A chemotherapy-immunity combination drug and its application

Abstract

This invention discloses a chemotherapy-immunotherapy combination drug and its application, comprising a first mixture and a second mixture. The first mixture contains the immunoadjuvant imiquimod R837, and the second mixture contains the chemotherapeutic drug oxaliplatin, which induces immunogenic death. The first mixture is a uniformly dispersed imiquimod emulsion obtained by ball milling imiquimod and a surfactant, with imiquimod particles having a particle size of 0.5-3 micrometers. The imiquimod emulsion is sterilized by high-temperature moist heat. The second mixture is prepared by mixing alginate and oxaliplatin with water, followed by filtration and sterilization. This invention produces a synergistic anticancer effect in treating tumors, with optimized production process and good product stability. For tumors with multiple mutations and high antigen expression, checkpoint inhibition therapy is significantly effective. Chemotherapy kills tumors and provides antigens, and the adjuvant amplifies the immune response, turning most tumors into "hot" tumors, greatly improving the effectiveness of checkpoint inhibitors.

Description

[0001] This application is a divisional application of a patent application with the invention name “A chemotherapy-immunocombination drug and its preparation method”, the application date of which is September 26, 2019 and the application number is 201910915738.3. Technical Field

[0002] The present invention relates to the field of tumor treatment drugs, and in particular to a chemotherapy-immunotherapy drug composition, as well as a preparation method and application thereof. Background Art

[0003] Chemotherapy is one of the three main treatment methods for tumors in clinical practice. Most cancer patients need to undergo chemotherapy to a certain extent. For tumors that have a tendency to metastasize or have already metastasized, chemotherapy is the main treatment method. However, traditional chemotherapy drugs also damage normal organs, and the commonly used chemotherapy mode in clinical practice is systemic administration, which is not very selective for the lesion site, and the toxic side effects of chemotherapy are very large.

[0004] Although tumor immunotherapy represented by immune checkpoint blockade has achieved encouraging results in recent years, this therapy still has important limitations, including low clinical response rate (about 20%) and side effects caused by nonspecific immune response. In particular, the low clinical response rate of clinical immune checkpoint blockade therapy means that most patients do not respond to this expensive therapy. In order to further improve the efficacy and response rate of tumor treatment, it is necessary to improve the drug administration route of existing therapies and develop chemotherapy-immunotherapy for tumors to achieve synergistic effects. For example, it is necessary to consider how to better limit the killing of tumor cells by ICD chemotherapy drugs to the tumor site to avoid damage to the whole body; how to better amplify the immunogenicity of tumor-associated antigens after cancer cell death to obtain a stronger tumor-specific immune response; how to more effectively combine the effects of immune checkpoint inhibitors (such as CTLA-4, PD-1 / PD-L1 antibodies) or IDO inhibitors to further enhance the specific immune response to tumors by regulating immune balance. A series of problems. The development of new technologies in this area is of great practical significance to China, where cancer is prevalent and the research and development of original anti-cancer drugs is relatively backward. In addition, how to inhibit tumor metastasis and prevent its recurrence while providing local treatment has always been a difficult problem that has plagued the world.

[0005] At the same time, the operability of related drug production, as well as the sterilization and subsequent stability of drug products, are also difficult problems. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new type of chemotherapeutic immunodrug composition, a new anticancer drug composition that can produce a synergistic anti-cancer effect and reduce side effects, reduce the probability of cancer metastasis, and reduce the probability of cancer recurrence. It can effectively kill the original tumor while inhibiting the immune response and reducing the growth of distant metastatic tumors and the probability of tumor recurrence. At the same time, the production process is relatively optimized and the product stability is good.

[0007] In order to solve the related technical problems, the present invention provides the following technical solutions:

[0008] A chemotherapy-immunity combination drug, comprising a chemotherapy drug and an immune adjuvant capable of causing immunogenic death, characterized in that: the chemotherapy-immunity combination drug comprises a first mixture and a second mixture, the first mixture contains an immune adjuvant, and the second mixture contains a chemotherapy drug capable of causing immunogenic death; the chemotherapy drug capable of causing immunogenic death is oxaliplatin, the immune adjuvant is imiquimod R837, and further comprises poloxamer 188 and sodium alginate ALG;

[0009] The first mixture is prepared by mixing the imiquimod R837 and poloxamer 188 and ball-milling to obtain a uniformly dispersed imiquimod emulsion, wherein the imiquimod particles have a particle size of 0.5-3 microns, and the imiquimod emulsion is sterilized by high temperature and wet heat;

[0010] The second mixture is prepared by mixing the sodium alginate ALG, oxaliplatin and water, and filtering and sterilizing the mixture through a micron filter membrane;

[0011] The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

[0012] A chemotherapy-immunity combination drug, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant: the chemotherapy-immunity combination drug comprises a first mixture and a second mixture, the first mixture comprises an immune adjuvant, and the second mixture comprises a chemotherapy drug capable of causing immunogenic death; the chemotherapy drug capable of causing immunogenic death is oxaliplatin, the immune adjuvant is imiquimod R837, and further comprises poloxamer 188 and sodium alginate ALG;

[0013] The first mixture is: the imiquimod R837 and poloxamer 188 are mixed and ball-milled to obtain a uniformly dispersed imiquimod emulsion, the imiquimod particles have a particle size of 0.5-3 microns, and the imiquimod emulsion is mixed with oxaliplatin and water, stirred evenly, and sterilized by high temperature and wet heat;

[0014] The second mixture is a mixture of the sodium alginate and water, which is then filtered and sterilized through a micron filter membrane to form a mixture;

[0015] The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

[0016] As a preferred embodiment of the chemotherapy-immunotherapy combination drug: the mass ratio of imiquimod R837 to poloxamer 188 is 1:(0.1-5), and the high temperature sterilization is wet heat sterilization at 105°C to 150°C for 10-15 minutes;

[0017] The second mixture is sterilized by filtration through a 0.22 micron filter membrane and freeze-dried to obtain a freeze-dried powder.

[0018] As a preferred embodiment of the chemotherapy-immunotherapy combination drug: the mass ratio of imiquimod R837 to poloxamer 188 is 1:(0.1-5), and the high temperature sterilization is wet heat sterilization at 105°C to 150°C for 10-15 minutes;

[0019] The second mixture is sterilized by filtration through a 0.22 micron filter membrane and freeze-dried to obtain a freeze-dried powder.

[0020] A chemotherapy-immunity combination drug, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant, the chemotherapy-immunity combination drug comprising a first mixture and a second mixture, the first mixture comprising the immune adjuvant, and the second mixture comprising the chemotherapy drug capable of causing immunogenic death;

[0021] The first mixture is, the immune adjuvant is imiquimod, imiquimod and a surfactant are mixed and ball-milled to obtain a uniformly dispersed imiquimod emulsion, the imiquimod particles have a particle size of 0.5-300 microns, the imiquimod emulsion is sterilized by high temperature and wet heat, and the surfactant is poloxamer 407, or polysorbate 80 (Tween 80), or polyethylene glycol-12-hydroxystearate (Solutol HS 15), or egg yolk lecithin, or polyoxyethylene (35) castor oil, or vitamin E succinate polyethylene glycol ester, or sodium hydroxymethylcellulose;

[0022] The second mixture is prepared by mixing sodium alginate, oxaliplatin and water, and filtering and sterilizing through a micron filter membrane to prepare a mixture;

[0023] The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

[0024] As a preferred embodiment of the chemotherapy-immunotherapy combination drug: the sodium alginate is replaced by chitosan, or fibrinogen, or alginate, or hyaluronic acid;

[0025] The imiquimod R837 is replaced by imidazoquinoline or glucopyranoside lipid;

[0026] The oxaliplatin Oxa is replaced by anthracyclines, or cyclophosphamide, or bortezomib, or gemcitabine, or 5-fluorouracil, or toxins.

[0027] A chemotherapy-immunity combination drug, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant, characterized in that: the chemotherapy-immunity combination drug comprises a first mixture and a second mixture, the first mixture comprising an immune adjuvant, and the second mixture comprising a chemotherapy drug capable of causing immunogenic death;

[0028] The first mixture is: the imiquimod R837 is mixed with a surfactant and ball-milled to obtain a uniformly dispersed imiquimod emulsion, wherein the imiquimod particles have a particle size of 0.5-3 microns, and the imiquimod emulsion is mixed with oxaliplatin and water, stirred evenly, and sterilized by high temperature and wet heat;

[0029] The surfactant is one or more of poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), egg yolk lecithin, polyoxyethylene (35) castor oil, vitamin E succinate polyethylene glycol ester, or sodium hydroxymethylcellulose;

[0030] The second mixture is a mixture of the sodium alginate and water, which is then filtered and sterilized through a micron filter membrane to form a mixture;

[0031] The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

[0032] As a preferred embodiment of the chemotherapy-immunotherapy combination drug: the sodium alginate can be replaced by chitosan, or fibrinogen, or alginate, or hyaluronic acid;

[0033] The imiquimod R837 can be replaced by imidazoquinoline or glucopyranoside lipid;

[0034] The oxaliplatin Oxa can be replaced by anthracyclines, or cyclophosphamide, or bortezomib, or gemcitabine, or pentafluorouracil, or toxins.

[0035] A method for preparing a chemotherapy-immunotherapy combination drug, characterized by comprising the following steps:

[0036] Step 1: Weigh imiquimod R837 and surfactant poloxamer 188 in a ratio of 1: (0.1-5), add water and ball mill for 2-3 hours, take out the homogenate after the end, add water, stir and mix, and sterilize at 105℃~150℃ for 10-15 minutes;

[0037] Step 2: Weigh sodium alginate ALG and oxaliplatin, add water, stir, and filter the obtained solution through a micron filter membrane for sterilization; after precooling, freeze-dry;

[0038] Step 3: When using, add the freeze-dried powder of mixture 2 into the solution of mixture 1, shake thoroughly to mix and dissolve, and then inject.

[0039] A chemotherapy-immunity combination drug for treating colon cancer tumors, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant, characterized in that: the chemotherapy-immunity combination drug comprises a first mixture and a second mixture, the first mixture comprising an immune adjuvant, and the second mixture comprising a chemotherapy drug capable of causing immunogenic death;

[0040] The first mixture is, the immune adjuvant is imiquimod, imiquimod and a surfactant are mixed and ball-milled to obtain a uniformly dispersed imiquimod emulsion, the imiquimod particles have a particle size of 0.5-3 microns, the imiquimod emulsion is sterilized by high temperature and wet heat, and the surfactant is poloxamer 407, or polysorbate 80 (Tween 80), or polyethylene glycol-12-hydroxystearate (Solutol HS 15), or egg yolk lecithin, or polyoxyethylene (35) castor oil, or vitamin E succinate polyethylene glycol ester, or sodium hydroxymethylcellulose;

[0041] The second mixture is prepared by mixing sodium alginate, oxaliplatin and water, and filtering and sterilizing through a micron filter membrane to prepare a mixture;

[0042] The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

[0043] The present invention also provides an in situ gelling chemotherapy-immunotherapy biopolymer drug composition, which contains: a first component is alginate, the alginate can form a porous gel with calcium ions in the body, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate;

[0044] The second group consists of chemotherapeutic agents that can cause immunogenic death;

[0045] The third category of components is immune adjuvants.

[0046] As a preferred embodiment of the in situ gelation chemotherapy-immunotherapy biopolymer pharmaceutical composition: the immune adjuvant is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod.

[0047] As a preferred embodiment of the in situ gelation chemotherapy-immunotherapy biopolymer drug composition: the second component of the chemotherapy drug that can cause immunogenic death is one or more of anthracyclines such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine.

[0048] As a preferred embodiment of the in situ gelation chemotherapy-immunotherapy biopolymer drug composition: it also includes a fourth component immune checkpoint inhibitor or IDO inhibitor, the fourth component immune checkpoint inhibitor antibodies usually include anti-CTLA-4, anti-PD-1 and anti-PD-L1, small molecule inhibitors usually include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1, and peptide inhibitors include DPPA-1;

[0049] The IDO inhibitors include small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.

[0050] As a preferred solution of the in situ gelling chemotherapy-immunotherapy biopolymer drug composition: the first component is sodium alginate, the second component is doxorubicin hydrochloride; the third component is imiquimod, and the mass ratio of the sodium alginate, doxorubicin hydrochloride and imiquimod is 50-800:1-100:1-100.

[0051] As a preferred solution of the in situ gelling chemotherapy-immunotherapy biopolymer drug composition: the mass ratio of the sodium alginate, doxorubicin hydrochloride and imiquimod is 200-400: 10-75: 10-75.

[0052] As a preferred solution of the in situ gelation chemotherapy-immunotherapy biopolymer pharmaceutical composition: the concentration of the sodium alginate is above 5 mg / ml.

[0053] A method for preparing an in situ gelling chemotherapy-immunotherapy biopolymer pharmaceutical composition, the method comprising:

[0054] Dissolving sodium alginate, imiquimod hydrochloride freeze-dried powder and doxorubicin hydrochloride in an aqueous solution, stirring until the solution is clear and transparent, and then freeze-drying the solution to obtain a freeze-dried powder of the composition;

[0055] Alternatively, sodium alginate and doxorubicin hydrochloride are dissolved in an aqueous solution, stirred until the solution is clear and transparent, and then freeze-dried to obtain a freeze-dried powder, which is then mixed with imiquimod hydrochloride freeze-dried powder by solid-solid shaking to obtain a composite freeze-dried powder;

[0056] Alternatively, doxorubicin hydrochloride and imiquimod hydrochloride are dissolved in an aqueous solution, stirred until the solution is clear and transparent, and then the sodium alginate aqueous solution is dropped into the continuously stirred mixed solution to ensure that the mixed solution is clear and transparent without flocculent precipitation, and the mixed solution is taken out and lyophilized to obtain a lyophilized powder of the composition.

[0057] As a preferred scheme of the in situ gelling chemotherapy-immunotherapy biopolymer drug composition: the first component is sodium alginate, the second component is oxaliplatin; the third component is imiquimod hydrochloride, and the mass ratio of sodium alginate, oxaliplatin and imiquimod is 50-800:1-75:1-100.

[0058] As a preferred solution of the in situ gelling chemotherapy-immunotherapy biopolymer pharmaceutical composition: the mass ratio of the sodium alginate, oxaliplatin and imiquimod is 200-400: 10-75: 10-75.

[0059] The method for preparing the in situ gelling chemotherapy-immunotherapy biopolymer pharmaceutical composition comprises:

[0060] Dissolving sodium alginate, imiquimod hydrochloride freeze-dried powder and oxaliplatin in an aqueous solution, stirring until the solution is clear and transparent, and then freeze-drying the solution to obtain a freeze-dried powder of the composition;

[0061] Alternatively, sodium alginate and oxaliplatin are dissolved in an aqueous solution and stirred until the solution is clear and transparent, and then freeze-dried to obtain a freeze-dried powder, which is then mixed with imiquimod hydrochloride freeze-dried powder by solid-solid shaking to obtain a composite freeze-dried powder.

[0062] Alternatively, oxaliplatin and imiquimod hydrochloride are dissolved in an aqueous solution and stirred until the solution is clear and transparent, then sodium alginate is dissolved in the aqueous solution and dropped into the continuously stirred mixed solution to ensure that the mixed solution is clear and transparent without flocculent precipitation, and the mixed solution is taken out and lyophilized to obtain a lyophilized powder of the composition.

[0063] As a preferred solution of the in situ gelation chemotherapy-immunotherapy biopolymer drug composition: the first component is sodium alginate, the second component is pentafluorouracil; the third component is imiquimod hydrochloride;

[0064] Alternatively, the first component is sodium alginate, the second component is cyclophosphamide; and the third component is imiquimod hydrochloride.

[0065] As a preferred embodiment of the in situ gelling chemotherapy-immunotherapy biopolymer drug composition: the first component is sodium alginate; the second component is doxorubicin hydrochloride or oxaliplatin; the third component is imiquimod hydrochloride; and the fourth component is anti-PDL1 antibody.

[0066] As a preferred embodiment of the in situ gelling chemotherapy-immunotherapy biopolymer drug composition: the first component is potassium alginate or ammonium alginate; the second component is doxorubicin hydrochloride or oxaliplatin; the third component is imiquimod hydrochloride; and the fourth component is anti-PDL1 antibody.

[0067] The invention discloses an in situ gelling chemotherapy-immunotherapy biopolymer drug composition, which is composed of: a first component is alginate, which can form a porous gel with calcium ions in the body, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; a second component is a chemotherapy drug that can cause immunogenic death; and a third component is an immune adjuvant.

[0068] The invention discloses an in situ gel-forming chemotherapy-immunotherapy biopolymer drug composition, which comprises: a first component is alginate, which can form a porous gel with calcium ions in the body, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; a second component is a chemotherapy drug that can cause immunogenic death, which is one or more of anthracyclines such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine; a third component is an immune adjuvant, which is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod.

[0069] An in situ gelling chemotherapy and immunotherapy biopolymer drug composition, comprising: a first component is alginate, the alginate can form a porous gel with calcium ions in the body, the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; a second component is a chemotherapy drug that can cause immunogenic death, which is one or more of anthracycline drugs such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine; a third component is an immune adjuvant, which is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod;

[0070] The fourth component is an immune checkpoint inhibitor or IDO inhibitor. The fourth component immune checkpoint inhibitor antibodies usually include anti-CTLA-4, anti-PD-1 and anti-PD-L1, small molecule inhibitors usually include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1, and peptide inhibitors include DPPA-1;

[0071] The IDO inhibitors include BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole small molecules.

[0072] An in situ gelling chemotherapy-immunotherapy biopolymer drug composition comprises: a first component is sodium alginate, a second component is doxorubicin hydrochloride; a third component is imiquimod, and the mass ratio of the sodium alginate, doxorubicin hydrochloride and imiquimod is 50-800:1-100:1-100.

[0073] The present invention provides a series of pharmaceutical compositions. In this composition system, there are mainly four types of components, and the first type of components can be combined with other types of components in different ways according to actual conditions, including:

[0074] The first type of components: sodium alginate excipients, which can produce gels with calcium ions and other ions in the human or animal body;

[0075] Category II components: chemotherapeutic drugs that cause immunogenic death;

[0076] The third component: immune adjuvant;

[0077] The fourth category of components: immune checkpoint inhibitors or IDO inhibitors.

[0078] The first category of components, excipients, usually include sodium alginate, potassium alginate and ammonium alginate. These polysaccharides will cross-link with each other to form a gel when encountering divalent ions such as calcium ions. Therefore, when the drug is encapsulated in it, the formed gel can effectively release the drug in it, thereby enhancing the efficacy and reducing side effects.

[0079] Sodium alginate is a natural polysaccharide that has the stability, solubility, viscosity and safety required for pharmaceutical excipients. Sodium alginate has been widely used in the food industry and the pharmaceutical field. Sodium alginate is the most widely used water-soluble alginate. Sodium alginate can quickly undergo ion exchange and form gel when it encounters calcium ions. There are sufficient calcium ions in the human body or animal body, so it can form gel in situ in the body.

[0080] Although potassium alginate and ammonium alginate contain different cations from sodium alginate, they can also cross-link with calcium ions to form porous gels, thereby playing a role in sustained release of drugs. Currently, sodium alginate is usually extracted from seaweed, so sodium alginate is a better choice.

[0081] The second category of components, chemotherapy drugs that can cause immunogenic death, include anthracyclines such as doxorubicin, epirubicin, mitoxantrone, etc., as well as oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, 5-fluorouracil and toxins such as maytansine, etc. These drugs have been clinically approved, and studies in recent years have shown that these drugs can cause immunogenic death of cancer cells. These dead cancer cells will express calreticulin that is easily recognized and taken up by immune cells, especially antigen-presenting cells, helping immune cells to recognize tumor cells and induce effective anti-tumor immune responses.

[0082] The third type of component, immune adjuvant, referred to as adjuvant, is a non-specific immunoproliferative agent, which refers to auxiliary substances that can enhance the body's immune response to antigens or change the type of immune response when injected into the body together with antigens or in advance. There are many types of immune adjuvants, and there is no unified classification method at present. Freund's adjuvant and cytokine adjuvant are more commonly used. The immunobiological effects of immune adjuvants are to enhance immunogenicity, enhance antibody titers, change the type of antibody production, and cause or enhance delayed hypersensitivity reactions. However, the specific mechanism of action of immune adjuvants is not yet fully understood, and the mechanisms of action of different adjuvants are also different. Usually there are imiquimod (R837), CpG oligonucleotides, monophosphoryl lipid A and resiquimod, etc. They are all agonists of Toll-like receptors (TLR for short), which can help antigen-presenting cells present antigens. Therefore, immune adjuvants can better present tumor-associated antigens produced by chemotherapy to T cells, thereby amplifying the immune response.

[0083] The fourth category of components, immunomodulators, include immune checkpoint inhibitors or IDO inhibitors. Immune checkpoint inhibitors include antibody inhibitors or small molecule inhibitors. Antibody inhibitors usually include anti-CTLA-4, anti-PD-1 and anti-PD-L1. Small molecule inhibitors usually include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1. Peptide inhibitors include DPPA-1. IDO inhibitors include small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole, which can inhibit IDO enzymes and enhance the role of antigen presenting cells. Since tumor cells can deceive the immune system and escape the immune response, these antibodies are needed to suppress the immune response that protects the tumor so that immune cells can better kill tumor cells.

[0084] The first category of components, excipients, can also be referred to as component one; the second category of components, ICD chemotherapy drugs, can also be referred to as component two; the third category of components, immune adjuvants, can also be referred to as component three; the fourth category of components, immune checkpoint inhibitors, can also be referred to as component four.

[0085] Lyophilized powder is a sterile powder injection made by freezing the drug solution into a solid state in a sterile environment and then vacuuming the water to sublimate and dry it.

[0086] The preparation process of the mixed liquid and freeze-dried preparation of component one, component two, component three and component four is as follows.

[0087] This patent mainly involves four types of raw materials: the first type of component auxiliary material sodium alginate (solid powder), the second type of component ICD chemotherapy drugs (solid powder), the third type of component immune adjuvant (solid powder), and the fourth type of component immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies, commercial products for clinical use, in the form of lyophilized powder or injection solution as raw materials).

[0088] Preparation scheme 1: Mix the solid powders of component 1 excipients, component 2 ICD chemotherapy drugs, and component 3 immune adjuvants in a certain proportion and put them into a large beaker, add deionized water (or physiological saline, or phosphate buffer solution), and stir with a stirring paddle at a speed of 50 to 500 rpm at room temperature of 25 degrees Celsius until the solution is clear and transparent; as needed, prepare component 4 immune checkpoint inhibitors into injection according to the product instructions and add them to the above mixed solution; after stirring the above solution evenly, take out the sub-bottles and freeze-dry. After the freeze-dried powder is reconstituted, turbidity and flocculent precipitation should not appear.

[0089] Preparation scheme 2: Weigh the target mass of component 1, component 2, and component 3 respectively, and add deionized water (or physiological saline, or phosphate buffer solution) to prepare three independent solutions; as needed, prepare component 4 into an injection solution according to the product instructions; mix the above solutions in an appropriate volume ratio, stir with a stirring paddle at a speed of 50 to 500 rpm at room temperature of 25 degrees Celsius until the solution is uniform and not turbid, and take out the sub-bottles for freeze-drying. After the freeze-dried powder is reconstituted, turbidity and flocculent precipitation should not appear. There is not much difference between scheme 1 and scheme 2.

[0090] Preparation scheme three: Weigh the target mass of component one, component two, and component three respectively, and add deionized water (or saline or phosphate buffer solution) to prepare three independent solutions; as needed, prepare component four into an injection according to the product instructions; for ICD drugs containing hydrochloride, it is necessary to stir and mix the solutions of component two, component three, and component four first, and slowly drip the solution of component one during the stirring process (25 degrees Celsius, stirring paddle at a speed of 50 to 300 rpm) until the entire solution is stirred evenly. Compared with schemes one and two, when component two or component three is a hydrochloride drug, considering the effect of pH value on solution stability, the composition needs to be prepared according to this scheme, so that the prepared composition mixed solution has no flocculent precipitation phenomenon, ensures uniform mixing, and the solution is clear and transparent, and then takes out the bottle for lyophilization. After the lyophilized powder is reconstituted, there will be no turbidity and flocculent precipitation.

[0091] Instructions for use of mixed solutions and lyophilized preparations of four types of components.

[0092] Scheme 1: After the freeze-dried powder injection of the above four components is reconstituted with physiological saline, the composition solution is directly injected into the patient's tumor site through clinical intervention administration and direct puncture administration. The multi-point injection method is used during injection to ensure that the composition solution is evenly filled with the entire tumor. After the composition is injected into the tumor, firstly, the first component alginate forms a gel when it encounters calcium ions. The first component will quickly gel after encountering calcium ions in the tissue to form a porous mesh cross-linked structure, so that the other three components mixed in the alginate can be slowly released, thereby enhancing its effect and reducing toxic side effects; secondly, the second component ICD chemotherapy drug can not only effectively kill tumor cells but also cause them to produce immunogenic death, produce tumor-associated antigens, and activate tumor-specific immune responses; thirdly, the third component immune adjuvant enhances the ability of antigen-presenting cells and further amplifies the corresponding immune response; finally, the fourth component immune checkpoint inhibitor or IDO inhibitor is used to prevent the metastatic tumor from escaping the immune response, so that immunotherapy can more effectively kill the tumor, thereby inhibiting the metastasis and recurrence of the tumor. (Example 12)

[0093] Usage plan two: After the freeze-dried powder injection of the composition of the above-mentioned first, second and third components is reconstituted with physiological saline, the composition solution is directly injected into the patient's tumor site through clinical intervention administration and direct puncture administration. The injection is carried out in a multi-point injection manner to ensure that the composition solution is evenly filled in the entire tumor. This treatment method is recommended to be used in combination with the fourth component immune checkpoint inhibitor: the combination scheme includes adding immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies) or IDO inhibitors to the injection solution according to the individual situation of the patient, and a one-time local injection into the tumor; it can also be an intravenous injection of immune checkpoint inhibitors after local treatment with injection of the mixed solution of the first, second and third components. (Reference example in Example 12)

[0094] Scheme 3 (spraying the wound with the combination of the four components, and then spraying the calcium ion solution to form a gel): After the tumor patient has normal surgery to remove the lesion, considering that the surgical resection cannot completely remove the tumor cells in the lesion, the freeze-dried powder injection of the above four components can be reconstituted with physiological saline, and then sprayed on the wound after surgical resection with a syringe or spray bottle, and then an appropriate amount of calcium chloride solution can be sprayed on the site to make it gel, and finally the wound is sutured. This scheme helps to eliminate residual cancer cells and can inhibit tumor metastasis and recurrence. (Example 16)

[0095] Scheme 4 (spraying the wound with the combination of the three components, then spraying the calcium ion solution to form a gel + adding the fourth component for combined use): After the tumor patient has normal surgical resection of the lesion site, considering that surgical resection cannot completely remove the tumor cells in the lesion site, the freeze-dried powder injection of the above-mentioned first, second and third components can be reconstituted with physiological saline, and then sprayed on the wound site after surgical resection with a syringe or spray bottle, and then an appropriate amount of calcium chloride solution can be sprayed on the site to make it gel, and finally the wound is sutured. This scheme helps to eliminate residual cancer cells and can inhibit tumor metastasis and recurrence. This treatment method is recommended to be used in combination with the fourth component immune checkpoint inhibitor or IDO inhibitor after treatment: the combined scheme includes adding immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies) or IDO inhibitors to the injection solution according to the individual situation of the patient, and a one-time local injection into the tumor; it can also be intravenous injection of immune checkpoint inhibitors after local treatment with injection of the mixed solution of the first, second and third components.

[0096] The use of the technical solution of this patent will have the following beneficial technical effects:

[0097] 1. Alginate is a natural polysaccharide, which is safe and non-toxic, has good biocompatibility, can be degraded, and is a good biomaterial. However, in the field of medicine, the common way of using it is to combine alginate and calcium ions in vitro to form a gel implantable material. This method of use not only limits its application in vivo, but also often requires surgery or intervention, which is difficult to operate, causes great damage to patients, and is not conducive to combined drug treatment. Therefore, we use the method of injecting alginate into the tumor, using the calcium ions in the tumor tissue to make sodium alginate gel in situ in the tumor, and using the formed cross-linked network structure to slowly release the drugs mixed in the alginate, with a better slow release effect. This technology has broad prospects for use. It can be directly injected with a syringe to treat tumors, which is simple to operate and less invasive; it can also be sprayed on the wound site after surgery with a sprayer to clean up the residual cancer cells with surgery. It is expected to provide personalized treatment for different patients at a low cost.

[0098] Second: Most conventional chemotherapy in clinical practice is administered intravenously or by infusion. This treatment method does not have good selectivity and targeting, and it damages both lesions and normal tissues, with significant side effects. Patients will suffer great physical and psychological harm. In addition, conventional chemotherapy requires maintaining a certain blood drug concentration, using large doses and multiple administrations, which not only greatly increases side effects, but also increases the cost of administration. We adopt a strategy of direct intratumoral administration and combine it with gel sustained-release technology to allow chemotherapy drugs to stay in the lesion site for a longer time, maximize the effect of the drug, and greatly reduce the damage of the drug to normal tissues. Direct intratumoral administration and sustained release allow the effective drug concentration in the lesion to be maintained at a very high range for a long time, while ensuring the efficacy of the drug, the number of administrations can be reduced, further reducing side effects and costs.

[0099] 3: The third type of component immune adjuvant mentioned in this technical solution has not been used clinically to directly treat tumors. These small molecule immunomodulators do not have antiviral and anti-tumor effects themselves, and are often only used as auxiliary adjuvants of vaccines to enhance the immunogenicity of antigens. For example, imiquimod (R873), which is usually used as an ointment preparation to treat adult genital and perianal condyloma acuminatum, has not been used in clinical tumor treatment. The technology of this patent adopts the method of injecting ICD chemotherapy drugs and immune adjuvants together. While the ICD drugs kill tumors and produce tumor antigens, the antigens and adjuvants play a role similar to tumor vaccines, which can not only inhibit metastatic tumors, but also prevent tumor recurrence. The technology of this patent has created a new strategy for the direct treatment of tumors with immune adjuvants in combination with chemotherapy drugs.

[0100] Four: Currently, immune checkpoint inhibition therapy has received much attention both in the scientific research front and in the clinic. However, although this type of antibody has a miraculous effect on some patients, its effectiveness is not 100%. In other words, the effect of checkpoint inhibitors on different patients with different indications needs further study. Current studies have shown that tumors can be divided into hot tumors and cold tumors. For those tumors with many mutations and high antigen expression, checkpoint inhibition therapy is often effective. Our solution uses chemotherapy to kill tumors to provide antigens, and then uses adjuvants to amplify their immune response, which means that for most tumors, this patented solution can turn them into hot tumors, greatly improving the effectiveness of checkpoint inhibitors.

[0101] 5. The pharmaceutical composition mixed with the relevant components of this patent can produce a unique and unexpected synergistic anti-cancer effect, and can reduce the side effects of conventional treatments, reduce the probability of cancer metastasis, and reduce the probability of cancer recurrence. It provides a highly effective tumor-specific immunotherapy program that can effectively kill in situ tumors while inhibiting the immune response and reducing the growth of distant metastatic tumors and the probability of tumor recurrence. It can help patients prolong their survival period and improve their quality of life while relatively controlling costs.

[0102] Six: Further, the relevant technical solutions in this patent can solve the problem of imiquimod being poorly soluble in water, the problem of imiquimod's stability after sterilization, and the problem of sterilization of sodium alginate. From the perspective of sterilization, sodium alginate and imiquimod R837 cannot be matched together. Sodium alginate ALG needs to be filtered and sterilized, but R837 particles cannot be filtered (the minimum particle diameter after ball milling is 500nm, and filtration sterilization requires a 220nm filter membrane, so it cannot pass); on the other hand, R837 requires wet heat sterilization, while sodium alginate ALG will degrade at high temperature. R837 and ALG cannot be sterilized together. Sodium alginate is a relatively special natural biopolymer material. Since it decomposes at high temperatures, traditional high temperature and wet heat sterilization cannot be used. In this patent, filtration sterilization is used to retain the properties of sodium alginate. The dosage form and preparation method of the relevant embodiments of this patent solve the relevant technical problems. At the same time, surfactants are selectively added, and poloxamer 188 is particularly preferred. Without the addition of Poloxamer 188, the R837 ball-milled emulsion will become unstable and produce obvious precipitation and particles after wet heat sterilization at 121°C, and its water dispersibility will be greatly reduced. Poloxamer 188 can greatly help R837 ensure its water dispersibility and stability after sterilization.

[0103] According to the technical solution of this patent, sodium alginate, chemotherapeutic drugs, and immune adjuvants combined with PD-1 can achieve relatively excellent therapeutic effects. However, further optimization, sodium alginate, chemotherapeutic drugs and immune adjuvants, plus poloxamer 188 to form a composition, can produce better therapeutic effects without the need for PD-1, and the effect is better, but the treatment cost is lower, see Fig.30 , Fig.31 , we can get the specific experimental data. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The invention discloses a preparation process of a lyophilized powder injection of a sodium alginate and imiquimod hydrochloride composition in Example 1, and instructions for use thereof.

[0105] Figure 2 This is a scanning electron microscope picture of the freeze-dried powder injection of the sodium alginate and imiquimod hydrochloride composition in Example 1 after reconstitution into gel.

[0106] Figure 3 The imiquimod drug release curve and data statistics at different sodium alginate concentrations in Example 1 are shown.

[0107] Figure 4 The imiquimod drug release curve and data statistics at different imiquimod concentrations in Example 1 are shown.

[0108] Figure 5 This is a scanning electron microscope picture of the lyophilized powder injection of the sodium alginate and CpG oligonucleotide composition after reconstitution into gel in Example 2.

[0109] Figure 6 The CpG drug release curve and data statistics at different sodium alginate concentrations in Example 2.

[0110] Figure 7 The CpG drug release curves and data statistics at different CpG concentrations in Example 2.

[0111] Figure 8 This is a scanning electron microscope picture of the lyophilized powder injection of the sodium alginate and doxorubicin hydrochloride composition after reconstitution into gel in Example 3.

[0112] Fig. 9 The drug release curves and data statistics of doxorubicin hydrochloride at different sodium alginate concentrations in Example 3 are shown.

[0113] Fig.10 The drug release curves and data statistics of doxorubicin hydrochloride at different doxorubicin hydrochloride concentrations in Example 3 are shown.

[0114] Fig.11 This is a scanning electron microscope picture of the freeze-dried powder injection of the sodium alginate and oxaliplatin combination after reconstitution into gel in Example 4.

[0115] Fig.12 The drug release curves and data statistics of oxaliplatin at different sodium alginate concentrations in Example 4 are shown.

[0116] Fig.13 The oxaliplatin drug release curve and data statistics at different oxaliplatin concentrations in Example 4 are shown.

[0117] Fig.14 This is a scanning electron microscope picture of the sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder injection after reconstitution into gel in Example 5.

[0118] Fig.15 This is a rheological property test of the sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder injection after reconstitution in Example 5.

[0119] Fig.16 This is a scanning electron microscope image of the sodium alginate, oxaliplatin and imiquimod hydrochloride lyophilized powder injection after reconstitution into gel in Example 6.

[0120] Fig.17 This is a scanning electron microscope picture of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody lyophilized powder injection after reconstitution into gel in Example 9.

[0121] Fig.18 This is the antibody activity detection after the sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody lyophilized powder injection are reconstituted in Example 9.

[0122] Fig.19 The tumor growth curve and data statistics of the sodium alginate and imiquimod hydrochloride combination combined with radiofrequency ablation therapy and anti-PDL1 antibody therapy in the mouse colon cancer tumor model in Example 13.

[0123] Fig. 20 The tumor growth curve and data statistics of the sodium alginate and imiquimod hydrochloride combination combined with HIFU treatment and anti-PDL1 antibody treatment in the mouse colon cancer tumor model in Example 13.

[0124] Fig.21 The growth curve and data statistics of the second implanted tumor induced by the sodium alginate and imiquimod hydrochloride combination combined with HIFU treatment and anti-PDL1 antibody treatment in the mouse colon cancer tumor model in Example 13.

[0125] Fig. 22 This is the tumor growth curve and data statistics after the freeze-dried powder of the sodium alginate and oxaliplatin combination was used to treat colon cancer in mice in Example 14.

[0126] Fig.23 This is the weight curve and data statistics of mice after colon cancer treatment with the freeze-dried powder of the sodium alginate and oxaliplatin combination in Example 14.

[0127] Fig.24 This is the in situ tumor growth curve after the treatment of sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in the mouse bilateral tumor model in Example 15.

[0128] Fig.25 This is the distal tumor growth curve and data statistics after the treatment of sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in the mouse bilateral tumor model in Example 15.

[0129] Fig.26 The tumor growth curve and data statistics of sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in Example 15 after the bilateral tumor model in mice was cured and then inoculated with the tumor again.

[0130] Fig. 27 The data are the fluorescence imaging data of mice after the treatment of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody in the mouse orthotopic breast cancer tumor model in Example 16.

[0131] Fig.28 The tumor growth curve and data statistics after the treatment of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody in the mouse brain cancer model in Example 17.

[0132] Fig.29 These are the data of fluorescence imaging of mice after treatment with sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody in the mouse tumor surgical resection model in Example 18.

[0133] Fig.30 This is the tumor growth of different treatment methods for direct drug injection of larger tumors (starting volume>120 cubic millimeters) in Example 19.

[0134] Fig.31 This is the tumor growth of the contralateral small tumor (initial volume <50 cubic millimeters) in Example 19 without direct drug injection and different treatment methods. DETAILED DESCRIPTION

[0135] Example 1: Preparation and use of a lyophilized powder injection of a combination of sodium alginate (first component) and imiquimod (third component) hydrochloride

[0136] Step 1: Preparation of imiquimod (third-class component) hydrochloride. Weigh 50-100 mg of imiquimod in a 50 ml glass mixing container, add 1 ml of 1M dilute hydrochloric acid, and add deionized water to dilute the white powdered imiquimod until it is fully dissolved to colorless and transparent, so that the final concentration of imiquimod is 2.5-5 mg per ml. The solution is freeze-dried to obtain imiquimod hydrochloride freeze-dried powder. The purpose of this step is to convert the water-insoluble imiquimod into a water-soluble hydrochloride form. A sufficiently long freeze-drying time is required to ensure the complete removal of residual hydrochloric acid.

[0137] Step 2: Preparation of lyophilized powder injection of sodium alginate (first component) and imiquimod (third component) hydrochloride composition can be carried out by the following three methods.

[0138] Method 1: Weigh 10-80 mg of sodium alginate and 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and dissolve them in 1 ml of aqueous solution, stir with a stirring paddle at 50-300 rpm until the solution becomes clear and transparent, maintain the temperature at 20-40 degrees Celsius, and pH at 6.5. Freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0139] Method 2: Weigh 10-80 mg of sodium alginate and dissolve it in 1 ml of aqueous solution. Stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, freeze-dry to obtain a lyophilized powder injection, and then mix it with 0.1-10 mg of imiquimod hydrochloride lyophilized powder by solid-solid shaking to obtain a combined lyophilized powder injection.

[0140] Method 3: 0.1-10 mg of imiquimod hydrochloride freeze-dried powder is dissolved in 1 ml of aqueous solution, stirred with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, then 10-80 mg of sodium alginate is dissolved in the aqueous solution, and the solution is dripped into the continuously stirred imiquimod hydrochloride solution at a volume ratio of 1:20 to ensure that the mixed solution is clear and transparent without flocculent precipitation. After the sodium alginate solution is completely added, the mixed solution is taken out and freeze-dried to obtain a freeze-dried powder injection of the composition.

[0141] Figure 1 The invention discloses a preparation process of a lyophilized powder injection of a composition of sodium alginate (first-class component) and imiquimod (third-class component) hydrochloride, and an instruction for use thereof.

[0142] Figure 2 for Figure 1 Scanning electron microscope image of a freeze-dried powder injection of a composition prepared by the method after gelation. It can be seen from the image that the composition still has good gelation ability after freeze-drying and re-dissolving, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0143] Step 3: Release curve of imiquimod in the freeze-dried powder injection of the composition of sodium alginate (first-class component) and imiquimod (third-class component) hydrochloride. The drug sustained-release carrier refers to a drug that allows the drug to slowly enter the blood and reduce the blood drug concentration. The preparation of a sustained-release long-acting drug that can slowly release the drug component is often very necessary in treatment. The drug release curve refers to the release of the encapsulated drug after we simulate the composition into gel in vitro.

[0144] The following is the release curve obtained by fixing the dosage of imiquimod and changing the dosage of sodium alginate.

[0145] A lyophilized powder injection of a composition of sodium alginate (a first component) and imiquimod (a third component) hydrochloride is prepared, wherein the concentration of sodium alginate is 1, 10, 20, 40 and 80 mg, and the concentration of imiquimod is 2 mg. The lyophilized powder injection of the composition is respectively redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 200 μl of a 5 mg / ml calcium chloride solution is added to make it gel. The reason for adding calcium chloride is to simulate in vitro the situation of sustained release of the drug after the composition encounters calcium ions after gelation after being injected into a tumor, and the colloid is immersed in 1 ml of a phosphate buffer solution and stirred. The content of the drug in the phosphate buffer solution is measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which is the release of imiquimod.

[0146] Figure 3 The imiquimod drug release curve and statistical table at different sodium alginate concentrations are shown in the figure. It can be seen from the figure that when the sodium alginate concentration is 5 mg / ml or above, imiquimod has an obvious sustained release phenomenon, so the concentration of sodium alginate in the composition is preferably 5 mg / ml to 80 mg / ml. When the sodium alginate concentration is 10 mg / ml, it is already relatively optimized, and when the sodium alginate concentration is 20 mg / ml, it basically reaches the peak value. When the concentration is further increased, the effect is not significantly improved.

[0147] The following is the release curve obtained by fixing the dosage of sodium alginate and changing the dosage of imiquimod.

[0148] A lyophilized powder injection of a composition of sodium alginate (a first component) and imiquimod (a third component) hydrochloride is prepared, wherein the concentration of imiquimod is 1, 2.5, 5, 7.5 and 10 mg (maximum solubility), and the concentration of sodium alginate is 20 mg. The lyophilized powder injection of the composition is redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 5 mg / ml of a calcium chloride solution is added to form a gel, and the colloid is immersed in 1 ml of a phosphate buffer solution and stirred. The content of the drug in the phosphate buffer solution is measured on days 0, 0.25, 0.5, 1, 2, 4 and 8, which is the release of imiquimod.

[0149] Figure 4The imiquimod drug release curve and statistical table at different imiquimod concentrations are shown in the figure. It can be seen from the figure that when the imiquimod concentration is higher than 7.5 mg / ml, the composition has obvious rapid drug release during gelation, and the subsequent release is also faster than that at low concentrations. However, the sustained release effect is still obvious, so the concentration of imiquimod in the lyophilized powder injection of the composition is selected to be 0.1 to 10 mg / ml. This shows that regardless of the concentration of imiquimod, it is effective. Although the high concentration releases quickly, it also has an obvious sustained release effect.

[0150] Through the above experiments, it is obtained that the preferred mass ratio of sodium alginate to imiquimod hydrochloride is 50-800 to 1-100, and the more preferred mass ratio is 200-400 to 10-75.

[0151] Example 2: Lyophilized powder injection of sodium alginate (first component) and CpG oligonucleotide (third component) composition

[0152] Step 1: Preparation of lyophilized powder injection of sodium alginate and CpG oligonucleotide combination

[0153] Weigh 10-80 mg of sodium alginate and 0.1-5 mg of CpG oligonucleotide and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0154] Figure 5 The scanning electron microscope image of the freeze-dried powder injection of the composition after reconstitution into gel. It can be seen from the image that the composition still has a good gelling ability after freeze-drying and reconstitution, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0155] Step 2: CpG release curve in the lyophilized powder injection of sodium alginate and CpG oligonucleotide combination

[0156] The following is the release curve obtained by fixing the amount of CpG oligonucleotide and changing the amount of sodium alginate.

[0157] A lyophilized powder injection of a sodium alginate and CpG oligonucleotide composition was prepared, wherein the concentration of sodium alginate was 1, 10, 20 and 40 mg, and the concentration of CpG oligonucleotide was 0.2 mg. The lyophilized powder injection of the composition was respectively redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 200 μl of a 5 mg / ml calcium chloride solution was added to make it gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which was the release of the CpG oligonucleotide.

[0158] Figure 6The CpG drug release curves at different sodium alginate concentrations are shown in the figure. As can be seen from the figure, when the sodium alginate concentration is 20 mg or more, the CpG oligonucleotide has an obvious sustained release phenomenon, so the concentration of sodium alginate in the lyophilized powder injection of the composition is selected to be 5 mg / ml to 80 mg / ml. When the sodium alginate concentration is 1 mg / ml, the effect is not very obvious. When the sodium alginate concentration is 10 mg / ml, the effect is already relatively optimized. When the sodium alginate concentration is 20 mg / ml, it basically reaches the peak value. When the concentration is further increased, the effect is not significantly improved.

[0159] The following is the release curve obtained by fixing the amount of sodium alginate and changing the amount of CpG oligonucleotide.

[0160] A lyophilized powder injection of a sodium alginate and CpG oligonucleotide composition was prepared, wherein the concentration of the CpG oligonucleotide was 0.1, 0.25, 0.5, 1 and 2 mg, and the sodium alginate was 20 mg. The lyophilized powder injection of the composition was respectively redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 5 mg / ml of a calcium chloride solution was added to form a gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which was the release of the CpG oligonucleotide.

[0161] Figure 7 is the CpG drug release curve at different CpG oligonucleotide concentrations. Figure 8 It can be seen that when the concentration of CpG oligonucleotide is higher than 1 mg / ml, a relatively obvious acute release occurs, while the subsequent release rate does not change much. For cost considerations, the price of CpG oligonucleotide is about RMB 10,000 / mg, so the concentration of CpG oligonucleotide in the lyophilized powder injection of the composition is selected to be 0.1-2 mg / ml, and preferably the concentration of CpG oligonucleotide is selected to be 0.1-0.5 mg / ml.

[0162] The above experiments show that the optimal mass ratio of sodium alginate to CpG oligonucleotide is 50-800 to 1-20, and the more preferred mass ratio is 200-400 to 1-20.

[0163] Example 3: Lyophilized powder injection of sodium alginate (first component) and doxorubicin hydrochloride (second component)

[0164] Step 1: Preparation of lyophilized powder injection of sodium alginate and doxorubicin hydrochloride composition:

[0165] Method 1: Weigh 20-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. Stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent. Then freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0166] Method 2: Dissolve 0.1-10 mg of doxorubicin hydrochloride in 1 ml of aqueous solution, stir with a stirring paddle at 50-300 rpm until the solution is clear and transparent, then dissolve 10-80 mg of sodium alginate in the aqueous solution, and drop into the continuously stirred doxorubicin hydrochloride solution at a volume ratio of 1:20, ensuring that the mixed solution is clear and transparent without flocculent precipitation. After all the sodium alginate solution is added, take out the mixed solution and freeze-dry it to obtain the freeze-dried powder injection of the composition.

[0167] Figure 8 The scanning electron microscope image of the freeze-dried powder injection of the composition after reconstitution into gel. It can be seen from the image that the composition still has a good gelling ability after freeze-drying and reconstitution, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0168] Step 2: Release curve of doxorubicin in the freeze-dried powder injection of sodium alginate and doxorubicin hydrochloride combination

[0169] A lyophilized powder injection of a sodium alginate and doxorubicin hydrochloride composition was prepared, wherein the concentration of sodium alginate was 1, 10, 20 and 40 mg, and the concentration of doxorubicin hydrochloride was 2 mg. The lyophilized powder injection of the composition was respectively redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 200 μl of a 5 mg / ml calcium chloride solution was added to make it gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, i.e., the release of doxorubicin hydrochloride.

[0170] Fig. 9 3 is the drug release curve of doxorubicin hydrochloride at different sodium alginate concentrations. It can be seen from the figure that when the sodium alginate concentration is 10 mg or more, doxorubicin hydrochloride has an obvious sustained release phenomenon, so the concentration of sodium alginate in the lyophilized powder injection of the composition is preferably 5 mg / ml to 80 mg / ml.

[0171] A lyophilized powder injection of a sodium alginate and doxorubicin hydrochloride composition was prepared, wherein the concentration of doxorubicin hydrochloride was 1, 2.5, 5, 7.5 and 10 mg (maximum solubility), and the amount of sodium alginate was 20 mg. The lyophilized powder injection of the composition was redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 5 mg / ml of a calcium chloride solution was added to form a gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which was the release of doxorubicin hydrochloride.

[0172] Fig.10The drug release curves of doxorubicin hydrochloride at different doxorubicin hydrochloride concentrations. It can be seen from the figure that when the doxorubicin hydrochloride concentration is higher than 7.5 mg, a relatively obvious rapid release occurs, and the subsequent release is also faster than that at low concentrations. However, the sustained release effect is still obvious, so the concentration of doxorubicin in the lyophilized powder injection of the composition is selected to be 0.1 to 10 mg per milliliter.

[0173] Through the above experiments, it is obtained that the preferred mass ratio of sodium alginate to doxorubicin hydrochloride is 50-800:1-100, and the more preferred mass ratio is 200-400:10-75.

[0174] Example 4: Lyophilized powder injection of sodium alginate (first component) and oxaliplatin (second component)

[0175] Step 1: Preparation of lyophilized powder injection of sodium alginate and oxaliplatin combination

[0176] Weigh 10-80 mg of sodium alginate and 1-7.5 mg of oxaliplatin and dissolve them in 1 ml of aqueous solution. Stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent. Then freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0177] Fig.11 The scanning electron microscope image of the freeze-dried powder injection of the composition after reconstitution into gel. It can be seen from the image that the composition still has a good gelling ability after freeze-drying and reconstitution, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0178] Step 2: Release curve of oxaliplatin in lyophilized powder injection of sodium alginate and oxaliplatin combination

[0179] A lyophilized powder injection of a sodium alginate and oxaliplatin composition was prepared, wherein the concentration of sodium alginate was 1, 10, 20 and 40 mg, and the concentration of oxaliplatin was 2 mg. The lyophilized powder injection of the composition was respectively redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 200 μl of a 5 mg / ml calcium chloride solution was added to form a gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which was the release of oxaliplatin.

[0180] Fig.12 The figure shows the drug release curve of oxaliplatin at different sodium alginate concentrations. It can be seen from the figure that when the sodium alginate concentration is 10 mg or above, oxaliplatin has an obvious sustained release phenomenon, so the concentration of sodium alginate in the lyophilized powder injection of the composition is selected to be 5 mg / ml to 80 mg / ml.

[0181] A lyophilized powder injection of a sodium alginate and oxaliplatin composition was prepared, wherein the oxaliplatin concentration was 1, 2.5, 5 and 7.5 mg (maximum solubility), and the sodium alginate concentration was 20 mg. The lyophilized powder injection of the composition was redissolved in 1 ml of an aqueous solution and shaken until clear and transparent, and then 5 mg / ml of a calcium chloride solution was added to form a gel, and the colloid was immersed in 1 ml of a phosphate buffer solution and stirred. The drug content in the phosphate buffer solution was measured on days 0, 0.25, 0.5, 1, 2, 4, and 8, which was the release of oxaliplatin.

[0182] Fig.13 The figure shows the drug release curve of oxaliplatin at different oxaliplatin concentrations. It can be seen from the figure that when the oxaliplatin concentration is higher than 7.5 mg, a relatively obvious acute release occurs, and the subsequent release is also faster than that at low concentrations. However, the sustained release effect is still obvious, so the concentration of oxaliplatin in the lyophilized powder injection of the composition is selected to be 0.1-7.5 mg / ml.

[0183] Through the above experiments, it is obtained that the preferred mass ratio of sodium alginate to oxaliplatin is 50-800 to 1-75, and the more preferred mass ratio is 200-400 to 10-75.

[0184] Example 5: Sodium alginate (first component) and doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) lyophilized powder injection

[0185] Step 1: Preparation of lyophilized powder injection of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride

[0186] Method 1 (components 1, 2 and 3 are dissolved in an aqueous solution, stirred, and then the mixed solution is freeze-dried): 10 to 80 mg of sodium alginate (first component), 0.1 to 10 mg of imiquimod (third component) hydrochloride freeze-dried powder and 0.1 to 10 mg of doxorubicin hydrochloride (second component) are weighed and dissolved in 1 ml of an aqueous solution, stirred with a stirring paddle at a speed of 50 to 300 rpm until the solution becomes clear and transparent, and then the solution is freeze-dried to obtain a composite freeze-dried powder injection.

[0187] Method 2 (components one and two are dissolved in an aqueous solution, the mixed solution is stirred and freeze-dried to obtain a freeze-dried powder, which is solid-solid mixed with the freeze-dried powder of component three): 10 to 80 mg of sodium alginate (first component) and 0.1 to 10 mg of doxorubicin hydrochloride (second component) are weighed and dissolved in 1 ml of an aqueous solution, stirred with a stirring paddle at a speed of 50 to 300 rpm until the solution is clear and transparent, then freeze-dried to obtain a freeze-dried powder, and mixed with 0.1 to 10 mg of imiquimod hydrochloride freeze-dried powder (third component) by solid-solid shaking to obtain a composite freeze-dried powder injection.

[0188] Method 3 (components 2 and 3 are dissolved in an aqueous solution, stirred to obtain a clear solution, the solution of component 1 is dripped into the aforementioned mixed solution at a ratio of 1:20, and the final mixed solution is freeze-dried to obtain a freeze-dried powder): 0.1-10 mg of doxorubicin hydrochloride and 0.1-10 mg of imiquimod hydrochloride are dissolved in 1 ml of an aqueous solution, stirred with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, then 20-80 mg of sodium alginate is dissolved in 1 ml of an aqueous solution, and dripped into the continuously stirred mixed solution at a volume ratio of 1:20 to ensure that the mixed solution is clear and transparent without flocculent precipitation. After all the sodium alginate solution is added, the mixed solution is taken out and freeze-dried to obtain a composite freeze-dried powder injection.

[0189] The preferred mass ratio of the first component, the second component and the third component in the composition is 50-800: 1-100: 1-100, and the more preferred mass ratio is 200-400: 10-75: 10-75.

[0190] Fig.14 The scanning electron microscope image of the freeze-dried powder injection of the composition after reconstitution into gel. It can be seen from the image that the composition still has a good gelling ability after freeze-drying and reconstitution, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0191] Step 2: Determination of rheological properties of sodium alginate after reconstitution with doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder injection

[0192] Sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride freeze-dried powder injection were dissolved in 1 ml of phosphate buffer solution. The rheological properties of 20 μl of 1, 5, 10 and 20 mg / ml sodium alginate compositions were tested after mixing with 20 μl of 10 mg / ml calcium ion solution.

[0193] Fig.15 The rheological properties of the lyophilized powder injection of sodium alginate and doxorubicin and imiquimod combination at different concentrations after reconstitution and contact with calcium ions. As can be seen from the figure, when the concentration of sodium alginate is 1 mg / ml, its storage modulus is less than the loss modulus, showing the behavior of a fluid. When the concentration of sodium alginate reaches more than 10 mg / ml, its storage modulus is greater than the loss modulus, showing the behavior of a gel, proving that sodium alginate will form a colloid when it encounters calcium ions at a concentration of more than 10 mg / ml.

[0194] Example 6: Lyophilized powder injection of sodium alginate (first component), oxaliplatin (second component) and imiquimod hydrochloride (third component)

[0195] Method 1: Weigh 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, and 0.1-7.5 mg of oxaliplatin, dissolve in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, then freeze-dry the solution to obtain a composite freeze-dried powder injection.

[0196] Method 2: Weigh 10-80 mg of sodium alginate and 0.1-7.5 mg of oxaliplatin, dissolve in 1 ml of aqueous solution, stir with a stirring paddle at 50-300 rpm until the solution is clear and transparent, then freeze-dry to obtain a freeze-dried powder, and then mix with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder by solid-solid shaking to obtain a combined freeze-dried powder injection.

[0197] Method 3: Dissolve 0.1-10 mg of oxaliplatin and 0.1-10 mg of imiquimod hydrochloride in 1 ml of aqueous solution, stir with a stirring paddle at 50-300 rpm until the solution is clear and transparent, then dissolve 20-80 mg of sodium alginate in 1 ml of aqueous solution, and drop into the continuously stirred mixed solution at a volume ratio of 1:20 to ensure that the mixed solution is clear and transparent without flocculent precipitation. After all the sodium alginate solution is added, take out the mixed solution and freeze-dry it to obtain a freeze-dried powder injection of the composition.

[0198] The mass ratio of the first component, the second component and the third component in the composition is 50-800: 1-75: 1-100, and the more preferred mass ratio is 200-400: 10-75: 10-75.

[0199] Fig.16 The scanning electron microscope image of the freeze-dried powder injection of the composition after gelation. It can be seen from the image that the composition still has a good gelation ability after freeze-drying and re-dissolving, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0200] Example 7: Lyophilized powder injection of sodium alginate (first component) with pentafluorouracil (second component) and imiquimod hydrochloride (third component)

[0201] Method 1: Weigh 10-80 mg of sodium alginate, 1-5 mg of pentafluorouracil and 0.1-10 mg of imiquimod hydrochloride and dissolve them in 1 ml of 2 mg / ml sodium hydroxide solution. Shake thoroughly until the solution is clear and transparent, then freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0202] Method 2: Weigh 10-80 mg of sodium alginate and 1-5 mg of pentafluorouracil, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then freeze-dry to obtain freeze-dried powder, and mix with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder by shaking to obtain a composite freeze-dried powder injection.

[0203] Example 8: Lyophilized powder injection of sodium alginate (first component), cyclophosphamide (second component) and imiquimod hydrochloride (third component)

[0204] Method 1: Weigh 10-80 mg of sodium alginate, 1-5 mg of cyclophosphamide and 0.1-10 mg of imiquimod hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, freeze-dry the solution to obtain a freeze-dried powder injection of the composition.

[0205] Method 2: Weigh 10-80 mg of sodium alginate and 1-5 mg of cyclophosphamide, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then freeze-dry to obtain a lyophilized powder, and mix with 0.1-10 mg of imiquimod hydrochloride lyophilized powder by shaking to obtain a combined lyophilized powder injection.

[0206] Example 9: Lyophilized powder injection of sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component), and anti-PDL1 antibody (fourth component)

[0207] Method 1: Weigh 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride lyophilized powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of anti-PDL1 solution, mix well, and lyophilize the solution to obtain a lyophilized powder injection of the composition.

[0208] Method 2: Weigh 10-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then add 100 μg-5 mg of anti-PDL1 solution, mix evenly, lyophilize to obtain lyophilized powder, and add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0209] Fig.17 The scanning electron microscope image of the freeze-dried powder injection of the composition after gelation. It can be seen from the image that the composition still has a good gelation ability after freeze-drying and re-dissolving, and it can be seen from the electron microscope image that there are many micron-level pores after gelation, which is of great help to the sustained release of drugs.

[0210] Fig.18 This is an activity test of the anti-PDL1 antibody after lyophilization. From the experimental results, it can be seen that the peak value of the anti-PDL1 antibody binding to the cell surface PDL1 antibody flow cytometry after lyophilization is consistent with the peak value of the pure anti-PDL1 antibody, indicating that lyophilization does not affect the activity of the anti-PDL1 antibody.

[0211] Example 10: Lyophilized powder injection of sodium alginate (first component), oxaliplatin (second component), imiquimod hydrochloride (third component), and anti-PDL1 antibody (fourth component)

[0212] Method 1: Weigh 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride lyophilized powder, and 0.1-7.5 mg of oxaliplatin and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of anti-PDL1 solution, mix well, and lyophilize the solution to obtain a lyophilized powder injection of the composition.

[0213] Method 2: Weigh 10-80 mg of sodium alginate and 0.1-10 mg of oxaliplatin, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then add 100 μg-5 mg of anti-PDL1 solution, mix evenly, lyophilize to obtain lyophilized powder, and add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0214] Example 11: Lyophilized powder injection of other alginates (first type component) and doxorubicin hydrochloride (second type component) and imiquimod hydrochloride (third type component) and anti-PDL1 antibody (fourth type component)

[0215] Preparation: Potassium alginate (first component) and doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) lyophilized powder injection

[0216] Method 1: Weigh 10-80 mg of potassium alginate, 0.1-10 mg of imiquimod hydrochloride lyophilized powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of anti-PDL1 solution, mix well, and lyophilize the solution to obtain a lyophilized powder injection of the composition.

[0217] Method 2: Weigh 10-80 mg of potassium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 μg-5 mg of anti-PDL1 solution, mix evenly, and lyophilize to obtain a lyophilized powder, and then add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0218] Preparation: freeze-dried powder injection of ammonium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component), and anti-PDL1 antibody (fourth component)

[0219] Method 1: Weigh 10-80 mg of ammonium alginate, 0.1-10 mg of imiquimod hydrochloride lyophilized powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of anti-PDL1 solution, mix well, and lyophilize the solution to obtain a lyophilized powder injection of the composition.

[0220] Method 2: Weigh 10-80 mg of ammonium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then add 100 μg-5 mg of anti-PDL1 solution, mix evenly, lyophilize to obtain lyophilized powder, and add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0221] Alginate with different cations can form a good combination with the other three components and still have the ability of gelation and sustained release.

[0222] Example 12: Lyophilized powder injection of alginate (first component) with doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) and IDO inhibitor 4-phenylimidazole (fourth component)

[0223] Preparation: Sodium alginate (first component) and doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) and 4-phenylimidazole (fourth component) freeze-dried powder injection

[0224] Method 1: Weigh 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride lyophilized powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of 4-phenylimidazole solution and mix evenly. The solution is lyophilized to obtain a lyophilized powder injection of the composition.

[0225] Method 2: Weigh 10-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 μg-5 mg of 4-phenylimidazole solution, mix evenly, and freeze-dry to obtain a freeze-dried powder, and then add 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, shake and mix evenly to obtain a combined freeze-dried powder injection.

[0226] Preparation: Potassium alginate (first component) and doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) and 4-phenylimidazole (fourth component) freeze-dried powder injection

[0227] Method 1: Weigh 10-80 mg of potassium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of 4-phenylimidazole solution and mix evenly. The solution is freeze-dried to obtain a composite freeze-dried powder injection.

[0228] Method 2: Weigh 10-80 mg of potassium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then add 100 μg-5 mg of 4-phenylimidazole solution, mix evenly, lyophilize to obtain lyophilized powder, and add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0229] Preparation: freeze-dried powder injection of ammonium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component), and 4-phenylimidazole (fourth component)

[0230] Method 1: Weigh 10-80 mg of ammonium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, and 0.1-10 mg of doxorubicin hydrochloride and dissolve them in 1 ml of aqueous solution. After fully shaking until the solution is clear and transparent, add 100 μg-5 mg of 4-phenylimidazole solution, mix well, and freeze-dry the solution to obtain a composite freeze-dried powder injection.

[0231] Method 2: Weigh 10-80 mg of ammonium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then add 100 μg-5 mg of 4-phenylimidazole solution, mix evenly, lyophilize to obtain lyophilized powder, and add 0.1-10 mg of imiquimod hydrochloride lyophilized powder, shake and mix evenly to obtain a combined lyophilized powder injection.

[0232] The following are experiments and data statistics related to the synergistic therapeutic effects of different compositions.

[0233] Example 13: Study on the efficacy of a lyophilized powder injection of a combination of sodium alginate (first-class component) and imiquimod hydrochloride (third-class component) in a colon cancer model

[0234] Step 1: Study on the efficacy of a lyophilized powder injection of a combination of sodium alginate (first-class component) and imiquimod hydrochloride (third-class component) combined with radiofrequency ablation therapy and immune checkpoint inhibition therapy with anti-PDL1 antibody.

[0235] Mouse colon cancer tumors were implanted on the left and right ends of the mouse back (the left one was considered as the in situ tumor, and the right one was considered as the distal tumor). The tumor-bearing mice were divided into four groups, with 5 mice in each group for treatment experiments.

[0236] Group 1: Radiofrequency ablation alone for the left in situ tumor (reference case);

[0237] Group 2: RFA treatment of the left in situ tumor followed by tail vein injection of anti-pdl1 antibody (reference case);

[0238] Group 3: Radiofrequency ablation treatment was performed after intratumoral injection of a sodium alginate and imiquimod hydrochloride combination freeze-dried powder injection (Example 1) in the left in situ tumor;

[0239] Group 4: The left in situ tumor was treated with intratumoral injection of a sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) followed by radiofrequency ablation and tail vein injection of anti-PDL1 antibody.

[0240] After different treatments, the length and width of the distal tumor on the right side of the mice were measured with a vernier caliper every two days. The volume of the tumor was (length multiplied by (width squared)) divided by 2. The experimental results showed that the in situ tumors on the left side of the mice were eliminated by radiofrequency ablation, while the distal tumors on the right side of the fourth group of mice were significantly inhibited, indicating that the combination of sodium alginate and imiquimod hydrochloride can better stimulate anti-tumor immune response after radiofrequency ablation, and has a good synergistic effect with anti-PDL1 antibodies.

[0241] ( Fig.19 )

[0242] Step 2: Study on the efficacy of a lyophilized powder injection of a combination of sodium alginate (first-class component) and imiquimod hydrochloride (third-class component) combined with high-intensity focused ultrasound (HIFU) and immune checkpoint inhibition therapy with anti-PDL1 antibody.

[0243] Mouse colon cancer tumors were implanted on the left and right ends of the mouse back (the left one was considered as the in situ tumor, and the right one was considered as the distal tumor). The tumor-bearing mice were divided into four groups, with 5 mice in each group for treatment experiments.

[0244] Group 1: left in situ tumor treated with HIFU alone (reference case);

[0245] Group 2: left orthotopic tumor treated with HIFU and then injected with anti-pdl1 antibody into tail vein (reference case);

[0246] Group 3: Intratumoral injection of a sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) followed by HIFU treatment in the left in situ tumor;

[0247] Group 4: The left in situ tumor was injected with a lyophilized powder injection of a sodium alginate and imiquimod hydrochloride composition (Example 1) and then treated with HIFU plus tail vein injection of anti-PDL1 antibody.

[0248] After different treatments, the length and width of the distal tumor on the right side of the mice were measured with a vernier caliper every two days. The volume of the tumor is (length multiplied by (width squared)) divided by 2. The experimental results showed that the in situ tumors on the left side of the mice were eliminated by HIFU treatment, while the distal tumors on the right side of the fourth group of mice were significantly inhibited, indicating that the sodium alginate and imiquimod hydrochloride combination can better stimulate anti-tumor immune response after HIFU treatment, and has a good synergistic effect with anti-PDL1 antibodies. ( Fig. 20 )

[0249] Step 3: Study on the immune memory effect induced by the lyophilized powder injection of the composition of sodium alginate (first-class component) and imiquimod hydrochloride (third-class component) combined with high-intensity focused ultrasound (HIFU) and immune checkpoint inhibition therapy with anti-PDL1 antibody.

[0250] The colon cancer-bearing mice were divided into six groups, with 5 mice in each group.

[0251] Group 1: normal saline group;

[0252] Group 2: tail vein anti-PDL1 antibody treatment (reference case);

[0253] Group 3: HIFU treatment alone (reference case);

[0254] Group 4: anti-pdl1 antibody therapy in the tail vein after HIFU treatment (reference case);

[0255] Group 5: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate and imiquimod hydrochloride (Example 1) followed by HIFU treatment;

[0256] Group 6: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate and imiquimod hydrochloride (Example 1) followed by HIFU treatment and caudal vein anti-PDL1 antibody treatment.

[0257] 40 days after the mouse tumors were eliminated by HIFU treatment, colon cancer tumor cells were implanted again in these mice that had received different treatments, and the length and width of the distal tumor on the right were measured with a vernier caliper. The volume of the tumor was (length multiplied by (width squared)) divided by 2. The experimental results showed that the growth of the tumors implanted again in the fifth and sixth groups of mice was significantly slower than that in the control group and was significantly inhibited. The tumor growth in the sixth group of mice was even slower than that in the fifth group, and some mice could not grow tumors anymore. This indicates that the lyophilized powder injection of the composition of sodium alginate (first-class component) and imiquimod hydrochloride (third-class component) combined with high-intensity focused ultrasound knife (HIFU) and immune checkpoint inhibition therapy anti-PDL1 antibody can significantly induce immune memory in mice and prevent tumor recurrence. ( Fig.21 )

[0258] Example 14: Study on the efficacy of a combination of sodium alginate (first component) and oxaliplatin (second component) lyophilized powder injection in a colon cancer model

[0259] The colon cancer-bearing mice were divided into 6 groups, with 5 mice in each group for treatment experiments.

[0260] Group 1: mice were injected intratumorally with normal saline (reference);

[0261] Group 2: Oxaliplatin (1.5 mg / kg body weight) (chemotherapy alone reference);

[0262] Group 3: intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (0.375 mg / kg body weight) (Example 4);

[0263] Group 4: intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (0.75 mg / kg body weight) (Example 4);

[0264] Group 5: intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (1.5 mg / kg body weight) (Example 4);

[0265] Group 6: Tail vein injection of oxaliplatin (3 mg / kg body weight) (reference example of chemotherapy alone).

[0266] After intratumoral injection, the length and width of the tumor were measured with a vernier caliper every two days. The tumor volume was (length multiplied by (width squared)) divided by 2. From the tumor growth curve ( Fig. 22 ) It can be seen that the therapeutic effect of intratumoral injection of sodium alginate and oxaliplatin combined freeze-dried powder injection at a dose of 0.75 mg / kg body weight has exceeded that of tail vein injection of 3 mg / kg body weight and simple drug injection of 1.5 mg / kg body weight; and the tumor growth was significantly inhibited when the intratumoral injection of sodium alginate and oxaliplatin combined freeze-dried powder injection was measured at 1.5 mg / kg body weight, and the therapeutic effect was significant. From the weight of mice ( Fig.23 ) It can be seen that the weight of mice in the tail vein injection of oxaliplatin group decreased significantly in the first four days, indicating that intravenous injection has certain toxic side effects, while intratumoral injection does not show obvious toxic side effects. It is obvious that the side effects of intratumoral administration of the composition of the patent technical solution are lower than those of intravenous administration.

[0267] Example 15: Study on the efficacy of sodium alginate (first type component), oxaliplatin (second type component), imiquimod hydrochloride (third type component) and anti-PDL1 antibody (fourth type component) lyophilized powder injection in a bilateral tumor (one tumor on each side) model.

[0268] Mouse colon cancer tumors were implanted at the left and right ends of the mouse back (the left one was considered as the in situ tumor, and the right one was considered as the distal tumor). The tumor-bearing mice were divided into 7 groups, with 6 mice in each group, for combined immune treatment experiments.

[0269] Group 1: mice were injected intratumorally with normal saline (reference);

[0270] Group 2: oxaliplatin and imiquimod combined with anti-PDL1 solution (reference example);

[0271] Group 3: Sodium alginate and oxaliplatin combination freeze-dried powder injection (Example 4) combined with anti-PDL1 intravenous injection (reference example);

[0272] Group 4: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, oxaliplatin and anti-PDL1 (Example 4);

[0273] Group 5: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, oxaliplatin and imiquimod (Example 6);

[0274] Group 6: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, oxaliplatin and imiquimod anti-PDL1 (Example 10);

[0275] Group 7: Sodium alginate, oxaliplatin and imiquimod composition freeze-dried powder injection (Example 6) combined with anti-PDL1 intravenous injection (reference example).

[0276] After the left orthotopic tumor was injected intratumorally, the right distal tumor was not injected. The length and width of the orthotopic tumor and the distal tumor were measured with a vernier caliper every two days. The volume of the tumor was (length multiplied by (width squared)) divided by 2. From the growth curve of the orthotopic tumor and the growth curve of the distal tumor ( Fig.24 and Fig.25 ) It can be seen that the in situ tumors and distal tumors of the mice in groups 6 and 7 were effectively inhibited and almost stopped growing. After the mice in groups 6 and 7 survived for two months, colon cancer cells were implanted again and it was found that the tumor growth was significantly inhibited, indicating that the recurrence of the tumor was effectively prevented ( Fig.26 ).

[0277] Example 16: Study on the efficacy of sodium alginate (first-class component) and doxorubicin hydrochloride (second-class component) and imiquimod hydrochloride (third-class component) and anti-PDL1 antibody (fourth-class component) lyophilized powder injection in breast cancer metastasis model

[0278] Mice bearing 4T1 breast cancer orthotopic tumors in the mammary pad were divided into 6 groups, with 6 mice in each group for treatment experiments in the metastatic tumor model.

[0279] Group 1: mice were injected intratumorally with normal saline (reference);

[0280] Group 2: doxorubicin and imiquimod hydrochloride with anti-PDL1 antibody (reference example);

[0281] Group 3: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride (Example 5);

[0282] Group 4: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, doxorubicin hydrochloride and anti-PDL1 antibody (Example 5);

[0283] Group 5: intratumoral injection of a lyophilized powder injection of a combination of sodium alginate, doxorubicin, imiquimod and anti-PDL1 antibody (Example 9);

[0284] Group 6: Sodium alginate, doxorubicin and imiquimod hydrochloride composition freeze-dried powder injection (Example 5) combined with anti-PDL1 antibody intravenous injection (reference example).

[0285] The first control group had their tumors removed directly by surgery. The mice were treated on the fifteenth day, and live fluorescence imaging of the animals was taken every five days. From the experimental results, it can be seen that both the fifth and sixth groups had good treatment effects. Fig. 27 )

[0286] Example 17: Study on the efficacy of sodium alginate (first-class component) and doxorubicin hydrochloride (second-class component) and imiquimod hydrochloride (third-class component) and anti-PDL1 antibody (fourth-class component) lyophilized powder injection on mouse brain cancer

[0287] The brain cancer mice were divided into nine groups, with six mice in each group for brain cancer treatment experiments.

[0288] Group 1: mice were injected intracranially with normal saline (reference);

[0289] Group 2: mice were intraperitoneally injected with temozolomide (reference example);

[0290] Group 3: mice were intracranial injected with a combination of imiquimod hydrochloride, anti-PDL1 antibody and sodium alginate (reference example);

[0291] Group 4: mice were intracranially injected with a lyophilized powder injection of a combination of sodium alginate and doxorubicin (Example 3);

[0292] Group 5: mice were intracranial injected with a combination of doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody (reference example);

[0293] Group 6: mice were intracranially injected with a freeze-dried powder injection of a composition of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride (Example 5);

[0294] Group 7: mice were intracranial injected with a combination of sodium alginate, doxorubicin hydrochloride and anti-PDL1 antibody (reference example);

[0295] Group 8: mice were intracranially injected with a freeze-dried powder injection of a composition of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody (Example 9);

[0296] Group 9: Mice were treated with intracranial injection of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder injection combined with anti-PDL1 antibody (reference example).

[0297] The mice were observed for death. Fig.28 This is the mortality curve of mice. It can be seen from the figure that the survival time of mice in groups 8 and 9 is twice as long as that of the control group, indicating that their treatment effect is better.

[0298] Example 18: Study on the efficacy of sodium alginate (first component) and doxorubicin hydrochloride (second component) and imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection in a mouse tumor surgical resection model

[0299] The mice with subcutaneous breast cancer were randomly divided into three groups, with six mice in each group for the treatment experiment of sodium alginate combined with doxorubicin, imiquimod and anti-PDL1 antibody. The mice underwent surgery to remove most of the subcutaneous tumors (removal of the subcutaneous tumors of mice retained their adjacent skin and muscles).

[0300] The first group received no treatment (reference case);

[0301] Group 2: surgery alone (reference group);

[0302] After the surgery, the third group applied a composite gel of sodium alginate, doxorubicin, imiquimod and anti-PDL1 antibody to the wound site (Example 9).

[0303] The treatment effect is judged by observing the metastasis and recurrence of the tumor after surgery, and conclusions are drawn through small animal bioluminescence imaging. Fig.29 It can be seen that the tumors of the third group of mice in the key group were well inhibited from metastasis and recurrence, which proved the effect of our sodium alginate, doxorubicin, imiquimod and anti-PDL1 antibody composite gel.

[0304] Example 19: The pharmaceutical composition of this example can be prepared in two dosage forms.

[0305] The first dosage form is as follows:

[0306] Component one, according to the ratio of imiquimod R837: surfactant poloxamer 188 1: (0.1-5). Weigh imiquimod R837 and surfactant poloxamer 188. Preferably 1g R837, add appropriate amount of poloxamer 188 (0.15g, 0.3g, 0.5g, 1g, 2g, 3g, 4g, 5g), add 10ml water and ball mill for 3 hours. After the end, take out 9ml of the homogenate and transfer it to a 250ml beaker, add 141ml of first-grade water, and stir at 500rpm for 1 hour to mix. Use a syringe to draw the suspension and fill it into 10ml syringe bottles, 5ml per bottle, for a total of 30 bottles. Cover with a rubber cap and seal it with an aluminum cap, and sterilize it with wet heat at 121℃ for 12 minutes;

[0307] Component 2: Prepare 0.1-5% sodium alginate and 0.1-1% oxaliplatin solution. Preferably, weigh sodium alginate ALG (3g, or 1.5g), an appropriate amount of oxaliplatin (159mg, 300mg, 450mg, 90mg), add 300ml of first-grade water, and stir in a 500ml beaker for 2-6 hours (25℃-40℃, 200-850rpm, seal the bottle mouth with plastic wrap). The obtained solution is sterilized by filtering through a 0.22 micron filter membrane. Fill into 20ml vials with a 60ml syringe, 10ml per bottle, a total of 30 bottles. After precooling in a -80℃ refrigerator for 30 minutes, freeze-dry for 30 hours. After freeze-drying, cover with a rubber cap and seal with an aluminum cap.

[0308] When in use, add the freeze-dried powder of component two to the solution of component one, mix thoroughly by shaking, and then inject.

[0309] The second dosage form is as follows:

[0310] Component 1: Weigh R837 and surfactant according to the ratio of imiquimod R837: surfactant poloxamer 188 of 1: (0.1-5), and add 0.1-1% oxaliplatin. Preferably, 1g of imiquimod R837 is added with an appropriate amount of poloxamer 188 (0.15g, 0.3g, 0.5g, 1g, 2g, 3g, 4g, 5g), and ball milled with 10ml water for 3 hours. After the end, take out 9ml of the homogenate and transfer it to a 250ml beaker, add 141ml of first-grade water, an appropriate amount of oxaliplatin (159mg, 300mg, 450mg, 900mg), and stir at 100-500rpm for 0.5-3 hours to mix. Use a 50ml syringe to draw the suspension and fill it into a 10ml syringe bottle, 5ml per bottle, for a total of 30 bottles. Cover with a rubber cap and seal with an aluminum cap, and sterilize with moist heat at 121°C for 12 minutes;

[0311] Component 2: Prepare a 0.1-5% sodium alginate solution. Preferably, weigh 3g or 1.5g of sodium alginate ALG, add 300ml of first-grade water, and stir in a 500ml beaker for 2-6 hours (25℃-40℃, 200-850rpm, seal the bottle mouth with plastic wrap). Filter the obtained solution through a 0.22 micron filter membrane for sterilization. Fill into 20ml vials with a 60ml syringe, 10ml per bottle, for a total of 30 bottles. Precool in a -80℃ refrigerator for 30 minutes, and freeze-dry for 30 hours. After freeze-drying, cover with a rubber cap and seal with an aluminum cap.

[0312] When in use, add the freeze-dried powder of component two to the solution of component one, mix thoroughly by shaking, and then inject.

[0313] The specific effects of this embodiment for treatment are as follows: using the first dosage form, the second dosage form has a similar therapeutic effect to the first dosage form.

[0314] Experimental method: Mouse colon cancer tumors were implanted on the left and right ends of the mouse's back (the left side was considered as the in situ tumor, and the right side was considered as the distal tumor), and the tumor-bearing mice were divided into 5 groups, with 6 mice in each group for combined immune treatment experiments.

[0315] Group 1: mice were injected intratumorally with normal saline (reference);

[0316] Group 2: intratumoral injection of oxaliplatin + poloxamer 188-dispersed imiquimod particles;

[0317] Group 3: intratumoral injection of oxaliplatin + sodium alginate;

[0318] Group 4: intratumoral injection of imiquimod particles dispersed with poloxamer 188 + sodium alginate;

[0319] Group 5: intratumoral injection of oxaliplatin + poloxamer 188-dispersed imiquimod particles + sodium alginate;

[0320] The left orthotopic tumor was injected. After the orthotopic tumor was injected intratumorally, the right distal tumor was not injected. The length and width of the orthotopic tumor and the distal tumor were measured with a vernier caliper every two days. The volume of the tumor was (length multiplied by (width squared)) divided by 2.

[0321] Treatment effect: From the in situ tumor growth curve and the distal tumor growth curve ( Fig.30 and Fig.31 ) It can be seen that the in situ tumors and distal tumors of the mice in group 5 were effectively inhibited and almost stopped growing. More importantly, this was a good therapeutic effect without the combination of anti-PDL1 antibodies, so it has very good application prospects and value. Some of the other corresponding treatment groups had certain therapeutic effects, while some experimental groups had very limited therapeutic effects.

[0322] Cancer treatment is a very complex comprehensive result, because both the body's immune system and the growth mechanism of cancer cells are very complex. The reason why this experiment can achieve relatively excellent treatment results, in addition to the explanations in other parts of this patent, may also include the following reasons: the imiquimod R837 ball milling method is used to ball mill the water-insoluble R837 powder in water to make the powder finer and finer, so that it has good water dispersibility.

[0323] Poloxamer P188 is added to the R837 ball-milled emulsion for ball milling. Poloxamer 188 is a new type of high molecular non-ionic surfactant with multiple uses including: as an emulsifier, stabilizer and solubilizer, which can further enhance the water dispersibility and stability of the R837 ball-milled emulsion.

[0324] The solubilizing pharmaceutical excipients used include: one or more of poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), egg yolk lecithin, polyoxyethylene (35) castor oil, vitamin E succinate polyethylene glycol ester, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 800, or sodium hydroxymethylcellulose. Among them, one or more of poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), egg yolk lecithin, polyoxyethylene (35) castor oil, vitamin E succinate polyethylene glycol ester, or sodium hydroxymethylcellulose have good solubilizing effects on R837.

[0325] However, the solubilization effects of polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 800 are average. In recent years, the number of adverse reaction cases reported has gradually increased. Polysorbate 80 (Tween 80), as its commonly used solubilizing excipient, has become the focus of research on the causes of adverse reactions. Polyoxyethylene castor oil, as a non-ionic surfactant, can increase the water solubility of most poorly soluble drugs, but it can lead to the release of histamine and cause a variety of toxic and side effects, such as severe allergic reactions, toxic kidney damage, neurotoxicity, cardiovascular toxicity, etc. According to literature reports, Solutol HS 15 has the greatest toxic and side effects in muscle stimulation and hemolysis experiments, while polyethylene glycol 200 has the least toxic and side effects. Polyethylene glycol is a stable hydrophilic substance that is non-toxic and non-irritating. It has solubilizing, stability-enhancing and prolonged effects on many drugs.

[0326] Poloxamer 188 is a series of multi-purpose pharmaceutical excipients. Due to its non-toxic, non-antigenic, non-sensitizing, non-irritating, non-hemolytic and stable chemical properties, Poloxamer 188 is one of the excipients with better safety. It has been used clinically as an emulsifier and solubilizer for intravenous administration.

[0327] Further experiments found that without the addition of Poloxamer 188, the R837 ball-milled emulsion would become unstable after wet heat sterilization at 121°C, resulting in obvious precipitation and particles, and the water dispersibility was greatly reduced. Poloxamer 188 can help R837 ensure water dispersibility and stability after sterilization.

[0328] Table 1: Water dispersibility of ball-milled imiquimod R837 before and after addition of surfactant

[0329]

[0330]

[0331] Table 2: Redispersibility of R837 ball-milled with different surfactants after autoclaving

[0332]

[0333] Table 3: Long-term stability of ball-milled R837 after autoclaving with different proportions of surfactants

[0334]

[0335]

[0336] a: Suitable surfactants include: poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), polyoxyethylene (35) castor oil, vitamin E succinate polyethylene glycol ester, and sodium hydroxymethylcellulose.

[0337] Surfactant b: R837 Long-term stability after autoclaving 0.15:1 A large number of granular aggregates appear 0.3:1 A large number of granular aggregates appear 0.5:1 A large number of granular aggregates appear 1:1 Evenly dispersed without granular aggregates 2:1 Evenly dispersed without granular aggregates 3:1 Evenly dispersed without granular aggregates 4:1 Evenly dispersed without granular aggregates 5:1 Evenly dispersed without granular aggregates

[0338] b: Applicable surfactants include: egg yolk lecithin.

[0339] Surfactant c: R837 Long-term stability after autoclaving 0.15:1 A large number of flocculent aggregates appear 0.3:1 A large number of flocculent aggregates appear 0.5:1 A large number of flocculent aggregates appear 1:1 A large number of flocculent aggregates appear 2:1 A large number of flocculent aggregates appear 3:1 A large number of flocculent aggregates appear 4:1 A large number of flocculent aggregates appear 5:1 A large number of flocculent aggregates appear

[0340] c: Applicable surfactants include: polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 800.

[0341] Although in theory, the more dispersants there are, the better the dispersibility, the ratio is generally not more than 5:1. The reason is that Poloxamer 188 (P188) itself is viscous, and too high a concentration will result in high viscosity, and the introduction of impurities should be avoided.

[0342] Sodium alginate is a relatively special natural biopolymer material. Since it decomposes at high temperatures, it cannot be sterilized by traditional high-temperature moist heat sterilization. This patent adopts filtration sterilization to retain the properties of sodium alginate. In addition, from the perspective of sterilization, sodium alginate and imiquimod R837 cannot be combined. Sodium alginate ALG needs to be sterilized by filtration, but R837 particles cannot be filtered (the minimum particle diameter after ball milling is 500nm, and filtration sterilization requires a 220nm filter membrane, so it cannot pass); on the other hand, R837 needs moist heat sterilization, while sodium alginate ALG will degrade at high temperatures. Therefore, R837 and ALG cannot be sterilized together.

[0343] In addition, the reason why imiquimod R837 has to be made into a ball-milled granule dosage form is that if R837 is formulated into a hydrochloride salt, it is incompatible with OXA, and OXA will react with chloride ions and become inactivated; secondly, the hydrochloride salt of R837 will increase the viscosity of sodium alginate ALG, making it inconvenient to use.

[0344] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art. The present invention will not be limited to the embodiments shown herein, but only needs to be consistent with the principles and features disclosed herein.

Claims

1. A chemotherapy-immunity combination drug, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant, characterized in that: The chemotherapy-immunity combination drug comprises a first mixture and a second mixture, wherein the first mixture contains an immune adjuvant, and the second mixture contains a chemotherapy drug that can cause immunogenic death; the immune adjuvant is imiquimod, and the chemotherapy drug that can cause immunogenic death is oxaliplatin; The first mixture is imiquimod and a surfactant mixed and ball-milled to obtain a uniformly dispersed imiquimod emulsion, wherein the imiquimod particles in the obtained imiquimod emulsion have a particle size of 0.5-3 microns, and the imiquimod emulsion is sterilized by high temperature and wet heat; The surfactant is one or more of poloxamer 407, egg yolk lecithin, or vitamin E succinate polyethylene glycol ester; The second mixture is prepared by mixing sodium alginate, oxaliplatin and water, and filtering and sterilizing through a micron filter membrane to prepare a mixture; The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

2. The chemotherapy-immunotherapy combination drug according to claim 1, characterized in that: It also includes an immunomodulator; the immunomodulator is mixed with the first mixture and the second mixture to form a chemotherapy-immunotherapy combination drug.

3. The chemotherapy-immunotherapy combination drug according to claim 2, characterized in that: The immunomodulators include immune checkpoint inhibitors or IDO inhibitors, and immune checkpoint inhibitors include antibody inhibitors, small molecule inhibitors or peptide inhibitors.

4. The chemotherapy-immunotherapy combination drug according to claim 3, characterized in that: The antibody inhibitor is selected from anti-CTLA-4, anti-PD-1 and anti-PD-L1, the small molecule inhibitor is selected from CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, the peptide inhibitor is selected from DPPA-1, and the IDO inhibitor is selected from BMS-986205, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.

5. The chemotherapy-immunotherapy combination drug according to claim 1 or 2, characterized in that: The sodium alginate is replaced by potassium alginate or ammonium alginate.

6. A chemotherapy-immunity combination drug, comprising a chemotherapy drug capable of causing immunogenic death and an immune adjuvant, characterized in that: The chemotherapeutic immunotherapy combination drug includes a first mixture and a second mixture; The first mixture is: imiquimod R837 is mixed with a surfactant and ball-milled to obtain a uniformly dispersed imiquimod emulsion, wherein the imiquimod particles in the obtained imiquimod emulsion have a particle size of 0.5-3 microns; the imiquimod emulsion is mixed with oxaliplatin and water, stirred evenly, and sterilized by high temperature and wet heat; The surfactant is one or more of poloxamer 407, egg yolk lecithin, or vitamin E succinate polyethylene glycol ester; The second mixture is a mixture of sodium alginate and water, which is then sterilized by filtering through a micron filter membrane to form a mixture; The first mixture and the second mixture are mixed to form a chemotherapeutic immunotherapy combination drug.

7. The chemotherapy-immunotherapy combination drug according to claim 6, characterized in that: It also includes an immunomodulator; the immunomodulator is mixed with the first mixture and the second mixture to form a chemotherapy-immunotherapy combination drug.

8. The chemotherapy-immunotherapy combination drug according to claim 7, characterized in that: The immunomodulators include immune checkpoint inhibitors or IDO inhibitors, and immune checkpoint inhibitors include antibody inhibitors, small molecule inhibitors or peptide inhibitors.

9. The chemotherapy-immunotherapy combination drug according to claim 8, characterized in that: The antibody inhibitor is selected from anti-CTLA-4, anti-PD-1 and anti-PD-L1, the small molecule inhibitor is selected from CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, the peptide inhibitor is selected from DPPA-1, and the IDO inhibitor is selected from BMS-986205, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.

10. The chemotherapy-immunotherapy combination drug according to claim 6 or 7, characterized in that: The sodium alginate can be replaced by potassium alginate or ammonium alginate.

11. Use of the chemotherapy-immunotherapy combination drug according to claims 1-10 in the preparation of drugs for treating colon cancer, breast cancer and brain cancer.

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