fluor 488 (1:2,000; A-11008), a goat anti-rabbit IgG antibody, as a secondary antibody for 45 minutes at room temperature.

[0057] Step 6: Wash with PBST, treat with 1 μg / ml DAPI for 1 minute, wash again with PBST, and finally mount the cells with Fluoromount-G (0100-20, Southern Biotech).

[0058] Step 7: Quantitative and qualitative analysis of fluorescent spots within cells was performed using an Axio Observer (Zeiss, Oberkochen, Germany) or a BZ-X700 (Keyence, Osaka, Japan) microscope. These spots are thought to be focus images that occur when RAD52 is involved in repair.

[0059] In this way, the primary assay system tested whether the test compound had inhibitory activity against the DNA repair pathway mediated by RAD52, while the secondary assay system tested whether compounds that inhibit the RAD52 repair pathway suppressed RAD52 focus formation.

[0060] Based on the results of the primary assay, suramin was identified as a compound that inhibits the DNA repair pathway mediated by RAD52.

[0061] Figure 1A shows the results of step 6 of the primary assay. Suramin was shown to inhibit RAD52-mediated DNA repair pathway activity in a concentration-dependent manner. Secondary assays also showed inhibition of RAD52 focus formation (Figure 1B). This indicates that suramin exhibits inhibitory activity of the RAD52-mediated DNA repair pathway through the inhibition of RAD52.

[0062] (Chronogenic assay using cell culture) HCT116 cells (RRID:CVCL_0291; RCB2979, RIKEN) and MCF7 cells (RRID:CVCL_0031; RCB1904, RIKEN) were seeded into 12-well (353043, Corning) and 24-well (353047, Corning) plates to achieve an appropriate number of colonies. For example, as a control, a total of 50 or 100 cells were seeded into 24-well or 12-well plates, respectively. Simultaneously, the same number of cells (50 or 100) were seeded into 24-well or 12-well plates, respectively, for suramin treatment. The following day, the attached cells were treated with various concentrations of suramin (193-10611, Wako) and incubated in a 37°C incubator for 1-2 weeks. To prevent colony detachment, methanol was slowly added along the sidewall after discarding the medium and held securely for several minutes. After discarding methanol, cells were stained by adding 3-5% Giemsa solution (509-01865, Wako) for at least 15 minutes. After staining, the cells were thoroughly washed and dried, and the number of colonies was counted. By measuring the relative number of colonies with the untreated control as the baseline value of 1, the survival rate of cancer cells was obtained, and the long-term antitumor effect of suramin treatment was determined. Reproducibility was verified by performing multiple independent experiments. The results are shown in Figure 2. As shown in Figure 2, suramin showed a concentration-dependent effect of reducing cell viability in RECQL4-deficient cells. This reduction in cell viability was observed similarly in both HCT116 cells and MCF7 cells.

[0063] In RECQL4-deficient cells, RAD52-dependent repair is activated, and it is thought that cancer cells utilize this mechanism to avoid death. RAD52 inhibitors (e.g., suramin) are understood to exert their antitumor effect by inhibiting DNA repair through this pathway. Therefore, for example, the antitumor effect of RAD52 inhibitor treatment is thought to be enhanced when used in combination with radiation therapy or platinum-based drug treatment, and conversely, the antitumor effect of radiation therapy or platinum-based drug treatment is thought to be enhanced by RAD52 inhibitor treatment.

[0064] Example 2: Therapeutic effect on an ectopic xenograft model 5 x 10 6 One wild-type (WT) MCF7 cell and one RECQL4-deficient MCF7 cell were each suspended in 50 μL of PBS and chilled on ice. This mixture was then thoroughly mixed with 50 μL of ice-chilled Matrigel (354234, Corning) to make a total volume of 100 μL. This mixture was then subcutaneously injected into the lateral flank of nude mice using a syringe (1 mL SS-01T, Terumo). Nude mice have an abnormal immune system and a low ability to eliminate cancer cells by the immune system. While this makes it easy to create a xenograft model, it also means that the immune system cannot be assisted in therapeutic experiments. To eliminate the influence of individual differences among mice, WT MCF7 cells were transplanted into the left flank and RECQL4-deficient MCF7 cells were transplanted into the right flank. Seven weeks after transplantation, when the establishment of a small mass of cancer cells was confirmed, oral administration of suramin was started. Based on past literature, the recommended dosage of suramin for intraperitoneal administration is 20 mg / kg weekly for the treatment of autism (Naviaux et al, PLoS One, 2013;8, e57380), 10-60 mg / kg weekly for cancer treatment (Song et al, Cancer Res, 2001, 61, 6145-50), and a non-toxic dose of 15 mg / kg (Song et al, Clin Cancer Res, 2004, 10, 6058-65, doi: 10.1158 / 1078-0432.CCR-04-0595). In this study, with the aim of obtaining an antitumor effect through RAD52 inhibition with minimal toxicity, we set the intraperitoneal administration doses to 0.3 mg / kg and 1 mg / kg, which are more than 15 times lower than the 15 mg / kg dose considered non-toxic in previous literature. When these doses were administered intraperitoneally once a week starting 7 weeks after transplantation of cancer cells, no antitumor effect against MCF7 cells was observed in this dose range (Figure 3A).

[0065] In response, various doses of suramin (3 mg / kg, 10 mg / kg, 30 mg / kg) were administered orally. In this experiment as well, oral administration was performed once a week starting 7 weeks after transplantation of cancer cells. As a result, as shown in Figure 3B, an antitumor effect was observed specifically on RECQL4-deficient MCF7 cells transplanted to the right flank in a dose-dependent manner. Furthermore, as shown in Figure 4, complete remission of RECQL4-deficient MCF7 cancer cells was observed in the group administered suramin orally. Due to its low absorption rate when administered orally, it was expected to be non-toxic (Ogden et al, Pharm Res, 2004, 21, 2058-63, doi: 10.1023 / b:pham.0000048197.77546.75). For example, according to Ogden et al., the mean maximum plasma concentration in rats orally administered 100 mg / kg of suramin was 9.04 μg / mL. When body weight changes were examined, no changes from the control group were observed in any of the dose groups, indicating extremely low levels of side effects (Figure 3B). This experiment was performed three times independently, and its reproducibility was confirmed.

[0066] Example 3: Therapeutic effect on an orthotopic xenograft model Next, we conducted an experiment in which human breast cancer cells were transplanted into the milk fat body of mice to investigate whether low-dose oral administration of suramin could produce an antitumor effect in an orthotopic model. 5×10 6One wild-type (WT) MCF7 cell and one RECQL4-deficient MCF7 cell were each suspended in 50 μL of PBS and chilled on ice. This mixture was then thoroughly mixed with 50 μL of ice-chilled Matrigel (354234, Corning) to make a total volume of 100 μL, which was then transplanted into the milk fat bodies of mice. In this experiment, to minimize the influence of individual differences, the third and fourth of the five pairs of mouse milk fat bodies were used, and the WT MCF7 cells and RECQL4-deficient MCF7 cells were transplanted in an alternating manner (see illustration in Figure 6). Perhaps because it reflects the environment in which the cells originally engrafted, the proliferation of MCF7 cells was faster in the orthotopic xenotransplantation experiment compared to the ectopic xenotransplantation experiment, and the timing of suramin administration was started earlier accordingly. Specifically, suramin administration was started 5 weeks after transplantation of cancer cells, when the establishment of a small cluster of cancer cells was confirmed. In this experiment as well, suramin was administered orally once a week starting 5 weeks after transplantation of cancer cells. In this experiment, various doses (1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg), including a lower dose (1 mg / kg), were administered orally to investigate the antitumor effect. A RECQL4-deficient MCF7 cell-specific antitumor effect was confirmed (see Figures 5 and 6). While a dose-dependent antitumor effect was observed from 1 mg / kg to 10 mg / kg, unlike the ectopic xenograft experiment, the 30 mg / kg oral administration group unexpectedly showed a reduced antitumor effect. This suggests that higher doses of suramin are not necessarily better, and that there may be an optimal dose that provides a RECQL4-deficient MCF7 cell-specific antitumor effect in vivo. Body weight changes were observed in all dose groups, similar to the control group, indicating extremely low side effects. This experiment was performed independently three times, confirming its reproducibility.

[0067] According to common understanding, an oral administration of 1 mg / kg body weight of suramin to mice is equivalent to 0.08 mg / kg body weight in humans (FASEB J, 2008, 22, 659-61). For a 60 kg human, the dose is approximately 4.86 mg. Compared to the 20 mg / kg body weight dose of suramin used to treat African sleeping sickness, administered intravenously, the oral dose is remarkably small. Furthermore, considering the very low absorption rate of suramin when administered orally, these results are quite surprising.

[0068] Example 4: Antitumor effect of suramin in the low-dose range The antitumor effect of low-dose suramin was confirmed using the same cell experimental system as in Example 2. Suramin doses were measured in the μM (Figure 7A), nM (Figure 7B), pM (Figure 7C), and fM (Figure 7D) regions. Suramin showed a dose-dependent antitumor effect in the μM region. However, it was revealed that suramin also exhibits a definite antitumor effect in the extremely low-dose region (nM to fM). Furthermore, the effect of suramin on RAD52 gene expression in the low-dose region was investigated using a standard quantitative RT-PCR method. It was shown that suramin reduced RAD52 expression at a dose of only 5 pM (Figure 8). RAD52 is involved in DNA repair, including the SSA pathway. Therefore, it was suggested that suramin inhibits DNA repair, including the SSA pathway, thereby preventing the proliferation of cancer cells. In contrast, suramin did not show any effect on the expression of factors in pathways other than the SSA pathway, such as RAD51.

[0069] Example 5: In vivo antitumor effect experiment in C57BL / 6 mice In the above example, immunodeficient nude mice were used, but in this example, the same experiment was performed using mice with a normal immune system.

[0070] Specifically, the procedure was as follows: FBS (Sigma, F9423-500ML) was immobilized by heating at 56°C for 30 minutes. Mouse colon cancer MC38 cells (Kerafast, ENH204-FP) were cultured in DMEM medium (Wako, 043-30085) containing 10% immobilized FBS and Penicillin-Streptomycin Solution (Wako, 168-23191) in a 5% CO2 incubator at 37°C. The cultured MC38 cells were detached with trypsin (Wako, 201-16945), washed, and then 5 × 10⁶ cells were removed. 5 Individual cells were suspended in 100 μL of PBS. This cell solution was transplanted into both flanks of 6-12 week old C57BL / 6 mice. The transplantation day was designated as day 0. Oral administration of RAD52 inhibitors was started on the third day after tumor transplantation. Oral administration was performed at the timings indicated by the arrowheads at the top of the time course shown in Figure 9A. Since tumors could not yet be confirmed at this point, mice were randomly assigned to each treatment group to avoid experimental bias. The RAD52 inhibitors EGC (TCI, E1084) and Suramin (Wako, 193-10611) were administered orally approximately every two days, three times a week, for three weeks (a total of nine doses). Poly I:C (GE Healthcare, 27473201) was administered intravenously at a concentration of 200 μg / mouse one week after tumor transplantation. This is the time when individuals with confirmed tumors begin to appear. Tumor size and body weight were measured twice a week. Measurements were performed by a measurement staff member who had no information about the administered drugs, using the same criteria each time. Mice with tumors of a certain size or those nearing death were euthanized by cervical dislocation, and metastatic lesions in the body were confirmed by autopsy. The control group was administered PBS without any drugs. The results are shown in Figures 9B and 9C. As shown in Figures 9B and 9C, tumor growth was suppressed in the RAD52 inhibitor (EGC or suramin) group compared to the control group. In other words, it was revealed that suramin does not require the presence of a RECQL4-reducing mutation in subjects with a normal immune system (especially the T-cell immune system).

[0071] Example 6: Changes in DNA repair pathways in oncogene overexpressing cells We created U2OS cells capable of expressing Cyclin E in a tetracycline-inducible manner. Using this cell line as the parent, we established a RECQL4-deficient U2OS cell line by knocking in the C-terminal domain-deficient RECLQ4 gene.

[0072] Furthermore, various DNA repair activities were quantified in this RECQL4-deficient U2OS cell line. Specifically, vectors capable of quantifying DNA repair activity by GFP reporter assays were introduced (DNA repair activity by homologous recombination (HR) was pDRGFP; Genes Dev, 1999, 13, 2633-8 / DNA repair activity by SSA was hprtSAGFP; Mol Cell Biol, 2004, 24, 9305-16 / DNA repair activity by c-NHEJ was pimEJ5GFP, DNA repair activity by Alt-EJ was EJ2GFP-puro; PLoS Genet. 2008, 4, :e1000110 / DNA repair activity by BIR was pBIR-GFP; Science, 2014, 343, 88-91), and reporter cells were established by drug selection with 2-3 μg / ml puromycin (see Figure 12). To briefly explain each of these non-limiting examples: In the BIR detection system, a portion of the N-terminal and C-terminal parts of GFP are positioned at different locations and in opposite directions. These N-terminal and C-terminal parts share a common sequence. A restriction enzyme cleavage site (e.g., I-SceI) is located at the very end of the N-terminal part. When the reporter system is cleaved by I-SceI, repair is induced, and functional GFP can be produced by the functional promoter. The efficiency of BIR can then be estimated from the correlation between the incidence of BIR and the luminescence intensity of GFP. In the HR detection system, a GFP (e.g., SceGFP) exists with a restriction enzyme cleavage site in between under the control of a functional promoter, and the correct GFP exists at a different location. When the restriction enzyme cleavage site is cleaved, recombination repair repairs SceGFP, making it translatable, and functional GFP can be produced by the functional promoter. The efficiency of HR can then be estimated from the correlation between the incidence of HR and the luminescence intensity of GFP. In the Alt-EJ detection system, a restriction enzyme cleavage site and a stop codon are inserted between the N-terminal tag and the entire length of GFP. Furthermore, the restriction enzyme cleavage site and the stop codon are flanked by two identical sequences of 8 nucleotides each. When Alt-EJ occurs, 35 nucleotides, including one of the 8 nucleotides, are deleted, allowing the tag and GFP to be linked in-frame, resulting in the expression of functional GFP.Furthermore, the efficiency of Alt-EJ can be estimated from the correlation between the incidence of Alt-EJ and the luminescence intensity of GFP. In the NHEJ detection system, a cassette exists between the promoter and GFP, flanked by two restriction enzyme cleavage sites (e.g., the I-SceI cleavage site). When two sites are cleaved by restriction enzymes, the two restriction enzyme cleavage sites are cleaved, forming complementary overhangs. When these overhangs are repaired by NHEJ, functional GFP is expressed. The efficiency of NHEJ can then be estimated from the correlation between the incidence of NHEJ and the luminescence intensity of GFP. In addition, in NHEJ, the restriction enzyme cleavage sites may be restored upon repair, or they may not be restored, resulting in restriction enzyme-resistant sites. NHEJ activity can be detected regardless of which of these occurs.

[0073] These reporter cells were transiently introduced with eGFP vectors (for measuring transfection efficiency) and pCBASceI vectors (for expressing the restriction enzyme ISceI) using Lipofectamine LTX Reagent with PLUS Reagent (Thermo Scientific, A12621). The DNA repair ability of double-strand breaks induced by ISceI expression was quantified 24–48 hours after transfection. A pCBA vector (empty vector as a negative control; no restriction enzyme ISceI expression) was used as a negative control.

[0074] To analyze the DNA repair capacity under Cyclin E overexpression, Cyclin E expression was induced by completely removing tetracycline 2 μg / ml from the culture medium. After culturing the cells for at least 4 days, the three vectors were transiently introduced, and the DNA repair capacity for double-strand breaks induced by ISceI expression was quantified at 24-48 hours post-transfection. The results are shown in Figure 10.

[0075] As shown in Figure 10, it was revealed that inducing Cyclin E expression enhances homologous recombination (HR), SSA, and BIR-mediated DNA repair activity.

[0076] Example 7: Combination therapy with a PARP inhibitor and a RAD52 inhibitor PARP inhibitors inhibit the DNA damage response (DDR) in cells or tumors with homologous recombination repair (HRR) deficiencies. Olaparib is a PARP inhibitor and an anticancer drug used to treat cancers with HRR deficiencies, such as BRCA1 and / or BRCA2 gene mutations. In this example, olaparib was administered as the PARP inhibitor, and EGC, D-103, or suramin (Sur) was administered as the RAD52 inhibitor.

[0077] MCF7 cells were cultured for 14 days in the presence of 0.5 μM olaparib and the indicated concentration of a RAD52 inhibitor (test group) or in its absence (control group). Both the test and control groups were then cultured until day 22 in the presence of 0.5 μM olaparib and the RAD52 inhibitor. Cell counts were measured on days 14 and 22 to evaluate the effect of the RAD52 inhibitor on cell proliferation. EGC, D-103, or suramin (Sur) were used as the RAD52 inhibitor. The results are shown in Figure 13. As shown in Figure 13, in WT cells, no increase in cell count was observed from day 14 to day 22 in the olaparib treatment (control group), whereas in RECQL4-deficient cells, the number of viable cells increased with olaparib treatment (control group). This result suggests that RECQL4-deficient cells acquired resistance to the PARP inhibitor upon PARP inhibitor treatment. In contrast, both the groups treated with a combination of PARP inhibitors and RAD52 inhibitors showed high antitumor efficacy. Furthermore, a comparison of cell counts on day 14 and day 22 showed that, for example, the suramin (Sur) administration group did not experience an increase in cell count, suggesting that the RAD52 inhibitor inhibited the acquisition of resistance to PARP inhibitors in RECQL4-deficient cells. Moreover, the RAD52 inhibitor showed sufficient efficacy at low concentrations (e.g., 1 nM). From this, it became clear that combining olaparib with a RAD52 inhibitor can neutralize the resistance acquired by olaparib treatment.

[0078] Example 8: Efficacy of RAD52 inhibitors on cancer cells with HRR deficiency The ovarian cancer cell line UWB1.289 is a BRCA1-deficient ovarian cancer cell line with HRR deficiency. Therefore, we treated UWB1.289 cells with a RAD52 inhibitor to investigate the effect of RAD52 inhibitors on BRCA1 deficiency. As a control, we used UWB1.289 cells that overexpressed functional BRCA1 (UWB1.289 cells + BRCA1). Suramin was used as the RAD52 inhibitor.

[0079] In the experimental setup described above, BRCA1-deficient cancer cells were cultured in the presence of various concentrations of RAD52 inhibitors, and the effects of RAD52 inhibitors on BRCA1-deficient cancer cells were investigated. The ratio of the number of BRCA1-deficient cancer cells in the presence of various concentrations of RAD52 inhibitors to the number of BRCA1-deficient cancer cells in the absence of RAD52 inhibitors was calculated. If the ratio is less than 1, the RAD52 inhibitor can be evaluated as having an antitumor effect. The results are shown in Figure 14. As shown in Figure 14, RAD52 inhibitors exhibited an antitumor effect on BRCA1-deficient cancer cells in the extremely low concentration range. Similar results were obtained when EGC or D-103 were used as the RAD52 inhibitor.

[0080] Example 9: Combination effect of platinum-based drugs or PARP inhibitors with RAD52 inhibitors on cancer cells with HRR deficiency UWB1.289 cells were seeded in 24-well plates at a rate of 1000 cells each. Subsequently, the cells were cultured in medium with the RAD52 inhibitors D-103, EGC, or suramin at the concentrations shown in the figure. After 2-3 weeks, the medium was removed, and the cells were subjected to methanol fixation followed by Giemsa staining. The number of stained colonies was then counted. For the platinum-based drug group, the number of colonies was counted as follows: UWB1.289 cells were seeded in 24-well plates at a rate of 6000 cells each. As described above, 0.05 μM cisplatin was added to the medium containing the RAD52 inhibitor at the concentrations shown in the figure, and the cells were cultured. After 2-3 weeks, the medium was removed, and the cells were subjected to methanol fixation followed by Giemsa staining. The number of stained colonies was then counted. Cisplatin was used as the platinum-based drug. The results are shown in Figure 15. As shown in Figure 15, the RAD52 inhibitor, when used in combination with cisplatin, showed a stronger antitumor effect against BRCA1-deficient cancer cells.

[0081] The combined effect of low concentrations (i.e., 0.1 μM or 0.05 μM) of platinum-based drugs and RAD52 inhibitors was confirmed using the experimental system described in Example 8. Cisplatin was used as the platinum-based drug, and suramin was used as the RAD52 inhibitor. After culturing, the number of viable cells was counted. Subsequently, the ratio of viable cells to the number of viable cells in the cisplatin-only treatment group was calculated. The results are shown in Figure 16. As shown in Figure 16, suramin exerted an antitumor effect even in the extremely low concentration range (sub-nM and sub-pM) in BRCA1-deficient cancer cells. Similar results were obtained when EGC or D-103 were used as the RAD52 inhibitor. Next, the same experiment was performed with increased concentrations of platinum-based drugs. 0.4 μM cisplatin was used as the platinum-based drug. As cells, UWB1.289 cells that forcibly expressed BRCA1 were used as a model of BRCA1 wild-type cells. The results are shown in Figure 17. As shown in Figure 17, platinum-based drugs and RAD52 inhibitors showed a synergistic effect when used in combination, even in cells with BRCA1 function. Similar results were obtained when EGC or D-103 were used as the RAD52 inhibitor. This result is thought to be because cells that underwent DNA damage induced by platinum-based drugs attempted to survive by activating the SSA pathway, while the RAD52 inhibitor inhibited the activation of the SSA pathway, thereby reducing the viability of cancer cells. This result is consistent with the fact that RECQL4-deficient cells have normal HR, but RAD52 inhibitors are effective against them. Furthermore, it suggests the possibility that RAD52 inhibitors may also be effective against cancer cells with normal HR function.

[0082] The combined efficacy of a PARP inhibitor and a RAD52 inhibitor was investigated for cancer cells with HRR deficiency. Olaparib was used as the PARP inhibitor at concentrations of 0.125 μM or 0.625 μM. Suramin was used as the RAD52 inhibitor. UWB1.289 cells were used as BRCA1-deficient cancer cells. BRCA1-overexpressing UWB1.289 cells were used as control cells. The results are shown in Figure 18. As shown in Figure 18, the RAD52 inhibitor showed a strong combined efficacy with the PARP inhibitor for BRCA1-deficient cancer cells. Similar results were obtained when EGC or D-103 were used as the RAD52 inhibitor. This suggests the efficacy of combination therapy with a PARP inhibitor and a RAD52 inhibitor for cancer cells with HRR deficiency. Furthermore, the efficacy of combination therapy with a PARP inhibitor and a RAD52 inhibitor was also demonstrated for cancer cells with HRR.

[0083] UWB1.289 cells, which overexpress BRCA1, were used as cancer cells possessing HRR. The combined effect of a PARP inhibitor and a RAD52 inhibitor was confirmed for these HRR-possessing cancer cells. Olaparib was used as the PARP inhibitor. The olaparib concentration was 0.625 μM or 2.5 μM. Suramin was used as the RAD52 inhibitor. As shown in Figure 19, the combined therapy of PARP inhibitor treatment and RAD52 inhibitor treatment was effective even for cancer cells possessing HRR. Similar results were obtained when EGC or D-103 were used as the RAD52 inhibitor.

[0084] Furthermore, the effect of long-term combination therapy on cancer cell survival was confirmed by long-term combination treatment of HRR-deficient cancer cells with a platinum-based drug and a RAD52 inhibitor. 0.05 μM cisplatin was used as the platinum-based drug. UWB1.289 cells were used as the HRR-deficient cancer cells. Specifically, 2000 to 6000 UWB1.289 cells were seeded in 24-well plates and cultured in the presence of a platinum-based drug or in the presence of a platinum-based drug and suramin (as shown in the figure). Cell counts were measured after 2 to 3 weeks. Subsequently, the ratio of viable cells to the number of viable cells in the cisplatin-only treatment group was calculated. The results are shown in Figure 20. As shown in Figure 20, it is known that long-term treatment with platinum-based drugs leads to the acquisition of resistance to the drug; however, combination therapy with a platinum-based drug and a RAD52 inhibitor did not show a reduction in antitumor effect due to resistance acquisition. This suggests that the RAD52 inhibitor suppresses the acquisition of resistance in cancer cells to platinum-based drug treatment.

[0085] Similarly, UWB1.289 cells, which are cancer cells with HRR, were cultured for 8, 27, 42, or 56 days in the presence of platinum-based drugs and RAD52 inhibitors, and cell proliferation was confirmed. Specifically, cells were cultured for 8 days in or without the RAD52 inhibitor at the concentrations shown (Control group), and the number of viable cells (day 8) was measured by detaching the cells with trypsin. As a control (Ctrl), cells were cultured in the presence of platinum-based drugs but without the RAD52 inhibitor. Cisplatin at 20 ng / mL was used as the platinum-based drug. Using the number of viable cells as a baseline, the same number of UWB1.289 cells were seeded into the wells of a 24-well plate, and the cells were cultured for 19 days in or without the RAD52 inhibitor at the concentrations shown (Control group). The number of viable cells (day 27) was measured by detaching the cells with trypsin. Similarly, the cells were cultured up to day 56 while repeating the passage, and the number of cells was measured. The vertical axis shows the percentage of viable cells. The results are shown in Figures 21 and 22. In the control group, the viability of cancer cells significantly improved after culturing for 42 days or more. This result indicates that cancer cells have acquired resistance to platinum-based drugs. In contrast, it was shown that this resistance was neutralized by combination therapy with platinum-based drugs and RAD52 inhibitors (1nM to 1fM) (Figures 21 and 22).

Claims

1. A pharmaceutical composition comprising suramin for use in treating cancer, wherein the cancer comprises cancer cells with enhanced RAD52-mediated DNA repair pathways.

2. The pharmaceutical composition according to claim 1, wherein the cancer cells with enhanced RAD52-mediated DNA repair pathway are cancers having a RECQL4-decreasing mutation.

3. A pharmaceutical composition according to claim 1 or 2, wherein the cancer is a cancer comprising cancer cells having a homologous recombination repair (HRR) deficiency.

4. A pharmaceutical composition according to any one of claims 1 to 3, which is an orally administered preparation.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer cells are cells having DNA damage to their genomic DNA.

6. A pharmaceutical composition according to any one of claims 1 to 5, to be used in combination with radiotherapy or chemotherapy with a platinum-based agent.

7. To prepare a composition comprising a compound selected from the group consisting of suramin, or its analogues, related compounds, varieties, and derivatives, Contacting cancer cells with enhanced RAD52-mediated DNA repair pathways with the compound, Select a composition containing a compound that suppresses the proliferation of cancer cells with enhanced RAD52-mediated DNA repair pathways. A method for selecting a composition that includes the following:

8. A pharmaceutical composition containing suramin for use in treating cancer, wherein the cancer is a cancer containing cancer cells with enhanced RAD52-mediated DNA repair pathway, formulated as an orally administered pharmaceutical composition.

9. A pharmaceutical composition for use in treating cancer by inhibiting the acquisition of resistance to platinum-based and / or PARP inhibitor treatment, comprising a RAD52 inhibitor, wherein the cancer comprises cancer cells with enhanced RAD52-mediated DNA repair pathways, and the RAD52 inhibitor is suramin.

10. A pharmaceutical composition for use in treating cancer in patients who have undergone platinum-based and / or PARP inhibitor treatment, the pharmaceutical composition for use in combination with said treatment, comprising a RAD52 inhibitor, wherein the cancer comprises cancer cells with enhanced RAD52-mediated DNA repair pathways, and the RAD52 inhibitor is suramin.

11. A pharmaceutical composition according to any one of claims 1 to 6 and 8 to 10, which is used in combination with a PARP inhibitor.

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