Ribavirin and pd-1 inhibitor combination drug composition and use thereof

Abstract

The application discloses a ribavirin and PD-1 inhibitor combined drug composition and application thereof. Based on cell experiments and animal model research, it is proved that the ribavirin and PD-1 inhibitor combined use has a remarkable treatment effect on lung cancer. The ribavirin and PD-1 inhibitor combined drug composition provided by the application overcomes the problem of unsatisfactory single treatment effect of tumor diseases, and the combined treatment remarkably enhances the anti-tumor treatment effect, especially the 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 combined use of ribavirin and a 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 immune checkpoint inhibitors, have demonstrated remarkable efficacy in anti-tumor immunotherapy. However, due to the limitations of monotherapy, combination therapies have become an important approach to address drug resistance and enhance efficacy. Ribavirin, a broad-spectrum antiviral drug, has been shown to have some anti-tumor potential in combination with PD-1 inhibitors for the treatment of lung cancer. Summary of the Invention

[0003] In order to overcome the shortcomings of existing single-drug treatments in the field of tumor treatment, the present invention aims to provide a pharmaceutical composition for the combined use of ribavirin and a 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 such as camrelizumab, nivolumab, pembrolizumab and tislelizumab that have inhibitory effects on PD-1 targets.

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

[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 composition.

[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 includes 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, and the like.

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

[0016] Based on cell experiments and animal model studies, this invention verifies the significant therapeutic effect of ribavirin combined with PD-1 on lung cancer.

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

[0018] Figure 1 These are 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 on the effect of ribavirin on LCC cell clone formation.

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

[0022] Figure 5 Photos of lung tumors in mice in each group.

[0023] Figure 6 is the average 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 with reference to 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 assay was used to detect cell viability.

[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 the digestion with trypsin, centrifuge and remove the supernatant. Add 1 mL of complete culture medium to fully resuspend the cell pellet. Take a 1.5 mL centrifuge tube and 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 culture 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 a 96-well plate. Add 150 μL of PBS to each of the outermost wells to minimize edge effects. Inoculate 100 μL of cell suspension into each of the remaining wells. Let the 96-well plate rest for 5 minutes before transferring it to a 5% CO2, 37°C incubator for continued incubation.

[0031] Drug Action: Preheat the culture medium at 37°C and prepare the desired drug concentration. After 12 hours, allow cells to adhere and reach approximately 60% confluence. Discard the top layer of culture medium from each well. For the control group, culture medium without drug was added. For the experimental group, 100 μL of culture medium containing 10 μM ribavirin was added. Set up six replicate wells per group and transfer to a hypoxic incubator at 1% O2, 5% CO2, and 37°C for an additional 48 hours.

[0032] After the culture, the cell growth of each group was observed under a microscope. In a dark environment, CCK-8 and basal culture medium were mixed at a ratio of 1:10 to prepare a working solution. The original culture solution was discarded, and 100 μL of the working solution was added to each well. The cells were incubated in an incubator. When the color of the control group turned obviously orange-yellow, the absorbance (OD) value at a wavelength of 450 nm was detected using a microplate reader.

[0033] Figure 1 This is the experimental result 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 A549 and LCC cells reach the logarithmic growth phase, seed 1000 cells per well of a 6-well plate according to the experimental method of Example 1. Inoculate each well with 2.5 mL of cell suspension and transfer to a 5% CO2, 37°C incubator for culture.

[0038] Drug effect: After 24 hours, cells were observed to have adhered to the wall. 2 mL of complete medium containing 10 μM ribavirin was added to the experimental group, and 2 mL of complete medium containing 1‰ DMSO was added to the control group. Culture was continued in a hypoxic incubator with 1% O2, 5% CO2, and 37°C for 24 hours. After that, the medium was changed and the cells were cultured normally. During this period, the medium was changed according to the cell status. The culture was carried out for a total of approximately 10 days.

[0039] Cell staining: Discard the original culture medium and wash multiple times with 2 mL of PBS per well. Fix with 1 mL of formaldehyde for 20 minutes, aspirate the formaldehyde, and wash with PBS to remove any residual residue. After fixation, stain with 1 mL of crystal violet solution per well for 15 minutes. Remove the dye, rinse again with PBS to remove any excess stain, and allow to dry before photographing under good light.

[0040] Figure 3 This is the experimental result of the effect of ribavirin on the clone formation of LCC cells.

[0041] Figure 4 This is the experimental result 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.

[0043] Fifty 6-8-week-old SPF-grade BALB / C male mice weighing 20 ± 2 g were selected. The mice were housed in a 12-h light / dark cycle at a temperature of 20–26°C and a humidity of 40–70% with good ventilation and natural light. They had free access to food and water. The 55 mice were randomly divided into groups of 7 mice each, except for the control group, which consisted of 10 mice per group.

[0044] Preparation of cell suspension

[0045] Cell inoculation: LLC cells were cultured to the logarithmic growth phase, washed with PBS, and trypsinized. The cells were resuspended in DMEM medium and the cell concentration was adjusted to 1-5×10^6 cells / mL.

[0046] Cell counting: Count cells using trypan blue staining to ensure that each mouse is injected with the same number of cells.

[0047] After one week of adaptive feeding, the mice were then intraperitoneally injected with LLC cell suspensions weekly to induce lung cancer models in the control, PD-1, ribavirin, and combination groups. Dosing regimens included 10 mg / kg of PD-1 and 10 mg / kg of ribavirin, and a combination of 10 mg / kg of PD-1 and 10 mg / kg of ribavirin every two days. After 13 weeks of successful model establishment, PD-1, ribavirin, or the combination therapy were administered concurrently with LLC subcutaneous tumor stimulation. Lung tissues were collected from each group three weeks later for subsequent analysis.

[0048] Outcome Assessment: Mice were sacrificed at week 5. One day before the end of the experiment, mice were fasted and drug intervention was discontinued. Tumors were promptly removed after sacrifice, rinsed with saline until blood was absent, and photographed and recorded. Tumor size was observed and weighed. Calipers were used to regularly measure the maximum long diameter (L) and minimum short diameter (W) of the tumors.

[0049] Calculate tumor volume (V): V=L*W 2 / 2

[0050] Tumor photos Figure 5 The volume and weight of the tumors in each group are shown in Figure 6 and Figure 7 The results show that the combination of ribavirin and PD-1 inhibitors can significantly reduce the tumor size of lung cancer mice and has a significant anti-tumor effect.

[0051] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of an anti-lung cancer drug, characterized in that: The pharmaceutical composition comprises ribavirin and a PD-1 inhibitor; the PD-1 inhibitor is carrelizumab.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of anti-lung cancer drugs, characterized in that: The dosage of ribavirin is a clinically acceptable safe dose, and the dosage of the PD-1 inhibitor is a clinical therapeutic dose.

3. Use of a pharmaceutical preparation in the preparation of an anti-lung cancer drug, characterized in that: The pharmaceutical preparation includes a pharmaceutical composition and a pharmaceutically acceptable carrier; the pharmaceutical composition includes ribavirin and a PD-1 inhibitor, and the PD-1 inhibitor is carrelizumab.

4. Use of the pharmaceutical preparation according to claim 3 in the preparation of anti-lung cancer drugs, characterized in that: The pharmaceutical preparation may be in the form of tablets, granules, capsules, emulsions, aerosols, injections, and powder injections.

5. Use of the pharmaceutical preparation according to claim 3 in the preparation of anti-lung cancer drugs, characterized in that: The administration forms of the pharmaceutical preparation include oral administration, injection administration or inhalation administration.

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