Ribavirin and PD-1 inhibitor combined pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition combining ribavirin and a PD-1 inhibitor and application of the pharmaceutical composition. Based on cell experiments and animal model researches, the invention proves that the combined use of ribavirin and the PD-1 inhibitor has a remarkable treatment effect on lung cancer. The ribavirin and PD-1 inhibitor combined pharmaceutical composition provided by the invention overcomes the problem that the curative effect is not ideal due to single treatment of tumor diseases, remarkably enhances the anti-tumor treatment effect due to combined treatment, and particularly has a remarkable treatment effect on lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of anti-tumor drugs, and specifically relates to a pharmaceutical composition for the combination of ribavirin and PD-1 inhibitor and its application. Background Art

[0002] Lung cancer is one of the leading causes of cancer death worldwide. In recent years, PD-1 (programmed death protein 1) inhibitors, as an immune checkpoint inhibitor, have shown significant effects in anti-tumor immunotherapy. However, due to the limitations of single treatment methods, combination therapy has become an important means to address drug resistance and enhance efficacy. Ribavirin is a broad-spectrum antiviral drug, and studies have shown that it has certain anti-tumor potential, but there has been no clear report on its combined use with PD-1 inhibitors for the treatment of lung cancer. Summary of the invention

[0003] In order to overcome the shortcomings of the existing single drug treatment in the field of tumor treatment, the present invention aims to provide a pharmaceutical composition for the combination of ribavirin and PD-1 inhibitor and its application.

[0004] To achieve the above-mentioned object of the invention, the present invention first provides a pharmaceutical composition, which comprises ribavirin and a PD-1 inhibitor.

[0005] Wherein, the PD-1 inhibitor includes PD-1 antibody.

[0006] Furthermore, the PD-1 antibodies include monoclonal antibodies that have inhibitory effects on PD-1 targets, such as camrelizumab, nivolumab, pembrolizumab and tislelizumab.

[0007] Furthermore, in the above-mentioned pharmaceutical composition, the dosage of ribavirin is a clinically acceptable safe dosage, and the dosage of the PD-1 inhibitor is a clinical therapeutic dosage.

[0008] The present invention also provides an application of the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.

[0009] Furthermore, the tumor is lung cancer.

[0010] Furthermore, the lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).

[0011] The present invention also provides a medicine combination package, which includes the above-mentioned medicine combination.

[0012] As one of the preferred technical solutions, the dosage of ribavirin in the pharmaceutical combination package is a clinically acceptable safe dosage, and the dosage of the PD-1 inhibitor is a clinical therapeutic dosage.

[0013] The present invention further provides an anti-tumor drug preparation, which comprises ribavirin, a PD-1 inhibitor and a pharmaceutically acceptable carrier.

[0014] Furthermore, the anti-tumor drug preparation can be in the form of tablets, ointments, granules, capsules, emulsions, aerosols, injections, powder injections, etc.

[0015] Furthermore, the anti-tumor drug preparation can be administered orally, by injection, by inhalation, or the like.

[0016] The present invention is based on cell experiments and animal model studies, which verify the significant therapeutic effect of ribavirin combined with PD-1 on lung cancer.

[0017] The pharmaceutical composition for combined use of ribavirin and PD-1 inhibitor provided by the present invention overcomes the problem of unsatisfactory therapeutic effect of single treatment of tumor diseases, and the combined treatment significantly enhances the anti-tumor therapeutic effect, especially the therapeutic effect on lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the experimental results of the effect of ribavirin on the proliferation rate of LLC cells.

[0019] Figure 2 The experimental results of ribavirin on the proliferation rate of A549 cells.

[0020] Figure 3 These are the experimental results of the effect of ribavirin on the clone formation of LCC cells.

[0021] Figure 4 These are the experimental results of the effect of ribavirin on the clone formation of A549 cells.

[0022] Figure 5 The following are photos of lung tumors in each group of mice.

[0023] Figure 6 The mean volume of lung tumor in each group of mice.

[0024] Figure 7 is the average weight of lung tumor in each group of mice. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] The PD-1 antibody used in the embodiments of the present invention is carrelizumab (R&D code: SHR-1210) from Hengrui Medicine Co., Ltd. Example 1

[0027] Using mouse Lewis lung cancer cells (LLC cells) and human non-small cell lung cancer cells (A549 cells) as examples, the CCK-8 method was used to detect cell survival rate.

[0028] Cell culture: Prepare complete culture medium: add 10% fetal bovine serum and 1% streptomycin penicillin mixture to DMEM high glucose medium.

[0029] Cell counting: When A549 and LCC cells reach the logarithmic growth phase, discard the stock solution, rinse with PBS, terminate with trypsin digestion, centrifuge and remove the supernatant, add 1 mL of complete medium to fully resuspend the cell pellet, take a 1.5 mL centrifuge tube, add 2 μL of trypan blue dye and 18 μL of cell suspension, mix well, stain at room temperature, and count using a cell counter. Take an appropriate amount of cell suspension and dilute it with fresh complete medium to adjust the cell concentration to 8×10 4 Pieces / mL.

[0030] Cell plating: Add 100 μL of complete medium to the top row of the 96-well plate, add 150 μL of PBS to each well in the outermost circle to reduce edge effects, and inoculate 100 μL of cell suspension to each well. After the 96-well plate is left to stand for 5 min, transfer it to a constant temperature incubator at 5% CO2 and 37°C for further culture.

[0031] Drug action: The culture medium was preheated at 37°C and the required concentration of drugs was prepared. After 12 hours, the cells adhered to the wall and the cell confluence reached about 60%. The upper culture medium of each well was discarded. The culture medium of the control group did not contain drugs. The experimental groups were added with 100 μL of culture medium containing 10uM ribavirin. Each group had 6 replicate wells and was transferred to a low-oxygen incubator with 1% O2, 5% CO2, and 37°C for further culture for 48 hours.

[0032] After the culture, observe the cell growth of each group under a microscope. Prepare a working solution with CCK-8 and basal culture medium at a ratio of 1:10 in a light-proof environment. Discard the original culture solution, add 100 μL of the working solution to each well, and incubate in an incubator. When the color of the control group turns obviously orange-yellow, use an enzyme-labeled instrument to detect the absorbance (OD) value at a wavelength of 450 nm.

[0033] Figure 1 These are the experimental results of the effect of ribavirin on the proliferation rate of LLC cells.

[0034] Figure 2 This is the experimental result of the effect of ribavirin on the proliferation rate of A549 cells. Example 2

[0035] Mouse Lewis lung cancer cells (LLC cells) and human non-small cell lung cancer cells (A549 cells) were used as examples to conduct cell monoclonal experiments.

[0036] Cell culture: Prepare complete culture medium: add 10% fetal bovine serum and 1% streptomycin penicillin mixture to DMEM high glucose medium.

[0037] Cell plating: When the A549 and LCC cells reach the logarithmic growth phase, 1000 cells are inoculated per well of a 6-well plate according to the experimental method of Example 1, and each well is inoculated with 2.5 mL of cell suspension, and the plates are transferred to a 5% CO2, 37°C incubator for culture.

[0038] Drug effect: After 24 hours, the cells were observed to have attached to the wall. 2 mL of complete medium containing 10uM ribavirin was added to the experimental group, and 2 mL of complete medium containing 1‰ DMSO was added to the control group. The cells were cultured in a hypoxic incubator with 1% O2, 5% CO2, and 37°C for 24 hours, then the medium was changed and the cells were cultured normally. The medium was changed according to the cell status during the period, and the total culture was about 10 days.

[0039] Cell staining: discard the original culture medium, add 2 mL PBS to each well for multiple washing, then add 1 mL formaldehyde for 20 min, remove the formaldehyde and wash with PBS to remove the residue. After the cells are fixed, add 1 mL crystal violet solution to each well for 15 minutes, then remove the dye, add PBS again to wash the floating color, and take pictures under good light after drying.

[0040] Figure 3 These are the experimental results of the effect of ribavirin on the clone formation of LCC cells.

[0041] Figure 4 These are the experimental results of the effect of ribavirin on the clone formation of A549 cells. Example 3

[0042] Establish a Lewis lung cancer model in mice to investigate the effects of combined drug therapy on lung cancer. Fifty 6-8 week old SPF BALB / C male mice weighing 20 ± 2 g were selected. The light / dark cycle was 12 h, the temperature was set at 20 ~ 26 °C, the humidity was 40 ~ 70%, good ventilation, natural light, free access to food and water, and the 55 mice were randomly divided into groups, except for the control group with 10 mice in each group, and the remaining groups with 7 mice in each group.

[0043] Preparation of cell suspension Cell inoculation: LLC cells were cultured to the logarithmic growth phase, washed with PBS, and digested with trypsin. The cells were resuspended in DMEM medium and the cell concentration was adjusted to 1-5×10^6 cells / mL.

[0044] Cell counting: Count cells using trypan blue staining to ensure that each mouse is injected with the same number of cells.

[0045] First, after one week of adaptive feeding of mice, LLC cell suspension was intraperitoneally injected into the control group, PD-1 drug group, ribavirin group, and combination group mice once a week to induce mouse lung cancer model. The dosing regimen was to inject 10 mg / kg of PD-1 and 10mg / Kg ribavirin and 10 mg / kg PD-1 and 10mg / Kg ribavirin combined every two days. After the 13-week model was successful, PD-1 or ribavirin and combined treatment were given while LLC subcutaneous tumor stimulation was given, and lung tissues of mice in each group were taken for subsequent analysis after 3 weeks.

[0046] Result evaluation: The mice were killed in the fifth week. The mice were fasted and drug intervention was stopped one day before the end of the experiment. The tumors were quickly removed after the mice were killed, washed with saline until there was no blood, and photographed and recorded. The size of the tumor was observed and weighed. The maximum long diameter (L) and the minimum short diameter (W) of the tumor were measured regularly using calipers.

[0047] Calculate tumor volume (V): V=L*W 2 / 2 Tumor photos Figure 5 The volume and weight of the tumors in each group are shown in Figure 6 and Figure 7 ,From the results, it can be seen that the combination of ribavirin and PD-1 inhibitor can significantly reduce the tumor size of lung cancer mice and has a significant anti-tumor effect.

[0048] 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

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises ribavirin and a PD-1 inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that The PD-1 inhibitor is a PD-1 antibody.

3. The pharmaceutical composition according to claim 1, characterized in that The PD-1 antibody is selected from one of carrelizumab, nivolumab, pembrolizumab and tislelizumab.

4. The pharmaceutical composition according to claim 1, characterized in that The dosage of ribavirin is a clinically acceptable safe dosage, and the dosage of PD-1 inhibitor is a clinical therapeutic dosage.

5. A drug combination package, characterized in that: A pharmaceutical composition comprising any one of claims 1 to 4.

6. A pharmaceutical preparation, characterized in that The invention comprises the pharmaceutical composition according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

7. The pharmaceutical preparation according to claim 6, characterized in that The pharmaceutical preparations may be in the form of tablets, ointments, granules, capsules, emulsions, aerosols, injections, and powder injections.

8. The pharmaceutical preparation according to claim 6, characterized in that The administration form of the pharmaceutical preparation includes oral administration, injection administration or inhalation administration.

9. Use of the pharmaceutical composition according to any one of claims 1 to 4, the pharmaceutical combination package according to claim 5, or the pharmaceutical preparation according to any one of claims 6 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is lung cancer.

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