CN116637195A - Application of CCR4 antagonist as preparation of medicine for treating alveolar echinococcosis - Google Patents

Abstract

The invention discloses the application of a CCR4 antagonist as a medicine for treating alveolar echinococcosis. The present invention finds that the CCR4 antagonist can inhibit the growth of lesions in animals infected with Echinococcus multilocularis or alveolar coccus, can alleviate the liver damage of animals infected with Echinococcus multilocularis or alveolar coccus, and can also restore Echinococcus multilocularis or Echinococcus multilocularis. Effector functions of hepatic T cells in alveolar coccoid-infected animals. It shows that CCR4 antagonist can be used as a new type of anti-alveolar echinococcosis drug, thereby providing a new way for the treatment of alveolar echinococcosis, and has a good clinical application prospect.

Description

Application of CCR4 antagonist as preparation of medicine for treating alveolar echinococcosis

technical field

The invention relates to the application of a CCR4 antagonist as a medicine for treating alveolar echinococcosis in the field of biomedicine.

Background technique

Alveolar Echinococcoisis (AE) is a lethal parasitic disease caused by the larvae of Echinococcus multilocularis (E.m) parasitizing the human body. They all originate in the liver, and the lesions grow malignantly and invasively in the liver, similar to "liver cancer". The disease is distributed globally and seriously threatens human health and life safety.

At present, the treatment of AE patients is mainly based on surgical resection of lesions. However, most patients are often at an advanced stage when they seek medical attention. Only 30% of AE patients can be treated surgically. For patients, drug treatment is particularly important for this disease. Albendazole (ABZ) is the first choice clinical drug recommended by WHO for the treatment of echinococcosis. For AE patients undergoing surgery, ABZ needs to be taken continuously for more than 2 years after surgery, and regular reexamination within 10 years; Surgery patients can only take ABZ drug for long-term treatment, but the blood concentration of ABZ is low, the intestinal absorption is poor, and long-term use may cause various adverse reactions and drug resistance, etc., resulting in limited use and poor efficacy of the drug good. Therefore, in-depth research and development of effective drugs for the treatment of AE is of great significance for the prevention and treatment of echinococcosis.

CC chemokine receptor 4 (CCR4) is a G protein-coupled receptor encoded by the CCR4 gene and belongs to the CC chemokine receptor (CCR) family. Many studies have confirmed that the high expression of CCR4 is closely related to the invasion and poor clinical prognosis of hematological tumors and malignant solid tumors. In this regard, in recent years, targeting the chemokine receptor CCR4 blockade treatment strategy, the development of anti-CCR4 antibodies and antagonist drugs has shown great potential for the treatment of malignant tumors, and has entered clinical phase I or II test.

There is no relevant report on the use of CCR4 antagonists as targeted drugs to block alveolar echinococcosis.

Contents of the invention

The technical problem to be solved by the invention is how to treat alveolar echinococcosis.

In order to solve the problems of the technologies described above, the present invention firstly provides any of the following applications:

1. The application of substances that inhibit CCR4 gene expression or knock out CCR4 gene in the preparation of products for the treatment and/prevention of alveolar echinococcosis, or in the treatment of alveolar echinococcosis;

2. The application of substances that reduce the content or activity of CCR4 in the preparation of products for the treatment of alveolar echinococcosis, or the application in the treatment of alveolar echinococcosis;

3. The application of the CCR4 antagonist in the preparation of products for the treatment of alveolar echinococcosis, or the application in the treatment of alveolar echinococcosis.

In the above application, the CCR4 antagonist may be C-021.

In the above application, the treatment and/prevention of alveolar echinococcosis can be embodied in inhibiting the growth of lesions in animals infected with Echinococcus multilocularis or alveolar coccidia, or reducing the liver damage of animals infected with Echinococcus multilocularis or alveolar coccoccus , or restore the effector function of liver T cells in animals infected with Echinococcus multilocularis or alveolus.

In the above application, the product can be a drug.

The present invention also provides the application of the substance for inhibiting the expression of CCR4 gene or knocking out the CCR4 gene in the preparation of products for inhibiting the infection of Echinococcus multilocularis or alveolar coccidia, or the application in inhibiting the infection of Echinococcus multilocularis or alveolar coccoccus .

The present invention also provides the application of the substance for reducing the content or activity of CCR4 in the preparation of products for inhibiting Echinococcus multilocularis or alveolar coccidia infection, or the application in inhibiting the infection of Echinococcus multilocularis or alveolar coccoccus infection.

The invention also provides the application of the CCR4 antagonist in the preparation of products for inhibiting the infection of Echinococcus multilocularis or alveolar coccidia, or the application in inhibiting the infection of Echinococcus multilocularis or alveolar coccoccus.

Wherein, the CCR4 antagonist can be C-021.

In the above application, the product can be medicine or vaccine.

In the present invention, the liver damage can be reflected in the content of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in serum.

The recovery of the effector function of the liver T cells can be reflected in the enhanced ability of the liver CD4 ⁺ T cells (NK1.1 ^- CD3 ⁺ CD4 ⁺ ) to secrete effector molecules TNF-α, CD107a and IL-2, and/or secrete IL-10 Decreased capacity; and/or, decreased capacity of CD8 ⁺ T cells (NK1.1 ^- CD3 ⁺ CD8 ⁺ ) to secrete IL-10.

Experiments have proved that CCR4 antagonists have therapeutic effects on the growth inhibition of alveolar echinococcosis lesions and the recovery of liver function, which means that CCR4 antagonists can be used as new anti-alveolar echinococcosis drugs, thus providing a new way for the treatment of alveolar echinococcosis. It provides a new way and has a good clinical application prospect.

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

Description of drawings

Fig. 1 is a diagram showing the operation of CCR4 antagonist C-021 in treating alveolar echinococcosis mouse model.

Figure 2 is the evaluation of the therapeutic effect of CCR4 antagonist C-021 on alveolar echinococcosis mouse model. A: The general diagram of the parasitic lesion; B: The weight of the parasitic liver; C: The total weight of the lesion.

Figure 3 is the evaluation of the liver function of the alveolar echinococcosis mouse model treated with the CCR4 antagonist C-021.

Figure 4 shows the effect of CCR4 antagonist C-021 treatment on the ability of T cells to secrete cytokines in the liver of a mouse model of alveolar echinococcosis.

Detailed ways

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Example 1. Application of CCR4 antagonist C-021 as a drug for the treatment of alveolar echinococcosis.

Establishment of 1 alveolar echinococcosis-infected mouse animal model

1.1 Preparation of Echinococcus multilocularis protoscole

A breeder mouse that had been infected with alveolar coccus for more than 6 months in the abdominal cavity was killed by cervical dislocation, and then completely immersed in 75% ethanol aqueous solution for 1 minute to sterilize the body surface. Separate the lesion tissue of abdominal infection under aseptic conditions, wash the residual blood with sterile PBS, place it in a sterilized 100-mesh steel cell mesh (steel mesh), and put the steel mesh into a plate containing PBS solution In the process, cut up the lesion tissue, use the piston handle of a 5 milliliter (mL) syringe to fully grind it on the steel mesh, add PBS solution to the steel mesh, stir and grind the lesion tissue continuously, and collect the alveolar coccus protoscoleum by filtration . Use 0.1% methylene blue staining for 1 minute to calculate the insect body activity, and the activity above 95% can be used for subsequent model establishment.

1.2 Establishment of mouse liver animal model of alveolar coccus infection by portal vein

Adjust the concentration of the isolated alveolar coccoid protoscole, so that the total system is 200-300 microliters (μL) containing 2000 protoscole. Select 6- to 8-week-old female C57BL/6J mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), inject the alveolar coccus protoscoleum through the hepatic portal vein, and press the puncture point with a dry cotton swab for about 2 minutes (min) until hemostasis. The postoperative mice were resuscitated on a heating platform at 30°C until they woke up, and then they were put back into the cage for normal feeding.

2 CCR4 antagonist C-021 treatment can inhibit the growth of alveolar echinococcosis mouse model lesion and restore liver function

2.1 After the completion of step 1, 12 weeks after the alveolar coccidia infection, mice with uniform infection degree were selected to enter the pharmacodynamic experiment. The experimental groups were CCR4 antagonist C-021 treatment group (C-021, Tocris Bioscience, Catalog No.: 3581) and solvent control group (Con, sterile water), with 5 mice in each group; C-021 treatment group was given The dose was 10 μmol/kg mice, intraperitoneally injected once every 3 days, and the administration was maintained for 24 weeks (ie, 12 weeks of treatment), and the solvent control group was injected with an equal volume of sterile water. The specific operation is shown in Figure 1.

2.2 Evaluation of the therapeutic effect after 12 weeks of treatment with CCR4 antagonist C-021:

Liver weight (including the weight of the lesion): the mice were sacrificed, and the complete liver (including the parasitic lesion) of the mouse was removed by laparotomy, and the weight of the whole liver was weighed;

Lesion weight: kill the mice, open the abdomen to remove all lesion tissues in the mouse abdominal cavity, including liver parasitic lesions and parasitic lesions metastasized to the abdominal cavity, remove the host tissue, and weigh all the lesions;

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content: Take the peripheral blood of the mouse in a 1.5mL centrifuge tube, let it stand at 4°C for 60min, centrifuge at 3000rpm for 15min, transfer the separated serum to a clean centrifuge tube, take 150μL of serum and Mix with 150 μL sterilized pure water, and use Shenzhen Mindray Biomedical Electronics Co., Ltd. automatic biochemical analyzer BS-120 for detection. The ALT and AST detection kits are all products of Shenzhen Mindray Biomedical Electronics Co., Ltd. (ALT batch number: 140122001; AST lot number 140222001).

The experimental results are shown in Figure 2 and Figure 3. Compared with the solvent control group, the liver weight (including lesion weight) and lesion weight of the mouse model in the C-021 treatment group were significantly reduced (P<0.05), and the serum trough of the C-021 treatment group The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly lower than those in the solvent control group. The results indicated that the CCR4 antagonist C-021 could inhibit the growth of lesions in the mouse model of alveolar echinococcosis and alleviate liver injury through intraperitoneal administration.

3CCR4 antagonist C-021 treatment can restore the effector function of alveolar echinococcosis CD4 ⁺ T lymphocytes and promote the killing of Em

Isolation of mouse liver mononuclear cells: Step 2 After 12 weeks of treatment with the CCR4 antagonist C-021, the liver tissue samples of the mice in the C-021 treatment group (C-021) and the solvent control group (Con) were collected respectively, and used to contain Grind 0.2% BSA in PBS in a plate, filter through a stencil, and centrifuge at 4°C, 1500rpm for 5min, take the precipitate, and resuspend it with 6ml of 40% Percoll (GE Healthcare company) solution, and collect the lower layer of cell pellet by density gradient centrifugation (25° C., 2000 rpm, 20 min), lysed with erythrocyte lysate (Biolegend Company) on ice and protected from light for 10 min, and finally collected the cells and performed cell counting to obtain mouse liver mononuclear cells.

Take 1× ¹⁰⁶ hepatic mononuclear cells of C-021 treatment group and control group respectively, put them in 1.5mL centrifuge tube, and culture them in 200μL culture medium RPMI1640 (Clonetech) including 10% fetal bovine serum at 37℃ Cocktail stimulant (500-fold dilution, ebioscience) was added at the same time for 4 hours. Cells were collected and washed with PBS, blocked with CD16/32 antibody (Biolegend), and then labeled with fluorescent antibodies against NK1.1, CD3, CD4, and CD8 (Biolegend); , staining and marking intracellular cytokines IFN-γ, TNF-α, CD107, IL-2 and IL-10, etc., and detected by flow cytometry.

The experimental results are shown in Figure 4. Compared with the solvent control group mice (Con), the CCR4 antagonist C-021 treatment group mice (C-021) model liver CD4 ⁺ T cells secreted effector molecules TNF-α, CD107a and IL-2 The ability of CD4 ⁺ T and CD8 ⁺ T cells to secrete IL-10 decreased significantly (P<0.05).

These results suggest that intraperitoneal treatment with the CCR4 antagonist C-021 can restore the effector functions of hepatic T cells.

The above results show that the CCR4 antagonist C-021 treatment can effectively inhibit the growth of lesions in animals infected with Echinococcus multilocularis or alveolus, relieve liver damage, and enhance the secretion of effector factors TNF-α, CD107a and IL by hepatic CD4 ⁺ T cells. -2 ability, reducing the ability of liver CD4 ⁺ T and CD8 ⁺ T cells to secrete IL-10, which means that the CCR4 antagonist C-021 has a therapeutic effect on alveolar echinococcosis.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application.

Claims (9)

1. Use of a substance that inhibits CCR4 gene expression or knocks out CCR4 gene for the preparation of a product for the treatment and/or prevention of echinococcosis, or for the treatment of echinococcosis.

2. Use of a substance that reduces CCR4 content or activity in the preparation of a product for the treatment of, or in the treatment of, vesicular echinococcosis.

Use of a ccr4 antagonist in the preparation of a product for the treatment of, or in the treatment of, blepharospermia.

4. A use according to claim 3, characterized in that: the CCR4 antagonist is C-021.

5. Use according to any one of claims 1-4, characterized in that: the treatment and/or prevention of echinococcosis is characterized in that focus growth of echinococcus multilocularis or echinococcosis infected animals is inhibited, or liver injury of echinococcus multilocularis or echinococcosis infected animals is relieved, or effect function of liver T cells of echinococcus multilocularis or echinococcosis infected animals is recovered.

6. The application of a substance for inhibiting CCR4 gene expression or knocking out CCR4 gene in the preparation of products for inhibiting echinococcus multilocularis or metacercaria infection or in inhibiting echinococcus multilocularis or metacercaria infection.

7. Use of a substance that reduces CCR4 content or activity for the preparation of a product for inhibiting infection by echinococcus multilocularis or metacercaria, or for inhibiting infection by echinococcus multilocularis or metacercaria.

Use of a ccr4 antagonist for the preparation of a product for inhibiting infection by echinococcus multilocularis or metacercaria, or for inhibiting infection by echinococcus multilocularis or metacercaria.

9. The use according to claim 8, characterized in that: the CCR4 antagonist is C-021.