CN114681401A

CN114681401A - Self-sustained-release immunoadjuvant suspension, and preparation method and application thereof

Info

Publication number: CN114681401A
Application number: CN202011612051.1A
Authority: CN
Inventors: 刘庄; 邓中清; 吴宇辰
Original assignee: Suzhou Baimai Biomedical Co ltd
Current assignee: Suzhou Baimai Biomedical Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-01
Also published as: CN115137813A

Abstract

The invention discloses a self-sustained-release immunologic adjuvant suspension, which consists of fat-soluble immunologic adjuvant and surfactant to form micron particles, and the balance is dispersant, wherein the surfactant coats the fat-soluble immunologic adjuvant to form the micron particles, and the micron particles are dispersed in the dispersant to form a suspension. The invention also provides a preparation method of the self-sustained-release immunoadjuvant suspension, and application of the self-sustained-release immunoadjuvant suspension in sensitization preparations for tumor treatment such as radiotherapy, chemotherapy or thermotherapy.

Description

Self-sustained-release immunologic adjuvant suspension, and preparation method and application thereof

Technical Field

The invention relates to the field of sensitizers for radiotherapy treatment of tumors, in particular to a self-sustained-release immunoadjuvant suspension and a preparation method and application thereof.

Background

Chemotherapy, radiotherapy and microwave thermal ablation are effective methods for treating malignant tumors, play a great role in clinical tumor treatment, and are widely used for common tumors such as liver cancer, lung cancer, kidney cancer and the like. Radiotherapy (radiotherapy for short) is external radiation radiotherapy based on rays, which is widely used in clinic, but is a local treatment scheme, and only can irradiate local tumors, and remote metastatic tumors cannot be effectively irradiated (such as remote hidden tumors). The external radiation radiotherapy widely used in clinic uses rays (such as X-rays) to perform local fixed-point radiation on a tumor part, so that the aim of killing tumor cells is fulfilled, and even tumors without far-end metastasis can be cured. However, when the tumor has distant metastasis, it is difficult to cover all tumor cells, especially metastatic tumor cells, in the human body by local therapy, and these "fish with net leakage" may grow new tumor metastasis at the distant end.

In radiotherapy clinical applications, it has been found that for a small proportion of patients there is a possibility of a "distal effect", i.e. local treatment of a tumour will sometimes also inhibit the growth of a tumour which is not irradiated at the distal end. This radiotherapy-induced "distal effect" has attracted considerable interest to researchers in recent years. Studies have shown that the mechanism of the "distal effect" is to induce immunogenic cell death of tumor cells, expose tumor-associated antigens, thereby activating an immune response against the tumor, and further achieve immunosuppression of the distal tumor by infiltration of the distal tumor by tumor-specific CD8+ T cells. Although the induced "distal effect" is of significant clinical value, the individualised variation in this effect is very large and the radiotherapy-induced "distal effect" is not very significant for most patients in the clinic. The important reason for this is that the immunogenicity of the tumor-associated antigen in the "cadavers" of tumor cells, which are generated after the induction of the death of the immunogenic cells of the tumor cells, is not always strong, and cannot be used as an effective "tumor vaccine", and it is difficult to activate a sufficiently effective anti-tumor immune response in many cases.

In modern medical technology, vaccines are often composed of two parts, an antigen and an adjuvant. The adjuvant is used for stimulating immune cells to amplify the immune response generated by antigen in order of magnitude. Therefore, if the immune adjuvant can be locally injected into the tumor before the tumor is treated, and then the tumor is treated, the immunogenicity of the tumor-associated antigen generated after radiotherapy is expected to be remarkably amplified through the immune stimulation effect of the adjuvant, for example, antigen presenting cells are recruited to the tumor residue part to recognize, phagocytize and present the tumor antigen, so that endogenous 'tumor vaccine' is generated in vivo, a strong anti-tumor immune response is obtained, and the far-end tumor is more effectively inhibited. The mature dosage form of imiquimod at the present stage is a cream preparation, usually acts on an epidermis lesion part in a smearing mode, clinically treats diseases caused by local virus infection such as condyloma acuminatum and the like, and also has an attempt of being used for treating skin superficial tumors in clinical tests.

Clinical radiotherapy is mostly multi-time fractional dose irradiation, the injected immunostimulant needs to be retained and slowly released in the tumor for a long time, and the long-time retention and slow release performance in the tumor is very important for the application of sensitization radiotherapy. At present, most of water-soluble immunoadjuvants have the problems that the immunoadjuvants are easy to be eliminated through blood circulation and cannot be retained at the action site for a long time to realize long-acting stimulation; the fat-soluble immunologic adjuvant has poor dispersibility and is difficult to directly use. The problems that an immunostimulant is properly designed, an operable production and manufacturing method is designed, and the sterilization and long-term storage stability of a drug product are difficult, for example, when micro-nano particles are prepared by a ball milling method, ceramic particles are generated to remain in the product, so that injection risks are brought, the problem of impurities in the preparation and processing of common micro-nano materials is not great, but the impurities are high in risk when the impurities are used for human body injection, and the problems in the drug forming stage cannot be solved, so that many experimental drugs cannot be truly applied to clinical application.

Disclosure of Invention

The invention provides a self-sustained-release immunoadjuvant suspension, and provides an anticancer pharmaceutical composition which has a good in-situ dispersion effect, can realize self sustained release to assist chemotherapy, radiotherapy or thermotherapy to generate immunological memory, activate the immunological characteristics of a human body, reduce the probability of cancer metastasis and recurrence, effectively kill in-situ tumors, and simultaneously inhibit and reduce the growth of distal metastatic tumors and the probability of tumor recurrence through immunoreaction.

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

the self-sustained-release immunologic adjuvant suspension consists of a fat-soluble immunologic adjuvant and a surfactant, and the balance is a dispersant, wherein the surfactant coats the fat-soluble immunologic adjuvant to form micron-sized particles, and the micron-sized particles are dispersed in the dispersant to form a suspension.

Further, the dispersing agent is water or normal saline.

Further, the lipid-soluble immunoadjuvant comprises at least one of imiquimod (R837), ranisimmod (R848), or glucopyranoside lipid a (mpla).

Further, the hydrophobic structure portion of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188(P188), poloxamer 237, poloxamer 338 and poloxamer 407 are included.

In a juxtaposition alternative, the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the oil-in-water emulsion concretely comprises at least one of sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000, or lecithin.

Furthermore, the self-sustained-release immunoadjuvant suspension is a composite particle with the particle size of 0.5-5 microns, and the surfactant coats the fat-soluble immunoadjuvant. Preferably, the particle size of the self-sustained-release immunoadjuvant suspension is 1-2 microns.

Further alternatively, the surfactant may be a mixture of two surfactants having different hydrophilic-lipophilic balance (HLB values). After the composite particles enter tumor bodies, the two surfactants with different hydrophilic-lipophilic balance values can be dissolved firstly, so that the surfactants with higher HLB values are coated on the surfaces of the fat-soluble immunologic adjuvant microparticles to form a plurality of open or tiny defect areas, the surface area of the inner layer imiquimod microparticles is changed step by step, effective components are released step by step, and a more personalized medicament scheme can be prepared according to the actual requirements of different tumor bodies and human bodies through the matching relation of the surfactants.

The invention provides a preparation method of a self-sustained-release immunoadjuvant suspension, which is characterized by comprising the following steps:

s1: the fat-soluble immunologic adjuvant is subjected to airflow pulverization to form primary powder;

s2: adding a water solution of a surfactant into the primary powder obtained in the step S1, carrying out high-pressure homogenization treatment, and taking out homogenate after the treatment is finished;

or S2': adding a water solution of a surfactant into the primary powder obtained in the step S1, carrying out high-shear process treatment, and taking out homogenate after the treatment is finished;

s3: and (5) sterilizing.

Further, the aqueous solution of the surfactant described in step S1 includes two surfactants with different hydrophilic-lipophilic balance values.

Preferably, the concentration of the aqueous solution of the surfactant in the step S1 is 6-30 mg/mL.

Further, the sterilization treatment in the step S3 is a wet heat treatment performed under a condition of 105 to 150 ℃ for 10 to 15 minutes.

The invention also provides a self-sustained-release immunologic adjuvant composition, which comprises a first composition and a second composition; the first composition consists of a fat-soluble immunologic adjuvant and a surfactant, and the balance is a dispersant, wherein the surfactant coats the fat-soluble immunologic adjuvant to form micron-sized particles, and the micron-sized particles are dispersed in the dispersant to form a suspension; the second composition comprises soluble alginate, a protective filler and a pH regulator to form freeze-dried powder.

The second composition may further optimize the sustained release characteristics of the first composition.

Further, the dispersing agent is water or normal saline.

Further, the hydrophobic moiety of the surfactant contains not less than 20 oxypropylene units, including poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407; or one or more hydrocarbon chains containing a total of no less than 15 carbon atoms, including at least one of sorbitan sesquioleate, soybean lecithin, glyceryl monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl (40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl 1000, or lecithin.

The invention also provides application of the self-sustained release immunoadjuvant suspension in preparation of a radiotherapy sensitizer.

The invention also provides application of the self-sustained release immunoadjuvant suspension in preparing a chemosensitizer.

The invention also provides application of the self-sustained release immunoadjuvant suspension in preparation of a thermotherapy sensitizer.

By adopting the technical scheme of the invention, the following beneficial technical effects can be achieved:

the self-sustained-release immunologic adjuvant suspension is a suspension formed by micron-sized particles of a fat-soluble immunologic adjuvant, the surface of the fat-soluble immunologic adjuvant is coated with a surfactant, and compared with hydrochloride of the immunologic adjuvant or other water-soluble immunologic adjuvant molecules (such as CpG, polyIC and the like), the self-sustained-release immunologic adjuvant suspension can be retained in tumors and slowly released after local injection, so that a self-sustained-release effect is formed, and the immunostimulation effect is stable and durable. Because clinical radiotherapy is mostly multi-time divided dose irradiation (such as 5 times of irradiation in a week), the injected immunostimulant needs to be retained and slowly released in the tumor for a long time, so that the induced immunogenic cell death can be effectively enhanced, and the anti-tumor immune response is induced.

The self-sustained-release immunologic adjuvant suspension overcomes the technical problems that the water solubility of the fat-soluble immunologic adjuvant is poor, and although the water solubility of the fat-soluble immunologic adjuvant hydrochloride is good, the fat-soluble immunologic adjuvant hydrochloride can be quickly diffused to other organs as small molecules when being locally injected into tumors and is quickly metabolized from the body. The fat-soluble immunologic adjuvant is made into micron-sized suspension, is a novel dosage form of the fat-soluble immunologic adjuvant, has a self-sustained release effect, increases the retention time of fat-soluble immunologic adjuvant microparticles in tumors, and slows down the release speed of immunologic adjuvant molecules, and the characteristic is very important for sensitizing external irradiation radiotherapy. In addition, since the micron-sized particle suspension needs to be subjected to standard autoclaving operation before being injected into a tumor to meet the requirement of sterility, the micron-sized particles need to be ensured not to be significantly agglomerated at about 121 ℃, a surfactant is required to have a sufficiently strong adsorption capacity with the particle surface, and mainly depends on hydrophobic interaction, so that the hydrophobic structure of the selected surfactant has an important effect on protecting the stability of the micron-sized suspension under autoclaving, and the hydrophobic structure part of the selected surfactant contains one or more hydrocarbon chains with a total number of not less than 15 carbon atoms or the hydrophobic structure part of the selected surfactant contains not less than 20 oxypropylene units.

The self-sustained-release immunoadjuvant suspension can further select the combination of two or more surfactants with different hydrophilic-lipophilic balance values (HLB values) as the coating layer of the micron particles. The two surfactants with different solubilities are not completely and homogeneously dispersed in a microscopic scale, but are locally and intensively dispersed, so that after the formed composite particle coating enters a tumor body, the surfactant with a high HLB value is firstly dissolved, and a plurality of tiny openings or tiny defect regions are formed on the surface of the coating of the micrometer particles, so that the surface area of the inner-layer fat-soluble immunologic adjuvant micrometer particles is gradually changed, effective components are gradually released, and more personalized medicament combination schemes (which accord with the actual conditions of different patients) are obtained for doctors to select by adjusting the selection or regulation and control proportioning relationship of the two or more surfactants according to the actual requirements of different tumor bodies and human bodies. And the combination of two or more surfactants with different hydrophilic-lipophilic balance values (HLB values) can further improve the stability of the micron particles in the process of autoclaving.

The invention also provides a novel preparation method of the self-sustained-release immunoadjuvant suspension, and research and development teams find that ceramic particles are generated in the ball milling process when the ball milling process is amplified, so that the injection risk is brought, and the impurities have little problem in the preparation of common micro-nano materials, but have greater risk when used for human body injection; the applicant research and development team carries out trial and error and improvement on a large number of experimental schemes in order to replace the technical scheme of obtaining imiquimod to be prepared into micron particles by using a ball milling method in the prior art, further provides a novel technical route of combining air flow grinding with high-pressure homogenization or air flow grinding with a high-shear method, and prepares micron-scale fat-soluble immunologic adjuvant micron particle suspension. The preparation method overcomes the technical prejudice in the preparation process of the micron particles and the actual technical problem in the technical improvement process, namely, the high-pressure homogenization process or the high-shear process is a liquid-phase processing method, while the fat-soluble immunologic adjuvant is a semi-solid medicament, and experiments show that if the fat-soluble immunologic adjuvant is directly subjected to the high-pressure homogenization or the high-shear process, the viscosity of the fat-soluble immunologic adjuvant is far higher than that of a solution or a common solid nano material, the blockage of a homogenizing valve can be caused, so that the micron particles can not be obtained; although micrometer particles can be partially obtained by directly adopting a high-shear method, the uniformity of the obtained particles is extremely poor, and most of the particles cannot achieve the expected granulation and pulverization effects and yield; in the invention, the primary powder is obtained by a pre-airflow crushing process, and the high-pressure homogenization or high-shear method is carried out under the condition of adding the aqueous solution of the surfactant, so that the high-pressure homogenization or high-shear micron particles can be subjected to rapid surface modification. Due to the existence of the surfactant, the fat-soluble immunologic adjuvant can be dispersed in a liquid phase in a discretization manner, so that primary powder of the fat-soluble immunologic adjuvant can be processed by a liquid-phase micro-nano process, and a fat-soluble immunologic adjuvant micron-sized particle suspension with good size uniformity can be obtained.

Compared with micro-nano particles obtained by various conventional processes, the self-sustained-release immunoadjuvant suspension can further adapt to more severe sterilization conditions, can withstand autoclaving treatment, still maintains the stability of the suspension and the stability of the particle size, and improves the production efficiency and the safety of the self-sustained-release immunoadjuvant suspension.

The self-sustained-release immunoadjuvant suspension is injected into the tumor, so that immunogenic cell death induced by radiotherapy, chemotherapy or thermotherapy can be effectively enhanced, anti-tumor immune response is induced, the embodied treatment effect can improve the curative effect of radiotherapy on in-situ tumor on one hand, and stronger far-end effect is obtained on the other hand, and the growth of tumor which is not irradiated at the far end is inhibited.

Drawings

FIG. 1 is a schematic diagram of the preparation of self-sustained release immunoadjuvant suspensions;

FIG. 2 is a graph comparing the tumor retention of different forms of self-sustained release immunoadjuvant suspensions over time after injection into tumors;

FIG. 3 is a graph comparing the time course of the drug concentration in blood of different forms of self-sustained-release immunoadjuvant suspensions injected into tumors;

FIG. 4 is a graph comparing the growth curves of tumors in situ following radiation therapy with imiquimod in different forms injected into the tumor;

FIG. 5 is a graph comparing the growth curves of distal tumors following radiation therapy with different forms of imiquimod injected into the tumors;

FIG. 6 is a graph comparing the change in body weight of mice receiving radiation therapy after injection of different forms of imiquimod into the tumor;

FIG. 7 is a graph showing the growth of in situ tumors in mice treated with microwave ablation after injection of imiquimod microparticles into the tumors;

FIG. 8 is a graph showing the growth of a tumor distal to a mouse treated with microwave ablation after injecting imiquimod microparticles into the tumor;

FIG. 9 is a graph of in situ tumor growth in mice treated with imiquimod microparticle potentiating tumor chemotherapy;

figure 10 is a graph of distal tumor growth in mice treated with imiquimod microparticle potentiating tumor chemotherapy.

Detailed Description

Example A: preparation of the formulations

Example a 1:

fig. 1 is a schematic diagram of preparation of self-sustained release immunoadjuvant suspension, prepared from sustained release imiquimod microparticles with reference to fig. 1, and prepared by the following method:

weighing a certain amount of fat-soluble immune adjuvant imiquimod R837 solid, and carrying out air flow crushing treatment under the crushing air pressure of 6-10bar to obtain micron-sized imiquimod R837 powder.

The weight ratio of 1: (0.025-5) weighing a micron-sized immune adjuvant imiquimod R837 and a surfactant poloxamer 188, preferably 2g R837, adding a proper amount of poloxamer 188(0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g and 10g), adding 100mL of water for injection, and stirring at 500rpm for 0.5-2 hours to obtain a suspension.

Homogenizing the suspension at 750-1200bar pressure for 2-4 times to obtain suspension, adding water for injection to constant volume until imiquimod concentration is 6.0mg/mL, and pumping the suspension by peristaltic pump to fill into 10mL ampoules, each containing 6mL, and 30 ampoules. And (3) carrying out heat-moisture sterilization at 105-150 ℃ for 15-20 minutes.

Poloxamer 188 is a new class of polymeric nonionic surfactants, and has multiple uses including: as an emulsifier, stabilizer and solubilizer, water dispersibility and stability of R837 can be further enhanced.

The hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included. In parallel, optionally, the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the oil-in-water emulsion concretely comprises at least one of sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000, or lecithin.

Poloxamers are a series of multipurpose pharmaceutical excipients, and are non-toxic, non-antigenic, non-allergenic, non-irritating, non-blood-soluble and chemically stable. Poloxamer 188 is one of the series of adjuvants with good safety. Poloxamer 188 can enable micro-scale powder obtained after airflow crushing of imiquimod to be processed by a liquid-phase micro-nano process to obtain imiquimod micro-scale particle suspension with good size uniformity, and poloxamer 188 can also help the imiquimod micro-scale particle suspension (6.0mg/mL or less) to ensure water dispersibility and stability after autoclaving.

However, while poloxamer 188-coated imiquimod microparticle suspensions maintain good suspension stability after autoclaving at lower concentrations (6.0mg/mL), if the imiquimod concentration is too high during sterilization, the imiquimod agglomerates after sterilization and can no longer stabilize the suspension. Lecithin is a natural surfactant, and imiquimod micro-particles which are subjected to high-pressure homogenization treatment by using lecithin as a stabilizer have good stability, and even if the imiquimod micro-particles are sterilized at high temperature under high concentration, suspension liquid of the imiquimod micro-particles can not agglomerate and can be kept to be stably suspended.

Table 1: preparation process and data of imiquimod/surfactant suspension;

a new technical route of combining air flow grinding with high-pressure homogenization or air flow grinding with a high-shear method is adopted to prepare micron-scale fat-soluble immunologic adjuvant micron particle suspension. The preparation method overcomes the technical prejudice and the actual technical problem in the preparation process of the micron particles, the high-pressure homogenization process or the high-shear process is a liquid-phase processing method, the fat-soluble immunologic adjuvant is a semi-solid medicament, and experiments show that if the high-pressure homogenization or the high-shear process is directly carried out on the fat-soluble immunologic adjuvant, the blockage of a homogenizing valve can be caused, so that the micron particles can not be obtained; although the high-shear method can partially obtain micron particles, the uniformity of the obtained particles is extremely poor, and most particles cannot achieve the expected granulation and pulverization effects and yield; according to the invention, the primary powder is obtained by an air flow crushing process, and then the high-pressure homogenization or high-shear method is carried out under the condition of adding a solution of a surfactant, so that the high-pressure homogenization or high-shear micron particles can be subjected to rapid surface modification and surface modification.

Table 2: adding the micro-particle powder of the imiquimod after airflow crushing into different surfactant aqueous solutions (the mass ratio of the imiquimod to the surfactant is 1:3), and then carrying out high-pressure homogenization treatment on the obtained mixture to obtain the water dispersible property of the imiquimod

Table 3: redispersibility of imiquimod suspension (6.0mg/mL) with different surfactants after autoclaving as described above (imiquimod: surfactant mass ratio 1:3)

Because the micron-sized particle suspension needs to be subjected to standard autoclaving operation before being injected into tumors to meet the requirement of sterility, the micron-sized particles need to be ensured not to be significantly agglomerated at about 121 ℃, a surfactant is required to have a sufficiently strong adsorption capacity with the particle surface, and mainly depends on hydrophobic interaction, the hydrophobic structure of the selected surfactant plays an important role in protecting the stability of the micron-sized suspension under autoclaving, and the hydrophobic structure part of the selected surfactant contains one or more hydrocarbon chains with the total number of not less than 15 carbon atoms or the hydrophobic structure part of the selected surfactant contains not less than 20 oxypropylene units. As in tables 2 and 3, poloxamer P124, due to insufficient hydrophobic structure, was unstable after autoclaving.

Table 4: suspension stability after autoclaving of different P188-dispersed imiquimod suspensions (R837 concentration 6.0mg/mL upon sterilization) added

Poloxamer 188R 837	Suspension stability after autoclaving
		0.5:1	A large amount of granular aggregates appear
1:1	Small amount of granular aggregates appeared
		2:1	Small amount of granular aggregates appeared
3:1	Uniformly dispersed without the occurrence of granular aggregates
		5:1	Uniformly dispersed without the appearance of granular aggregates

Although theoretically, the more dispersant the better the dispersion, the ratio is generally not more than 5:1, because: poloxamer 188(P188) is viscous, and has high viscosity when being excessively concentrated; and avoids excessive introduction of impurities into the dispersant.

Table 5: suspension stability of P188 dispersed imiquimod suspensions of different concentrations after autoclaving (P188: imiquimod R837 mass ratio 3: 1). The P188-coated imiquimod suspension maintained good stability when autoclaved at low R837 concentrations, but had significantly reduced autoclave stability at high R837 concentrations.

Concentration of R837 upon sterilization	Suspension stability after autoclaving
		3.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
		9.0mg/mL	The appearance of partially particulate aggregates
12.0mg/mL	A large amount of granular aggregates appear
		15.0mg/mL	A large amount of granular aggregates appear
18.0mg/mL	A large amount of granular aggregates appear

Table 6: suspension stability after autoclaving of imiquimod suspensions (R837 concentration: 6.0mg/mL or 18mg/mL at sterilization) dispersed with lecithin at different ratios was added. Lecithin can keep good suspension stability even at a lower proportion after high-concentration imiquimod suspension is autoclaved.

Lecithin R837	R837 concentration upon Sterilization	Suspension stability after autoclaving
			0.025:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.05:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.1:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.25:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.5:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
1:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.025:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.05:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.1:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.25:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.5:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
1:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates

Example a 2:

weighing a certain amount of solid of the liposoluble immunologic adjuvant Rasimethide (R848), and carrying out air flow crushing treatment under the crushing air pressure of 6-10bar to obtain the micron-sized Rasimethide (R848).

The proportion is 1: (0.025-5) weighing the micron-sized immune adjuvant Rasimethide (R848) and poloxamer 407 surfactant, preferably 0.2g R848, adding a proper amount of poloxamer 407(0.005g, 0.01g, 0.2g, 0.4g, 0.8g, 1g), adding 200mL of water for injection, and stirring at 100-500rpm for 0.5-2 hours to obtain a suspension.

Homogenizing the suspension at 750-1200bar pressure for 2-4 times to obtain suspension, and filling the suspension into 10mL ampoules (6 mL each) with a peristaltic pump for 30 bottles. And (3) carrying out heat sealing to obtain a micron suspension, and carrying out moist heat sterilization at 105-150 ℃ for 15-20 minutes.

Poloxamer 407 is a novel class of polymeric nonionic surfactants, having a variety of uses including: as an emulsifier, stabilizer and solubilizer, the water dispersibility and stability of R848 can be further enhanced.

Example a 3:

weighing a certain amount of liposoluble immunologic adjuvant glucopyranoside lipid A (MPLA); the selected surfactant is a mixed surfactant of poloxamer 188 and lecithin in a mass ratio of 9:1, and the other preparation methods are the same as those of the surfactant in the example A2.

Example a 4:

otherwise, as in example A1, a quantity of the lipid-soluble immunoadjuvant imiquimod (R837) is weighed out; the selected surfactant is a mixed surfactant of poloxamer 188 and lecithin in a mass ratio of 3: 1. The dosing concentrations of the different surfactants had a certain effect on the suspension stability after autoclaving of R837, the results are shown in table 7. Long term stability after R837 autoclaving the effect of P188 alone solubilizing R837 in the presence of lecithin, resulting in smaller particle size and better uniformity of the particles. And the influence of the feeding concentration can be enlarged in equal proportion, so that the technical effect of increasing the final concentration of R837 is achieved.

Table 7: suspension stability after R837 autoclaving with different concentrations of surfactant

R837 poloxamer 188 lecithin	Long term stability after autoclaving
		12mg/mL:36mg/mL:0mg/mL	A large amount of granular aggregates appear
12mg/mL:36mg/mL:12mg/mL	Uniformly dispersed without the appearance of granular aggregates
		18mg/mL:54mg/mL:0mg/mL	A large amount of granular aggregates appear
18mg/mL:54mg/mL:18mg/mL	Uniformly dispersed without the appearance of granular aggregates

Therefore, the mixing of the two surfactants can further increase the suspension stability performance of the self-sustained-release immunoadjuvant suspension in autoclaving, and the performance is particularly remarkable at higher surfactant concentration. Two or more surfactant combinations different in hydrophilic-lipophilic balance (HLB value) or two surfactants different in hydrophobic structure portion (for example, one surfactant containing not less than 20 oxypropylene units, or one surfactant containing one or more hydrocarbon chains not less than 15 carbon atoms in total) are used as the coating layer of the microparticles. The two surfactants with different solubilities are not completely and homogeneously dispersed with each other, but form a relatively uniform and locally gathered dispersion structure, after the formed coating composite particles enter tumor bodies, the surfactant with a larger HLB value is firstly dissolved, so that a plurality of tiny openings or tiny defect regions are formed on the surface of the coating of the microparticles, the surface area of the inner layer fat-soluble immunologic adjuvant microparticles is gradually changed, effective components are gradually released, and a medicament combination scheme with various models can be obtained by adjusting the selection or proportioning relation of the two or more surfactants according to the actual requirements of different tumor bodies and human bodies.

Table 8: variation in particle size after R837 autoclaving with addition of different proportions of surfactant

Meanwhile, as shown in table 8, the presence of both lecithin and P188 resulted in the minimal change in particle size before and after sterilization of R837 and a smaller particle size distribution range, i.e., the presence of both lecithin and P188 more contributed to the stability of the sample during the sterilization process. Wherein D50 is the corresponding particle size when the cumulative particle size distribution in the sample reaches 50%, D90 is the corresponding particle size when the cumulative particle size distribution in the sample reaches 90%, Dmax is the maximum particle size of the particles in the sample, and the smaller the difference between the three is, the higher the uniformity of the sample particles is. In the experiment, the suspension sample with the simultaneous existence of the P188 and the lecithin can not generate wall hanging after being stored for a long time. It is worth noting that microparticle size uniformity is an important parameter to ensure stable and reproducible drug release behavior in vivo.

Example a 5:

preparation of the first composition:

The proportion is 1: (0.025-5) weighing a micron-sized immune adjuvant imiquimod R837 and a surfactant poloxamer 188, preferably 2g R837, adding a proper amount of poloxamer 188(0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g and 10g), adding 100mL of water for injection, and stirring at 500rpm for 0.5-2 hours to obtain a suspension.

Homogenizing the suspension at 750-1200bar pressure for 2-4 times to obtain suspension, adding water for injection to constant volume until imiquimod concentration is 6.0mg/mL, and pumping the suspension by peristaltic pump to fill into 10mL ampoules, each containing 6mL, and 30 ampoules. And (3) carrying out heat sealing to obtain a micron suspension, and carrying out moist heat sterilization at 105-150 ℃ for 15-20 minutes.

Preparation of the second composition:

preparing a sodium alginate/mannitol or sodium alginate/lactose solution according to the proportion 1 (1-5), wherein the concentration of the sodium alginate solution is 10mg/mL, 20mg/mL or 40mg/mL, the final concentration of mannitol or lactose is 1-50 mg/mL, 20-100 mg/mL or 40-200 mg/mL, the mannitol or lactose is added after the sodium alginate solution is uniformly stirred, the sodium alginate solution is subpackaged in penicillin bottles, and after precooling, freeze-drying, filling nitrogen gas and sealing the bottles.

Before the experiment, the two compositions were mixed well, placed in dialysis bags (permeation molecular weight of 12000-14000Da) and then dialyzed in buffer solutions of different pH. In the control group, the release of imiquimod was monitored by directly placing the imiquimod suspension in dialysis bags (with a molecular weight of 12000-14000Da) and dialyzing the solution in buffers with different pH values. Wherein the buffer solution with pH of 7.4 is added with 2mM CaCl₂The pH of the phosphate buffer solution of (3) is 4.0, and the buffer solution is acetic acid-sodium acetate buffer solution.

The release rate of imiquimod from sodium alginate/calcium ion hydrogel (ALG) as a function of time is shown in table 9. Under the condition of acid, the imiquimod has a faster release speed, and under the condition of two pH values, the sodium alginate/calcium ion gel can obviously reduce the release rate of the imiquimod so as to achieve the effect of slow release.

Table 9, release data of imiquimod from sodium alginate/calcium ion hydrogel:

example B: animal experiments and comparisons of microparticles

Example B1:

the distribution behavior in vivo of the imiquimod formulation illustrated in this example is as follows: .

The experimental method comprises the following steps: mouse colon cancer (CT26) tumors were implanted on the backs of the mice, and the mice were randomly divided into 3 groups, and 3 mice per group were studied for drug distribution behavior.

A first group: mice were injected intratumorally with small molecule imiquimod hydrochloride at 6mg/kg

Second group: mice were injected intratumorally with poly (lactic-co-glycolic acid) (PLGA) encapsulated imiquimod nanoparticles (average particle size about 100nm) at an injection dose of 6 mg/kg;

third group: mice were injected intratumorally with imiquimod microgranules (this preparation) at 6 mg/kg; mice were sacrificed 72h after injection and the major organs and tumor bodies were dissected and the drug content in the organs and tumor tissues was examined.

The experimental results are as follows: from the content graph (fig. 2) of imiquimod in mouse major organs and tumor tissues, it can be seen that both small-molecule imiquimod hydrochloride and imiquimod/PLGA nano-preparations can not ensure most of the imiquimod hydrochloride to stay at the tumor site, wherein the retention of the small-molecule imiquimod hydrochloride after intratumoral injection for 72 hours is extremely low, and the imiquimod nano-preparations stay more in other organs. Compared with small-molecule imiquimod hydrochloride and imiquimod nano preparations, the invention has the advantages that the retention amount of the imiquimod micro preparation in tumor tissues is obviously improved, and the retention time of the imiquimod micro preparation in tumors is longest. The imiquimod micron preparation for intratumoral injection is more beneficial to subsequent treatment.

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 experiment was able to achieve superior therapeutic results, and in addition to the explanations in the other parts of the patent, it may also include the following reasons, namely the use of imiquimod R837 micron particles. The water-insoluble R837 powder is prepared into micron particles with the particle size of 1-3 microns, pharmacokinetics and intratumoral retention time are monitored after intratumoral injection, and the results are shown in table 8, and the results show that the micron particles can obviously prolong the retention time and blood circulation half-life of imiquimod at a tumor part, so that the slow release effect is achieved, and the immune system is stimulated for a long time.

Table 10: comparing the residence time of imiquimod in different dosage forms;

example B2:

the pharmacokinetics in vivo of the imiquimod formulation illustrated in this example are as follows: .

The experimental method comprises the following steps: mouse colon cancer (CT26) tumors were implanted on the backs of the mice and the mice were randomized into 3 groups of 3 drugs each for pharmacokinetic studies.

A first group: intratumoral injection of small molecule imiquimod hydrochloride in mice; the injection dosage is 6mg/kg, blood is collected intravenously at 5h, 6h, 12h, 24h, 48h and 72h after injection, and the concentration of imiquimod is determined uniformly to detect the concentration of imiquimod in blood.

Second group: mice are injected with imiquimod/PLGA nano-particles (the average particle size is about 100nm) intratumorally, the injection dose is 6mg/kg, blood is collected intravenously at 5h, 6h, 12h, 24h, 48h and 72h after injection, and the concentration of the imiquimod is determined uniformly to detect the concentration of the imiquimod in blood.

Third group: mice are injected with imiquimod micron particles (the preparation) intratumorally, the injection dose is 6mg/kg, blood is collected intravenously at 5h, 6h, 12h, 24h, 48h and 72h after injection, and the concentration of imiquimod is determined uniformly to detect the concentration of imiquimod in blood.

The experimental results are as follows: from the time-dependent blood drug concentration curve (fig. 3) and the blood circulation statistical analysis table (table), it can be seen that the small-molecule imiquimod hydrochloride is cleared quickly, the blood drug concentrations of 48h and 72h are lower than the detection limit, and the blood circulation half-life period and the drug mean residence time of the micrometer preparation are longer than those of the imiquimod nanometer preparation.

Example B3:

the specific effects of this example for enhancing radiation therapy are as follows: .

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

A first group: mouse tumor is not treated, and injection of reagent and radiotherapy treatment are not carried out;

second group: carrying out simple radiotherapy treatment on the mouse in-situ tumor, carrying out radiotherapy on the mouse in-situ tumor for 5 consecutive days 1 time per day, and carrying out no treatment on the far-end tumor;

third group: carrying out intratumoral injection of small-molecule imiquimod hydrochloride on the mouse in-situ tumor, wherein the dose is 6 mg/kg; 1.5Gy of radiotherapy is carried out on days 0, 1, 2, 3 and 4 after administration, and the left tumor of the mouse is not treated;

and a fourth group: injecting PLGA nano-particles (with the particle size of about 100nm) of imiquimod into the tumor of the mouse in situ, wherein the dosage is 6 mg/kg; radiotherapy of 1.5Gy is carried out every time on 0, 1, 2, 3 and 4 days after administration, and the tumor at the far end of the mouse is not treated;

and a fifth group: mice were injected intratumorally with imiquimod microgranules (this preparation) at a dose of 6 mg/kg; radiotherapy of 1.5Gy is carried out every time on 0, 1, 2, 3 and 4 days after administration, and the tumor at the far end of the mouse is not treated;

a sixth group: mice were injected intratumorally with imiquimod microgranules (this preparation) at a dose of 12 mg/kg; 1.5Gy radiotherapy is carried out every time on 0, 1, 2, 3 and 4 days after administration, and the tumor at the far end of the mouse is not treated;

the length and width of the in situ and distal tumors were measured every two days with a vernier caliper, and the volume of the tumor was (length multiplied by (width squared)) divided by 2.

Fig. 4 is a graph comparing growth curves of in-situ tumors after injecting imiquimod hydrochloride, nano-particles and micro-particles into tumors and performing radiotherapy, and fig. 5 is a graph comparing growth curves of far-end tumors after injecting imiquimod hydrochloride, nano-particles and micro-particles into tumors and performing radiotherapy.

The treatment effect is as follows: as can be seen from the in situ tumor growth curve (FIG. 4) and the distal tumor growth curve (FIG. 5), both the in situ tumor and the distal tumor of the mice in the sixth group are effectively inhibited, and hardly grow any more, so that the method has 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. The body weight change curves of all mice in all groups are shown in figure 6 and are in a normal range, and the body weights of the mice in the experimental group and the mice in the control group are not different, so that the preparation has certain safety.

Radiotherapy can induce a distal effect although it has been reported, this effect is not very significant. In experiments, the immune adjuvant is injected into the tumor, and then the tumor is irradiated by rays, so that the immunogenic cell death induced by radiotherapy can be effectively enhanced; the specific effect is that on one hand, the curative effect of radiotherapy on in-situ tumor can be improved, on the other hand, stronger far-end effect is obtained, and the growth of far-end tumor which is not irradiated is inhibited.

Example B4:

the effect of the present embodiment for the microwave ablation combined treatment of tumors is as follows:

the experimental method comprises the following steps: the colon cancer tumors of the mice (the right side is regarded as the in-situ tumor, and the left side is regarded as the far-end tumor) are respectively planted at the left and the right ends of the back of the mice, the tumor-bearing mice are divided into 3 groups, and 5 mice in each group are subjected to microwave treatment and immunotherapy combined treatment experiments.

A first group: mouse tumor is not treated, and injection of reagent and microwave treatment are not carried out;

second group: carrying out simple microwave thermal ablation treatment on the mouse in-situ tumor, wherein the microwave power is 7W, the local temperature of the tumor reaches 53 ℃, and the tumor on the left side is not treated;

third group: injecting imiquimod micron preparation into the tumor on the right side of the mouse, wherein the dosage is 6 mg/kg; after administration, performing microwave thermal ablation treatment, wherein the microwave power is 7W, the local temperature of the tumor reaches 53 ℃, and no treatment is performed on the tumor on the left side of the mouse;

The treatment effect is as follows: from the in situ tumor growth curve (fig. 7) and the distal tumor growth curve (fig. 8), it can be seen that bilateral tumors of the third group of mice were well suppressed, while only the in situ tumor was destroyed and the distal tumor was still growing in the mice that were treated with microwave thermal ablation alone, indicating that the present invention has significant gain in distal effect on microwave thermal ablation.

Example B5:

the effect of the combination therapy of the present example for tumor chemotherapy and immunotherapy is as follows:

the experimental method comprises the following steps: mouse colon cancer CT26 tumors (the right side is regarded as an in-situ tumor, and the left side is regarded as a far-end tumor) are respectively planted at the left and right ends of the back of the mouse, the tumor-bearing mice are divided into 3 groups, and 5 mice in each group are subjected to chemotherapy and immunotherapy combined treatment experiments.

A first group: normal saline is injected into the tumor of the mouse in situ, and the tumor at the far end is not treated;

second group: injecting oxaliplatin chemotherapeutic drug into the mice in situ tumor, and treating the distal tumor without any treatment;

third group: injecting a mixture of oxaliplatin chemotherapeutic drug and imiquimod micron preparation into the mice in situ tumor, and treating the distal tumor without any treatment;

the length and width of the mice neutral were measured periodically and the tumor volume was (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 (fig. 9) and the distal tumor growth curve (fig. 10) that the use of the chemotherapeutic drug can inhibit the growth of the in situ tumor, but the growth inhibition of the distal tumor is not obvious, and the imiquimod micron preparation can effectively inhibit the growth of the distal tumor after being added, which indicates that the imiquimod micron preparation can effectively enhance the distal effect of the chemotherapy.

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 self-sustained release immunoadjuvant suspension characterized by: the emulsion is characterized by comprising a fat-soluble immunologic adjuvant and a surfactant, wherein the balance is a dispersant, the surfactant coats the fat-soluble immunologic adjuvant to form micron-sized particles, and the micron-sized particles are dispersed in the dispersant to form a suspension.

2. Self-sustained release immunoadjuvant suspension according to claim 1, characterized in that: the fat-soluble immunoadjuvant comprises at least one of imiquimod (R837), ranitidine (R848), or glucopyranoside lipid a (mpla).

3. Self-sustained release immunoadjuvant suspension according to claim 1, characterized in that: the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included.

4. Self-sustained release immunoadjuvant suspension according to claim 1, characterized in that: the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the compound is characterized by specifically comprising sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000 and lecithin.

5. Self-sustained release immunoadjuvant suspension according to claim 1, characterized in that: the surfactant is a mixture of two surfactants with different hydrophilic-lipophilic balance values.

6. Self-sustained release immunoadjuvant suspension according to claim 1, characterized in that: the self-sustained release fat-soluble immunologic adjuvant nano-particles are core-shell composite particles with the particle size of 0.5-5 microns, and preferably, the self-sustained release fat-soluble immunologic adjuvant core-shell composite particles have the particle size of 1-2 microns.

7. A preparation method of a self-sustained-release immunoadjuvant suspension is characterized by comprising the following steps:

s1: the fat-soluble immunologic adjuvant is subjected to airflow pulverization to form primary micron-sized powder;

s2: adding the fat-soluble immune adjuvant into the primary micron-sized powder of the fat-soluble immune adjuvant obtained in the step S1 according to the weight ratio of the fat-soluble immune adjuvant: adding a surfactant aqueous solution according to a surfactant mass ratio of (1: 0.025-5), carrying out high-pressure homogenization treatment, and taking out homogenate after the treatment is finished;

or S2': adding the fat-soluble immunologic adjuvant into the fat-soluble immunologic adjuvant micron powder obtained in the step S1 according to the weight ratio of the fat-soluble immunologic adjuvant: adding a surfactant aqueous solution according to a surfactant mass ratio of (1: 0.025-5), carrying out high-shear process treatment, and taking out homogenate after the treatment is finished;

s3: and (5) carrying out high-pressure sterilization treatment.

8. The method according to claim 7, wherein the surfactant in step S2 comprises two surfactants having different solubilities.

9. The method of claim 7, wherein the sterilization treatment is carried out under high pressure, preferably at 105-150 ℃ for 10-20 minutes.

10. Use of a self-sustained release immunoadjuvant suspension obtained by the method according to any one of claims 7 to 9 for the manufacture of a medicament for the adjuvant treatment of tumors.

11. A self-sustained release immunoadjuvant composition, comprising a first composition and a second composition; the first composition consists of a fat-soluble immunologic adjuvant and a surfactant, and the balance is a dispersant, wherein the surfactant coats the fat-soluble immunologic adjuvant to form micron-sized particles, and the micron-sized particles are dispersed in the dispersant to form a suspension; the second composition comprises soluble alginate, a protective filler and a pH regulator to form freeze-dried powder.

12. Self-sustained release immunoadjuvant suspension according to claim 11, characterized in that: the fat-soluble immunoadjuvant comprises at least one of imiquimod (R837), ranitidine (R848), or glucopyranoside lipid a (mpla).

13. Self-sustained release immunoadjuvant suspension according to claim 11, characterized in that: the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included.

14. Self-sustained release immunoadjuvant suspension according to claim 11, characterized in that: the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the compound is characterized by specifically comprising sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000 and lecithin.

15. Self-sustained release immunoadjuvant suspension according to claim 11, characterized in that: the surfactant is a mixture of two surfactants with different hydrophilic-lipophilic balance values.

16. Use of a self-sustained release immunoadjuvant suspension according to any one of claims 1 to 6 or claims 11 to 15, for the preparation of a radiosensitizer.

17. Use of a self-sustained release immunoadjuvant suspension according to any one of claims 1 to 6 or claims 11 to 15, for the preparation of a chemosensitizer.

18. Use of a self-sustained release immunoadjuvant suspension according to any one of claims 1 to 6 or claims 11 to 15, for the preparation of a hyperthermia sensitizer.