WO2023185681A1 - Use of recombinant hirudin in preparation of drug for treating liver cancer - Google Patents

Abstract

Provided in the present invention is the use of recombinant hirudin in the preparation of a drug for treating liver cancer, in the preparation of a drug for inhibiting the growth of liver cancer, in the preparation of a drug for inhibiting liver cancer metastasis, and in the preparation of a drug for inhibiting liver cancer migration and/or inhibiting liver cancer invasion. The present invention finds that recombinant hirudin can inhibit the proliferation, migration, adhesion and invasion of liver cancer cells by means of targeting thrombin, and can also inhibit angiogenesis while inducing tumor cell apoptosis.

Description

Application of recombinant hirudin in the preparation of drugs for the treatment of liver cancer Technical field

The invention belongs to the field of biotechnology and relates to the application of recombinant hirudin in the preparation of drugs for treating liver cancer.

Background technique

Hepatocellular carcinoma (HCC, referred to as liver cancer) is the most common primary liver cancer worldwide, and the cause of death ranks second among various cancers. Although significant progress has been made in the diagnosis and treatment of liver cancer, its prognosis remains poor, with a 5-year overall survival (OS) rate of only 12% for all stages combined. Therefore, it is particularly necessary to explore new treatment methods to further improve patient prognosis and quality of life.

Hirudin is the main active ingredient in traditional Chinese medicine leeches. It has the effects of breaking blood, removing blood stasis, eliminating symptoms, and stimulating menstruation. It is a good medicine for treating lumps, blood stasis, and amenorrhea. Since hirudin only exists in the salivary glands of leeches, the content is very small. In 1986, Harvey used genetic engineering methods to synthesize recombinant hirudin (Recombinant Hirudin, rH), overcoming the difficulty of sources of natural hirudin. The emergence of recombinant hirudin has opened up new avenues for the research of anticoagulant and antithrombotic drugs.

invention disclosure

The object of the present invention is to provide the application of recombinant hirudin in the preparation of drugs for treating liver cancer.

The present invention provides the use of recombinant hirudin in preparing drugs for treating liver cancer.

The present invention also provides the use of recombinant hirudin in preparing drugs for inhibiting the growth of liver cancer tumors.

The invention also provides the use of recombinant hirudin in preparing drugs for inhibiting liver cancer metastasis.

The present invention also provides the use of recombinant hirudin in preparing drugs that inhibit liver cancer migration and/or inhibit liver cancer invasion.

The invention also provides the application of recombinant hirudin in the preparation of products; the functions of the products are as follows (a) or (b) or (c) or (d):

(a) Inhibit the hematogenous spread of liver cancer cells;

(b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

(c) Inhibit vascular invasion of liver carcinoma in situ;

(d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.

The invention also provides the application of recombinant hirudin in the preparation of products; the function of the product is to inhibit the proliferation of liver cancer cells and/or promote the apoptosis of liver cancer cells and/or inhibit the survival of liver cancer cells and/or inhibit the migration of liver cancer cells and/or Inhibit liver cancer cell invasion and/or inhibit liver cancer cell adhesion.

In any of the above applications, the recombinant hirudin acts through targeting thrombin.

In any of the above applications, the recombinant hirudin exerts its effects through targeting PAR-1.

In any of the above applications, the recombinant hirudin exerts its effects through the PI3K/Akt pathway.

The invention also provides a medicine for treating liver cancer, which contains recombinant hirudin.

The present invention also provides a drug containing recombinant hirudin; the function of the drug is to inhibit liver cancer tumor growth and/or inhibit liver cancer metastasis and/or inhibit liver cancer migration and/or inhibit liver cancer invasion.

The invention also provides a product containing recombinant hirudin; the functions of the product are as follows (a) or (b) or (c) or (d):

(a) Inhibit the hematogenous spread of liver cancer cells;

(b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

(c) Inhibit vascular invasion of liver carcinoma in situ;

(d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.

The invention also provides a product, which contains recombinant hirudin; the function of the product is to inhibit the proliferation of liver cancer cells and/or promote the apoptosis of liver cancer cells and/or inhibit the survival of liver cancer cells and/or inhibit the migration of liver cancer cells and/or Inhibit liver cancer cell invasion and/or inhibit liver cancer cell adhesion.

The invention also provides the application of recombinant hirudin in treating liver cancer.

The present invention also provides the application of recombinant hirudin in inhibiting the growth of liver cancer tumors.

The present invention also provides the application of recombinant hirudin in inhibiting liver cancer metastasis.

The present invention also provides the application of recombinant hirudin in inhibiting liver cancer migration and/or inhibiting liver cancer invasion.

The invention also provides the application of recombinant hirudin, which is as follows (a) or (b) or (c) or (d):

(a) Inhibit the hematogenous spread of liver cancer cells;

(b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

(c) Inhibit vascular invasion of liver carcinoma in situ;

(d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.

The present invention also provides the application of recombinant hirudin; the application is to inhibit the proliferation of liver cancer cells and/or promote the apoptosis of liver cancer cells and/or inhibit the survival of liver cancer cells and/or inhibit the migration of liver cancer cells and/or inhibit the invasion of liver cancer cells and/or Or inhibit liver cancer cell adhesion.

In any of the above medicines, the recombinant hirudin exerts its effects through targeting thrombin.

In any of the above products, the recombinant hirudin exerts its effects through targeting thrombin.

In any of the above applications, the recombinant hirudin acts through targeting thrombin.

In any of the above drugs, the recombinant hirudin exerts its effects through targeting PAR-1.

In any of the above products, the recombinant hirudin exerts its effects through targeting PAR-1.

In any of the above applications, the recombinant hirudin exerts its effects through targeting PAR-1.

In any of the above drugs, the recombinant hirudin exerts its effects through the PI3K/Akt pathway.

In any of the above products, the recombinant hirudin exerts its effects through the PI3K/Akt pathway.

In any of the above applications, the recombinant hirudin exerts its effects through the PI3K/Akt pathway.

Any of the above liver cancers may specifically be hepatocellular carcinoma.

Specifically, the recombinant hirudin is a product of Tianjin Hemu Jianmin Biotechnology Co., Ltd.

Specifically, the recombinant hirudin is shown in Sequence 1 of the sequence list in the patent announcement text of CN101250530B.

Specifically, the recombinant hirudin is expressed from a DNA molecule as shown in sequence 2 of the sequence list in the patent announcement text of CN101250530B.

Specifically, the recombinant hirudin is the recombinant hirudin expressed by Hansenula polymorpha 205-17CGMCC No. 2424 in the patent announcement text of CN101250530B.

Specifically, the recombinant hirudin is the recombinant hirudin obtained in paragraph 0084 of the patent announcement text of CN101250530B.

In practical applications, recombinant hirudin can be administered directly to patients, or mixed with a suitable carrier or excipient and then administered to patients to achieve therapeutic purposes. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, freeze-dried powder injections, etc. It can be ordinary preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems. To formulate unit dosage forms into tablets, a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbing agents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and adhesives, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia glue, gelatin slurry, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, agar powder, fucoid starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promotion Agents, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets. To formulate unit dosage forms into pills, a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, gum tragacanth, and gelatin. , ethanol, honey, liquid sugar, rice cereal or batter, etc.; disintegrating agents, such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc. To formulate a unit dosage form as a suppository, a wide variety of carriers known in the art may be used. Examples of the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like. In order to formulate unit dosage forms into injection preparations, such as solutions, emulsions, lyophilized powder injections and suspensions, all diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation. In addition, conventional co-solvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection.

The dosage of recombinant hirudin depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route and frequency of administration wait. The above dosage may be in the form of a single dose or divided into several, for example two, three or four doses Dosage form.

Description of drawings

Figure 1 shows the cell proliferation rate results and IC ₅₀ value results in Example 1.

Figure 2 shows the results of cell proliferation rate in Example 2.

Figure 3 shows the results of the scratch test to measure tumor cell migration in Example 2.

Figure 4 is the results of the Transwell assay to measure tumor cell invasion in Example 2.

Figure 5 shows the results of adhesion of recombinant hirudin to BEL-7402 cells in Example 2.

Figure 6 is the detection results of the expression of caspase-3, Bax and Bcl-2 in Example 2.

Figure 7 is the detection result of the number of TUNEL-positive cells in Example 2.

Figure 8 shows the results of the role of the PI3K/Akt pathway in recombinant hirudin-induced cell apoptosis in Example 2.

Figure 9 shows the results of cell proliferation rate in Example 3.

Figure 10 shows the results of the scratch test in Example 3.

Figure 11 shows the results of the Transwell experiment in Example 3.

Figure 12 shows the results of the adhesion test in Example 3.

Figure 13 is the detection results of caspase-3 and Bax expression in Example 3.

Figure 14 is the detection result of the number of TUNEL-positive cells in Example 3.

Figure 15 shows the results in Example 3 that recombinant hirudin can inhibit thrombin-induced tumor angiogenesis.

Figure 16 shows the changes in animal body weight and transplanted tumor volume with administration time in step 1 of Example 4.

Figure 17 is a photograph of the animal sacrificed and a photograph of the tumor tissue in step 1 of Example 4.

Figure 18 is a photo of H&E staining of tumor tissue in step 1 of Example 4.

Figure 19 shows the changes in animal body weight with administration time in step 2 of Example 4.

Figure 20 is a photo of H&E staining in step 2 of Example 4.

Figure 21 shows the changes in animal body weight over time in step three of Example 4.

Figure 22 shows the results of animal liver/body ratio and liver volume in step three of Example 4.

Figure 23 is a photograph of the liver in step three of Example 4 and a photograph of H&E staining.

Figure 24 is a photograph of the liver and the absorbance results of the homogenate supernatant in step three of Example 4.

Figure 25 shows the results of Westren Blot detection (caspase-3, Bax and Bcl-2) in step three of Example 4.

Figure 26 shows the effect of recombinant hirudin on the TUNEL-positive area of orthotopic carcinoma model mice in step three of Example 4.

Figure 27 shows the results of Westren Blot detection in step three of Example 4 (phosphorylated PI3K and phosphorylated Akt).

Figure 28 is the result of the bleeding time of the mouse tail tip in step three of Example 4.

Best way to implement your invention

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples were repeated three times, and the results were averaged. In the embodiment, TUNEL apoptosis detection kit is used to detect cell apoptosis. 5-Fluorouracil (CAS number: 51-21-8): Beijing Solebao Technology Co., Ltd. The recombinant hirudin used in the examples is a product of Tianjin Hemu Jianmin Biotechnology Co., Ltd. (For the preparation method, please refer to the patent document with the announcement number CN101250530B, that is, the recombinant hirudin obtained in paragraph 0084, the application number of the patent document is 200810103154.8) . BEL-7402 cells (human liver cancer cells), Huh-7 cells (human liver cancer cells), HepG2 cells (human liver cancer cells), L-O2 cells (human normal liver cells), Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cell culture conditions: in an incubator at 37°C, 5% CO ₂ and 100% humidity. Complete culture medium: containing 1% penicillin-streptomycin and 10% fetal calf serum, the balance is RPMI-1640 medium. Serum-free culture medium: contains 1% penicillin-streptomycin, and the balance is RPMI-1640 medium. The experimental data in the examples are expressed as mean ± standard error (Mean ± SD), and SPSS 21.0 statistical software is used for analysis; the mean between the two groups is tested using t, and p<0.05 is statistically significant.

Example 1. IC ₅₀ value of recombinant hirudin against three types of liver cancer cells

Test cells: BEL-7402 cells, Huh-7 cells, HepG2 cells.

Test drug: recombinant hirudin.

1. Inoculate the test cells into a 96-well cell culture plate (4000-5000 cells per well) and culture them in complete culture medium until the cells adhere to the wall and the growth density is about 50%.

2. After completing step 1, discard the culture supernatant, add 100 μL of serum-free culture medium containing the test drug to each well, and culture for 24 hours. Set different concentrations of the test drug and set a control without adding the test drug. Set up 6 duplicate wells for each concentration treatment.

3. After completing step 2, discard the culture supernatant, wash with PBS buffer, then add 100 μL CCK-8 solution to each well, and incubate for 1-2 hours.

4. After completing step 3, use a microplate reader to measure the absorbance value (OD value) at 450 nm and calculate the cell proliferation rate.

Cell proliferation rate = (OD _{control well} – OD _{drug well} ) ÷ OD _{control well} × 100%.

The drug concentration at which the cell proliferation rate is 50% is the IC ₅₀ value.

The results of cell proliferation rate and IC ₅₀ value are shown in Figure 1. The IC ₅₀ value of recombinant hirudin against BEL-7402 cells is 192.8 μmol/L. The IC ₅₀ value of recombinant hirudin against HepG2 cells is 241.9 μmol/L. The IC ₅₀ value of recombinant hirudin against Huh-7 cells is 387 μmol/L.

Example 2. Effect of recombinant hirudin on liver cancer cells (cell test)

1. Effect of recombinant hirudin on cell proliferation

Test cells: BEL-7402 cells, L-O2 cells.

Test drug: recombinant hirudin.

The test method is the same as in Example 1.

The results of cell proliferation rate are shown in Figure 2 (compared with the control group, ^* p<0.05). Recombinant hirudin on L-O2 cells There is not much impact on the proliferation and growth (left picture), and only a certain inhibitory effect is produced at higher doses (160μM and 320μM). Recombinant hirudin has an inhibitory effect on BEL7402 cells at a dosage concentration of 20 μM (right picture). The inhibitory rate increases with the concentration, showing a dose-dependent effect.

2. Effect of recombinant hirudin on migration and invasion of BEL-7402 cells

Test drugs: recombinant hirudin or 5-fluorouracil (positive control drug).

1. Scratch assay to measure tumor cell migration

(1) Use a marker to draw three horizontal lines on average on the back of the 6-well plate, and then inoculate BEL-7402 cells in the logarithmic growth phase into the 6-well plate (3×10 ⁵ -5×10 ⁵ cells per well) , use complete culture medium to culture until the cell density is greater than 70%.

(2) After completing step (1), discard the culture supernatant, use a sterile pipette tip to scratch the cell surface perpendicularly to the well line, and then wash it 2-3 times with PBS buffer to remove the scratched cells and debris. .

(3) After completing step (2), add serum-free culture medium containing the test drug, mark the intersection of the horizontal line and the scratch with a marker, and take photos under an inverted microscope to record the 0h situation. Set different test drug concentrations and set a control (Control) without adding the test drug. Three duplicate wells were set for each concentration treatment.

(4) After completing step (3), continue to culture for 6h, 12h or 24h, take photos and observe under an inverted microscope, and record the cell migration.

The results are shown in Figure 3 ( ^* p<0.05, ^** p<0.01 compared with the control group).

2. Transwell assay to measure tumor cell invasion

(1) Select a transwell chamber with a membrane pore size of 8 μm, dilute Matrigel to 8 times the volume with serum-free culture medium, then coat the upper chamber surface of the bottom membrane with a dosage of 40 μl/chamber, and let stand at 37°C for 3 hours to allow Matrigel to dissolve. polymerize to form a gel.

(2) After completing step (1), add 600 μl of RPMI-1640 medium containing 10% FBS to the lower chamber, sterilize the chamber with tweezers and gently place it into the well plate, and add BEL-7402 in the logarithmic growth phase to the upper chamber. Cells (1.5×10 ⁵ cells per well) were cultured for 24 h in serum-free culture medium containing test drugs. Set different test drug concentrations and set a control (Control) without adding the test drug. Three duplicate wells were set for each concentration treatment.

(3) After completing step (2), suck up the remaining culture medium liquid in the upper chamber, gently wipe off the cells in the upper chamber with a cotton swab, add 1ml of methanol to fix for 10min-20min, discard the fixative, and wash with PBS buffer for 2-3 once, stain with crystal violet dye for 30 minutes, and then wash with PBS buffer 3 times to wash away the crystal violet and non-specific binding dye that are not bound to the cells. Then place the chamber on a glass slide and observe it under an inverted microscope. Randomly 5 Count of photos taken on each interface.

The results are shown in Figure 4 ( ^* p<0.05, ^** p<0.01 compared with the control group).

The results of step two showed that compared with the control group, recombinant hirudin at concentrations of 40, 80 and 160 μM significantly reduced the wound healing of BEL-7402 cells, reduced the number of transmembrane cells, and was more effective than 5-fluorouracil. This shows that recombinant hirudin inhibits the migration and invasion abilities of cancer cells in a dose-dependent manner.

3. Effect of recombinant hirudin on BEL-7402 cell adhesion

Test drugs: recombinant hirudin or 5-fluorouracil (positive control drug).

1. Dilute FN fibronectin (Beijing Solebao Technology Co., Ltd.) to 10 in serum-free culture medium μg/ml is the FN dilution.

2. Take a 96-well plate, add 50 μl FN diluent to each well, and let stand at 4°C overnight.

3. After completing step 1, discard the liquid in the wells, wash with 1% BSA solution, then add 50 μl of 1% BSA solution to each well, and incubate at 37°C for 1 hour.

4. After completing step 3, discard the liquid in the wells, add 4×10 ⁴ BEL-7402 cells to each well, and culture for 2 hours in serum-free culture medium containing the test drug. Set different test drug concentrations and set a control (Control) without adding the test drug. Three duplicate wells were set for each concentration treatment.

5. After completing step 4, discard the liquid in the well, wash 3 times with PBS buffer to wash away unadhered cells, fix with cell fixative for 15-30 minutes, wash off the fixative, and then use 0.5% crystal violet solution Stain for 20 minutes, then wash 2-3 times with PBS buffer, randomly select 4 fields of view in the 96-well plate to take pictures of the adherent cells and count the cells.

6. After completing step 5, soak the cells stained with crystal violet with 30% acetic acid solution, extract the dye, and measure the absorbance value with a microplate reader at 600 nm.

The results are shown in Figure 5 ( ^* p<0.05, ^** p<0.01 compared with the control group). The results showed that BEL-7402 cells showed obvious central aggregation and adhesion phenomena when induced by adhesion factors. Recombinant hirudin treatment could significantly alleviate the adhesion of cancer cells to the matrix in a dose-dependent manner.

4. Recombinant hirudin induces apoptosis in BEL-7402 cells

In order to further explore the mechanism by which recombinant hirudin induces BEL-7402 cell death and exerts a protective effect, the inventors detected the expression of anti-apoptotic proteins and pro-apoptotic proteins. It was found that recombinant hirudin significantly increased the expression of cleaved caspase-3 and Bax and decreased the expression of Bcl-2, which was accompanied by an increase in the number of TUNEL-positive cells. The results are shown in Figure 6 ( ^* p<0.05, ^** p<0.01 compared with the control group) and Figure 7 ( ^** p<0.01 compared with the control group).

The PI3K/Akt signaling pathway plays an important role in regulating cell cycle, proliferation and apoptosis. To study the role of the PI3K/Akt pathway in apoptosis induced by recombinant hirudin, the activation of PI3K and Akt was detected by Western blotting using phosphoantibodies. The results are shown in Figure 8 ( ^* p<0.05, ^** p<0.01 compared with the control group). Treatment with recombinant hirudin reduced the levels of phosphorylated PI3K and phosphorylated Akt in BEL-7402 cells.

The results of Example 2 show that recombinant hirudin (rH) can inhibit the proliferation of liver cancer cells BEL-7402, but does not affect the growth and proliferation of normal liver cells; at the same time, the administration of rH can significantly inhibit the migration, invasion and adhesion of tumor cells. Attachment, and induces apoptosis of tumor cells in a dose-dependent manner.

Example 3. Cellular inhibition is mediated by targeting thrombin

Thrombin: Beijing Solebao Technology Co., Ltd.

1. Recombinant hirudin can inhibit the abnormal growth of tumor cells induced by thrombin

In order to clarify the specific mechanism by which recombinant hirudin exerts its protective effect, the inventor further used thrombin (1U/mL) to induce BEL-7402 cells in vitro and conducted experiments related to cell proliferation and migration.

See Example 2 for test methods.

The results of cell proliferation rate are shown in Figure 9 (compared with the thrombin induction group, ^** p<0.01; compared with the control group, ^## p<0.01). The scratch test results are shown in Figure 10 (compared with the thrombin induction group, ^** p<0.01; compared with the control group, ^#p <0.05). The results of the Transwell experiment are shown in Figure 11 (compared with the thrombin induction group, ^** p<0.01; compared with the control group, ^## p<0.01). The adhesion test results are shown in Figure 12 (compared with the thrombin induction group, ^** p<0.01; compared with the control group, ^## p<0.01).

The results show that thrombin induction can significantly promote the proliferation and wound healing of tumor cells, improve the invasion ability of tumor cells through the matrix and the adhesion effect to the matrix; with the induction of thrombin, the protective effect of recombinant hirudin can be partially antagonized. , causing the protective effect originally produced at a lower dose (20 μM) to disappear, but as the concentration of recombinant hirudin increases, it can effectively reverse the abnormal growth of tumor cells caused by thrombin induction, and at a certain dose dependency.

2. Recombinant hirudin can reverse the reduction of tumor cell apoptosis induced by thrombin

The apoptosis-promoting protein Bax and cleaved caspase-3 protein were significantly reduced under the action of thrombin, indicating that the induction of thrombin can reduce apoptosis in tumor cells and inhibit the apoptosis-promoting effect of recombinant hirudin on tumor cells; however, With the administration of recombinant hirudin, the above phenomenon was effectively improved, accompanied by an increase in the number of TUNEL-positive cells, indicating that recombinant hirudin can effectively reverse the reduction of tumor cell apoptosis induced by thrombin.

The results are shown in Figure 13 (compared with the thrombin induction group, ^* p<0.05, ^** p<0.01; compared with the control group, ^## p<0.01) and Figure 14.

3. Recombinant hirudin can inhibit thrombin-induced tumor angiogenesis

Abnormal angiogenesis is an important influencing factor in the tumor microenvironment. It is precisely because of this characteristic that there are a large number of growth factors, cell chemokines and various proteolytic enzymes in the tumor microenvironment that produce immune and inflammatory reactions. , these characteristics are very conducive to tumor proliferation, invasion, adhesion, angiogenesis and resistance to radiotherapy and chemotherapy, promoting the generation and metastasis of malignant tumors. Among them, Ang-I, Ang-Ⅱ and VEGFA are closely related to angiogenesis and blood vessel maturation. Close albumen. In order to determine the effect of recombinant hirudin on reversing thrombin-induced angiogenesis, the inventors further detected the changes in the above indicators.

The results are shown in Figure 15 (compared with the thrombin induction group, ^* p<0.05, ^** p<0.01; compared with the control group, ^## p<0.01). In response to thrombin induction, thrombin receptor PAR-1 was significantly increased compared with the non-induced group, accompanied by a significant increase in the expression of Ang-I, Ang-II and VEGFA, indicating that thrombin can induce the expression of the above proteins and further promote Tumor neovascularization. The administration of recombinant hirudin can significantly inhibit the expression of PAR-1 protein and alleviate the above-mentioned phenomenon, reducing angiogenesis-related proteins to the original level or even below the original level, indicating that recombinant hirudin can effectively inhibit angiogenesis induced by thrombin.

The results of Example 3 show that recombinant hirudin (rH) can inhibit the proliferation, migration, invasion and adhesion of BEL-7402 cells, and induce apoptosis of tumor cells. Treatment with the drug can effectively inhibit angiogenesis-related processes in tumor cells. The expression of the protein is dose-dependent; moreover, the anti-tumor effect of recombinant hirudin is exerted by targeted inhibition of thrombin.

Example 4. Recombinant hirudin inhibits tumor growth and angiogenesis in vivo

The experimental animals were male BALB/c-nude mice 4-6 weeks old; Nanjing University-Nanjing Institute of Biomedicine, animal use license number: SCXK (Su) 2015-0001. Raised in SPF environment.

1. Recombinant hirudin inhibits the growth of axillary xenograft tumors in nude mice

1. Establishment of nude mouse xenograft tumor model

(1) Take BEL-7402 cells in the logarithmic growth phase, digest them, and then centrifuge to collect the cells. Wash the cells with PBS buffer, and then resuspend the cells in Matrigel diluent (dilute Matrigel to 8 times the volume with serum-free culture medium). , to obtain a cell suspension of 5×10 ⁸ cells/mL.

(2) Inoculate the cell suspension prepared in step (1) subcutaneously into the right armpit of the test animal, 100 μL/animal. After normal feeding, in about a week, indurations the size of rice grains can be observed under the skin at the inoculation site, indicating that the nude mouse xenograft tumor model was successfully established.

2. Administration and efficacy evaluation

Take a nude mouse xenograft tumor model and raise it normally until the tumor volume reaches 100mm ³ . The animals were then divided into six groups and administered drugs in groups. Tumor volume V = ab ² /2; a: the maximum long diameter of the transplanted tumor, b: the maximum transverse diameter of the transplanted tumor.

Model group (Model): subcutaneously administered twice a day (9:00/17:30), 0.4ml of normal saline each time;

Recombinant hirudin treatment group 1 (0.2mg/kg): The single dose of recombinant hirudin is 0.2mg/kg;

Recombinant hirudin treatment group 2 (0.4mg/kg): The single dose of recombinant hirudin is 0.4mg/kg;

Recombinant hirudin treatment group 3 (0.8mg/kg): The single dose of recombinant hirudin is 0.8mg/kg;

Recombinant hirudin treatment group 4 (1.6mg/kg): The single dose of recombinant hirudin is 1.6mg/kg;

5-Fu positive control group (20 mg/kg): The single dose of 5-fluorouracil is 20 mg/kg.

The recombinant hirudin treatment group was administered subcutaneously twice a day (9:00/17:30), and the volume of each administration was 0.4 ml (prepared with physiological saline).

The 5-Fu positive control group was administered intraperitoneally once a day.

Dosing is done in groups for 28 consecutive days. The animals were weighed every 4 days and the transplanted tumor volume was measured. After 28 days of administration in groups, the animals were sacrificed by cervical dislocation and photographed. The tumor tissue was peeled off and photographed. The tumor tissue was stained with H&E.

The changes in animal body weight and transplanted tumor volume with administration time are shown in Figure 16 (n=2).

Photos of the sacrificed animals and tumor tissue are shown in Figure 17.

Photos of H&E staining of tumor tissue are shown in Figure 18.

The results showed that the body weight of mice in the model group showed a downward trend, and the weight changes of each drug group remained basically stable during the drug administration period. The tumor volume growth trend was significantly weakened compared with the model group, indicating that the administration of recombinant hirudin can protect nude mice from transplantation. It can reduce the weight caused by tumors and at the same time slow down the growth trend of tumors, and has a good anti-tumor effect. The H&E staining results of tumor tissue showed that cells in the tumor tissue of the model group showed obvious phenomena such as chromatin edges and nuclear pyknosis, and the cancer cells had obvious atypia. Recombinant hirudin treatment can significantly alleviate the occurrence of the above phenomena.

2. Recombinant hirudin inhibits hematogenous metastasis of BEL-7402 liver cancer cells in nude mice

1. Take BEL-7402 cells in the logarithmic growth phase, digest them, and then centrifuge to collect the cells. Resuspend the cells in physiological saline to obtain a cell suspension.

2. Inject the cell suspension prepared in step 1 into the tail vein of the test animal, 5×10 ⁵ cells/animal.

3. Twelve hours after completing step 2, divide the medicine into five groups and administer the medicine in groups.

Model group (Model): subcutaneously administered twice a day (9:00/17:30), 0.4ml of normal saline each time;

Recombinant hirudin treatment group 1 (0.4mg/kg): The single dose of recombinant hirudin is 0.4mg/kg;

Recombinant hirudin treatment group 2 (0.8mg/kg): The single dose of recombinant hirudin is 0.8mg/kg;

Recombinant hirudin treatment group 3 (1.6mg/kg): The single dose of recombinant hirudin is 1.6mg/kg;

5-Fu positive control group (20 mg/kg): The single dose of 5-fluorouracil is 20 mg/kg.

The recombinant hirudin treatment group was administered subcutaneously twice a day (9:00/17:30), and the volume of each administration was 0.4 ml (prepared with physiological saline).

The 5-Fu positive control group was administered intraperitoneally once a day.

Dosing is done in groups for 28 consecutive days. The animals were weighed every 4 days. After 28 days of administration in groups, the animals were sacrificed by cervical dislocation, and liver, lung, and brain tissues were harvested. Parts were fixed with 4% formaldehyde, embedded in paraffin, and stained with H&E.

The changes in animal body weight with administration time are shown in Figure 19 (n=6). Within 14 days, the weight of mice in each group remained unchanged or increased steadily; after 14 days, the weight of mice in the model group decreased significantly. Although the weight of the low-dose and medium-dose groups of recombinant hirudin decreased, the trend was lower than that of the model group. The weight of the positive control group remained stable. This shows that after 14 days, the mice developed an obvious cachexia state due to hematogenous metastasis of tumor cells. The administration of recombinant hirudin can weaken or even reverse the above situation.

Photos of H&E staining are shown in Figure 20. All the organs of the mice in the model group had metastasis to varying degrees. The tumor cells were arranged in nests, sheets, and cords, with multi-nucleated fission. As the concentration of recombinant hirudin increased, the above symptoms were significantly improved.

3. Recombinant hirudin inhibits liver cancer in situ in BEL-7402 cells in nude mice

1. Take BEL-7402 cells in the logarithmic growth phase, digest them, and then centrifuge to collect the cells. Resuspend the cells in physiological saline to obtain a cell suspension.

2. Test animals (weighing about 20g) are anesthetized and fixed on the operating table in a supine position. Use iodophor and 70% ethanol solution to disinfect the skin of the upper abdominal surgical area, and then make an incision on the left liver of the upper abdomen. Make an oblique incision about 1 cm in length to expose the abdominal cavity layer by layer, and drag the left lobe of the liver out of the abdominal cavity with a sterile cotton swab for exposure. Then inject the cell suspension prepared in step 1 into the liver (4×10 ⁶ cells/animal), press with cotton swabs until hemostasis, suture with 5-0 sutures, and wipe the wound with gentamicin to prevent infection.

3. Group administration

The test animals in the control group were treated according to step 2, but no cell suspension was injected.

Model group (Model): Experimental animals that completed step 2 were administered subcutaneously twice a day (9:00/17:30), with 0.4ml of normal saline each time;

Recombinant hirudin treatment group 1 (0.4mg/kg): For experimental animals that have completed step 2, the single dose of recombinant hirudin is 0.4mg/kg;

Recombinant hirudin treatment group 2 (0.8mg/kg): For experimental animals that have completed step 2, the single dose of recombinant hirudin is 0.8mg/kg;

Recombinant hirudin treatment group 3 (1.6mg/kg): For experimental animals that have completed step 2, the single dose of recombinant hirudin is 1.6mg/kg;

5-Fu positive control group (20 mg/kg): For experimental animals that have completed step 2, the single dose of 5-fluorouracil is 20 mg/kg.

The recombinant hirudin treatment group was administered subcutaneously twice a day (9:00/17:30), and the volume of each administration was 0.4 ml (prepared with physiological saline).

The 5-Fu positive control group was administered intraperitoneally once a day.

Dosing is done in groups for 28 consecutive days.

4. During the group administration process, weigh the animals every 4 days. The changes in body weight over time are shown in Figure 21 (n=8). Compared with the blank group, the nude mice in the model group showed a significant downward trend in body weight and died due to cachexia. The administration of recombinant hirudin could significantly improve the above situation. The weight loss trend of nude mice increased at medium and high doses. .

5. After 28 days of group administration, the animals were sacrificed by cervical dislocation, liver tissue was taken, the liver/body ratio was calculated, and the liver volume was calculated using the drainage method. The results are shown in Figure 22 (compared with the model tumor-bearing group, ^* p<0.05, ^** p<0.01; compared with the blank control, ^## p<0.01) (n=8). Compared with the blank group, the liver/body ratio and liver volume increased significantly in the model group. With the administration of recombinant hirudin, the above situation was improved and showed a dose-dependent manner. High-dose recombinant hirudin can adjust the liver-body ratio of tumor-bearing mice. and liver volume approached normal levels.

6. After 28 days of administration in groups, the animals were sacrificed by cervical dislocation. The livers were removed and photographed. The liver, lung, and brain tissues were removed, fixed with 4% formaldehyde, embedded in paraffin, and stained with H&E. Photos of the liver and H&E stained photos are shown in Figure 23. As shown in the figure, compared with the blank group, the model group showed large-area tumor growth and infiltration and multiple tumor growth foci in the liver, and obvious metastasis and inflammatory damage occurred in the lungs and brain tissue. With the increase in the concentration of recombinant hirudin, the above symptoms were significantly improved. The area of liver tumors in the medium-dose and high-dose groups was significantly reduced, the number of growth lesions was significantly reduced, and the tumor metastasis in lung and brain tissue was significantly reduced, indicating that the recombinant hirudin was significantly reduced. The hormone can improve the symptoms of liver cancer in situ in BEL-7402 cells and improve the survival rate.

7. After 28 days of group administration, Evans Blue dye solution was injected into the tail vein (100 μl per mouse. The Evans Blue dye solution was obtained by diluting Evans Blue with physiological saline to 20 μg/μL; the eyes of the mice can be observed after the injection. , ears and limbs immediately turn blue), 4 hours after injection, the experimental animals were anesthetized and laparotomized, and normal saline (10 mL for each animal) was injected into the right ventricle for liver perfusion until the fluid flowing out of the right ventricle was bloodless. Take the liver and weigh it, drain and weigh the volume, soak it in N,N-dimethylformamide solution at a ratio of 150mg/2mL, homogenize it, extract it in a water bath at 60°C for 24 hours, centrifuge it at 12000g for 20 minutes, and take the supernatant Measure the absorbance at 600 nm with a UV spectrophotometer. The photos of the liver and the absorbance results of the homogenate supernatant are shown in Figure 24 (compared with the model tumor-bearing group, ^** p<0.01; compared with the blank control group, ^## p<0.01) (n=3). The results showed that the vascular leakage of nude mice in the model group was more obvious due to the influence of tumors. The administration of recombinant hirudin significantly changed the tumor size and also significantly reduced the extravasation of Evans Blue dye, indicating that recombinant hirudin can inhibit the vascular invasion of BEL-7402 cell liver carcinoma in situ in a dose-dependent manner.

8. After 28 days of group administration, the animals were sacrificed by cervical dislocation, the tumor tissue on the liver was taken, the total protein was extracted, and Westren Blot detection was performed. The results of the Westren Blot test are shown in Figure 25 (compared with the model tumor-bearing group, ^* p < 0.05, ^** p < 0.01) (n = 3) and Figure 27 (compared with the model tumor-bearing group, ^* p < 0.05, ^** p<0.01) (n=3). The effect of recombinant hirudin on the TUNEL-positive area in orthotopic carcinoma model mice is shown in Figure 26. The results showed that the pro-apoptotic protein Bax and cleaved caspase-3 were lowly expressed in tumor tissues, but with the treatment of recombinant hirudin, the protein levels increased significantly compared with the unmedicated model group, and the anti-apoptotic protein Bcl -2 shows an opposite trend; at the same time, it was found that recombinant hirudin can reduce the levels of phosphorylated PI3K and phosphorylated Akt in vivo, accompanied by tumor tissue The increase of TUNEL-positive cells was dose-dependent, and the above results showed the same trend as the in vitro results.

9. Recombinant hirudin changes the tail bleeding time of in situ cancer-bearing mice

Nude mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) 2 hours after the last administration. Use a vernier caliper to measure the tail diameter of each animal at a distance of 1 cm from the tail tip and record it. Mice with a tail diameter of 1.108-1.110 mm were selected for the next experiment. Use surgical scissors to quickly cut the animal 1.5cm away from the tip of the tail. Start timing when blood appears. Then dip the prepared filter paper into the tail every 30 seconds until the tail stops bleeding. The bleeding time of the tail tips of mice in each group was recorded (i.e., the time when the tail of the mice stopped bleeding - the time when the bleeding started).

The results are shown in Figure 28 (compared with the model tumor-bearing group, ^* p<0.05, ^** p<0.01; compared with the blank control group, ^## p<0.01) (n=8). Due to the influence of the tumor microenvironment, the blood of the tumor-bearing mice in the model group was in a hypercoagulable state. Compared with the blank group, which was dipped in a piece of filter paper with bleeding tail tips, the time for blood stains to appear on the filter paper piece in the model group was significantly reduced. After administration of recombinant hirudin , compared with the model group, the tail bleeding time of nude mice was significantly longer, but there were no subcutaneous bleeding points or uncontrollable bleeding, indicating that recombinant hirudin can alleviate the hypercoagulable state of the tumor microenvironment and has a certain degree of safety.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Industrial applications

The invention disclosed has the following effects: recombinant hirudin (rH) can inhibit the proliferation, migration, adhesion and invasion of liver cancer cells by targeting thrombin, inhibit angiogenesis and induce tumor cell apoptosis, has certain pharmacological activity, and is safe. efficient. The present invention provides a new strategy for developing therapeutic intervention drugs for hepatocellular carcinoma.

Claims (16)

1. Application of recombinant hirudin in the preparation of drugs for the treatment of liver cancer.
2. Application of recombinant hirudin in the preparation of drugs that inhibit the growth of liver cancer tumors.
3. Application of recombinant hirudin in the preparation of drugs that inhibit liver cancer metastasis.
4. Application of recombinant hirudin in the preparation of drugs that inhibit liver cancer migration and/or inhibit liver cancer invasion.
5. Application of recombinant hirudin in the preparation of products; the function of the product is as follows (a) or (b) or (c) or (d):

   (a) Inhibit the hematogenous spread of liver cancer cells;

   (b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

   (c) Inhibit vascular invasion of liver carcinoma in situ;

   (d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.
6. Application of recombinant hirudin in the preparation of products; the function of the product is to inhibit the proliferation of liver cancer cells and/or promote the apoptosis of liver cancer cells and/or inhibit the survival of liver cancer cells and/or inhibit the migration of liver cancer cells and/or inhibit the invasion of liver cancer cells and /or inhibit liver cancer cell adhesion.
7. A drug for treating liver cancer containing recombinant hirudin.
8. A medicine containing recombinant hirudin; the function of the medicine is to inhibit liver cancer tumor growth and/or inhibit liver cancer metastasis and/or inhibit liver cancer migration and/or inhibit liver cancer invasion.
9. A product containing recombinant hirudin; the function of the product is as follows (a) or (b) or (c) or (d):

   (a) Inhibit the hematogenous spread of liver cancer cells;

   (b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

   (c) Inhibit vascular invasion of liver carcinoma in situ;

   (d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.
10. A product containing recombinant hirudin; the function of the product is to inhibit liver cancer cell proliferation and/or promote liver cancer cell apoptosis and/or inhibit liver cancer cell survival and/or inhibit liver cancer cell migration and/or inhibit liver cancer cell invasion and /or inhibit liver cancer cell adhesion.
11. Application of recombinant hirudin in the treatment of liver cancer.
12. Application of recombinant hirudin in inhibiting liver cancer tumor growth.
13. Application of recombinant hirudin in inhibiting liver cancer metastasis.
14. Application of recombinant hirudin in inhibiting liver cancer migration and/or inhibiting liver cancer invasion.
15. The application of recombinant hirudin is as follows (a) or (b) or (c) or (d):

    (a) Inhibit the hematogenous spread of liver cancer cells;

    (b) Inhibit lung metastasis and/or brain metastasis of liver cancer;

    (c) Inhibit vascular invasion of liver carcinoma in situ;

    (d) Alleviate the hypercoagulable state of the tumor microenvironment of liver cancer.
16. The application of recombinant hirudin; the application is to inhibit the proliferation of liver cancer cells and/or promote the apoptosis of liver cancer cells Apoptosis and/or inhibit the survival of liver cancer cells and/or inhibit the migration of liver cancer cells and/or inhibit the invasion of liver cancer cells and/or inhibit the adhesion of liver cancer cells.