CN114917337A - Targeted pharmaceutical composition for treating colorectal cancer and application thereof - Google Patents

Abstract

The invention relates to a targeted pharmaceutical composition for treating colorectal cancer, which consists of a water extract of a levo-golden pill and cetuximab, wherein the ratio of the water extract of the levo-golden pill to the cetuximab is as follows: 22.5:1. The invention also provides application of the pharmaceutical composition in preparing a targeted medicament for treating colorectal cancer. The targeted pharmaceutical composition of the invention is used for the research on pharmacodynamics, pharmacology and early safety of the in-vivo tumor-bearing nude mouse model level synergistic multi-target EGFR resistance, and is proved to be capable of improving the general condition of the colorectal cancer animal model and inhibiting the growth and transfer of colorectal cancer. The targeted pharmaceutical composition can block the activation of signal protein closely related to EGFR and inhibit the expression of downstream transcription factors. The activity of proteins such as p-EGFR, p-Akt, p-mTOR and the like in the colorectal cancer cells after the combined CET and the left-gold pill is adopted for dry prognosis is reduced, and compared with the single CET, the combined CET and the left-gold pill can inhibit the expression of phosphorylation of proteins such as EGFR, Akt, mTOR and the like related to an EGFR signal pathway in the colorectal cancer cells.

Description

Targeted pharmaceutical composition for treating colorectal cancer and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a targeted medicine composition for treating colorectal cancer and application thereof.

Background

Colorectal cancer (CRC) is a malignant tumor with high global morbidity and mortality. Epidermal Growth Factor Receptor (EGFR) is one of the most important protooncogenes in epithelial tumors, and high expression of EGFR occurs in 72 to 89% of CRC tissues. Therefore, Cetuximab (CET) with the epidermal growth receptor as a target achieves better clinical curative effect. CET is IgG monoclonal antibody, can be specifically combined with epidermal growth factor EGF receptor EGFR on the cell surface, competitively blocks the combination of EGF and other ligands, and accordingly blocks biological effects such as EGFR-dependent tumor cell proliferation, metastasis, invasion, angiogenesis and the like. However, with the clinical application of CET, the problem of drug resistance is highlighted, which not only increases the economic burden of patients, but also shortens the overall survival time of patients, and has become the biggest obstacle for metastatic colorectal cancer patients to benefit from CET treatment.

With the gradually clarified role of tumor metabolism specificity in tumor development and development, the energy metabolism phenotype of drug-resistant cells is also more and more emphasized by people and is probably a key factor for inducing tumor drug resistance. Recent studies have shown that glycolysis is directly related to tumor resistance, and aerobic glycolysis in resistant cells is significantly enhanced compared to non-resistant cells. Otto Warburg in 1920 first proposed that tumor cells, even when oxygen was sufficient, still supply energy in a glycolytic manner, namely: the Warburg effect is an important mechanism for maintaining the biological function of tumor cells and is also an important therapeutic target for reversing the drug resistance of tumors.

Long-term clinical practice proves that the traditional Chinese medicine and the targeted medicine have obvious advantages in the aspects of improving curative effect and reducing toxic and side effects of the medicine, and are one of effective means for comprehensive treatment of colorectal cancer. Subject group preliminary studies prove that the ZUOJIN pill has clinical effects of reversing multidrug resistance of tumors, improving the life quality of colorectal cancer patients and prolonging the life, but the specific action mechanism is not clear yet.

ZJW, originally originated from the book of Danxi Xin Fa of the Ming Dynasty, is a classic formula of traditional Chinese medicine theory that the cold is hot and the hot is cold. Coptis root, rhizoma Coptidis, being bitter and cold in flavor, clears heart and purges liver, evodia fruit, being pungent and hot in flavor, draws heat downward, the two herbs are primary and secondary, the cold and the heat are in opposition. For the indications of Zuojin Wan, the original book only has the function of treating liver fire, but the modern application is very extensive. Modern pharmacodynamics and pharmacology holds that the drug resistance of tumors is mostly caused by the fact that chemotherapy drugs are often biased to be hot, so that breakthrough gains are obtained in the research of medicine micro-calorimetry by adopting the levo-gold pills or anti-levo-gold pills biased to be cold to be matched with chemotherapy. According to traditional Chinese medicine, the bitter and cold coptis chinensis is mainly used as a monarch drug in the formula for clearing away fire heat and clearing away the adverse flow of qi, and clearing away liver fire and stomach heat, the liver fire does not transversely flow to the stomach when the liver fire is cleared away, and the stomach fire is descending so that the qi can be automatically neutralized. The combination of the pungent and hot fructus evodiae can not only soothe the liver and relieve depression to make liver qi reach, but also counteract the cold of coptis root and protect spleen yang.

However, no report is found about a targeted pharmaceutical composition for treating colorectal cancer at present.

Disclosure of Invention

The first purpose of the present invention is to provide a targeted pharmaceutical composition for treating colorectal cancer, which overcomes the defects in the prior art.

The second purpose of the invention is to provide the application of the pharmaceutical composition.

In order to achieve the first purpose, the invention adopts the technical scheme that:

a targeted pharmaceutical composition for treating colorectal cancer comprises a water extract of Zuojin pills and cetuximab.

As a preferred example, the ratio of the aqueous extract of zuojin pills to cetuximab is as follows: 22.5:1.

As a preferred example, the aqueous extract of Zuojin pill is obtained by the following method: taking coptis chinensis: the evodia rutaecarpa 6 is extracted at a ratio of 1, and the yield of the aqueous extract of the Zuojin pill is 1g of crude drug/0.071 g of aqueous extract.

In order to achieve the second object, the invention adopts the technical scheme that: the application of the pharmaceutical composition in preparing a targeted medicament for treating colorectal cancer.

The invention has the advantages that: the targeted pharmaceutical composition of the invention is used for the research on pharmacodynamics, pharmacology and early safety of the in-vivo tumor-bearing nude mouse model level synergistic multi-target EGFR resistance, and is proved to be capable of improving the general condition of the colorectal cancer animal model and inhibiting the growth and transfer of colorectal cancer. The targeted pharmaceutical composition can block the activation of signal protein closely related to EGFR and inhibit the expression of downstream transcription factors. The activity of proteins such as p-EGFR, p-Akt, p-mTOR and the like in the colorectal cancer cells after the combined CET and the left-gold pill is adopted for dry prognosis is reduced, and compared with the single CET, the combined CET and the left-gold pill can inhibit the expression of phosphorylation of proteins such as EGFR, Akt, mTOR and the like related to an EGFR signal pathway in the colorectal cancer cells. The research also observes that the combination of the zuojin pill and the cetuximab can obviously reduce the glucose intake, the lactic acid production, the ATP content and the ECAR in the cells, and further proves that the zuojin pill reverses the targeted drug resistance of the cetuximab and can play a role by inhibiting the glycolysis pathway of drug-resistant cells.

Drawings

FIG. 1: the influence of the Zuojin pill extract and the cetuximab on the body weight of nude mice with colorectal cancer orthotopic transplantation tumor.

FIG. 2 is a schematic diagram: the combination of the Zuojin pill extract and the cetuximab has the effect of treating lung metastasis of nude mice with colorectal cancer orthotopic transplantation tumor.

FIG. 3: effect of aqueous extract of levo-gold pills on survival of SW620 cells.

FIG. 4: inhibition of SW620 cell proliferation by levo-gold bolus in combination with CET.

FIG. 5: the left golden pill aqueous extract combines with CET to act on EGFR, Akt and mTOR proteins of SW620 cells and phosphorylation thereof. P <0.01 compared to SW620 group.

FIG. 6A: glucose uptake was compared for each group. P <0.01 compared to Control group; comparison with CET group # P < 0.05.

FIG. 6B: and comparing the lactic acid content of each group. P <0.01 compared to Control group; # P <0.01 compared to CET group.

FIG. 6C: ATP content of each group is compared. P <0.01 compared to Control group; # P <0.01 compared to CET group.

FIG. 6D: comparison of extracellular acidity for each group.

FIG. 6E: comparing the basal oxygen consumption rate of each group of cells.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

1. preparation of Zuojin pill extract

The method for extracting the aqueous extract of the ZUOJIN pill (coptis chinensis: evodia rutaecarpa 6:1) comprises the following steps: weighing 30g of fully dried coptis chinensis medicinal material powder and 5g of fructus evodiae medicinal material, soaking and extracting for 3 hours in 0.2% sulfuric acid warm water, extracting for three times, each time for 350ml, concentrating an extracting solution to 100ml under reduced pressure, adding lime milk to adjust the pH value to 10, standing for 2 hours in a refrigerator, centrifuging to remove precipitates, extracting a water layer for five times by using chloroform, each time for 100ml, and recovering the chloroform to be dry to obtain powder I. Hydrochloric acid is added into the water layer to adjust the pH value to 2, 8g of salt is added, the mixture is placed in a refrigerator for 24 hours, and precipitates are collected and dried to obtain powder. The powder (i) and the powder (ii) were combined to obtain 2.50g of a dry total powder. The yield of the water extract of Zuojin pill is 1g crude drug/0.071 g water extract. For cell experiments, a proper amount of dry powder of the water extract is weighed before the experiments, is fully dissolved in an L-15 culture medium by methods such as vortex, ultrasonic, water bath and the like, and then is filtered by a 0.22 mu m filter to prepare a solution with required concentration, and is stored at 4 ℃ for later use.

2. Nude mouse model of colorectal cancer orthotopic transplantation tumor

Single cell suspension was prepared from colon cancer cells SW620 in logarithmic growth phase, and PBS was used to adjust the cell concentration to 1X 10 ⁷ and/mL. After the cell activity is determined to be more than or equal to 95% by trypan blue staining, an orthotopic transplantation tumor model is started to be manufactured: anesthetizing mouse with sodium pentobarbital or diethyl ether, cutting abdomen with ophthalmic scissors, sucking SW620 single cell suspension with l mL microsyringe and 23-gauge needle, and placing at a ratio of 1 × 10 ⁷ Cell numbers per mL were inoculated into the colon under the cecum of 6-8 week old male BALB/c mice, followed by gentle swabbing with an alcohol cotton swab to ensure no efflux of cells into the peritoneal cavity. Inoculating cell suspension to cecum of each nude mouse with micro syringe, introducing cecum back into abdominal cavity, suturing with absorbable thread, closing abdomen, sterilizing wound, and suturing with sterile suture.

3. Grouping and administration of animal experiments

The tumor body to be grown is about 100mm ³ The groups were randomly divided into 4 groups of 8 individuals. Corresponding treatment is given to each group, the traditional Chinese medicine (ZJW group) is given to the traditional Chinese medicine for intragastric administration, the clinical equivalent dose is referred, the equivalent dose ratio of normal adults and mice is 9:1 according to the conversion of the body surface area of modern medical experimental zoology, the daily dose of the mice is 3.15 g/kg/day when the weight of the normal adults is calculated according to 60kg, and the dose converted into the water extract of the ZJW group is 0.225 g/kg/day, which is the concentration of the ZJW group. The dosage is 0.2ml once daily for 28 days. Targeted drug group (CET): by adopting CET, the injection is injected into the abdominal cavity at the dose of 10mg/kg for 1 time/3 days, and the total time is 28 days. Levo-jin pill in combination with cetuximab group (ZJW + CET): the gazettus sinicus pill is used for intragastric administration, 0.2ml is taken once a day for 28 days according to the specified dosage, CET is simultaneously used, 0.2ml is intraperitoneally injected according to the dosage of 10mg/kg for 1 time/3 days, and the continuous 28 days (for animal experiments, the ratio of the water extract of the gazettus sinicus pill to the cetuximab is 22.5:1(0.225 g/kg: 10 mg/kg)), and the equivalent physiological saline is intragastric administered every day for the model group.

After the treatment, the nude mice are sacrificed, tumor bodies are quickly stripped, the tumor weight and the body weight are weighed, and the tumor inhibition rates of different treatment groups are calculated. The tumor growth inhibition ratio (% control group average tumor weight-experimental group average tumor weight)/control group average tumor weight × 100%. The morphology of the cells and the organ metastasis were observed by H & E staining.

CCK8 Kit (Cell Counting Kit-8) for detecting Cell proliferation

According to the comparison group: SW620 cells; CET group: SW620+ CET (different doses); ③ Zuojin Wan union CET group: SW620+ CET (different doses) + ZJW (15mg/mL) groups, each group adjusted to 1X 10 ⁵ mL, 100. mu.L per well in 96 well cell culture plates in 5% CO ₂ And conventionally culturing in an incubator at the saturation humidity of 37 ℃, and after cells adhere to the wall for 3-5 hours, respectively adding 100 mu L of culture medium containing different concentrations of CET (0.5,1,2,4,8) except a control group, wherein each group is provided with 4 multiple wells. After 48h of culture, adding 10% of CCK8 culture medium into each well, continuously culturing for 1.5h in an incubator, and detecting the absorbance OD value at the wavelength of 450nm by using an enzyme-labeling instrument. The growth inhibition rate and the drug Resistance index (RF) of the cells were calculated. Calculating the formula: the growth inhibition rate (1-OD average of experimental group/OD average of control group) × 100%. The above experiment was repeated 3 times.

Western Blot for detecting expression of EGFR, Akt and mTOR proteins in human colon cancer cells

Washing each group of cells with precooled PBS for 2 times, removing PBS by suction, adding precooled protein extraction reagent containing inhibitor, and gently shaking for 5 min; then scraping off cells on the wall of the culture flask by using precooled rubber and plastic cells, transferring the cell suspension into a centrifuge tube, and carrying out cracking for 15min in ice bath. Centrifuging the lysate in 14000g precooled centrifuge for 15min, and discarding the supernatant; BCA assay reagents determine protein concentration. After separation of 50. mu.g of total protein by SDS polyacrylamide gel electrophoresis, the proteins were electrotransferred to a PVDF membrane, which was incubated in 5% BSA solution for 1h at room temperature to block non-specific binding on the membrane. The antigen-antibody binding was achieved by adding the primary antibody to the blocked membrane overnight at 4 ℃. Washing the membrane with TBS/T for 5min for 3 times, adding HRP-labeled secondary antibody to combine the primary antibody and HRP-labeled anti-biotin antibody to combine with molecular weight standard, incubating the membrane for 1h at room temperature, and washing the membrane with TBS/T for 3 times, 5min for each time; the same method was used to label murine monoclonal anti-GAPDH as a control. After washing the membrane slightly dry, AB developer (conjugated with secondary antibody HRP) was added at 1:1 and developed on a Bio-Rad chemiluminescence imager. Then, the gray value is analyzed, and the gray coefficient ratio is calculated.

6. Cellular glucose uptake, lactate production and ATP content detection

And detecting the glucose uptake, the lactic acid production and the ATP content of the cells according to the glucose uptake colorimetric determination kit, the lactic acid determination kit II and the ATP colorimetric determination kit specification respectively. For the glucose uptake colorimetry, cells were seeded in 96-well plates at a density of 1500 per well. Cells were glucose starved by pre-incubation with 100mL KRPH buffer containing 2% BSA for 40min, followed by incubation with 10mL 10 mmol/L2-DG for 20 min. For lactic acid and ATP assays, 1X 10 was collected ⁶ Cells were homogenized in 100mL of assay buffer provided in the corresponding kit, samples were centrifuged, and the soluble fraction content was determined.

Seahorse energy metabolism analyzer for detecting relevant indexes of human colon cancer cell metabolism phenotype

The extracellular acidification rate (ECAR) and cellular Oxygen Consumption Rate (OCR) were measured using a SeahorseXF 96 extracellular flux analyzer. Cells were plated at 1X 10 according to ECAR kit instructions ⁴ The cells were inoculated in a density of one ml onto a plate dedicated to Seahorse Xfe96 and allowed to stand at room temperature for 1 hour. After baseline measurements were taken, glucose, the oxidative phosphorylation inhibitor oligomycin, and the glycolytic inhibitor 2-DG were injected into each well in sequence at the indicated time points, incubated at 37 ℃ for 30min, and tested on the machine. For OCR, oligomycin, a reversible oxidative phosphorylation inhibitor p-trifluoromethylcarbonylcyanide phenylhydrazone (FCCP) and a mitochondrial complex I inhibitor rotenone plus mitochondrial complex III inhibitor antimycin A (Rote/AA) were injected sequentially. Data were evaluated by Seahorse XF-96Wave software. OCR is shown in pmols/min and ECAR is shown in mpH/min.

The results show that

1. Influence of Zuojin pill extract and cetuximab on weight condition of colorectal cancer orthotopic transplantation tumor nude mice

The abdomen of the mice in the normal saline control group after molding gradually swells, the activity is less, and the sensitivity is poor. Compared with a normal saline control group, the mice in the traditional Chinese medicine group and the cetuximab treatment group are agile in activity, good in mental state and agile in activity. After 15 days of administration, the body mass of each group of mice began to decrease compared to the normal saline control group; when the composition is administrated for 28 days, the body mass of mice in the treatment group of the zuojin pill and the cetuximab is obviously different from that of a normal saline control group, and the composition has statistical significance.

2. Influence of Zuojin pill extract and cetuximab on colorectal cancer in-situ transplantation tumor quality and tumor inhibition rate

Compared with a model group, the traditional Chinese medicine group and the cetuximab treatment group have unobvious change of tumor volume, but the tumor volume of the combined group is greatly reduced (P is less than 0.01);

TABLE 1 influence of Zuojin pill extract on the quality and tumor inhibition rate of in-situ transplanted tumor of nude mice with colorectal cancer in each group

Note: comparison with model group P <0.01

3. Effect of Zuojin pill extract and cetuximab on lung metastasis of colorectal cancer orthotopic transplantation tumor nude mice

From the lung tissue HE staining result after the mouse is dissected, obvious metastasis foci can be seen on the lung of the mouse in the model group, and the tumor focus areas in the lung tissues of the mouse in the traditional Chinese medicine group and cetuximab treatment group are smaller than those of the normal saline control group and the cetuximab treatment group, which shows that the traditional Chinese medicine group and the cetuximab treatment group can inhibit lung metastasis of the intestinal cancer orthotopic transplantation tumor animal model. See fig. 2.

Determination of dosage of water extract of Zuojin pill

According to the formula of the cell survival rate, the IC of the SW620 cells acted by the Zuojin pill for 24h is calculated by using the detection result of CCK-8 ₅₀ 48h IC 100.4 ug/mL ₅₀ 73.09 ug/mL, 72h IC ₅₀ 65.29 μ g/mL. The proliferation effect on SW620 cells is gradually enhanced along with the increase of the action time of the Zuojin pill, which indicates that the Zuojin pill inhibits the proliferation of colorectal cancer cells in a time and dose dependent manner. In accordance withAccording to the previous experimental results, IC lower than 72h is adopted ₅₀ And IC ₁₀ 15 μ g/mL as a non-toxic dose (IC) ₁₀ 18.45 μ g/mL) was used in subsequent experiments (see fig. 3).

Regulation effect of 5-zuojin pill aqueous extract and CET on SW620 cell targeted therapy

The CCK-8 test result shows that the inhibition rate of the ZJW combined CET group on SW620 cells is higher than that of the CET group (10 mu g/mL) under each concentration, and the inhibition rate of the ZJW combined CET group on the SW620 cells is obviously dose-dependent along with the increase of the dose of the CET at 24h, 48h and 72h (as shown in a figure 4).

Influence of aqueous extract of Zuojin pill combined with CET on EGFR signaling pathway of SW620 cells of human colon cancer

The Western Blot result shows that the EGFR, Akt, mTOR proteins and phosphorylated proteins thereof are highly expressed in SW620 cells, which indicates that upstream and downstream proteins of an EGFR signaling pathway are in an activated state in Kras mutated SW620 cells. Compared with the control group, the expression of EGFR, Akt and mTOR proteins is not obviously changed after CET is added (P is more than 0.05). However, the phosphorylation levels of P-EGFR, P-Akt and P-mTOR were significantly inhibited 72h after the combined group of levo-gold pellets and CET interfered with SW620 cells (P <0.01), as shown in FIG. 5. The result indicates that the combination of the Zuojin pill and the CET can reduce the phosphorylation degree of EGFR, Akt and mTOR proteins in SW620 cells and inhibit the activation of EGFR signaling pathway.

7 Regulation effect of Zuojin pill water extract and CET on SW620 cell glycolysis

Cells can produce large quantities of H during glycolysis ⁺ And lactic acid, these H ⁺ And lactic acid entering the intercellular space results in a decrease in PH and extracellular acidification, thus ECAR is an important indicator for detecting cellular glycolysis. Compared with the control group, the glucose uptake, the lactic acid production, the ATP content and the ECAR in the cells of the CET group are obviously reduced, which indicates that the glycolysis phenomenon of SW620 cells is inhibited after the CET treatment; compared with the CET group alone, the ZJW combined CET group has obvious reduction in glucose uptake, lactic acid production, ATP content and ECAR, and the difference has statistical significance P<0.01。

See specifically fig. 6A; FIG. 6B; FIG. 6C; FIG. 6D; fig. 6E.

Aiming at the defect that the curative effect of the targeted medicament taking the epidermal growth receptor as the target in the prior art is reduced, the invention provides a novel targeted medicament for treating colorectal cancer, and particularly relates to a targeted medicinal composition for treating colorectal cancer.

The targeted pharmaceutical composition of the invention is used for the research on pharmacodynamics, pharmacology and early safety of the in vivo tumor-bearing nude mouse model level synergistic multi-target anti-EGFR, and proves that the targeted pharmaceutical composition can improve the general condition of a colorectal cancer animal model and inhibit the growth and transfer of colorectal cancer.

The targeted pharmaceutical composition can block the activation of signal protein closely related to EGFR and inhibit the expression of downstream transcription factors. The activity of proteins such as p-EGFR, p-Akt, p-mTOR and the like in colorectal cancer cells is reduced after the combination of the levorotary pill and the CET, and compared with the single CET, the levorotary pill can relieve the expression of protein phosphorylation such as EGFR, Akt, mTOR and the like related to an EGFR signal pathway in the colorectal cancer cells caused by the CET.

The research also observes that the combination of the zuojin pill and the cetuximab can obviously reduce the glucose intake, the lactic acid production, the ATP content and the ECAR in the cells, and further proves that the zuojin pill reverses the targeted drug resistance of the cetuximab and can play a role by inhibiting the glycolysis pathway of drug-resistant cells.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (4)

1. A targeted pharmaceutical composition for treating colorectal cancer is characterized by consisting of a water extract of Zuojin pills and cetuximab.

2. The pharmaceutical composition of claim 1, wherein the ratio of aqueous extract of zuojin pill to cetuximab is: 22.5:1.

3. The pharmaceutical composition of claim 1, wherein the aqueous extract of Zuojin pill is obtained by the following method: taking coptis chinensis: the evodia rutaecarpa 6 is extracted at a ratio of 1, and the yield of the aqueous extract of the Zuojin pill is 1g of crude drug/0.071 g of aqueous extract.

4. Use of a pharmaceutical composition according to any one of claims 1-3 for the manufacture of a targeted medicament for the treatment of colorectal cancer.

Images