CN118806770A - Application of Carboprost in the preparation of anti-tumor drugs - Google Patents

Abstract

The present invention relates to the field of biomedical technology, and in particular to the use of Carboprost in the preparation of anti-tumor drugs. The present invention investigates the anti-tumor effects of Carboprost alone and Carboprost combined with immune checkpoint inhibitors through in vivo experiments. The results show that Carboprost alone and Carboprost combined with immune checkpoint inhibitors can significantly inhibit the growth of tumor volume and have a significant inhibitory effect on tumors.

Description

Application of Carboprost in the preparation of anti-tumor drugs

This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on April 20, 2023, with application number 202310426538.8 and invention name “Application of Carboprost in the Preparation of Anti-tumor Drugs”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present invention relates to the technical field of biomedicine, and in particular to the application of Carboprost in the preparation of anti-tumor drugs.

Background Art

Tumor immunotherapy has become a new means of tumor treatment. Various types of immunotherapy drugs have emerged, including tumor vaccines, cell-based immunotherapy, immunomodulatory drugs targeting T cells, and immune checkpoint inhibitors. At the same time, more and more tumor immunotherapy drugs have been approved for second-line or first-line treatment of various tumors. Therefore, research on tumor immunotherapy will provide new strategies for the prevention and treatment of tumors.

NKT cells are a subset of lipid- and glycolipid-responsive T lymphocytes that co-express NK cell-related markers (NKp46, NK1.1). They are a unique group of T lymphocytes located between the innate and adaptive immune systems. NKT cells have the characteristics of both T cells and NK cells and are important mediators of immune response and tumor monitoring. As an important immunoregulatory cell, NKT cells affect innate immunity by recruiting and activating effector cells such as NK cells, macrophages, dendritic cells (DCs), and traditional CD4 ⁺ and CD8 ⁺ T cells. Activated NKT cells can promote the production of IFN-γ, activate NK and CD8 ⁺ T cells, and connect innate and adaptive immunity. NKT cells can also directly kill tumor cells through the perforin/granzymes pathway and the Fas/FasL pathway. On the one hand, NKT cells activate CD8 ⁺ T cells to kill tumor cells positive for major histocompatibility complex (MHC) molecules, and on the other hand, they also activate NK cells to eliminate MHC-negative tumor cells, so that the tumor is completely eradicated without recurrence. Therefore, NKT cell targeted therapy can be used for any type of tumor and any individual, regardless of the human leukocyte antigen (HLA) type. NKT cells are 7 to 8 times larger than NK cells and 4 to 5 times larger than cytotoxic T lymphocytes (CTL). NKT cells store thousands of times more killing enzymes than NK cells and CD8 ⁺ T cells, and can kill tumor cells very quickly. Studies have used NKT cell-deficient mice to demonstrate that NKT cells play an important role in anti-tumor responses. In fact, the importance of NKT cells in anti-tumor immunity has been further emphasized, and a reduction in the number and function of NKT cells has been confirmed in a large number of cancer patients, including patients with progressive malignant multiple myeloma. Due to the important role of NKT cells in immune surveillance and anti-tumor immunity, NKT cell-based tumor immunotherapy has become an important research field. At present, there are no clinical drugs that promote the increase in the number or function of NKT cells to fight tumors.

In recent years, immunotherapy has brought a major revolution to cancer treatment. Tumor immunotherapy has become an effective anti-tumor method by activating the body's own immune system to achieve the purpose of tumor suppression. Compared with traditional chemotherapy and radiotherapy, it has the advantages of significant efficacy, lasting effect and few toxic side effects. Immune checkpoint inhibitor therapy represented by PD-1/PD-L1 and CTLA-4 antibodies has shown good clinical efficacy in a variety of tumors. However, most existing tumor immunotherapy drugs are antibodies, which need to be injected, are inconvenient to use and expensive. Moreover, only 20% to 40% of patients with immune checkpoint inhibitors such as PD-1 antibodies respond to treatment and benefit from it. Therefore, it is of great significance to continue to provide an anti-tumor drug that is effective for most patients, low in price and easy to take.

Summary of the invention

In view of this, the present invention provides the use of Carboprost in the preparation of anti-tumor drugs, which has the effect of inhibiting the growth of various tumors.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides the use of Carboprost in any of the following items:

(I) promoting the increase of lymphocyte count; and/or

(II), increasing the proportion of lymphocytes in spleen cells; and/or

(III), increasing the number of lymphocytes in the tumor microenvironment; and/or

(IV) Increase the proportion of lymphocytes in the tumor microenvironment;

The lymphocytes include one or more of T cells, B cells or NKT cells.

The present invention also provides the use of Carboprost in any of the following items:

(I) preparing a drug for promoting the increase of lymphocyte count; and/or

(II), preparing a drug for preventing and/or inhibiting tumor cell growth; and/or

(III) preparing anti-tumor drugs;

The lymphocytes include one or more of T cells, B cells or NKT cells.

The present invention also provides one or a combination of two or more of (I) to (V), and use of Carboprost in the preparation of anti-tumor drugs:

(I), antibody drugs; and/or

(II), oncolytic viruses; and/or

(III), chemotherapy drugs; and/or

(IV), immune checkpoint inhibitors; and/or

(V) Nucleic acid for transducing chimeric antigen receptor genes.

In some specific embodiments of the present invention, the antibody drug in the above application includes PD1 antibody and/or CTLA4 antibody; and/or

The oncolytic virus comprises adenovirus, herpes virus and/or reovirus; and/or

The herpes virus includes HSV; and/or

The immune checkpoint inhibitor comprises one or a combination of two or more of PD1 antibody, PDL1 antibody or CTLA4 antibody; and/or

The chemotherapy drugs include one or more of cisplatin, pemetrexed, and cyclophosphamide; and/or

The chemotherapy drugs also include other platinum chemotherapy drugs.

In some specific embodiments of the present invention, the platinum chemotherapy drug in the above application includes one or more of carboplatin, nedaplatin, lobaplatin or oxaliplatin.

The present invention also provides a composition comprising one or a combination of two or more of (I) to (V), and Carboprost:

(I), antibody drugs; and/or

(II), oncolytic viruses; and/or

(III), chemotherapy drugs; and/or

(IV), immune checkpoint inhibitors; and/or

(V) Nucleic acid for transducing chimeric antigen receptor genes.

In some specific embodiments of the present invention, the antibody drug in the above composition includes PD1 antibody and/or CTLA4 antibody; and/or

The oncolytic virus comprises adenovirus, herpes virus and/or reovirus; and/or

The herpes virus includes HSV; and/or

The immune checkpoint inhibitor comprises one or a combination of two or more of PD1 antibody, PDL1 antibody or CTLA4 antibody; and/or

The chemotherapy drugs include one or more of cisplatin, pemetrexed, and cyclophosphamide; and/or

The chemotherapy drugs also include other platinum chemotherapy drugs.

In some specific embodiments of the present invention, the platinum chemotherapy drug in the above composition includes one or more of carboplatin, nedaplatin, lobaplatin or oxaliplatin.

The present invention also provides application of the above composition in preparing anti-tumor drugs.

In some specific embodiments of the present invention, the tumor in the above application also includes tumor cells;

The tumor cells include one or more of B16F10 cells, MC38 cells, LLC cells, E0771 cells or AKR cells; and/or

The dosage form of the drug includes injection or oral preparation; and/or

The dosage of Carboprost in the drug is 0.2 mg/kg/d;

The dosage of Carboprost in the drug in a 70 kg human body is 1.14 mg/d.

The dosage forms of the above drugs include: enteral dosage form or parenteral dosage form;

The gastrointestinal dosage forms include: powders, tablets, granules, capsules, emulsions or suspensions;

The parenteral dosage forms include injections, sprays, drops, patches or ointments.

The dosage forms of the above-mentioned drugs also include: liquid dosage form, solid dosage form, semi-solid dosage form or gas dosage form;

The liquid dosage form includes a solution, a lotion or a liniment;

The solid dosage form includes a pill or a film;

The semisolid dosage forms include gels, suppositories or pastes;

The gaseous dosage form includes aerosols.

The present invention also provides a medicine, comprising the above composition and acceptable adjuvants or auxiliary agents.

Excipients and/or auxiliary agents include, but are not limited to, binders, fillers, disintegrants, lubricants, isotonicity regulators, solubilizers, solubilizers, preservatives, colorants, suspending agents, wetting agents, emulsifiers and/or surfactants.

In some specific embodiments of the present invention, the above-mentioned Carboprost, the above-mentioned composition or the above-mentioned drug is used in combination with antibody drugs, CAR-T, oncolytic viruses or even chemotherapy drugs and/or radiotherapy.

In some specific embodiments of the present invention, the above-mentioned combination includes combination with PD1 antibody, PDL1 antibody, and CTLA4 antibody.

In some specific embodiments of the present invention, the tumors described in the above application include: squamous cell head & neck cancer, gastrointestinal malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, colorectal cancer, small cell lung cancer, triple-negative breast cancer, stomach cancer, urothelial cancer, hematologic malignancy, esophageal cancer, bile tract cancer, prostate cancer, cervical cancer, pancreatic cancer, bladder cancer, endometrial cancer, esophageal cancer or ovarian cancer. The application and medicine of the present invention have the following effects:

The present invention uses a model of tumor cell lines derived from subcutaneous tumor-bearing mice in female C57 mice aged 6 to 8 weeks to investigate the anti-tumor effects of Carboprost alone and Carboprost combined with immune checkpoint inhibitors (such as PD1 antibodies, PDL1 antibodies, and CTLA4 antibodies). The results show that Carboprost alone and Carboprost combined with immune checkpoint inhibitors (such as PD1 antibodies, PDL1 antibodies, and CTLA4 antibodies) can significantly inhibit the growth of tumor volume and have a significant inhibitory effect on tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

Figure 1A and Figure 1B show the effect of Carboprost on NKT cells detected by flow cytometry;

FIG2 shows the effect of Carboprost on tumor volume in subcutaneous tumor-bearing B16F10 mice;

FIG3 shows the effect of Carboprost on the body weight of subcutaneous tumor-bearing B16F10 mice;

FIG4 shows the effect of Carboprost on the number of NKT cells that promote anti-tumor immunity in the tumor microenvironment of mice;

Figure 5 shows the results of a combined trial of Carboprost and immune checkpoint inhibitors;

FIG6 shows the effect of Carboprost on tumor volume in subcutaneous MC38 tumor-bearing mice;

FIG7 shows the effect of Carboprost on tumor volume in subcutaneous LLC tumor-bearing mice;

FIG8 shows the effect of Carboprost on tumor volume in mice orthotopically inoculated with E0771 breast cancer in the mammary fat pad;

FIG9 shows the effect of Carboprost on tumor volume in subcutaneous tumor-bearing AKR mice;

FIG10 shows the effect of Carboprost on tumor volume in subcutaneous PC-3 tumor-bearing mice;

FIG11 shows the effect of Carboprost on tumor volume in subcutaneous tumor-bearing SK-OV-3 mice;

FIG. 12 shows the results of a combination test of Carboprost and chemotherapeutic drugs.

DETAILED DESCRIPTION

The present invention discloses the use of Carboprost in the preparation of anti-tumor drugs. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Prostaglandins are a class of lipid mediators produced by enzymatic metabolism of the twenty-carbon unsaturated fatty acid arachidonic acid. Arachidonic acid is released from cell membrane phospholipids by phospholipase A2 (PLA2) under various physiological and pathological stimuli, and is converted into prostaglandin intermediate metabolites PGG2 and PGH2 in sequence under the action of the epoxidation activity and peroxidation activity of prostaglandin H synthase (PGHS), also known as cyclooxygenase (COX), and then metabolized by different downstream prostaglandin synthases to produce various biologically active prostaglandins, including PGI2, PGE2, PGF2α, PGD2, and thromboxane A2 (TxA2). Some articles have reported that PGF2α stimulates the movement and invasion of colorectal tumor cells, and is significantly increased in the serum of breast cancer patients. PGE2 and PGF2α are the main prostaglandins that mediate various behaviors such as cancer cell proliferation and invasion. There are no patents and articles related to Carboprost. In addition, patents and articles report that PGI2 (PGI2 is produced by the vascular endothelium, inhibits platelet aggregation and promotes vasodilation, and is the most effective platelet aggregation inhibitor) and its derivatives Iloprost and Eptaloprost can inhibit lung metastasis of tumors including lung cancer, melanoma, and prostate cancer cell lines, although no effect on primary tumors was detected. In addition, articles report that PGE2 can promote the growth of gastric cancer, colon cancer, and prostate cancer cells.

Natural prostaglandin F2α is widely present in various tissues and body fluids of the human body. Carboprost is a derivative of natural prostaglandin F2α and is relatively stable. This drug has the effects of softening and dilating the cervix, increasing the frequency and amplitude of uterine contractions, and enhancing the contraction force of the uterus. It has a strong anti-fertility effect. It is mainly used for mid-term abortion, late-term pregnancy to promote cervical ripening and induction of labor. It can also be combined with other drugs to prevent early pregnancy. It is a better non-surgical medical abortion method. The molecular formula of Carboprost is C ₂₁ H ₃₆ O ₅ , the molecular weight is 368.50800, and the structural formula is shown in Formula I.

Formula I

The present invention provides the use of Carboprost in the preparation of anti-tumor drugs, and the use of Carboprost combined with an immune checkpoint inhibitor in the preparation of anti-tumor drugs.

In some embodiments, the immune checkpoint inhibitor is at least one of PD1 antibody, PDL1 antibody, and CTLA4 antibody.

In some embodiments, the tumor is at least one of squamous cell carcinoma of the head and neck, melanoma, primary small cell carcinoma, hepatocellular carcinoma, colon and rectal cancer, small cell lung cancer, triple-negative breast cancer, gastric cancer, urothelial carcinoma, hematological malignancies, esophageal cancer, biliary tract cancer, prostate cancer, cervical cancer, pancreatic cancer, bladder cancer, endometrial cancer, esophageal cancer, and epithelial ovarian cancer.

In some embodiments, the Carboprost is capable of inhibiting the growth of tumor cell volume, inhibiting the increase of tumor cell number and/or inhibiting the migration of tumor cells.

In the above application provided by the present invention, the administration of Carboprost includes injection and oral administration, wherein the oral administration dosage is 0.2 mg/kg/d body weight.

The present invention also provides an anti-tumor drug, comprising Carboprost and a pharmaceutically acceptable carrier.

In some embodiments, the anti-tumor drug further comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is at least one of PD1 antibody, PDL1 antibody, and CTLA4 antibody.

In some embodiments, the tumor is at least one of squamous cell carcinoma of the head and neck, melanoma, primary small cell carcinoma, hepatocellular carcinoma, colon and rectal cancer, small cell lung cancer, triple-negative breast cancer, gastric cancer, urothelial carcinoma, hematological malignancies, esophageal cancer, biliary tract cancer, prostate cancer, cervical cancer, pancreatic cancer, bladder cancer, endometrial cancer, esophageal cancer, and epithelial ovarian cancer.

The present invention uses a model of tumor cell lines derived from subcutaneous tumor-bearing mice in female C57 mice aged 6 to 8 weeks to investigate the anti-tumor effects of Carboprost alone and Carboprost combined with immune checkpoint inhibitors (such as PD1 antibodies, PDL1 antibodies, and CTLA4 antibodies). The results show that Carboprost alone and Carboprost combined with immune checkpoint inhibitors (such as PD1 antibodies, PDL1 antibodies, and CTLA4 antibodies) can significantly inhibit the growth of tumor volume and have a significant inhibitory effect on tumors.

The present invention uses a model of a tumor cell line derived from subcutaneous tumor-bearing mice of 6-8 week-old female C57 mice to clarify the inhibitory effect of Carboprost on tumors. The cell line used is B16F10 melanoma.

Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

The present invention will be further described below in conjunction with embodiments:

Example 1

1. Materials and Methods

1. Cells

Spleen cells from 8-week-old female C57 mice were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Drug treatment

Carboprost (Manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), concentration: 0 μM, 5 μM, 10 μM.

3. Experimental Methods

a. Spleens from 8-week-old female C57 mice were used and spleen cells were treated with Carboprost in vitro.

The spleen of C57 mice was removed under sterile conditions, ground, filtered (200 mesh filter), and lysed on ice for 5 min with red blood cell lysis buffer (Beijing Solebow Technology Co., Ltd.), terminated by adding PBS, and centrifuged (4°C, 500 g, 5 min). After the spleen cells were mixed, they were evenly seeded into 24-well plates at 1×10 ⁶ /well and incubated with three concentrations of Carboprost, namely 0 μM, 5 μM, and 10 μM, in a 37°C incubator for 48 h.

b. After 48 h of drug incubation, cells were collected and flow cytometry was used to detect the effect of Carboprost on NKT cells.

① Collect the cells in each well into a 1.5 ml EP tube and centrifuge (4°C, 500 g, 5 min).

② Wash cells with PBS: Add 1 mL PBS to each tube, mix the cells by pipetting, and centrifuge (4°C, 500 g, 5 min).

③ Discard the supernatant and add 50 μL PBS to resuspend the precipitate.

④ Add 0.5 μL Zombie NIR dye and incubate at room temperature in the dark for 7 min.

⑤ Add 0.5 μL CD16/32 antibody for blocking and incubate at room temperature in the dark for 7 min.

⑥ Prepare antibody mixture (total system is 50 μL): select appropriate antibodies according to the antibody instructions

The antibody mixture was prepared with the amount, added to the sample after blocking, and incubated at room temperature in the dark for 7 minutes.

⑦ After incubation, add 1 mL of PBS and mix by inverting. Centrifuge at 500 g, 4°C for 5 min.

⑧ Discard the supernatant, keep the cell pellet, add 200 μL PBS buffer to resuspend. Transfer the sample to a flow tube for flow cytometry analysis.

Table 1: Antibodies related to flow cytometry

c. Streaming on-machine analysis.

2. Results Analysis

As shown in Figures 1A and 1B (the corresponding data are shown in Table 2), compared with the control group (Carboprost is 0 μM), Carboprost (abbreviated as C) treatment increased the proportion of NKT cells in the spleen cells of wild-type mice in vitro, suggesting that Carboprost (abbreviated as C) can increase the proportion of NKT cells.

Table 2

Example 2

1. Materials and Methods

1. Cell lines

B16F10 melanoma cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

a. Use 6-8 week old female C57 mice to establish tumor mouse model.

Melanoma (B16F10), subcutaneously inoculated with 2×10 ⁴ / mouse, about 5-6 days later, when the tumor volume was measured to be 50-100 mm ³ , the drug was started. If it was in the range of 50-100 mm ³ , the mice were randomly divided into two groups, the experimental group and the control group, according to the tumor size. The day was recorded as Day 0 and drug C was gavaged every day at a dose of 0.2 mg/kg. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume and mouse weight were measured every three days.

If you start from the smallest to the largest and label them A, B, B, A, A, B, B, A..., the mice labeled A are grouped together, and the mice labeled B are grouped together. This is the first batch of DAY 0. The remaining mice that have not been measured are measured on the second day, using the same method as before. This is the second batch of DAY 0. The mice that have not been measured are measured on the third day, using the same method as before. This is the third batch of DAY 0. DAY 3, DAY 6, DAY 9, DAY 12, the measurement method is the same as before.

On DAY 12, the two groups of mice were treated and the tumors were collected.

b. 6-7 days after tumor loading, the tumor volume of mice was observed and measured. When the tumor volume reached 50-100 mm ³ , the mice were randomly divided into an experimental group and a control group, a total of 2 groups. The day was recorded as Day 0. The experimental group was gavaged with drug C every day from Day 0, with a daily dose of 0.2 mg/kg, once a day, and the control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume and mouse weight were measured every three days. The results are shown in Figures 2, 3 and Tables 3-6.

c. Two groups of mice were treated 11 days after administration.

2. Results Analysis

As shown in Figure 2 and Tables 3-6, in the subcutaneous B16F10 tumor-bearing mouse model, the tumor growth volume of the mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Table 3

Table 4

Example 3

1. Materials and Methods

1. Cells

B16F10 melanoma cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Drug treatment

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

a. Construction of tumor mouse model

Before tumor loading, the area around the armpit of the right forelimb of the mice was shaved, and B16F10 melanoma cells were subcutaneously injected into the armpit of the right forelimb of the mice at 2×10 ⁴ /mouse. 5~6 days after the mice were subcutaneously inoculated with tumor cells, the tumor volume of the mice was observed and measured. When the tumor volume reached 50~100 mm ³ , the mice were randomly divided into a control group and an experimental group. The day was recorded as Day 0. The experimental group was gavaged with Carboprost (manufacturer: Shanghai Taoshu Biochemical Technology Co., Ltd.) every day from Day 0, with a daily dose of 0.2 mg/kg, once a day, and the control group was gavaged with an equal volume of drug solvent 0.5% sodium carboxymethyl cellulose solution every day until the end of the experiment. The mice were randomly grouped, and the tumor volume of the mice was sorted from small to large, and marked A, B, B, A, A, B, B, A... at the back. The mice marked with A were grouped into one group, and the mice marked with B were grouped into one group. The two groups of mice were killed on Day 6.

b. Preparation of single cell suspension from tumor tissue

(1) Sampling: Mice were killed on Day 6, and the subcutaneous tumor tissue was completely removed. The tumor was immersed in pre-cooled DMEM.

(2) Grinding tumor tissue: Add 3-4 mL of PBS buffer to a 6 cm small dish, place the tumor piece on a 200-mesh filter, and grind the tumor tissue with the rubber tip of a 5 mL syringe on ice.

(3) Filtration: Use a Pasteur pipette to mix the ground tissue suspension, and filter it through a 200-mesh filter into a 15 mL tube.

(4) Centrifugation: 4°C, 500 g, 5 min.

(5) Discard the supernatant and add 1 mL of red blood cell lysis buffer to each tube to resuspend the cell pellet. Place on ice for 5 min to lyse the red blood cells.

(6) Add 9 mL of 1× PBS to each tube to stop red blood cell lysis and centrifuge (4°C, 500 g, 5 min).

(7) Discard the supernatant, add 1 mL of 1× PBS to resuspend the cell pellet, aspirate 10 μL of the cell suspension and dilute it 100 times in 990 μL of PBS buffer and mix well. Then aspirate 10 μL of the diluted cell suspension into a cell counting plate and count the cells under an optical microscope. Centrifuge the remaining cells (4°C, 500 g, 5 min).

(8) Discard the supernatant and add 100 μL 1× PBS to resuspend the cells for subsequent flow cytometry.

c. Flow cytometry

(1) The single-cell suspension sample preparation process is as described above.

(2) Cell death/alive: Add 0.5 μL Zombie NIR to each tube, pipette and mix thoroughly, and incubate at room temperature in the dark for 7 min.

(3) Blocking: Add 0.5 μL CD16/32 to each tube and mix thoroughly by pipetting. Incubate at room temperature in the dark for 7 min.

(4) Surface staining: In a dark environment, select the appropriate amount of the target antibody according to the instructions and the number of samples to prepare a surface staining antibody mixture (add 50 μL of mix to each sample). After blocking, add 50 μL of the surface staining antibody mixture to the sample and mix by pipetting. Incubate at room temperature in a dark environment for 7 min.

(5) After staining, add 1 mL of 1× PBS to each tube and centrifuge (4°C, 500 g, 5 min).

(6) Discard the supernatant and add 200 μL 1× PBS to each tube to resuspend the cell pellet. Transfer the pellet to a flow cytometry glass tube for flow cytometry detection and analysis.

2. Results Analysis

As shown in Figure 4 (corresponding data are shown in Table 7), compared with the control group, the number of NKT cells that promote anti-tumor immunity in the tumor microenvironment of mice in the Carboprost (abbreviated as C) treatment group was significantly increased.

Example 4: Combination of Carboprost and Immune Checkpoint Inhibitors

1. Materials and Methods

1. Cell lines

B16F10 melanoma cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

PD-1 mAb (Bioxcell, #BE0146), dose 200 μg/mouse, route: intraperitoneal injection.

3. Experimental Methods

(1) Use 6- to 8-week-old female C57 mice to construct a tumor mouse model.

B16F10 melanoma, mice were subcutaneously inoculated with 2×10 ⁴ B16F10 melanoma. About 5-6 days later, when the tumor volume reached 50-100 mm ³ , drugs and antibodies were administered. If the tumor volume was within the range of 50-100 mm ³ , the mice were randomly divided into four groups: C drug-0.2 mg/kg group, PD-1 mAb group, PD-1 mAb and C drug-0.2 mg/kg combined treatment group, and control group. The day was recorded as Day 0. C drug-0.2 mg/kg group and PD-1 mAb and C drug-0.2 mg/kg combined treatment group were gavaged with C drug every day at a dose of 0.2 mg/kg. PD-1 mAb was intraperitoneally injected once every 3 days. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day and IgG2b 200 μg/mouse was intraperitoneally injected once every 3 days. The tumor volume was measured every three days.

For example, start sorting from small to large and label them A, B, C, D, D, C, B, A, A, B, C, D, ... at the end, those marked with A are grouped together, those marked with B are grouped together, those marked with C are grouped together, and those marked with D are grouped together. This is DAY0, and the following DAY3, DAY6, DAY9, and DAY12 are measured in the same way as before.

On DAY12, the mice in four groups were treated and the tumors were collected.

(2) The experimental group was divided into the following 4 groups:

1) IgG2b group;

2) PD-1 antibody group;

3) Carboprost group;

4) PD-1 antibody + Carboprost group.

2. Results Analysis

As shown in Figure 5 and Tables 8 to 15, in the subcutaneous B16F10 tumor-bearing mouse model, the results showed that Carboprost (abbreviated as C) or PD-1 antibody alone or in combination inhibited the growth of B16F10 melanoma in mice, but the combination of C and PD-1 antibody did not have a synergistic anti-tumor effect.

Example 5

1. Materials and Methods

1. Cell lines

MC38 colorectal cancer cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.1 and 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

Female C57 mice aged 6 to 8 weeks were used to construct tumor mouse models.

MC38 colorectal cancer, mice were subcutaneously inoculated with 2×10 ⁵ MC38, and about 3 to 4 days later, when the tumor volume reached 50-100 mm ³ , the drug was administered. If it was in the range of 50-100 mm ³ , the mice were randomly divided into three groups: C drug-0.1 mg/kg, C drug-0.2 mg/kg, and control group according to the tumor size. The day was recorded as Day 0 and C drug was gavaged every day, with doses of 0.1 mg/kg and 0.2 mg/kg, respectively. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume was measured every three days.

If we start sorting from small to large and label them A, B, C, C, B, A, A, B, C, ... at the end, those marked with A are grouped together, those marked with B are grouped together, and those marked with C are grouped together. This is DAY0, and the following DAY3, DAY6, DAY9, and DAY12 are measured in the same way as before.

On DAY12, mice in three groups were treated and their tumors were collected.

2. Results Analysis

As shown in Figure 6 and Tables 16-21, in the mouse model bearing subcutaneous tumor MC38, the tumor growth volume of mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Example 6

1. Materials and Methods

1. Cell lines

LLC small cell lung cancer cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

Female C57 mice aged 6 to 8 weeks were used to construct tumor mouse models.

Small cell lung cancer (LLC), mice were subcutaneously inoculated with 1×10 ⁶ LLC, and the drug was started after about 3-4 days when the tumor volume reached 50-100 mm ^3. If it was in the range of 50-100 mm ³ , the mice were randomly divided into an experimental group and a control group according to the tumor size. The day was recorded as Day 0 and drug C was gavaged every day at a dose of 0.2 mg/kg. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume was measured every three days.

If you start sorting from small to large and label them A, B, B, A, A, B, ... at the end, those marked with A are grouped together, and those marked with B are grouped together. This is DAY0, and the following DAY3, DAY6, DAY9, and DAY12 are measured in the same way as before.

The two groups of mice were treated on DAY12 and the tumors were collected.

2. Results Analysis

As shown in Figure 7 and Tables 22-25, in the subcutaneous LLC tumor-bearing mouse model, the tumor growth volume of the mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Example 7

1. Materials and Methods

1. Cell lines

E0771 breast cancer cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

(1) Use 6- to 8-week-old female C57 mice to construct a tumor mouse model.

Breast cancer (E0771), 1×10 ⁶ E0771 were inoculated in the mammary fat pad of mice. After about 3-4 days, when the tumor volume reached 50-100 mm ³ , the drug was administered. If it was in the range of 50-100 mm ³ , the mice were randomly divided into an experimental group and a control group according to the tumor size. The day was recorded as Day 0 and drug C was gavaged daily at a dose of 0.2 mg/kg. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume was measured every three days.

If you start sorting from small to large and label them A, B, B, A, A, B, ... at the end, those labeled A are grouped together, those labeled B are grouped together, and so on. This is DAY0, and the following DAY3, DAY6, DAY9, and DAY12 are measured in the same way as before.

The two groups of mice were treated on DAY12 and the tumors were collected.

2. Results Analysis

As shown in Figure 8 and Tables 26-29, in the mouse model of E0771 breast cancer in situ inoculated in the mouse mammary fat pad, the tumor growth volume of the mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Example 8

1. Materials and Methods

1. Cell lines

AKR esophageal carcinoma cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dosage: 0.1 mg/kg and 0.2 mg/kg, administration method: oral gavage.

3. Experimental Methods

Female C57 mice aged 6 to 8 weeks were used to construct tumor mouse models.

AKR esophageal cancer, mice were subcutaneously inoculated with 5×10 ⁵ AKR esophageal cancer cells. About 5-6 days later, when the tumor volume reached 50-100 mm ³ , the drug was administered. If it was in the range of 50-100 mm ³ , the mice were randomly divided into three groups: C drug-0.1 mg/kg, C drug-0.2 mg/kg, and control group according to the tumor size. The day was recorded as Day 0 and C drug was gavaged every day, with doses of 0.1 mg/kg and 0.2 mg/kg, respectively. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume was measured every six days.

If we start sorting from small to large and label them A, B, C, C, B, A, A, B, C, ... at the end, those marked with A are grouped together, those marked with B are grouped together, and those marked with C are grouped together. This is DAY0, and the following DAY6, DAY12, DAY18, and DAY24 are measured in the same way as before.

On DAY24, the three groups of mice were treated and the tumors were collected.

2. Results Analysis

As shown in Figure 9 and Tables 30-35, in the subcutaneous AKR tumor-bearing mouse model, the tumor growth volume of the mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Embodiment 9

The anti-tumor effects of Carboprost were observed in a variety of humanized mouse tumor models.

1. Materials and Methods

1. Cell lines

Human prostate cancer PC-3 cells were cultured in DMEM-F12 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin, and human ovarian cancer SK-OV-3 cells were cultured in McCoys 5A medium (Gibco) containing 10% fetal bovine serum and 1% penicillin/streptomycin.

2. Construction method

a. 6-8 week old NCG-X mice were used and subcutaneously loaded with PC-3 tumors (2×10 ⁶ /mouse) and SK-OV-3 tumors (1×10 ⁷ /mouse). Simultaneously, 5×10 ⁶ human peripheral blood mononuclear cells (hPBMC) were intraperitoneally injected into each mouse.

b. 6-7 days after tumor loading, the tumor volume of mice was measured and observed. When the tumor volume reached 50-100 ^mm3 , the mice were randomly divided into three groups, and the day was recorded as day 0. On the second day, two of the groups began to receive C every day, with doses of 0.1 mg/kg and 0.2 mg/kg, respectively, and the remaining control group was given drug solvent every day. The tumor volume and mouse weight were measured every three days.

c. 11 days after administration, three groups of mice were treated.

2. Results Analysis

As shown in Figure 10 and Tables 36-39, in the mouse model bearing subcutaneous PC-3 tumors, the tumor growth volume of mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Table 36

Table 37

Table 38

Table 39

As shown in Figure 11 and Tables 40 to 43, in the subcutaneous tumor-bearing mouse model of SK-OV-3, the tumor growth volume of mice in the drug-treated group was significantly smaller than that in the control group, indicating that Carboprost (abbreviated as C) has a significant anti-tumor effect.

Table 40

Table 41

Table 42

Table 43

Carboprost was administered to the above humanized mouse tumor model, and it was found that the tumor volume of mice in the Carboprost-administered group was smaller than that in the control group, indicating that Carboprost can inhibit human tumor cells.

Example 10: Combination of Carboprost and the chemotherapy drug cisplatin

1. Materials and Methods

1. Cell lines

LLC non-small cell lung cancer cells were cultured in RPMI medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin.

2. Treatment drugs

Carboprost (manufacturer: Shanghai Taosu Biochemical Technology Co., Ltd.), dose: 0.2 mg/kg, administration method: oral gavage.

Cisplatin (Aladdin, # D109812), dose 5 mg/kg, route: intraperitoneal injection.

3. Experimental Methods

(1) Use 6- to 8-week-old female C57 mice to construct a tumor mouse model.

LLC non-small cell lung cancer, mice were subcutaneously inoculated with 1×10 ⁶ LLC non-small cell lung cancer cells. About 5-6 days later, when the tumor volume reached 50-100 mm ³ , drugs and antibodies were administered. If the tumor volume was within the range of 50-100 mm ³ , the mice were randomly divided into four groups: C drug-0.2 mg/kg group, ciaplatin group, ciaplatin and C drug-0.2 mg/kg combined treatment group, and control group. The day was recorded as Day 0. C drug-0.2 mg/kg group and ciaplatin and C drug-0.2 mg/kg combined treatment group began to be gavaged with C drug every day at a dose of 0.2 mg/kg. Cisplatin was injected intraperitoneally once on Day 0 in the ciaplatin group and ciaplatin and C drug-0.2 mg/kg combined treatment group at a dose of 5 mg/kg. The control group was gavaged with an equal volume of drug solvent (0.5% CMCNa sodium carboxymethyl cellulose) every day. The tumor volume was measured every three days.

For example, start sorting from small to large and label them A, B, C, D, D, C, B, A, A, B, C, D, ... at the end, those marked with A are grouped together, those marked with B are grouped together, those marked with C are grouped together, and those marked with D are grouped together. This is DAY0, and the measurement method for DAY3, DAY6, DAY9, DAY12, and DAY15 is the same as before.

On DAY15, the mice in the four groups were treated and the tumors were collected.

(2) The experimental group was divided into the following 4 groups:

1) IgG2b group;

2) ciplatin group;

3) Carboprost group;

4) cisplatin+Carboprost group.

2. Results Analysis

As shown in Figure 12 and Tables 44 to 51, in the subcutaneous LLC tumor-bearing mouse model, the results showed that Carboprost (abbreviated as C) or cisplatin alone and their combination inhibited the growth of LLC non-small cell lung cancer in mice, and the combination of C and cisplatin had a synergistic anti-tumor effect.

Table 44

Table 45

Table 46

Table 47

Table 48

Table 49

Table 50

Table 51

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (11)

1. Use of carboprost to promote an increase in lymphocyte count;

   The lymphocytes comprise one or more of T cells, B cells, or NKT cells.
2. Use of carboprost in any of the following:

   (I) Preparing a medicament for promoting the increase of lymphocyte number; and/or

   (II) preparing a medicament for preventing and/or inhibiting the growth of tumor cells; and/or

   (III) preparing an anti-tumor drug;

   The lymphocytes comprise one or more of T cells, B cells, or NKT cells.
3. 3. (I) One or more than two of (V), and Carboprost in the preparation of antitumor drugs:

   (I) Antibody drugs; and/or

   (II), oncolytic viruses; and/or

   (III) chemotherapeutic agents; and/or

   (IV), immune checkpoint inhibitors; and/or

   (V) nucleic acids for transduction of chimeric antigen receptor genes.
4. 4. The use of claim 3, wherein the antibody drug comprises a PD1 antibody and/or a CTLA4 antibody; and/or

   The oncolytic virus comprises adenovirus, herpes virus and/or reovirus, wherein the herpes virus comprises HSV; and/or

   The chemotherapeutic drug comprises one or more of cisplatin, pemetrexed or cyclophosphamide; and/or

   The immune checkpoint inhibitor includes one or more of a PD1 antibody, a PDL1 antibody, or a CTLA4 antibody.
5. 5. The use of any one of claims 2 to 4, wherein the tumour comprises one or more of head and neck squamous cell carcinoma, hematological malignancy, melanoma, merkel cell carcinoma, triple negative breast cancer, small cell lung cancer, non-small cell lung cancer, oesophageal cancer, stomach cancer, gastrointestinal malignant melanoma, hepatocellular carcinoma, biliary tract cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer or urothelial cancer.
6. 6. Use according to any one of claims 2 to 5, comprising:

   The tumor also includes tumor cells; and/or

   The tumor cells include one or more of B16F10 cells, MC38 cells, LLC cells, E0771 cells, or AKR cells.
7. 7. The composition is characterized by comprising one or more than two of (I) - (V), and Carboprost:

   (I) Antibody drugs; and/or

   (II), oncolytic viruses; and/or

   (III) chemotherapeutic agents; and/or

   (IV), immune checkpoint inhibitors; and/or

   (V) nucleic acids for transduction of chimeric antigen receptor genes.
8. 8. The composition of claim 7, wherein the antibody drug comprises a PD1 antibody and/or a CTLA4 antibody; and/or

   The oncolytic virus comprises adenovirus, herpes virus and/or reovirus, the herpes virus comprises HSV; and/or

   The chemotherapeutic drug comprises one or more of cisplatin, pemetrexed or cyclophosphamide; and/or

   The immune checkpoint inhibitor comprises one or a combination of more than two of PD1 antibody, PDL1 antibody or CTLA4 antibody.
9. 9. Medicament, characterized in that it comprises a composition according to claim 7 or 8, and acceptable auxiliary materials or auxiliaries.
10. 10. The medicament of claim 9, wherein the medicament is in a dosage form comprising an injection or an oral administration.
11. 11. The medicament of claim 9, wherein the dosage of Carboprost in a human of 70: 70 kg is 1.14: 1.14 mg/d.