CN114522168A

CN114522168A - Pharmaceutical composition for treating gastric cancer and application

Info

Publication number: CN114522168A
Application number: CN202210230795.XA
Authority: CN
Inventors: 柴杰; 王龙刚; 刘冰; 程序瑞
Original assignee: Cancer Hospital of Shandong First Medical University
Current assignee: Cancer Hospital of Shandong First Medical University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-05-24

Abstract

The invention belongs to the field of medicines, and particularly discloses application of arctigenin and 5-fluorouracil in treatment of gastric cancer. The method comprises the synergistic effect of the combination of arctigenin and 5-fluorouracil on inhibiting the proliferation, invasion and transfer of SGC-7901 cells, provides a new thought for designing a synergistic anti-gastric cancer medicament, has a certain guiding significance for the clinical treatment of gastric cancer by the combination of Chinese and western medicines, and provides a theoretical basis for the development of medicaments for treating gastric cancer.

Description

Pharmaceutical composition for treating gastric cancer and application

Technical Field

The invention belongs to the field of tumor biological treatment, and relates to application of a traditional Chinese medicine monomer arctigenin and western medicine 5-fluorouracil in combination in treatment of gastric cancer.

Background

The advanced gastric cancer is an invasive disease, the prognosis is still poor, and the survival time is short. Although various drugs and combination therapies have been developed and the mortality rate of gastric cancer has been gradually reduced in recent decades, no suitable drug has been available so far to effectively eliminate gastric cancer cells. Therefore, a new therapeutic strategy is urgently needed in clinical and practical applications.

The arctigenin is extracted and purified from fructus Arctii, and has the following structural formula:

the burdock seed is pungent and bitter in taste and cold in nature, and can disperse wind and heat, ventilate lung and promote eruption, dissipate stagnation and remove toxicity. Root, bitter and pungent in flavor, cold in nature, clear heat and remove toxicity, dispel wind and relieve sore throat. In clinical application, the burdock and other Chinese medicines form a compound preparation for treating lung cancer, colorectal cancer and other metastatic cancers, the phenomenon of cancer swelling disappearance or reduction is found, and the life cycle can be prolonged. The medicine is also used for treating throat tumor with accumulation of toxic heat and stagnation of qi and blood. In addition, it is also commonly used to treat common cold, red and swollen throat, epidemic parotitis, eruptions, carbuncle, furuncle, pyocutaneous disease, eczema, etc.

5-fluorouracil of formula C₄H₃FN₂O₂The pyrimidine analog belongs to one of antimetabolites, is mainly used for treating tumor agents, and has the following structural formula:

5-fluorouracil is firstly changed into fluorouracil deoxynucleotide through a series of reactions in vivo, and then effects are exerted (DNA synthesis is influenced); can be converted into fluorouracil to be incorporated into RNA in vivo, thereby interfering with protein synthesis. Mainly acts on S phase, but also has certain effect on other cells in each phase. It is easy to penetrate blood brain barrier and enter brain tissue and tumor metastasis. About 10% to 30% of the prototype is excreted in urine60-80% of the active ingredients are inactivated in the liver and changed into CO₂And urea, excreted by the respiratory tract and urine, respectively. After continuous use, leukopenia and thrombocytopenia are mainly generated. The side effects of alopecia, erythematous dermatitis, skin pigmentation hand-foot syndrome and transient cerebellar movement disorder, which influence the heart function, etc. are frequent.

Disclosure of Invention

In order to overcome the technical defects of larger side effect and poor treatment effect of 5-fluorouracil, the invention provides a pharmaceutical composition for treating gastric cancer, which is prepared by combining 5-fluorouracil and arctigenin. The two active ingredients of the medicine have obvious synergistic effect on the aspects of inhibiting the proliferation, invasion and metastasis of gastric cancer cells, so that the medicine can play a good role in treating the gastric cancer, and the treatment effect and the life quality of tumor patients are improved. The pain of the patient is relieved, and the service life of the patient is prolonged.

In order to achieve the above purpose, the invention adopts the following technical scheme to realize:

a pharmaceutical composition for treating gastric cancer metastasis, which comprises arctigenin and 5-fluorouracil.

The pharmaceutical composition can also contain gimeracil and oteracil potassium.

Calculated by molar volume ratio, the arctigenin: 5-fluorouracil 20-50: 1.

calculated by molar volume ratio, the arctigenin: 5-fluorouracil ═ 30: 1.

the use of arctigenin and 5-fluorouracil in preparing a medicament for inhibiting proliferation, invasion and metastasis of gastric cancer cells. The gastric cancer cells are cell lines including but not limited to SGC-7901, HGC27 and MGC 803.

Application of arctigenin and 5-fluorouracil in preparing medicine for treating gastric cancer.

The arctigenin and 5-fluorouracil are separately/mixedly packaged in the medicament.

The pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials.

The dosage form of the pharmaceutical composition is an oral preparation, and further preferably tablets and capsules.

The inventor unexpectedly discovers that the 5-fluorouracil and the traditional Chinese medicine extract, particularly arctigenin, have a synergistic interaction effect when being used for treating the gastric cancer in a combined way, so that the inventor has a certain guiding significance for clinical treatment of the gastric cancer by combining the traditional Chinese medicine with the western medicine.

Drawings

FIG. 1: toxicity study of arctigenin and 5-fluorouracil on the SGC-7901 gastric cancer cell line, and the survival rates of the single group and the combined group of arctigenin and 5-fluorouracil on the SGC-7901 gastric cancer cell line were respectively recorded by MTT experiments. A graph shows the survival rates of different concentrations of arctigenin on SGC-7901 gastric cancer cell line;

b shows the survival of the SGC-7901 gastric cancer cell line with different concentrations of 5-fluorouracil;

c shows that the arctigenin and 5-fluorouracil have proliferation inhibition effect on SGC-7901 and show obvious concentration dependence.

FIG. 2: effect of arctigenin of different concentrations on growth and proliferation of SGC-7901 cells.

FIG. 3: as can be seen from tumor cell invasion and metastasis experiments, the number of cells invading to the lower layer by SGC-7901 cells is obviously reduced along with the increase of the concentration of the medicament, and the research shows that the medicament combination has the effect of inhibiting the invasion and metastasis of tumor cells.

FIG. 4: the effects of arctigenin and 5-fluorouracil concentration ratio of 0 and 30:1 on SGC-7901 cell proliferation are respectively. Wherein arctigenin is 300 μ M/ml, and 5-fluorouracil is 10 μ M/ml.

Detailed Description

The technical solutions in the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, belong to the scope of the present invention.

Example 1: the influence of the combination of arctigenin and 5-fluorouracil on the growth and proliferation of SGC-7901 cells is detected by an MTT experimental method.

Thiazole blue (MTT) is a yellow dye which reacts only with dehydrogenases of living cells to produce blue-violet crystals (formazan) and deposits in cells, but does not react with dead cells, formazan is dissolved in DMSO, and the absorbance at a specific wavelength is measured by an elisa detector to reflect the proportion of living cells.

1. Preparation of the test:

1) culture of test cell lines

Recovering SGC-7901 cells from liquid nitrogen tank, digesting, counting, diluting with culture medium to obtain suspension, standing at 37 deg.C and 5% CO₂Culturing in an incubator.

2) Preparation of drug mother liquor

Arctigenin and 5-fluorouracil raw material medicines are taken, dissolved by DMSO and 0.9% of normal saline respectively, and then diluted by culture media with proper volume respectively to prepare mixtures with different proportions.

2. Procedure of the test

The present inventors prepared SGC-7901 cells into 4X 10 cells⁴The cells/mL, 100. mu.L per well of 96-well plate medium, 96-well cell culture plates were placed at 37 ℃ in 5% CO₂Culturing in an incubator for 24 hours; diluting the drug with culture medium to required working solution concentration, adding 100 μ l corresponding drug-containing culture medium into each well, and setting up negative control group; the 96-well cell culture plate is placed at 37 ℃ and 5% CO₂After 72 hours in the incubator, the 96-well plate was MTT stained with λ 490 n.

The OD was measured as follows: mu.l MTT (5mg/ml) was added to each well and incubation was continued in the incubator for 4 hours; discarding the supernatant, adding 150 μ l DMSO into each well, and gently mixing by shaking for 10 min; λ 490nm, OD value per well was read by microplate reader, and cell inhibition rate was calculated according to the following formula.

The cell inhibition ratio (%) (negative control group OD value-experimental group OD value)/negative control group OD value. times.100%, IC of arctigenin and 5-fluorouracil were calculated according to the above method₅₀The values are as follows:

TABLE 1 combination of arctigenin and 5-fluorouracil for inhibiting gastric cancer cell proliferation

5-Fluorouracil (μ M)	Arctiin (mu M)	Inhibition ratio (%)	Ratio of
				0.5	30	19.7	60
1.5	75	36.2	50
				4.5	135	42.8	30
13.5	270	61.5	20
				40.5	405	75.8	10
121.5	1215	82.6	10

TABLE 2 synergistic effect of arctigenin and 5-fluorouracil in SGC-7901 cells

Note: the synergistic effect is strong: (CI ═ 0.3-0.7), moderate synergy: (CI-0.7-0.85)

In a certain concentration range, arctigenin and 5-fluorouracil have proliferation inhibition effects on gastric cancer SGC-7901 cells and show a remarkable concentration dependence phenomenon, and arctigenin and 5-fluorouracil have proliferation inhibition effects on SGC-7901 and show a remarkable concentration dependence, which is shown in figure 1(A, B, C).

Example 2: the effect of different amounts of arctigenin and 5-fluorouracil on the growth of gastric cancer cells was performed as in example 1

TABLE 3 arctigenin IC of different ratio composition of arctigenin and 5-fluorouracil₅₀Comparison

As can be seen from the above table, the arctigenin of the above ratio: the proportion of 5-fluorouracil shows that the arctigenin: 5-fluorouracil 20-50: 1 hour IC₅₀The value is small, especially arctigenin: IC when 5-fluorouracil is 30:1₅₀The value is minimum, and the medicine is safest.

Example 3: effect of different aglycones in combination with 5-Fluorouracil on gastric carcinoma cell growth as described in example 1

TABLE 4 IC of the different aglycones₅₀Comparison

Note: aglycone: 5-Fluorouracil ═ 30:1

As can be seen from the above table, the aglycone: the molar volume ratio of 5-fluorouracil is 30:1, and the different aglycones are subjected to IC₅₀The comparative ratio shows that arctigenin and 5-fluorouracil IC₅₀The value is minimum, which indicates that the combined use of the arctigenin and the icariin is superior to the combination of the existing arctigenin or icariin.

Example 4: effect of combination of arctigenin and 5-fluorouracil under different concentrations on SGC-7901 cell invasion capacity

The influence of the drug combination on the invasion capacity of tumor cells is briefly described as follows by adopting a Transwell experimental method:

1) coating a substrate: uniformly mixing the frozen and thawed Matrigel matrix by using a precooled pipette tip, diluting the Matrigel by using a precooled serum-free culture medium according to a volume ratio of 4:1, placing a Transwell chamber with the pore diameter of 8 mu m into a 24-pore plate matched with the Transwell chamber, coating a filter membrane at the bottom of the Transwell chamber by using the diluted Matrigel, and incubating for 1h at room temperature. After coating, the Matrigel matrix not bound to the filter was removed and gently washed 2 times with serum-free medium.

2) Cell treatment: 250 μ L of gastric cancer cells SGC-7901 (medium without serum) were digested and plated on the upper layer of matrix-coated Transwell chamber, and arctigenin of the present invention was added: the concentration of arctigenin in 5-fluorouracil is 0, 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, respectively, and the concentration of 5-fluorouracil is 1. mu.M/mL.

3) And (3) invasive culture: to the culture well in the lower chamber was added 750. mu.L of a medium containing 20% FBS as a chemotactic agent for the lower chamber, and the plate was placed in a cell culture chamber to continue culturing for 24 hours.

4) Dyeing: after 24 hours of incubation, the plates were removed and the medium in the wells was removed, washed 2 times with PBS, and the chamber was fixed in 4% paraformaldehyde solution for 30 min. The cells were then washed twice with PBS and the cells on the side of the chamber facing up were wiped off with a cotton swab and washed with PBS until all cells on the side of the chamber facing up were washed away. The cell was placed in 0.1% crystal violet staining solution for 30 min. After the staining is finished, the cells are washed for three times by PBS, pictures are taken by a microscope, and the difference of the invasion capacity of SGC-7901 cells of different groups is calculated.

The inhibition rate of invasion was (1-number of invaded cells in experimental group/number of invaded cells in control group) × 100%.

The experimental result is shown in figure 3, the number of cells invading into the lower layer of SGC-7901 cells is obviously reduced along with the increase of the concentration of the drug, and the research shows that the drug combination of the invention has the function of inhibiting the invasion and metastasis of tumor cells.

Claims

1. The pharmaceutical composition for treating the gastric cancer is characterized by comprising arctigenin and 5-fluorouracil.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises gimeracil and oteracil potassium.

3. The pharmaceutical composition of claim 1, wherein the arctigenin: 5-fluorouracil 20-50: 1.

4. the pharmaceutical composition of claim 1, wherein the arctigenin: 5-fluorouracil ═ 30: 1.

5. the pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is in the form of an oral preparation, further preferably a tablet or capsule.

6. Application of arctigenin and 5-fluorouracil in preparing medicine for treating gastric cancer.

7. The use of arctigenin and 5-fluorouracil for preparing a medicament for inhibiting proliferation, invasion and transfer of gastric cancer cells.

8. The use of claim 7, wherein the gastric cancer cells include but are not limited to SGC-7901, HGC27 and MGC803 cell lines.

9. The use according to claim 6 or 7, wherein the arctigenin and 5-fluorouracil are packaged separately/mixed in the medicament.

10. The use of claim 6 or 7, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.