WO2023172032A1 - Composition for preventing or treating cancer comprising nmur2 inhibitor as active ingredient - Google Patents

Abstract

The present application relates to a pharmaceutical composition for preventing or treating cancer comprising an NMUR2 expression inhibitor or an NMUR2 activity inhibitor as an active ingredient. A pharmaceutical composition of the present invention comprising an NMUR2 inhibitor according to the present invention as an active ingredient is advantageous in that the composition can be used as an alternative anticancer agent in cases where unresponsiveness or resistance to conventional anticancer agents is exhibited.

Description

Composition for preventing or treating cancer containing an NMUR2 inhibitor as an active ingredient

[Cross-citation with related applications]

This application claims the benefit of priority based on Korean Patent Application No. 2022-0029570 filed on March 8, 2022, and all contents disclosed in the document are included as part of this specification.

[Technology field]

The present invention relates to a composition for preventing or treating cancer.

Cancer has a high mortality rate worldwide and is the most common cause of death in Western societies after cardiovascular disease. In particular, colon cancer, breast cancer, and prostate cancer are continuously increasing due to the aging of the population, the generalization of high-fat diet due to westernization of diet, rapid increase in environmental pollutants, and increase in alcohol consumption. In this situation, there is an urgent need for the creation of anticancer substances that can prevent and treat cancer and contribute to the improvement of human health, healthy quality of life, and human health.

Among them, brain and central nervous system cancers are ranked 3rd in men and 2nd in women, and 3rd among men and women among the crude incidence rates for the 0 to 14-year-old group, according to national cancer registration statistics. Brain tumors occur in approximately 3,000 people per year in Korea. Benign brain tumors can be cured through curative surgery, but malignant brain tumors are treated with a combination of surgery, radiation therapy, and chemotherapy. Commonly occurring brain tumors include glioma, meningoencephalopathy, pituitary adenoma, metastatic brain tumor, and acoustic schwannoma. Among these, glioma (glioblastoma) is known to be the most common malignant tumor, grade 4 in the WHO brain tumor classification, and has an incidence rate of 1 It is said to be about 3 to 4 per 100,000 people per year.

Treatment methods for brain tumors include surgery, radiation therapy, and chemotherapy. Generally, chemotherapy is used after removing the cancer through surgery and radiation therapy. The first-line chemotherapy treatment for brain tumors includes Temozolomide, which alkylates or methylates intracellular DNA, and the second-line drug is Avastin-irinotecan. Irinotecan).

However, due to drug refractoriness and resistance to temozolomide, which is used to treat brain tumors, the cure rate of patients is low and recurrence and metastasis occur frequently, resulting in poor patient prognosis. As a result, the 5-year survival rate of malignant brain tumors is low. As many patients die early in the tumor treatment process (about 20-30%), there is a need to discover new targets to treat this.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

Another object of the present invention is to provide a method for screening agents for treating cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer containing an NMUR2 inhibitor as an active ingredient.

The NMUR2 inhibitor may be an NMUR2 expression inhibitor or an NMUR2 activity inhibitor.

The NMUR2 inhibitor may be a compound having the following formula (I) or a pharmaceutically acceptable salt thereof.

<Formula I>

The cancer may be one in which NMUR2 is overexpressed.

Additionally, the cancer may be a brain tumor.

In addition, in order to achieve the above object, the present invention includes the steps of treating cells overexpressing NMUR2 with an anticancer drug candidate; and measuring the expression or activity level of NMUR2 in cells treated with the candidate substance.

NMUR2, which is targeted by the pharmaceutical composition of the present invention, is a novel target that has not been previously known as a target for the prevention or treatment of cancer. The pharmaceutical composition of the present invention, which contains an NMUR2 expression or activity inhibitor as an active ingredient, is a conventional anticancer agent. It has the advantage of being able to be used as an alternative anticancer agent in cases of refractoriness or resistance.

In particular, NMUR2 activity inhibitors have a significantly lower IC ₅₀ value compared to temozolomide, a conventional anticancer drug, and can be used alone as an effective ingredient for the prevention or treatment of cancer. In addition, the pharmaceutical composition of the present invention, when used in combination with conventional anticancer drugs, exhibits a synergistic effect compared to when each is used alone, so it has the advantage of being able to be used as an anticancer adjuvant. In addition, NMUR2 expression inhibitors can inhibit cancer cell proliferation and cell migration, promote cancer cell death, and inhibit metastasis, so they can be used as effective anticancer agents.

Figures 1A, 1B, and 1C show the structure of the pcDNA3.1 plasmid vector expressed by the CMV promoter and inserted with the base sequence encoding NMUR2 and the base sequence encoding IRES-GFP, the bright field and fluorescence of GFP expression, and the expression of the vector in the HTLA cell line. This is a graph showing the intensity of luciferase when introduced into and treated with NMU-25, the ligand of NMUR2.

Figure 2a is a graph showing the intensity of luciferase when the HTLA cell line was treated with NMU-25 and the compound of Formula I.

Figure 2b is a graph showing the intensity of luciferase when the HTLA cell line was treated with NMU-25 and the compound of Formula I at various concentrations.

Figures 3A and 3B are graphs measuring the IC ₅₀ of temozolomide and the compound of Formula I against brain tumor cell lines U-87MG, T-98G, and U-373MG, and graphs showing the activity of caspase 3/7 according to concentration. .

Figures 4A and 4B are graphs confirming the OD450, caspase 3/7 activity, and CI values when temozolomide and the compound of Formula I are co-treated.

Figure 5 is a graph confirming IC ₅₀ when gastric cancer, colon cancer, and skin cancer cell lines SNU-638, HCT116, and SK-MEL-2 were treated with the compound of Formula I.

Figure 6 is a diagram analyzing the correlation between genetic mutations and NMUR2 expression in glioblastoma and glioma samples.

Figure 7 is a diagram confirming that, unlike the glioblastoma cell line into which the pcDNA empty vector was introduced, the glioblastoma cell line into which the pcDNA3.1 NMUR2-overexpression vector was introduced expresses NMUR2 at a high level.

Figure 8 is a diagram confirming that the proliferation rate of glioblastoma cells in the NMUR2 overexpressing cell line is increased compared to the control group.

Figure 9 is a diagram confirming that the migration of glioblastoma cells in the NMUR2 overexpressing cell line is increased compared to the control group.

Figure 10 is a diagram confirming that the expression of NMUR2 was significantly reduced in glioblastoma cell lines treated with siRNA against NMUR2.

Figure 11 is a diagram confirming that the proliferation rate of cells in glioblastoma cell lines treated with siRNA against NMUR2 was reduced compared to the control group.

Figure 12 is a diagram confirming the decrease in cell migration in glioblastoma cell lines treated with siRNA against NMUR2 compared to the control group.

Figure 13 is a diagram confirming that the compound of Formula 1, a compound that inhibits NMUR2, inhibited cell migration of a glioblastoma cell line.

Hereinafter, the present invention will be described in detail.

1. Pharmaceutical composition for the treatment or prevention of cancer

One aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer.

The pharmaceutical composition for treating or preventing cancer of the present invention contains an inhibitor of NMUR2 (Neuromedin U Receptor 2; NCBI GenBank ID: 56923) as an active ingredient.

The “NMUR2 (Neuromedin U Receptor 2)” is used as the name of the gene or the name of the protein encoded by the gene. The NMUR2 protein, encoded by the NMUR2 gene, is a type of G-protein coupled receptor (GPCR) and is responsible for the neuropeptide hormones NMU (neuromedin U) and NMS (neuromedin S). It plays a role in activating various cellular reactions such as cell growth, proliferation, and death by combining with.

The NMUR2 inhibitor may be an NMUR2 expression inhibitor or an NMUR2 activity inhibitor.

In the present invention, the term "NMUR2 expression inhibitor or NMUR2 activity inhibitor" is used to collectively refer to all agents that reduce the expression or activity of NMUR2, and specifically, NMUR2 at the transcription, mRNA, or translation level. It may include any agent that inhibits the activity of NMUR2 by suppressing its expression by reducing the expression level of the gene or by interfering with the activity of the NMUR2 protein.

The “NMUR2 expression inhibitor or NMUR2 activity inhibitor” can be used without limitation in its form, such as a compound, nucleic acid, peptide, virus, or vector containing the nucleic acid that can inhibit the expression or activity of NMUR2 by targeting NMUR2. For example, the NMUR2 expression inhibitor may be a miRNA, siRNA or shRNA, or an antisense oligonucleotide that binds complementary to the mRNA of the NMUR2 gene. In particular, it may be siRNA, and the NMUR2 activity inhibitor is specific for the NMUR2 protein. It may be an aptamer, antibody, or low-molecular-weight compound that binds to.

In the present invention, the terms "miRNA", "siRNA", and "shRNA" are nucleic acid molecules that can mediate RNA interference or gene silencing, and can inhibit the expression of target genes, thereby producing efficient gene knockdown. down) method or gene therapy method. For the purpose of the present invention, miRNA, siRNA, and shRNA can inhibit NMUR2 by acting specifically on NMUR2 to cleave the NMUR2 mRNA molecule and induce RNA interference (RNAi). miRNAs, siRNAs and shRNAs can be synthesized chemically or enzymatically. In particular, the siRNA may include the nucleotide sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6, or the nucleotide sequences represented by SEQ ID NO: 7 and SEQ ID NO: 8.

In the present invention, the term “aptamer” refers to a single-stranded oligonucleotide, about 20 to 60 nucleotides in size, and a nucleic acid molecule that has binding activity to a given target molecule. It has a variety of three-dimensional structures depending on its sequence, and can have high affinity for specific substances, like an antigen-antibody reaction. An aptamer can inhibit the activity of a certain target molecule by binding to it. The aptamer of the present invention may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may also be in a linear or circular form. Preferably, the aptamer may bind to NMUR2 and serve to inhibit the activity of NMUR2. Such an aptamer can be prepared from the sequence of NMUR2 by a method known to those skilled in the art.

As used herein, the term “antibody” refers to a polypeptide that includes at least one immunoglobulin variable region sequence and specifically binds to a given antigen. For example, an antibody may comprise a heavy (H) chain variable region and a light (L) chain variable region.

In the present invention, the term "low molecular weight compound" is a natural or synthetic compound with a molecular weight of 1000 Da or less, such as 800 Da or less, especially 500 Da or less, and may bind to the NMUR2 protein and interfere with the activity of NMUR2. In particular, the low molecular weight compound may be a compound having the formula of <Formula I> below or a pharmaceutically acceptable salt thereof.

<Formula I>

The meaning of “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond a level acceptable to the subject of application (prescription). The term “salt” refers to inorganic and organic acid addition salts of the compound. The “pharmaceutically acceptable salt” may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound, and may be an inorganic acid salt, an organic acid salt, or a metal salt. The inorganic acid salt may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salts include formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl salt, trichloroacetic acid, and trifluoroacetate. , benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspart. It may be an acid salt. The metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

In the present invention, the term "cancer" refers to colorectal cancer, including colon cancer and rectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, liver cancer, Esophageal cancer, small intestine cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, and neck cancer. , cutaneous melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, or central nervous system (CNS) cancer, such as colon cancer, stomach cancer, and lung cancer. , it may be pancreatic cancer, liver cancer, small intestine cancer, skin cancer, or central nervous system cancer, especially central nervous system cancer.

The central nervous system cancer may be a brain tumor. The “brain tumor” refers to any tumor that occurs within the skull and includes all tumors that occur in the brain and structures surrounding the brain. In particular, the brain tumor may be glioma, meningoencephalopathy, pituitary adenoma, metastatic brain tumor, acoustic schwannoma, glioblastoma, etc., but is not limited thereto.

Meanwhile, the cancer may be one in which NMUR2 is overexpressed. Since the pharmaceutical composition of the present invention contains an NMUR2 inhibitor as an active ingredient, it can exhibit a particularly excellent prevention or treatment effect against cancer in which NMUR2 is overexpressed.

As used herein, the term “treatment” refers to clinical intervention to alter the natural processes of the individual or cell being treated, which may be performed during the course of the clinical pathology or to prevent it. The desired therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, and alleviating the disease state. or temporarily alleviating, remission, or improving prognosis. The present invention includes all actions to improve the course of cancer by administering a composition containing a substance that inhibits NMUR2. In addition, “prevention” refers to all actions that inhibit or delay the onset of cancer by administering a composition containing a substance that inhibits NMUR2 according to the present invention.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration may include tablets, powders, granules, capsules, etc., and such solid preparations may contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. It can be prepared by mixing (lactose), gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

In addition, the pharmaceutical composition of the present invention is not limited thereto, but includes tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, and freezing. It may have any one formulation selected from the group consisting of dry formulations.

The pharmaceutical composition of the present invention may be administered in a daily dose of about 0.0001 mg/kg to about 10 g/kg, and may be administered in a daily dosage of about 0.001 mg/kg to about 1 g/kg. However, the dosage may vary depending on the degree of purification of the mixture, the patient's condition (age, gender, weight, etc.), and the severity of the condition being treated. For convenience, the total daily dose may be divided and administered several times during the day, if necessary.

In a specific example of the present invention, it was confirmed that the compound of formula (I) is an NMUR2 inhibitor that inhibits the activity of NMUR2 (see Figure 2), and it was confirmed that the conventional brain tumor cell lines U-87MG, T-98G, and U-373MG were used as brain tumor cell lines. It was confirmed that it exhibited a much better therapeutic effect than the therapeutic agent temozolomide (see Figures 3A and 4A).

In another embodiment of the present invention, in order to confirm the effect of the compound of Formula I on other cancer types, the compound of Formula I was administered alone to SNU-638, HCT116, and SK-MEL-2, which are gastric cancer, colon cancer, and skin cancer cell lines, respectively. and was confirmed to have an IC ₅₀ similar to that of brain tumor cell lines.

2. Kit for preventing or treating cancer

Another aspect of the present invention provides a kit for preventing or treating cancer.

The kit for preventing or treating cancer of the present invention may include the pharmaceutical composition and an existing anticancer agent. The kit for preventing or treating cancer of the present invention may additionally include a carrier, diluent, excipient, or a combination of two or more commonly used in pharmaceutical compositions. In addition, the kit may further include a notice in a form established by a government agency for regulating the manufacture, use, or sale of pharmaceuticals or biological products.

Since the pharmaceutical composition is the same as described in ' 1. Pharmaceutical composition for treating or preventing cancer ', further description is omitted.

The “existing anticancer agent” refers to a chemotherapy treatment agent that has been previously used to inhibit the proliferation of cancer cells. The existing anticancer agent as described above may be an anticancer agent targeting a target other than NMUR2, for example, temozolomide or Avastin-irinotecan, and especially temozolomide.

Since the pharmaceutical composition contains an NMUR2 inhibitor as an active ingredient, it can further increase the anticancer effect by acting on a different target than the existing anticancer agent.

The pharmaceutical composition and the existing anticancer agent may be administered simultaneously or sequentially. When the pharmaceutical composition and the existing anticancer drug are administered sequentially, the pharmaceutical composition may be administered first and then the existing anticancer drug may be administered later, or the existing anticancer drug may be administered first and then the pharmaceutical composition. It may also be administered later. Therefore, the kit may further include an instruction manual containing instructions on the above administration method.

The cancer is the same as described in ‘ 1. Pharmaceutical composition for treating or preventing cancer ’. In particular, the cancer may overexpress NMUR2, and may further be refractory or resistant to the existing anticancer drugs.

In a specific example of the present invention, it was confirmed that when temozolomide and the compound of formula (I) were treated in combination, the growth of brain tumor cells was significantly inhibited and cell death was increased compared to when each drug was treated alone. . In addition, in order to confirm the effect of the combination treatment of the temozolomide and the compound of Formula I, the two drugs were treated in combination and the CI (Combination Index) was checked, and it was found that there was a synergism during the combination treatment. Confirmed.

3. Anticancer drug screening method

Another aspect of the present invention provides a method for screening anticancer drugs.

The anticancer drug screening method of the present invention includes treating cells overexpressing NMUR2 with an anticancer drug candidate; And measuring the expression or activity level of NMUR2 in cells treated with the candidate substance.

The “anticancer drug candidate” refers to a substance expected to exhibit an effect of inhibiting the proliferation of cancer cells.

The anticancer drug candidate may be treated at a concentration of 1 μM to 40 μM. For example, the anticancer drug candidate may be treated at a concentration of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, or 35 μM.

The term “expression level measurement” may refer to the process of confirming the presence and expression level of mRNA of a gene, or the presence and expression level of protein expressed from the gene. The presence or expression level of mRNA of the gene can be confirmed by measuring the amount of mRNA of the gene. Analysis methods for this include RT-PCR and competitive RT-PCR using primer pairs, probes, or anti-sense nucleotides for the mRNA of NMUR2. PCR), real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, etc., but are not limited to these. Additionally, the presence or expression level of the protein expressed from the gene can be confirmed by confirming the amount of the protein of the gene. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, and Ouktero using an antibody that specifically binds to the NMUR2 protein. Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. are limited to these. It doesn't work. In addition, the “activity level measurement” is a process of confirming whether a protein expressed from a gene is active, and is accomplished by confirming whether and to what extent the protein is functional.

In addition, the anticancer drug screening method may further include determining the candidate substance as an anticancer drug when the measured expression or activity level of NMUR2 is significantly lower than that of cells not treated with the candidate substance.

The "significantly low case" means that the expression or activity level of NMUR2 is lower by 10% or more compared to cells not treated with the candidate substance, such as 20% or more, 30% or more, 40% or more, 50% or more. It may be above or below 60%, and especially may be below 70%. The screening method can be performed in vivo or in vitro, and is not particularly limited. Candidate substances may be known substances or new substances, and large-scale screening can be performed through, for example, plant extracts or chemical libraries. Through this, it is possible to discover agents that can suppress cancer, especially brain tumors, by inhibiting the expression or activity of NMUR2.

Below, preferred preparation examples and experimental examples are presented to aid understanding of the present invention. However, the following manufacturing examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the manufacturing examples and experimental examples.

[Example 1]

Confirmation of NMUR2 inhibitory activity of compounds of formula I

To confirm the NMUR2 inhibitory activity of the compound of Formula I, a cell line into which an NMUR2 expression vector was introduced was constructed. Specifically, an NMUR2-IRES-GFP expression vector was constructed by inserting a base sequence encoding NMUR2 and a base sequence encoding IRES-GFP into the pcDNA3.1 plasmid vector expressed by the CMV promoter, and expressing the NMUR2-IRES-GFP. HTLA_NMUR2-IRES-GFP cells were created by transducing the vector into the PRESTO-TANGO HTLA cell line, which expresses luciferase in a manner dependent on beta-arrestin signaling activity.

By selecting a single clone expressing the GFP fluorescent protein using flow cytometry (FACS), a cell line into which the expression vector was successfully transduced was secured. The obtained cell line was treated with NMU-25, a NMUR2 ligand, and 6 hours later, the activity of luciferase was observed to increase, confirming that the activity of NMUR2 was increased, and a drug screening cell line selective for NMUR2 was created.

On a 96-well white plate, the cell lines into which the expression vector was successfully transduced were cultured at a density of 20,000 cells/well in a 5% CO ₂ incubator at 37°C for 24 hours. Afterwards, 10 μl of DMSO (100 μM) and the compound of Formula I (100 μM) diluted in DPBS (Dulbecco's Phosphate Buffered Saline) were treated so that the final concentration was 10 μM, or the compound of Formula I was treated at 0.1 and 1, respectively. After diluting to 10, 50, 100, and 200μM, 10ul was applied per well so that the final concentrations were 0, 0.01, 1, 5, 10, and 20μM, respectively. 15 minutes after treatment with DMSO and the compound of Formula I, 10 μl of NMU-25, a ligand for NMUR2, was treated to a final concentration of 20 nM.

Afterwards, the cells were cultured in a 37°C, 5% CO ₂ incubator for 6 hours, then luciferase activity solution (One-glo, Promega) was added at a density of 50 μl/well, reacted at room temperature for 5 minutes, and then luciferase Activity was measured. Compared to the group treated with DMSO alone, the luciferase activity significantly increased in the group treated with DMSO and NMU-25, a ligand of NMUR2, while the group treated simultaneously with NMU-25, a ligand of NMUR2, and the compound of Formula I showed luciferase activity. No change in rase activity was observed, confirming that the compound of Formula I inhibits NMUR2 (see Figure 2a). When the experiment was repeated three times under the above conditions and methods, the degree of inhibition of NMUR2 by the compound of Formula I in the experimental group repeatedly showed a high inhibition effect of more than 70%.

Inhibition effect by NNC 05-2090 (% inhibition) Primary 78.06 Secondary 80.79 3rd 74.26

In addition, when the compound of Formula I was treated at different concentrations, it was confirmed that luciferase activity decreased depending on the concentration (see Figure 2b). The above results show that the compound of Formula I can effectively inhibit NMUR2. It shows.

[Example 2]

IC of compounds of formula I in brain tumor cell lines ₅₀ check

The IC ₅₀ of the compound of Formula I and temozolomide was confirmed in brain tumor cell lines U-87MG, T-98G, and U-373MG (Korea Cell Line Bank).

In a 96-well culture plate, U-87MG, U-373MG were cultured at a density of 8,000/well, and T-98G was 5,000/well in a 37°C, 5% CO ₂ incubator for 24 hours, and the compound of Formula I was cultured in a 96-well culture plate. It was diluted in DPBS so that the final concentrations were 0, 1, 2, 5, 10, and 20 μM. Temozolomide was dissolved directly in the medium at concentrations of 0, 106, 213, 425, 850, and 1700 μM and then treated by changing the medium. 48 hours after drug treatment, cell viability was measured using the CCK8 kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions, and the IC ₅₀ value was derived based on this.

At this time, the IC ₅₀ of temozolomide was confirmed to be 1000 μM or more, showing a minimal inhibitory effect on brain tumor cells even at high concentrations, while the IC ₅₀ of the compound of Formula I was confirmed to be 6 to 7 μM. In other words, the compound of Formula I can inhibit brain tumor cell growth even when used alone at a lower concentration than temozolomide, which is currently used as a brain tumor treatment (see Figure 3A).

Temozolomide IC ₅₀ (μM) Compound IC of Formula I ₅₀ (μM) U-87MG 14621 6.412 T-98G 1666 6.160 U-373MG 2630 7.994

U-87MG and U-373MG were placed in a 96-well white plate at a density of 8,000 cells/well each, and T-98G was placed at a density of 5,000 cells/well, and then cultured in an incubator at 37°C and 5% CO ₂ for 24 hours, and the chemical formula Compound I was treated at concentrations of 0, 5, and 10 μM, respectively. After 48 hours, the cells were treated with 30 μl of Caspase 3/7-Glo (Promega) solution, waited for 30 minutes at room temperature and in the dark, and changes in cell death rate were measured. Significant induction of cell death was observed when treated with 10 μM compound of formula I in all three brain tumor cell lines (see Figure 3B).

[Example 3]

Confirmation of synergistic effect by combined treatment of temozolomide and compound of formula I in brain tumor cell lines

Cell growth and cell death were observed in T-98G, a brain tumor cell line, when temozolomide and the compound of formula (I) were treated alone or in combination.

Compared to when the brain tumor cell line T98G was cultured in a 96-well culture plate and a 96-well white plate at a density of 3000 cells/well for 24 hours and then treated alone with temozolomide at a final concentration of 1mM, the final concentration of the compound of Formula I was When treated alone at a concentration of 10 μM, the cell growth rate and cell death rate increased. In addition, higher cell growth and cell death rates were observed when treated in combination with temozolomide at the same dose as above, compared to when the compound of Formula I was treated alone (see Figure 4A).

Through this, it can be expected that the treatment effect of brain tumor patients will be increased by the combined treatment of temozolomide and the compound of formula (I).

[Example 4]

Confirmation of combination index by combined treatment of temozolomide and compound of formula I in brain tumor cell lines

To check the combination index (CI), which indicates the degree of drug-drug interaction, the brain tumor cell line T-98G was cultured in a 96-well plate at 3000 cells/well for 24 hours, and then temozolomide was added at a concentration of 0. to 1000 mM and simultaneously treated with the compound of formula I at a concentration of 1 to 20 μM. The degree to which they influence each other when diluted to 1/2 of the highest concentration was calculated as the value of the combination index. A combination index value of less than 1.0 indicates a synergistic effect, and when temozolomide and the compound of Formula I were treated in combination, CI = 0.686 was calculated, confirming the existence of a synergistic effect (see Figure 4B).

[Example 5]

IC of compounds of formula I in gastric, colon and skin cancer cell lines ₅₀ check

SNU-638, HCT116, and SK-MEL-2, which are cell lines for stomach cancer, colon cancer, and skin cancer, were cultured in a 96-well plate at a density of 3000 cells/well for 24 hours in a 5% CO2 incubator at 37°C, and then HCT116 and SK-MEL-2 was treated with a compound of formula I (final concentration 0~20 μM) diluted to 1/2 of the highest concentration, and for SNU-638, a compound of formula I (final concentration 0~100 μM) was treated. It was diluted to 1/2 of the highest concentration and treated with 10 μl each. After culturing for 48 hours after drug treatment, cell growth rate was measured using the CCK8 kit according to the manufacturer's instructions.

As shown in Table 3 below, IC ₅₀ values were similar to those in brain tumor cell lines (see Figure 5).

cell line IC ₅₀ (μM) SNU-638 9.657 HCT116 8.326 SK-MEL-2 5.156

These values indicate that the compound of Formula I has an effect of inhibiting cell growth in cancer types other than brain tumors.

[Example 6]

Correlation analysis between NUMR2 expression and genetic mutations in glioma and glioblastoma patients

Genetic mutations and NMUR2 were identified in glioblastoma and glioma samples using The Cancer Genome Atlas (TCGA), which catalogs cancer-causing genetic mutations using genome sequencing and bioinformatics. The correlation between expressions was analyzed.

As a result, as shown in Figure 6, there are 236 samples in cases where the cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP) occurs at a high level, whereas there are 236 samples in cases where the cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP) occurs at a low level. was found to be 17, and it was confirmed that NMUR2 expression was significantly increased in cases where G-CIMP occurred at high levels compared to cases where G-CIMP occurred at low levels.

The above results show that in the case of glioblastoma or glioma with a high level of G-CIMP, the expression of NUMR2 is also high.

[Example 7]

Construction of NMUR2 overexpressing cell lines

In order to create a cell line overexpressing NMUR2, a cell line into which an NMUR2 expression vector was introduced was created.

Specifically, the nucleotide sequence encoding NMUR2 was inserted into the pcDNA3.1 plasmid vector to construct a pcDNA3.1 NMUR2-overexpression vector, and the pcDNA empty vector and pcDNA3.1 NMUR2-overexpression vector were added to the glioblastoma cell line T-98G, respectively. was transduced using Lipofectamine 2000 (Thermo Scientific), and after RNA extraction and cDNA synthesis 72 hours later, overexpression of NMUR2 was confirmed by qPCR. The nucleotide sequences of the primers used in qPCR are shown in Table 4 below.

designation Sequence (5'→3') sequence number NMUR2 forward AGA TGT GGC GCA ACT ACC C One NMUR2 reverse CGA AGC ACA CGG TCT CAA AGA 2 GAPDH forward CTC TGC TCC TCCTGT TCG AC 3 GAPDH reverse TTA AAA GCA GCC CTG GTG AC 4

As a result, as shown in Figure 7, unlike the glioblastoma cell line into which the pcDNA empty vector was introduced, the glioblastoma cell line into which the pcDNA3.1 NMUR2-overexpression vector was introduced was confirmed to express NMUR2 at a high level.

[Example 8]

Cell proliferation analysis of NMUR2 overexpressing cell lines

Cell proliferation was analyzed in the glioblastoma cell line introduced with the pcDNA empty vector prepared in Example 7 and the glioblastoma cell line introduced with the pcDNA3.1 NMUR2-overexpression vector.

Specifically, the glioblastoma cell line into which the pcDNA empty vector was introduced and the glioblastoma cell line into which the pcDNA3.1 NMUR2-overexpression vector was introduced were distributed in a 96-well plate at a density of 3000 cells/well and incubated at 37°C with 5% CO _{2 .} After 24, 48, 72, and 96 hours of culture, cell proliferation was analyzed using the CCK8 kit (Dojindo Molecular Technologies, Inc.).

As a result, as shown in Figure 8, it was confirmed that the proliferation rate of glioblastoma cells in the NMUR2 overexpressing cell line increased compared to the control group.

The above results show that cancer cells can proliferate at a rapid rate in glioblastoma where NMUR2 is overexpressed.

[Example 9]

Cell migration analysis of NMUR2 overexpressing cell lines

Cell migration was analyzed in the glioblastoma cell line introduced with the pcDNA empty vector prepared in Example 7 and the glioblastoma cell line introduced with the pcDNA3.1 NMUR2-overexpression vector.

Specifically, the glioblastoma cell line introduced with the pcDNA empty vector and the glioblastoma cell line introduced with the pcDNA3.1 NMUR2-overexpression vector were placed in the upper chamber of an 8μm 24-well 'insert' (FALCON) at ^5x10 cells each in 500μl of serum-free medium. It was put in with. The lower chamber was filled with 500 μl of medium containing 10% FBS. Afterwards, the transwell was cultured at 37°C and 5% CO ₂ for 18 hours, fixed with formaldehyde diluted to 4% using distilled water and 100% methanol, and then diluted to 0.1% using 20X PBS (LPS solution). Stained with crystal violet (SIGMA).

As a result, as shown in Figure 9, it was confirmed that the migration of glioblastoma cells in the NMUR2 overexpressing cell line increased compared to the control group.

The above results show that the migration of cancer cells in glioblastoma overexpressing NMUR2 is increased and can metastasize to surrounding tissues.

[Example 10]

Confirmation of NMUR2 expression changes in glioblastoma introduced with siRNA against NMUR2

To confirm changes in NMUR2 expression in glioblastoma into which siRNA against NMUR2 was introduced, siNMUR2-1 and siNMUR2-2 were transduced into T-98G, a glioblastoma cell line, respectively, using lipofectamineRNAiMAX (Thermo Scientific) (Table 5 below). ), 72 hours later, qPCR was performed using the method described in Example 7 above to measure the expression level of NMUR2.

designation Sequence (5'→3') sequence number siNMUR2-1 sense AGA AUG CUU CCU GGA UCU A 5 siNMUR2-1 anti-sense UAG AUG CAG GAA GCA UUC U 6 siNMUR2-2 sense GCU GAC CGA AGA UAU AGG 7 siNMUR2-2 anti-sense ACC UAU AUC UUC GGU CAG C 8

As a result, as shown in Figure 10, it was confirmed that the expression of NMUR2 was significantly decreased in glioblastoma cell lines treated with siRNA against NMUR2.

[Example 11]

Analysis of cell proliferation of glioblastoma introduced with siRNA against NMUR2

Cell proliferation was analyzed in the cell lines transduced with siNMUR2-1 and siNMUR2-2, respectively, into T-98G, a glioblastoma cell line prepared in Example 10.

Specifically, the cell lines transduced with siNMUR2-1 and siNMUR2-2, respectively, into T-98G, a glioblastoma cell line, were distributed at a density of 3000 cells/well in a 96-well plate, and incubated at 37°C with 5% CO _{2 .} After 24, 48, 72, and 96 hours of culture, cell proliferation was analyzed using the CCK8 kit (Dojindo Molecular Technologies, Inc.).

As a result, as shown in Figure 11, it was confirmed that the proliferation rate of cells in glioblastoma cell lines treated with siRNA against NMUR2 was decreased compared to the control group.

The above results show that siRNA against NMUR2 inhibits the growth of glioblastoma and can be used as a preventive or therapeutic agent for glioblastoma.

[Example 12]

Cell migration analysis of glioblastoma introduced with siRNA against NMUR2

Cell migration was analyzed in T-98G, a glioblastoma cell line prepared in Example 10, in which siNMUR2-1 and siNMUR2-2 were transduced, respectively.

Specifically, the cell lines transduced with siNMUR2-1 and siNMUR2-2, respectively, into T-98G, a glioblastoma cell line, were placed in the upper chamber of an 8μm 24-well 'insert' (FALCON), ^5x104 each, along with 500μl of serum-free medium. I put it in. The lower chamber was filled with 500 μl of medium containing 10% FBS. Afterwards, the transwell was cultured at 37°C and 5% CO ₂ for 18 hours, fixed with formaldehyde diluted to 4% using distilled water and 100% methanol, and then diluted to 0.1% using 20X PBS (LPS solution). Stained with crystal violet (SIGMA).

As a result, as shown in Figure 12, it was confirmed that cell migration was decreased in glioblastoma cell lines treated with siRNA against NMUR2 compared to the control group.

The above results show that siRNA against NMUR2 can inhibit the migration of glioblastoma and metastasis of cancer, and can be used as a preventive or therapeutic agent for glioblastoma.

[Example 13]

Analysis of the effect of compounds of formula 1 on cell migration of glioblastoma

The effect of the compound of Formula 1, a compound that inhibits NMUR2, on cell migration of glioblastoma cell lines was analyzed.

Specifically, glioblastoma cell lines U-87MG, T-98G, and U-373MG were placed in an upper chamber at 1x10 ^cells each with 500 ㎕ of serum-free medium, and the concentration of the compound of Formula 1 in the medium was 0 (DMSO). , the compound of Formula 1 was treated to concentrations of 5, 10, and 20 μM. The lower chamber was filled with 500 μl of medium containing 10% FBS. Afterwards, the transwell was cultured at 37°C and 5% CO ₂ for 20 hours, fixed with formaldehyde diluted to 4% using distilled water and 100% methanol, and then diluted to 0.1% using 20X PBS (LPS solution). Stained with crystal violet (SIGMA).

As a result, as shown in Figure 13, it was confirmed that the number of cells migrating between 10 μM and 20 μM, which is above the IC ₅₀ value concentration, decreased rapidly.

The above results show that the compound of Formula 1, which is an inhibitor for NMUR2, can inhibit the migration of glioblastoma and inhibit cancer metastasis, and can be used as a preventive or therapeutic agent for glioblastoma.

Claims (13)

A pharmaceutical composition for preventing or treating cancer comprising an NMUR2 expression inhibitor or an NMUR2 activity inhibitor as an active ingredient. In claim 1, The NMUR2 expression inhibitor is selected from the group consisting of miRNA, siRNA, and shRNA, and the NMUR2 activity inhibitor is selected from the group consisting of antibodies, aptamers, and small molecule compounds. A pharmaceutical composition for preventing or treating cancer. In claim 1, The NMUR2 expression inhibitor is a pharmaceutical composition for preventing or treating cancer, comprising the base sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6. In claim 1, The NMUR2 expression inhibitor is a pharmaceutical composition for preventing or treating cancer, comprising the base sequences represented by SEQ ID NO: 7 and SEQ ID NO: 8. In claim 1, A pharmaceutical composition for preventing or treating cancer, wherein the NMUR2 activity inhibitor includes a compound having the following formula or a pharmaceutically acceptable salt thereof. <Formula I>

The method according to any one of claims 1 to 5, The cancer is selected from the group consisting of brain tumor, central nervous system cancer, glioma, glioblastoma, meningitis, pituitary adenoma, metastatic brain tumor, acoustic neuroma, stomach cancer, colon cancer, and skin cancer, and pharmaceuticals for the prevention or treatment of cancer. Composition. The method according to any one of claims 1 to 5, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is selected from the group consisting of glioma, glioblastoma, meningitis, pituitary adenoma, metastatic brain tumor, and acoustic neuroma. The method according to any one of claims 1 to 5, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is selected from the group consisting of stomach cancer, colon cancer, and skin cancer. The method according to any one of claims 1 to 5, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is one in which NMUR2 (Neuromedin U Receptor 2) is overexpressed. The pharmaceutical composition of any one of claims 1 to 5; and anticancer drugs; A pharmaceutical kit for preventing or treating cancer, comprising: In claim 10, The kit, wherein the anticancer agent is temozolomide. Treating cells overexpressing NMUR2 with an anticancer drug candidate; and Measuring the expression or activity level of NMUR2 in cells treated with the candidate substance; Anticancer drug screening method comprising. In claim 12, If the measured expression or activity level of NMUR2 is lower than that of cells not treated with the candidate substance, determining the candidate substance as an anticancer drug.

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