Application of combination of cordyceps sinensis extract and PD-1 monoclonal antibody in preparation of medicine for treating tumors

Abstract

The invention provides application of a combination of a cordyceps sinensis extract and a PD-1 monoclonal antibody in preparation of a medicine for treating tumors. Through combination of the cordyceps sinensis extract and the PD-1 monoclonal antibody in a specific proportion, infiltration of CD8 + T cells can be enhanced, the weight and size of tumors are remarkably reduced, and the treatment effect of the PD-1 monoclonal antibody on the tumors is effectively improved. The combination of the cordyceps sinensis extract and the PD-1 monoclonal antibody has a good prospect in synergistic lung cancer tumor immunotherapy.

Description

Technical Field

[0001] The present invention specifically relates to the use of a Cordyceps sinensis extract in combination with PD-1 monoclonal antibody in the preparation of a medicament for treating tumors. Background Art

[0002] A tumor is a neoplasm formed by the proliferation of local tissue cells in response to various tumorigenic factors. It is divided into two major categories: benign tumors and malignant tumors. Lung cancer is a common malignant tumor. The first national retrospective survey on causes of death conducted in the mid-1970s showed that lung cancer ranked fifth in my country after gastric cancer, esophageal cancer, liver cancer, and cervical cancer, accounting for 7.43% of all cancer deaths. However, it has now become the leading cause of death from malignant tumors in my country's urban population. Non-small cell lung cancer includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, its cancer cells grow and divide more slowly, and spread and metastasize relatively late. Non-small cell lung cancer accounts for approximately 80% of all lung cancers, and approximately 75% of patients are diagnosed in the middle or late stages, with a very low five-year survival rate.

[0003] Cordyceps sinensis is a dried complex of the fungus Cordyceps sinensis (Berk.) Sacc. of the family Clavicipitaceae that parasitizes the fruiting bodies and larval corpses of insect larvae of the family Bat Moth. It has a wide range of pharmacological effects, including immunomodulation, antibacterial, anti-tumor, antioxidant, anti-aging, and hypoglycemic and lipid-lowering. Patent CN111658679A, which was applied for based on previous research results, discloses that Cordyceps sinensis extract is a PD-1 immune checkpoint inhibitor that can play a role in treating lung cancer. PD-1 monoclonal antibodies are monoclonal antibodies against PD-1 and are commonly used in the clinical treatment of complex tumors, including lung cancer. There are currently no reports on the combined use of Cordyceps sinensis extract and PD-1 monoclonal antibodies for the treatment of tumors, especially lung cancer. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides the use of Cordyceps sinensis extract combined with PD-1 monoclonal antibody in the preparation of a drug for treating tumors.

[0005] Furthermore, the tumor includes lung cancer; the lung cancer includes small cell lung cancer, large cell lung cancer and lung adenocarcinoma.

[0006] Furthermore, the mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.25, preferably 270:1.25.

[0007] Furthermore, the Cordyceps sinensis extract is the methanol extraction residue after Cordyceps sinensis is extracted with water and methanol in sequence, extracted with ethyl acetate, the ethyl acetate extract and the methanol extract are combined, dispersed with water, and then extracted with petroleum ether and ethyl acetate in sequence, the petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of Cordyceps sinensis is obtained by mixing.

[0008] The present invention also provides a combined pharmaceutical composition for treating tumors, which contains a Cordyceps sinensis extract and a PD-1 monoclonal antibody for simultaneous or separate administration.

[0009] Furthermore, the mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.25, preferably 270:1.25.

[0010] Furthermore, the Cordyceps sinensis extract is the methanol extraction residue after Cordyceps sinensis is extracted with water and methanol in sequence, extracted with ethyl acetate, the ethyl acetate extract and the methanol extract are combined, dispersed with water, and then extracted with petroleum ether and ethyl acetate in sequence, the petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of Cordyceps sinensis is obtained by mixing.

[0011] Finally, the present invention provides a combined preparation for treating tumors, which consists of an oral preparation and an injection; The oral preparation is prepared with Cordyceps sinensis extract as the active ingredient and pharmaceutically acceptable excipients; The injection is prepared with PD-1 monoclonal antibody as the active ingredient and pharmaceutically acceptable excipients; The mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.25.

[0012] Furthermore, the mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 270:1.25.

[0013] Furthermore, the Cordyceps sinensis extract is the methanol extraction residue after Cordyceps sinensis is extracted with water and methanol in sequence, extracted with ethyl acetate, the ethyl acetate extract and the methanol extract are combined, dispersed with water, and then extracted with petroleum ether and ethyl acetate in sequence, the petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of Cordyceps sinensis is obtained by mixing.

[0014] The "low-polarity part of Cordyceps sinensis" of the present invention is prepared according to the following method: 1) Dried Cordyceps sinensis was ground into powder, added to ultrapure water at a material-liquid ratio of 1:10, and soaked at 4°C for 4 h. Ultrasonic extraction was performed twice in an ice-water bath for 10 min each, followed by centrifugation at 4000 rpm for 10 min. The residue was then added to 60°C ultrapure water at a material-liquid ratio of 1:10, and ultrasonic extraction was performed twice at 60°C for 10 min each, followed by centrifugation at 4000 rpm for 10 min to obtain the hot water extraction residue. 2) adding methanol to the hot water extraction residue obtained in step 1) at a solid-liquid ratio of 1:20, soaking at 37°C for 2 h, ultrasonicating the mixture five times for 30 min each, and centrifuging at 4000 rpm for 10 min. The supernatant was concentrated to obtain a methanol extract. Adding ethyl acetate to the residue at a solid-liquid ratio of 1:20, soaking the mixture at 37°C for 2 h, ultrasonicating the mixture three times for 30 min each, and concentrating the supernatant to obtain an ethyl acetate extract. 3) Dispersing the methanol extract and the ethyl acetate extract obtained in step 2) in 60°C ultrapure water, extracting with petroleum ether and then ethyl acetate, evaporating the petroleum ether extract and the ethyl acetate extract to dryness, and mixing to obtain the low-polarity fraction of Cordyceps sinensis.

[0015] The invention provides a method for preparing a drug for treating tumors by combining the Cordyceps sinensis extract with the PD-1 monoclonal antibody. The method can enhance the activity of CD8 T cells by combining the Cordyceps sinensis extract and the PD-1 monoclonal antibody in a specific ratio. + The infiltration of T cells effectively increases the therapeutic effect of PD-1 monoclonal antibody on tumors. Animal experiments have shown that the combination of Cordyceps sinensis extract and PD-1 monoclonal antibody can increase the CD8 + The infiltration effect of T cells significantly reduced the weight and volume of tumors. Combining Cordyceps sinensis extract with PD-1 monoclonal antibody has great prospects in enhancing the immunotherapy of lung cancer tumors.

[0016] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0017] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Diagram of drug administration steps; Figure 2 Animal experimental results on the synergistic effect of Cordyceps sinensis combined with PD-1 monoclonal antibody; Figure 3 The low-polarity fraction of Cordyceps sinensis can increase the infiltration of CD8+ T cells in mouse tumors. DETAILED DESCRIPTION

[0019] Example 1 Study on the Effect of Cordyceps Sinensis Extract on PD-1 Monoclonal Antibody in the Treatment of Lung Cancer 1. Preparation of the Cordyceps Sinensis Extract of the Present Invention 1. Weigh 2 kg of dried Cordyceps sinensis and grind it into powder. Use ultrapure water as the solvent with a solid-liquid ratio of 1:10. Soak the powder at 4°C for 4 h. Insulate it in an ice-water bath and perform ultrasonic extraction twice, each time for 10 min. Centrifuge the extract at 4000 rpm for 10 min. Filter the supernatant and concentrate it under reduced pressure to obtain the cold water extract (DCXC_LS).

[0020] 2. Take the cold water extraction residue, use 60℃ ultrapure water as the solvent, with a solid-liquid ratio of 1:10, and place it at 60℃ for ultrasonic extraction twice, each time for 10 min. Centrifuge the extract at 4000 rpm for 10 min. Filter the supernatant and concentrate under reduced pressure to obtain the hot water extract (DCXC_RS).

[0021] 3. The hot water extract was dispersed in ultrapure water at 60°C and extracted three times with water-saturated n-butanol. The three extracts were combined and evaporated to dryness under reduced pressure to obtain an extract. Together with the remaining aqueous phase and emulsion layer, a total of three samples were obtained, namely the water portion of the hot water extract (DCXC_RS_Water), the emulsion portion of the hot water extract (DCXC_RS_Emulsion), and the n-butanol portion of the hot water extract (DCXC_RS_NBA).

[0022] 4. Take hot water to extract the residue, use pure methanol as the solvent, with a solid-liquid ratio of 1:20, soak it at 37°C for 2 h, keep warm and ultrasonicate it 5 times, each time for 30 min, centrifuge the extract at 4000 rpm for 10 min, filter the supernatant and concentrate it under reduced pressure to obtain the methanol extract (DCXC_MT).

[0023] 5. Take the methanol extraction residue, use ethyl acetate as the solvent, with a solid-liquid ratio of 1:20, soak it at 37°C for 2 hours, keep warm and ultrasonicate 3 times, each time for 30 minutes, centrifuge the extract at 4000 rpm for 10 minutes, filter the supernatant and concentrate under reduced pressure to obtain ethyl acetate extract (DCXC_EA).

[0024] 6. The ethyl acetate extract and the methanol extract were combined and dispersed in 60°C ultrapure water. Extraction was performed three times, sequentially with petroleum ether, ethyl acetate, and water-saturated n-butanol. The three extracts were combined and evaporated to dryness under reduced pressure to obtain an extract. Together with the remaining aqueous phase, four samples were obtained: the petroleum ether extract of the methanol extract (DCXC_MT_PE), the ethyl acetate extract of the methanol extract (DCXC_MT_EA), the n-butanol extract of the methanol extract (DCXC_MT_NBA), and the water extract of the methanol extract (DCXC_MT_Water). Finally, the petroleum ether extract of the methanol extract (DCXC_MT_PE) and the ethyl acetate extract of the methanol extract (DCXC_MT_EA) were combined to obtain the low-polarity fraction of Cordyceps sinensis.

[0025] 2. Effect of treating lung cancer Based on the previous research results on the efficacy of Cordyceps sinensis extracts, we attempted to combine the prepared low-polarity fraction of Cordyceps sinensis with PD-1 monoclonal antibodies for the treatment of lung cancer in mice. The combination of the low-polarity fraction of Cordyceps sinensis and PD-1 monoclonal antibodies achieved significant results. The specific animal experiments on the combination of the low-polarity fraction of Cordyceps sinensis and PD-1 monoclonal antibodies are as follows: 1 Experimental Materials 1.1 Cell lines Mouse lung cancer cells LLC-Luc were purchased from Subic (Shanghai) Biotechnology Co., Ltd.

[0026] 1.2 Animals Thirty SPF-grade male C57 mice, aged 6 to 8 weeks and weighing (20 ± 2) g, were purchased from Chengdu Yaokang Biotechnology Co., Ltd. (certificate number SCXK (Sichuan) 2020-0034). They were housed in the Department of Traditional Chinese Medicine Animal Laboratory of Chengdu University of Traditional Chinese Medicine, which maintains optimal conditions such as a temperature of 20–25°C, a humidity of 50%–55%, and a light-drying cycle of 12 L / 12 D. Under these conditions, mice were maintained on regular feed and drinking water for one week.

[0027] 1.3 Drugs Cordyceps sinensis low polarity fraction; InVivoMAb anti-mouse PD-1 (CD279) (BE0146) was purchased from Bioxcell, USA; 1.4 Reagents Dulbecco's modified Eagle's medium (DMEM), 0.25% trypsin, fetal bovine serum, and penicillin-streptomycin (lot numbers 8122145, 2323491, 15140122, and 10099141C, respectively) were purchased from Gibco, USA. PE Anti-Mouse CD3 Antibody [17A2] and APC Anti-Mouse CD8a Antibody [53-6.7] (lot numbers E-AB-F1013D, E-AB-F1097C, and E-AB-F1104E, respectively) were purchased from Elabscience, China.

[0028] 1.5 Instruments The clean bench was purchased from ESCO, Singapore, model SVE-6A1; the constant temperature and humidity cell incubator was purchased from Thermo Fisher Scientific, USA, model 3111; and the CytoFLEX flow cytometer was purchased from Beckman Coulter.

[0029] 2 Experimental methods 2.1 Cell culture LLC-Luc was cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture in a 37°C, 5% CO2 cell culture incubator. After the cells covered the bottom of the culture dish, they were digested and passaged using 0.25% trypsin containing EDTA.

[0030] 2.2 Establishment of subcutaneous transplant tumor model LLC-Luc cells in the logarithmic growth phase were harvested by trypsinization. After counting the cells, they were resuspended in PBS solution and adjusted to 2.5×10 6 Gently shake the centrifuge tube to mix the cells evenly. Gently draw up the cell suspension with a syringe. Secure the mouse with your left hand, fully exposing the mouse's axilla. Insert the needle horizontally slightly above the right waist, accurately injecting 0.2 mL of cell suspension into the axilla. Carefully withdraw the needle and observe for any cell leakage.

[0031] 2.3 Animal grouping and drug administration C57 mice were randomly divided into five groups: blank group (CK, normal mice gavaged with normal saline), model group (M, mice with subcutaneous tumor xenografts gavaged with normal saline and then injected with normal saline), low-polarity fraction group (TQ, mice with subcutaneous tumor xenografts gavaged with low-polarity fraction of Cordyceps sinensis and then injected with normal saline), PD-1 group (PD, mice with subcutaneous tumor xenografts gavaged with normal saline and then injected with PD-1 monoclonal antibody), and low-polarity fraction + PD-1 group (TP, mice with subcutaneous tumor xenografts gavaged with low-polarity fraction of Cordyceps sinensis and then injected with PD-1 monoclonal antibody). The low-polarity fraction dose was set at 600 mg / kg (i.e., 15 mg per mouse per dose) based on the dose conversion. In addition, the PD-1 monoclonal antibody dose was set at 250 μg per mouse per dose. Drug preparation was as follows: the low-polarity fraction was diluted with normal saline to a concentration of 150 mg / mL, mixed after sonication, and then used. The PD-1 monoclonal antigen solution was aseptically diluted with normal saline to a concentration of 1.25 mg / mL. The administration time and volume are as follows: the low polarity site is administered by oral gavage starting on the 5th day after inoculation, 0.1 ml / day per mouse, for a total of 18 days; PD-1 monoclonal antibody is administered by intraperitoneal injection on the 9th, 12th, 15th, 18th, and 21st days after inoculation, 0.2 ml / day per mouse. Figure 1 During the entire medication cycle, the dosage of the low-polarity part of Cordyceps sinensis is 15 mg*18=270 mg, and the dosage of PD-1 monoclonal antibody is 0.25 mg*5=1.25 mg.

[0032] 2.4 Collection of materials After the experiment, the mice in each group were anesthetized and then blood was collected from the eyeballs. The blood was placed in a centrifuge tube and centrifuged at 3000 r / min. -1 After centrifugation for 15 minutes, serum was collected into a new, labeled centrifuge tube and stored at -80°C for later use. Tumor tissue was then dissected from the right axilla of the mouse, cleaned with pre-chilled sodium chloride solution, photographed, and cut in half. One portion was quickly frozen in a cryovial placed on liquid nitrogen and stored at -80°C. The other portion was fixed in 4% paraformaldehyde.

[0033] 2.5 Mouse body weight, tumor weight and volume The weight of mice was measured every 7 days. After the experiment, the weight of the mouse tumor was measured and photographed. The long and short diameters of the subcutaneous transplanted tumor were recorded with a vernier caliper to calculate the tumor volume (v). The calculation formula is: v = (a × b 2 ) / 2, where a is the maximum surface diameter and b is the minimum surface diameter.

[0034] 2.6 Flow cytometry detection of T lymphocyte infiltration in tumor tissue Mouse tumor specimens were minced with sterile scissors, ground into a homogenate using a homogenizer, and filtered through a 40 μm filter to prepare a single-cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube, added with PBS, and centrifuged at 800 g for 5 minutes. The supernatant was discarded, the cells were resuspended in PBS, centrifuged at 800 g for 5 minutes, and resuspended in PBS again. 100 μL of the cells were transferred to a flow cytometer tube and incubated with the appropriate flow cytometer antibody for staining for 20 minutes before analysis.

[0035] The following flow cytometry antibodies were added in this study: PE-labeled CD3 antibody and APC-labeled CD8a antibody.

[0036] 3 Experimental results 3.1 Results of mouse body weight, tumor weight and volume Specific results can be found in Figure 2 ,from Figure 2 It can be seen that there was no significant difference in the body weight of mice among the model group, low-polarity site group, PD-1 group, and low-polarity site + PD-1 group. The body weight of mice in the blank group decreased significantly in the later stage of the experiment. This may be because normal mice were gavaged and injected with normal saline and did not form tumors, so there was no effect on tumor weight.

[0037] The average tumor weight in the model group was 3.04 g, and the average tumor volume was 2227.64 mm 3 The average tumor weight in the low-polarity group was 2.89 g, and the average tumor volume was 1580.18 mm 3 The average tumor weight in the PD-1 group was 2.3 g, and the average tumor volume was 1053.96 mm 3 The average tumor weight of the low-polarity site + PD-1 group was 1.38 g, and the average tumor volume was 379.7 mm 3 Compared with the model group, the tumor weight in the low-polarity site group decreased by 4.9% and the tumor volume decreased by 29%; the tumor weight in the PD-1 group decreased by 24.3% and the tumor volume decreased by 52.7%; and the tumor weight in the low-polarity site + PD-1 group decreased by 54.6% and the tumor volume decreased by 83%. The tumor weight reduction rate in the low-polarity site + PD-1 group was nearly twice the combined reduction rate of the PD-1 and low-polarity site groups, and the volume reduction rate was also greater than the combined reduction rate of the PD-1 and low-polarity site groups. Cordyceps sinensis extract combined with PD-1 monoclonal antibody achieved a synergistic effect in the treatment of non-small cell lung cancer.

[0038] 3.2 T lymphocyte infiltration results in tumor tissue See Figure 3 ,from Figure 3 Visible: Cordyceps sinensis low polarity fraction can increase CD8 in mouse tumors +The infiltration effect of T cells can significantly enhance the therapeutic effect of PD-1 monoclonal antibody on lung cancer.

[0039] In summary, the present invention can enhance the CD8 + The infiltration of T cells significantly reduced the weight and volume of lung tumors, effectively enhancing the therapeutic efficacy of PD-1 monoclonal antibodies against lung cancer. Combining Cordyceps sinensis extract with PD-1 monoclonal antibodies has great potential in enhancing tumor immunotherapy.

Claims

1. Use of Cordyceps sinensis extract combined with PD-1 monoclonal antibody in the preparation of drugs for treating tumors.

2. The use according to claim 1, characterized in that: The tumor includes lung cancer; the lung cancer includes small cell lung cancer, large cell lung cancer and lung adenocarcinoma.

3. The use according to claim 1, characterized in that: The mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.25, preferably 270:1.

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4. The use according to any one of claims 1 to 3, characterized in that: The cordyceps sinensis extract is obtained by extracting the cordyceps sinensis with water and methanol in sequence, adding ethyl acetate to the methanol extraction residue, combining the ethyl acetate extract and the methanol extract, adding water to disperse the extract, and then sequentially extracting with petroleum ether and ethyl acetate. The petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of the cordyceps sinensis is obtained by mixing them.

5. A combined pharmaceutical composition for treating tumors, characterized in that: It contains Cordyceps sinensis extract and PD-1 monoclonal antibody for simultaneous or separate administration.

6. The combined pharmaceutical composition according to claim 5, characterized in that: The mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.25, preferably 270:1.

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7. The combined pharmaceutical composition according to claim 5, characterized in that: The cordyceps sinensis extract is obtained by extracting the cordyceps sinensis with water and methanol in sequence, adding ethyl acetate to the methanol extraction residue, combining the ethyl acetate extract and the methanol extract, adding water to disperse the extract, and then sequentially extracting with petroleum ether and ethyl acetate. The petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of the cordyceps sinensis is obtained by mixing them.

8. A combined preparation for treating tumors, characterized by: It consists of an oral preparation and an injection; The oral preparation is prepared with Cordyceps sinensis extract as the active ingredient and pharmaceutically acceptable excipients; The injection is prepared with PD-1 monoclonal antibody as the active ingredient and pharmaceutically acceptable excipients; The mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 200-400:1.

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9. The composition according to claim 8, characterized in that: The mass ratio of the Cordyceps sinensis extract to the PD-1 monoclonal antibody is 270:1.

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10. The composition according to claim 9, characterized in that: The cordyceps sinensis extract is obtained by extracting the cordyceps sinensis with water and methanol in sequence, adding ethyl acetate to the methanol extraction residue, combining the ethyl acetate extract and the methanol extract, adding water to disperse the extract, and then sequentially extracting with petroleum ether and ethyl acetate. The petroleum ether extract and the ethyl acetate extract are evaporated to dryness, and the low-polarity part of the cordyceps sinensis is obtained by mixing them.

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