KR20230021987A - Composition for treating, alleviating or preventing brain cancer comprising siRNA targeting NK1R - Google Patents

Abstract

The present invention relates to a composition for treating brain cancer that exhibits chemical synthetic lethality, and the like. The combined administration of any one selected from a group consisting of hydrazinobenzoylcurcumin (HBC), berbamine, and siCaMKIIγ and siRNA (siNK1R) targeting NK1R has a synergistic synthetic lethality against cancer stem-like cells compared to individual components and has shown to significantly inhibit the formation of neurospheres, thereby being usefully used for the treatment, prevention, or improvement of brain cancer.

Description

Composition for treating, alleviating or preventing brain cancer comprising siRNA targeting NK1R {Composition for treating, alleviating or preventing brain cancer comprising siRNA targeting NK1R}

The present invention relates to a composition for treating brain cancer exhibiting chemical synthetic lethality, and the like.

Cancer is one of the most common causes of death worldwide. Approximately 10 million new cases occur each year, accounting for approximately 12% of all deaths, making it the third leading cause of death. Among them, brain cancer occurs regardless of age and is largely classified into primary brain tumor and metastatic brain tumor. Symptoms of brain cancer include local symptoms such as motor paralysis, sensory paralysis, speech difficulties, visual disturbances, and balance disorders, and symptoms of intracranial hypertension.

Despite advances in the field of cancer treatment, the current leading treatments are surgery, radiation and chemotherapy. Chemotherapy approaches are used primarily to treat metastatic or particularly aggressive cancers. Most of the cancer chemotherapy drugs currently used clinically are cytotoxins. Cytotoxic agents work by injuring or killing rapidly growing cells.

On the other hand, cancer stem cells (CSC or Tumor Initiating Cells: TICs) refer to cancer cells (found in tumors or blood cancers) that have the ability to generate tumors. Cancer stem cells have the same characteristics as normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. That is, cancer stem cells are tumorigenic, unlike cancer cells that do not form tumors. Cancer stem cells generate tumors through self-renewal and differentiation capabilities, which are characteristics of stem cells, in various cell types.

In addition, it is differentiated from other groups in the tumor and causes recurrence and metastasis by generating new tumors. Therefore, the development of a specific treatment method targeting cancer stem cells can be a hope for increasing the survival rate and improving the quality of life of cancer patients, especially in patients with metastatic disease.

Accordingly, the inventor of the present invention completed the present invention as a result of studying a treatment target differentiated from existing targets for selectively controlling the self-renewal, proliferative, and differentiation-inducing abilities of cancer stem cells.

Republic of Korea Patent Registration No. 10-1892078

An object of the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a pharmaceutically acceptable salt thereof, berbamine or a pharmaceutically acceptable salt thereof, and siCaMKIIγ; And to provide a pharmaceutical composition for preventing or treating brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

Another object of the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; And to provide a food composition for preventing or treating brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

In order to achieve the object of the present invention, any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a pharmaceutically acceptable salt thereof, berbamine or a pharmaceutically acceptable salt thereof, and siCaMKIIγ; And it provides a pharmaceutical composition for preventing or treating brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

In addition, the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; And it provides a food composition for preventing or improving brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

In one embodiment of the present invention, the molar concentration ratio of the HBC and siNK1R may be 20 to 80:1.

In one embodiment of the present invention, the molar concentration ratio of berbamine and siNK1R may be 40 to 80:1.

In one embodiment of the present invention, the molar concentration ratio of siCaMKIIγ and siNK1R may be 3 to 5:1.

In one embodiment of the present invention, the composition can kill brain cancer stem cells.

In one embodiment of the present invention, the composition can inhibit the formation of neurospheres in brain cancer stem cells.

any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; and NK1R-targeting siRNA (siNK1R) have synergistic synergistic lethal interactions on cancer stem-like cells compared to the individual components, and show results that significantly inhibit neurosphere formation, thus treating and preventing brain cancer. Or it can be usefully used for improvement purposes.

1 shows the degree of apoptosis of U87MG cancer stem-like cells when HBC and siNK1R are co-administered.
Figure 2 is a graph showing the results of Figure 1.
3 is a graph showing the inhibitory effect of U87MG cancer stem-like cells on neurosphere formation upon co-administration of HBC and siNK1R.
Figure 4 shows the degree of apoptosis of U87MG cancer stem-like cells when berbamin and siNK1R were co-administered.
Figure 5 is a graph showing the results of Figure 4.
6 is a graph showing the inhibitory effect of neurosphere formation in U87MG cancer stem-like cells when berbamin and siNK1R are co-administered.
7 shows the degree of apoptosis of U87MG cancer stem-like cells when co-administered with siCaMKIIγ and siNK1R.
8 is a graph showing the results of FIG. 4 above.
9 is a graph showing the inhibitory effect of U87MG cancer stem-like cells on neurosphere formation when co-administered with siCaMKIIγ and siNK1R.

The inventor of the present invention discovers a treatment target differentiated from existing targets for selectively controlling the self-renewal ability, proliferation ability, and differentiation induction ability of cancer stem cells, and various types of “customized anticancer agents for cancer stem cells”. With the goal of “suggesting a new strategy for development”, “screening for anti-cancer drugs that exhibit chemical synthetic lethality specific to cancer stem cells” without affecting non-cancer stem cells will lead to new cancer lines. A biomolecule that interacts with Ca ²⁺ /CaM-dependent protein kinase II (CaMKII) and synthetic lethality was newly discovered using a chemical synthetic lethal approach, and differentiated from existing treatment methods. As a result of conducting a study on “CaMKII-targeted therapy for more effective removal of cells (glioblastoma stem cells)”, the present invention was completed.

Accordingly, the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a pharmaceutically acceptable salt thereof, berbamine or a pharmaceutically acceptable salt thereof, and siCaMKIIγ; And it provides a pharmaceutical composition for preventing or treating brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

In the present invention, the HBC is a Ca2+/CaM antagonist and has a structure represented by Formula 1 below.

[Formula 1]

In the present invention, berbamin is a kind of alkaloid, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, and has the structure of Formula 2 below,

[Formula 2]

In the present invention, the siCaMKIIγ is a siRNA that silences the gene expression of Ca2+/calmodulin-dependent protein kinase II gamma, CaMKIIγ.

In the present invention, the siNK1R is a siRNA that silences the gene expression of NK1R, a neurokinin-1 receptor.

In the present invention, "brain cancer" refers to any type of abnormally increased proliferation of nerve cells of any type, also referred to as primary brain cancer, or any other cancer that metastasizes to the central nervous system (CNS), also referred to as brain metastases. . That is, in the present invention, brain cancer collectively refers to primary brain cancer generated in brain tissue and the meninges surrounding the brain and secondary brain cancer metastasized from cancer generated in the skull or other parts of the body.

In the present invention, "pharmaceutically acceptable salt" means a salt formed with any inorganic or organic acid or base that does not cause serious irritation to the organism to which it is administered and does not impair the biological activity and physical properties of the compound. As the salt, a salt commonly used in the art, such as an acid addition salt formed by a pharmaceutically acceptable free acid, may be used.

As used herein, the term "prevention" refers to any activity that inhibits or delays the onset of brain cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms caused by brain cancer by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a pharmaceutically acceptable salt thereof, berbamine or a pharmaceutically acceptable salt thereof, and siCaMKIIγ; and siRNA (siNK1R) targeting NK1R as an active ingredient, and may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulation and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. It is not, and if necessary, other conventional additives such as antioxidants and buffers may be further included.

In addition, diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection or oral intake.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously or subcutaneously) depending on the desired method, and the dosage is the condition and weight of the patient, the severity of the disease, the form of the drug, Depending on the route and time of administration, it can be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, and activity of the drug in the patient. , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, and the like.

In the present invention, the molar concentration ratio of the HBC and siNK1R may be 20 to 80:1. Specifically, the molar concentration ratio of HBC and siNK1R may be 20:1, 40:1 or 80:1.

In one embodiment of the present invention, the molar concentration ratio of berbamine and siNK1R may be 40 to 80:1. Specifically, the molar concentration ratio of berbamine and siNK1R may be 40:1 or 80:1.

In one embodiment of the present invention, the molar concentration ratio of siCaMKIIγ and siNK1R may be 3 to 5:1. Specifically, the molar concentration ratio of siCaMKIIγ and siNK1R may be 4:1.

In the present invention, the composition can kill brain cancer stem cells.

In the present invention, the composition can inhibit the formation of neurospheres in brain cancer stem cells.

In another aspect, the present invention is any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; And it provides a food composition for preventing or improving brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.

The food composition is a concept including health functional food.

As used herein, the term "improvement" refers to all actions that at least reduce the parameters related to the condition to be treated, for example, the severity of symptoms.

In the food composition of the present invention, any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; And siRNA targeting NK1R (siNK1R) can be added to food as it is or used together with other foods or food ingredients, and can be appropriately used according to conventional methods.

The mixed amount of any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or its salts, berbamine or its salts, and siCaMKIIγ and siRNA (siNK1R) targeting NK1R is suitable according to its purpose of use (for prevention or improvement) can be determined

In general, when preparing food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.

The health functional food composition of the present invention is not particularly limited in other components other than containing the above components as essential components in the indicated proportions, and may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. there is.

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The ratio of the natural carbohydrates can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The ratio of these additives can also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1: Materials and Preparation>

1.1. experimental material

Hydrazinobenzoylcurcumin (HBC) used in the experiment was purchased from US Biological (Salem, MA, USA), berbamine dihydrochloride (berbamine) was purchased from Sigma-Aldrich (St. Louis, MA, USA), and siCaMKIIγ was used for silencing the CaMKIIγ gene. After informing Bioneer (Daejeon) of possible double-stranded RNA oligo sequence information, synthesis was requested.

The sense and antisense sequences of CaMKIIγ siRNA are 5'-GUA GAG UGC UUA CGC AAA U-3' and 5'-A UUU GCG UAA GCA CUC UAC-3', respectively. siNK1R is a pool of three siRNAs capable of silencing the NK1R gene (sc-36069) and was purchased from Santa Cruz Biotechnology.

All drugs were dissolved at a concentration of 100 mM using DMSO, stored at -20 ° C, and used in experiments after thawing at room temperature during experiments. CellTiter-Glo® luminescent cell viability assay kit was purchased from Promega (Madison, WI, USA).

1.2. Cancer stem-like cell culture

The human glioblastoma cell line U87MG was purchased from Korea Cell Line Bank. U87MG-derived cancer stem-like cells were supplemented with 1× B-27 serum-free supplement (Gibco), 5μg/mL heparin (Sigma-Aldrich), 2mM L-glutamine (Gibco), and 20ng/mL epidermal growth factor (EGF; Prospecbio, East Brunswick, NJ, USA), 20ng/mL basic fibroblast growth factor (bFGF; Prospecbio, East Brunswick, NJ, USA), Dulbecco modified Eagle medium/nutrient mixture F-12 (DMEM) with 1% penicillin/streptomycin (Gibco) /F12; HyClone, Marlborough, MA, USA) culture medium was used for spheroid culture. The cells were cultured at 37°C in a 5% CO2 cell incubator where appropriate humidity was maintained.

1.3. Cell viability assay (synthetic lethality-luminescence assay)

Human glioblastoma U87MG cancer stem-like cells transfected with siRNA (siCaMKIIγ or siNK1R) with Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) reagent were 96-white at a concentration of 3×10 ³ cells per well. After seeding in well culture plates, compounds of various concentrations were treated alone or in combination and cultured for 7 days.

To measure cell viability, 20 μL of CellTiter-Glo® luminescent cell viability assay kit was added to each well, and the culture plate was shaken for 2 minutes and reacted for 8 minutes at room temperature in a dark place. Luminescence was measured using a multimode microplate reader (Biotek).

1.4. Analysis of neurosphere formation

Human glioblastoma U87MG cancer stem-like cells transfected with siRNA (siCaMKIIγ or siNK1R) with Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) reagent were prepared in 96-white wells at a concentration of 3×10 ³ cells per well. After dispensing to the culture plate, the compounds at various concentrations were treated alone or in combination. After culturing for 7 days, the size and number of formed neurospheres were observed and counted under a 200x light microscope (Olympus).

<Example 2: Confirmation of cancer stem cell death and suppression of neurosphere formation by co-administration of HBC and siNK1R>

siNK1R is known as siRNA that silences the gene expression of NK1R, a neurokinin-1 receptor. siNK1R was treated alone at a concentration of 500 nM or in combination with HBC at a concentration of 0-40 μM and measured by luminescence analysis after 7 days. was significantly suppressed, showing a strong synthetic lethal interaction (see Figs. 1 and 2).

In addition, 500 nM siNK1R and 20 μM HBC strongly suppressed the survival and formation of neurospheres of U87MG cancer stem-like cells when compared to the single treatment (see FIG. 3).

<Example 3: Confirmation of inhibition of cancer stem cell death and neurosphere formation by co-administration of berbamin and siNK1R>

siNK1R is known as siRNA that silences the gene expression of NK1R, a neurokinin-1 receptor. siNK1R was treated alone at a concentration of 500 nM or in combination with berbamine at a concentration of 0-40 μM and measured by luminescence analysis after 7 days. Significantly inhibited cell survival, showing a strong synthetic lethal interaction (see Figs. 4 and 5).

In addition, 500 nM siNK1R and 20 μM berbamin strongly suppressed the survival and formation of neurospheres of U87MG cancer stem-like cells when compared to the single treatment (refer to FIG. 6).

<Example 4: Confirmation of inhibition of cancer stem cell death and neurosphere formation by co-administration of siCaMKIIγ and siNK1R>

siNK1R is known as siRNA that silences the gene expression of NK1R, a neurokinin-1 receptor. siNK1R was treated alone at a concentration of 500nM or in combination with siCaMKIIγ at a concentration of 2μM and measured by luminescence analysis after 7 days. , showed strong synthetic lethal interactions (see Figs. 7 and 8).

In addition, 500 nM siNK1R and 2 μM siCaMKIIγ strongly suppressed the survival and formation of neurospheres of U87MG cancer stem-like cells when compared to the single treatment (see FIG. 9).

Claims (12)

Any one selected from the group consisting of hydrazinobenzoylcurcumin (HBC) or a pharmaceutically acceptable salt thereof, berbamine or a pharmaceutically acceptable salt thereof, and siCaMKIIγ; and
A pharmaceutical composition for preventing or treating brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the molar concentration ratio of HBC and siNK1R is 20 to 80:1.
The pharmaceutical composition according to claim 1, wherein the molar concentration ratio of berbamine and siNK1R is 40 to 80:1.
The pharmaceutical composition according to claim 1, wherein the molar concentration ratio of siCaMKIIγ and siNK1R is 3 to 5:1.
The pharmaceutical composition according to claim 1, wherein the composition kills brain cancer stem cells.
The pharmaceutical composition according to claim 1, wherein the composition inhibits formation of neurospheres in brain cancer stem cells.
any one selected from the group consisting of HBC (hydrazinobenzoylcurcumin) or a salt thereof, berbamine or a salt thereof, and siCaMKIIγ; and
A food composition for preventing or improving brain cancer comprising siRNA (siNK1R) targeting NK1R as an active ingredient.
The method of claim 7, wherein the molar concentration ratio of the HBC and siNK1R is 20 to 80: 1
A food composition, characterized in that.
The food composition according to claim 7, wherein the molar concentration ratio of berbamine and siNK1R is 40 to 80:1.
The food composition according to claim 7, wherein the molar concentration ratio of siCaMKIIγ and siNK1R is 3 to 5:1.
The food composition according to claim 7, wherein the composition kills brain cancer stem cells.
The food composition according to claim 7, wherein the composition inhibits formation of neurospheres in brain cancer stem cells.

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