Pharmaceutical composition for preventing or treating cancer-related diseases comprising antisense oligonucleotide and anticancer agent as active ingredients

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer-related diseases, comprising an antisense oligonucleotide and an anticancer agent as active ingredients. By confirming that an antisense oligonucleotide having a specific base sequence enhances the anticancer effect of a chemotherapeutic agent, the present invention can be effectively utilized as a pharmaceutical composition for preventing or treating cancer-related diseases, or as an anticancer adjuvant for enhancing the anticancer effect of an anticancer agent.

Description

Pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide and an anticancer agent as active ingredients

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide and an anticancer agent as active ingredients.

[0002] Cancer is one of humanity's incurable diseases, and massive investments are being made worldwide to develop treatments for it. In Korea, it is the leading cause of death, with over 100,000 new cases diagnosed annually and over 60,000 deaths. Carcinogens, including smoking, ultraviolet rays, chemicals, food, and other environmental factors, are known to cause cancer. However, the diverse causes make the development of treatments challenging, and the effectiveness of these treatments varies depending on the site of origin. Despite recent rapid advances in medicine and the development of various cancer treatments, including surgery, radiation therapy, and chemotherapy, many cancer patients still struggle with the complete removal of cancer cells and the prevention of metastasis.

[0003] Currently, three main treatment methods are used to treat cancer: radiation therapy, surgery, and chemotherapy, and cancer is treated through one or a combination of these. Among them, chemotherapy refers to treatment using an appropriate anticancer drug after diagnosing the patient's condition. It is broadly categorized into chemotherapy (cytotoxic anticancer drugs), targeted anticancer drugs, and immunotherapy (immune checkpoint inhibitors). Chemotherapy drugs are substances that exhibit anticancer effects by blocking cell division of cancer cells themselves, thereby preventing cell proliferation and then inducing apoptosis. Examples include cisplatin, oxaliplatin, and doxorubicin. Targeted anticancer drugs include erlotinib, gefitinib, and trastuzumab, which exhibit anticancer effects by selectively attacking specific molecular biological targets that play a key role in the development or growth of cancer. Immunotherapy drugs do not cause the death of cancer cells themselves, but bind to PD-1 expressed on cancer cells, block the interaction between PD-1 / PD-L1, and induce T cell activation through this, thereby attacking cancer cells through T cells and exhibiting an anticancer effect. Examples include pembrolizumab and nivolumab.

[0004] Although the anticancer effects of many anticancer drugs have been reported to date, many problems with the prescription of anticancer drugs have also been reported, such as the reduced effectiveness of anticancer drugs in anticancer drug-resistant cancer cells and the occurrence of anticancer drug side effects. Therefore, active research is being conducted to develop anticancer adjuvants that enhance the anticancer effects of anticancer drugs or reduce the side effects of anticancer drugs.

[0005] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide and an anticancer agent as active ingredients.

[0006] Another object of the present invention is to provide an anticancer adjuvant for enhancing the anticancer effect of an anticancer agent containing an antisense oligonucleotide as an active ingredient.

[0007] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide having a base sequence represented by SEQ ID NO: 1; and an anticancer agent as an active ingredient.

[0008] In addition, the present invention provides an anticancer adjuvant for enhancing the anticancer effect of an anticancer agent, which comprises an antisense oligonucleotide having a base sequence represented by sequence number 1 as an active ingredient.

[0009] According to the present invention, by confirming that an antisense oligonucleotide having a specific base sequence enhances the anticancer effect of a chemotherapeutic agent, the anticancer agent can be usefully utilized as a pharmaceutical composition for preventing or treating cancer; or as an anticancer adjuvant for enhancing the anticancer effect of an anticancer agent.

[0010] Figure 1 shows the results of analyzing the anticancer effect of an antisense oligonucleotide (hereinafter referred to as a sample) disclosed in Experimental Example 1-1 below and the effect of using it in combination with a chemotherapy agent.

[0011] Figure 2 shows the results of analyzing the effect of the sample on the expression of col1a1 (Collagen Type I Alpha 1 Chain) and vimentin.

[0012] Figure 3 shows the results of analyzing the combined effect of samples and standard chemotherapy (gemcitabine + paclitaxel).

[0013] Hereinafter, the present invention will be described in more detail.

[0014]

[0015] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide having a base sequence represented by SEQ ID NO: 1; and an anticancer agent as an active ingredient.

[0016] The sequence of the above antisense oligonucleotide is as follows.

[0017] [Sequence number 1] (DNA sequence, 5' → 3')

[0018] GGCGG CATGT CTATT TTGTA

[0019] The above antisense oligonucleotide may be a 2'-O-methoxyethyl (MOE) modified nucleotide.

[0020] The above antisense oligonucleotide may enhance the anticancer effect of an anticancer agent.

[0021] Additionally, the antisense oligonucleotide may inhibit the expression of col1a1 (Collagen Type I Alpha 1 Chain) or vimentin.

[0022] The above anticancer agent may be a chemotherapy agent, and the chemotherapy agent may be at least one selected from the group consisting of oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, albumin-bound paclitaxel, vincristine, etoposide, and doxorubicin, preferably gemcitabine; or gemcitabine and paclitaxel, and may also be a formulation containing paclitaxel, for example, a solubilizing agent, particularly a micelle, containing paclitaxel, but is not limited thereto.

[0023] The above paclitaxel is a drug used to treat ovarian, breast, lung, pancreatic, and other cancers, and was isolated from the bark of the Pacific yew tree (Taxus brevifolia) and commercially developed by Bristol-Myers Squibb under the trademark Taxol. The paclitaxel may be administered in any form, including a formulation comprising paclitaxel, whether in the form of Abraxane®, an albumin-bound paclitaxel in nanoparticle form, or nab paclitaxel.

[0024] The above cancer disease may be one or more selected from the group consisting of melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hawkins' disease, esophageal cancer, small intestine cancer, colon cancer, colon cancer, rectal cancer, anal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0025] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0026] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0027] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0028] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0029] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0030] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0031] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0032] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition. A person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0033]

[0034] In addition, the present invention provides an anticancer adjuvant for enhancing the anticancer effect of an anticancer agent, which comprises an antisense oligonucleotide having a base sequence represented by sequence number 1 as an active ingredient.

[0035] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0036]

[0037] [Experimental Example 1] Experimental Preparation

[0038] 1-1. Sample

[0039] The antisense oligonucleotide (hereinafter referred to as ASO) used as a sample of the present invention is an oligonucleotide in the form of a '5+4 Gapmer' having the sequence of 5'-GGCGG CATGT CTATT TTGTA-3', and the Gapmer is an oligonucleotide substituted in the form of 2'-O-methoxyethyl (MOE). The oligonucleotide was manufactured through an organic chemical synthesis method known in the technical field to which the present invention pertains.

[0040]

[0041] 1-2. Cells and Organoids

[0042] Examples of the present invention (Examples 1 to 3) were performed through a test request to Organoid Science Co., Ltd. (Seongnam, Gyeonggi-do, Republic of Korea), a testing agency. The CAF (Cancer-associated fibroblasts) and hPCO (Human Pancreatic Cancer Organoids) used in Examples 1 and 2 below were cells for evaluation and organoids constructed and stored by Organoid Science Co., Ltd., the testing agency.

[0043] In addition, the pancreatic cancer cell line AsPC-1 cells used in Example 3 of the present invention were stored at Organoid Science Co., Ltd. (Seongnam, Gyeonggi-do, Republic of Korea). The cells were cultured in RPMI-1640 medium containing 10% FBS (Fetal Bovine Serum) and 1% AA (antibiotic-antimycotic solution).

[0044]

[0045] [Example 1] Analysis of anticancer effects and combined effects with chemotherapy drugs

[0046] To determine the anticancer effect of the sample and the effect in combination with chemotherapy, 1 × 10 cancer-associated fibroblasts (CAF) were seeded per well in a 48-well plate. 5 cells / well and hPCO (Human Pancreatic Cancer Organoid) 3.3×10 4 Cells were seeded by mixing them (3:1 ratio) per well. At this time, 20 μl per well was dispensed in a resuspended culture solution mixed with Matrigel: Organoid media = 6:4. After 2-3 days, it was confirmed that CAFs were mixed with the organoids and fused to form a dark border around the edge of the dome. After 48 hours of stabilization after hPCO and CAF co-culture, CIPCOs (CAF-integrated pancreatic cancer organoids) formed in the Matrigel dome were treated with test substances. The experimental groups were set up as follows.

[0047] 1) Normal group: Untreated with test substance

[0048] 2) Negative control group: ASO 3μM treatment

[0049] 3) Sample treatment group: 3 μM sample treatment

[0050] 4) Gemcitabine treatment group: 40 nM gemcitabine treatment

[0051] 5) Sample + gemcitabine treatment group: 3 μM sample + 40 nM gemcitabine treatment

[0052] ASO (Antisense Oligonucleotide) was used as a negative ASO with a random sequence. Each experimental group was treated with the test substance and cultured for 72 hours under 5% CO2 and 37°C conditions. The viability of CIPCO was measured using the CellTiterGlo 3D kit (Promega, Cat. No.: G9681) according to the product manual. Statistical significance between experimental groups was verified using Student's T-test, and *p<0.05 was considered significant.

[0053] As a result, as shown in Fig. 1, the survival rate of CIPCO decreased in the negative control group, sample treatment group, and gemcitabine treatment group compared to the normal group, but no statistically significant difference was observed. On the other hand, the survival rate of CIPCO significantly decreased in the sample + gemcitabine treatment group compared to the normal group. From the above results, it was confirmed that a significant anticancer effect (anticancer synergy effect) occurred when the sample and gemcitabine were combined.

[0054]

[0055] [Example 2] Analysis of fibrosis biomarker gene expression

[0056] col1a1 (Collagen Type I Alpha 1 Chain) and vimentin are fibrosis biomarker genes and are known as defense mechanisms of cancer cells. Therefore, qRT-PCR analysis was performed to confirm the effect of the sample on the expression of col1a1 and vimentin. Five experimental groups were set up in the same manner as in Example 1, but the qRT-PCR analysis was performed using organoids separate from the experiment performed in Example 1. The supernatant was removed from the CIPCO present in the Matrigel dome, only the pellet was collected, washed to remove the Matrigel, and RNA was obtained using the RNeasy Plus Mini Kit (QIAGEN, cas. 74136). RNA was synthesized into cDNA using Primescript RT master mix (TAKARA, cas. RR036A-1). When making the qRT-PCR mixture, the experiment was performed by quantifying it so that 100 ng of cDNA was included per sample. qRT-PCR analysis in organoids was performed using equipment and programs provided by the CFX Connect Real-time system (BIO-RAD). Gene expression was assessed by confirming the expression of the col1a1 and vimentin genes and correcting for GAPDH expression. Statistical significance between experimental groups was verified using the Student's T-test, and a p<0.05 value was considered significant. The primers for the genes used in this analysis are listed in Table 1.

[0057] geneforward sequencereverse sequencecol1a1CCC CGA GGC TCT GAA GGTGCA ATA CCA GGA GCA CCA TTGvimentinCCT GAA CCT GAG GGA AAC TAATCGT TGA TAA CCT GTC CAT CTC TGAPDHGAG AAG GCT GGG GCT CAT TTAGT GAT GGC ATG GAC TGT GG

[0058] As a result, as shown in Fig. 2, there was no significant change in the expression of col1a1 and vimentin in the sample treatment group compared to the normal group (vehicle-untreated group), (vimentin expression increased, but the difference was not statistically significant). In contrast, the expression of col1a1 and vimentin increased in the gemcitabine treatment group compared to the normal group (vimentin showed a statistically significant difference). On the other hand, the expression of col1a1 and vimentin significantly decreased in the sample and gemcitabine treatment groups compared to the gemcitabine treatment group. From the above results, it was confirmed that when an anticancer drug (chemotherapy drug, gemcitabine) and the sample were combined, the sample enhanced the anticancer effect of the anticancer drug by suppressing the defense mechanism of cancer cells activated by the anticancer drug.

[0059]

[0060] [Example 3] Analysis of the effect of combination therapy with standard chemotherapy.

[0061] There are many different types of cancer treatment prescriptions, and among them, one of the chemotherapy prescriptions for pancreatic cancer is the combination of gemcitabine and paclitaxel. Therefore, in order to confirm the combined effect of the sample and the standard chemotherapy (gemcitabine + paclitaxel), the cultured AsPC-1 cells (Experimental Example 1-2) were first harvested through 0.25% trypsin solution and culture medium washing, centrifuged at 1,500 rpm for 3 minutes, and then resuspended in RPMI-1640 medium containing 5% FBS and 1% AA. After that, the harvested cells were counted in the culture medium, and 5 × 10 per well were seeded in a 96-well plate. 3 / 100 μL of the medium was dispensed. Approximately 24 hours after dispensing, cell attachment was confirmed, and the culture medium in each well was removed by suction. Then, 100 μL of RPMI-1640 medium containing 5% FBS, 1% AA, and the test substance was added. The experimental groups were set up as follows.

[0062] 1) Normal group (Con): Untreated with test substance

[0063] 2) Sample treatment group (ASO): 30 μM sample treatment

[0064] 3) Chemotherapy group (Gem+PTX): Gemcitabine 200nM + paclitaxel 25nM treatment

[0065] 4) Sample + chemotherapy treatment group (Gem+PTX+ASO): Sample 30 μM + Gemcitabine 200 nM + Paclitaxel 25 nM treatment

[0066] After treatment with the test substance, the cells were cultured for 48 hours under 5% CO2 and 37°C conditions, and then cell viability was measured using the D-plus CCK assay kit (Dong-In LS, Cat. no: CCK-1000) according to the product manual. Statistical significance between the experimental groups was verified using the Student's T-test, and *p<0.05 was considered significant.

[0067] As a result, as shown in Fig. 3, there was no significant difference in cell viability between the normal group and the sample treatment group, and the viability was significantly reduced in the chemotherapy treatment group and the sample + chemotherapy treatment group compared to the normal group, and the viability was significantly lower in the sample + chemotherapy treatment group than in the chemotherapy treatment group. From the above results, it was confirmed that the sample enhances the anticancer effect of the chemotherapy agent.

[0068]

[0069] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising an antisense oligonucleotide having a base sequence represented by sequence number 1; and an anticancer agent as an active ingredient.

2. A pharmaceutical composition according to claim 1, characterized in that the antisense oligonucleotide is a 2'-O-methoxyethyl (MOE) modified nucleotide.

3. A pharmaceutical composition according to claim 1, wherein the antisense oligonucleotide enhances the anticancer effect of an anticancer agent.

4. A pharmaceutical composition according to claim 1, wherein the antisense oligonucleotide inhibits the expression of col1a1 (Collagen Type I Alpha 1 Chain) or vimentin.

5. A pharmaceutical composition according to claim 1, characterized in that the anticancer agent is a chemotherapy anticancer agent.

6. A pharmaceutical composition according to claim 5, wherein the chemotherapeutic agent is at least one selected from the group consisting of oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, albumin-bound paclitaxel, vincristine, etoposide, and doxorubicin.

7. A pharmaceutical composition according to claim 6, wherein the chemotherapeutic anticancer agent is gemcitabine; or gemcitabine and paclitaxel.

8. A pharmaceutical composition according to claim 5, wherein the chemotherapeutic agent is a formulation containing albumin-bound paclitaxel or paclitaxel.

9. A pharmaceutical composition according to claim 1, wherein the cancer disease is at least one selected from the group consisting of melanoma, skin cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, bone cancer, head or neck cancer, uterine cancer, ovarian cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hawkins' disease, esophageal cancer, small intestine cancer, colon cancer, colon cancer, rectal cancer, anal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

10. An anticancer adjuvant for enhancing the anticancer effect of an anticancer agent, comprising as an active ingredient an antisense oligonucleotide having a base sequence represented by sequence number 1.

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