KR20200031735A - An infusion solution for treating cancer - Google Patents

Abstract

Disclosed is an infusion composition for treating cancer containing a specific sugar. The infusion composition of the present invention contains sugars of glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. The present invention is to provide the infusion composition for treating cancer containing the specific sugar. The infusion composition according to the present invention exhibits an excellent anticancer effect.

Description

{An infusion solution for treating cancer}

The present invention relates to a fluid composition for the treatment of cancer.

The main materials covered in the life sciences are proteins, amino acids, nucleic acids, fats, fatty acids, carbohydrates, polysaccharides and sugars. Life science and medical researchers have studied and researched protein as a major communication molecule in vivo through a long period of research, but came to the conclusion that protein alone is not sufficient to deliver all messages inside cells, and accordingly, protein molecule and carbohydrate molecule Began to study the bound glycoproteins.

Glucose (Glu), Galactose (Gal), Mannose (Man), L-Fucose (Fuc), Xylose (Xy), N-acetylglucosamine (N-Acetylglucosamine: GlcNAc), N-acetylgalactosamine (N-Acetylgalactosamine: GalNAc), N-Acetylneuraminic acid (NANA; or sialic acid) 8 sugars maintain bioactivity in the body Plays a very important role in

The present inventors confirmed that the sap composition containing 8 kinds of sugars as described above has an anticancer effect, and completed the present invention.

Korean Patent Publication No. 10-2011-0131699

The present invention is to provide an infusion composition for treating cancer comprising a specific sugar.

In order to solve the above problems, the present invention discloses an infusion composition for treating cancer containing a specific sugar.

Specifically, the infusion composition of the present invention includes glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sugars of N-acetylneuraminic acid.

The infusion composition of the present invention may further include one or more amino acids.

Here, the amino acid is at least one selected from the group consisting of isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, proline, serine, tyrosine, and glycine. Can be.

Alternatively, as the amino acid, one or more of 20 amino acids capable of constituting a human protein may be selected.

The infusion composition according to the present invention exhibits excellent anticancer effects.

1A and 1B are graphs showing the results of cell proliferation inhibition experiments for the compositions of the present invention, respectively.
Figure 2a is a photograph of cells metastasized in the cell invasion inhibition experiment for the composition of the present invention, Figure 2b is a graph comparing the infiltration inhibition.
3A and 3B are photographs of well plates taken in a cell mobility inhibition experiment for the composition of the present invention, and FIGS. 3C and 3D are graphs comparing mobility inhibition.

The present invention will be described in detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  1: Preparation of sugar-containing infusion solution

Glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid from Sigma-Aldrich (St. Louis, Missouri, USA) 8 sugars were dissolved in water for injection (foreign pharmaceutical). As a stabilizer, aspartic acid (L-Aspartic acid), a type of amino acid, was used.

In order to adjust the pH of the prepared sugar-containing aqueous solution, a NaOH solution of 1N concentration (Samjeon Pure Chemical Co., Ltd.) was used. The pH was adjusted to 7.0 using a pH meter (METTLER TOLEDO Seven Compact pH / Ion).

Table 1 shows the composition of the sugar-containing solution.

ingredient Sugar content
Infusion Sugar (mg) L-(-)-Fucose 31 D-(+)-Galactose 31 D-(+)-Xylose 31 D-(+)-Glucose 31 D-(+)-Mannose 31 N-Acetylneuraminic acid 31 N-Acetyl-D-galactosamine 31 N-Acetyl-D-glucosamine 31 Stabilizer (mg) Aspartic acid 20 solvent Water for injection q.s. pH adjuster
(μL) 1N NaOH q.s. Total 10 mL

* Total 10mL standard composition table (actually 50mL was prepared)

Example  2: Preparation of sap solutions containing sugars and amino acids

Glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid from Sigma-Aldrich (St. Louis, Missouri, USA) After dissolving 8 kinds of sugars in water for injection (foreign pharmaceutical), it was mixed with a commercially available amino acid solution, 89.4% furoamine (Proamin injection, Hanol Biopharma). The furoamine strain contains amino acids such as isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, proline, serine, tyrosine, and glycine. As a stabilizer, aminoguanidine (Sigma-Aldrich) was used.

In order to adjust the pH of the prepared aqueous solution containing sugar and amino acids, a 1N NaOH solution (Samjeon Pure Chemical Co., Ltd.) was used. The pH was adjusted to 7.0 using a pH meter (METTLER TOLEDO Seven Compact pH / Ion).

Table 2 shows the composition of the aqueous solution containing the sugar and amino acid.

ingredient Sugars and amino acids
contain Infusion Sugar (mg) L-(-)-Fucose 31 D-(+)-Galactose 31 D-(+)-Xylose 31 D-(+)-Glucose 31 D-(+)-Mannose 31 N-Acetylneuraminic acid 31 N-Acetyl-D-galactosamine 31 N-Acetyl-D-glucosamine 31 Amino acid (mg) L-Isoleucine 28 L-Leucine 62.5 L-Lysine Acetate 62 L-Methionine 17.5 L-Phenylalanine 46.75 L-Threonine 32.5 L-Tryptophan 6.5 L-Valine 22.5 L-Alanine 31.25 L-Arginine 39.5 L-Aspartic Acid 19 L-Cysteine 5 L-Glutamic Acid 32.5 L-Histidine 30 L-Proline 16.5 L-Serine 11 L-Tyrosine 1.75 Aminoacetic Acid 53.5 Stabilizer (mg) Aminoguanidine 40 solvent Water for injection q.s. pH adjuster
(μL) 1N NaOH q.s. Total 10 mL

* Total 10mL standard composition table (actually 50mL was prepared)

Example  3: cell Proliferative capacity  Inhibition test

end. Experimental method

HeLa cells derived from cells of cervical cancer patients were cultured at 37 ° C. 5% CO ₂ , and 5,000 to 10,000 cells (in Opti-MEM media) were placed in 96 well plates, followed by a negative control (Normal). , Sugar-containing infusion solution stock, sugar-containing infusion solution 10-fold dilution, sugar-containing infusion solution stock solution 100-fold dilution, sugar and amino acid-containing infusion solution stock solution, sugar and amino acid-containing infusion solution stock solution, sugar and amino acid-containing solution infusion solution 100 The effect of 6 samples on the cell proliferation capacity by treatment with fold dilution was confirmed by MTT test method.

After adding 100 μl of HeLa cells (5 × 10 ⁵ cells / ml) into a 96 well plate and pre-incubating for 18 hours, a 10-fold dilution of the sugar-containing infusion solution stock, a sugar-containing infusion solution stock solution, and a 100-fold dilution of the sugar-containing solution solution stock solution, Stock solutions of sugar and amino acid-containing solutions, 10-fold dilutions of sugar and amino acid-containing solutions, and 100-fold dilutions of sugar and amino acid-containing solutions are applied to the medium every 0, 24, 48, 72 and 96 hours. Each time the sample was treated, some of the cells to be analyzed were treated with 10 μl / well of the MTT solution. Cells treated with MTT solution were incubated at 37 ° C 5% CO ₂ for 3-4 hours, the reaction was stopped by adding 15% sodium dodecyl sulfate, and formazan was dimethyl sulfoxide (DMSO). After dissolution, the sample was analyzed by measuring absorbance at a wavelength of 570 nm with a spectrophotometer (ELISA reader) the next day.

Through these cell proliferation assays, the effect of the fluid composition of the present invention on cell proliferation and cytotoxicity was tested.

I. Experiment result

The results of cell proliferation inhibition experiments at 48 hours and 72 hours are shown in FIGS. 1A and 1B.

In the case of treatment with Sample No. 1 (sugar solution containing sugar and amino acids) and Sample No. 4 (sugar solution containing sugar and amino acids), inhibition of the division of HeLa cells by about 20% to 50% was observed.

The effect of suppressing cell division was higher at higher concentrations. Therefore, the effect of inhibiting cell division of the infusion solution of the present invention appears to be concentration dependent.

As such, the sap composition according to the present invention exhibited excellent anti-cancer effects.

Example  4: Experiment to confirm the effect on cell infiltration

end. Experimental method

In order to confirm the effect of the infusion solution of the present invention on the mobility and infiltration of cells, it was divided into matrigel substrates for about 3 days to test whether the cells penetrated through the substrates and passed.

On the first day of the experiment, the matrigel stored at 20 ° C was moved to 4 ° C to dissolve.

On the second day, set the dilution factor according to the experimental conditions, dilute the matrigel in medium (no FBS, no Antibiotics) about 2 times, put 100 μl of the diluted matrigel in a transwell, put it in a 24 well plate, sterilize it by being exposed to UV for more than 1 hour It was hardened in the form of a gel, and the surface of the transwell was coated with matrigel. 5000 to 10000 cancer cells (in Opti-MEM media) were coated on a transwell matrigel, and a certain amount of a sugar-containing solution or a sugar and amino acid-containing solution was added. Under the transwell, 1 mL of 10% FBS bovine serum medium was added.

On the last day of the experiment, the matrigel was sucked with a pipette, and the cells were fixed by transferring the transwell with 3.7% formaldehyde. After washing the cells 3 times with PBS, staining with hematoxylin for 5 minutes, washing with distilled water 5 times, staining with eosin for 10 minutes, washing with distilled water 3 times, 70% It was washed 3 times with ethanol, and finally with 100% ethanol.

After cutting the bottom surface of the cells attached to the transwell with a blade, the cells were adhered and fixed with a fixed solution so that no bubbles were generated in the slide glass, and the metastasis of the cancer cells was observed with an optical microscope.

I. Experiment result

FIG. 2A is a photograph of metastasized cancer cells taken with an optical microscope, and FIG. 2B is a graph comparing the number of metastasized cells with each sample and comparing it with a negative control.

In the invasive process essential for the metastasis of cancer cells, the metastasis of cancer cells was inhibited by 20% when treated with the sap solution containing sugar and amino acids, and the metastasis of cancer cells was inhibited by 50% when treated with the sac solution containing sugar and amino acids.

As described above, the fluid composition according to the present invention is excellent in suppressing metastasis of cancer cells.

Example  5: Experiment to confirm the mobility of cells

end. Experimental method

In order to confirm the mobility of the cells in relation to the metastasis and viability of cancer cells, a mobility experiment was performed to compare how much the cells move in the scratched portion when the sample is processed by scratching the cells.

Cells were placed in a 12 well plate at 80% confluency in six test compositions (i.e., a sugar-containing infusion solution stock, a 10-fold dilution of the sugar-containing infusion solution stock, a sugar-containing infusion solution stock solution in 100-fold dilution, a sugar and amino acid-containing solution stock solution, sugar and It was treated with a 10-fold dilution of a stock solution containing amino acids and a 100-fold dilution of a stock solution containing sugars and amino acids).

After 24 hours of treatment with 6 samples, a 200 μl yellow pipette tip was used to scratch the center of the 12 well plate, and then a 12 well plate with scratches on the cell population was mounted with a camera equipped with an optical microscope. Filmed (0 hour). After 12 hours and 24 hours, the change in the width of the scratched portion of the cell group was measured using a graduated lens mounted on an optical microscope. Through this, the amount of change in the width of the scratched portion of the cell group was compared with the width at 0 hour. Mobility was evaluated. Since cancer cells are used in this experiment, the rate of metastasis in cancer can be lowered when the amount of cell migration is reduced.

I. Experiment result

3A and 3B are photographs of well plates taken in a cell mobility inhibition experiment for the composition of the present invention. Here, the two yellow lines are imaginary lines in the well plate indicating the boundary between the non-cell part and the cell part. Therefore, in the case of 0H when the plate is scratched, the portion where the cells are removed by the scratch is displayed between the yellow lines, and as time passes, cells existing outside the yellow line move to the scratched area, (the cell is present. The width of the imaginary yellow line (meaning the boundary between the part and the cell-free part) gradually decreases.

3C and 3D show a change in the width (the value of the changed width) after 12 hours (FIG. 3C) and 24 hours (FIG. 3D), when compared with the width of the cell scratches occurring at 0 hour when treated with each sample. It is a graph that is calculated by calculating the percentage of the cell's movement distance). Therefore, it can be said that the mobility of cells is small when the change in width is small.

Referring to Figure 3c and Figure 3d, the movement of cancer cells is inhibited by 50% to 70% during the treatment of the undiluted solution containing sugar and amino acids, and the movement of cancer cells is inhibited by 20% to 30% during the treatment of the undiluted solution containing the sugar and amino acid. In addition, the cancer cell migration inhibitory effect was also observed in the diluted solution of the infusion solution of the present invention.

The smaller the migration value (the distance the cell travels), the smaller the mobility of cancer cells, so it can be seen that it has anti-cancer effects by reducing the metastasis of cancer. As a result of the experiment, it can be seen that when treated with the infusion composition of the present invention, the mobility of the cells was low, and thus the anticancer effect was excellent.

The infusion composition of the present invention is excellent in anticancer effect.

Claims (4)

Infusion composition for the treatment of cancer comprising glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid. The composition of claim 1, further comprising an amino acid. The method of claim 2, wherein the amino acid is isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, proline, serine, tyrosine, and glycine Infusion composition, characterized in that one or more selected. The composition of claim 2, wherein the amino acid is at least one of 20 amino acids capable of constituting a protein.

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