KR102522963B1 - Composition for Antiobesity Comprising Capmatinib - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating obesity, including a compound of capmatinib (Cabmatinib). Since the present invention has the effect of decomposing fat and inhibiting fat synthesis, the present invention can be widely used for preventing or treating obesity.

Description

Anti-obesity composition containing capmatinib {Composition for Antiobesity Comprising Capmatinib}

The present invention relates to an anti-obesity composition comprising capmatinib.

Obesity generally means a state in which adipose tissue is excessive in the body, and is a phenomenon in which energy consumed from food is not balanced with energy consumed through physical activities, etc., and thus surplus energy is accumulated as body fat. The degree of obesity is evaluated by body mass index (BMI), which is calculated by dividing a person's weight (kg) by the square of height (m ² ).

According to recent research results, there is a research result that the higher the body mass index, which is the criterion for obesity, the higher the prevalence of carcinoma. In the above study, leukemia, multiple myeloma, pancreatic cancer, endometrial cancer, rectal cancer, renal cell carcinoma, malignant melanoma, non-Hodgkin's lymphoma, esophageal cancer, brain tumor and central nervous system tumor, breast cancer, colon cancer, gallbladder cancer, lung cancer, liver cancer, ovarian cancer, As a result of studying the correlation with body mass index for 20 types of cancer, including thyroid, bladder, gastric, and prostate cancers, the body mass index was associated with cancer prevalence in all cancers except for three types of cancer (bladder, stomach, and prostate cancer). It turns out that this could be a factor.

Therefore, it was expected that if a drug that is a cancer treatment and suppresses obesity at the same time is developed, obesity can be prevented, and the occurrence of related cancers can be prevented in advance.

Republic of Korea Registration No. 10-1900244 (2018.08.03)

One aspect provides a pharmaceutical composition for the prevention or treatment of obesity containing capmatinib as an active ingredient.

One aspect provides a pharmaceutical composition for preventing or treating obesity comprising a compound of Formula 1 as an active ingredient.

[Formula 1]

In the present invention, the compound is Capmatinib. The capmatinib may be a compound used for the treatment of non-small cell lung cancer (NSCLC) characterized by metastasized MET exon 14 skipping (METex14) mutation as a c-Met inhibitor. The c-Met is also called thyroxine-protein kinase Met or hepatocyte growth factor receptor (HGFR), and may be a factor that can cause cancer.

According to one embodiment, the compound may inhibit cancer and at the same time prevent or treat obesity.

According to one embodiment, the concentration of the compound may be 0.3nM to 1.5nM. For example, it may be 0.8 nM to 1.2 nM.

According to an embodiment of the present invention, it was confirmed that the cell viability decreased rapidly when the concentration of the compound was 2 nM. The above results may indicate that when the compound of the present invention is used at a high concentration, it can attack cells themselves rather than inhibiting fat cell production or decomposing neutral fat.

According to one embodiment, the compound may inhibit lipid deposition or inhibit fat synthesis. For example, the compound may inhibit protein expression of SREBP1, C/EBPa, FAS and SCD1 involved in fat synthesis.

According to one embodiment, the compound may decompose fat. For example, the compound may decompose neutral fat into fatty acids and glycerol, and may release glycerol.

The term "prevention" of the present invention refers to any action that suppresses or delays the onset of obesity by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to the onset of obesity and symptoms of a subject by administration of the pharmaceutical composition. means any action that ameliorates or changes favorably.

According to one embodiment, the pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations or suppositories. there is.

In this case, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits characteristics that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like. In addition, the pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient in the capmatinib, for example, starch, calcium carbonate, sucrose It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogeratin and the like may be used.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals, such as rats, mice, livestock, and the like, including humans who have or may develop obesity. Preferably, it may be a mammal including a human. Obesity can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to a subject, and the pharmaceutical composition of the present invention can be used alone or as an agent for preventing or treating obesity, including surgery, hormone therapy, drug therapy, and biological response modifiers. It can be used in combination with methods using .

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's sex, age, and weight , health condition, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, factors including drugs used in combination or concomitantly, and other medical fields well known. It can be easily determined by a person skilled in the art according to known factors.

Since the composition containing capmatinib of the present invention exhibits effects of decomposing fat and inhibiting fat synthesis, the present invention can be widely used for preventing or treating obesity.

Figure 1 shows the survival rate of cells according to the treatment concentration of capmatinib.
Figure 2 is data confirming that the accumulation of neutral fat is inhibited in the differentiation of adipocytes when capmatinib is administered, confirming the accumulation of neutral fat through Oil-Red O staining technique on days 4 and 7 will be.
Figure 3 is data quantifying the neutral fat content confirmed in Figure 2. (A: Day 4, B: Day 7)
Figure 4 is data confirming the degree of triglyceride decomposition of capmatinib in differentiated 3L3-L1.
5 is data showing the numerical values of SREBP1 protein related to early lipogenesis expressed in differentiated adipocytes through Western blotting.
6 is data showing the numerical values of C/EBPa protein related to early lipogenesis expressed in differentiated adipocytes through Western blotting.
Figure 7 is data showing the numerical values of the FAS protein related to late lipogenesis expressed in differentiated adipocytes through Western blotting.
8 is data showing the numerical values of SCD1 protein related to late lipogenesis expressed in differentiated adipocytes through Western blotting.

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

Experiment method 1. Reagents and processing time

Capmatinib (Capmatinib, CAP) was purchased from Selleckchem and used. Capmatinib (Capmatinib, CAP) concentration was treated for 24 hours, respectively, 0.3, 1, 2nM.

A combination of 3-Isobutyl-1-methylxanthine (M; 0.5mM), dexamethasone (D; 1μM), and insulin (insulin, I; 10ug/ml) It was used as an adipose differentiation inducer (MDI) for 3 days, and insulin (10 ug/ml) was additionally treated for 3 days to complete differentiation.

Experimental method 2. Cell preparation and sample processing

Mouse preadipocytes, 3T3-L1, were purchased from ATCC and used. The cells were cultured in a CO ₂ incubator maintained at 37° C. using DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal calf serum (FCS) and 1% penicillin streptomycin. . Differentiation was performed for 6 to 7 days using an adipocyte differentiation kit (Sigma).

Experiment method 3. Check cell viability

In order to confirm the toxicity of capmatinib, 3T3-L1 adipocytes were treated with each concentration, and cell viability was confirmed through flow cytometry analysis.

To this end, 100 μl PBS was added to the cells to resuspend, and 200 μl of 95% ethanol was added to vortex the cells, followed by incubation at 4° C. for 1 hour. Thereafter, the cells were washed with PBS, resuspended by adding 250 μl of a 1.12% sodium citrate solution at pH 8.4 containing 12.5 μg ribonuclease (RNase), and further incubated at 37° C. for 30 minutes. did

Then, 100 μl of 35.4 μg/ml MTT solution was added to the cells, and the cells were stained for 1 hour at room temperature. Cell viability was measured by dissolving the stained cells in dimethyl sulfoxide (DMSO) and measuring absorbance at 470 nm.

Experimental method 4. Oil red-O staining

3T3-L1 adipocytes were stained with Oil red-O to measure the degree of triglyceride (TG) accumulation in the cells. After fixation with 10% formalin for 40 minutes, hepatocytes were stained with Oil-Red-O solution (Sigma) at 37°C for 1 hour. Oil red-O stained triglyceride (TG) content was quantified by adding isopropanol to each sample. The mixture was stirred gently at 25 °C for 8 minutes. Finally, 100 μl of isopropanol extraction sample was analyzed spectrophotometrically at 510 nm.

Experimental method 5. Measurement of glycerol release

It was performed using the Glycerol Assay Kit (Abcam) for cells that had been differentiated into adipocytes and treated with drugs.

Experimental method 6. Western blotting

Differentiated 3T3-L1 adipocytes were obtained, and proteins were extracted with a lysis buffer (PRO-PREP; Intron Biotechnology, Seoul Korea) at 4°C for 60 minutes. 35 μg of the extracted protein was subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Bioscience, Westborough, MA, USA). After detection with the primary antibody, it was probed with a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Samples were detected with an ECL kit.

Example 1: Cell viability of Capmatinib

In order to confirm the effect on cells when capmatinib is administered in excess, an experiment was performed according to Experimental Method 3. Capmatinib was administered to the cells at concentrations of 0.3nM, 1nM, and 2nM, respectively, and cell viability was confirmed.

As shown in Figure 1, it was confirmed that cell viability was not affected in cells administered with 0.3nM and 1nM of capmatinib, but cell viability significantly decreased at 2nM.

Example 2: Confirmation of fat accumulation inhibitory effect

Oil red-O staining was performed to confirm that the accumulation of neutral fat was suppressed in the differentiation of adipocytes when capmatinib was administered.

As shown in Figures 2 and 3, it was confirmed that neutral fat was more suppressed according to the increase in capmatinib treatment concentration and treatment period.

Example 3: Confirmation of neutral fat decomposition effect

In order to confirm the lipolysis effect of capmatinib in differentiated adipocytes, glycerol release was measured.

As shown in Figure 4, it was confirmed that the amount of glycerol released was increased in a concentration-dependent manner when treated with capmatinib. Through the above results, it can be seen that capmatinib can induce the decomposition of neutral fat accumulated in adipocytes.

Example 4: Confirmation of suppression of early lipogenesis protein expression in differentiated adipocytes

Western blotting was performed to determine whether protein expression of genes related to early lipogenesis was inhibited by capmatinib.

5 and 6, it was confirmed that the expression of both SREBP1 and C/EBPα proteins, which are key transcription factors in the lipogenesis process, decreased depending on the treatment concentration of capmatinib, and in the case of capmatinib 1nM treatment, SREBP1 It was confirmed that the protein expression was inhibited to a degree similar to that of the control group not treated with MDI.

Example 5: Confirmation of inhibition of late lipogenesis protein expression in differentiated adipocytes

Western blotting was performed to determine whether the protein expression of FAS, a fatty acid synthase involved in late lipogenesis, and SCD1, a steroyl-CoA desaturase, were suppressed in differentiated adipocytes.

It was confirmed that both FAS and SCD1 protein expressions decreased depending on the capmatinib concentration, and in particular, when the concentration of capmatinib was 1 nM, it was confirmed that the FAS and SCD1 protein expressions significantly decreased.

Claims (3)

A pharmaceutical composition for preventing or treating obesity comprising a compound represented by Formula 1 as an active ingredient.
[Formula 1]

The pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the compound has activity in inhibiting lipid deposition, inhibiting fat synthesis, or lipolysis.
The method of claim 1, wherein the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, freeze-dried preparations, or suppositories. A pharmaceutical composition for preventing or treating obesity, characterized in that it has any one formulation.

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