CN113730570A

CN113730570A - Chemotherapy immune combined medicine and application thereof

Info

Publication number: CN113730570A
Application number: CN202111061706.5A
Authority: CN
Inventors: 刘庄; 许欢; 陶惠泉; 巢宇; 赵琪
Original assignee: Suzhou Baimai Biomedical Co ltd
Current assignee: Suzhou Baimai Biomedical Co ltd
Priority date: 2019-09-26
Filing date: 2019-09-26
Publication date: 2021-12-03
Also published as: CN110917345A; WO2021056815A1; CN110917345B

Abstract

The invention discloses a chemotherapy immune combination drug and application thereof, which is formed by mixing a first mixture and a second mixture, wherein the first mixture contains an immune adjuvant imiquimod R837, and the second mixture contains a chemotherapeutic drug oxaliplatin capable of causing immunogenic death; the first mixture is that imiquimod and surfactant are mixed and ball-milled to prepare uniformly dispersed imiquimod emulsion, the obtained imiquimod particles in the imiquimod emulsion have the particle size of 0.5-3 microns, the imiquimod emulsion is sterilized by high-temperature moist heat, and the second mixture is that alginate, oxaliplatin and water are stirred, mixed and filtered to remove bacteria to prepare a mixture; the invention has the advantages of generating synergistic anticancer effect to treat tumor, optimizing production process and having good product stability. The check point inhibition therapy has obvious effect on tumors with more mutation and high antigen expression, and chemotherapy kills the tumors to provide antigens and is matched with an adjuvant to amplify the immune response of the tumors, so that most of the tumors are changed into hot tumors, and the effectiveness of the check point inhibitor is greatly improved.

Description

Chemotherapy immune combined medicine and application thereof

The application is a divisional application of patent application with the application number of 201910915738.3 and the application date of 09 and 26 in 2019.

Technical Field

The invention relates to the field of medicaments for treating tumors, in particular to a chemotherapeutic and immune combination treatment pharmaceutical composition, a preparation method and application thereof.

Background

Chemotherapy is one of three main treatment methods for treating tumors clinically at present, most cancer patients need to receive a certain degree of chemotherapy, and the chemotherapy is the main treatment method for tumors which are prone to metastasis or have metastasized. However, the traditional chemotherapeutic drugs also have damage to normal organs, and the chemotherapy modes commonly used in clinic are systemic administration, so that the traditional chemotherapeutic drugs do not have good selectivity on pathological parts, and have very large toxic and side effects in chemotherapy.

Although tumor immunotherapy represented by immune checkpoint blockade has been encouraging in recent years, there are important limitations to this therapy, including low clinical response rates (around 20%), side effects due to non-specific immune reactions, and the like. In particular, the low clinical response rate of current clinical immune checkpoint blockade therapies means that most patients are unresponsive to this costly therapy. In order to further improve the curative effect and response rate of tumor treatment, it is necessary to improve the administration route of the existing therapy and develop the chemotherapy-immune combination therapy for tumor to realize synergistic effect. For example, how to better limit the killing of the ICD chemotherapeutic drugs on tumor cells to tumor in situ and avoid damaging the whole body needs to be considered; how to better amplify the immunogenicity of tumor-associated antigens of cancer cells after death so as to obtain stronger tumor-specific immune response; how to more effectively combine the action of an immune checkpoint inhibitor (such as CTLA-4, PD-1/PD-L1 antibody) or an IDO inhibitor so as to further enhance the specific immune response against the tumor by regulating the immune balance. The development of the new technology in the aspect is of great practical significance to China with high cancer incidence and relatively lagged research and development of the anti-cancer original drug. In addition, how to inhibit tumor metastasis and prevent recurrence while local treatment is a problem all over the world.

At the same time, the operability of the related drug production, as well as the sterilization and subsequent stability of the drug product, are also difficult problems.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel chemotherapeutic immune pharmaceutical composition, which can generate a synergistic anti-cancer effect, reduce side effects, reduce the probability of cancer metastasis and reduce the probability of cancer recurrence, can effectively kill in-situ tumors, inhibit the immune reaction and reduce the growth of distal metastatic tumors and the probability of tumor recurrence, and has the advantages of optimized production process and good product stability.

In order to solve the related technical problems, the invention provides the following technical scheme:

a chemotherapeutic-immunoadjuvant combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant, wherein: the chemotherapy immune 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 capable of causing immunogenic death; the chemotherapeutic drug capable of causing immunogenic death is oxaliplatin, the immunologic adjuvant is imiquimod R837, and the chemotherapeutic drug further comprises poloxamer 188 and sodium alginate ALG;

the first mixture is prepared by mixing and ball-milling imiquimod R837 and poloxamer 188 to obtain uniformly dispersed imiquimod emulsion, wherein the particle size of the imiquimod particles is 0.5-3 microns, and the imiquimod emulsion is subjected to high-temperature moist heat sterilization;

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

the first mixture and the second mixture are mixed to form the chemotherapy immune combination drug.

A chemotherapeutic-immunoadjuvant combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant: the chemotherapy immune 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 capable of causing immunogenic death; the chemotherapeutic drug capable of causing immunogenic death is oxaliplatin, the immunologic adjuvant is imiquimod R837, and the chemotherapeutic drug further comprises poloxamer 188 and sodium alginate ALG;

the first mixture is that imiquimod R837 and poloxamer 188 are mixed and ball-milled to obtain evenly dispersed imiquimod emulsion, the particle size of the imiquimod particles is 0.5-3 microns, the imiquimod emulsion, oxaliplatin and water are mixed and evenly stirred, and high-temperature moist heat sterilization is carried out;

the second mixture is prepared by mixing the sodium alginate with water, and then filtering and sterilizing the mixture through a micron filter membrane;

As a preferred scheme of the chemotherapy immune combination drug: the mass ratio of the imiquimod R837 to the poloxamer 188 is 1: (0.1-5), and the high-temperature sterilization is performed for 10-15 minutes at the temperature of 105-150 ℃ by moist heat;

filtering the second mixture with 0.22 μm filter membrane for sterilization, and lyophilizing to obtain lyophilized powder.

A chemotherapeutic-immune combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immune adjuvant, said chemotherapeutic-immune combination comprising a first mixture comprising the immune adjuvant and a second mixture comprising the chemotherapeutic agent capable of causing immunogenic death;

the first mixture is that the immunologic adjuvant is imiquimod, the imiquimod and a surfactant are mixed and ball-milled to obtain evenly dispersed imiquimod emulsion, the particle size of the imiquimod is 0.5-300 microns, the imiquimod emulsion is sterilized by high-temperature damp heat, and 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 succinic acid polyethylene glycol ester or sodium hydroxymethyl cellulose;

the second mixture is prepared by stirring and mixing sodium alginate, oxaliplatin and water, and filtering and sterilizing the mixture by a micron filter membrane;

As a preferred scheme of the chemotherapy immune combination drug: the sodium alginate is replaced by chitosan, fibrinogen, alginate or hyaluronic acid;

the imiquimod R837 is replaced with imidazoquinoline, or glucopyranoside lipid;

the oxaliplatin Oxa is replaced by an anthracycline drug, or cyclophosphamide, or bortezomib, or gemcitabine, or pentafluorouracil, or a toxin.

A chemotherapeutic-immunoadjuvant combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant, wherein: the chemotherapy immune 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 capable of causing immunogenic death;

the first mixture is that imiquimod R837 and surfactant are mixed and ball-milled to obtain evenly dispersed imiquimod emulsion, the particle size of the imiquimod particles is 0.5-3 microns, the imiquimod emulsion, oxaliplatin and water are mixed and stirred evenly, and high-temperature moist heat sterilization is carried out;

the surfactant is one or more of 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 polyethylene glycol succinate, or sodium hydroxymethyl cellulose;

As a preferred scheme of the chemotherapy immune combination drug: the sodium alginate can be replaced by chitosan, fibrinogen, alginate or hyaluronic acid;

the imiquimod R837 can be replaced with imidazoquinoline, or glucopyranoside lipid;

the oxaliplatin Oxa can be replaced by an anthracycline drug, or cyclophosphamide, or bortezomib, or gemcitabine, or pentafluorouracil, or a toxin.

The preparation method of the chemo-immune combination drug is characterized by comprising the following steps:

the first step is as follows: the proportion is 1: (0.1-5) weighing imiquimod R837 and a surfactant poloxamer 188, adding water, ball-milling for 2-3 hours, taking out homogenate after finishing, adding water, stirring and uniformly mixing, and carrying out moist heat sterilization for 10-15 minutes at 105-150 ℃;

the second step is that: weighing sodium alginate ALG and oxaliplatin, adding water, stirring, and filtering the obtained solution through a micron filter membrane for sterilization; after precooling, freeze-drying;

the third step: when in use, the mixture II lyophilized powder is added into the mixture I solution, fully shaken, uniformly mixed and dissolved, and then injected.

A chemotherapeutic-immunoadjuvant combination for the treatment of colon cancer tumors comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant, characterized in that: the chemotherapy immune 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 capable of causing immunogenic death;

the first mixture is that the immunologic adjuvant is imiquimod, the imiquimod and a surfactant are mixed and ball-milled to obtain uniformly dispersed imiquimod emulsion, the particle size of the imiquimod is 0.5-3 microns, the imiquimod emulsion is subjected to high-temperature wet heat sterilization, and 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 succinic acid polyethylene glycol ester or sodium hydroxymethyl cellulose;

The invention also provides an in-situ gelling chemotherapeutic immune combination treatment biopolymer pharmaceutical composition, which comprises: the first component is alginate which can form porous gel with calcium ions in vivo, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate;

the second group of components are chemotherapeutic drugs that cause immunogenic death;

the third group of components is immunological adjuvants.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the immunological adjuvant is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the chemotherapeutic drug capable of causing immunogenic death of the second component is one or more of anthracyclines such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: also included are a fourth class of component immune checkpoint inhibitors, typically anti-CTLA-4, anti-PD-1 and anti-PD-L1, or IDO inhibitors, typically small molecule inhibitors, typically CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, and peptidic inhibitors, typically DPPA-1;

the IDO inhibitor comprises BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat, 4-phenylimidazole and other small molecules.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the first component is sodium alginate, and the second component is doxorubicin hydrochloride; the third component is imiquimod, and the mass ratio of the sodium alginate to the doxorubicin hydrochloride to the imiquimod is 50-800: 1-100.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the mass ratio of the sodium alginate to the doxorubicin hydrochloride to the imiquimod is 200-400 to 10-75.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the concentration of the sodium alginate is more than 5 mg per ml.

A method of preparing an in situ gelling chemotherapeutic immune combination therapeutic biopolymer pharmaceutical composition, said method comprising:

dissolving sodium alginate, imiquimod hydrochloride freeze-dried powder and doxorubicin hydrochloride in an aqueous phase solution, stirring until the solution is clear and transparent, and freeze-drying the solution to obtain composition freeze-dried powder;

or dissolving sodium alginate and doxorubicin hydrochloride in the aqueous solution, stirring until the solution is clear and transparent, freeze-drying to obtain freeze-dried powder, and uniformly mixing the freeze-dried powder with imiquimod hydrochloride freeze-dried powder through solid and solid shaking to obtain composition freeze-dried powder;

or dissolving adriamycin hydrochloride and imiquimod hydrochloride in the aqueous phase solution, stirring until the solution is clear and transparent, dripping the continuously stirred mixed solution into the sodium alginate aqueous phase solution to ensure that the mixed solution is clear and transparent without flocculent precipitates, taking out the mixed solution, and freeze-drying to obtain the composition freeze-dried powder.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the first component is sodium alginate, and the second component is oxaliplatin; the third component is imiquimod hydrochloride, and the mass ratio of the sodium alginate to the oxaliplatin to the imiquimod is 50-800: 1-75: 1-100.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the mass ratio of the sodium alginate to the oxaliplatin to the imiquimod is 200-400 to 10-75.

A method of preparing said in situ gelling chemotherapeutic immune combination therapy biopolymer pharmaceutical composition, said method comprising:

dissolving sodium alginate, imiquimod hydrochloride freeze-dried powder and oxaliplatin in an aqueous phase solution, stirring until the solution is clear and transparent, and freeze-drying the solution to obtain composition freeze-dried powder;

or dissolving sodium alginate and oxaliplatin in the aqueous solution, stirring until the solution is clear and transparent, freeze-drying to obtain freeze-dried powder, and then uniformly mixing with the imiquimod hydrochloride freeze-dried powder through solid-solid shaking to obtain the composition freeze-dried powder.

Or dissolving oxaliplatin and imiquimod hydrochloride in the aqueous phase solution, stirring until the solution is clear and transparent, dissolving sodium alginate in the aqueous phase solution, dripping the continuously stirred mixed solution into the solution to ensure that the mixed solution is clear and transparent and has no flocculent precipitate, taking out the mixed solution, and freeze-drying to obtain the composition freeze-dried powder.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the first component is sodium alginate, and the second component is pentafluorouracil; the third component is imiquimod hydrochloride;

or the first component is sodium alginate, and the second component is cyclophosphamide; the third group of components is imiquimod hydrochloride.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the first component is sodium alginate; the second component is adriamycin hydrochloride or oxaliplatin; and a third class of components is imiquimod hydrochloride; the fourth group of components is anti-PDL1 antibody.

As a preferred scheme of the in-situ gelling chemotherapy immune combination treatment biopolymer pharmaceutical composition: the first component is potassium alginate or ammonium alginate; the second component is adriamycin hydrochloride or oxaliplatin; and a third class of components is imiquimod hydrochloride; the fourth group of components is anti-PDL1 antibody.

An in-situ gelling chemotherapeutic immune combination treatment biopolymer pharmaceutical composition comprises the following components: the first component is alginate which can form porous gel with calcium ions in vivo, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; the second group of components are chemotherapeutic drugs that cause immunogenic death; the third group of components is immunological adjuvants.

An in-situ gelling chemotherapeutic immune combination treatment biopolymer pharmaceutical composition comprises the following components: the first component is alginate which can form porous gel with calcium ions in vivo, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; the second group of chemotherapeutic agents which cause immunogenic death are one or more of anthracyclines such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine; the third group of components is immunological adjuvants, and the immunological adjuvants are one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod.

An in-situ gelling chemotherapeutic immune combination treatment biopolymer pharmaceutical composition comprises the following components: the first component is alginate which can form porous gel with calcium ions in vivo, and the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate; the second group of chemotherapeutic agents which cause immunogenic death are one or more of anthracyclines such as doxorubicin, epirubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine; the third component is an immunological adjuvant, and the immunological adjuvant is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod;

a fourth class of component immune checkpoint inhibitors, typically anti-CTLA-4, anti-PD-1 and anti-PD-L1, or IDO inhibitors, the fourth class of component immune checkpoint inhibitor antibodies typically being CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, and the peptide inhibitors typically being DPPA-1;

the IDO inhibitor comprises BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole small molecules.

An in-situ gelling chemotherapeutic immune combination treatment biopolymer pharmaceutical composition comprises the following components: the first component is sodium alginate, and the second component is doxorubicin hydrochloride; the third component is imiquimod, and the mass ratio of the sodium alginate to the doxorubicin hydrochloride to the imiquimod is 50-800: 1-100.

The invention provides a series of pharmaceutical compositions. In the composition system, four types of components are mainly used, and the first type of component and other types of components can be combined differently according to actual conditions, and the composition system comprises the following components:

a first type of component: the sodium alginate auxiliary materials can generate gel with calcium ions and the like in the human body or the animal body;

the second type of components: chemotherapeutic agents that cause immunogenic death;

the third kind of components: an immunological adjuvant;

the fourth group of components: an immune checkpoint inhibitor or an IDO inhibitor.

The first component and the auxiliary materials usually comprise sodium alginate, potassium alginate, ammonium alginate and the like, and the polysaccharides can be crosslinked with each other to form gel after meeting divalent ions such as calcium ions, so that the formed gel can effectively and slowly release the medicine in the gel after the medicine is wrapped in the gel, thereby enhancing the curative effect and weakening the side effect.

Sodium alginate is a natural polysaccharide, and has the stability, solubility, viscosity and safety required by pharmaceutical preparation adjuvants. Sodium alginate has been widely used in the food industry and in the medical field. Sodium alginate is the most widely used water-soluble alginate. Sodium alginate can quickly generate ion exchange when meeting calcium ions to generate gel, and sufficient calcium ions exist in human bodies or animal bodies, so that the gel can be formed in situ in the bodies.

The two alginates of potassium alginate and ammonium alginate, although the cations contained in the alginates are different from those of sodium alginate, can be crosslinked with calcium ions to form porous gel, thereby playing the role of slowly releasing the medicine. At present, sodium alginate is usually extracted from seaweed, so the sodium alginate is a more preferable choice.

The second group of components, chemotherapeutic drugs that cause immunogenic death, include anthracyclines such as doxorubicin, epirubicin, mitoxantrone, etc., as well as oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins such as maytansine, etc. These drugs have been clinically approved and studies in recent years have shown that these drugs cause immunogenic death of cancer cells, which express calreticulin that is easily recognized and taken up by immune cells, especially antigen-presenting cells, helping immune cells recognize tumor cells, causing an effective anti-tumor immune response.

The third group of components, immunoadjuvants, short for adjuvants, i.e. non-specific immunoproliferative agents, refer to auxiliary substances that are injected into the body together with or in advance with an antigen, and that enhance the body's ability to respond to the antigen or alter the type of immune response. The immune adjuvants are various in types, a uniform classification method is not available at present, and Freund's adjuvants and cytokine adjuvants are more applied. The immunobiological effect of the immunological adjuvant is to enhance immunogenicity, enhance antibody titer, change the type of antibody production, and cause or enhance delayed hypersensitivity, but the specific action mechanism of the immunological adjuvant is not completely understood, and the action mechanisms of different adjuvants are different. Generally, imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A, and resiquimod, etc., which are agonists of Toll-like receptors (TLRs for short), can help antigen presenting cells to present antigens, so that the immunoadjuvant can better present tumor-associated antigens generated by chemotherapy to T cells, thereby amplifying immune response.

In a fourth class, the immune modulator comprises an immune checkpoint inhibitor or an IDO inhibitor. The immune checkpoint inhibitor comprises antibody inhibitor or small molecule inhibitor, wherein the antibody inhibitor is usually selected from anti-CTLA-4, anti-PD-1 and anti-PD-L1, the small molecule inhibitor is usually selected from CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, and the peptide inhibitor is DPPA-1. The IDO inhibitor comprises BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole and other small molecules, and can inhibit IDO enzyme so as to enhance the effect 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, allowing the immune cells to better kill the tumor cells.

The first type of component auxiliary materials can also be called component one for short; the second group of component ICD chemotherapeutic drugs can also be called component two for short; the third component immunologic adjuvant can also be called component three for short; the fourth class of component immune checkpoint inhibitors may also be referred to as component four for short.

The lyophilized powder is sterile powder injection prepared by freezing the medicinal liquid into solid state under sterile environment, and vacuum-pumping to sublimate and dry water.

The preparation process of the mixed liquid medicine and freeze-dried preparation of the component I, the component II, the component III and the component IV is as follows.

This patent mainly involves four kinds of raw materials composition: the first component auxiliary materials comprise sodium alginate (solid powder), the second component ICD chemotherapeutic drug (solid powder), the third component immunological adjuvant (solid powder) and the fourth component immunological check point inhibitor (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibody, a commercial product for clinical use, and freeze-dried powder or injection is used as a raw material).

The preparation scheme I comprises the following steps: mixing solid powder of the component I adjuvant, the component II ICD chemotherapeutic drug and the component III immunological adjuvant according to a certain proportion, putting the mixture into a big beaker, adding deionized water (or normal saline or phosphoric acid buffer solution), and stirring the mixture at room temperature of 25 ℃ by using a stirring paddle at a rotating speed of 50-500 rpm until the solution is clear and transparent; preparing the component IV immune checkpoint inhibitor into injection according to the product specification according to the requirements, and adding the injection into the mixed solution; and (3) after the solution is uniformly stirred, taking out the solution, and performing bottle separation and freeze drying. After the freeze-dried powder is redissolved, turbidity and flocculent precipitate can not appear.

The second preparation scheme is as follows: respectively weighing a component I, a component II and a component III with target mass, and respectively adding deionized water (or normal saline or phosphoric acid buffer solution) to prepare three independent solutions; according to the requirements, the four components are prepared into injection according to the product specification; mixing the solutions according to a proper volume ratio, stirring the mixed solution at room temperature of 25 ℃ by a stirring paddle at a rotating speed of 50-500 revolutions per minute until the solution is uniform and not turbid, taking out the mixed solution, and bottling and freeze-drying the mixed solution. After the freeze-dried powder is redissolved, turbidity and flocculent precipitate can not appear. Scheme one and scheme two do not differ much.

The preparation scheme is three: respectively weighing a component I, a component II and a component III with target mass, and respectively adding deionized water (or normal saline or phosphoric acid buffer solution) to prepare three independent solutions; according to the requirements, the four components are prepared into injection according to the product specification; for ICD medicine containing hydrochloride, the solution of the second component, the third component and the fourth component needs to be stirred and mixed firstly, and the solution of the first component is slowly dripped into the solution of the second component and the fourth component in the stirring process (25 ℃, the stirring paddle rotates at the speed of 50 to 300 revolutions per minute) until the whole solution is uniformly stirred. After the freeze-dried powder is redissolved, turbidity and flocculent precipitate can not appear.

The application scheme of the mixed liquid medicine and the freeze-dried preparation of the four components is described.

The first use scheme is as follows: after the composition freeze-dried powder injection of the four components is redissolved by normal saline, the composition solution is directly injected to the tumor part of a patient in a mode of clinical intervention administration and direct puncture administration, and the composition solution is ensured to be uniformly filled in the whole tumor by adopting a mode of multi-point injection during injection. After the composition is injected into tumors, firstly, the characteristic that the first component alginate can form gel when meeting calcium ions is utilized, and the first component can be quickly gelated when meeting calcium ions in tissues to form a porous reticular cross-linked structure, so that other three components mixed in the alginate can be slowly released, the effect is enhanced, and the toxic and side effects are reduced; secondly, the second group of ICD chemotherapeutic drugs can not only effectively kill tumor cells but also cause the tumor cells to generate immunogenicity and die, generate tumor-associated antigens and activate tumor specific immunoreactions; thirdly, the third group of component immunoadjuvants enhances the capacity of antigen presenting cells to further amplify the corresponding immune response; finally, the use of a fourth class of components immune checkpoint inhibitors or IDO inhibitors prevents the metastatic tumor from escaping the immune response, allowing immunotherapy to more effectively kill the tumor, thereby inhibiting metastasis and recurrence of the tumor. Example twelve

The use scheme II comprises the following steps: after the composition freeze-dried powder injection of the first, second and third components is redissolved by normal saline, the composition solution is directly injected to the tumor part of a patient in a mode of clinical intervention administration and direct puncture administration, and the composition solution is ensured to be uniformly filled in the whole tumor by adopting a mode of multi-point injection during injection. This treatment regimen is recommended in combination with a fourth class of component immune checkpoint inhibitors: the combined scheme comprises adding an immune checkpoint inhibitor (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibody) or an IDO inhibitor into injection according to individual conditions of patients, and carrying out one-time intratumoral local injection; the immune checkpoint inhibitor can also be administered intravenously after local treatment by injection of a mixture of the first, second and third components. (example twelve reference example)

Using protocol three (spraying the wound with a composition of four components followed by spraying a calcium ion solution to form a gel): after the focus part of a tumor patient is removed by normal operation, considering the problem that tumor cells at the focus part can not be completely removed by the operation removal, the freeze-dried powder injection of the four components can be redissolved by normal saline, then the freeze-dried powder injection is sprayed on the wound part after the operation removal by using a syringe or a spray bottle, then a proper amount of calcium chloride solution can be sprayed on the wound part to be gelatinized, and finally the wound is sutured. The scheme helps to eliminate residual cancer cells and can inhibit tumor metastasis and recurrence. Example sixteen

Using protocol four (spraying the wound with the composition of three types of components, then spraying the calcium ion solution to form a gel + adding the fourth type of component in combination): after the focus part of a tumor patient is removed in a normal operation, considering the problem that tumor cells at the focus part cannot be completely removed in the operation removal, the freeze-dried powder injection of the first, second and third components can be redissolved by normal saline, then the freeze-dried powder injection is sprayed on the wound part after the operation removal by using a syringe or a spray bottle, then a proper amount of calcium chloride solution can be sprayed on the wound part to be gelatinized, and finally the wound is sutured. The scheme helps to eliminate residual cancer cells and can inhibit tumor metastasis and recurrence. This treatment regimen is recommended to be combined with a fourth class of component immune checkpoint inhibitors or IDO inhibitors after treatment: the combined scheme comprises adding an immune checkpoint inhibitor (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibody) or an IDO inhibitor into injection according to individual conditions of patients, and carrying out one-time intratumoral local injection; the immune checkpoint inhibitor can also be administered intravenously after local treatment by injection of a mixture of the first, second and third components.

Adopt the technical scheme of this patent, can have following beneficial technological effect:

firstly, the method comprises the following steps: alginate is a natural polysaccharide, is safe and nontoxic, has good biocompatibility, can be degraded, and is a good biological material. However, in the field of medicine, the alginate and calcium ions are combined in vitro to form a gel implantable material, and such a use mode not only limits the application in vivo, but also often requires an operation or intervention, has great operation difficulty and great damage to patients, and is not beneficial to combined drug therapy. Therefore, the alginate is injected into the tumor, the sodium alginate is gelatinized in situ in the tumor by utilizing the calcium ions in the tumor tissue, and the formed cross-linked network structure is utilized to slowly release the medicine mixed in the alginate, so that the slow release effect is better. The technology has wide application prospect, can be directly injected by an injector to treat the tumor, and has simple operation and small invasiveness; the sprayer can be used for spraying the wound part after the operation, and the residual cancer cells are cleaned in cooperation with the operation, so that personalized treatment is expected to be performed on different patients, and the cost is low.

II, secondly: most of the clinical conventional chemotherapy is intravenous administration or perfusion administration, the treatment mode has no good selectivity and targeting property, the lesion and normal tissues are damaged, the side effect is great, and patients can bear great physical and psychological damage. In addition, the conventional chemotherapy needs to maintain a certain blood concentration, has large dosage and more administration times, not only greatly increases the side effect, but also improves the administration cost. The strategy of direct intratumoral administration is adopted and the gel slow-release technology is matched, so that the chemotherapeutic drug stays at a focus part for a longer time, the effect of the drug is exerted to the maximum extent, and the damage of the drug to normal tissues is greatly reduced. The direct intratumoral administration and slow release enable the effective drug concentration in the focus to be kept in a very high range for a long time, ensure the drug effect and reduce the administration times, further reduce the side effect and the cost.

Thirdly, the method comprises the following steps: the third group of component immunoadjuvants mentioned in the technical scheme has no precedent for directly treating tumors clinically. These small molecule immunomodulators do not have antiviral and antitumor effects on their own, and often serve only as adjuvants for vaccines to enhance the immunogenicity of antigens. For example imiquimod (R873), is a drug commonly used as an ointment formulation for the treatment of adult external genitalia and perianal condyloma acuminata, and has not been used clinically for tumor treatment. The technology of the patent adopts a mode of injecting the ICD chemotherapeutic drug and the immunologic adjuvant together, when the ICD drug kills tumor to generate tumor antigen, the antigen and the immunologic adjuvant play a role similar to tumor vaccine, not only can inhibit metastasis tumor, but also can prevent tumor recurrence. The technology of the patent creates a new strategy for directly treating tumors by combining immunologic adjuvants with chemotherapeutic drugs.

Fourthly, the method comprises the following steps: currently, immune checkpoint suppression therapies are of great interest, both in advance of research and clinically. However, although such antibodies have shown dramatic effects in some patients, their effectiveness is not one hundred percent. That is, the effect of checkpoint inhibitors for different patients with different indications remains to be investigated further. The current research shows that the tumors can be divided into hot tumors and cold tumors, and the checkpoint inhibition therapy effect is obvious for the tumors with multiple mutations and high antigen expression. According to the scheme, the antigen is provided by killing the tumor through chemotherapy, and the immune response is amplified by matching with the adjuvant, so that most tumors can be changed into the heat tumors through the scheme, and the effectiveness of the checkpoint inhibitor is greatly improved.

Fifthly: the pharmaceutical composition formed by mixing the related components of the patent can generate distinctive and unexpected synergistic anticancer effect, can reduce the side effect of conventional treatment, reduce the cancer metastasis probability and reduce the cancer recurrence probability, provides an efficient tumor specific immunotherapy scheme, can effectively kill in-situ tumors, inhibit and reduce the growth of distal metastatic tumors and the tumor recurrence probability through immunoreaction, and can help patients to prolong the life cycle and improve the life quality on the premise of relatively controlling the cost.

Sixthly, the method comprises the following steps: furthermore, the related technical scheme in the patent can solve the problems that imiquimod is difficult to dissolve in water, the stability of the imiquimod after sterilization and the sterilization problem of sodium alginate. From the viewpoint of sterilization, sodium alginate and imiquimod R837 cannot be matched together, sodium alginate ALG needs to be subjected to filtration sterilization, but R837 particles cannot be subjected to filtration sterilization (the diameter of the particles after ball milling is 500nm at the lowest, and a filter membrane with the diameter of 220nm is required for filtration sterilization, so that the particles cannot pass through); on the other hand, R837 requires moist heat sterilization, while sodium alginate ALG degrades at high temperature. Both R837 and ALG cannot be sterilized together. Sodium alginate is a special natural biological high molecular material, and can be decomposed at high temperature, so that the traditional high-temperature damp-heat sterilization cannot be adopted, and the filtering sterilization is adopted in the patent, so that the property of the sodium alginate is reserved. The related embodiments of the dosage form and the preparation method solve the related technical problems. At the same time, the selectivity is increased by a surfactant, and poloxamer 188 is particularly preferred. In the case of no addition of poloxamer 188, after the R837 ball-milled emulsion is subjected to moist heat sterilization at 121 ℃, the emulsion is unstable, obvious precipitates and particles are generated, the water dispersibility is greatly reduced, and poloxamer 188 can greatly help R837 to ensure the water dispersibility and stability after sterilization.

According to the technical scheme of the patent, the sodium alginate, the chemotherapeutic agent and the immunologic adjuvant are combined to use the PD-1 to obtain a relatively excellent treatment effect, but the sodium alginate, the chemotherapeutic agent and the immunologic adjuvant, and the poloxamer 188 are further optimized to form the composition, so that a better treatment effect can be generated without combining the PD-1, the effect is better, the treatment cost is lower, and specific experimental data can be obtained by referring to fig. 30 and fig. 31.

Drawings

Fig. 1 shows the preparation process and the application of the lyophilized powder injection of the composition of sodium alginate and imiquimod hydrochloride in the first embodiment.

FIG. 2 is a scanning electron microscope image of the lyophilized powder for injection prepared by dissolving sodium alginate and imiquimod hydrochloride in gelatin in the example I.

Figure 3 is a graph of imiquimod drug release curves and statistics for varying concentrations of sodium alginate in one example.

Figure 4 is a graph of the release profile and data for imiquimod drug at different concentrations of imiquimod in the first example.

FIG. 5 is the scanning electron microscope picture of the lyophilized powder injection of sodium alginate and CpG oligonucleotide composition of the second embodiment after being redissolved into gel.

FIG. 6 is the CpG drug release profile and statistics for different concentrations of sodium alginate in example two.

FIG. 7 is the CpG drug release profile and statistics for different concentrations of CpG in example two.

FIG. 8 is the scanning electron microscope picture of the lyophilized powder injection of the combination of sodium alginate and doxorubicin hydrochloride in the third embodiment after redissolving into gel.

FIG. 9 shows the release profile and statistics of the doxorubicin hydrochloride drug at different concentrations of sodium alginate in the third example.

FIG. 10 is the release curve and statistics of the doxorubicin hydrochloride drug of example three at different doxorubicin hydrochloride concentrations.

FIG. 11 is a scanning electron microscope image of a lyophilized powder for injection of sodium alginate and oxaliplatin combination of the example IV after reconstitution in gel.

Figure 12 is the oxaliplatin drug release profile and statistics for varying concentrations of sodium alginate in example four.

Figure 13 is the oxaliplatin drug release profile and statistics for different oxaliplatin concentrations in example four.

FIG. 14 is the SEM image of the sodium alginate in the form of a gel with doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder for injection in example V.

FIG. 15 shows the rheological property test of the lyophilized powder for injection prepared by dissolving sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride in the fifth embodiment.

FIG. 16 is the scanning electron microscope picture of sodium alginate, oxaliplatin and imiquimod hydrochloride freeze-dried powder injection after being redissolved into gel in the embodiment.

FIG. 17 is a scanning electron microscope image of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride, and anti-PDL1 lyophilized powder for injection prepared in example Jiu after being redissolved into gel.

FIG. 18 shows the antibody activity detection of sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride, and anti-PDL1 antibody lyophilized powder for injection after reconstitution in EXAMPLE Jiu.

Figure 19 is a graph of tumor growth curves and statistics for the combination of sodium alginate and imiquimod hydrochloride composition of thirteen example with radiofrequency ablation therapy and anti-PDL1 antibody treatment on a mouse colon cancer tumor model.

Figure 20 is a graph of tumor growth curves and data statistics for the combination of sodium alginate and imiquimod hydrochloride composition of the thirteen example with HIFU treatment and anti-PDL1 antibody treatment in a mouse colon cancer tumor model.

Figure 21 is a graph of growth curves and data statistics for the sodium alginate and imiquimod hydrochloride composition of example thirteen in combination with HIFU treatment and anti-PDL1 antibody treatment to cause a second implantation of tumors on a mouse colon cancer tumor model.

FIG. 22 shows the tumor growth curves and statistics of the lyophilized powder of sodium alginate and oxaliplatin combination of the example of colon cancer treatment in mice.

FIG. 23 shows the body weight curves and statistics of mice treated with lyophilized powder of sodium alginate and oxaliplatin combination of the example, colon cancer.

Figure 24 is a graph of tumor growth in situ following treatment of sodium alginate with oxaliplatin and imiquimod hydrochloride and anti-PDL1 antibody in a mouse bilateral tumor model in example fifteen.

Figure 25 is a graph of distal tumor growth curves and statistics for sodium alginate with oxaliplatin and imiquimod hydrochloride and anti-PDL1 antibody after treatment in a mouse bilateral tumor model in example fifteen.

Fig. 26 is a graph of tumor growth curves and statistics for sodium alginate with oxaliplatin and imiquimod hydrochloride and anti-PDL1 antibody after tumor re-inoculation after healing in the mouse bilateral tumor model in example fifteen.

FIG. 27 is data of fluorescence imaging of mice after treatment of example hexadecimal alginate with doxorubicin hydrochloride and imiquimod hydrochloride, and anti-PDL1 antibody on a mouse orthotopic breast cancer tumor model.

FIG. 28 is a graph of tumor growth curves and statistics for sodium alginate and doxorubicin hydrochloride and imiquimod hydrochloride and anti-PDL1 antibody after treatment in a mouse brain cancer model in seventeen example.

Figure 29 is data of fluorescence imaging of mice after treatment of sodium alginate with doxorubicin hydrochloride and imiquimod hydrochloride and anti-PDL1 antibody on a mouse tumor surgical resection model, example eighteen.

FIG. 30 shows tumor growth with different treatment modalities for the nineteen examples, with direct drug injection to larger tumors (starting volume >120 cubic millimeters).

Fig. 31 shows tumor growth in nineteen contralateral small tumors (starting volume <50 cubic millimeters) without direct injection of drug, for different treatment modalities.

Detailed Description

The first embodiment is as follows: preparation and application of sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection

The method comprises the following steps: preparation of imiquimod (third class of component) hydrochloride. 50-100 mg of imiquimod is weighed into a 50ml glass mixing container, 1ml of 1M diluted hydrochloric acid is added into the glass mixing container, and deionized water is added for dilution after white powdery imiquimod is fully dissolved to be colorless and transparent, so that the final concentration of the imiquimod is 2.5-5 mg per ml. And (4) freeze-drying the solution to obtain the imiquimod hydrochloride freeze-dried powder. This step is intended to bring the water-insoluble imiquimod into the water-soluble hydrochloride form. A sufficiently long lyophilization time is required to ensure complete removal of the hydrochloric acid residues.

Step two: the sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection can be prepared by the following three methods.

The method comprises the following steps: 10-80 mg of sodium alginate and 0.1-10 mg of imiquimod hydrochloride freeze-dried powder are weighed and dissolved in 1ml of aqueous phase solution, stirring is carried out by a stirring paddle at the speed of 50-300 rpm until the solution is clear and transparent, the temperature is kept at 20-40 ℃, and the pH value is kept at-6.5. And freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate, dissolving in 1ml of water phase solution, stirring with a stirring paddle at the speed of 50-300 rpm until the solution is clear and transparent, freeze-drying to obtain freeze-dried powder injection, and then mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through solid and solid shaking to obtain the composition freeze-dried powder injection.

Dissolving 0.1-10 mg of imiquimod hydrochloride freeze-dried powder in 1ml of aqueous phase solution, stirring with a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, then dissolving 10-80 mg of sodium alginate in the aqueous phase solution, and dripping the constantly stirred imiquimod hydrochloride solution in the volume ratio of 1: 20 to ensure that the mixed solution is clear and transparent and has no flocculent precipitate. And after the sodium alginate solution is completely added, taking out the mixed solution and freeze-drying to obtain the composition freeze-dried powder injection.

Fig. 1 is a preparation process of a sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection and an application description thereof.

Fig. 2 is a scanning electron microscope picture of the composition lyophilized powder for injection prepared by the method shown in fig. 1 after gelling. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Step three: the release curve of imiquimod in the freeze-dried powder injection of the composition of sodium alginate (the first component) and imiquimod (the third component) hydrochloride. The drug sustained-release carrier is used for making drugs slowly enter blood to reduce the concentration of the drugs in the blood, and is very needed for preparing sustained-release long-acting drugs capable of slowly releasing drug components in treatment. The drug release profile refers to the release profile of the encapsulated drug after the in vitro simulated gelling of the composition.

The release profile obtained with fixed imiquimod dosage and varying sodium alginate dosage was as follows.

Preparing sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection, wherein the concentration of sodium alginate is 1, 10, 20, 40 and 80 mg, the concentration of imiquimod is 2 mg, the composition freeze-dried powder injection is respectively re-dissolved in 1ml of aqueous phase solution and shaken until the mixture is clear and transparent, then 200 microliter of 5 mg per ml of calcium chloride solution is added to gelatinize the mixture, the reason for adding calcium chloride is to simulate the condition that the composition meets the gelatinization of calcium ions after being injected into a tumor in vitro and slowly releases the medicament, the colloid is soaked in 1ml of phosphoric acid buffer solution and stirred, and the content of the medicament in the phosphoric acid buffer solution is determined to be the release of the imiquimod on days 0, 0.25, 0.5, 1, 2, 4 and 8.

Fig. 3 shows the release curve and statistical table of imiquimod drug at different concentrations of sodium alginate, and it can be seen that at concentrations of 5 mg/ml or above, imiquimod has a significant slow release, so the concentration of sodium alginate in the composition is preferably 5 mg/ml to 80 mg/ml. The concentration of the sodium alginate is 10 mg per ml, the concentration is optimized, the concentration of the sodium alginate reaches a peak value basically when the concentration is 20 mg per ml, and the effect is not obviously improved when the concentration is increased.

The release profile obtained with fixed sodium alginate and varying amounts of imiquimod was as follows.

Preparing sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection, wherein the concentration of the imiquimod is 1, 2.5, 5, 7.5 and 10 mg (maximum solubility), the concentration of the sodium alginate is 20 mg, respectively dissolving the composition freeze-dried powder injection in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphoric acid buffer solution, stirring, and measuring the content of the medicine in the phosphoric acid buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to obtain the release of the imiquimod.

Fig. 4 is a graph showing the release curve and statistical table of imiquimod drugs at different concentrations of imiquimod, and it can be seen from the graph that when the concentration of imiquimod is higher than 7.5 mg per ml, the composition is significantly and rapidly released when gelling, and the subsequent release is faster than that at low concentration, but the sustained release effect is still significant, so the concentration of imiquimod in the composition freeze-dried powder injection is selected to be 0.1-10 mg per ml. This indicates that the formulation is effective regardless of the concentration of imiquimod, and that a high concentration, although released quickly, also has a significant sustained release effect.

The preferable mass ratio of the sodium alginate to the imiquimod hydrochloride obtained through the experiment is 50-800 to 1-100, and the more preferable mass ratio is 200-400 to 10-75.

Example two: sodium alginate (first class component) and CpG oligonucleotide (third class component) composition freeze-dried powder injection

The method comprises the following steps: preparation of sodium alginate and CpG oligonucleotide composition freeze-dried powder injection

Weighing 10-80 mg of sodium alginate and 0.1-5 mg of CpG oligonucleotide, dissolving in 1ml of water phase solution, fully shaking until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

FIG. 5 is the scanning electron microscope picture of the composition after the freeze-dried powder injection is redissolved into glue. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Step two: CpG release curve in sodium alginate and CpG oligonucleotide composition freeze-dried powder injection

The following release curves were obtained with varying amounts of sodium alginate for fixed CpG oligonucleotide.

Preparing sodium alginate and CpG oligonucleotide composition freeze-dried powder injection, wherein the concentration of sodium alginate is 1, 10, 20 and 40 mg, the concentration of CpG oligonucleotide is 0.2 mg, respectively dissolving the composition freeze-dried powder injection in 1ml of water phase solution, shaking to be clear and transparent, adding 200 microliter of 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphate buffer solution, stirring, and measuring the content of the medicine in the phosphate buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to be the release of the CpG oligonucleotide.

Fig. 6 is a CpG drug release curve when the sodium alginate concentration is different, and it can be known from the graph that when the sodium alginate concentration is 20 mg or more, the CpG oligonucleotide has an obvious slow release phenomenon, so the concentration of the sodium alginate in the composition lyophilized powder for injection is selected from 5 mg/ml to 80 mg/ml. The effect is not obvious when the concentration of the sodium alginate is 1 mg per ml, the effect is optimized when the concentration of the sodium alginate is 10 mg per ml, the concentration of the sodium alginate reaches a peak value basically when the concentration of the sodium alginate is 20 mg per ml, and the effect is not obviously improved when the concentration is increased.

The release profile obtained with varying amounts of CpG oligonucleotide for a fixed amount of sodium alginate was as follows.

Preparing sodium alginate and CpG oligonucleotide composition freeze-dried powder injection, wherein the concentration of CpG oligonucleotide is 0.1, 0.25, 0.5, 1 and 2 mg, the concentration of sodium alginate is 20 mg, respectively dissolving the composition freeze-dried powder injection in 1ml of water phase solution, shaking to be clear and transparent, adding 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphate buffer solution, stirring, and measuring the content of the medicine in the phosphate buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to be the release of the CpG oligonucleotide.

Fig. 7 is a release curve of CpG drugs when the concentration of CpG oligonucleotide is different, and it can be seen from fig. 8 that when the concentration of CpG oligonucleotide is higher than 1 mg per ml, a relatively significant acute release occurs, and the subsequent release rate is not changed much, and in view of cost, the price of CpG oligonucleotide is as much as 1 ten thousand renminbi/mg, so the concentration of CpG oligonucleotide in the composition lyophilized powder injection is selected to be 0.1-2 mg per ml, and preferably the concentration of CpG oligonucleotide is selected to be 0.1-0.5 mg per ml.

The optimal mass ratio of the sodium alginate to the CpG oligonucleotide obtained through the experiment is 50-800 to 1-20, and the more preferable mass ratio is 200-400 to 1-20.

Example three: sodium alginate (first component) and doxorubicin hydrochloride (second component) composition freeze-dried powder injection

The method comprises the following steps: the preparation of the sodium alginate and doxorubicin hydrochloride composition freeze-dried powder injection comprises the following steps:

the method comprises the following steps: weighing 20-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, stirring by using a stirring paddle at the speed of 50-300 rpm until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: dissolving 0.1-10 mg of adriamycin hydrochloride into 1ml of aqueous phase solution, stirring by using a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, then dissolving 10-80 mg of sodium alginate into the aqueous phase solution, and dripping the continuously stirred adriamycin hydrochloride solution into the aqueous phase solution according to the volume ratio of 1: 20 to ensure that the mixed solution is clear and transparent and has no flocculent precipitate. And after the sodium alginate solution is completely added, taking out the mixed solution and freeze-drying to obtain the composition freeze-dried powder injection.

FIG. 8 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is redissolved into gel. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Step two: adriamycin release curve in sodium alginate and adriamycin hydrochloride composition freeze-dried powder injection

Preparing sodium alginate and doxorubicin hydrochloride composition freeze-dried powder injection, wherein the concentration of sodium alginate is 1, 10, 20 and 40 mg, and the concentration of doxorubicin hydrochloride is 2 mg, respectively re-dissolving the composition freeze-dried powder injection in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, then adding 200 microliter of 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphoric acid buffer solution, stirring, and measuring the content of the medicine in the phosphoric acid buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to obtain the release of the doxorubicin hydrochloride.

Fig. 9 is a release curve of the adriamycin hydrochloride drug at different concentrations of sodium alginate, and it can be seen from the graph that adriamycin hydrochloride has an obvious slow release phenomenon at a concentration of 10 mg or more of sodium alginate, so the concentration of sodium alginate in the composition lyophilized powder injection is preferably 5 mg/ml to 80 mg/ml.

Preparing sodium alginate and doxorubicin hydrochloride composition freeze-dried powder injection, wherein the concentration of doxorubicin hydrochloride is 1, 2.5, 5, 7.5 and 10 mg (maximum solubility), the concentration of sodium alginate is 20 mg, respectively dissolving the composition freeze-dried powder injection in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, then adding 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphoric acid buffer solution, stirring, and measuring the content of the drug in the phosphoric acid buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to obtain the release of doxorubicin hydrochloride.

Fig. 10 is a release curve of an doxorubicin hydrochloride drug at different doxorubicin hydrochloride concentrations, and it can be seen from the graph that when the doxorubicin hydrochloride concentration is higher than 7.5 mg, a relatively significant rapid release occurs, and the subsequent release is also faster than at low concentrations, but the sustained release effect is still significant, so that the doxorubicin concentration in the composition lyophilized powder for injection is selected to be 0.1-10 mg per ml.

The preferable mass ratio of the sodium alginate to the doxorubicin hydrochloride obtained through the experiment is 50-800 to 1-100, and the more preferable mass ratio is 200-400 to 10-75.

Example four: sodium alginate (first component) and oxaliplatin (second component) composition freeze-dried powder injection

The method comprises the following steps: preparation of sodium alginate and oxaliplatin composition freeze-dried powder injection

Weighing 10-80 mg of sodium alginate and 1-7.5 mg of oxaliplatin, dissolving in 1ml of aqueous phase solution, stirring with a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

FIG. 11 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is redissolved into gel. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Step two: oxaliplatin release curve in sodium alginate and oxaliplatin composition freeze-dried powder injection

Preparing sodium alginate and oxaliplatin composition freeze-dried powder injection, wherein the concentration of sodium alginate is 1 mg, 10 mg, 20 mg and 40 mg, and the concentration of oxaliplatin is 2 mg, respectively re-dissolving the composition freeze-dried powder injection in 1ml of aqueous phase solution, shaking to be clear and transparent, then adding 200 microliter of 5 mg per ml of calcium chloride solution to gelatinize, soaking the colloid in 1ml of phosphoric acid buffer solution, stirring, and measuring the content of the medicament in the phosphoric acid buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to obtain the release of oxaliplatin.

Fig. 12 is a release curve of oxaliplatin drugs at different concentrations of sodium alginate, and it can be seen from the graph that oxaliplatin has an obvious sustained release phenomenon at a concentration of sodium alginate of 10 mg or more, so the concentration of sodium alginate in the composition lyophilized powder for injection is selected from 5 mg/ml to 80 mg/ml.

Preparing sodium alginate and oxaliplatin composition freeze-dried powder injection, wherein the concentration of oxaliplatin is 1, 2.5, 5 and 7.5 mg (maximum solubility), the concentration of sodium alginate is 20 mg, re-dissolving the composition freeze-dried powder injection in 1ml of aqueous phase solution respectively, shaking until the solution is clear and transparent, adding 5 mg per ml of calcium chloride solution to gelatinize the solution, soaking the colloid in 1ml of phosphoric acid buffer solution, stirring, and measuring the content of the medicament in the phosphoric acid buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to obtain the release of oxaliplatin.

Fig. 13 is an oxaliplatin drug release curve when the oxaliplatin concentrations are different, and it can be seen from the graph 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 a low concentration, however, the sustained release effect is still obvious, so the concentration of oxaliplatin in the composition freeze-dried powder injection is selected to be 0.1-7.5 mg per ml.

The preferable mass ratio of the sodium alginate to the oxaliplatin is 50-800 to 1-75, and the more preferable mass ratio is 200-400 to 10-75.

Example five: sodium alginate (first component), doxorubicin hydrochloride (second component) and imiquimod (third component) hydrochloride freeze-dried powder injection

The method comprises the following steps: preparation of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride freeze-dried powder injection

Method one (dissolving the first, second and third components in the aqueous solution, stirring, and then freeze-drying the mixed solution): weighing 10-80 mg of sodium alginate (first component), 0.1-10 mg of imiquimod (third component) hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride (second component) and dissolving in 1ml of aqueous phase solution, stirring by using a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

And a second method (dissolving the first component and the second component in the aqueous phase solution, stirring, freeze-drying the mixed solution to obtain freeze-dried powder, and mixing the freeze-dried powder with the freeze-dried powder of the third component in a solid-solid manner): weighing 10-80 mg of sodium alginate (first component) and 0.1-10 mg of doxorubicin hydrochloride (second component) and dissolving in 1ml of aqueous solution, stirring with a stirring paddle at the speed of 50-300 rpm until the solution is clear and transparent, freeze-drying to obtain freeze-dried powder, and shaking and mixing the freeze-dried powder with 0.1-10 mg of imiquimod hydrochloride (third component) freeze-dried powder through solid and solid to obtain the composition freeze-dried powder injection.

Method three (dissolving component two and component three in water phase solution, stirring to obtain clear solution, dripping solution of component one into the mixed solution according to the ratio of 1: 20, and freeze-drying the final mixed solution to obtain freeze-dried powder): dissolving 0.1-10 mg of adriamycin hydrochloride and 0.1-10 mg of imiquimod hydrochloride in 1ml of aqueous phase solution, stirring by a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, then dissolving 20-80 mg of sodium alginate in 1ml of aqueous phase solution, and dripping the mixed solution which is continuously stirred into the solution in a volume ratio of 1: 20 to ensure that the mixed solution is clear and transparent and does not have flocculent precipitates. And after the sodium alginate solution is completely added, taking out the mixed solution and freeze-drying to obtain the composition freeze-dried powder injection.

The preferable mass ratio of the first component, the second component and the third component in the composition is 50-800 to 1-100, and the more preferable mass ratio is 200-400 to 10-75. .

FIG. 14 is the scanning electron microscope picture of the composition after the freeze-dried powder injection is redissolved into gel. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Step two: determination of rheological property of sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride freeze-dried powder injection after redissolution

Sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride freeze-dried powder injection are dissolved in 1ml of phosphoric acid buffer solution. The rheological properties of the compositions of 20. mu.l of 1, 5,10 and 20 mg per ml of sodium alginate mixed with 20. mu.l of 10 mg per ml of calcium ion solution were examined.

FIG. 15 shows the rheological mechanical properties of lyophilized powder for injection of different concentrations of sodium alginate and doxorubicin and imiquimod composition after reconstitution and contact with calcium ions. As can be seen from the figure, the storage modulus is smaller than the loss modulus at a sodium alginate concentration of 1 mg per ml, showing a fluid behavior, and the storage modulus is larger than the loss modulus at a sodium alginate concentration of more than 10 mg per ml, showing a gel behavior, demonstrating that sodium alginate forms a gel when encountering calcium ions at more than 10 mg per ml.

Example six: sodium alginate (first component), oxaliplatin (second component) and imiquimod hydrochloride (third component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-7.5 mg of oxaliplatin, dissolving in 1ml of aqueous phase solution, stirring by using a stirring paddle at the speed of 50-300 rpm until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate and 0.1-7.5 mg of oxaliplatin, dissolving in 1ml of aqueous phase solution, stirring with a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, freeze-drying to obtain freeze-dried powder, and then uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through solid-solid shaking to obtain the composition freeze-dried powder injection.

The third method comprises the following steps: dissolving 0.1-10 mg of oxaliplatin and 0.1-10 mg of imiquimod hydrochloride in 1ml of aqueous phase solution, stirring by a stirring paddle at the speed of 50-300 revolutions per minute until the solution is clear and transparent, then dissolving 20-80 mg of sodium alginate in 1ml of aqueous phase solution, and dripping the mixed solution which is continuously stirred in a volume ratio of 1: 20 to ensure that the mixed solution is clear and transparent and does not have flocculent precipitates. And after the sodium alginate solution is completely added, taking out the mixed solution and freeze-drying to obtain the composition freeze-dried powder injection.

The mass ratio of the first component, the second component and the third component in the composition is 50-800 to 1-75 to 1-100, and the more preferable mass ratio is 200-400 to 10-75. .

FIG. 16 is the scanning electron microscope picture of the composition after the freeze-dried powder injection is gelatinized. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

Example seven: sodium alginate (first component), pentafluorouracil (second component) and imiquimod hydrochloride (third component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 1-5 mg of pentafluorouracil and 0.1-10 mg of imiquimod hydrochloride, dissolving in 1ml of 2 mg per ml of sodium hydroxide solution, fully shaking until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate and 1-5 mg of pentafluorouracil, dissolving in 1ml of aqueous solution, shaking until the solution is clear and transparent, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

Example eight: sodium alginate (first component), cyclophosphamide (second component) and imiquimod hydrochloride (third component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 1-5 mg of cyclophosphamide and 0.1-10 mg of imiquimod hydrochloride, dissolving in 1ml of water phase solution, fully shaking until the solution is clear and transparent, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate and 1-5 mg of cyclophosphamide, dissolving in 1ml of water phase solution, shaking until the solution is clear and transparent, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

Example nine: sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous solution, shaking until the solution is clear and transparent, adding 100 microgram-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

FIG. 17 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is gelled. As can be seen from the figure, the composition still has good gelling capability after freeze-drying and redissolving, and an electron microscope picture shows that the composition has a plurality of micron-level pore channels after gelling, thereby being of great help for drug sustained release.

FIG. 18 shows the activity test of antibody anti-PDL1 after freeze-drying, and it can be seen from the experimental results that the peak value of antibody flow of anti-PDL1 after freeze-drying combined with cell surface PDL1 is consistent with the peak value of antibody of anti-PDL1 alone, which indicates that freeze-drying does not affect the activity of anti-PDL1 antibody.

Example ten: sodium alginate (first component), oxaliplatin (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-7.5 mg of oxaliplatin, dissolving in 1ml of aqueous phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: 10-80 mg of sodium alginate and 0.1-10 mg of oxaliplatin are weighed, dissolved in 1ml of aqueous phase solution, shaken until the solution is clear and transparent, added with 100-5 mg of anti-PDL1 solution, uniformly mixed and lyophilized to obtain lyophilized powder, and then uniformly mixed with 0.1-10 mg of imiquimod hydrochloride lyophilized powder through shaking to obtain the composition lyophilized powder injection.

Example eleven: other alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection

Preparation: potassium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of potassium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of water phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of potassium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 100 micrograms-5 mg of anti-PDL1 solution, uniformly mixing, freeze-drying to obtain freeze-dried powder, then mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, and uniformly mixing through solid-solid shaking to obtain the composition freeze-dried powder injection.

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

The method comprises the following steps: weighing 10-80 mg of ammonium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of water phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of ammonium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 100-5 mg of anti-PDL1 solution, uniformly mixing, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

The alginate with different cations can form a good composition with other three components and still has the capability of gelling and slow release.

Example twelve: alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and IDO inhibitor 4-phenylimidazole (fourth component) freeze-dried powder injection

Preparation: sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and 4-phenylimidazole (fourth component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of sodium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, fully shaking until the solution is clear and transparent, adding 100-microgram-5 mg of 4-phenylimidazole solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of sodium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 100 micrograms-5 mg of 4-phenylimidazole solution, uniformly mixing, freeze-drying to obtain freeze-dried powder, then mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder, and uniformly mixing through solid-solid shaking to obtain the composition freeze-dried powder injection.

Preparation: potassium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and 4-phenylimidazole (fourth component) freeze-dried powder injection

The method comprises the following steps: weighing 10-80 mg of potassium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of 4-phenylimidazole solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of potassium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 100-5 mg of 4-phenylimidazole solution, uniformly mixing, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

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

The method comprises the following steps: weighing 10-80 mg of ammonium alginate, 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of water phase solution, fully shaking until the solution is clear and transparent, adding 100-5 mg of 4-phenylimidazole solution, uniformly mixing, and freeze-drying the solution to obtain the composition freeze-dried powder injection.

The second method comprises the following steps: weighing 10-80 mg of ammonium alginate and 0.1-10 mg of doxorubicin hydrochloride, dissolving in 1ml of aqueous phase solution, shaking until the solution is clear and transparent, adding 100-5 mg of 4-phenylimidazole solution, uniformly mixing, and freeze-drying to obtain freeze-dried powder, and uniformly mixing with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder through shaking to obtain the composition freeze-dried powder injection.

The following are experiments and data statistics relating to the synergistic therapeutic effect of different compositions.

Example thirteen: curative effect research of sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection on colon cancer model

The method comprises the following steps: the sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection is combined with the curative effect research of the anti-PDL1 antibody in the radio frequency ablation treatment and immune checkpoint inhibition therapy.

The colon cancer tumor of the mice (the left side is regarded as the in situ tumor, the right side is regarded as the far-end tumor) is planted at the left and the right ends of the back of the mice respectively, and the tumor-bearing mice are divided into four groups, and 5 mice in each group are treated.

A first group: left in situ tumor independent radiofrequency ablation therapy (reference example);

second group: the anti-pdl1 antibody (reference example) is injected into the tail vein after the left in-situ tumor radiofrequency ablation treatment;

third group: performing radiofrequency ablation treatment after intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (example one) on the left in situ tumor;

and a fourth group: after the intratumoral injection of the sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (example one) into the left in situ tumor, the treatment of radiofrequency ablation is carried out, and the treatment is carried out by adding the anti-PDL1 antibody into the tail vein.

Mice were measured with a vernier caliper for the length and width of their right distal tumor, the volume of the tumor (length times (width squared)) divided by 2, every two days after the different treatments. The experimental result shows that the in-situ tumor on the left side of the mouse is eliminated by the radiofrequency ablation treatment, and the far-end tumor on the right side of the fourth group of mice is obviously inhibited, so that the sodium alginate and imiquimod hydrochloride composition can better stimulate the anti-tumor immune response after the radiofrequency ablation treatment, and has a good synergistic effect with the anti-PDL1 antibody.

(FIG. 19)

Step two: the curative effect research of the sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection combined with the high-energy focused ultrasound (HIFU) and the anti-PDL1 antibody of the immune checkpoint inhibition therapy is carried out.

A first group: left in situ tumor HIFU alone treatment (reference example);

second group: the anti-pdl1 antibody (reference example) is injected into the tail vein after the left in-situ tumor HIFU treatment;

third group: HIFU treatment after intratumoral injection of sodium alginate and imiquimod hydrochloride composition lyophilized powder for injection (example one) to the left in situ tumor;

and a fourth group: the left in-situ tumor is treated by adding anti-PDL1 antibody into tail vein after injecting sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (example one) into the tumor.

Mice were measured with a vernier caliper for the length and width of their right distal tumor, the volume of the tumor (length times (width squared)) divided by 2, every two days after the different treatments. The experimental result shows that the in-situ tumor on the left side of the mouse is eliminated by HIFU treatment, and the far-end tumor on the right side of the fourth group of mice is obviously inhibited, which shows that the composition of sodium alginate and imiquimod hydrochloride can better stimulate the anti-tumor immune response after the HIFU treatment, and has good synergistic effect with anti-PDL1 antibody. (FIG. 20)

Step three: the sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection combined with a high-energy focused ultrasound (HIFU) and an anti-PDL1 antibody of immune checkpoint inhibition therapy causes the research of the immunological memory effect.

Colon cancer tumor-bearing mice were divided into six groups of 5 mice each.

A first group: a physiological saline solution group;

second group: tail vein anti-PDL1 antibody treatment (reference example);

third group: HIFU therapy alone (reference example);

and a fourth group: tail vein anti-pdl1 antibody treatment after HIFU treatment (reference example);

and a fifth group: HIFU treatment after intratumoral injection of sodium alginate and imiquimod hydrochloride composition lyophilized powder injection (example one);

a sixth group: sodium alginate and imiquimod hydrochloride composition lyophilized powder for injection (example one) intratumorally followed by HIFU treatment and caudal vein anti-PDL1 antibody treatment.

After 40 days of tumor elimination by HIFU treatment, colon cancer tumor cells were replanted on these differently treated mice, and the length and width of the distal tumor to the right was measured with a vernier caliper, and the tumor volume was (length multiplied by (width squared)) divided by 2. The experimental results show that the tumor growth of the mice of the fifth group and the sixth group planted again is obviously slower than that of the control group and is obviously inhibited, and the tumor growth of the mice of the sixth group is slower than that of the mice of the fifth group, even a part of the mice can not grow the tumor any more. The combination of the sodium alginate (first component) and the imiquimod hydrochloride (third component) composition freeze-dried powder injection and a high-energy focused ultrasound (HIFU) and an anti-PDL1 antibody of immune checkpoint inhibition therapy can obviously cause the immune memory of mice so as to prevent tumor recurrence. (FIG. 21)

Example fourteen: research on curative effect of sodium alginate (first component) and oxaliplatin (second component) composition freeze-dried powder injection on colon cancer model

The colon cancer tumor-bearing mice were divided into 6 groups, and 5 mice per group were subjected to treatment experiments.

A first group: mice were injected intratumorally with physiological saline (reference example) respectively;

second group: oxaliplatin (1.5 mg per kg body weight) (reference example of chemotherapeutic alone);

third group: lyophilized powder injection (0.375 mg per kg body weight) of composition of oxaliplatin and sodium alginate is injected intratumorally (example four);

and a fourth group: lyophilized powder injection (0.75 mg per kg body weight) of composition of oxaliplatin and sodium alginate is injected intratumorally (example four);

and a fifth group: lyophilized powder injection (1.5 mg per kg body weight) of composition of oxaliplatin and sodium alginate is injected intratumorally (example four);

a sixth group: oxaliplatin (3 mg per kg body weight) was injected into the tail vein (reference example for chemotherapeutic alone).

After intratumoral injection, the length and width of the tumor were measured with a vernier caliper every two days, and the volume of the tumor was (length times (width squared)) divided by 2. From the tumor growth curve (fig. 22), it can be seen that the therapeutic effect of the composition lyophilized powder injection of sodium alginate and oxaliplatin injected in tumor is already higher than that of tail vein injection by 3 mg/kg body weight and that of single drug injection by 1.5 mg/kg body weight at the dosage of 0.75 mg/kg body weight; when the composition freeze-dried powder injection of sodium alginate and oxaliplatin for intratumoral injection is measured by 1.5 milligrams kilogram of body weight, the tumor growth is obviously inhibited, and the curative effect is obvious. From the body weight of the mice (fig. 23), the body weight of the mice in the group of oxaliplatin for tail vein injection is obviously reduced in the first four days, which indicates that the mice have certain toxic and side effects in intravenous injection, but have no obvious toxic and side effects in intratumoral injection. It is evident that the compositions of the present patent regimen have less side effects when administered intratumorally than when administered intravenously.

Example fifteen: the curative effect research of sodium alginate (first component), oxaliplatin (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection on bilateral tumor models (one tumor is arranged on each of the left side and the right side).

The colon cancer tumors of the mice (the left side is regarded as the in-situ tumor, and the right side is regarded as the far-end tumor) are planted at the left and the right ends of the back of the mice respectively, the tumor-bearing mice are divided into 7 groups, and 6 mice in each group are subjected to combined immune treatment experiments.

second group: oxaliplatin and imiquimod complexed with anti-PDL1 solution (reference example);

third group: sodium alginate and oxaliplatin composition freeze-dried powder injection (example four) are combined with anti-PDL1 for intravenous injection (reference example);

and a fourth group: sodium alginate, oxaliplatin and anti-PDL1 composition freeze-dried powder injection for intratumoral injection (example four);

and a fifth group: sodium alginate, oxaliplatin and imiquimod composition freeze-dried powder injection (example six) for intratumoral injection;

a sixth group: lyophilized powder for injection of composition of sodium alginate, oxaliplatin and imiquimod anti-PDL1 for intratumoral injection (example ten);

a seventh group: sodium alginate was injected intravenously (reference) in combination with oxaliplatin and imiquimod composition lyophilized powder for injection (example six) and anti-PDL 1.

Injection of left in situ tumor after intratumoral injection of in situ tumor, the right distal tumor was not injected and the length and width of the in situ and distal tumors were measured every two days with a vernier caliper, the volume of the tumor being (length multiplied by (width squared)) divided by 2. As can be seen from the in situ and distal tumor growth curves (fig. 24 and 25), both the in situ and distal tumors of group 6 and group 7 mice were effectively inhibited and almost no longer grew. Two months after the mice in

groups

6 and 7 survived, and were replanted with colon cancer cells, significant inhibition of tumor growth was found, indicating effective prevention of tumor recurrence (fig. 26).

Example sixteen: curative effect research of sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection on breast cancer metastasis tumor model

The mice with breast pad in situ tumor 4T1 breast cancer were divided into 6 groups, and 6 mice per group were used for treatment experiments of the metastatic tumor model.

second group: doxorubicin and imiquimod hydrochloride with anti-PDL1 antibody (reference);

third group: lyophilized powder for injection of intratumoral sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride composition (example five);

and a fourth group: sodium alginate, doxorubicin hydrochloride and anti-PDL1 antibody composition freeze-dried powder injection for intratumoral injection (example five);

and a fifth group: sodium alginate, adriamycin, imiquimod and anti-PDL1 antibody composition freeze-dried powder injection for intratumoral injection (example nine);

a sixth group: sodium alginate and adriamycin and imiquimod hydrochloride composition freeze-dried powder injection (example five) is combined with anti-PDL1 antibody for intravenous injection (reference example).

The first control group directly removed the mouse tumor by surgery. Mice were treated on day fifteen and live fluorescence imaging of the mice was taken every five days. As can be seen from the experimental results, the fifth and sixth groups had excellent therapeutic effects. (FIG. 27)

Example seventeen: the curative effects of sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) freeze-dried powder injection on mouse brain cancer are researched

The brain cancer mice are divided into nine groups, and each group is provided with six mice for brain cancer treatment experiment.

A first group: mice were injected with physiological saline intracranially (reference example);

second group: umirolium was injected intraperitoneally by mice (reference);

third group: mice were injected intracranially with imiquimod hydrochloride and anti-PDL1 antibody, as well as with sodium alginate composition (reference example);

and a fourth group: a mouse is injected with the sodium alginate and adriamycin composition freeze-dried powder injection in an intracranial way (example three);

and a fifth group: mice were injected intracranially with doxorubicin hydrochloride and imiquimod hydrochloride, as well as with the anti-PDL1 antibody composition (reference example);

a sixth group: mouse intracranial injection sodium alginate, doxorubicin hydrochloride and imiquimod hydrochloride composition freeze-dried powder injection (example five);

a seventh group: intracranial injection of sodium alginate and doxorubicin hydrochloride and anti-PDL1 antibody composition (reference example) in mice;

and an eighth group: mouse intracranial injection sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody composition freeze-dried powder injection (example nine);

ninth group: mice were treated with intracranial sodium alginate injection in combination with doxorubicin hydrochloride and imiquimod hydrochloride lyophilized powder injection with anti-PDL1 antibody (reference example).

Mice were observed for mortality. FIG. 28 is a graph showing the mortality of mice, and it can be seen that the mice in the eighth and ninth groups survived twice as long as the control group, indicating that they were more effective.

Example eighteen: research on curative effect of sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) lyophilized powder for injection on model after surgical resection of tumor of mouse

Subcutaneous breast cancer mice are randomly divided into three groups, and each group is provided with six sodium alginate, adriamycin, imiquimod and anti-PDL1 antibody compound gel treatment experiments. Mice were post-surgical resection of most subcutaneous tumors (resection of subcutaneous tumors in mice retained their paracancerous skin and muscle).

First group no treatment (reference);

a second group of simple surgeries (reference example);

the third group of surgery was followed by applying a complex gel of sodium alginate with doxorubicin and imiquimod and anti-PDL1 antibody to the wound site (example nine).

The treatment effect is judged by observing the metastasis and recurrence of the tumor after the operation, and the conclusion is drawn by biological self-luminous imaging of the small animal. As can be seen from FIG. 29, the tumors of the third group of mice in the key group have good effects of inhibiting metastasis and recurrence, and the effect of the complex gel of sodium alginate, adriamycin, imiquimod and anti-PDL1 antibody is proved.

Example nineteenth: the pharmaceutical composition of this example can be prepared in two dosage forms.

The first dosage form is as follows:

component one, according to imiquimod R837: ratio 1 of surfactant poloxamer 188: (0.1-5). Imiquimod R837 and surfactant poloxamer 188 were weighed. Preferably 1g R837, an appropriate amount of poloxamer 188(0.15g, 0.3g, 0.5g, 1g, 2g, 3g, 4g, 5g) is added, 10ml of water is added for ball milling for 3 hours, after that, 9ml of homogenate is taken out and transferred into a 250ml beaker, 141ml of primary water is added, and the mixture is stirred at 500rpm for 1 hour and mixed evenly. The suspension is sucked up by a syringe and filled into 10ml penicillin bottles, 5ml each bottle and 30 bottles in total. Covering a rubber cover, sealing with an aluminum cover, and performing damp-heat sterilization at 121 ℃ for 12 minutes;

and (2) component two: preparing 0.1-5% sodium alginate and 0.1-1% oxaliplatin solution, preferably weighing sodium alginate ALG (3g or 1.5g) and proper amount of oxaliplatin (159mg,300mg,450mg and 90mg), adding 300ml of primary water, and stirring in a 500ml beaker for 2-6 hours (25-40 ℃, 200 and 850rpm, sealing the bottle mouth with a preservative film). The resulting solution was sterilized by filtration through a 0.22 micron filter. Filling into 20ml penicillin bottles with 10ml each by using a 60ml syringe, and obtaining 30 bottles. Precooling the mixture for 30 minutes in a refrigerator at the temperature of minus 80 ℃, and freeze-drying the mixture for 30 hours. After freeze-drying, the rubber cover is covered, and the aluminum cover is sealed.

When in use, the component two freeze-dried powder is added into the component one solution, fully and uniformly mixed in a shaking mode and then injected.

The second dosage form was as follows:

the component one: according to imiquimod R837: the ratio of the surfactant poloxamer 188 was 1: (0.1-5) weighing R837 and surfactant, and adding 0.1-1% oxaliplatin. Preferably 1g of imiquimod R837, adding a proper amount of poloxamer 188(0.15g, 0.3g, 0.5g, 1g, 2g, 3g, 4g and 5g), adding 10ml of water, ball-milling for 3 hours, taking out 9ml of homogenate after finishing, transferring into a 250ml beaker, adding 141ml of primary water and a proper amount of oxaliplatin (159mg,300mg,450mg and 900mg), and stirring at 500rpm for 0.5-3 hours for uniform mixing. The suspension is sucked up by a 50ml syringe and filled into 10ml penicillin bottles, 5ml each bottle, and 30 bottles in total. Covering a rubber cover, sealing with an aluminum cover, and performing damp-heat sterilization at 121 ℃ for 12 minutes;

and (2) component two: preparing 0.1-5% sodium alginate solution. Preferably, sodium alginate ALG3g or 1.5g is weighed, added with 300ml of primary water and stirred in a 500ml beaker for 2-6 hours (25 ℃ -40 ℃, 200-. The resulting solution was sterilized by filtration through a 0.22 micron filter. Filling into 20ml penicillin bottles with 10ml each by using a 60ml syringe, and obtaining 30 bottles. Precooling the mixture for 30 minutes in a refrigerator at the temperature of minus 80 ℃, and freeze-drying the mixture for 30 hours. After freeze-drying, the rubber cover is covered, and the aluminum cover is sealed.

The specific therapeutic effect of this example is as follows, using a first dosage form, the second dosage form being as effective as the first dosage form.

The experimental method comprises the following steps: the colon cancer tumors of the mice (the left side is regarded as the in-situ tumor, and the right side is regarded as the far-end tumor) are planted at the left and the right ends of the back of the mice respectively, the tumor-bearing mice are divided into 5 groups, and 6 mice in each group are subjected to combined immune treatment experiments.

second group: oxaliplatin + poloxamer 188 dispersed imiquimod particles are injected into the tumor;

third group: injecting oxaliplatin + sodium alginate into the tumor;

and a fourth group: injecting poloxamer 188 dispersed imiquimod particles and sodium alginate into the tumor;

and a fifth group: oxaliplatin, poloxamer 188 dispersed imiquimod particles and sodium alginate are injected into the tumor;

the left in situ tumor was injected, after intratumoral injection of the in situ tumor, the right distal tumor was not injected, and the length and width of the in situ and distal tumors were measured every two days with a vernier caliper, the volume of the tumor being (length multiplied by (width squared)) divided by 2.

The treatment effect is as follows: it can be seen from the in situ tumor growth curve and the distal tumor growth curve (fig. 30 and fig. 31) that both the in situ tumor and the distal tumor of the group 5 mice are effectively inhibited and hardly grow any more, and more importantly, the anti-PDL1 antibody is not combined to obtain a good therapeutic effect, so the method has a very good application prospect and value. Other corresponding treatment groups, in part, had some therapeutic effect, and some experimental groups had very limited therapeutic effect.

Cancer treatment is a very complex result, because both the immune system of the body, as well as the growth mechanisms of cancer cells, are very complex. The reason why the experiment can obtain a relatively excellent therapeutic effect includes, in addition to the explanation of the other parts of the patent, the reason that the R837 powder insoluble in water is ball-milled in water by the imiquimod R837 ball milling method, so that the powder is more finely milled, and the water-dispersible powder is excellent in water dispersibility.

Poloxamer P188 is added into the R837 ball-milling emulsion for ball milling, and poloxamer 188 is a novel high-molecular nonionic surfactant and has multiple purposes: as emulsifier, stabilizer and solubilizer, the water dispersibility and stability of the R837 ball milling emulsion can be further enhanced.

The solubilizing pharmaceutic adjuvant comprises: poloxamer 188, poloxamer 407, polysorbate 80 (tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), egg yolk lecithin, polyoxyethylene (35) castor oil, vitamin E polyethylene succinate, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 800, or sodium hydroxymethyl cellulose. Wherein one or more of poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (SolutolHS 15), egg yolk lecithin, polyoxyethylene (35) castor oil, vitamin E polyethylene succinate, or sodium hydroxymethyl cellulose has good solubilizing effect on R837.

However, polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 800 are generally effective in solubilizing. In recent years, adverse reaction cases are reported more and more. Polysorbate 80 (Tween 80) is used as a commonly used solubilizer auxiliary material, and becomes a research focus of adverse reaction causes. Although the polyoxyethylene castor oil serving as a nonionic surfactant can increase the water solubility of most of insoluble medicines, the polyoxyethylene castor oil can cause the release of histamine and cause various toxic and side effects, such as severe anaphylactic reaction, toxic kidney injury, neurotoxicity, cardiovascular toxicity and the like. According to literature reports, the Solutol HS 15 has the largest toxic and side effects on muscle stimulation and hemolysis experiments, and the polyethylene glycol 200 has the smallest toxic and side effects. Polyethylene glycol is a stable hydrophilic substance, has no toxicity or irritation, and has solubilization, stability increasing and effect prolonging effects on many medicines.

Poloxamer 188 poloxamer is a series of multipurpose medicinal auxiliary materials, and has the advantages of no toxicity, no antigenicity, no sensitization, no irritation, no blood dissolution and stable chemical properties. Poloxamer 188 is one of the series of auxiliary materials with better safety, and is currently used in clinic as an emulsifier and solubilizer for intravenous administration.

Further experiments show that in the case of no poloxamer 188, after the R837 ball-milled emulsion is subjected to moist heat sterilization at 121 ℃, the emulsion is unstable, obvious precipitates and particles are generated, and the water dispersibility is greatly reduced. Poloxamer 188 may help R837 to ensure water dispersibility and stability after sterilization.

Table 1: water dispersibility of imiquimod R837 by ball milling before and after addition of surfactant

Table 2: redispersibility of ball milled R837 with addition of different surfactants after autoclaving

Table 3: long term stability after ball milling of R837 autoclaved with different proportions of surfactant

Suitable surfactants include: poloxamer 188, poloxamer 407, polysorbate 80 (tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), polyoxyethylene (35) castor oil, vitamin E polyethylene succinate, and sodium hydroxymethyl cellulose.

Surfactant b: r837	Long term stability after autoclaving
		0.15:1	A large amount of granular aggregates appear
0.3:1	A large amount of granular aggregates appear
		0.5:1	A large amount of granular aggregates appear
1:1	Uniformly dispersed without the appearance of granular aggregates
		2:1	Uniformly dispersed without the appearance of granular aggregates
3:1	Uniformly dispersed without the appearance of granular aggregates
		4:1	Uniformly dispersed without the appearance of granular aggregates
5:1	Uniformly dispersed without the appearance of granular aggregates

Suitable surfactants include: egg yolk lecithin.

Surfactant c: r837	Long term stability after autoclaving
		0.15:1	A large amount of flocculent aggregates appear
0.3:1	A large amount of flocculent aggregates appear
		0.5:1	A large amount of flocculent aggregates appear
1:1	A large amount of flocculent aggregates appear
		2:1	A large amount of flocculent aggregates appear
3:1	A large amount of flocculent aggregates appear
		4:1	A large amount of flocculent aggregates appear
5:1	A large amount of flocculent aggregates appear

Suitable surfactants include: polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 800.

Although theoretically, the more dispersant the better the dispersion, the ratio is generally not more than 5:1, because: poloxamer 188(P188) is inherently viscous, has high viscosity at too high a concentration, and avoids introducing impurities.

Sodium alginate is a special natural biological high molecular material, and can be decomposed at high temperature, so that the traditional high-temperature damp-heat sterilization cannot be adopted. In addition, from the viewpoint of sterilization, sodium alginate and imiquimod R837 cannot be matched together, sodium alginate ALG needs to be subjected to filtration sterilization, but R837 particles cannot be subjected to filtration sterilization (the diameter of the particles after ball milling is 500nm at the lowest, and a filter membrane of 220nm is required for filtration sterilization, so that the particles cannot pass through); on the other hand, R837 requires moist heat sterilization, while sodium alginate ALG degrades at high temperature. Thus, both R837 and ALG cannot be sterilized together.

In addition, the reason why the imiquimod R837 is prepared into a ball-milled particle dosage form is that if the R837 is prepared into hydrochloride, the compatibility of the hydrochloride and the OXA exists contraindication, and the OXA reacts with chloride ions to be inactivated; secondly, the hydrochloride salt of R837 increases the viscosity of sodium alginate ALG, making it inconvenient to use.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Claims

1. A chemotherapeutic-immunoadjuvant combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant, wherein: the chemotherapy immune 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 capable of causing immunogenic death;

the first mixture is that the immune adjuvant is imiquimod, the imiquimod and a surfactant are mixed and ball-milled to obtain evenly dispersed imiquimod emulsion, the obtained imiquimod particles in the imiquimod emulsion have the particle size of 0.5-3 microns, and the imiquimod emulsion is sterilized by high temperature and moist heat;

2. The chemotherapeutic-immune combination of claim 1, wherein: further comprising an immune modulating agent; the immune regulating agent is mixed with the first mixture and the second mixture to form the chemotherapy immune combination drug.

3. The chemotherapeutic-immune combination of claim 2, wherein: the immune regulating agent comprises an immune checkpoint inhibitor or an IDO inhibitor, the immune checkpoint inhibitor comprises an antibody inhibitor or a small molecule inhibitor, the antibody inhibitor is usually anti-CTLA-4, anti-PD-1 and anti-PD-L1, the small molecule inhibitor is usually CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, the peptide inhibitor is DPPA-1, and the IDO inhibitor comprises small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.

4. The chemotherapeutic-immune combination according to claim 1 or 2, characterized in that: the sodium alginate is replaced by potassium alginate, ammonium alginate, chitosan, or fibrinogen, or alginate, or hyaluronic acid;

the imiquimod R837 is replaced with imidazoquinoline, or glucopyranoside lipid;

5. A chemotherapeutic-immunoadjuvant combination comprising a chemotherapeutic agent capable of causing immunogenic death and an immunoadjuvant, wherein: the chemotherapy immune 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 capable of causing immunogenic death;

the first mixture is that imiquimod R837 and surfactant are mixed and ball-milled to obtain evenly dispersed imiquimod emulsion, the obtained imiquimod particles in the imiquimod emulsion have the particle size of 0.5-3 microns, and the imiquimod emulsion, oxaliplatin and water are mixed and stirred evenly and are sterilized by high temperature and moist heat;

6. The chemotherapeutic-immune combination of claim 5, wherein: further comprising an immune modulating agent; the immune regulating agent is mixed with the first mixture and the second mixture to form the chemotherapy immune combination drug.

7. The chemotherapeutic-immune combination of claim 6, wherein: the immune regulating agent comprises an immune checkpoint inhibitor or an IDO inhibitor, the immune checkpoint inhibitor comprises an antibody inhibitor or a small molecule inhibitor, the antibody inhibitor is usually anti-CTLA-4, anti-PD-1 and anti-PD-L1, the small molecule inhibitor is usually CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ1, the peptide inhibitor is DPPA-1, and the IDO inhibitor comprises small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.

8. The chemotherapeutic immuno-combination drug of claim 5 or 6, wherein: the sodium alginate can be replaced by potassium alginate, ammonium alginate, chitosan, or fibrinogen, or alginate, or hyaluronic acid;

9. Use of a chemotherapeutic immuno-combination drug according to claims 1-8 for the preparation of a medicament for the treatment of a tumor-related indication.

10. The use according to claim 9, wherein the tumor-related indication is colon cancer, lung cancer, liver cancer, breast cancer, ovarian cancer and brain cancer.