CN115154612B

CN115154612B - Pharmaceutical composition and application thereof in preparing anti-tumor combined therapeutic drug

Info

Publication number: CN115154612B
Application number: CN202210973034.3A
Authority: CN
Inventors: 陆金健; 陈宇驰
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2024-01-19
Anticipated expiration: 2042-08-15
Also published as: CN115154612A

Abstract

The invention discloses a pharmaceutical composition and application thereof in preparing an anti-tumor combined therapeutic drug. The pharmaceutical composition comprises a medicament for inhibiting PD-1 or PD-L1 and radix tetrastigme polysaccharide. The medicine combination of the radix tetrastigme polysaccharide and the medicine (such as PD-L1 antibody) for inhibiting PD-1 or PD-L1 is used for enhancing the curative effect of anti-PD-1 or anti-PD-L1 immunotherapy by inhibiting M2 polarization of macrophages and promoting M1 polarization and changing tumor microenvironment, so that the synergistic anti-tumor effect is obtained. Therefore, the application of the pharmaceutical composition in preparing anti-tumor combined therapeutic drugs provides a new potential strategy for tumor immunotherapy.

Description

Pharmaceutical composition and application thereof in preparing anti-tumor combined therapeutic drug

Technical Field

The invention relates to the technical field of anti-tumor, in particular to a pharmaceutical composition and application thereof in preparing an anti-tumor combined therapeutic drug.

Background

Malignant tumors generally refer to cancers, which have biological characteristics such as abnormal cell differentiation and proliferation, loss of control of growth, infiltration, and metastasis. Malignant tumors seriously affect the healthy life of people and bring serious challenges to the operation of urban medical systems and the social and economic development. Immunotherapy, represented by the programmed death receptor 1 (programmed cell death 1, pd-1) and programmed death ligand 1 (programmed death ligand-1, pd-L1) immune checkpoint inhibitors, significantly improves survival of tumor patients, but still suffers from low clinical response rates, and the like, and there is a need to find new and effective combination strategies.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition and application thereof in preparing anti-tumor combined therapeutic drugs so as to improve the technical problems.

The invention is realized in the following way:

in a first aspect, the invention provides a pharmaceutical composition comprising a medicament that inhibits PD-1 or PD-L1 and a tetrastigme polysaccharide.

Optionally, the drug that inhibits PD-1 or PD-L1 is a polypeptide or an antibody, optionally, the drug is a PD-1 antibody or a PD-L1 antibody.

In a second aspect, the invention also provides application of the pharmaceutical composition in preparing an anti-tumor combined therapeutic drug.

In a third aspect, the invention also provides a kit for anti-tumor comprising the pharmaceutical composition.

Alternatively, in the method of using the kit, the single dose of the drug for inhibiting PD-1 or PD-L1 is 1 to 10. Mu.g/g, preferably 4 to 6. Mu.g/g, and the single dose of the radix tetrastigme polysaccharide is 50 to 400mg/kg, preferably 150 to 250mg/kg.

The invention has the following beneficial effects: the radix tetrastigme polysaccharide is combined with a drug combination of drugs (such as PD-L1 antibodies) for inhibiting PD-1 or PD-L1, and the radix tetrastigme polysaccharide can enhance the curative effect of anti-PD-1 or anti-PD-L1 immunotherapy by inhibiting M2 polarization of macrophages and promoting M1 polarization and changing tumor microenvironment, so that a synergistic anti-tumor effect is obtained. Therefore, the application of the pharmaceutical composition in preparing anti-tumor combined therapeutic drugs provides a new potential strategy for tumor immunotherapy.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a diagram of pharmacodynamics evaluation results of a subcutaneous tumor-transplanted mouse model, in fig. 1, a is a diagram of tumor volume statistics, B is a diagram of mouse weight statistics, and C is a photograph taken of a mouse tumor; THP: radix tetrastigme polysaccharide; anti-PD-L1: PD-L1 antibody. * : p <0.05,: p <0.01,: p <0.001;

FIG. 2 is a graph showing the effect of co-administration of tetrastigme polysaccharide and PD-L1 antibody on macrophages in tumor tissue; in FIG. 2, A is a graph showing the effect of infiltration on tumor macrophages, B is a graph showing the effect of M1 on tumor macrophages, C is a graph showing the effect of M2 on tumor macrophages, and D is a graph showing the effect of the ratio of M1/M2 on tumor macrophages. THP: radix tetrastigme polysaccharide; anti-PD-L1: PD-L1 antibody. * : p <0.05;

FIG. 3 is a graph showing the effect of co-administration of tetrastigme polysaccharide and PD-L1 antibodies on T cells in tumor tissue; in fig. 3, a is a graph of the effect of infiltration of total T cells of tumor tissue, and B is a graph of the effect of infiltration of CD8 positive T cells of tumor tissue; c is an infiltration influence diagram of CD4 positive T cells of tumor tissues; d is an infiltration influence graph of Treg on tumor tissues; THP: radix tetrastigme polysaccharide; anti-PD-L1: PD-L1 antibody,: p <0.05,: p <0.01.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

The pharmaceutical composition and the application thereof in preparing the anti-tumor combined therapeutic drug are specifically described below.

First, some embodiments of the present invention provide a pharmaceutical composition comprising a drug that inhibits PD-1 or PD-L1 and a tetrastigme polysaccharide.

Specifically, the radix tetrastigme is named as radix tetrastigme, namely, radix tetrastigme climbing vine (Tetrastigma hemsleyanum Diels & Gilg), and also named as gold thread hoist, radix hedyotis, radix aconiti carmichaeli, stone-like fleshy, and the like, is a unique precious medicinal material in China, and is mainly distributed in areas such as Zhejiang, guizhou, yunnan and Taiwan. Radix tetrastigme is used as a medicine by using root tuber or whole herb, and has the effects of clearing heat and detoxicating, dispelling wind and resolving phlegm, promoting blood circulation and relieving pain and the like; the long-term medical history in the folk indicates that the medicine has better safety. The radix tetrastigme polysaccharide is polysaccharide active ingredient extracted and separated from radix tetrastigme. The radix tetrastigme polysaccharide is an existing raw material, can be prepared by extraction by an existing method, and can be purchased by an existing market purchase way and the like.

The inventor finds that the radix tetrastigme polysaccharide and the medicament for inhibiting PD-1 or PD-L1 are combined through a large number of researches and practices, the medicament can effectively inhibit M2 polarization of macrophages and promote M1 polarization, and change tumor microenvironment so as to enhance the curative effect of anti-PD-1 or PD-L1 immunotherapy and achieve the coordinated and synergistic anti-tumor effect.

In some embodiments, the agent that inhibits PD-1 or PD-L1 includes, but is not limited to, a polypeptide or antibody, e.g., the agent may be a PD-1 antibody or a PD-L1 antibody, preferably the agent is a PD-L1 antibody. The drug may also be a PD-1 antibody or a functional fragment of a PD-L1 antibody, for example any one selected from the group consisting of VHH, F (ab ') 2, fab', fab, fv and scFv of a PD-1 antibody or a PD-L1 antibody.

The functional fragment of the antibody generally has the same binding specificity as the antibody from which it is derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The functional fragments described above are readily available to those skilled in the art based on the prior art disclosing the structure of the intact antibody. Functional fragments of the above antibodies may also be obtained synthetically by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

Further, PD-L1 antibodies include, but are not limited to, any of Abelimumab, dewar Lu Shankang, abikovimumab, CS1001, KN035, SHR-1316, HLX20, MSB2311, KL-A167, JS003, P003, STI-A1014, HLX-10, and MCLA-145. PD-1 antibodies include, but are not limited to, any of pembrolizumab, nivolumab, cimetidine Li Shan, terlipressin Li Shan, xindi Li Shan, kari Li Zhushan, and tirelimumab. It should be noted that various inhibitors targeting PD-1 or PD-L1 are also within the scope of the present invention.

In some embodiments, when the drug is a PD-L1 antibody, in order to achieve better coordination effect, the single administration ratio of the two is optimized, i.e. the dosage ratio of radix tetrastigme polysaccharide to the PD-L1 antibody is 10-400:1, preferably 60-350:1. In this case, the pharmaceutical composition is used as a single-dose pack. The two components of the composition may not be in a mixed state, but may be administered separately by different routes.

Further, some embodiments of the present invention also provide the use of the pharmaceutical composition in the above embodiments in the preparation of a combination therapeutic agent for anti-tumor.

The combination therapy may be a pharmaceutical pack with individually packaged components or a pharmaceutical pack mixed together, i.e., the active components of the combination therapy include the pharmaceutical composition in the above embodiment, and may further include pharmaceutically acceptable additives or auxiliary materials, and the individual components or the formulations corresponding to the component mixtures include, but are not limited to, tablets, pills, powders, creams, granules, nanoparticles, suspensions, gels, emulsions, suppositories, injections, capsules, sprays or injections, and the like.

In some embodiments, the combination therapeutic is an agent that increases the ratio of macrophage M1/M2 in tumor tissue. That is, some embodiments of the present invention provide for the use of the pharmaceutical composition of the above embodiments in the preparation of a medicament for increasing the ratio of macrophages M1/M2 in tumor tissue.

In some embodiments, the combination therapy may also be an agent that increases T cell infiltration and decreases the proportion of tregs in tumor tissue. That is, some embodiments of the present invention provide for the use of the pharmaceutical composition of the above embodiments in the preparation of a medicament for increasing infiltration of T cells in tumor tissue and decreasing the proportion of tregs.

The combination therapeutic agent of the present application has a uniform and good anti-tumor effect, and is directed against tumors including, but not limited to, colorectal cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, renal cancer, fibrosarcoma, multiple myeloma, tube cancer, esophageal cancer, ovarian cancer, malignant melanoma, bladder cancer, glioma, breast cancer, and the like. In a preferred embodiment, the tumor is colorectal cancer.

Then, some embodiments of the present invention also provide a kit for anti-tumor comprising the pharmaceutical composition of the above embodiments.

Specifically, in the method of using the above kit, the single dose of the drug for inhibiting PD-1 or PD-L1 may be 1 to 10. Mu.g/g, for example, 4 to 6. Mu.g/g, once every three days, three times in total. A single dose of radix tetrastigme polysaccharide may be 50-400 mg/kg, e.g., 150-250 mg/kg, once daily.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

In the following examples, the PD-L1 antibody was anti-mouse PD-L1 (clone 10F.9G2, #BE0101, bioXcell), and the radix tetrastigme polysaccharide was prepared as follows: cleaning aerial parts of radix Trifolii Pratentis, drying, and pulverizing to obtain radix Trifolii Pratentis powder; adding radix tetrastigme powder into double distilled water, carrying out reflux extraction, filtering, adding a filtrate into an ethanol water solution with the volume concentration of 95%, fully and uniformly mixing, standing at room temperature for 12 hours, centrifuging, taking a precipitate, washing with absolute ethanol, carrying out suction filtration, volatilizing the ethanol solution, and carrying out precipitation freeze-drying to obtain radix tetrastigme crude polysaccharide; dissolving radix tetrastigme crude polysaccharide in double distilled water, removing protein by Sevag method, desalting, passing through anion exchange column, collecting 0.3-0.6mol/L NaCl eluting peak, and removing NaCl to obtain radix tetrastigme polysaccharide.

Example 1

This example was prepared by establishing a subcutaneous engrafting tumor mouse model, purchasing C57BL/6 male mice (six weeks old) from Australian university animal center, and performing the experiment in this center SPF-class laboratory. The relevant study will be approved by the central animal ethics experimental committee. The specific animal experiment scheme is as follows: mice with substantially consistent body weight, vitality, etc. were selected for the experiment. MC38 cells in the logarithmic growth phase were resuspended to appropriate concentrations in PBS and the MC38 subcutaneous tumors were constructed subcutaneously on the right back of 100 ten thousand/injection into C57BL/6 male mice. Tumors were measured 7 days after the tumor (volume = length-width-height/2) and the tumor volume was taken to be relatively homogeneous (≡100 mm) ³ ) All animals were weighed before starting the administration and the tumor volume was measured, and the animals were randomly grouped into a blank group, a shaggy-leaf polysaccharide administration group, a PD-L1 antibody single administration group, and a PD-L1 antibody-shaggy-leaf polysaccharide combination group, each group being 8 animals, based on the tumor size and the weight of the mice.

After grouping, the corresponding administration dose of the radix tetrastigme polysaccharide is 200mg/kg, and the administration is intragastric administration, and the administration time is from once a day to the sacrifice of the mice. The control group was given an equal volume of ddH by the same administration ₂ O. The PD-L1 antibody was administered at a dose of 75 μg/mouse, intraperitoneally, once every three days, three times in total. The control group was given an equal quality isotype control with the same dosing regimen. The administration time was 15 days. The body weight, food intake, water intake, defecation, hair color, motility, mental state and the like of the experimental animals are detected regularly, and the tumor volume and the body weight of the mice are observed and recorded every other day. Tumor bodyThe product calculation formula is: volume = length x width x height/2.

As can be seen from fig. 1, the combined administration of the tetrastigme polysaccharide and the PD-L1 antibody significantly enhances the inhibition of MC38 tumors by the PD-L1 antibody alone. The combination administration had more excellent antitumor effect (see a, C in fig. 1) than the PD-L1 antibody alone, while having no effect on the body weight of the mice (see B in fig. 1).

Example 2

The embodiment provides the research on the improvement effect of the radix tetrastigme polysaccharide combined with the PD-L1 antibody on the immune microenvironment.

After the MC38 subcutaneous tumor model mice are sacrificed, tumor tissues are dissected and removed, and single cell suspensions are prepared from the tumor tissues for flow cytometry detection. Flow cytometry analyzed changes in macrophage infiltration for each of the groups M1 and M2: anti-CD45 (immune cells), anti-CD11b (myeloid immune cells), anti-F4/80 (macrophages), anti-CD206 (M2 cells) staining to detect the number of M1 and M2 macrophages. Cell debris was removed, single cell clusters were selected, CD45, CD11b and F4/80 positive cells were circled as macrophages, and on this basis, M1 and M2 macrophages were distinguished by CD206 negative and positive, and each group was analyzed for infiltration changes.

The experimental results are shown in fig. 2, and it can be seen from fig. 2 that the co-administered group had no significant effect on infiltration of macrophages in tumor tissue (see a in fig. 2) but significantly increased the ratio of macrophages M1/M2 (see B-D in fig. 2) compared to the PD-L1 antibody alone.

Analysis of other immune cell abundance changes: anti-CD45 (immune cells), anti-CD3 (T cells), anti-CD8 (CD8+ T cells), anti-CD4 (CD4+ T cells), anti-FOXP3 (Treg cells) staining detects tumor infiltrating CD4, CD8 positive T cells, and Treg cells. Removing cell debris, selecting single cell clusters, circling CD45 and CD3 positive cells as T cells, taking CD8 positive cells as CD8+ T cells on the basis, taking CD4 positive cells as CD4+ T cells, taking FOXP3 positive cells as Treg cells on the basis of the CD4+ T cells, and analyzing infiltration change of each group.

As shown in fig. 3, it can be seen from fig. 3 that the co-administration significantly increased infiltration of total T cells (a in fig. 3), CD8 positive T cells (B in fig. 3) and CD4 positive T cells (C in fig. 3), reducing the proportion of tregs (D in fig. 3).

In summary, the pharmaceutical composition and the application of the pharmaceutical composition in preparing the anti-tumor combined therapeutic drug provided by the embodiment of the invention can inhibit the M2 polarization of macrophages and promote the M1 polarization through the combined action of the radix tetrastigme polysaccharide and the anti-PD-L1 antibody, have no obvious influence on the infiltration of tumor tissue macrophages, increase the infiltration of total T cells, CD8 positive T cells and CD4 positive T cells, reduce the proportion of tregs, and further improve the tumor microenvironment. Achieves the effect of enhancing the anti-PD-L1 immunotherapy and provides a new potential strategy for tumor immunotherapy.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of a pharmaceutical composition in preparing an anti-tumor combined therapeutic drug, which is characterized in that the pharmaceutical composition comprises a PD-L1 antibody and radix tetrastigme polysaccharide; the PD-L1 antibody is clone 10F.9G2, BE0101 and BioXcell;

the tumor is colorectal cancer.

2. The application of the pharmaceutical composition according to claim 1, wherein when the drug is a PD-L1 antibody, the dosage ratio of the radix tetrastigme polysaccharide to the PD-L1 antibody is 10-400:1.

3. The use of the pharmaceutical composition according to claim 2, wherein when the drug is a PD-L1 antibody, the dosage ratio of the radix tetrastigme polysaccharide to the PD-L1 antibody is 60-350:1.

4. The use of the pharmaceutical composition according to claim 2, wherein when the drug is a PD-L1 antibody, the dosage ratio of the radix tetrastigme polysaccharide to the PD-L1 antibody is 300-350:1.

5. A kit for anti-tumour comprising the pharmaceutical composition of any one of claims 1 to 4.

6. The kit according to claim 5, wherein in the method of using the kit, the single dose of the PD-L1 antibody is 1-10 μg/g.

7. The kit of claim 6, wherein in the method of using the kit, the single dose of the PD-L1 antibody is 4-6 μg/g.

8. The kit according to claim 5, wherein in the method of using the kit, the single administration dose of the radix tetrastigme polysaccharide is 50-400 mg/kg.

9. The kit of claim 8, wherein in the method of using the kit, the single dose of the radix tetrastigme polysaccharide is 150-250 mg/kg.