CN118436639A - Application of warfarin and/or collagenase in the preparation of drugs for treating tumors expressing collagen and/or collagen-related diseases - Google Patents

Abstract

The present invention provides the use of warfarin and/or collagenase in the preparation of drugs for treating tumors expressing collagen and/or collagen-related diseases. Warfarin and/or collagenase are used in tumor treatment, and it is found that they have a good therapeutic effect in combination with ICI in tumors expressing collagen. The mechanism is that warfarin and/or collagenase disrupt the arrangement of collagen on the tumor surface, increase the infiltration of T cells, and enhance the immune effect related to T cells. This not only provides a new application direction for warfarin and/or collagenase, but also provides a new drug option for the treatment of tumor diseases.

Description

Application of warfarin and/or collagenase in the preparation of drugs for treating tumors expressing collagen and/or collagen-related diseases

Technical Field

The present invention relates to a new application field of drugs, and in particular to the application of warfarin and/or collagenase in the preparation of drugs for treating tumors expressing collagen and/or collagen-related diseases.

Background technique

Cancer is a leading cause of death worldwide, accounting for nearly one in six deaths. In recent years, immune checkpoint inhibitors (ICIs), represented by anti-PD1/PD-L1 monoclonal antibodies, have brought breakthrough progress in tumor treatment. aPD-1/PD-L1 monoclonal antibodies have been approved for the treatment of multiple tumor types, including melanoma, lung cancer, bladder urothelial carcinoma, and esophageal cancer. The clinical success of immunotherapy has completely changed the field of cancer immunotherapy and made immunotherapy a mainstay of cancer treatment along with traditional treatments such as surgery, chemotherapy, and radiotherapy.

Immunotherapy has brought lasting clinical benefits and greatly extended the life of cancer patients. The addition of immune checkpoint blockers (ICBs) to first-line chemotherapy based on platinum chemotherapy can significantly improve the overall survival of patients with non-small cell lung cancer (NSCLC). However, clinical findings show that more than 60% of patients who initially respond will later develop acquired resistance. The average overall survival of patients is extended by less than 3 years. Currently, our understanding of the mechanism of acquired resistance to ICIs is very limited, and there is a lack of effective treatments to break through acquired resistance.

Warfarin is an anticoagulant drug that exerts its anticoagulant effect by inhibiting the synthesis of vitamin K-dependent coagulation factors and anticoagulant proteins C and S. It is clinically used to prevent and treat thromboembolic diseases, especially in moderate to severe mitral stenosis (usually rheumatic diseases), mechanical valve replacement, and bioprosthetic valve replacement (the first 3 months after surgery). Its therapeutic status is irreplaceable by other new oral anticoagulants. At present, there are no reports on its treatment of tumors.

The chemical name of collagenase is collagenase, which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. Collagenase can be divided into two types according to its existence mode: endogenous collagenase and medicinal collagenase. Endogenous collagenase refers to the collagenase that the human body has itself, such as epithelial tissues such as gums and fascia, synovial membranes of joints, and intervertebral discs. This collagenase exists to varying degrees, and it plays an indispensable role in the decomposition of collagen in the body. Medicinal collagenase refers to a white or off-white sterile freeze-dried powder injection biological preparation extracted, purified and refined from the fermentation broth of Clostridium histolyticum using high-tech means of biopharmaceuticals. At present, there are no reports on its related research on the treatment of tumors.

Summary of the invention

The purpose of the present invention is to provide the use of warfarin and/or collagenase in the preparation of drugs for treating tumors expressing collagen and/or collagen-related diseases in view of the deficiencies in the prior art.

The present invention has found that warfarin and/or collagenase can be used in tumor treatment, and their combination with ICI has a better therapeutic effect in tumors expressing collagen. The mechanism is that warfarin and/or collagenase break the arrangement of collagen on the tumor surface, increase T cell infiltration, and enhance T cell-related immune effects. This not only provides a new application direction for warfarin and/or collagenase, but also provides a new drug option for the treatment of tumor diseases.

In order to achieve the above object, the technical solution adopted by the present invention is:

Use of warfarin in preparing medicine for treating collagen-expressing tumors and/or collagen-related diseases.

Application of collagenase in the preparation of medicines for treating tumors expressing collagen and/or collagen-related diseases.

This application established an immunotherapy-resistant subcutaneous tumor model and studied that the tumor growth of mice in the warfarin or collagenase combined with anti-PD1/PD-L1 administration group was significantly slower than that in the control group, indicating that collagen-related drugs (warfarin and/or collagenase) have a good therapeutic effect on immunotherapy-resistant tumors.

IF staining (Immuno Fluorescence staining) showed that warfarin reduced COL3A1 and COL6A1, and scanning electron microscopy and transmission electron microscopy analysis showed that most of the collagen fibril structures on the surface of resistant tumor cells were eliminated by warfarin treatment. When combined with anti-PD1 antibodies, warfarin and/or collagenase treatment significantly inhibited tumor growth in mice, and the area of tumor tissue necrosis increased. In particular, T cell Gzmk+ cytotoxic CD8+ T cells increased significantly after warfarin and/or collagenase treatment, indicating that warfarin and/or collagenase can enhance T cell infiltration and activation by removing collagen fibrils and reversing acquired resistance to immunotherapy.

At the same time, warfarin and/or collagenase were used to test the killing of immunotherapy-resistant non-small cell lung cancer (NSCLC) organoids in vitro, and it was found that warfarin had a weak killing effect on organoids and was highly safe. The experimental test results show that warfarin and/or collagenase can be used to prepare drugs for the treatment of tumor diseases, which not only provides a new application direction for warfarin/collagenase, but also provides a new drug option for the treatment of tumor diseases. Collagen-related diseases are diseases caused by abnormal accumulation of collagen. Collagen is the main component of various connective tissues, maintaining the integrity of tissues and organs, and is closely related to functions such as early human development, organ formation, cell-to-cell connections, cell chemotaxis, platelet aggregation, and membrane permeability. Excessive or insufficient production of collagen, as well as defects in collagen structure, can lead to disease. Many diseases are caused by abnormally high expression of collagen, including idiopathic pulmonary fibrosis, systemic lupus erythematosus, rheumatoid arthritis, myelofibrosis, scleroderma, Crohn's disease, ulcerative colitis, etc. The present invention also proposes that warfarin and collagenase can be used to degrade collagen, thereby treating related diseases.

Studies have found that the types of tumors that can be treated with warfarin and/or collagenase are tumor cells that highly express collagen or tumors that are rich in stromal cells that highly express collagen. Warfarin and collagenase treatment can remove the collagen of the tumor cells themselves or the collagen of the stromal cells inside the tumor, promote the infiltration of immune-killing T cells, and simultaneously use immune checkpoint inhibitors anti-PD1/anti-PDL1, anti-CTLA4 or CART cell transfusions that activate the body's immune system, thereby better treating tumors.

Furthermore, the tumor expressing collagen is at least one of melanoma, lung cancer, bladder urothelial carcinoma, esophageal cancer, and gastric cancer.

Use of warfarin and/or collagenase in preparing medicine for inhibiting collagen fibril formation on anti-tumor cells.

Use of warfarin and/or collagenase in the preparation of a drug for increasing T cell infiltration.

Furthermore, the minimum concentration of warfarin in mice is 1 mg/kg, and the minimum concentration of warfarin in humans is 0.1 mg/kg.

Use of a pharmaceutical composition in the preparation of a drug corresponding to any one or more of the following diseases or conditions a-c, wherein the pharmaceutical composition comprises a therapeutically effective amount of warfarin and/or collagenase, and a pharmaceutically acceptable excipient;

a. Treat NSCLC tumors, or treat NSCLC tumors resistant to immunotherapy;

b. Inhibit the formation of collagen fibrils on anti-tumor cells;

c. Increase the infiltration and activation of T cells.

By making warfarin and/or collagenase into a pharmaceutical composition as a pharmaceutical raw material, a targeted pharmaceutical product can be obtained, and the corresponding pharmaceutical product has a good preventive and therapeutic effect on the above three situations a-c. a. For NSCLC tumors, it can increase tumor sensitivity and enhance the killing power of bioengineering treatment methods on tumor cells. b. For anti-tumor cell therapy, it can inhibit collagen fibril formation, reduce tumor defense, and increase drug killing sensitivity. c. Warfarin and/or collagenase reduce the collagen binding strength on the surface of tumor cells, increase the infiltration and activation of T cells, and enhance the killing power.

Furthermore, the pharmaceutical composition also includes an effective dose of anti-PD1 and/or Car-T. Warfarin and/or collagenase and PD1 or car-T used in biological therapy synergistically exert a better tumor killing effect and achieve a higher therapeutic effect.

Furthermore, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and pigments.

Furthermore, the pharmaceutical composition is a liquid preparation, a solid preparation or a spray preparation.

In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

During the experimental research of the present invention, an immunotherapy-resistant subcutaneous tumor model was established, and the tumor growth of mice in the warfarin or collagenase combined with anti-PD1 administration group was significantly slower than that in the control group, indicating that collagen-related drugs (warfarin and/or collagenase) have a good therapeutic effect on immunotherapy-resistant tumors; IF staining showed that warfarin reduced COL3A1 and COL6A1, and scanning electron microscopy and transmission electron microscopy analysis showed that most of the structure of collagen fibrils on the surface of resistant tumor cells was eliminated by warfarin treatment.

The experimental study of the present invention shows that when combined with anti-PD1 antibody, the application of warfarin and/or collagenase improves the therapeutic effect, significantly inhibits the growth of tumors in mice, and increases the area of tumor necrosis. In particular, T cell Gzmk+ cytotoxic CD8+ T cells significantly increase after treatment with warfarin and/or collagenase, indicating that warfarin and/or collagenase can enhance the infiltration and activation of T cells by removing collagen fibrils and reversing acquired resistance to immunotherapy.

At the same time, in the experiment of the present invention, warfarin and/or collagenase were used to test the killing of immunotherapy-resistant non-small cell lung cancer (NSCLC) organoids in vitro, and it was found that warfarin had a weak killing effect on organoids and high safety. The experimental test results show that warfarin and/or collagenase can be used to prepare drugs for treating tumor diseases, which not only provides a new application direction for warfarin/collagenase, but also provides a new drug option for the treatment of tumor diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the process of effective warfarin treatment of immune-resistant tumors, a chart of experimental results, and a fluorescent staining photograph.

FIG. 2 is a fluorescent staining photograph and analysis diagram showing that warfarin disrupts the collagen arrangement on the surface of drug-resistant tumors.

FIG3 is a staining photograph and result analysis chart of the test results of warfarin increasing the infiltration of T cells.

FIG4 is a process analysis chart and mechanism analysis schematic diagram of single-cell transcriptome sequencing revealing the mechanism of warfarin treatment of drug-resistant tumors.

Figure 5 is a schematic diagram of the experimental process of the combination of collagenase and Anti-PD1 for the treatment of immunotherapy-resistant tumors in animal models, result analysis photos and experimental data analysis charts.

Figure 6 shows cell fluorescence staining photos and experimental data analysis charts after the combined use of collagenase and Anti-PD1 to treat tumors.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings.

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

The purchase source and specifications of warfarin in the following examples are: (Selleck, Cat# S4545).

Purchase source and specifications of collagenase: Collagenase I (Gibico, Cat#17100017).

Example 1

1. Warfarin kills NSCLC organoids resistant to immunotherapy in vitro

1.1. Methods

(1) Take fresh immunotherapy-resistant NSCLC tumor tissue and cut it into pieces on ice;

(2) Resuspend the minced tissue with collagenase (1 mg/mL collagenase I and 0.5 mg/mL collagenase IV). The amount of minced tissue used is 1 to 2 grams, and the amount of collagenase used is 10 mL.

(3) The collagenase-treated tissue blocks were shaken at 37°C, 220 rpm, for 30 min. The cells were dissociated from the tissue by pipetting every 10 min.

(4) Filter the digested cell suspension in step (3) through a 100 μm cell mesh, centrifuge at 1500 rpm for 5 min at room temperature, and remove the supernatant;

(5) Add 5 mL of DMEM/F12 to resuspend the cells, centrifuge at 1500 rpm for 5 min at room temperature, and remove the supernatant;

(6) After cell counting, approximately every 4000 cells were mixed with 10 μL of Martrigel and seeded into a 96-well plate;

(7) Transfer to an incubator at 37°C and 5 wt.% CO ₂ to solidify the Martrigel for 10-20 min;

(8) Add 10 μL of cell culture medium to each well and culture in a cell culture incubator for 48 h;

(9) Warfarin was added to each well of cells at final concentrations of 0, 2 μM, 10 μM, and 50 μM in cell culture medium, with 3 wells added for each concentration (parallel samples). The drug solvent (DMSO) was used as the control experimental group.

(12) After 72 h of drug treatment, CCK8 was used to detect the activity of cells in each well, and a microscope was used to take photos and count the number of organoids;

(13) Compared with the solvent water control group, calculate the relative percentage of CCK8 activity detection in each well (% vs control experimental group (vehicle). The calculation method is as follows:

% vehicle = (cell activity of warfarin wells)/(cell activity of vehicle group).

1.2. Results

A in Figure 1 is a data chart showing the killing effect of warfarin on immunotherapy-resistant tumor organoids, which shows that warfarin has a weak inhibitory effect on immunotherapy-resistant NSCLC tumor organoids, and a concentration of 50 μM has a weak killing effect on the organoids.

2. In vivo therapeutic effect of warfarin on the growth of immunotherapy-resistant NSCLC tumors

2.1. Methods

Organoid culture of mouse tumors resistant to immunotherapy was performed and transplanted subcutaneously into mice (5×10 ⁴ cells/mouse). The tumor burden of mice was measured by vernier caliper. When the tumor volume was approximately 80-100 mm ³ , intratumoral injection was started. The mice were randomly divided into two groups, one group received anti-PD1 combined with solvent, and the other group received anti-PD1 combined with warfarin.

The dosing pattern is: anti-PD1 antibody (100ug/mouse) is given on Day 1, and warfarin (dose of 1mg/kg)/solvent is given at the same time. Anti-PD1 antibody is given every three days, and warfarin/solvent is given every other day. The administration is continued for 9 days. Finally, the tumor tissue is dissected for volume measurement and pathological analysis.

2.2 Results

B in Figure 1 is a diagram of the in vivo treatment model of warfarin combined with Anti-PD1; C in Figure 1 is a diagram of tumor volume changes after in vivo treatment with warfarin combined with Anti-PD1; D in Figure 1 is a diagram of tumor pathological changes after in vivo treatment with warfarin combined with Anti-PD1.

The results showed that in the immunotherapy-resistant subcutaneous tumor model, the tumor growth of mice in the warfarin combined with anti-PD1 group was significantly slower than that in the control group, indicating that warfarin has a better therapeutic effect on immunotherapy-resistant tumors.

3. Warfarin disrupts the collagen arrangement on the surface of drug-resistant tumors

3.1 Methods

Immunofluorescence staining was performed on tumors treated with warfarin combined with anti-PD1 and those in the control group to detect the expression of collagen 3 (COLA31) and collagen 6 (COLA6A1). Scanning electron microscopy and transmission electron microscopy were performed on tumors in both groups to detect the expression of collagen on the tumor surface and between cells.

3.2 Results

A in Figure 2 shows the expression of collagen 3 and collagen 6 in resistant tumors after warfarin and solvent combined with anti-PD1 treatment; B in Figure 2 shows the arrangement of collagen on the surface of resistant tumors after warfarin and solvent combined with anti-PD1 treatment detected by scanning electron microscopy and transmission electron microscopy.

The results of immunofluorescence staining showed that the expression levels of collagen 3 and collagen 6 were significantly reduced in the warfarin combined with anti-PD1 treatment group compared with the control group (A in Figure 2); at the same time, the scanning electron microscopy and transmission electron microscopy results in Figure 2 (B) also showed that the collagen on the surface of tumor cells in the warfarin combined with anti-PD1 treatment group was significantly reduced.

4. Warfarin increases T cell infiltration

4.1 Methods

The tumors after warfarin combined with anti-PD1 treatment and the tumors in the control group were subjected to immunofluorescence staining to detect the number of T cells expressing CD3 and CD8.

4.2 Results

A in Figure 3 shows CD3 and CD8 staining in resistant tumors after warfarin and solvent combined with anti-PD1 treatment; B in Figure 3 shows the statistics of CD3 and CD8 T cells in resistant tumors after warfarin and solvent combined with anti-PD1 treatment.

The results of immunofluorescence staining showed that the number of CD3 and CD8 T cells in the tumor increased significantly after warfarin combined with anti-PD1 treatment, indicating that warfarin promoted the infiltration of T cells.

5. Single-cell transcriptome sequencing reveals the mechanism of warfarin treatment of drug-resistant tumors

5.1 Methods

The tumor cells after warfarin combined with anti-PD1 treatment and the tumor cells in the control group were digested and single-cell transcriptome sequencing was performed to construct their single-cell transcriptome maps and analyze the composition and differential gene changes of tumor cells and microenvironment cells.

5.2 Results

A in Figure 4 is the single-cell transcriptome map of resistant tumors after warfarin and solvent combined with anti-PD1 treatment; B in Figure 4 is the changes in various cellular components in resistant tumors after warfarin and solvent combined with anti-PD1 treatment; C in Figure 4 is the enrichment pathway of differentially expressed genes in resistant tumor cells after warfarin and solvent combined with anti-PD1 treatment; D in Figure 4 is the change in the number of T cells in resistant tumors after warfarin and solvent combined with anti-PD1 treatment.

Single-cell transcriptome sequencing analysis revealed that after warfarin combined with anti-PD1 treatment, tumor cells were significantly reduced, while T cells were significantly increased (A and B in Figure 4); gene pathway enrichment analysis revealed that collagen-related pathways were downregulated, while immune activation-related pathways were upregulated in tumor cells after warfarin combined with anti-PD1 treatment (C in Figure 4), further confirming that warfarin enhances the efficacy of immunotherapy by regulating the distribution of collagen and promoting immune cell infiltration.

6. In vivo therapeutic effect of collagenase on the growth of immunotherapy-resistant NSCLC tumors

Methods

The mouse tumors resistant to immunotherapy were cultured in organoids and transplanted into the subcutaneous tissue of mice (5×10 ⁴ cells/mouse). The tumor load of the mice was measured by vernier calipers. When the tumor volume was about 80-100 mm ³ , intratumoral injection was started. The mice were randomly divided into two groups, one group was anti-PD1 combined with solvent, and the other group was anti-PD1 combined with collagenase. The dosing mode was: anti-PD1 antibody (100ug/mouse) was first given on Day 1, and collagenase (dose of 20mg/kg)/solvent (DPBS) was given at the same time. Anti-PD1 antibody was given once every three days, and collagenase/solvent was given every other day. The drugs were given continuously for 9 days, and the tumor tissue was finally dissected for volume measurement and pathological analysis.

6.2 Results

A in Figure 5 is a diagram of the in vivo treatment model of the combination of collagenase and Anti-PD1; B in Figure 5 is a diagram of the change in tumor volume after the in vivo treatment of collagenase and Anti-PD1; C in Figure 5 is a gross image of the tumor after solvent/collagenase and Anti-PD1 treatment; D in Figure 5 is a statistical diagram of tumor pathological changes and necrotic areas after the in vivo treatment of collagenase and Anti-PD1.

The above results show that in the immunotherapy-resistant subcutaneous tumor model, the tumor growth of mice in the collagenase combined with anti-PD1 administration group was significantly slower than that in the control group, and the tumor necrosis area was larger, indicating that collagenase has a good therapeutic effect on immunotherapy-resistant tumors.

6.3. Collagenase disrupts collagen arrangement on the surface of drug-resistant tumors

6.3.1 Methods

Immunofluorescence staining was performed on the tumors treated with collagenase combined with anti-PD1 and the tumors in the control group to detect the expression of collagen 3 and 6. At the same time, scanning electron microscopy and transmission electron microscopy were performed on the tumors in both groups to detect the expression of collagen on the tumor surface and between cells.

6.3.2 Results

Figure 6 A shows the expression of collagen 3 and 6 in resistant tumors after collagenase and solvent combined with anti-PD1 treatment; Figure 6 B shows the collagen arrangement on the surface of resistant tumors after collagenase and solvent combined with anti-PD1 treatment detected by transmission electron microscopy.

The results of immunofluorescence staining showed that the expression levels of collagen 3 and 6 in the collagenase combined with anti-PD1 treatment group were significantly lower than those in the control group (A in Figure 6 ). At the same time, the transmission electron microscopy results in Figure 6 B also showed that the collagen on the surface of tumor cells in the collagenase combined with anti-PD1 treatment group was significantly reduced.

6.4. Collagenase increases T cell infiltration

6.4.1 Methods

Immunofluorescence staining was performed on the tumors treated with collagenase combined with anti-PD1 and the tumors in the control group to detect the number of T cells expressing CD3 and CD8.

6.4.2 Results

D in Figure 6 shows the statistics of the number of CD3 and CD8 T cells in resistant tumors after collagenase and solvent combined with anti-PD1 treatment.

The number of CD3 and CD8 T cells in the tumor increased significantly after collagenase combined with anti-PD1 treatment, indicating that collagenase promoted the infiltration of T cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Application of warfarin in preparing medicine for treating tumor expressing collagen and/or collagen related diseases is provided.

2. Use of collagenase in the manufacture of a medicament for the treatment of a collagen-expressing tumour and/or a collagen-associated disease.

3. The use according to claim 1 or 2, wherein the collagen-expressing tumor is at least one of melanoma, lung cancer, urothelial carcinoma of the bladder, esophageal cancer, pancreatic cancer, gastric cancer.

4. The use according to claim 1 or 2, wherein the collagen-related disorder is at least one of idiopathic pulmonary fibrosis, systemic lupus erythematosus, rheumatoid arthritis, myelofibrosis, scleroderma, crohn's disease, and ulcerative colitis.

5. Use of warfarin and/or collagenase in the manufacture of a medicament for inhibiting collagen fibril formation on an anti-tumour cell.

6. Use of warfarin and/or collagenase in the manufacture of a medicament for increasing T cell infiltration.

7. The application of a pharmaceutical composition in preparing medicines for treating any one or more of the following a-c diseases is characterized in that the pharmaceutical composition comprises warfarin and/or collagenase with effective treatment dose and pharmaceutically acceptable auxiliary materials;

a. treating NSCLC tumors, or, immunotherapeutic drug-resistant NSCLC tumors;

b. inhibiting collagen fibril formation on anti-tumor cells;

c. increase infiltration and activation of T cells.

8. The use of claim 7, the pharmaceutical composition further comprising an effective dose of anti-PD1 and/or Car-T.

9. The use according to claim 7, wherein the pharmaceutically acceptable excipients comprise at least one of diluents, excipients, fillers, binders, humectants, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, pigments.

10. The use according to any one of claims 7 to 9, wherein the pharmaceutical composition is a liquid formulation, a solid formulation or a spray formulation.