CN112438944A

CN112438944A - Temperature-sensitive gel pharmaceutical composition for treating tumors

Info

Publication number: CN112438944A
Application number: CN201910828820.2A
Authority: CN
Inventors: 刘庄; 陶惠泉; 巢宇; 赵琪
Original assignee: Suzhou Baimai Biomedical Co ltd
Current assignee: Suzhou Baimai Biomedical Co ltd
Priority date: 2019-09-03
Filing date: 2019-09-03
Publication date: 2021-03-05
Anticipated expiration: 2039-09-03
Also published as: WO2021042778A1

Abstract

The patent discloses a temperature-sensitive gel pharmaceutical composition for treating tumors, which is characterized in that: the composition comprises an active component and a gelling auxiliary material, wherein the active component comprises a chemotherapeutic drug causing immunogenic death of tumor cells, or an immunologic adjuvant, or an immune checkpoint inhibitor, and the gelling auxiliary material comprises a gelling matrix; the gel-forming matrix comprises natural biological materials: chitosan, dextran, cellulose, sodium alginate, hyaluronic acid and synthetic polymers: the temperature-sensitive type gel medicine composition is prepared by one or more of poly N-isopropylacrylamide copolymer, polyoxyethylene/polyoxypropylene, polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer and polyethylene oxide sodium glycerophosphate, is simple in preparation method, can slowly release medicines by forming gel, and has a good application prospect in the anti-tumor field.

Description

Temperature-sensitive gel pharmaceutical composition for treating tumors

Technical Field

The patent relates to a medicine for treating tumor, in particular to a medicine composition for treating tumor.

Background

The malignant tumor (cancer) has become one of the main public health problems seriously threatening the health of Chinese population, according to the latest statistical data, the death of the malignant tumor accounts for 23.91 percent of the total death causes of residents, and the morbidity and the mortality of the malignant tumor are in a continuously rising state in recent ten years, the medical cost caused by the malignant tumor exceeds 2200 hundred million every year, and the prevention and control situation is severe. The etiology of cancer is: the result of the body canceration of normal cells of the body under the action of various carcinogenic substances and carcinogenic factors, such as environmental pollution, chemical pollution (chemical toxin), ionizing radiation, free radical toxin, microorganism (bacteria, fungi, virus, etc.) and metabolic toxin thereof, genetic characteristics, endocrine imbalance, immune dysfunction, etc., is often expressed as: local lumps are formed by abnormal proliferation of cells in local tissues. Cancer is a large group of diseases caused by multiple causes, multiple stages and multiple mutations of normal cells of the body. The characteristics of cancer cells are: unlimited and endless proliferation, so that the nutrient substances in the body of the patient are greatly consumed; the cancer cells release various toxins, so that a series of symptoms are produced by a human body; cancer cells can also be transferred to all parts of the body to grow and reproduce, which causes emaciation, weakness, anemia, inappetence, fever, serious organ function impairment and the like. Compared with benign tumor, the benign tumor is easy to remove and clean, generally does not transfer and relapse, only has extrusion and blocking effects on organs and tissues, but cancer (malignant tumor) can also destroy the structures and functions of the tissues and the organs to cause necrotizing hemorrhage and infection, and finally the patient dies due to exhaustion of the organ functions.

The various characteristics of cancer present a significant challenge to cancer treatment. Three common clinical cancer treatments are surgery, chemotherapy and radiotherapy, which are also called as "treatment of cancer. The operation treatment of cancer is the most widely applied means in modern western medicine clinical, and the operation treatment can rapidly remove the tumor, so that the large and small tumors can be separated from the human body within a few hours. Surgery is often used for early treatment of cancer because early tumor cells are mostly in situ and no metastasis occurs. The tumor can be quickly stripped off the human body, and the harm of the tumor to the human body can be directly blocked. Most of the middle and late stage cancer patients lose the chance of operation, most of the cancer is spread and metastasized, and the surgical knife is not used. It is known that surgery can only remove macroscopic tumors, which leads to the potential for re-scaring of hidden cancer cells under certain conditions, and is why many cancer patients relapse after surgery. In addition, patients with poor physical quality, such as elderly patients and patients with poor cardiopulmonary function, are not suitable for surgical treatment. The operation process can cause the injury of primordial qi of a human body, and if the immunity of a patient is too low, residual cancer cells are likely to explode again. Radiotherapy is the treatment of tumors by physical damage of rays, is also a local treatment, and is suitable for cancer diseases with tumor types sensitive to radiotherapy. The application of radiotherapy first needs to be in line with the fact that the tumor is sensitive to radiotherapy, and then the condition that radical resection cannot be performed on the tumor for surgery is considered. Radiotherapy can be used as adjuvant therapy for surgery, chemotherapy, etc. This is true, and radiotherapy is still in the position of adjuvant therapy for most solid tumors, however, radiotherapy has significant toxic side effects and the treatment regimen remains to be improved. .

Chemotherapy is a common treatment for cancer following surgery. In the middle and late stage cancer patients, the cancer is already transferred, and the transferred cancer cells may be hidden in other parts or spread throughout the body, and new focuses may be formed at the original parts and other parts at any time. Chemotherapeutic drugs are used as a systemic treatment by delivering the drug through the blood to various parts of the body and then acting on cancer cells. However, most chemotherapy drugs are classified into enemies and self-help, and when entering the human body, the chemotherapy drugs kill cancer cells and simultaneously determine normal cells of the human body. More unfortunately, most chemotherapy drugs are directed against rapidly proliferating cells, including not only cancer cells, but also immune cells that have important functions in humans. This makes the original cancer patients with poor immune function suffer serious injury again, even bone marrow suppression is initiated, and the white blood cells are sharply reduced. Therefore, patients with poor physical quality, such as older age and poor cardiopulmonary function, are not suitable to be treated by chemotherapy.

In recent years, with the rise of tumor immunotherapy and the ever-increasing efforts of countless researchers, hundreds of chemotherapeutic drugs are screened, and it is found that some chemotherapeutic drugs can cause the body to generate significant anti-tumor immune response due to special molecular mechanisms. After killing cancer cells, these chemotherapeutic drugs cause the dead cancer cells to express sites that are easily recognized by immune cells and thus recognized by the human immune system. The human immune system utilizes these dead cancer cells as antigens to train a batch of targeted toxic T cells, which further migrate to attack the whole body and eliminate cancer cells with the same antigens, thereby inhibiting metastasis and recurrence of cancer cells.

Meanwhile, some chemotherapy drugs can improve or adjust the complex microenvironment of solid tumors, so that the tumors are more sensitive to immunotherapy and the immunotherapy can play a larger role. In conclusion, chemotherapy may also induce a certain degree of immune response specific to tumors in the body to further inhibit tumors, especially inhibit tumor metastasis and recurrence, which are the reasons why tumors are difficult to cure.

Tumor immunotherapy is considered to be the most promising method for treating tumors today, and has received much attention and has made unprecedented breakthroughs in recent years. The nobel prize in 2018 awarded two scientists with pioneering results in tumor immunotherapy, particularly in the field of checkpoint inhibitor therapy. Among the various tumor immunotherapies, checkpoint inhibitor therapy has made breakthrough in recent years of research and clinical trials. Checkpoint inhibitor therapy is directed at the mechanisms of tumor immune escape, and research shows that tumors utilize the unique immune brake mechanisms of the body, which are used by the body to protect the body from being attacked by the immune system, however, cancer cells are mostly caused by endogenous factors, and the mechanisms are also learned so that the immune system of the body cannot effectively control the cancer cells. Scientists have successfully discovered these mechanisms to open up the critical path for tumor immunotherapy, and three PD1 antibody drugs have been approved by the FDA for clinical tumor treatment in recent years, and have shown unusual therapeutic effects on melanoma. However, studies have shown that checkpoint inhibitors are not highly effective in other solid tumors, which is associated with the complex and harsh microenvironment of the solid tumor itself and the insufficient recognition of cancer cells by the body itself. These problems are solved by the chemotherapeutic drugs mentioned above, which can turn "cold tumors" that are not immune-sensitive into "hot tumors" that are immune-sensitive. Thus, the combination of chemotherapy and immunotherapy is well-documented.

However, chemotherapeutic drugs, immunological adjuvants and immune checkpoint inhibitors all have certain side effects, and conventional administration methods such as intravenous injection and the like at present have no way of fully exerting the drug effect of tumor treatment drugs and reducing the side effects. How to make the existing chemotherapeutic drugs, immune adjuvants and immune checkpoint inhibitors capable of causing immunogenic death of tumor cells can kill tumors more effectively and inhibit the metastasis and recurrence of tumors is a very challenging research and development direction.

Disclosure of Invention

The invention aims to provide a temperature-sensitive gel pharmaceutical composition for treating tumors, a sustained-release gel and a preparation method thereof. The temperature-sensitive gel pharmaceutical composition is loaded with chemotherapeutic drugs, immunologic adjuvants and immune check point inhibitors which cause immunogenic death of tumor cells, can kill tumors more effectively and inhibit metastasis and recurrence of the tumors. The temperature-sensitive gel pharmaceutical composition has a simple preparation method, can slowly release the drug by forming gel, and has a good application prospect in the anti-tumor field.

In order to achieve the purpose, the invention provides the following technical scheme: a temperature-sensitive gel pharmaceutical composition for treating tumors is characterized in that: the composition comprises an active component and a gelling auxiliary material, wherein the active component comprises a chemotherapeutic drug causing immunogenic death of tumor cells, or an immunologic adjuvant, or an immune checkpoint inhibitor, and the gelling auxiliary material comprises a gelling matrix;

the gel-forming matrix comprises natural biological materials: chitosan, dextran, cellulose, sodium alginate, hyaluronic acid and synthetic polymers: one or more of poly N-isopropyl acrylamide copolymer, polyoxyethylene/polyoxypropylene, polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer and polyethylene oxide sodium glycerophosphate.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating tumors, the gel-forming matrix is poloxamer 407, the concentration of the gel-forming matrix is 10 milligrams per milliliter to 500 milligrams per milliliter, and the buffer solution is an aqueous solution or a pure water solution;

or the gel-forming matrix is composed of chitosan and sodium glycerophosphate, the concentration of the chitosan is 10 mg per ml to 40mg per ml, the buffer solution is an acidic buffer solution such as acetic acid or hydrochloric acid, and the ion concentration is 0.1 mg per ml to 40mg per ml;

or the gel-forming matrix composition is a polylactic glycolic acid-polyethylene glycol-polylactic glycolic acid copolymer with the concentration of 0.1 to 100 milligrams per milliliter, and the buffer solution is an aqueous solution or a pure water solution.

As a preferred scheme of the temperature-sensitive gel pharmaceutical composition for treating the tumor, the temperature-sensitive gel pharmaceutical composition is a temperature-sensitive gel pharmaceutical composition for treating the colon cancer tumor, the gel-forming matrix is poloxamer 407, and the poloxamer 407 is dissolved by adding an aqueous solution to ensure that the final concentration is 100-400 mg per ml; the poloxamer 407 solution is added with oxaliplatin solution with the concentration of 5-200 mg per ml and imiquimod emulsion with the concentration of 10-400 mg per ml, and the medicine composition obtained by fully and uniformly mixing can rapidly form gel within 1-2 minutes at the temperature of over 25 ℃ in vivo.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating the tumor, the temperature-sensitive gel pharmaceutical composition is a temperature-sensitive gel pharmaceutical composition for treating the colon cancer tumor, the gel-forming matrix is formed by mixing chitosan and sodium glycerophosphate into gel, the temperature is controlled below 20 ℃, and the chitosan is added into a hydrochloric acid buffer solution for dissolution, so that the final concentration of the chitosan is 10-40 mg per ml; adding sodium glycerophosphate to a calcium ion buffer solution containing oxaliplatin and imiquimod R837 to make the final concentration of the sodium glycerophosphate be 50-800 mg per ml; the chitosan solution and the sodium glycerophosphate solution are uniformly mixed, and the gel can be rapidly formed in 1-2 minutes at the temperature of more than 30 ℃ in vivo.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating tumors, the gel-forming matrix is a polylactic glycolic acid-polyethylene glycol-polylactic glycolic acid copolymer, the temperature is controlled below 20 ℃, doxorubicin hydrochloride, imiquimod R837, a-PDL1 gel compound is prepared, a polylactic glycolic acid-polyethylene glycol-polylactic glycolic acid copolymer solution with the concentration of 0.1 to 100mg per ml is uniformly mixed with an aqueous solution containing doxorubicin hydrochloride, R837, a-PDL1 to obtain the temperature-sensitive gel pharmaceutical composition, and the temperature-sensitive gel pharmaceutical composition can be rapidly formed into gel within 1 to 2 minutes when the temperature exceeds 32 ℃ in vivo.

As a preferable scheme of the temperature-sensitive type gel pharmaceutical composition for treating the tumor, the imiquimod solution is an imiquimod hydrochloride solution, 1M dilute hydrochloric acid is added into the imiquimod, the imiquimod is fully dissolved to be colorless and transparent, and the final concentration of the imiquimod is 2.5-5 milligrams per milliliter through dilution of deionized water.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating tumors, the imiquimod solution is imiquimod emulsion, and imiquimod R837 and deionized water are added into a zirconia ball milling tank to be ball-milled for 2-3 hours to obtain the uniformly dispersed imiquimod emulsion, wherein the particle size of the imiquimod particles is 20-300 microns.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating the tumor, the gel-forming matrix is poloxamer 407, the concentration of the poloxamer 407 is more than 100mg per ml, the emulsion mass ratio of the poloxamer 407 to imiquimod is more than 0.1 to 1, and the composition forms gel within one minute at the temperature of more than 25 ℃.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating tumors, the gelling matrix is poloxamer 407, the temperature is controlled at 20 ℃, a poloxamer 407 aqueous solution with the concentration of 100-400 mg per ml is prepared, then oxaliplatin chemotherapeutic drug with the concentration of 5-200 mg per ml is added, and after the complete and uniform mixing, the composition can rapidly gel at the temperature of more than 25 ℃ for 1 minute.

As a preferable scheme of the temperature-sensitive gel pharmaceutical composition for treating the tumor, the pharmaceutical composition is in the form of ointment, gel, suppository, spray, solution or implant.

The invention provides a pharmaceutical composition for treating tumors, which comprises active components and auxiliary materials, wherein the active components comprise the following raw materials: chemotherapeutic drugs causing immunogenic death of tumor cells, immune adjuvants, immune checkpoint inhibitors.

Preferably, the auxiliary materials comprise the following raw materials, a gel-forming matrix and a buffer solution.

Preferably, the chemotherapeutic drug causing the immunogenic death of the tumor cells is one or more of anthracycline drugs (such as adriamycin, epirubicin, mitoxantrone and the like), oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins (such as maytansine), and can change the dead tumor cells into related antigens after clinical use and killing the tumor cells to activate the anti-tumor immune response; the immune adjuvant is one or more of agonists of Toll-like receptors such as imiquimod, CpG oligonucleotide, monophosphoryl lipid A and resiquimod, and can help antigen presenting cells to present antigens, so that the immune adjuvant can better present tumor-associated antigens generated by chemotherapy to T cells, thereby amplifying immune response; the checkpoint inhibitor antibody is one or more of anti-CTLA4, anti-PDL1 and anti-PD1, and can inhibit immune escape of tumors, so that immune cells can kill tumor cells better.

The invention provides a preparation method of a temperature-sensitive slow-release gel for treating tumors, which comprises the following steps: 1. mixing and dissolving an immune adjuvant imiquimod and a hydrochloric acid buffer solution, and freeze-drying to obtain a component A; 2. dissolving poloxamer 407 with purified water to obtain a component B; 3. and (3) stirring and mixing the chemotherapeutic drug causing immunogenic death, the component A and the immune checkpoint inhibitor with the component B obtained in the step (2) to obtain a component C, mixing at room temperature and injecting into an affected part to form the sustained-release gel in situ.

The invention provides a preparation method of a temperature-sensitive slow-release gel for treating tumors, which comprises the following steps: 1. taking an immune adjuvant imiquimod, and ball-milling in a ball mill until the imiquimod is uniformly dispersed to obtain a component A; 2. dissolving poloxamer 407 with purified water to obtain a component B; 3. mixing the chemotherapeutic drug causing immunogenic death, the component A and the immune checkpoint inhibitor with the component B obtained in the step 2) under stirring to obtain a component C, mixing at room temperature, and injecting into the affected part to form the sustained-release gel in situ.

The invention provides a preparation method of a temperature-sensitive slow-release gel for treating tumors, which comprises the following steps: 1. taking an immune adjuvant imiquimod, and ball-milling in a ball mill until the imiquimod is uniformly dispersed to obtain a component A; 2. mixing chitosan with hydrochloric acid buffer solution for dissolving to obtain a component B; 3. dissolving sodium glycerophosphate and purified water to obtain a component C; 4. mixing the chemotherapeutic drug causing immunogenic death, the component A, the immune checkpoint inhibitor and the component C obtained in the step 3) under stirring to obtain a component D; 5. mixing component D and component A under stirring, and injecting into affected part at room temperature to form sustained release gel in situ.

The method of the invention can form gel in situ at the affected part of the tumor, so that the medicine mixed in the solution can be slowly released, and the effect of inhibiting the tumor metastasis and recurrence is achieved. Firstly, the chemotherapeutic drugs kill tumor cells and cause the immunogenic death of the tumor cells, and activate the specific immune reaction of the tumor; secondly, the immune adjuvant enhances the capability of antigen presenting cells, and further enhances the corresponding immune response; finally, the use of immune checkpoint inhibitors allows immunotherapy to kill tumors more effectively, thereby inhibiting metastasis and recurrence of the tumor.

One reason for this is that chemotherapy alone is not sufficient to elicit a robust enough immune response, the body's immunity first requires antigens, i.e. those dead immune cells, for the immune system to recognize cancer cells, further these antigens are phagocytosed by the cells responsible for antigen presentation, then the antigen-presenting cells transfer the antigens to T cells, which are trained to cytotoxic T cells with specific killing ability after receiving these antigens, and finally these cytotoxic T cells search the whole body for cancer cells with recognized antigens and attack and eliminate these cancer cells. This is a cascade of reactions, each of which requires a certain efficiency to make the whole process efficient. While conventional chemotherapy only completes the first step, the presentation of the second antigen requires the help of an immunoadjuvant to proceed more effectively. The immune adjuvant is a special compound molecule which can specifically stimulate the maturation of antigen presenting cells and present antigens in the antigen presenting cells to T cells, and the antigen presenting capability of the immune adjuvant can be obviously improved with the help of the immune adjuvant. However, most of the immune adjuvants are used for treating some common immune diseases, such as lupus erythematosus, genital infection, skin diseases and the like, and are not yet used for treating tumors. Further, a combination of traditional chemotherapy and existing immunotherapy remains to be developed.

A hydrogel is a three-dimensional cross-linked network of polymers. It has many advantages, such as high water content, specific three-dimensional structure, variable mechanical strength, porous nature and good biocompatibility and degradability. Injectable hydrogels are a particular class of hydrogel materials. These materials tend to be responsive and crosslink with each other to form a specific crosslinked network upon a series of stimuli such as temperature, acid-base, ionic, shear, light, sound, magnetic, and electrical. The hydrogel is characterized in that the hydrogel can be injected to any part of a body in a fluid form and forms stable colloid with certain shape and mechanical strength within a certain period of time, so that the material is free from an invasive surgical implantation mode, the administration is more convenient, and the invasiveness is low, and the harm to a patient is small; on the other hand, the hydrogel can be efficiently loaded with hydrophilic drugs and allows controlled and slow release of the drugs therein due to its specific three-dimensional porous network structure and high water content.

The temperature-sensitive hydrogel is a common injectable hydrogel, and the gel forming mechanism is that the high molecular polymer in the gel is subjected to phase transition due to the temperature difference between the external environment and the administration part, so that the temperature-sensitive hydrogel has a wide application prospect in the field of drug sustained release. The hydrogel has unique gel-forming property, optimized mechanical strength, good biocompatibility and degradability, and has wide application in controlled release of medicaments. However, temperature-sensitive hydrogels are of various types, complex in classification, and different in applicable temperature and environment, and a series of comprehensive and deep exploration needs to be performed on quality control and dosage form selection of the temperature-sensitive hydrogels applied to local administration.

The medicine composition is selected through a large number of experiments and research, so that the medicine composition becomes an efficient medicine controllable release device, the duration of the medicine on the local focus is prolonged, the medicine release is delayed, the curative effect is enhanced, the administration times are effectively reduced, and the toxic and side effects are reduced. The patent scheme creatively uses the colloid forming raw material to wrap the chemotherapeutic drug, the immunologic adjuvant and the immune check point inhibitor, the colloid forming is carried out in situ and the components are slowly released, the specific immunoreaction of the tumor is synchronously induced and the effect of immunity is enhanced, the damage to a patient is low, the large-area preventive spraying of a potential target area after an operation can be assisted, and the metastasis and the recurrence of the tumor are further inhibited.

In the aspect of preparation process, firstly, the scheme ensures that imiquimod insoluble in immune adjuvant has good water solubility, water dispersibility and stability, secondly, the most suitable proportioning condition is obtained through the quality control of the proportioning ratio and concentration of the components of the colloid-forming matrix, and finally, the scheme optimizes the freeze-drying process and the sterilization process of the composition and has great help for the industrial production of subsequent products.

Drawings

Figure 1 is the release profile of imiquimod drug from example 3.

Figure 2 is the oxaliplatin drug release profile of example 4.

FIG. 3 is a graph of the in situ tumor growth curve in example 5.

FIG. 4 is a graph of the distal tumor growth curve in example 5.

Figure 5 is the oxaliplatin release profile following gelation of the gel complex of example 7.

FIG. 6 is the release profile of gemcitabine from gel formulations of example 8 after gelation.

FIG. 7 is the release profile of the pentafluorouracil after gelation of the gel complex of example 9.

Figure 8 is the release profile of the doxorubicin hydrochloride drug after gelling of the gel complex of example 10.

FIG. 9 is a graph of the release of a-PD1 after gelling of the gel composite of example 12.

FIG. 10 is a scanning electron micrograph of the gel composite of example 13 after gelling.

FIG. 11 is a graph of the rheology of the gel composition of example 14 at different temperatures.

FIG. 12 is a graph showing the growth of in situ and distal tumors on a bilateral tumor model of colon cancer in example 15.

FIG. 13 is a scanning electron micrograph of the gel composite of example 16 after gelling.

FIG. 14 is a graph of the rheology of the gel composite of example 17 at different temperatures.

Figure 15 is the drug and a-PDL1 drug release profiles after gelling of the gel complex in example 18.

Detailed Description

The temperature-sensitive gel pharmaceutical composition for treating tumor and the preparation method thereof according to the present invention will be described in detail with reference to the following examples and accompanying fig. 1-15, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of the immunoadjuvant hydrochloride (in the case of imiquimod). The immune adjuvant imiquimod is a hydrophobic small molecule drug which is completely insoluble in water and soluble in an organic solvent, namely dimethyl sulfoxide. However, the injection for clinical treatment of tumor is aqueous solution or physiological solution, so water-insoluble imiquimod cannot be directly used as injection, and the imiquimod needs to be further used as lyophilized powder after being hydrochlorinated.

Firstly, in the embodiment, a large number of experimental analyses are performed on the solubility, dispersibility and stability of imiquimod freeze-dried powder after acidification by different acidic buffer solutions, and hydrochloric acid and lactic acid are found to be suitable acidic buffer solutions. Table 1 shows the difference in water solubility, water dispersibility and stability of imiquimod lyophilized powder after acidification with different acidic buffers. As can be seen from Table 1, after acidification with hydrochloric acid or lactic acid, the imiquimod lyophilized powder has good water solubility and good stability. And the imiquimod freeze-dried powder acidified by acetic acid, oxalic acid or carbonic acid is unstable in water.

Acidic buffer solution	Water solubility	Water-dispersible	Stability of	Injectability
					Hydrochloric acid	Solution	Uniformity	Stabilization	√
Lactic acid	Solution	Uniformity	Stabilization	√
					Acetic acid	Insoluble matter	Unevenness of	Instability of the film	×
Oxalic acid	Insoluble matter	Unevenness of	Instability of the film	×
					Carbonic acid	Insoluble matter	Unevenness of	Instability of the film	×

Table 1 water solubility, water dispersibility and stability differences of imiquimod lyophilized powder after acidification with different acidic buffers.

Then, the mixture ratio of a series of imiquimod and hydrochloric acid buffer solution is investigated, and the optimal mixture ratio of the imiquimod and the hydrochloric acid buffer solution is determined. Table 2 shows the difference in water solubility and acidic residue of imiquimod lyophilized powder after acidification with hydrochloric acid buffers of different concentrations. As can be seen from Table 2, the acidified imiquimod lyophilized powder was poorly water soluble at hydrochloric acid concentrations below 0.1 moles per liter. When the concentration of hydrochloric acid is between 0.1 and 2 mol per liter, the acidified imiquimod freeze-dried powder has good water solubility and stability. The concentration of hydrochloric acid is between 2 and 12 mol per liter, the imiquimod freeze-dried powder after being acidified by strong acid has high hydrochloric acid residue, and the aqueous solution is acidic and can not be injected into a human body, thereby causing obvious toxic and side effects.

TABLE 2 difference in water solubility and acidic residue of imiquimod lyophilized powder after acidification with hydrochloric acid buffer solutions of different concentrations

The finally determined preparation process comprises the following steps: weighing 50-100mg of imiquimod in a 50mL serum bottle, adding 1M diluted hydrochloric acid to fully dissolve the imiquimod to be colorless and transparent, diluting the imiquimod by deionized water to ensure that the final concentration of the imiquimod is 0.5-10 mg per mL, and freeze-drying to obtain the imiquimod hydrochloride freeze-dried powder.

Example 2

Preparation of immune adjuvant emulsion (taking imiquimod as an example): in addition to acidifying imiquimod to dissolve it in water, the imiquimod emulsion is ball milled to reduce its particle size to micron order, which also allows imiquimod to possess good water dispersibility. In this example, imiquimod emulsion with different ball milling times was studied, and table 3 shows the particle size, water dispersibility and stability variation of the imiquimod emulsion after ball milling. As can be seen from table 3, the imiquimod emulsion showed good dispersibility after ball milling for more than 15 minutes.

Using a 50Ml ball mill jar, imiquimod R837 and deionized water were added and ball milled for 3 cycles for about 3 hours to obtain a uniformly dispersed imiquimod emulsion.

Ball milling time (minutes)	Water-dispersible	Stability of	Particle size (micron)
				1	Unevenness of	Instability of the film	2-5
15	Uniformity	Stabilization	2-3
				60	Uniformity	Stabilization	1-2
180	Uniformity	Stabilization	0.3-1

TABLE 3 particle size, Water dispersibility and stability Change of the ball milled imiquimod emulsion

Example 3

The method comprises the following steps: preparation of gel composite with immunoadjuvant (poloxamer 407 as an example of the gel forming matrix and imiquimod ball-milled emulsion as an example of the immunoadjuvant):

polyoxyethylene-polyoxypropylene copolymer (PEO-co-PEO), also known as poloxamer, is a non-ionic polymeric surfactant. In general, the lengths of the PEO and PPO blocks and the relative PEO/PPO content are controlled during preparation to yield ABA triblock copolymers with different properties PEO-PPO-PEO. Poloxamer can also form polymers with high self-assembly performance with polyesters such as PLA, PLGA, PCL and the like, and form hydrogel in selective solvent (generally water). Currently, the main research on PEO-PPO-PEO is the nature of its copolymers and aqueous solutions. The mechanism of gelation is that the polymer itself shows a tendency to form micelles, which leads to dehydration of the polymer block. The higher the temperature, the easier it is to gel the strands resulting in immobility of the chains and thus gel.

This example first investigated the water dispersibility, stability, gelation temperature, gelation rate and viscosity differences of two useful poloxamer polymers, poloxamer 188 and poloxamer 407, respectively, when mixed with a drug. Table 4 shows the properties of poloxamer 188 and 407 when mixed with the immune adjuvant imiquimod emulsion. As can be seen from table 4, poloxamer 407 makes the imiquimod emulsion more stable, more water dispersible, and has excellent gelling properties.

Poloxamers	Proportioning	Whether to glue	Water-dispersible	Stability of	Can be injected or not
						188	1:1	Can not	Uniformity	Instability of the film	Can be used for
407	1:1	Can be used for	Uniformity	Stabilization	Can be used for

TABLE 4 Properties of Poloxamers 188 and 407 when mixed with the immune adjuvant imiquimod emulsion

Then, in this example, the gelling properties, gelling speed, viscosity and injectability of the composition at different concentrations and different drug ratios were studied, and the appropriate concentration of gelling matrix and drug ratio were determined. Table 5 shows the gelling properties of different concentrations of poloxamer 407 and the immune adjuvant imiquimod composition. As can be seen from the table, at concentrations of poloxamer 407 exceeding 100mg per ml, the compositions gel at 25 degrees celsius and within one minute, suitable for injection and clinical use. When the mass ratio of the poloxamer 407 to the imiquimod emulsion is more than 0.1 to 1, the composition has good water dispersibility, good stability and injectability.

TABLE 5 gelling Properties of different concentrations of Poloxamer 407 and ImmunoQuinmod compositions

The preparation process comprises the following steps: when the temperature is controlled at 20 ℃, 100mg of poloxamer 407 is weighed in a 50mL serum bottle, and the water solution is added to dissolve the poloxamer so that the final concentration is 100-400 mg per mL. Further adding 5ml of imiquimod ball milling emulsion with the concentration of 10-400 mg per ml, fully and uniformly mixing, storing at 20 ℃, and when in use, injecting into a human body at the temperature of more than 25 ℃ to quickly form gel.

Step two: release of imiquimod in the composition (gel-forming matrix poloxamer 407 as an example):

placing the composition in a water bath at 37 ℃ to simulate the body temperature of a human body for 1 minute to form colloid, soaking the colloid in 1 ml of phosphoric acid buffer solution, stirring, controlling the temperature at 37 ℃ all the time, and determining that the content of the medicine in the phosphoric acid buffer solution is the release of the imiquimod on days 0, 0.25, 0.5, 1, 2, 4 and 8.

Fig. 1 shows the release curve of imiquimod drug, and it can be seen that imiquimod has an obvious slow release phenomenon.

Example 4

The method comprises the following steps: preparation of gel complexes containing chemotherapeutic agents (poloxamer 407 for the matrix and oxaliplatin for the chemotherapeutic agents).

The preparation process comprises the following steps: when the temperature is controlled at 20 ℃, 100mg of poloxamer 407 is weighed in a 50mL serum bottle, and the water solution is added to dissolve the poloxamer so that the final concentration is 100-400 mg per mL. Further adding 5ml of oxaliplatin chemotherapeutic drug with the concentration of 5-200 mg per ml, fully and uniformly mixing, storing at 20 ℃, and quickly forming gel when being injected into a human body at the temperature of more than 25 ℃ in use.

Step two: oxaliplatin release in the composition (gum-forming matrix exemplified by poloxamer 407).

Placing the composition in a water bath at 37 ℃ to simulate the body temperature of a human body for 1 minute to form colloid, soaking the colloid in 1 ml of phosphoric acid buffer solution, stirring, controlling the temperature at 37 ℃ all the time, 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 oxaliplatin.

Fig. 2 is a release curve of oxaliplatin, and it can be seen from the figure that oxaliplatin has an obvious sustained release phenomenon.

Example 5

The method comprises the following steps: preparation of gel complexes containing chemotherapeutic agents and immunoadjuvants (poloxamer 407 for the gelling matrix, oxaliplatin for the chemotherapeutic agents and imiquimod emulsion for the immunoadjuvants).

The preparation process comprises the following steps: when the temperature is controlled at 20 ℃, 100mg of poloxamer 407 is weighed in a 50mL serum bottle, and the water solution is added to dissolve the poloxamer so that the final concentration is 100-400 mg per mL. Further adding oxaliplatin chemotherapeutic drug and imiquimod emulsion 5ml, wherein the oxaliplatin concentration is 5-200 mg per ml, the imiquimod concentration is 10-400 mg per ml, fully mixing uniformly, storing at 20 ℃, and when in use, injecting into a human body at more than 25 ℃ to quickly form gel.

Step two: the therapeutic effect of the gel complex containing the chemotherapeutic agent, adjuvant and antibody (poloxamer 407 as an example of the gel-forming matrix, oxaliplatin as an example of the chemotherapeutic agent, imiquimod emulsion as an example of the immunological adjuvant, and a-PD1 as an example of the antibody).

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.

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

second group: intratumoral injection of oxaliplatin gel complex (example 4);

third group: intratumoral injection of oxaliplatin gel complex (example 4) in combination with intravenous injection of anti-PD1 (reference);

and a fourth group: intratumoral injection of oxaliplatin and imiquimod gel complex (example 5);

and a fifth group: intratumoral injection of oxaliplatin and imiquimod gel complex (example 5) in combination with intravenous injection of anti-PD1 (reference);

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. 3 and 4), both the in situ and distal tumors of group 5 mice were effectively inhibited and almost no longer grew.

Example 6

The gel-forming matrix is prepared by mixing chitosan and sodium glycerophosphate to form gel, and the immune adjuvant is prepared by taking imiquimod as an example.

Chitosan is a natural polysaccharide and is a commonly used temperature-sensitive hydrogel matrix. Sodium glycerophosphate is a weakly basic compound in which hydroxyl groups of a bifunctional anion coupling agent and phosphate anions are present. When the temperature of the hydrogel is higher than the low critical phase separation temperature, weakly alkaline sodium glycerophosphate can easily capture protons on chitosan NH3+, so that the hydrogen bonding between chitosan molecular chains is enhanced, the hydrogen bonding between chitosan and water is weakened, and the molecular chains are promoted to form crosslinking and gelation. Therefore, increasing either the amount of sodium beta-glycerophosphate or the amount of chitosan will increase the cross-linking between groups, thereby reducing the gel time. This example studies the gelling properties, gelling temperature, gelling speed and stability of the compositions at different ratios. Table 6 shows the gelling properties of the compositions at different ratios, and it can be seen from table 6 that the gelling properties are most suitable when the chitosan concentration is 10 to 40mg per ml and the sodium glycerophosphate concentration is 200 to 600 mg per ml.

TABLE 6 gelling Properties of the compositions at different ratios

The preparation process comprises the following steps: the temperature is controlled at 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to an aqueous solution containing R837 to give a final sodium glycerophosphate concentration of 50-800 mg per mL. The two solutions are mixed evenly and stored at 20 ℃ for use.

Example 7

The method comprises the following steps: the gel compound containing the chemotherapeutic drug is prepared by taking chitosan and sodium glycerophosphate as an example of a gel forming matrix to be mixed into gel, and taking oxaliplatin as an example of the chemotherapeutic drug.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to an aqueous solution containing oxaliplatin to give a final concentration of sodium glycerophosphate of 50-800 mg per mL. The two solutions are mixed evenly and then used.

Step two: release of chemotherapeutic agents in the gel-forming complex (the gel-forming matrix is prepared by mixing chitosan and sodium glycerophosphate into gel, and the chemotherapeutic agent is prepared by oxaliplatin):

placing the gel compound containing the oxaliplatin in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml 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 the oxaliplatin.

Fig. 5 is a release curve of oxaliplatin, and from the figure, it can be known that oxaliplatin has a remarkable slow release phenomenon.

Example 8

The method comprises the following steps: preparation of gel-forming compound containing chemotherapeutic agent (gel-forming base is prepared by mixing chitosan and sodium glycerophosphate into gel, and chemotherapeutic agent is prepared by mixing gemcitabine):

the preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and an aqueous solution containing gemcitabine was added to give a final concentration of 50-800 mg per mL of sodium glycerophosphate. The two solutions are mixed evenly and then used.

Step two: release of chemotherapeutic agents in gel complexes (gel-forming matrix is exemplified by chitosan and sodium glycerophosphate mixed into gel, chemotherapeutic agents are exemplified by gemcitabine):

placing the gel compound containing the gemcitabine in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml of phosphate buffer solution, stirring, and measuring the content of the drugs in the phosphate buffer solution on days 0, 0.25, 0.5, 1, 2, 4 and 8 to determine that the content of the drugs is the release of the gemcitabine.

FIG. 6 is a graph showing the release profile of gemcitabine, which shows a significant sustained release of gemcitabine.

Example 9

The method comprises the following steps: the gel-forming matrix is prepared by mixing chitosan and sodium glycerophosphate into gel, and the chemotherapeutic drug is prepared by taking pentafluorouracil as an example.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to an aqueous solution containing pentafluorouracil to give a final concentration of sodium glycerophosphate of 50-800 mg per mL. The two solutions are mixed evenly and then used.

Step two: release of chemotherapeutic drugs in the gel-forming complex (the gel-forming matrix is exemplified by chitosan and sodium glycerophosphate mixed into gel, and the chemotherapeutic drugs are exemplified by pentafluorouracil):

placing the gel compound containing the pentafluorouracil in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml of phosphoric acid buffer solution, stirring, and measuring the content of the drug in the phosphoric acid buffer solution on the 0 th, 0.25 th, 0.5 th, 1 th, 2 th, 4 th and 8 th days to determine that the content of the drug is the release of the pentafluorouracil.

FIG. 7 is a release curve of a pentafluorouracil drug, and it can be known that pentafluorouracil has an obvious sustained release phenomenon.

Example 10

The method comprises the following steps: the gel-forming matrix is prepared by mixing chitosan and sodium glycerophosphate to form gel, and the chemotherapeutic drug is doxorubicin hydrochloride.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to an aqueous solution containing doxorubicin hydrochloride to give a final concentration of 50-800 mg per mL of sodium glycerophosphate. The two solutions are mixed evenly and then used.

Step two: release of chemotherapeutic agents in the gel-forming complex (gel-forming matrix is exemplified by chitosan and sodium glycerophosphate mixed into gelatin, chemotherapeutic agents are exemplified by doxorubicin hydrochloride):

placing the gel compound containing the doxorubicin hydrochloride in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml 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 determine that the content of the drug in the phosphoric acid buffer solution is the release of the doxorubicin hydrochloride.

FIG. 8 is the release curve of the adriamycin hydrochloride drug, and it can be seen that adriamycin hydrochloride has a distinct slow release phenomenon.

Example 11

The gel compound containing chemotherapeutic medicine and adjuvant is prepared by mixing chitosan and sodium glycerophosphate into gel as gel matrix, oxaliplatin as chemotherapeutic medicine, imiquimod as immunological adjuvant, and R837 as imiquimod for short.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to a calcium ion buffer containing oxaliplatin, imiquimod R837 to a final concentration of 50-800 mg per mL of sodium glycerophosphate. The two solutions are mixed evenly and then used.

Example 12

The method comprises the following steps: preparation of gel complexes containing antibodies, the gel-forming matrix is exemplified by chitosan and sodium glycerophosphate mixed into a gel, and the antibodies are exemplified by immune checkpoint inhibitor a-PD 1.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to an aqueous solution containing a-PD1 to give a final concentration of 50-800 mg per mL of sodium glycerophosphate. The two solutions are mixed evenly and then used.

Step two: the release of the antibody drug in the gel compound takes chitosan and sodium glycerophosphate as an example of a gel forming matrix to be mixed into gel, and takes a-PD1 as an example of the antibody drug.

Placing the gel compound containing a-PD1 in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml 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 oxaliplatin.

Fig. 9 shows the drug release curve of a-PD1, and it can be seen that a-PD1 has a distinct slow release phenomenon.

Example 13

The gel compound containing the chemotherapeutic drug, the adjuvant and the antibody is prepared, wherein the gel matrix takes the case of mixing chitosan and sodium glycerophosphate into gel, the chemotherapeutic drug takes the case of oxaliplatin, the immunologic adjuvant takes the case of imiquimod, and the antibody takes the case of a-PD 1.

The preparation process comprises the following steps: the temperature is controlled below 20 ℃, 40mg of chitosan is weighed in a 5mL serum bottle, and hydrochloric acid buffer solution is added for dissolution, so that the final concentration is 10-40 mg per mL. 400mg of sodium glycerophosphate was weighed into a 5mL serum bottle and added to a calcium ion buffer containing oxaliplatin, R837, a-PD1 to give a final concentration of sodium glycerophosphate of 50-800 mg per mL. The two solutions are mixed evenly and then used. As can be seen from the figure, a clear porous three-dimensional network structure is visible after the composite is gelled. (FIG. 10)

Example 14

The gel-forming matrix takes chitosan and sodium glycerophosphate mixed into gel as an example, the chemotherapeutic drug takes oxaliplatin as an example, the immunologic adjuvant takes imiquimod as an example, and the antibody takes a-PD1 as an example.

Preparing oxaliplatin, R837, a-PDL1 temperature-sensitive gel compound, and detecting the rheological mechanical property of the gel formed by mixing 20 mg per ml of chitosan solution and 50 mg per ml to 400mg per ml of sodium glycerophosphate compound solution.

As can be seen from the figure, the storage modulus of the compound is smaller than the loss modulus at the temperature of lower than 26 ℃, the compound shows the behavior of fluid, and the storage modulus of the compound is larger than the loss modulus at the temperature of higher than 30 ℃, the behavior of gel is shown, and the compound temperature-sensitive gel is proved to form colloid at the temperature of higher than 26 ℃. (FIG. 11)

Example 15

The gel-forming matrix is prepared by mixing chitosan and sodium glycerophosphate to form gel, the chemotherapeutic drug is prepared by taking oxaliplatin as an example, the immune adjuvant is prepared by taking imiquimod as an example, and the antibody is prepared by taking a-PD1 as an example.

second group: intratumoral injection of oxaliplatin gel complex (example 7);

third group: intratumoral injection of oxaliplatin gel complex (example 7) in combination with intravenous injection of anti-PD1 (reference);

and a fourth group: intratumoral injection of oxaliplatin and imiquimod gel complex (example 11);

and a fifth group: intratumoral injection of oxaliplatin and imiquimod gel complex (example 11) in combination with intravenous injection of anti-PD1 (reference);

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, both the in situ and distal tumors in group 5 mice were effectively inhibited and almost no longer grew. (FIG. 12) if anti-PD1 is also administered in gel, the effect is better.

Example 16

The gel-forming matrix is a block copolymer of poly-N-isopropylacrylamide and polyethylene glycol, the chemotherapeutic agent is doxorubicin, the immunoadjuvant is imiquimod, and the antibody is a-PDL 1.

The preparation process comprises the following steps: the block copolymer of poly-N-isopropylacrylamide and polyethylene glycol (concentration of 0.1 mg per ml to 100mg per ml) was mixed well with an aqueous solution containing DOX, R837, a-PDL1 at a temperature below 20 ℃ for use. FIG. 13 is a scanning electron micrograph of the hybrid composition after it has been mixed into a gel. The porous three-dimensional network structure after gelling can be seen from the figure.

Example 17

The gel compound containing chemotherapeutic medicine, adjuvant and antibody is prepared, the gel-forming matrix takes poly (lactic-co-glycolic acid) -polyethylene glycol-poly (lactic-co-glycolic acid) as an example, the chemotherapeutic medicine takes doxorubicin hydrochloride as an example, the immune adjuvant takes imiquimod as an example, and the antibody takes a-PDL1 as an example.

The polylactic glycolic acid-polyethylene glycol-polylactic glycolic acid copolymer triblock copolymer is one of temperature-sensitive hydrogels, can be automatically degraded in vivo and has good biocompatibility. The phase transition temperature of the copolymer is controlled by the hydrophilic group and the hydrophobic group in the macromolecule, the phase transition temperature of the copolymer is increased along with the increase of the hydrophilic group in the molecular chain, and the phase transition temperature of the copolymer is correspondingly reduced when the hydrophobic group in the molecular chain is increased, so the phase transition temperature of the obtained copolymer can be changed by changing the feeding proportion and the relative molecular mass of PEG.

The preparation process comprises the following steps: controlling the temperature below 20 ℃, preparing doxorubicin hydrochloride, R837, a-PDL1 coagulated compound, and uniformly mixing polylactic glycolic acid-polyethylene glycol-polylactic glycolic acid copolymer (the concentration is 0.1 mg per milliliter to 100mg per milliliter) with an aqueous solution containing doxorubicin hydrochloride, R837, a-PDL1 for use. And detecting the rheological mechanical property of the mixed rubber. As can be seen from the figure, the storage modulus of the compound is smaller than the loss modulus at the temperature of lower than 32 ℃, the compound shows the behavior of fluid, and the storage modulus of the compound is larger than the loss modulus at the temperature of higher than 32 ℃, the compound shows the behavior of gel, and the compound temperature-sensitive gel can form colloid at the temperature of higher than 32 ℃. (FIG. 14)

Example 18

The drug release of the gel compound containing the chemotherapeutic drug, the adjuvant and the antibody (the gel-forming matrix takes poly (lactic-co-glycolic acid) -polyethylene glycol-poly (lactic-co-glycolic acid) as an example, the chemotherapeutic drug takes oxaliplatin, adriamycin, pentafluorouracil and gemcitabine as an example, the immune adjuvant takes imiquimod as an example, and the antibody takes a-PDL1 as an example):

the preparation process comprises the following steps: the temperature is controlled below 20 ℃, the gel compound containing the chemotherapeutic drug, the adjuvant and the antibody is prepared, and the poly (lactic-co-glycolic acid) -polyethylene glycol-poly (lactic-co-glycolic acid) (the concentration is 0.1 mg per ml to 100mg per ml) and the aqueous solution containing the drug, R837, a-PDL1 are uniformly mixed for use. Placing the gel compound in a water bath at 37 ℃ to simulate the human body temperature for 2 minutes to form gel, soaking the gel in 1 ml of phosphoric acid buffer solution, stirring, and measuring the content of the medicine in the phosphoric acid buffer solution, namely the release of the medicine and the alpha-PDL 1 on days 0, 0.25, 0.5, 1, 2, 4 and 8.

FIG. 15 shows the release profile of the drug and a-PDL1, which shows that various chemotherapeutic drugs and a-PDL1 have a significant sustained release effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A temperature-sensitive gel pharmaceutical composition for treating tumors is characterized in that: the composition comprises an active component and a gelling auxiliary material, wherein the active component comprises a chemotherapeutic drug causing immunogenic death of tumor cells, or an immunologic adjuvant, or an immune checkpoint inhibitor, and the gelling auxiliary material comprises a gelling matrix and a buffer solution;

2. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 1, wherein: the gel-forming matrix is poloxamer 407, the concentration is 10 mg per ml to 500 mg per ml, and the buffer solution is an aqueous solution or a pure water solution;

3. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 1, wherein:

the temperature-sensitive gel pharmaceutical composition is used for treating colon cancer tumor, the gel-forming substrate is poloxamer 407, and the poloxamer 407 is dissolved by adding aqueous solution to ensure that the final concentration is 100-400 mg per ml; the poloxamer 407 solution is added with oxaliplatin solution with the concentration of 5-200 mg per ml and imiquimod emulsion with the concentration of 10-400 mg per ml, and the medicine composition obtained by fully and uniformly mixing can rapidly form gel within 1-2 minutes at the temperature of over 25 ℃ in vivo.

4. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 1, wherein: the temperature-sensitive gel pharmaceutical composition is used for treating colon cancer tumors, the gel-forming matrix is formed by mixing chitosan and sodium glycerophosphate into gel, the temperature is controlled below 20 ℃, and the chitosan is added into a hydrochloric acid buffer solution to be dissolved so that the final concentration is 10-40 mg per ml; adding sodium glycerophosphate to a calcium ion buffer solution containing oxaliplatin and imiquimod R837 to make the final concentration of the sodium glycerophosphate be 50-800 mg per ml; the chitosan solution and the sodium glycerophosphate solution are uniformly mixed, and the gel can be rapidly formed in 1-2 minutes at the temperature of more than 30 ℃ in vivo.

5. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 1, wherein: the gelling matrix is polylactic glycolic acid-polyethylene glycol-polylactic acid glycolic acid copolymer, the temperature is controlled below 20 ℃, doxorubicin hydrochloride, imiquimod R837 and a-PDL1 gelling compound are prepared, polylactic acid glycolic acid-polyethylene glycol-polylactic acid glycolic acid copolymer solution with the concentration of 0.1-100 mg per ml and aqueous solution containing doxorubicin hydrochloride, R837 and a-PDL1 are uniformly mixed to obtain the temperature-sensitive gel medicine composition, and the temperature-sensitive gel medicine composition can be rapidly gelled in 1-2 minutes when the temperature in a body exceeds 32 ℃.

6. The thermo-sensitive gel pharmaceutical composition for the treatment of tumor according to claim 3, 4 or 5, wherein: the imiquimod solution is an imiquimod hydrochloride solution, 1M dilute hydrochloric acid is added into the imiquimod to fully dissolve the imiquimod to be colorless and transparent, and the final concentration of the imiquimod is 0.1-10 milligrams per milliliter by diluting with deionized water.

7. The thermo-sensitive gel pharmaceutical composition for the treatment of tumor according to claim 3, 4 or 5, wherein: the imiquimod solution is imiquimod emulsion, and imiquimod R837 and deionized water are added into a ball milling tank to be ball milled for 2-3 hours to obtain the uniformly dispersed imiquimod emulsion, wherein the particle size of the imiquimod particles is 0.3-1 micron.

8. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 3, wherein: the gel-forming matrix is poloxamer 407, the concentration of the poloxamer 407 is greater than 100 milligrams per milliliter, the emulsion mass ratio of the poloxamer 407 to the imiquimod is more than 0.1 to 1, and the composition can form gel within one minute when the temperature is higher than 25 ℃.

9. The temperature-sensitive gel pharmaceutical composition for treating tumor according to claim 3, wherein: the gelling matrix is poloxamer 407, the temperature is controlled at 20 ℃, poloxamer 407 aqueous solution with the concentration of 100-400 mg per ml is prepared, then oxaliplatin chemotherapeutic drug with the concentration of 5-200 mg per ml is added, and after the materials are fully and uniformly mixed, the composition can rapidly gel at the temperature of over 25 ℃ for 1 minute.

10. The thermo-sensitive gel pharmaceutical composition for the treatment of tumor according to any one of claims 1-9, wherein: the dosage form of the pharmaceutical composition is ointment, gel, suppository, spray, solution or implant.