WO2022143894A1 - 一种自缓释免疫佐剂混悬液及其制备方法和应用 - Google Patents

一种自缓释免疫佐剂混悬液及其制备方法和应用 Download PDF

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WO2022143894A1
WO2022143894A1 PCT/CN2021/143057 CN2021143057W WO2022143894A1 WO 2022143894 A1 WO2022143894 A1 WO 2022143894A1 CN 2021143057 W CN2021143057 W CN 2021143057W WO 2022143894 A1 WO2022143894 A1 WO 2022143894A1
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immune adjuvant
suspension
self
imiquimod
surfactant
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PCT/CN2021/143057
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English (en)
French (fr)
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刘庄
陶惠泉
邓中清
吴宇辰
柏上
周炫坊
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苏州百迈生物医药有限公司
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Priority claimed from CN202011612051.1A external-priority patent/CN114681401A/zh
Priority claimed from CN202111307908.3A external-priority patent/CN114010592B/zh
Application filed by 苏州百迈生物医药有限公司 filed Critical 苏州百迈生物医药有限公司
Priority to US18/265,987 priority Critical patent/US20240050560A1/en
Priority to CN202180088558.8A priority patent/CN116710073A/zh
Publication of WO2022143894A1 publication Critical patent/WO2022143894A1/zh

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Definitions

  • the present application relates to the field of biomedicine, in particular to a self-sustained-release immune adjuvant suspension and a preparation method and application thereof.
  • Radiotherapy is a ray-based external beam radiotherapy that is widely used in clinical practice, but it is a local treatment plan that can only irradiate local tumors, and distant metastases cannot be effectively irradiated (such as remote hidden tumors). tumor).
  • External beam radiation therapy which is widely used in clinical practice, uses rays (such as X-rays) to irradiate the tumor site locally, so as to achieve the goal of killing tumor cells.
  • rays such as X-rays
  • distal effect In the clinical application of radiotherapy, it is found that for a small number of patients, there may be a "distant effect", that is, local treatment of tumors sometimes inhibits the growth of distant unirradiated tumors.
  • This radiotherapy-induced “distal effect” has aroused great interest among researchers in recent years. Studies have shown that the mechanism of the "distal effect” lies in inducing immunogenic cell death of tumor cells, exposing tumor-associated antigens, thereby activating the immune response against the tumor, and further infiltrating distant tumors through tumor-specific CD8+ T cells. to achieve immunosuppression of distant tumors.
  • the induced “distal effect” has important clinical value, the individual differences of this effect are very large, and the "distal effect” induced by radiotherapy is not very significant for most patients in the clinic.
  • the important reason is that the tumor-associated antigens in the "corpses" of tumor cells generated after the induction of immunogenic cell death of tumor cells are not very immunogenic, so they cannot be used as effective "tumor vaccines” and are difficult to activate most of the time. Sufficiently effective antitumor immune response.
  • an effective immune response requires sufficient tumor antigen exposure and antigen presentation by immune adjuvants, wherein the role of immune adjuvants is to amplify the immune response generated by tumor antigens by an order of magnitude by stimulating immune cells. Therefore, if an immune adjuvant can be injected locally into the tumor during tumor treatment, and then the tumor is treated, it is expected to significantly amplify the immunogenicity of tumor-associated antigens generated after radiotherapy through the immunostimulatory effect of the adjuvant.
  • Imiquimod which has been clinically approved for use, is a typical lipid-soluble immune adjuvant. This small molecule immunomodulatory imidazoquelinamide is not a cytotoxic drug and has no significant direct killing of viruses or tumor cells. effect.
  • Imiquimod a ligand for Toll-like receptor 7 (TLR7), can stimulate macrophages, monocytes, dendritic cells, induce interferon alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) ) and stimulate the production of interleukin-2 (IL-2), IL-6, IL-8 and other cytokines, thereby further stimulating the activation of cellular immunity, recognizing viruses or other tumor antigens, stimulating relevant immune responses, and eliminating pathogenic disease factor.
  • TLR7 Toll-like receptor 7
  • the mature dosage form of imiquimod at this stage is a cream preparation, which is often applied to epidermal lesions by smearing. It is used to treat diseases caused by local viral infections such as condyloma acuminatum, and it is also used in clinical trials for superficial skin tumors. Attempts to treat. Imiquimod is currently approved for the treatment of actinic keratosis of the head and neck and superficial basal cell carcinoma. In addition, a number of clinical trials have confirmed that imiquimod plays an immune adjuvant effect in the treatment of squamous cell carcinoma, metastatic melanoma, vulvar intraepithelial neoplasia and other superficial tumors, and has certain application potential.
  • imiquimod itself is a small fat-soluble molecule and is hardly soluble in water. At the same time, imiquimod has strong skin irritation.
  • imiquimod cream By applying 5% imiquimod cream to the bare skin of mice, the The establishment of a mouse model of psoriasis-like skin lesions is sufficient to illustrate the irritation of imiquimod to normal tissues.
  • External administration has advantages and disadvantages. Although it has a good immune-enhancing effect on the immunotherapy of individual superficial lesions, it also limits the immunotherapy application of imiquimod in other tumors.
  • imiquimod-containing injections there are two main methods for preparing imiquimod-containing injections.
  • One is to directly dissolve imiquimod with acid, for example, imiquimod is dissolved in hydrochloric acid to form a hydrochloride salt, which is dispersed in the aqueous phase.
  • the pH value of the solution obtained by this method is relatively low, generally around 3.0-4.0, and the solution at this pH value is used for living organisms and has certain irritation.
  • imiquimod hydrochloride will rapidly exude from the tumor and enter the blood after being injected into the tumor, resulting in a high acute exposure in the blood after injection (bringing safety risks). ), at the same time, the half-life of imiquimod hydrochloride in tumors is very short, and it will be quickly cleared, so its immune activation effect after intratumoral administration cannot be maintained for a long enough time.
  • Another way to prepare imiquimod injection is to use amphiphilic polymers or other nanostructures capable of loading hydrophobic drugs to load R837.
  • the preparation process of these nanoparticles is often complicated, which is not conducive to process scale-up and standardized mass production.
  • these nanoparticle preparations are often difficult to exist stably under the conditions of terminal high temperature and high pressure sterilization (according to the "Guidelines for Research and Validation of Sterilization and Sterilization Processes for Chemical Injections", terminal high temperature and high pressure sterilization is the preferred sterilization strategy for injections) .
  • lipid-soluble immune adjuvants As immune adjuvants for the immunotherapy of non-superficial tumors.
  • the preparation should achieve long-term retention and sustained release of the adjuvant in the tumor, reduce its exposure in blood and normal tissues to ensure the safety of its clinical use; in addition, in order to meet the needs of industrial transformation, the preparation method of the preparation It needs to be scaled up, and the stability of the preparation needs to meet the requirements of terminal high temperature and autoclave sterilization.
  • the application provides a self-sustained-release immune adjuvant suspension, which is composed of a fat-soluble immune adjuvant and a surfactant, and the balance is a dispersion medium, and the surfactant coats the fat-soluble immune adjuvant to form The micron-sized particles are dispersed in the dispersion medium to form a suspension.
  • the lipid-soluble immune adjuvant comprises at least one of imiquimod (R837), resiquimod (R848), or glucopyranoside lipid A (MPLA).
  • the lipid-soluble immune adjuvant particles are core-shell composite particles with a particle size of 0.5-5 microns.
  • the particle size of the lipid-soluble immune adjuvant core-shell composite microparticles is 1-2 microns.
  • the lipid-soluble immune adjuvant is imiquimod microparticles.
  • the imiquimod microparticles have an average particle size of 0.5-5 microns.
  • the surfactant is a higher fatty acid chain-containing surfactant.
  • the higher fatty acid chain-containing surfactant includes an anionic surfactant.
  • the anionic surfactants include sodium oleate, sodium lauryl sulfate, sodium stearate, sodium N-lauroyl sarcosinate, sodium cocoyl methyl taurate, At least one of sodium N-lauroyl glutamate, sodium laureth carboxylate, and lauryl phosphate.
  • the higher fatty acid chain-containing surfactant includes an amphiphilic ionic surfactant.
  • the higher fatty acid chain-containing surfactant includes a phospholipid ionic surfactant.
  • the phospholipid ionic surfactant includes at least one of lecithin, soybean phospholipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.
  • the hydrophobic moiety of the surfactant contains no less than 20 oxypropylene units.
  • the surfactant comprises at least one of Poloxamer 188, Poloxamer 237, Poloxamer 338, and Poloxamer 407.
  • the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains totaling not less than 15 carbon atoms.
  • the surfactant comprises sorbitan sesquioleate, soybean lecithin, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, Polysorbate 85, Sorbitan Stearyl (Span 60), Stearate, Vitamin E Polyethylene Succinate, Polyoxyethylene Alkyl Ether, Polyoxyethylene Stearate, Polyhydrocarbon Stearate At least one of oxy(40) ester, sucrose stearate, polyoxyethylene castor oil derivative, polycitorol 1000, and lecithin.
  • the surfactant is a mixture of two surfactants with different hydrophilic-lipophilic balances.
  • the self-sustained release immune adjuvant suspension comprises an imiquimod suspension formulation comprising: imiquimod microparticles, higher fatty acid chains-containing Surfactant and dispersion medium.
  • the concentration of the imiquimod microparticles is 1-18 mg/mL, the surfactant containing higher fatty acid chains and imiquimod
  • the mass ratio of ultra-micron particles is 0.025 to 3:1.
  • the mass ratio of the surfactant containing higher fatty acid chains to the imiquimod microparticles is 0.1-1:1.
  • the present application provides a method for preparing a self-sustained-release immune adjuvant suspension, which includes the following steps: S1: dispersing a surfactant and a fat-soluble immune adjuvant in the same dispersion system, and stirring to obtain a mixed solution. Suspension; S2: Perform homogenization treatment/high shear process treatment on the suspension prepared above; S3: Sterilize the suspension after homogenization/high shear process treatment.
  • the preparation method includes: S1: forming a fat-soluble immune adjuvant into a primary micron-sized powder through an air-jet pulverization process; S2: adding the lipid-soluble immune adjuvant to the primary micron-sized powder obtained in step S1 Add the aqueous solution of surfactant according to the lipid-soluble immune adjuvant:surfactant mass ratio (1:0.025 ⁇ 5), carry out high-pressure homogenization process treatment, and take out the homogenate after the treatment; or S2': to the lipid obtained in step S1
  • the soluble immune adjuvant micron powder is added with an aqueous solution of surfactant according to the mass ratio of fat-soluble immune adjuvant:surfactant (1:0.025-5), and subjected to high shear process treatment, and the homogenate is taken out after the treatment; S3: Autoclave treatment.
  • the surfactant described in step S2 of the preparation method includes two surfactants with different solubility.
  • the conditions of the autoclaving treatment of the preparation method are treatment at 105°C to 150°C for 10-20 minutes.
  • the present application also provides a method for preparing an imiquimod suspension preparation, comprising the steps of: S1: dispersing the surfactant containing higher fatty acid chains and the imiquimod microparticles in the same dispersion In the system, stirring to obtain a suspension; S2: Homogenizing the suspension prepared above; S3: Filling the homogenized suspension, sealing and sterilizing at high temperature and high pressure.
  • the conditions of the high temperature and autoclave sterilization are: 110-145° C., 5-30 min.
  • the present application also provides the application of the self-sustained-release immune adjuvant suspension obtained by the preparation method described in the present application in the preparation of a drug for adjuvant therapy of tumors.
  • the present application also provides a self-sustained-release immune adjuvant composition, comprising a first composition and a second composition;
  • the first composition is composed of a fat-soluble immune adjuvant and a surfactant,
  • the balance is a dispersant, and the surfactant coats the fat-soluble immune adjuvant to form micron-sized particles, and is dispersed in the dispersant to form a suspension;
  • the second composition includes readily soluble alginate and protective filler to form a lyophilized powder.
  • the lipid-soluble immune adjuvant comprises imiquimod (R837), resiquimod (R848) or a glucopyranoside lipid At least one of A(MPLA).
  • the hydrophobic moiety of the surfactant contains no less than 20 oxypropylene units.
  • the surfactant comprises Poloxamer 188, Poloxamer 237, Poloxamer 338 and Poloxamer 407 at least one.
  • the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms.
  • the surfactant comprises sorbitan sesquioleate, soybean lecithin, glycerol monostearate, polysorbate 40, polysorbate Ester 60, Polysorbate 65, Polysorbate 80, Polysorbate 85, Sorbitan Stearyl (Span 60), Stearate, Vitamin E Polysuccinate, Polyoxyethylene Alkyl Ether, At least one of polyoxyethylene stearate, polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivative, polycitorol 1000 and lecithin.
  • the surfactant in the self-sustained release immune adjuvant composition, is a mixture of two surfactants with different hydrophilic-lipophilic balance values.
  • the dispersing agent in the self-sustained release immune adjuvant composition, is water or physiological saline.
  • the protective filler in the self-sustained release immune adjuvant composition, is mannitol or lactose.
  • the second composition further includes a pH adjusting agent.
  • the present application also provides the application of the self-sustained-release immune adjuvant suspension or the self-sustained-release immune adjuvant composition in the preparation of an anti-tumor combined immunotherapy preparation.
  • the self-sustained release immune adjuvant comprises an imiquimod suspension formulation.
  • the imiquimod suspension formulation is premixed with the platinum-based chemical to assist in the sustained release of the platinum-based chemical.
  • the imiquimod suspension formulation is premixed with the anthracycline to facilitate sustained release of the anthracycline.
  • the present application also provides the use of the self-sustained-release immune adjuvant suspension or the self-sustained-release immune adjuvant composition in the preparation of a radiosensitizer.
  • the present application also provides the use of the self-sustained-release immune adjuvant suspension or the self-sustained-release immune adjuvant composition in the preparation of a chemosensitizer.
  • the present application also provides the use of the self-sustained-release immune adjuvant suspension or the self-sustained-release immune adjuvant composition in the preparation of a hyperthermia sensitizer.
  • the present application also provides the application of the self-sustained-release immune adjuvant suspension or the self-sustained-release immune adjuvant composition in the preparation of an alcohol ablation sensitizer.
  • Fig. 1 is the schematic diagram of preparation of self-sustained release immune adjuvant suspension
  • Fig. 2 is the photo after adding the micron-scale imiquimod suspension sterilization of different surfactants containing higher fatty acid chains after shaking;
  • Figure 3 is a comparison chart of the changes in tumor retention over time after the self-sustained-release immune adjuvant suspensions of different forms were injected into the tumor;
  • Figure 4 is a comparison chart of the changes of drug concentration in the blood over time after different forms of self-sustained-release immune adjuvant suspensions are injected into the tumor;
  • Figure 5 is a comparison chart of the growth curves of in situ tumors after injection of different forms of imiquimod into tumors and radiotherapy;
  • Figure 6 is a comparison chart of the growth curves of distal tumors after imiquimod of different forms is injected into the tumor and then radiotherapy;
  • Figure 7 is a comparison chart of the body weight changes of mice that received radiotherapy after imiquimod of different forms was injected into the tumor;
  • Figure 8 is a graph showing the growth curve of orthotopic tumors in mice after microwave ablation treatment of imiquimod microparticles was injected into the tumor;
  • Figure 9 is a graph showing the growth curve of a distal tumor in a mouse after microwave ablation was performed after imiquimod microparticles were injected into the tumor;
  • Figure 10 is a graph showing the growth curve of orthotopic tumor in mice treated with imiquimod microparticles for enhanced tumor chemotherapy
  • Figure 11 is a graph showing the growth curve of distal tumors in mice treated with imiquimod microparticles for enhanced tumor chemotherapy
  • Figure 12 is the in situ tumor growth curve of different groups of mice in the experiment of micron-sized imiquimod suspension combined with radiotherapy;
  • Figure 13 is the distal tumor growth curve of different groups of mice in the experiment of micron-sized imiquimod suspension combined with radiotherapy;
  • Figure 14 is the tumor growth curve of different groups of mice in the experiment of micron-sized imiquimod suspension combined with alcohol ablation therapy.
  • Figure 15 is a statistical graph of the relative content of platinum in major tissues and organs before and after mixing oxaliplatin and micron-sized imiquimod suspension, and 72 hours after intratumoral injection;
  • Figure 16 is the curve of the drug concentration in blood over time before and after mixing oxaliplatin and micron-sized imiquimod suspension, after intratumoral injection;
  • Figure 17 is a mouse bilateral tumor model, the tumor growth curve of in situ tumors, the groups are the vehicle control group, the oxaliplatin monotherapy group, the imiquimod suspension preparation treatment group, and the oxaliplatin treatment group. Combined treatment group with imiquimod suspension preparation;
  • Figure 18 is a mouse bilateral tumor model, the tumor growth curve of the distal tumor, the groups are the vehicle control group, the oxaliplatin monotherapy group, the imiquimod suspension preparation treatment group, and the oxaliplatin treatment group. Combined treatment group with imiquimod suspension preparation.
  • Figure 19 is an in vitro release profile of doxorubicin after being mixed with a micron-sized imiquimod suspension formulation
  • Figure 20 is an in vitro release profile of doxorubicin after being mixed with micron-scale imiquimod suspension formulations of different concentrations;
  • Figure 21 is an in vitro release profile of epirubicin after being mixed with a micron-sized imiquimod suspension formulation
  • Figure 22 is a graph showing the in vitro release curve of epirubicin mixed with a micron-sized imiquimod suspension obtained by suspending with phosphatidylglycerol.
  • the present invention provides a self-sustained release immune adjuvant suspension, which can achieve good in situ dispersion effect, and can realize self-sustained release to assist chemotherapy, radiotherapy or hyperthermia to generate immune memory and activate human immune characteristics.
  • a new dosage form of immune adjuvant, an anticancer drug composition that reduces the probability of cancer metastasis and recurrence, can effectively kill the in situ tumor and at the same time inhibit the immune response and reduce the growth of distant metastatic tumors and the probability of tumor recurrence.
  • the present invention provides the following solutions:
  • a self-sustained-release immune adjuvant suspension consisting of a fat-soluble immune adjuvant and a surfactant, the balance being a dispersant, and the surfactant coats the fat-soluble immune adjuvant to form micron-sized particles, and Disperse in the dispersant to form a suspension.
  • the dispersing agent is water or physiological saline.
  • the fat-soluble immune adjuvant includes at least one of imiquimod (R837), resiquimod (R848) or glucopyranoside lipid A (MPLA).
  • imiquimod R837)
  • resiquimod R848
  • MPLA glucopyranoside lipid A
  • hydrophobic structure part of the surfactant contains no less than 20 oxypropylene units; specifically including Poloxamer 188 (P188), Poloxamer 237, Poloxamer 338, Poloxamer UM 407.
  • the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms; specifically including sorbitan sesquioleate, soybean lecithin, glyceryl monostearate , polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearyl (Span 60), stearate, vitamin E polyethylene succinate , polyoxyethylene alkyl ethers, polyoxyethylene stearate, polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, polycidol 1000, or lecithin at least one of them.
  • hydrocarbon chains with a total of not less than 15 carbon atoms
  • the self-sustained-release immune adjuvant suspension is composite particles with a particle size of 0.5-5 microns, and the surfactant coats the fat-soluble immune adjuvant.
  • the particle size of the self-sustained-release immune adjuvant suspension is 1-2 microns.
  • the surfactant may be a mixture of two surfactants with different hydrophilic-lipophilic balance values (HLB values).
  • HLB values hydrophilic-lipophilic balance values
  • Two surfactants with different hydrophilic and lipophilic balance values can dissolve the surfactant with the larger HLB value first after the composite particles enter the tumor, thereby coating the surface of the lipid-soluble immune adjuvant microparticles to form some openings or Tiny defect areas, so that the surface area of the inner layer of imiquimod micro-particles gradually changes, the active ingredients are gradually released, and more personalized formulations can be prepared according to the actual needs of different tumors and the human body through the ratio of surfactants. drug regimen.
  • the invention provides a preparation method of self-sustained release immune adjuvant suspension, which is characterized by comprising the following steps:
  • the fat-soluble immune adjuvant is formed into a primary powder by an air-jet pulverization process
  • step S2 adding an aqueous solution of surfactant to the primary powder obtained in step S1, performing high-pressure homogenization process treatment, and taking out the homogenate after the treatment;
  • step S2' add the aqueous solution of surfactant to the primary powder obtained in step S1, carry out high shear process treatment, and take out the homogenate after the treatment;
  • aqueous solution of the surfactant described in the step S1 contains two surfactants with different hydrophilic and lipophilic balance values.
  • the concentration of the aqueous solution of the surfactant in the step S1 is 6-30 mg/mL.
  • the sterilization treatment in the step S3 is a moist heat treatment at 105°C to 150°C for 10-15 minutes.
  • the present invention also provides a self-sustained release immune adjuvant composition, comprising a first composition and a second composition;
  • the first composition is composed of a fat-soluble immune adjuvant and a surfactant, and the balance is a dispersant,
  • the surfactant coats the fat-soluble immune adjuvant to form micron-sized particles, and is dispersed in the dispersant to form a suspension;
  • the second composition comprises a readily soluble alginate and a protective filler to form lyophilized powder.
  • the second composition can further optimize the sustained release properties of the first composition.
  • the dispersing agent is water or physiological saline.
  • the fat-soluble immune adjuvant includes at least one of imiquimod (R837), resiquimod (R848) or glucopyranoside lipid A (MPLA).
  • imiquimod R837)
  • resiquimod R848
  • MPLA glucopyranoside lipid A
  • the hydrophobic structure part of the surfactant contains no less than 20 oxypropylene units, including Poloxamer 188, Poloxamer 237, Poloxamer 338, Poloxamer 407; or One or more hydrocarbon chains with a total of not less than 15 carbon atoms, including sorbitan sesquioleate, soybean lecithin, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65, Polysorbate 80, Polysorbate 85, Sorbitan Stearyl (Span 60), Stearate, Vitamin E Polysuccinate, Polyoxyethylene Alkyl Ether, Polyoxyethylene Stearate, At least one of polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivative, polycitorol 1000 or lecithin.
  • Poloxamer 188 Poloxamer 237
  • Poloxamer 338 Poloxamer 407
  • One or more hydrocarbon chains with a total of not less than 15 carbon atoms including
  • the present invention also provides the application of the self-sustained-release immune adjuvant suspension in the preparation of a radiosensitizer.
  • the present invention also provides the application of the self-sustained release immune adjuvant suspension in the preparation of chemosensitizers.
  • the present invention also provides the application of the self-sustained-release immune adjuvant suspension in preparing a hyperthermia sensitizer.
  • the self-sustained-release immune adjuvant suspension of the present invention is a suspension composed of micron-sized particles of a fat-soluble immune adjuvant, and the surface active agent is coated on the surface of the fat-soluble immune adjuvant.
  • this preparation does not require other sustained-release adjuvants after local injection, that is, it can be retained in the tumor and slowly released, forming a self-sustained release effect.
  • the immune stimulating effect is stable and long-lasting.
  • the self-sustained-release immune adjuvant suspension of the present invention overcomes the poor water solubility of the fat-soluble immune adjuvant itself, and although the fat-soluble immune adjuvant hydrochloride can be well water-soluble, it will spread rapidly as a small molecule locally injected into the tumor The technical problem of transferring to other organs and metabolizing faster from the body.
  • Micron-sized suspension of lipid-soluble immune adjuvant is a new formulation of lipid-soluble immune adjuvant, which has the effect of self-sustained release, which increases the residence time of lipid-soluble immune adjuvant microparticles in the tumor and slows down the immune system.
  • the release rate of adjuvant molecules is crucial for sensitizing external beam radiation therapy.
  • the micron-sized particle suspension needs to be subjected to standard autoclaving operations to meet sterility requirements before being injected into the tumor, it is necessary to ensure that the micron-sized particles do not undergo significant agglomeration at about 121 degrees Celsius, requiring
  • the surfactant and the particle surface have a strong enough adsorption capacity, mainly relying on the hydrophobic interaction, so the hydrophobic structure of the selected surfactant plays an important role in protecting the stability of the micron-scale suspension under high pressure sterilization.
  • the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms or the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units.
  • two or more surfactant combinations with different hydrophilic-lipophilic balance values can be further selected as the coating layer of the microparticles.
  • the two surfactants with different solubility are not completely homogeneously dispersed with each other in the microcosm, but aggregated and dispersed locally. Therefore, after the formed composite particle coating enters the tumor, the surface with a larger HLB value will be formed.
  • the active agent dissolves first, thereby forming some tiny openings or tiny defect areas on the surface of the coating layer of the microparticles, so that the surface area of the inner layer of lipid-soluble immune adjuvant microparticles gradually changes, and the active ingredients are gradually released.
  • HLB values hydrophilic-lipophilic balance values
  • the present invention also provides a new preparation method of self-sustained release immune adjuvant suspension, because the research and development team found that when the ball milling process is enlarged, ceramic particles will be generated during the ball milling process, which will bring injection risks. There is little problem in the preparation of micro-nano materials, but there is a great risk in human injection; the applicant's research and development team has carried out a lot of work in order to replace the technical solution of obtaining imiquimod by ball milling and making micro-particles in the prior art. After trial and error and improvement of the experimental plan, a new technical route of jet pulverization combined with high pressure homogenization or jet pulverization combined with high shear method was proposed to prepare micron-scale lipid-soluble immune adjuvant microparticle suspension.
  • the preparation method overcomes the technical prejudice in the preparation process of micro-particles and the actual technical problem in the process of technological improvement, that is, the high-pressure homogenization process or the high-shear method is a liquid-phase processing method, while the fat-soluble immune adjuvant It is a semi-solid drug.
  • the high-pressure homogenization process or the high-shear method is a liquid-phase processing method
  • the fat-soluble immune adjuvant It is a semi-solid drug.
  • the blockage of the quality valve makes it impossible to obtain micron-sized particles; while the high-shear method can directly obtain micron-sized particles, but the uniformity of the obtained particles is extremely poor, and most of the particles cannot achieve the expected granulation and pulverization effect and yield; and
  • the primary powder is obtained after the pre-air jet pulverization process, and the high-pressure homogenization or high-shear method is carried out under the condition of adding an aqueous solution of surfactant, so that the high-pressure homogenization or high-shear micron particles can be quickly surfaced. retouch.
  • the presence of surfactants enables the fat-soluble immune adjuvant to be discretely dispersed in the liquid phase, so that the primary powder of the fat-soluble immune adjuvant can be processed by liquid phase micro-nano technology and obtain good size uniformity. Suspension of micron-sized particles of lipid-soluble immune adjuvant.
  • the self-sustained-release immune adjuvant suspension of the present invention can be further adapted to more severe sterilization conditions, can withstand high pressure sterilization treatment, and still maintain the suspension
  • the stability and particle size are stable, and the production efficiency and safety of self-sustained-release immune adjuvant suspensions are improved.
  • Injecting the self-sustained-release immune adjuvant suspension into the tumor can effectively enhance the immunogenic cell death induced by radiotherapy, chemotherapy or hyperthermia, and induce an anti-tumor immune response. On the other hand, a stronger distal effect is obtained, which inhibits the growth of distal unirradiated tumors.
  • the present invention also provides an imiquimod suspension preparation, comprising imiquimod microparticles, a surfactant containing higher fatty acid chains and a dispersion medium.
  • the dispersion medium is water, physiological saline or glucose solution.
  • the average particle size of the imiquimod microparticles is 0.5-5.0 ⁇ m.
  • the surfactant containing a higher fatty acid chain is an ionic surfactant containing a higher fatty chain.
  • the surfactants containing higher fatty acid chains include anionic surfactants and amphiphilic ionic surfactants.
  • the surfactants containing higher fatty acid chains include linear alkyl carboxylates, linear alkyl sulfonates, linear alkyl sulfates, linear alkanol sulfates, and the like.
  • the anionic surfactants containing higher fatty acid chains are sodium oleate, sodium lauryl sulfate, sodium stearate, sodium N-lauroyl sarcosinate, sodium cocoyl methyl taurate , Sodium N-lauroyl glutamate, sodium laureth carboxylate, lauryl phosphate.
  • the surfactant containing higher fatty acid chains is a phospholipid ionic surfactant.
  • the phospholipid ionic surfactant is lecithin, soybean phospholipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.
  • the dispersion medium is water.
  • the mass ratio of the surfactant containing a higher fatty acid chain to imiquimod is 0.025-3:1.
  • the mass ratio of the surfactant containing higher fatty acid chain to imiquimod is 0.1-1:1.
  • the imiquimod suspension preparation provided by the present invention can be retained and released slowly in the tumor for a long time, and can be further combined with chemotherapy, radiotherapy, alcohol ablation and other therapies that cause immunogenic death of tumor cells, thereby significantly enhancing the anti-tumor immune response , while effectively eliminating in situ tumors, it induces a systemic anti-tumor immune response and inhibits tumor metastasis and the growth of distant tumors.
  • the micron-scale imiquimod suspension preparation has good stability, can realize high temperature and high pressure sterilization, and reaches the preparation safety standard for clinical application.
  • the terminal sterilizable micron-scale imiquimod suspension of the invention has the characteristics of simple components, simple and convenient preparation, stable finished product, aseptic and low pyrogen.
  • the invention provides a preparation method of imiquimod suspension preparation.
  • the conditions of high temperature and high pressure sterilization are 110°C to 145°C for 5 to 30 minutes.
  • micron-sized imiquimod suspension preparation after the micron-sized imiquimod suspension preparation is sterilized by high temperature and high pressure, its state is no coagulation or agglomeration, or after agglomeration/coagulation, it can be re-dispersed into a uniform suspension by simple shaking liquid.
  • the present invention also provides the application of the imiquimod suspension preparation in the preparation of an anti-tumor combined immunotherapy preparation.
  • the sustained release of the chemical can be achieved.
  • the sustained release of the chemical can be achieved.
  • an effective dose of micron-sized imiquimod suspension preparation can be administered to a patient in need, wherein the use mode of the micron-sized imiquimod suspension preparation is intratumoral or peritumoral injection.
  • the terminally sterilizable micron-sized imiquimod suspension of the present invention provides an injectable imiquimod suspension dosage form, which can apply imiquimod to the immunotherapy of non-superficial tumors.
  • sterile, pyrogen-free stable dosage forms can be obtained by high temperature and high pressure sterilization, with good homogeneity and stability.
  • the micron-sized imiquimod suspension Compared with the imiquimod hydrochloride small molecule injection formulation, the micron-sized imiquimod suspension has a longer half-life in the tumor; compared with the imiquimod nanoparticle preparation, the micron-sized imiquimod suspension has The preparation process of quimod suspension is more feasible for scale-up production, can maintain long-term stability of the dosage form after high temperature and high pressure sterilization, and can meet the needs of clinical use.
  • the imiquimod suspension of the present invention can be applied to enhanced anti-tumor immunotherapy by intratumoral or peritumoral injection, combined with radiotherapy, chemical ablation and other treatment methods, and can be used in combination with platinum-based chemical drugs or anthracyclines
  • the premixed injection of the chemical drug can cause the sustained release of the chemical drug, prolong the action time of the drug at the lesion site, enhance the anti-tumor immune response of the combined chemical drug, effectively inhibit the growth of distant tumors, and prevent tumor metastasis and recurrence.
  • Fig. 1 is a schematic diagram of the preparation of self-sustained-release immune adjuvant suspension, with reference to Fig. 1 to prepare self-sustained-release imiquimod microparticles, and the preparation method is as follows:
  • a certain amount of fat-soluble immune adjuvant imiquimod R837 solid is weighed and subjected to jet pulverization, and the pulverization pressure is 6-10 bar to obtain micron-scale imiquimod R837 powder.
  • Proportion 1 (0.025 ⁇ 5) Weigh the micron-scale immune adjuvant imiquimod R837 and the surfactant poloxamer 188, preferably 2g R837, add an appropriate amount of poloxamer 188 (0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g, 10g), add 100mL of water for injection, and stir at 100-500rpm for 0.5-2 hours to obtain a suspension.
  • Poloxamer 188 is a new type of polymer nonionic surfactant, which has various uses including: as an emulsifier, stabilizer and solubilizer, which can further enhance the water dispersibility and stability of R837.
  • the hydrophobic structure part of the used surfactant contains no less than 20 oxypropylene units; specifically, it includes Poloxamer 188, Poloxamer 237, Poloxamer 338, and Poloxamer 407.
  • the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms; specifically including sorbitan sesquioleate, soybean lecithin, glyceryl monostearate , polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearyl (Span 60), stearate, vitamin E polyethylene succinate , polyoxyethylene alkyl ethers, polyoxyethylene stearate, polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, polycidol 1000, or lecithin at least one of them.
  • Poloxamer is a series of multi-purpose pharmaceutical excipients, which are non-toxic, non-antigenic, non-sensitizing, non-irritating, non-hemolytic, and chemically stable. Poloxamer 188 is one of the series of excipients with better safety. Poloxamer 188 can make the micron-sized powder obtained by jet pulverization of imiquimod to be processed by liquid phase micro-nano process to obtain imiquimod micron-sized particle suspension with good size uniformity, Poloxamer 188 can also help imiquimod micron-sized particle suspensions (6.0 mg/mL and below) ensure water dispersibility and stability after autoclaving.
  • the suspension of imiquimod microparticles coated with Poloxamer 188 maintains better suspension stability after autoclaving at a lower concentration (6.0 mg/mL), if imiquimod is sterilized during sterilization If the concentration is too high, it will cause imiquimod to agglomerate and clump after sterilization and can no longer be stably suspended.
  • Lecithin is a natural surfactant, and the imiquimod microparticles treated by high pressure homogenization with lecithin as a stabilizer have good stability. Still does not agglomerate and maintains a stable suspension.
  • the preparation method overcomes the technical prejudice and practical technical problems in the preparation process of micro-particles.
  • the high-pressure homogenization process or the high-shear process is a liquid-phase processing method, while the fat-soluble immune adjuvant is a semi-solid state.
  • the homogenization valve will be blocked and micro-particles cannot be obtained; while direct high-shear method can partially obtain micro-particles,
  • the uniformity of the obtained particles is extremely poor, and most of the particles cannot achieve the expected granulation and pulverization effect and yield; in the present invention, the primary powder is first obtained through the jet pulverization process, and then the solution is carried out under the condition of adding a surfactant.
  • High-pressure homogenization or high-shearing method can be used for rapid surface modification and surface modification of micro-particles under high-pressure homogenization or high shearing.
  • lipid-soluble immune adjuvants can be dispersed.
  • the lipid-soluble immune adjuvant is dispersed in the liquid phase, so that the primary powder of the lipid-soluble immune adjuvant can be processed by the liquid-phase micro-nano technology and the micron-sized particle suspension of the lipid-soluble immune adjuvant with good size uniformity can be obtained.
  • the surface active agent has a strong enough adsorption capacity with the particle surface, mainly relying on the hydrophobic interaction, so the hydrophobic structure of the selected surfactant plays an important role in protecting the stability of the micron-scale suspension under high pressure sterilization.
  • the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms or the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units. As shown in Table 2 and Table 3, Poloxamer P124, due to insufficient hydrophobic structure, was unstable after autoclaving.
  • Poloxamer 188:R837 Suspension stability after autoclaving 0.5:1 A large number of granular aggregates appear 1:1 A small amount of granular aggregates appear 2:1 A small amount of granular aggregates appear 3:1 Homogeneous dispersion and no granular aggregates 5:1 Homogeneous dispersion and no granular aggregates
  • a certain amount of fat-soluble immune adjuvant Resiquimod (R848) solid is weighed and subjected to jet pulverization, and the pulverization pressure is 6-10 bar to obtain micron-sized Resiquimod (R848).
  • Proportion 1 (0.025 ⁇ 5) Weigh micron-scale immune adjuvant Resiquimod (R848) and surfactant Poloxamer 407, preferably 0.2g R848, add an appropriate amount of Poloxamer 407 (0.005g, 0.01 g, 0.2 g, 0.4 g, 0.8 g, 1 g), add 200 mL of water for injection, and stir at 100-500 rpm for 0.5-2 hours to obtain a suspension.
  • the above suspension is homogenized under high pressure at 750-1200bar for 2-4 times to obtain the suspension, and the suspension is sucked by a peristaltic pump and filled into 10mL ampoule bottles, each bottle of 6mL, for a total of 30 bottles. After melting and sealing, a micron suspension is obtained, which is sterilized by moist heat at 105°C to 150°C for 15-20 minutes.
  • Poloxamer 407 is a new type of polymer nonionic surfactant, which has many uses including: as emulsifier, stabilizer and solubilizer, which can further enhance the water dispersibility and stability of R848.
  • lipid-soluble immune adjuvant glucopyranoside lipid A MPLA
  • the surfactant selected is a mixed surfactant with a mass ratio of Poloxamer 188 and lecithin of 9:1, and other preparation methods Same as Example A2.
  • Example A1 Other preparation methods are the same as in Example A1, take by weighing a certain amount of fat-soluble immune adjuvant imiquimod (R837); the surfactant selected is the mixed surface of poloxamer 188 and the mass ratio of lecithin 3:1 active agent.
  • the feeding concentration of different surfactants has a certain influence on the suspension stability of R837 after autoclaving, and the results are shown in Table 7. Long-term stability of R837 after autoclaving in the presence of lecithin The effect of solubilizing R837 with P188 alone resulted in smaller particle size and better uniformity of the obtained particles. And the influence of the feeding concentration can be expanded in equal proportion, so as to achieve the technical effect of increasing the final concentration of R837.
  • the mixing of the two surfactants can further increase the suspension stability performance of the self-sustained-release immune adjuvant suspension in autoclaving, especially at higher surfactant concentrations.
  • Two or more surfactant combinations with different hydrophilic-lipophilic balance (HLB values) or two surfactants with different hydrophobic structural moieties for example, one surfactant contains not less than 20 oxypropylene units
  • a surfactant containing one or more hydrocarbon chains with a total of not less than 15 carbon atoms as the coating layer of the microparticles.
  • the two surfactants with different solubility are not completely homogeneously dispersed with each other, but form a relatively uniform and locally aggregated dispersed structure.
  • the surfactant with a larger HLB value First dissolve, thereby forming some tiny openings or tiny defect areas on the surface of the coating layer of the microparticles, so that the surface area of the inner layer of lipid-soluble immune adjuvant microparticles gradually changes, and the active ingredients are gradually released.
  • various types of drug combination schemes can be obtained.
  • the R837 obtained by the coexistence of lecithin and P188 has the smallest particle size change before and after sterilization, and the particle size distribution range is smaller, that is, the coexistence of lecithin and P188 is more helpful for the sample to be sterilized.
  • stability 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. The higher the homogeneity of the sample particles. It was also observed in the experiment that the suspension samples in which P188 and lecithin coexisted would not hang on the wall after being left for a long time. It is worth noting that the size uniformity of microparticles is an important parameter to ensure stable and reproducible drug release behavior in vivo.
  • a certain amount of fat-soluble immune adjuvant imiquimod R837 solid is weighed and subjected to jet pulverization, and the pulverization pressure is 6-10 bar to obtain micron-scale imiquimod R837 powder.
  • Proportion 1 (0.025 ⁇ 5) Weigh the micron-scale immune adjuvant imiquimod R837 and the surfactant poloxamer 188, preferably 2g R837, add an appropriate amount of poloxamer 188 (0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g, 10g), add 100mL of water for injection, and stir at 100-500rpm for 0.5-2 hours to obtain a suspension.
  • sodium alginate/mannitol or sodium alginate/lactose solution Prepare sodium alginate/mannitol or sodium alginate/lactose solution according to ratio 1:(1 ⁇ 5), wherein, the concentration of sodium alginate solution is 10mg/mL, 20mg/mL, 40mg/mL, and the final concentration of mannitol or lactose It is 1 ⁇ 50mg/mL, 20 ⁇ 100mg/mL, 40 ⁇ 200mg/mL, the sodium alginate solution is stirred evenly, then mannitol or lactose is added, and packed in vials, after precooling, freeze-dried, and after nitrogen filling Seal the bottle.
  • the two compositions were thoroughly mixed, and then placed in a dialysis bag (permeable molecular weight 12000-14000 Da), and then dialyzed in buffer solutions of different pH.
  • the imiquimod suspension was directly placed in a dialysis bag (permeable molecular weight 12000-14000 Da), and dialyzed in buffers with different pH values to monitor the release of imiquimod.
  • the buffer solution of pH 7.4 is phosphate buffer solution added with 2 mM CaCl 2
  • the buffer solution of pH 4.0 is acetic acid-sodium acetate buffer solution.
  • the ratio of imiquimod released from sodium alginate/calcium ion hydrogel (ALG) over time is shown in Table 9.
  • Imiquimod has a faster release rate under acidic conditions, and the presence of sodium alginate/calcium ion gel can significantly reduce the release rate of imiquimod under both pH conditions, achieving sustained release Effect.
  • Imiquimod suspension formulations were prepared with various suspending agents.
  • an imiquimod suspension was prepared.
  • S1 prepare lecithin into uniform suspensions of different concentrations, add imiquimod powder to make the concentration of imiquimod 1-18 mg/mL, and stir the suspension;
  • step S2 homogenize the suspension obtained in step S1;
  • S3 Filling the homogenized suspension, sealing the cap, and performing high temperature and high pressure sterilization.
  • the conditions of high temperature and high pressure sterilization are: high temperature and high pressure sterilization at 110°C to 145°C for 10 to 30 minutes.
  • Embodiment B2 the influence of different suspending agent imiquimod suspension stability
  • the selection of the suspending agent should be based on several factors. First, as a suspending agent applied to the injection dosage form, the approved injection-grade pharmaceutical excipients are the first choice, so as to avoid the hidden safety hazards of the suspending agent itself; secondly, The suspending agent itself cannot chemically react with the drug molecule to change the drug activity or increase the toxicity.
  • suspending agent is helpful for the stability of imiquimod suspension after terminal sterilization is mainly judged from three aspects.
  • the appearance changes of the suspension before and after autoclaving were observed, and the samples were defined as good stability, average, and unstable according to the appearance changes. Specifically, observe whether there are particles or agglomerations visible to the naked eye, stick to the wall, and cannot be re-dispersed, and record the corresponding situation.
  • the sample does not have the above phenomenon, it is considered that the sample has good stability after sterilization; when the sample has the above phenomenon after sterilization, but can be re-dispersed to obtain a uniform suspension after shaking or shaking, it is regarded as The state of the sample after sterilization is normal; when the above phenomenon occurs after sterilization and no re-dispersed suspension can be obtained after shaking or shaking to different degrees, the sample is regarded as unstable.
  • D50 is the median diameter of the particles in the suspension, which means that 50% of the particles in the suspension have a particle diameter below this value.
  • D90 means that 90% of the particles in the system have a particle size below this value.
  • the difference between D50 and D90 can indicate the span of particle size distribution and the quality of particle size uniformity.
  • the sterilized sample for a long time, observe the state of the sample and detect the average particle size of the sample. If the sample can still be resuspended and the D50 and D90 do not increase significantly or the difference between D90 and D50 is small, it can be regarded as the auxiliary suspension.
  • the agent helps to increase the stability of the micron-sized imiquimod suspension. In this case, the long-term storage conditions are 2 to 8°C, and the time is 12 months.
  • the different types of surfactants added to the samples from 1° to 9° in Figure 2 are: lecithin, Tween-80, Tween-20, Poloxamer 188, Poloxamer 407, and polyoxyethylene castor oil. , Vitamin E polyethylene glycol succinate, sodium oleate, phosphatidylglycerol.
  • the samples at 1°, 8°, and 9° in Figure 2 are uniformly dispersed suspensions, and the remaining samples have varying degrees of sticking, caking and even precipitation.
  • Table 10 adds the particle size change of the imiquimod suspension of different types of suspending agents before and after sterilization and long-term placement after sterilization and the phenomenon record table (wherein, after sterilization is to within 1 week after sterilization; long-term Placement refers to placement for 12 months).
  • Selected 9 kinds of surfactants such as polyoxyethylene nonionic surfactants such as Tween-80 and Tween-20, polyoxyethylene castor oil, etc.
  • Phenomenon that is, the force between the surfactant and the water is destroyed by high temperature, and the solution becomes unstable.
  • poloxamers are generally considered to have good water solubility, and will not appear peak when heated under normal pressure.
  • ionic surfactants including anionic surfactants and zwitterionic surfactants, are added to the system as suspending agents to ensure that the imiquimod suspension is sterilized. long-term stability. Further analysis of the structures of the suspending agents that can stabilize the suspension shows that these ionic surfactants all contain higher-order aliphatic chain structures, and the molecular weight of the hydrophobic end is much larger than that of the hydrophilic end. Therefore, the selection of such ionic surfactants containing advanced aliphatic chains can effectively help the micron-sized imiquimod suspension to maintain the stability after terminal sterilization.
  • Example B3 Stabilization of imiquimod by other ratios of surfactants
  • the mass ratio of the surfactant containing higher fatty acid chain to imiquimod can be 0.025-3:1.
  • the mass ratio of the phospholipid ionic surfactant and the imiquimod may be 0.025-1:1.
  • Example B4 Study on the type of dispersion medium for imiquimod suspension preparations
  • isotonicity regulators When large-volume injection is administered clinically, the addition of isotonicity regulators is usually used to avoid local tissue damage or microenvironment disturbances caused by changes in osmotic pressure. Effects of formulation sterilization stability.
  • Example B1 The method of Example B1 was used to prepare an imiquimod suspension preparation with a concentration of 1 mg/mL, except that in step S1, a solution was prepared with normal saline or 5% glucose, and mixed with imiquimod microparticles to homogenize. The appearance state of the suspension preparation before and after autoclaving was observed, and no caking was found, indicating that the dispersion medium of the preparation can directly use physiological saline or 5% glucose solution.
  • the preparation scale was expanded, and the stability of the preparation product was investigated. After moist heat sterilization, there was no agglomeration, and it could still be dispersed well after being placed for a long time, and the particle size change was small, which further verified the feasibility of the aforementioned conditions.
  • the distribution behavior of the imiquimod preparation described in this example in the body is as follows:
  • mice Mouse colon cancer (CT26) tumor was implanted on the back of mice, and the mice were randomly divided into 3 groups, with 3 mice in each group for drug distribution behavior research.
  • Group 1 Mice received intratumoral injection of small molecule imiquimod hydrochloride at a dose of 6 mg/kg
  • mice were injected intratumorally with polylactic acid-co-glycolic acid (PLGA)-coated imiquimod nanoparticles (average particle size about 100 nm) at a dose of 6 mg/kg;
  • PLGA polylactic acid-co-glycolic acid
  • mice were injected intratumorally with imiquimod microparticles (this preparation) at a dose of 6 mg/kg; the mice were sacrificed 72 hours after injection, and the main organs and tumors were dissected to detect the presence of imiquimod in the organs and tumor tissues. drug content.
  • Cancer treatment is a very complex and comprehensive outcome, because both the body's immune system and the growth mechanism of cancer cells are very complex.
  • the reason why this experiment can achieve a relatively excellent therapeutic effect may include the following reasons, using imiquimod R837 micron particles.
  • the water-insoluble R837 powder was prepared into microparticles with a particle size of 1-3 microns, and the pharmacokinetics and intratumoral retention time were monitored after intratumoral injection. The results are shown in Table 12. The results show that microparticles can significantly prolong imiquine Mott's residence time at the tumor site and blood circulation half-life achieves a sustained release effect, thereby stimulating the immune system for a long time.
  • mice The mouse colon cancer (CT26) tumor was implanted on the back of the mice, and the mice were randomly divided into 3 groups, and the pharmacokinetics of the drug was studied with 3 mice in each group.
  • CT26 mouse colon cancer
  • mice were injected with small molecule imiquimod hydrochloride intratumorally; the injection dose was 6 mg/kg, venous blood was collected at 5h, 6h, 12h, 24h, 48h, and 72h after injection, and imiquimod was administered uniformly. The concentration of imiquimod in blood was measured.
  • mice were injected intratumorally with imiquimod/PLGA nanoparticles (average particle size about 100 nm) at a dose of 6 mg/kg, and venous blood was collected at 5 h, 6 h, 12 h, 24 h, 48 h, and 72 h after injection. And unified imiquimod concentration determination, detection of imiquimod concentration in blood.
  • mice were injected intratumorally with imiquimod microparticles (this preparation) at a dose of 6 mg/kg, venous blood collection was performed at 5h, 6h, 12h, 24h, 48h, and 72h after injection, and imiquimod was administered uniformly. Mott concentration assay, to detect the concentration of imiquimod in blood.
  • mice Colon cancer tumors were implanted on the left and right ends of the back of the mice (the right side was regarded as the in situ tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into 6 groups, 6 mice in each group were treated with radiotherapy Therapeutic trial of immunotherapy combination.
  • Group 1 Mice tumors were not treated with any treatment, and neither reagent injection nor radiotherapy was performed;
  • Group 2 The orthotopic tumors in mice were treated with radiotherapy alone, and the orthotopic tumors in mice were treated with radiotherapy once a day for 5 consecutive days, and the distal tumors were not treated.
  • Group 3 Intratumoral injection of small molecule imiquimod hydrochloride at a dose of 6 mg/kg to orthotopic tumors in mice; 1.5 Gy radiotherapy on days 0, 1, 2, 3, and 4 after administration , no treatment was performed on the left tumor of the mouse;
  • the fourth group intratumoral injection of PLGA nanoparticles of imiquimod (about 100 nm in diameter) into orthotopic tumors of mice, at a dose of 6 mg/kg; on days 0, 1, 2, 3, and 4 after administration 1.5Gy of radiotherapy was performed each time, and the distal tumors of the mice were not treated with any treatment;
  • mice were injected intratumorally with imiquimod microparticles (this preparation) at a dose of 6 mg/kg; 1.5 Gy of radiotherapy was performed on the 0th, 1st, 2nd, 3rd, and 4th days after administration. Distal tumors do not do any treatment;
  • mice were injected intratumorally with imiquimod microparticles (this preparation) at a dose of 12 mg/kg; on the 0, 1, 2, 3, and 4 days after administration, 1.5 Gy of radiotherapy was performed each time. Distal tumors do not do any treatment;
  • the length and width of in situ and distal tumors were measured with calipers every two days, and the tumor volume was (length times (width squared)) divided by 2.
  • Figure 5 is a comparison chart of the growth curves of in situ tumors after imiquimod hydrochloride, nanoparticles, and microparticles are injected into the tumor and then radiotherapy.
  • Figure 6 is a graph of imiquimod hydrochloride, nanoparticles, and microparticles. Comparison of the growth curve of the distal tumor after radiotherapy after injection into the tumor.
  • Radiotherapy can induce distant effects, although it has been reported, but this effect is not very significant.
  • injecting an immune adjuvant into the tumor and then irradiating the tumor with radiation can effectively enhance the immunogenic cell death induced by radiotherapy.
  • a stronger distal effect was obtained, inhibiting the growth of distal unirradiated tumors.
  • mice Colon cancer tumors of mice were implanted on the left and right ends of the back of the mice (the right side was regarded as the in situ tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into 3 groups, 5 mice in each group were microwaved Therapeutic experiments in combination with immunotherapy.
  • Group 1 Mice tumors were not treated with any treatment, reagent injection and microwave treatment were not performed;
  • Group 2 In situ tumors in mice were treated with simple microwave thermal ablation, the microwave power was 7W, the local tumor temperature reached 53°C, and the left tumor was not treated;
  • the third group intratumoral injection of imiquimod micro-preparation to the right tumor of the mice at a dose of 6 mg/kg; microwave thermal ablation treatment was performed after administration, the microwave power was 7 W, and the local tumor temperature reached 53 °C. The tumor on the left side of the mouse did not receive any treatment;
  • the length and width of in situ and distal tumors were measured with vernier calipers every two days, and the tumor volume was (length times (width squared)) divided by 2.
  • mice The mouse colon cancer CT26 tumor was implanted on the left and right ends of the back of the mouse (the right side was regarded as the in situ tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into 3 groups, with 5 mice in each group. Chemotherapy-immunotherapy combination therapy trial.
  • mice were injected with normal saline in the orthotopic tumor, and the distal tumor was not treated;
  • mice were injected with oxaliplatin chemotherapy drugs in orthotopic tumors, and the distal tumors were not treated.
  • mice were injected with a mixture of oxaliplatin chemotherapeutic drug and imiquimod micro-preparation into the orthotopic tumor tumor, and the distal tumor did not receive any treatment;
  • mice The neutral length and width of the mice were measured periodically, and the tumor volume was (length times (width squared)) divided by 2.
  • CT26 tumor cells were inoculated on the back of the mice to establish a mouse CT26 subcutaneous tumor model.
  • the mice with the same tumor size were equally divided into 3 groups with 3 mice in each group.
  • the grouping conditions are as follows:
  • PLGA-R837 PLGA nanoparticles loaded with R837
  • R837 ⁇ HCl Aqueous solution of R837 hydrochloride, R837 has excellent dispersibility in this system;
  • Imiquimod suspension preparation the terminal sterilized imiquimod micron-sized suspension (suspending agent is lecithin) of the present invention.
  • C max and T max reflect the speed of drug absorption from a certain preparation into the systemic blood circulation, and the time to peak (T max ) of the three dosage forms is consistent.
  • T max time to peak
  • Example E Enhanced radiotherapy and alcohol ablation therapy with micron-scale imiquimod suspension
  • Example E1 Therapeutic experiment of micron-sized imiquimod suspension preparation combined with radiotherapy
  • CT26 colon cancer tumor cells were inoculated on the back of mice to establish a mouse colon cancer subcutaneous double tumor model, which were orthotopic tumor and distal tumor respectively.
  • orthotopic tumor volume was about 100 mm 3
  • mice were randomly divided into 6 groups. The groups are as follows:
  • Vehicle vehicle control group, the operation of intratumoral injection of dispersion medium was performed on orthotopic tumors, and the injection volume was 25 ⁇ L;
  • RT in the external radiation therapy group, X-ray irradiation was performed on the in situ tumor, and the radiation dose was 4 Gy, which were performed on the first day and the third day of the treatment;
  • R837 imiquimod micron-sized suspension preparation of the present invention, wherein the concentration of R837 is 6 mg/mL, and the injection dose is 25 ⁇ L;
  • R837+RT After intratumoral injection of 25 ⁇ L of 6 mg/mL imiquimod micron-sized suspension preparation, the orthotopic tumor was subjected to the same X-ray irradiation treatment as the RT group.
  • the q value of in situ tumor was 1.17
  • the q value of distal tumor was 1.63, both of which had synergistic effect.
  • micron-sized imiquimod suspension preparation can be combined with external radiation therapy to enhance the anti-tumor immune response in vivo, especially to amplify the distal effect of radiotherapy and inhibit the growth of distal tumors.
  • Example E2 Therapeutic experiment of micron-sized imiquimod suspension formulation combined with alcohol ablation.
  • Alcohol ablation is a kind of local tumor ablation therapy.
  • the tumor tissue is coagulated and necrotic, so as to achieve the purpose of treatment.
  • it is difficult to completely remove the tumor by simply injecting alcohol or hydrochloric acid and other chemical ablation methods without affecting the surrounding normal tissue.
  • the micron-sized imiquimod suspension preparation was combined with alcohol ablation therapy to prove the anti-tumor effect of the micron-sized imiquimod suspension preparation of the present invention combined with chemotherapy.
  • mice were randomly divided into 5 groups with 5 mice in each group, and the groupings were as follows:
  • R837 Peritumoral injection of micron-sized imiquimod suspension
  • ETOH+R837(50) 50 ⁇ L imiquimod suspension was injected around the tumor, and absolute ethanol was injected intratumorally.
  • micron-sized imiquimod suspension concentration of micron-sized imiquimod suspension was 12 mg/mL, and the injection dose of absolute ethanol was 30 ⁇ L.
  • the micron-sized imiquimod suspension preparation was first injected into the subcutaneous site around the tumor, with an interval of about 10 minutes, and then anhydrous ethanol was administered by intratumoral injection. The changes of tumor volume in mice were monitored, and tumor growth curves were prepared. The results are shown in Figure 14.
  • mice in the group only administered with absolute ethanol had tumor fibrosis and scabs in the center of the tumor, but because the peripheral tumor tissue was not completely eliminated, it gradually developed and the outer diameter continued to increase, resulting in this group.
  • the value of tumor volume was not significantly different from that of the control group, so in the tumor growth curve, the tumor growth curve of the alcohol ablation group and the blank control group almost overlapped.
  • Figure 14 it can be seen from Figure 14 that the tumor growth of mice in the combined administration of micron-sized imiquimod suspension and alcohol ablation group was significantly inhibited. Both can improve the efficacy of alcohol ablation, achieve better tumor treatment effect, and inhibit tumor growth, indicating that the micron-sized imiquimod suspension can enhance the effect of alcohol ablation treatment of tumors.
  • Example F1 Verification of the sustained-release effect of oxaliplatin mixed with imiquimod suspension on oxaliplatin
  • Colon cancer (CT26) tumor cells were inoculated on the back of the mouse tumor to establish a mouse subcutaneous tumor model. After a mass was formed at the inoculation site for about a week, the mice were randomly divided into two groups:
  • OXA-R837 Oxaliplatin solution mixed with micron imiquimod suspension
  • oxaliplatin solution or oxaliplatin solution mixed with micron-sized imiquimod suspension preparation was injected intratumorally into each group of mice, and then at different time points (10min, 30min, 1h, 3h, 6h, 9h, 12h, 24h, 48h, 72h) to take blood samples from mice, and sacrifice the mice at the end point to obtain major organs and tumors.
  • Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect platinum in blood samples and organs. Relative content of ions and make a statistical graph. The results are shown in FIGS. 15 and 16 .
  • Figure 15 shows the biodistribution of oxaliplatin. Compared with free oxaliplatin, administration after premixing with imiquimod micron-sized suspension can significantly increase the retention of platinum in tumor sites , 72 hours after drug injection, the platinum content in the tumor site of the mice in the mixed injection group was dozens of times higher than that in the free group, indicating that the imiquimod micron-sized suspension can increase the retention of platinum-based chemicals in the tumor site and slow down the release of oxaliplatin.
  • Fig. 16 shows the time-dependent changes in the concentration of oxaliplatin in blood.
  • the oxaliplatin mixed with micron-sized imiquimod suspension has a more obvious sustained-release effect, which is manifested in a lower peak concentration and a later peak time. , remain in the blood longer.
  • the specific time to peak (Tmax), peak concentration (Cmax) and area under the curve (AUC) are shown in Table 14.
  • Tmax peak concentration
  • Cmax peak concentration
  • AUC area under the curve
  • Example F2 Combined use of oxaliplatin and micron-sized imiquimod suspension formulation to enhance anti-tumor effect
  • CT26 colon cancer
  • VEHICAL vehicle group, inject 30 ⁇ L of normal saline
  • OXA Injection of oxaliplatin solution 30 ⁇ L
  • OXA+R837 inject 30 ⁇ L of suspension mixed with micron-sized imiquimod suspension and oxaliplatin
  • the calculated q of the tumor inhibition rate of the in situ tumor was about 1.1, and the q of the distal tumor was about 1.27, indicating that the micron-sized imiquimod suspension has a synergistic effect of oxaliplatin chemotherapy.
  • oxaliplatin can cause tumor immunogenic death, and the addition of imiquimod will enhance the anti-tumor immune effect and induce a systemic anti-tumor immune response, thereby inhibiting the growth of distant tumors, which is different from the use of each component alone.
  • intratumoral injection of oxaliplatin and imiquimod at the same time could effectively inhibit the growth of distal tumors.
  • the tumor growth of the mice in the combination treatment group was the slowest and the intra-group differences were smaller.
  • Example G1 In vitro release experiment of doxorubicin after imiquimod micron-sized suspension formulation was mixed with doxorubicin (DOX).
  • DOX an aqueous solution of doxorubicin with a concentration of 3 mg/mL
  • DOX+R837 Dissolve 3 mg of doxorubicin with 1 mL of imiquimod micron-sized suspension preparation (sample containing lecithin and sterilized, wherein the concentration of imiquimod is 12 mg/mL).
  • the in vitro simulated release experiment can reflect the behavior of the drug in the body to a certain extent, indicating that the micron-sized imiquimod suspension preparation and doxorubicin are premixed and injected, which can slow down the release rate of the drug after in situ injection. Extend the retention time of chemotherapeutic drugs in the tumor site and increase the retention amount of the drug in the tumor site, thereby enhancing the effect of the drug in the tumor site and reducing the toxic and side effects of the drug on the system.
  • Example G2 Change the mixing ratio of imiquimod micron-sized suspension preparation and doxorubicin to verify the sustained-release effect.
  • DOX an aqueous solution of doxorubicin with a concentration of 3 mg/mL
  • DOX+R837 dissolve doxorubicin with micron-scale imiquimod suspension preparation, the concentration of imiquimod suspension preparation is 12mg/mL, and the final concentration of doxorubicin is 3mg/mL;
  • DOX+R837 (1/3) dissolve doxorubicin with micron-sized imiquimod suspension preparation, the concentration of imiquimod suspension preparation is 4 mg/mL, and the final concentration of doxorubicin is 3 mg/mL.
  • the drug release curves of the three groups were drawn using the same detection method and data processing method as in Example G1, and the results are shown in Figure 20.
  • the release rate of the drug increased, but compared with the simple doxorubicin aqueous solution, the release rate still slowed down, further indicating that the micron-sized imiquimod mixed Suspension preparations can slow down the release of doxorubicin, and the sustained-release effect is related to the ratio of the two.
  • the concentration ratio of quimod is 1:1 to 1:18.
  • Example G3 In vitro release experiment of epirubicin (EPI) mixed with micron-sized imiquimod suspension formulation.
  • EPI the aqueous solution of epirubicin, the concentration is 2mg/mL;
  • EPI+R837 Dissolve epirubicin with micron-scale imiquimod suspension preparation, and the final concentration of epirubicin is 2 mg/mL;
  • Example G1 Same as Example G1, put the sample in a dialysis bag (molecular weight cut-off is 3500D), and the sustained-release system is a 500mL PBS solution, detect the drug release amount at different time points, calculate the release percentage and draw a drug release curve, the results are shown in Figure 21 shown.
  • the results showed that in the early stage of the in vitro release experiment (before 6 h), the drug release trends of different groups were similar, but with the prolongation of time, the release of epirubicin mixed with the micron-sized imiquimod suspension formulation slowed down.
  • the sustained-release effect of epirubicin was slightly lower.
  • the inventor speculates that the anthracycline in the mixture may form a formation between the imiquimod microparticles.
  • There is a certain ⁇ - ⁇ stacking force and doxorubicin and epirubicin are isomers of each other. From the analysis of the structure, doxorubicin and imiquimod are easy to form a more stable ⁇ - ⁇ stacking force, Therefore, it will show a stronger sustained release effect.
  • Example G4 Sustained release of epirubicin by micron-sized imiquimod suspension preparation obtained as phosphatidylglycerol as suspending agent
  • EPI an aqueous solution of epirubicin with a concentration of 2 mg/mL
  • EPI+R837 (0.25PG): the micron-sized imiquimod suspension preparation obtained by suspending with phosphatidylglycerol dissolves epirubicin, wherein the mass ratio of phosphatidylglycerol and imiquimod is 0.25:1. The final concentration of rubicin was 2 mg/mL;
  • EPI+R837(3PG) The micron-sized imiquimod suspension preparation obtained by suspending with phosphatidylglycerol dissolves epirubicin, wherein the mass ratio of phosphatidylglycerol and imiquimod is 3:1, and the epirubicin is The final concentration of bicin was 2 mg/mL.

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Abstract

一种自缓释免疫佐剂混悬液，由脂溶性免疫佐剂和表面活性剂组成微米颗粒，余量为分散剂，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散剂中形成混悬液，该自缓释免疫佐剂混悬液可在肿瘤内的长时间滞留和保持缓释性能，还可以通过免疫反应抑制远端转移肿瘤的生长和降低肿瘤复发的概率。提供一种自缓释免疫佐剂混悬液的制备方法，以及自缓释免疫佐剂混悬液在放疗、化疗或热疗等肿瘤治疗所用的增敏制剂中的应用。

Description

一种自缓释免疫佐剂混悬液及其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种自缓释免疫佐剂混悬液及其制备方法和应用。
背景技术
化疗、放疗、微波热消融疗法是有效的恶性肿瘤治疗的方法，在临床肿瘤治疗中已经发挥了巨大的作用，已经广泛用于肝癌、肺癌、肾癌等常见肿瘤。放射治疗(简称放疗)是基于射线的外照射放疗在临床被广泛使用，但这是一种局部治疗方案，只能对局部的肿瘤进行照射，远端转移肿瘤不能得到有效照射(如远端隐匿的肿瘤)。临床中广泛使用的外照射放疗是利用射线(如X射线)对肿瘤部位进行局部定点照射，从而实现杀伤肿瘤细胞的目标，对于未发生远端转移的肿瘤甚至有机会实现治愈。然而，当肿瘤发生远端转移时，利用局部治疗很难覆盖人体内所有的肿瘤细胞，特别是转移肿瘤细胞，这些“漏网之鱼”有可能在远端生长出新的肿瘤转移灶。
在放射治疗临床应用中发现，对于少部分患者有可能产生“远端效应”，即对肿瘤的局部治疗有时也会抑制远端未被照射的肿瘤的生长。这种放疗诱导的“远端效应”近年来引起了研究者们的极大兴趣。研究表明，“远端效应”的机制在于诱导肿瘤细胞的免疫原性细胞死亡，暴露肿瘤相关抗原，从而激活针对肿瘤的免疫反应，并进一步通过肿瘤特异性CD8+T细胞对远端肿瘤的浸润来实现对远端肿瘤的免疫抑制。尽管诱导的“远端效应”具有重要的临床价值，但是这一效应的个体化差异非常大，对于临床中的大部分患者放疗诱导的“远端效应”并不十分显著。其中的重要原因，是诱导肿瘤细胞免疫原性细胞死亡后产生的肿瘤细胞“尸体”中的肿瘤相关抗原本身的免疫原性并不是很强，不能作为有效的“肿瘤疫苗”，多数时候难以激活足够有效的抗肿瘤免疫反应。
现代医学技术中，有效的免疫反应需要充分的肿瘤抗原暴露以及免疫佐剂的抗原呈递，其中免疫佐剂的作用是通过刺激免疫细胞，数量级地放大肿瘤抗原产生的免疫反应。因此，如能在肿瘤的治疗时在瘤内局部注射免疫佐剂，再对肿瘤进行治疗，将有望通过佐剂的免疫刺激效应从而显著性放大放疗后产生的肿瘤相关抗原的免疫原性，如招募抗原呈递细胞到肿瘤残留物部位来识别、吞噬和呈递肿瘤抗原，从而在体产生内源性的“肿瘤疫苗”，获得强有力的抗肿瘤免疫反应，实现对远端肿瘤的更有效抑制。
由于临床放疗大都是多次分剂量照射，因此需要注射的免疫刺激剂能够在肿瘤内实现长 时间滞留和缓释，这对于增敏放疗至关重要。目前大部分水溶性免疫佐剂本身存在的问题是很容易通过血液循环而被清除，不能够长期滞留在作用部位以实现长效刺激；而脂溶性免疫佐剂的分散性差，很难直接使用。怎样恰当的设计免疫刺激剂，并设计可操作的生产制造方法，以及药物产品的灭菌和储存长期稳定性，都是难题，例如在球磨法工艺制备微纳颗粒时，会产生陶瓷颗粒残留在产物中，这种杂质在普通的微纳材料制备加工中问题不大，但是用于人体注射则存在较大风险，这些成药阶段的问题无法被解决导致很多实验药物无法真正的走向临床的应用。
目前已被临床批准使用的咪喹莫特是一种典型的脂溶性免疫佐剂，这种咪唑奎琳胺小分子免疫调节剂不是一种细胞毒性药物，无显著的直接杀灭病毒或肿瘤细胞功效。咪喹莫特是Toll样受体7(TLR7)的配体，能够刺激巨噬细胞、单核细胞、树突状细胞，诱导干扰素α(IFN-α)和肿瘤坏死因子α(TNF-α)的产生，同时刺激白介素-2(IL-2)、IL-6、IL-8等细胞因子的产生，从而进一步刺激细胞免疫的激活，识别病毒或其他肿瘤抗原，激发相关免疫应答，清除致病因子。
咪喹莫特现阶段的成熟剂型为乳膏制剂，常通过涂抹的方式作用于表皮病变部位，临床治疗尖锐湿疣等局部病毒感染导致的疾病，也有在临床试验中在被用于皮肤浅表肿瘤治疗的尝试。目前，咪喹莫特已被批准应用于治疗头颈部光化性角化病和浅表性基底细胞癌。另外，多项临床试验证实，咪喹莫特在鳞状细胞癌、转移性黑色素瘤、外阴上皮内瘤变等浅表肿瘤治疗中，发挥免疫佐剂效果，具有一定的应用潜力。
然而，咪喹莫特本身为脂溶性小分子，难溶于水，同时，咪喹莫特具有较强的皮肤刺激性，通过给小鼠裸露皮肤涂抹5％的咪喹莫特乳膏，可以建立小鼠银屑样皮损模型，足以说明咪喹莫特对正常组织的刺激性。外部施用的方式有利有弊，虽然其对个别浅表病变的免疫治疗具有较好的免疫增强效果，但是也限制了咪喹莫特在其他肿瘤中的免疫治疗应用。
目前制备含有咪喹莫特的注射液主要有两种办法，一种是直接将咪喹莫特用酸溶解，例如将咪喹莫特溶于盐酸形成盐酸盐的形式，分散在水相中。但是，这种方法得到的溶液其pH值偏低，一般在3.0-4.0左右，该pH值下的溶液用于生物体，具有一定的刺激性。此外，咪喹莫特盐酸盐作为小分子在注射进入肿瘤内后，会快速从瘤内渗出并进入血液，使得其注射后在血液中有较高的急性暴露量(带来安全性风险)，同时咪喹莫特盐酸盐在肿瘤内的半衰期很短，会被快速清除，因而其瘤内给药后的免疫激活作用无法维持足够长的时间。
另一种制备咪喹莫特注射液的方式，是使用两亲高分子或其他可装载疏水药物的纳米结构装载R837。但这些纳米颗粒的制备过程往往比较复杂，不利于工艺放大和标准化批量生产。 此外，这些纳米颗粒制剂在终端高温高压灭菌的条件下往往难以稳定存在(根据《化学药品注射剂灭菌和灭菌工艺研究及验证指导原则》，终端高温高压灭菌为注射剂首选灭菌策略)。
类似的脂溶性免疫佐剂的进一步应用面临着类似的问题，因此，开发可注射的脂溶性免疫佐剂的注射制剂，作为免疫佐剂应用于非浅表肿瘤的免疫治疗具有重要意义。该制剂应实现佐剂在肿瘤内长时间的滞留和缓释，降低其在血液和正常组织中的暴露以保障其临床使用的安全性；此外，为了满足产业转化的需求，该制剂的制备方法需要可以实现规模化放大，并且该制剂的稳定性需要满足终端高温高压灭菌的要求。
发明内容
本申请提供了一种自缓释免疫佐剂混悬液，其由脂溶性免疫佐剂和表面活性剂组成，余量为分散介质，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散介质中形成混悬液。
在某些实施方式中，所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
在某些实施方式中，所述脂溶性免疫佐剂颗粒为粒径为0.5-5微米的核壳复合颗粒。
在某些实施方式中，所述脂溶性免疫佐剂核壳复合微米颗粒的粒径为1-2微米。
在某些实施方式中，所述脂溶性免疫佐剂为咪喹莫特微米颗粒。
在某些实施方式中，所述咪喹莫特微米颗粒的平均粒径为0.5～5微米。
在某些实施方式中，所述表面活性剂为含有高级脂肪酸链的表面活性剂。
在某些实施方式中，所述含有高级脂肪酸链的表面活性剂包括阴离子型表面活性剂。
在某些实施方式中，所述阴离子型表面活性剂包括油酸钠、十二烷基硫酸钠、硬脂酸钠、N-月桂酰肌胺酸钠、椰油酰基甲基牛磺酸钠、N-月桂酰基谷胺酸钠、月桂醇聚氧乙烯醚羧酸钠、十二烷基磷酸酯的至少一种。
在某些实施方式中，所述含有高级脂肪酸链的表面活性剂包括两亲性离子型表面活性剂。
在某些实施方式中，所述含有高级脂肪酸链的表面活性剂包括磷脂类离子型表面活性剂。
在某些实施方式中，所述磷脂类离子型表面活性剂包括卵磷脂、大豆磷脂、磷脂酰甘油、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇中的至少一种。
在某些实施方式中，所述表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。
在某些实施方式中，所述表面活性剂包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338，泊洛沙姆407中的至少一种。
在某些实施方式中，所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或 多条碳氢链。
在某些实施方式中，所述表面活性剂包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000，卵磷脂中的至少一种。
在某些实施方式中，所述表面活性剂为两种亲水亲油平衡值不同的表面活性剂的混合物。
在某些实施方式中，自缓释免疫佐剂混悬液包括咪喹莫特混悬液制剂，所述咪喹莫特混悬液制剂包括：咪喹莫特微米颗粒、含有高级脂肪酸链的表面活性剂和分散介质。
在某些实施方式中，在所述咪喹莫特混悬液中，所述咪喹莫特微米颗粒的浓度为1～18mg/mL，所述含有高级脂肪酸链的表面活性剂与咪喹莫特微米颗粒的质量比为0.025～3：1。
在某些实施方式中，在所述咪喹莫特混悬液中，所述含有高级脂肪酸链的表面活性剂与咪喹莫特微米颗粒的质量比为0.1～1：1。
另一方面，本申请提供了一种自缓释免疫佐剂混悬液的制备方法，其包括如下步骤：S1：将表面活性剂和脂溶性免疫佐剂分散在同一分散体系中，搅拌得到混悬液；S2：对上述制得的混悬液进行均质处理/进行高剪切工艺处理；S3：对均质后/高剪切工艺处理后的混悬液进行灭菌处理。
在某些实施方式中，所述制备方法包括：S1：将脂溶性免疫佐剂通过气流粉碎工艺形成初级微米级粉体；S2：向S1步骤获得的脂溶性免疫佐剂初级微米级粉体中按照脂溶性免疫佐剂：表面活性剂质量比(1：0.025～5)加入表面活性剂的水溶液，进行高压均质工艺处理，处理结束后取出匀浆；或S2’：向S1步骤获得的脂溶性免疫佐剂微米粉体中按脂溶性免疫佐剂：表面活性剂质量比(1：0.025～5)加入表面活性剂的水溶液，进行高剪切工艺处理，处理结束后取出匀浆；S3：高压灭菌处理。
在某些实施方式中，所述制备方法的步骤S2中所述的表面活性剂包括两种溶解度不同表面活性剂。
在某些实施方式中，所述制备方法的所述高压灭菌处理的条件为105℃～150℃下处理10-20分钟。
另一方面，本申请还提供了一种咪喹莫特混悬液制剂的制备方法，其包括如下步骤：S1：将含有高级脂肪酸链的表面活性剂和咪喹莫特微米颗粒分散在同一分散体系中，搅拌得到混悬液；S2：将上述制得的混悬液进行均质处理；S3：对均质后的混悬液进行灌装，封闭后进行高温高压灭菌。在某些实施方式中，所述高温高压灭菌的条件为：110～145℃，5～30min。
另一方面，本申请还提供了本申请所述制备方法获得的自缓释免疫佐剂混悬液在制备肿瘤辅助治疗药物中的应用。
另一方面，本申请还提供了一种自缓释免疫佐剂组合物，其包括第一组合物和第二组合物；所述第一组合物由脂溶性免疫佐剂和表面活性剂组成，余量为分散剂，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散剂中形成混悬液；所述第二组合物包括易溶性海藻酸盐和保护填充剂，形成的冻干粉。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述表面活性剂包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338和泊洛沙姆407中的至少一种。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述表面活性剂包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000和卵磷脂中的至少一种。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述表面活性剂为两种亲水亲油平衡值不同的表面活性剂的混合物。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述分散剂为水或生理盐水。
在某些实施方式中，在所述自缓释免疫佐剂组合物中，所述保护填充剂为甘露醇或乳糖。
在某些实施方式中，所述第二组合物还包括pH调节剂。
另一方面，本申请还提供了所述自缓释免疫佐剂混悬液或所述的自缓释免疫佐剂组合物在制备抗肿瘤联合免疫治疗制剂中的应用。
在某些实施方式中，所述自缓释免疫佐剂包含咪喹莫特混悬液制剂。
在某些实施方式中，所述咪喹莫特混悬液制剂与铂类化药预混，协助铂类化药的缓释。
在某些实施方式中，所述咪喹莫特混悬液制剂与蒽环类化药预混，协助蒽环类化药的缓释。
另一方面，本申请还提供了所述自缓释免疫佐剂混悬液或所述自缓释免疫佐剂组合物在制备放疗增敏剂中的应用。
另一方面，本申请还提供了所述自缓释免疫佐剂混悬液或所述自缓释免疫佐剂组合物在制备化疗增敏剂中的应用。
另一方面，本申请还提供了所述自缓释免疫佐剂混悬液或所述自缓释免疫佐剂组合物在制备热疗增敏剂中的应用。
另一方面，本申请还提供了所述自缓释免疫佐剂混悬液或所述自缓释免疫佐剂组合物在制备酒精消融增敏剂中的应用。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1是自缓释免疫佐剂混悬液制备示意图；
图2是加入不同含有高级脂肪酸链的表面活性剂的微米级咪喹莫特混悬液灭菌后经震摇后的照片；
图3是不同形态的自缓释免疫佐剂混悬液注射到肿瘤内后，其肿瘤滞留量随时间的变化对比图；
图4是不同形态的自缓释免疫佐剂混悬液注射到肿瘤内后，其血液中的药物浓度随时间的变化对比图；
图5是不同形态的咪喹莫特注射到肿瘤内后进行放疗，原位肿瘤的生长曲线对比图；
图6是不同形态的咪喹莫特注射到肿瘤内后进行放疗，远端肿瘤的生长曲线对比图；
图7是不同形态的咪喹莫特注射到肿瘤内后进行放疗的小鼠体重变化对比图；
图8是咪喹莫特微米颗粒注射到肿瘤内后进行微波消融治疗，小鼠原位肿瘤的生长曲线图；
图9是咪喹莫特微米颗粒注射到肿瘤内后进行微波消融治疗，小鼠远端肿瘤的生长曲线 图；
图10是咪喹莫特微米颗粒增效肿瘤化疗的小鼠原位肿瘤生长曲线图；
图11是咪喹莫特微米颗粒增效肿瘤化疗的小鼠远端肿瘤生长曲线图；
图12是微米级咪喹莫特混悬液联合放疗实验中不同组别的小鼠的原位肿瘤生长曲线；
图13是微米级咪喹莫特混悬液联合放疗实验中不同组别的小鼠的远端肿瘤生长曲线；
图14是微米级咪喹莫特混悬液联合酒精消融疗法实验中不同组别的小鼠的肿瘤生长曲线。
图15是奥沙利铂与微米级咪喹莫特混悬液混合前后，瘤内注射后72小时，主要组织器官中铂的相对含量统计图；
图16是奥沙利铂与微米级咪喹莫特混悬液混合前后，瘤内注射后，血液中的药物浓度随时间变化的曲线；
图17是小鼠双边肿瘤模型，原位瘤的肿瘤生长曲线图，组别分别为溶媒对照组、奥沙利铂单药治疗组、咪喹莫特混悬液制剂治疗组、奥沙利铂与咪喹莫特混悬液制剂联合治疗组；
图18是小鼠双边肿瘤模型，远端瘤的肿瘤生长曲线图，组别分别为溶媒对照组、奥沙利铂单药治疗组、咪喹莫特混悬液制剂治疗组、奥沙利铂与咪喹莫特混悬液制剂联合治疗组。
图19是与微米级咪喹莫特混悬液制剂混合后的多柔比星体外释放曲线图；
图20是与不同浓度的微米级咪喹莫特混悬液制剂混合后的多柔比星体外释放曲线图；
图21是与微米级咪喹莫特混悬液制剂混合后的表柔比星体外释放曲线图；
图22是与磷脂酰甘油助悬得到的微米级咪喹莫特混悬液混合的表柔比星体外释放曲线图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
发明详述
本发明提供一种自缓释免疫佐剂混悬液，提供一种能够在原位分散效果好的，且能够实现自缓释以辅助化疗、放疗或热疗产生免疫记忆，激活人体免疫特性的免疫佐剂新剂型，降低癌症转移及复发概率的抗癌药物组合物，在有效杀灭原位肿瘤的同时还可以通过免疫反应 抑制、降低远端转移肿瘤的生长和肿瘤复发的概率。
为解决相关技术问题，本发明提供了如下方案：
一种自缓释免疫佐剂混悬液，由脂溶性免疫佐剂和表面活性剂组成，余量为分散剂，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散剂中形成混悬液。
进一步的，所述分散剂为水或生理盐水。
进一步的，所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
进一步的，所述表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元；具体包括泊洛沙姆188(P188)，泊洛沙姆237，泊洛沙姆338，泊洛沙姆407。
并列可选地，所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链；具体包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000，或卵磷脂中的至少一种。
进一步的，所述自缓释免疫佐剂混悬液为粒径为0.5-5微米的复合颗粒，所述表面活性剂包覆所述脂溶性免疫佐剂。优选的，所述自缓释免疫佐剂混悬液的粒径为1-2微米。
进一步的可选的，所述表面活性剂可以为两种亲水亲油平衡值(HLB值)不同的表面活性剂的混合。两种不同亲水亲油平衡值的表面活性剂可以在复合颗粒进入瘤体后，HLB值较大的表面活性剂首先溶解，从而包覆在脂溶性免疫佐剂微米颗粒的表面形成一些开口或者微小的缺陷区域，从而使内层咪喹莫特微米颗粒的表面积逐步变化，有效成分逐步释放，更可根据不同瘤体及人体的实际需要，通过表面活性剂的配比关系调配更加个性化的药剂方案。
本发明提供了一种自缓释免疫佐剂混悬液的制备方法，其特征在于包括如下步骤：
S1：将脂溶性免疫佐剂通过气流粉碎工艺形成初级粉体；
S2：向S1步骤获得的初级粉体中加入表面活性剂的水溶液，进行高压均质工艺处理，处理结束后取出匀浆；
或S2’：向S1步骤获得的初级粉体中加入表面活性剂的水溶液，进行高剪切工艺处理，处理结束后取出匀浆；
S3：灭菌处理。
进一步的，所述步骤S1中所述的表面活性剂的水溶液中，包含两种亲水亲油平衡值不同 表面活性剂。
优选的，所述步骤S1中所述的表面活性剂的水溶液浓度为6-30mg/mL。
进一步的，所述步骤S3中所述灭菌处理为条件为105℃～150℃的湿热处理10-15分钟。
本发明还提供一种自缓释免疫佐剂组合物，包括第一组合物和第二组合物；所述第一组合物由脂溶性免疫佐剂和表面活性剂组成，余量为分散剂，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散剂中形成混悬液；所述第二组合物包括易溶性海藻酸盐和保护填充剂，形成的冻干粉。
第二组合物可进一步优化所述第一组合物的缓释特性。
进一步的，所述分散剂为水或生理盐水。
进一步的，所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
进一步的，所述表面活性剂的疏水结构部分含不少于20个的氧丙烯单元，包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338，泊洛沙姆407；或者含总数不少于15个碳原子的一条或多条碳氢链，包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000或卵磷脂中的至少一种。
本发明还提供了所述自缓释免疫佐剂混悬液在制备放疗增敏剂中的应用。
本发明还提供了所述自缓释免疫佐剂混悬液在制备化疗增敏剂中的应用。
本发明还提供了所述自缓释免疫佐剂混悬液在制备热疗增敏剂中的应用。
采用本发明的技术方案，会具有如下的有益技术效果：
本发明的自缓释免疫佐剂混悬液是脂溶性免疫佐剂的微米级颗粒构成的混悬液，表面活性剂包覆在所述脂溶性免疫佐剂表面，与免疫佐剂的盐酸盐或者其他的水溶性免疫佐剂分子(如CpG、polyIC等)相比，本制剂局部注射后不需要其他的缓释辅助剂，即可以在瘤内滞留且缓慢释放，形成自缓释效果，免疫刺激效应稳定而持久。由于临床放疗大都是多次分剂量照射(如一周照射5次)，需要使注射的免疫刺激剂在肿瘤内具有比较长时间的滞留和缓释，可以有效增强诱导的免疫原性细胞死亡，诱导抗肿瘤免疫反应，本制剂咪喹莫特微米级颗粒在肿瘤内的长时间滞留和缓释性能，对于增敏放疗、化疗或热疗的应用，诱导抗肿瘤免疫反应而言至关重要。
本发明的自缓释免疫佐剂混悬液克服了脂溶性免疫佐剂本身的水溶性差，而脂溶性免疫佐剂盐酸盐虽然可以水溶好，但是局部注射到肿瘤内作为小分子会迅速扩散到其他器官并较快从体内代谢的技术问题。将脂溶性免疫佐剂做成微米级悬液是一类脂溶性免疫佐剂的新剂型，具有自缓释的效果，增加了脂溶性免疫佐剂微米颗粒在肿瘤内的滞留时间，减缓了免疫佐剂分子的释放速度，这个特点对于增敏外照射放疗来说至关重要。此外，由于该微米级颗粒混悬液在注射到瘤内前需要进行标准的高压灭菌操作以满足无菌的要求，需要确保微米级颗粒在约121摄氏度的条件下不发生显著的团聚，要求表面活性剂与颗粒表面具有足够强的吸附能力，主要依靠疏水相互作用，因此所选表面活性剂的疏水结构对于保护该微米级混悬液高压灭菌下的稳定性有重要作用，本发明选择的所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链或表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。
本发明的自缓释免疫佐剂混悬液，可以进一步选择两种及以上的亲水亲油平衡值(HLB值)不同的表面活性剂组合作为微米颗粒的包覆层。两种不同溶解性的表面活性剂在微观中并不是完全均质的相互分散，而是局部区域性的聚集式分散，因此形成的复合颗粒包覆层进入瘤体后，HLB值较大的表面活性剂首先溶解，从而在微米颗粒的包覆层表面形成一些微小开口或者微小的缺陷区域，从而使内层脂溶性免疫佐剂微米颗粒的表面积逐步变化，有效成分逐步释放，更可根据不同瘤体及人体的实际需要，通过调配两种或多种表面活性剂的选择或调控配比关系，获得更加个性化(符合不同病人实际情况)的药剂组合方案供医生选择。且两种及以上的亲水亲油平衡值(HLB值)不同的表面活性剂组合可进一步提高微米颗粒在高压灭菌过程中的稳定性。
本发明还提供了一种新的自缓释免疫佐剂混悬液制备方法，因为本研发团队发现在球磨法工艺放大时，球磨过程会产生陶瓷颗粒从而带来注射风险，这种杂质在普通的微纳材料制备中问题不大，但是用于人体注射则存在较大风险；申请人研发团队为了替换现有技术中用球磨法获得咪喹莫特做成微米颗粒的技术方案进行了大量的实验方案的试错和改进，并进一步提出了一种气流粉碎联合高压均质或气流粉碎联合高剪切法的新型技术路线，制备了微米尺度的脂溶性免疫佐剂微米颗粒混悬液。该制备方法克服了微米颗粒的制备工艺中的技术偏见和技术改进过程中实际的技术问题，即高压均质工艺或高剪切法工艺是一种液相的加工方法，而脂溶性免疫佐剂是一种半固态药剂，实验发现如果直接对脂溶性免疫佐剂进行高压均质或高剪切工艺，由于脂溶性免疫佐剂的粘滞性远高于溶液或常用固态纳米材料，会造成均 质阀的堵塞从而无法获得微米级颗粒；而直接采用高剪切法虽然可以部分得到微米颗粒，但是得到的微粒均匀性极差，大部分微粒无法达到预期的颗粒化粉碎效果和产率；而本发明中通过预先的气流粉碎工艺后获得初级粉体，加入表面活性剂的水溶液的条件下进行高压均质或高剪切法，可以对高压均质或高剪切的微米颗粒进行快速的表面修饰。表面活性剂的存在，使得脂溶性免疫佐剂可以离散化的分散在液相之中，从而使得脂溶性免疫佐剂的初级粉体得以利用液相微纳工艺进行加工且获得尺寸均一性好的脂溶性免疫佐剂微米级颗粒混悬液。
本发明的自缓释免疫佐剂混悬液，相比于现有各类工艺获得的微纳颗粒，可进一步适应更严苛的灭菌条件，能够经受高压灭菌处理，依然保持混悬液的稳定性和颗粒尺寸的稳定，提高自缓释免疫佐剂混悬液的生产效率和安全性。
将自缓释免疫佐剂混悬液注射进入肿瘤内，可以有效增强放疗、化疗或热疗诱导的免疫原性细胞死亡，诱导抗肿瘤免疫反应，其体现的治疗效果一方面可以提升放疗对原位肿瘤的疗效，另一方面获得更强的远端效应，抑制远端没有被照射肿瘤的生长。
为了解决相关技术问题，本发明还提供一种咪喹莫特混悬液制剂，包括咪喹莫特微米颗粒、含有高级脂肪酸链的表面活性剂和分散介质。其中，所述分散介质为水，生理盐水或葡萄糖溶液。
具体的，咪喹莫特微米颗粒的平均粒径为0.5～5.0μm。
其中，所述含有高级脂肪酸链的表面活性剂为含有高级脂肪链的离子型表面活性剂。
具体的，所述含有高级脂肪酸链的表面活性剂包括阴离子型表面活性剂和两亲性离子型表面活性剂。
具体的，所述含有高级脂肪酸链的表面活性剂包括直链烷基羧酸盐，直链烷基磺酸盐，直链烷基硫酸盐，直链烷醇硫酸酯盐等。
具体的，所述含有高级脂肪酸链的阴离子型表面活性剂为油酸钠、十二烷基硫酸钠、硬脂酸钠、N-月桂酰肌胺酸钠、椰油酰基甲基牛磺酸钠、N-月桂酰基谷胺酸钠、月桂醇聚氧乙烯醚羧酸钠、十二烷基磷酸酯。
可选的，所述含有高级脂肪酸链的表面活性剂为磷脂类离子型表面活性剂。
具体的，所述磷脂类离子型表面活性剂为卵磷脂、大豆磷脂、磷脂酰甘油、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇。
可选的，所述分散介质为水。
可选的，所述含有高级脂肪酸链的表面活性剂与咪喹莫特的质量比为0.025～3：1。
可选的，所述含有高级脂肪酸链的表面活性剂与咪喹莫特的质量比为0.1～1：1。
本发明提供的咪喹莫特混悬液制剂，能够在肿瘤内长时间的滞留和缓释，进一步联合化疗、放疗、酒精消融等引起肿瘤细胞免疫原性死亡的疗法，显著增强抗肿瘤免疫反应，在有效消灭原位肿瘤的同时，诱发全身性抗肿瘤免疫反应，抑制肿瘤转移及远端肿瘤的生长。同时，该微米级咪喹莫特混悬液制剂具有较好的稳定性，能够实现高温高压灭菌，达到临床应用的制剂安全标准。本发明所述可终端灭菌的微米级咪喹莫特混悬液具有组分简单、制备简便、成品稳定、无菌低热原的特点。
本发明提出了一种咪喹莫特混悬液制剂的制备方法。
包括如下步骤：
S1：将含有高级脂肪酸链的表面活性剂和咪喹莫特微米颗粒分散在同一分散体系中，搅拌得到混悬液；
S2：将上述制得的混悬液进行均质；
S3：对均质后的混悬液进行灌装，封闭后进行高温高压灭菌。
其中高温高压灭菌的条件为110℃～145℃，5～30min。
具体的，所述微米级咪喹莫特混悬液制剂经过高温高压灭菌处理后，其状态为无聚沉或结块，或结块/聚沉后可通过简单晃动重新分散为均匀混悬液。
本发明还提供所述咪喹莫特混悬液制剂在制备抗肿瘤联合免疫治疗制剂中的应用。
具体的，本发明所述咪喹莫特混悬液制剂在和铂类化药混合后，可实现化药的缓释。
具体的，本发明所述咪喹莫特混悬液制剂在和蒽环类化药混合后，可实现化药的缓释。
具体的，本发明所述咪喹莫特混悬液制剂用于制备增强抗肿瘤免疫治疗的制剂的用途。在具体实施时，可向有需要的患者施用有效剂量的微米级咪喹莫特混悬液制剂，其中该微米级咪喹莫特混悬液制剂的使用方式为瘤内或瘤周注射。
采用本发明的技术方案，具有如下的技术效果：
本发明所述可终端灭菌的微米级咪喹莫特混悬液提供了可注射的咪喹莫特混悬液剂型，能够将咪喹莫特应用于非浅表肿瘤的免疫治疗中。在含有高级脂肪酸链的表面活性剂的帮助下，可通过高温高压灭菌得到无菌、无热原的稳定剂型，具有很好的均一性和稳定性。相比于咪喹莫特盐酸盐小分子注射剂型，该微米级咪喹莫特混悬液在肿瘤内的具有更长的半衰期；相比于咪喹莫特纳米颗粒制剂，该微米级咪喹莫特混悬液的制备工艺放大生产的可行性更强，可在高温高压灭菌后保持剂型的长期稳定，能够满足临床使用的需求。
本发明所述咪喹莫特混悬剂能够通过瘤内或瘤周注射的方式，联合放疗、化学消融等治疗手段应用于增强的抗肿瘤免疫治疗，并且在与铂类化药或蒽环类化药预混后注射，能够引 起化药的缓释，延长药物在病灶部位作用时间，增强联合化药的抗肿瘤免疫反应，有效抑制远端肿瘤的生长，防止肿瘤转移和复发。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的自缓释免疫佐剂混悬液、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例A 制剂的制备
实施例A1：
图1是自缓释免疫佐剂混悬液制备示意图，参考图1制备自缓释咪喹莫特微米颗粒，制备方法如下：
称取一定量的脂溶性免疫佐剂咪喹莫特R837固体进行气流粉碎处理，粉碎气压6-10bar，得到微米级咪喹莫特R837粉体。
按比例1：(0.025～5)称取微米级免疫佐剂咪喹莫特R837和表面活性剂泊洛沙姆188，优选2g R837，加入适量的泊洛沙姆188(0.05g，0.3g，0.6g，1g，2g，4g，6g，8g，10g)，加100mL注射用水，100-500rpm搅拌0.5-2小时，获得悬浊液。
将上述悬浊液于750-1200bar压力下高压均质2-4次获得混悬液，加入注射用水定容至咪喹莫特浓度6.0mg/mL，以蠕动泵吸取混悬液灌装到10mL安瓿瓶中，每瓶6mL，共30瓶。熔封后得到微米悬液，105℃～150℃湿热灭菌15-20分钟。
泊洛沙姆188是一种一类新型的高分子非离子表面活性剂，有多种用途包括：作为乳化剂，稳定剂和增溶剂，可以进一步增强R837的水分散性和稳定性。
所用表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元；具体包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338，泊洛沙姆407。并列可选地，所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链；具体包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000，或卵磷脂中的至少一种。
泊洛沙姆是一系列多用途的药用辅料，由于无毒，无抗原性，无致敏性，无刺激性、不溶血，化学性质稳定。泊洛沙姆188是系列辅料中具有较好安全性的一种。泊洛沙姆188 可以使得咪喹莫特气流粉碎后获得的微米级粉体得以利用液相微纳工艺进行加工获得尺寸均一性好的咪喹莫特微米级颗粒混悬液，泊洛沙姆188还可以帮助咪喹莫特微米级颗粒混悬液(6.0mg/mL及以下)在高压灭菌后保证水分散性和稳定性。
但是泊洛沙姆188包覆的咪喹莫特微米颗粒混悬液虽然在较低浓度(6.0mg/mL)高压灭菌后保持较好的混悬稳定性，如果灭菌时咪喹莫特浓度过高，则会导致灭菌后咪喹莫特团聚结块不能再稳定混悬。卵磷脂是一种天然表面活性剂，用卵磷脂作为稳定剂通过高压均质处理的咪喹莫特微米颗粒具有很好的稳定性，即使在高咪喹莫特浓度下高温灭菌，其混悬液依然不会团聚保持稳定混悬。
表1咪喹莫特/表面活性剂混悬液制备工艺与数据
Figure PCTCN2021143057-appb-000001
气流粉碎联合高压均质或气流粉碎联合高剪切法的新的技术路线，制备了微米尺度的脂溶性免疫佐剂微米颗粒混悬液。该制备方法克服了微米颗粒的制备工艺中的技术偏见和实际的技术问题，高压均质工艺或高剪切法工艺是一种液相的加工方法，而脂溶性免疫佐剂是一种半固态药剂，实验发现如果直接对脂溶性免疫佐剂进行高压均质或高剪切工艺，会造成均质阀的堵塞从而无法获得微米颗粒；而直接采用高剪切法虽然可以部分的得到微米颗粒，但是得到的微粒均匀性极差，大部分微粒无法达到预期的颗粒化粉碎效果和产率；而本发明中 首先通过气流粉碎工艺后获得初级粉体，再在加入表面活性剂的溶液条件下进行中进行高压均质或高剪切法，可以对高压均质或高剪切的微米颗粒进行快速的表面修饰和表面改性，因为有了表面活性剂的存在，使得脂溶性免疫佐剂可以离散化的分散在液相之中，从而使得脂溶性免疫佐剂的初级粉体得以利用液相微纳工艺进行加工且获得尺寸均一性好的脂溶性免疫佐剂微米级颗粒混悬液。
表2将气流粉碎后的咪喹莫特微米颗粒粉体加入不同表面活性剂水溶液(咪喹莫特：表面活性剂质量比＝1:3)再进行高压均质处理后咪喹莫特的水分散性
Figure PCTCN2021143057-appb-000002
表3上述添加不同表面活性剂的咪喹莫特混悬液(6.0mg/mL)高压灭菌后的再分散性(咪喹莫特：表面活性剂质量比＝1:3)
Figure PCTCN2021143057-appb-000003
Figure PCTCN2021143057-appb-000004
由于该微米级颗粒混悬液在注射到瘤内前需要进行标准的高压灭菌操作以满足无菌的要求，需要确保微米级颗粒在约121摄氏度的条件下不发生显著的团聚，要求表面活性剂与颗粒表面具有足够强的吸附能力，主要依靠疏水相互作用，因此所选表面活性剂的疏水结构对于保护该微米级混悬液高压灭菌下的稳定性有重要作用，本发明选择的所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链或面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。如表2和表3中，泊洛沙姆P124，因疏水结构不足，在高压灭菌后出现不稳定现象。
表4加入不同比例P188分散的咪喹莫特混悬液(灭菌时R837浓度＝6.0mg/mL)在高压灭菌后的混悬稳定性
泊洛沙姆188:R837 高压灭菌后的混悬稳定性
0.5:1 出现大量颗粒状聚集体
1:1 出现少量颗粒状聚集体
2:1 出现少量颗粒状聚集体
3:1 均匀分散且未出现颗粒状聚集体
5:1 均匀分散且未出现颗粒状聚集体
虽然理论上，分散剂越多，分散性越好，但比例一般不超过5:1，原因是：泊洛沙姆188(P188)本身有粘性，浓度过高粘度很大；且避免分散剂过多引入杂质。
表5 P188分散的不同浓度咪喹莫特混悬液在高压灭菌后的混悬稳定性(P188：咪喹莫特R837质量比＝3:1)。P188包覆的咪喹莫特混悬液在低R837浓度下高压灭菌能保持较好稳定性，但在高R837浓度下高压灭菌稳定性显著下降。
灭菌时R837浓度 高压灭菌后的混悬稳定性
3.0mg/mL 均匀分散且未出现颗粒状聚集体
6.0mg/mL 均匀分散且未出现颗粒状聚集体
9.0mg/mL 出现部分颗粒状聚集体
12.0mg/mL 出现大量颗粒状聚集体
15.0mg/mL 出现大量颗粒状聚集体
18.0mg/mL 出现大量颗粒状聚集体
表6加入不同比例卵磷脂分散的咪喹莫特混悬液(灭菌时R837浓度＝6.0mg/mL或18mg/mL)在高压灭菌后的混悬稳定性。卵磷脂哪怕在较低的比例下都可以使高浓度咪喹莫特悬液高压灭菌后保持很好的混悬稳定性。
卵磷脂:R837 灭菌时R837浓度 高压灭菌后的混悬稳定性
0.025:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
0.05:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
0.1:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
0.25:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
0.5:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
1:1 6.0mg/mL 均匀分散且未出现颗粒状聚集体
0.025:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
0.05:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
0.1:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
0.25:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
0.5:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
1:1 18.0mg/mL 均匀分散且未出现颗粒状聚集体
实施例A2：
称取一定量的脂溶性免疫佐剂雷西莫特(R848)固体进行气流粉碎处理，粉碎气压6-10bar，得到微米级雷西莫特(R848)。
按比例1：(0.025～5)称取微米级免疫佐剂雷西莫特(R848)和表面活性剂泊洛沙姆407，优选0.2g R848，加入适量的泊洛沙姆407(0.005g，0.01g，0.2g，0.4g，0.8g，1g)，加200mL注射用水，100-500rpm搅拌0.5-2小时，获得悬浊液。
将上述悬浊液于750-1200bar压力下高压均质2-4次获得混悬液，以蠕动泵吸取混悬液灌装到10mL安瓿瓶中，每瓶6mL，共30瓶。熔封后得到微米悬液，105℃～150℃湿热灭菌15-20分钟。
泊洛沙姆407是一种一类新型的高分子非离子表面活性剂，有多种用途包括：作乳化剂，稳定剂和增溶剂，可以进一步增强R848的水分散性和稳定性。
实施例A3：
称取一定量的脂溶性免疫佐剂吡喃葡糖苷脂质A(MPLA)；选用的表面活性剂为泊洛沙姆188与卵磷脂的质量比9:1的混合表面活性剂，其他制备方法与实施例A2相同。
实施例A4：
其他制备方法与实施例A1相同，称取一定量的脂溶性免疫佐剂咪喹莫特(R837)；选用的表面活性剂为泊洛沙姆188与卵磷脂的质量比3:1的混合表面活性剂。不同表面活性剂的投料浓度对R837高压灭菌后的混悬稳定性有一定影响，结果如表7所示。在有卵磷脂存在的条件下，R837高压灭菌后的长期稳定性单独P188增溶R837的效果，获得颗粒的粒径更小且均一性更好。并且投料浓度的影响可等比例扩大，从而达到增加R837最终浓度的技术效果。
表7加入不同浓度表面活性剂的R837高压灭菌后的混悬稳定性
R837:泊洛沙姆188:卵磷脂 高压灭菌后的长期稳定性
12mg/mL:36mg/mL:0mg/mL 出现大量颗粒状聚集体
12mg/mL:36mg/mL:12mg/mL 均匀分散且未出现颗粒状聚集体
18mg/mL:54mg/mL:0mg/mL 出现大量颗粒状聚集体
18mg/mL:54mg/mL:18mg/mL 均匀分散且未出现颗粒状聚集体
可见，两种表面活性剂的混合，可进一步增加自缓释免疫佐剂混悬液在高压灭菌中的混悬稳定性表现，尤其是较高的表面活性剂浓度时，表现突出。两种及以上的亲水亲油平衡值(HLB值)不同的表面活性剂组合或两种疏水结构部分不同的表面活性剂(例如，一种表面活性剂含不少于20个的氧丙烯单元，或一种表面活性剂含有总数不少于15个碳原子的一条或多条碳氢链)作为微米颗粒的包覆层。两种不同溶解性的表面活性剂并不是完全均质的相互分散，而是形成相对均匀和局部聚集的分散结构，形成的包覆层复合颗粒进入瘤体后，HLB值较大的表面活性剂首先溶解，从而在微米颗粒的包覆层表面形成一些微小开口或者微小的缺陷区域，从而使内层脂溶性免疫佐剂微米颗粒的表面积逐步变化，有效成分逐步释放，更可根据不同瘤体及人体的实际需要，通过调配两种或多种表面活性剂的选择或配比关系的调配，获得多种型号的药剂组合方案。
表8加入不同比例的表面活性剂的R837高压灭菌后的粒径变化
Figure PCTCN2021143057-appb-000005
同时，如表8所示，卵磷脂和P188同时存在得到的R837灭菌前后粒径变化最小，且粒径分布范围更小，即卵磷脂和P188同时存在更有助于样品在灭菌处理中的稳定性。其中，D50为样品中累计粒度分布达到50％时对应的粒径，D90为样品中累计粒度分布达到90％时对应的粒径，Dmax为样品中颗粒的最大粒径，三者差异越小，样品颗粒的均一度越高。在实验中还观察到，P188和卵磷脂同时存在的混悬液样品，久置后不会出现挂壁现象。值得说明的是，微米颗粒尺寸均一性是保证药物在体内具有稳定可重复的释放行为的一个重 要参数。
实施例A5：
第一组合物制备：
称取一定量的脂溶性免疫佐剂咪喹莫特R837固体进行气流粉碎处理，粉碎气压6-10bar，得到微米级咪喹莫特R837粉体。
按比例1：(0.025～5)称取微米级免疫佐剂咪喹莫特R837和表面活性剂泊洛沙姆188，优选2g R837，加入适量的泊洛沙姆188(0.05g，0.3g，0.6g，1g，2g，4g，6g，8g，10g)，加100mL注射用水，100-500rpm搅拌0.5-2小时，获得悬浊液。
将上述悬浊液于750-1200bar压力下高压均质2-4次获得混悬液，加入注射用水定容至咪喹莫特浓度6.0mg/mL，以蠕动泵吸取混悬液灌装到10mL安瓿瓶中，每瓶6mL，共30瓶。熔封后得到微米悬液，105℃～150℃湿热灭菌15-20分钟。
第二组合物制备：
按比例1:(1～5)配制海藻酸钠/甘露醇或海藻酸钠/乳糖溶液，其中，海藻酸钠溶液浓度为10mg/mL、20mg/mL、40mg/mL,甘露醇或乳糖最终浓度为1～50mg/mL、20～100mg/mL、40～200mg/mL，海藻酸钠溶液搅拌均匀后再加入甘露醇或乳糖，分装于西林瓶中，预冷后，冻干，充氮气后封瓶。
实验前，将两种组合物充分混合，再置于透析袋(透过分子量12000-14000Da)中，随即在不同pH的缓冲溶液中进行透析。对照组将咪喹莫特悬液直接置于透析袋(透过分子量12000-14000Da)中，在不同pH值的缓冲液中透析，监测咪喹莫特的释放情况。其中，pH7.4的缓冲溶液为加入2mM CaCl 2的磷酸缓冲液，pH4.0的缓冲溶液为醋酸-醋酸钠缓冲溶液。
咪喹莫特从海藻酸钠/钙离子水凝胶(ALG)释放的比例随时间的变化如表9所示。咪喹莫特在酸性条件下会有更快的释放速度，而在两种pH值条件下，海藻酸钠/钙离子凝胶的存在均能够明显降低咪喹莫特的释放速率，达到缓释的效果。
表9咪喹莫特从海藻酸钠/钙离子水凝胶中的释放数据
Figure PCTCN2021143057-appb-000006
Figure PCTCN2021143057-appb-000007
实施例B 助悬剂考察
实施例B1：咪喹莫特混悬液的制备
制备各种助悬剂的咪喹莫特混悬液制剂。
以含有高级脂肪酸链的表面活性剂卵磷脂为例，制备咪喹莫特混悬液。
S1：将卵磷脂制备为不同浓度的均匀悬浊液，加入咪喹莫特粉末，使咪喹莫特的浓度为1～18mg/mL，搅拌混悬液；
S2：将S1步骤得到的混悬液进行均质处理；
S3：将均质后的混悬液进行灌装，并封盖，进行高温高压灭菌处理。高温高压灭菌的条件为：110℃～145℃高温高压灭菌10～30分钟。
实施例B2：不同助悬剂咪喹莫特混悬液稳定性的影响
助悬剂的选择应基于几个因素的考量，首先，作为一种应用于注射剂型的助悬剂，首选已经批准的注射级药用辅料，从而避免助悬剂本身的安全性隐患；其次，助悬剂本身不能和药物分子发生化学反应使药物活性改变或者毒性增加。
主要从三方面判断助悬剂是否有助于咪喹莫特混悬液终端灭菌后的稳定。
其一，观察高压灭菌前后混悬液的外观变化，并根据外观变化定义样品为稳定性较好、一般、不稳定。具体的，观察是否出现肉眼可见的颗粒或结块、粘壁、无法重新分散的情况，并对相应情况进行记录。当该样品无上述现象出现时，视为该样品在灭菌后稳定性较好；当样品在灭菌后出现上述现象，但经晃动或震摇后可重新分散得到均匀混悬液时，视为该样品在灭菌后状态一般；当样品在灭菌后出现以上现象且经过不同程度的晃动或震摇后均得不到重新分散的混悬液时，视为该样品不稳定。
其二，检测实施例B1中S3步骤前后的咪喹莫特混悬液制剂中的粒径分布，检测手段为动态光散射。检测中关键参数为D50、D90。其中D50是混悬剂中颗粒的中值粒径，意为混悬液中有50％的颗粒粒径在该值以下，是表示粒度大小的经典值，常被用于表示颗粒的平均粒径；D90意为体系中有90％的颗粒粒径在该值以下。D50与D90的差值可以说明粒径分布的跨度，及粒径均一性的优劣。分析检测数据时，主要判断样品D50和D90数值的大小和在灭菌前后D50和D90的变化：D50和D90数值越大，说明颗粒分散性不好；D50和D90数 值增加越大，说明该样品稳定性越差；因此，D50和D90数值越大和这两个数值量增加越大都说明该样品中使用的助悬剂不能够有效助悬，以得到可湿热灭菌的制剂产品。
其三，将灭菌后的样品长期放置，观察样品的状态及检测样品平均粒径，如果样品仍能够重悬并D50、D90未明显增加或D90与D50差别较小，可视为该助悬剂有助于增加微米级咪喹莫特混悬液的稳定性。在本案中，长期放置的条件为2～8℃，时间为12个月。
基于以上两个评判标准，将不同样品灭菌前后、长期放置的粒径数值及现象记录如表1所示，并将不同样品状态拍照记录如图2所示。
图2中1°～9°样品中加入的不同类型表面活性剂分别为：卵磷脂、吐温-80、吐温-20、泊洛沙姆188、泊洛沙姆407、聚氧乙烯蓖麻油、维生素E聚乙二醇琥珀酸酯、油酸钠、磷脂酰甘油。
图2中1°、8°、9°样品为均匀分散的混悬液，其余样品出现不同程度的粘壁、结块甚至沉淀现象。
表10加入不同类型助悬剂的咪喹莫特混悬液在灭菌前后及灭菌后长期放置的粒径变化及现象记录表(其中，灭菌后为灭菌后到1周内；长期放置指放置12个月)。
Figure PCTCN2021143057-appb-000008
Figure PCTCN2021143057-appb-000009
Figure PCTCN2021143057-appb-000010
选取了9种表面活性剂，其中例如吐温-80和吐温-20、聚氧乙烯蓖麻油等聚氧乙烯类非离子表面活性剂，在溶液温度升高到一定程度时，会出现起昙现象，即表面活性剂与水之间的作用力被高温破坏，溶液变得不稳定，当体系温度下降至昙点以下，有些溶液会重新恢复透明，有些则不会。同样作为聚氧乙烯类表面活性剂，泊洛沙姆一般被认为具有较好的水溶性，在常压下加热不会出现昙点。但在实验中发现，使用泊洛沙姆188或泊洛沙姆407作为表面活性剂进行高压灭菌时，短期稳定性一般，长期稳定性不易控制，没有达到预期的稳定效果。综合来看，所有的非离子型表面活性剂均不能达到预期的稳定混悬液的效果，即不能帮助灭菌后的混悬液能够均匀分散。
与非离子型表面活性剂相比，离子型表面活性剂，包括阴离子型表面活性剂和两性离子型表面活性剂，作为助悬剂加入体系中，能够保证咪喹莫特混悬液灭菌后的长期稳定性。进一步分析可稳定混悬液的助悬剂的结构发现，这些离子型表面活性剂均包含高级脂肪链结构，并且，疏水端的分子量远大于亲水端。因此，选用此类含有高级脂肪链的离子型表面活性剂，能够有效地帮助微米级咪喹莫特混悬液保持终端灭菌后的稳定性。
实施例B3：其他比例的表面活性剂对咪喹莫特的稳定作用
通常情况下，在保证成药性的前提下，药物制剂的非活性成分越少，使用及储存的安全性风险越低。因此，我们进一步以卵磷脂为例，尝试使用更低比例的含有高级脂肪酸链的表面活性剂，验证助悬效果。用实施例B1的方法制备不同卵磷脂与R837微米颗粒质量比的混 悬液，R837的浓度为15mg/mL在高温高压灭菌后检测混悬液中微米颗粒的粒径，并观察混悬液稳定性状态，记录如表11所示。
表11低比例卵磷脂对R837混悬液的助悬效果评价。
Figure PCTCN2021143057-appb-000011
从结果可以看出，低比例的卵磷脂依然能够保证混悬液在高温高压灭菌后的稳定性，并且，咪喹莫特微米颗粒的粒径没有发生更大的变化，甚至较加入更高比例的离子型表面活性剂的样品，其粒径分布更加集中，即粒径更加均匀。因此，含有高级脂肪酸链的表面活性剂与咪喹莫特的的质量比可以在0.025～3：1。优选地，磷脂类离子型表面活性剂和咪喹莫特的质量比可以为0.025～1：1。
实施例B4：咪喹莫特混悬液制剂分散介质种类研究
临床上进行大体积注射液给药时，通常使用添加等渗调节剂来避免渗透压改变带来的局部组织损伤或微环境紊乱，因此，考察常用等渗调节剂对咪喹莫特混悬液制剂灭菌稳定性的影响。
使用实施例B1的方法制备咪喹莫特混悬液制剂，浓度为1mg/mL，区别在于：步骤S1中使用生理盐水或5％葡萄糖配制溶液，并与咪喹莫特微米颗粒混合均质。观察高压灭菌前后混悬液制剂的外观状态，均未发现结块情况，说明该制剂的分散介质可以直接使用生理盐水或5％葡萄糖溶液。
进一步的，扩大制备规模，并进行制剂产品稳定性的考察，湿热灭菌后未出现结块情况，且长期放置依然能够分散良好，粒径变化小，进一步验证了前述条件的可行性。
实施例C 微米颗粒的动物实验及对比
实施例C1：
本实施例说明的咪喹莫特制剂在体内的分布行为如下：
实验方法：在小鼠背部种植小鼠结肠癌(CT26)肿瘤，并将小鼠随机分为3组，每组3只小鼠做药物的分布行为研究。
第一组：小鼠进行瘤内注射小分子咪喹莫特盐酸盐，注射剂量为6mg/kg
第二组：小鼠进行瘤内注射聚乳酸-羟基乙酸共聚物(PLGA)包裹的咪喹莫特纳米颗粒(平均粒径约100nm)，注射剂量为6mg/kg；
第三组：小鼠进行瘤内注射咪喹莫特微米颗粒(本制剂)，注射剂量为6mg/kg；在注射后72h牺牲小鼠，并解剖主要器官和瘤体，检测器官和肿瘤组织中的药物含量。
实验结果：从咪喹莫特在小鼠主要器官和肿瘤组织的含量图(图3)可以看出，小分子咪喹莫特盐酸盐和咪喹莫特/PLGA纳米制剂均不能保证大部分滞留在肿瘤部位，其中小分子咪喹莫特盐酸盐在瘤内注射后72h整体的滞留量极低，咪喹莫特纳米制剂则更多的滞留在其他器官中。相较于小分子咪喹莫特盐酸盐和咪喹莫特纳米制剂，本发明中咪喹莫特微米制剂在肿瘤组织中滞留量显著提高，咪喹莫特微米制剂在瘤内滞留时间最长。说明瘤内注射咪喹莫特微米剂型更有利于后续治疗。
癌症治疗是一个非常复杂的综合结果，因为无论是机体的免疫系统，以及癌细胞的生长机制都是非常复杂的。本实验之所以能够取得比较优异的治疗效果，除了本专利其他部分的解释外，可能还包括如下原因，采用了咪喹莫特R837微米颗粒。将不溶于水的R837粉末制备为1-3微米粒径的微米颗粒，瘤内注射后监测药代动力学和瘤内滞留时间，结果如表12所示，结果显示微米颗粒能够显著延长咪喹莫特的在肿瘤部位的滞留时间和血液循环半衰期，达到了缓释的效果，从而长效地刺激免疫系统。
表12不同剂型咪喹莫特的滞留时间对比数据；
Figure PCTCN2021143057-appb-000012
实施例C2：
本实施例说明的咪喹莫特制剂在体内的药代动力学如下：
实验方法：在小鼠背部种植小鼠结肠癌(CT26)肿瘤，并将小鼠随机分为3组，每组3只做药物的药代动力学研究。
第一组：小鼠瘤内注射小分子咪喹莫特盐酸盐；注射剂量为6mg/kg，在注射后5h、6h、12h、24h、48h、72h进行静脉采血，并统一进行咪喹莫特浓度测定，检测血液中咪喹莫特浓度。
第二组：小鼠瘤内注射咪喹莫特/PLGA纳米颗粒(平均粒径约100nm)，注射剂量为6mg/kg，在注射后5h、6h、12h、24h、48h、72h进行静脉采血，并统一进行咪喹莫特浓度测定，检测血液中咪喹莫特浓度。
第三组：小鼠瘤内注射咪喹莫特微米颗粒(本制剂)，注射剂量为6mg/kg，在注射后5h、6h、12h、24h、48h、72h进行静脉采血，并统一进行咪喹莫特浓度测定，检测血液中咪喹莫特浓度。
实验结果：从血液药物浓度随时间的变化曲线(图4)及血液循环统计分析表(表)可以看出，小分子咪喹莫特盐酸盐很快被清除，其48h和72h的血药浓度已低于检测限，与咪喹莫特纳米制剂相比，微米制剂的血液循环半衰期和药物平均滞留时间更长。
实施例C3：
本实施例用于增强放射治疗的具体效果如下：
实验方法：在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(右边视为原位肿瘤，左边视为远端肿瘤)，并将荷瘤小鼠分为6组，每组6只做放疗免疫治疗联合的治疗实验。
第一组：小鼠肿瘤不做任何处理，不进行试剂的注射和放疗治疗；
第二组：小鼠原位肿瘤进行单纯放疗治疗，对小鼠原位肿瘤进行每天1次，连续5天的放射治疗，远端肿瘤不做任何处理；
第三组：对小鼠原位肿瘤进行瘤内注射小分子咪喹莫特盐酸盐，剂量为6mg/kg；在给药后第0、1、2、3、4天进行1.5Gy的放疗，对小鼠左侧肿瘤不做任何处理；
第四组：对小鼠原位肿瘤进行瘤内注射咪喹莫特的PLGA纳米颗粒(粒径100nm左右)，剂量为6mg/kg；在给药后第0、1、2、3、4天进行每次1.5Gy的放疗，小鼠远端肿瘤不做任何处理；
第五组：小鼠瘤内注射咪喹莫特微米颗粒(本制剂)，剂量为6mg/kg；给药后第0、1、2、3、4天进行每次1.5Gy的放疗，小鼠远端肿瘤不做任何处理；
第六组：小鼠瘤内注射咪喹莫特微米颗粒(本制剂)，剂量为12mg/kg；给药后第0、1、2、3、4天进行每次1.5Gy的放疗，小鼠远端肿瘤不做任何处理；
每两天用游标卡尺测量原位肿瘤和远端肿瘤的长和宽，肿瘤的体积为(长乘以(宽的 平方))除以2。
图5是咪喹莫特盐酸盐、纳米颗粒、微米颗粒注射到肿瘤内后进行放疗后，原位肿瘤的生长曲线对比图，图6是咪喹莫特盐酸盐、纳米颗粒、微米颗粒注射到肿瘤内后进行放疗后，远端肿瘤的生长曲线对比图。
治疗效果：从原位肿瘤生长曲线(图5)和远端肿瘤生长曲线(图6)可以看出，第六组小鼠的原位瘤和远端肿瘤都得到了有效的抑制，几乎不再生长，因此具有非常好的应用前景与价值。其他对应的治疗组，部分具有一定的治疗效果，也有一些实验组的治疗效果是非常有限的。所有组别小鼠的体重变化曲线如图7所示，均在正常范围，实验组小鼠与对照组小鼠体重无差异，说明本制剂具有一定的安全性。
放疗可以诱导远端效应虽然已有报道，但是这种效应并不是很显著。我们在实验中发现，将免疫佐剂注射进入肿瘤内，再用射线辐照肿瘤，可以有效增强放疗诱导的免疫原性细胞死亡；其体现的效果是一方面可以提升放疗对原位肿瘤的疗效，另一方面获得更强的远端效应，抑制远端没有被照射肿瘤的生长。
实施例C4：
本实施例用于肿瘤微波消融联合治疗的效果如下：
实验方法：在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(右边视为原位肿瘤，左边视为远端肿瘤)，并将荷瘤小鼠分为3组，每组5只做微波治疗免疫治疗联合的治疗实验。
第一组：小鼠肿瘤不做任何处理，不进行试剂的注射和微波治疗；
第二组：小鼠原位肿瘤进行单纯微波热消融治疗，微波功率为7W，肿瘤局部温度达到53℃，左侧肿瘤不做任何处理；
第三组：对小鼠右侧肿瘤进行瘤内注射咪喹莫特微米制剂，剂量为6mg/kg；在给药后进行微波热消融治疗，微波功率为7W，肿瘤局部温度达到53℃，对小鼠左侧肿瘤不做任何处理；
每两天用游标卡尺测量原位肿瘤和远端肿瘤的长和宽，肿瘤的体积为(长乘以(宽的平方))除以2。
治疗效果：从原位肿瘤生长曲线(图8)和远端肿瘤生长曲线(图9)可以看出，第三组小鼠肿瘤的双侧肿瘤都受到了很好的抑制，而单纯进行微波热消融治疗的小鼠，只有原位瘤被消灭，远端肿瘤仍然在生长，说明本发明的咪喹莫特微米制剂对微波热消融疗法的远端效应增益明显。
实施例C5：
本实施例用于肿瘤化疗免疫治疗联合治疗的效果如下：
实验方法：在小鼠背部左右两端分别种植小鼠结肠癌CT26肿瘤(右边视为原位肿瘤，左边视为远端肿瘤)，并将荷瘤小鼠分为3组，每组5只做化疗免疫治疗联合的治疗实验。
第一组：小鼠原位肿瘤瘤内注射生理盐水，远端瘤不做任何处理；
第二组：小鼠原位肿瘤瘤内注射奥沙利铂化疗药物，远端瘤不做任何处理；
第三组：小鼠原位肿瘤瘤内注射奥沙利铂化疗药物和咪喹莫特微米制剂混合物，远端瘤不做任何处理；
定期测量小鼠中立的长和宽，肿瘤的体积为(长乘以(宽的平方))除以2。
治疗效果：从原位肿瘤生长曲线(图10)和远端肿瘤生长曲线(图11)可以看出，化疗药物的使用能够抑制原位肿瘤的生长，但对远端肿瘤的生长抑制不明显，加入咪喹莫特微米制剂后，能够有效抑制远端肿瘤的生长，说明本发明所述咪喹莫特微米制剂能够有效增强化疗的远端效应。
实施例D 动物实验
实施例D1：生物分布
在小鼠背部接种CT26肿瘤细胞，建立小鼠CT26皮下瘤模型，将肿瘤大小一致的小鼠平均分为3组，每组3只小鼠，分组情况如下：
PLGA-R837：装载R837的PLGA纳米颗粒；
R837·HCl：R837盐酸盐的水溶液，该体系中R837分散性极好；
咪喹莫特混悬液制剂：本发明所述经终端灭菌的咪喹莫特微米级混悬液(助悬剂为卵磷脂)。
根据分组，向每组小鼠瘤内注射含有相同剂量R837的不同制剂，按照一般方法，研究其注射后72h内的药代动力学特征，统计不同组别小鼠血液中药物浓度的达峰时间(T max)、达峰浓度(C max)和半衰期(t 1/2)，结果如表13所示。
C max和T max反映药物从某制剂吸收进入全身血循环的速度，三种剂型的达峰时间(T max)一致，但是，PLGA纳米颗粒包裹的R837很快就在血液中大量暴露，而瘤内注射微米级咪喹莫特混悬液制剂后，未出现短时间内药物的大量暴露；此外，三种剂型的血液循环半衰期相差甚远，与纳米制剂和小分子制剂相比，微米级咪喹莫特混悬剂的半衰期显著延长，即微米级咪喹莫特混悬液经瘤内给药的方式施用，具有明显的缓释效果。
表13不同组别小鼠的药代动力学参数均值统计表。
Figure PCTCN2021143057-appb-000013
免疫佐剂在肿瘤内的长滞留，理论上能够更有效地刺激抗肿瘤免疫反应，为证明缓释剂型的抗肿瘤免疫增强效果，设计实验验证微米级咪喹莫特混悬液在肿瘤外放射或酒精消融疗法联合免疫治疗中的应用。
实施例E 微米级咪喹莫特混悬液增效放疗、酒精消融疗法
实施例E1：微米级咪喹莫特混悬液制剂联合放疗的治疗实验
在小鼠背部接种CT26结肠癌肿瘤细胞，建立小鼠结肠癌皮下双肿瘤模型，分别为原位肿瘤和远端肿瘤，当原位肿瘤体积约100mm 3时，将小鼠随机分为6组。分组如下：
Vehicle：溶媒对照组，对原位瘤进行瘤内注射分散介质的操作，注射体积为25μL；
RT：外放射治疗组，对原位瘤进行X射线辐照处理，放射剂量为4Gy，分别于治疗开始的当天和第3天进行；
R837：本发明所述咪喹莫特微米级混悬液制剂，其中R837的浓度为6mg/mL，注射剂量为25μL；
R837+RT：对原位瘤进行瘤内注射25μL的6mg/mL咪喹莫特微米级混悬液制剂后，进行与RT组相同的X射线辐照处理。
在R837+RT组别中，对原位肿瘤进行瘤内注射咪喹莫特混悬液制剂后半小时，对每只小鼠的原位肿瘤进行外放射治疗，远端肿瘤不做任何处理，监测小鼠原位肿瘤和远端肿瘤的体积，并制作肿瘤生长曲线，结果如图12(原位瘤生长曲线)和图13(远端瘤生长曲线)所示，并计算抑瘤率，结果如表14所示。表14为原位肿瘤和远端肿瘤的抑瘤率统计表。根据金氏公式q＝E(A+B)/(EA+EB-EA*EB)计算药物协同作用，其中E(A+B)为联合治疗组的抑瘤率，EA和EB分别为两种手段单独使用时的抑瘤率，当q≥1时，说明两种手段具有协同效果。计算可得，原位瘤的q值为1.17，远端瘤的q值为1.63，均具有协同效果。
同时，从图中可以观察到，多次放疗可以在一定程度上抑制肿瘤生长，瘤内注射咪喹莫特，能够进一步提高肿瘤放射治疗效果。微米级咪喹莫特混悬液制剂，由于其在肿瘤部位的 长滞留，和在体内的高生物利用度，刺激了最强烈的系统性抗肿瘤免疫反应，远端肿瘤生长被抑制，与外放射治疗手段达到了协同效果。
表14咪喹莫特混悬液制剂联合放疗治疗小鼠皮下肿瘤模型，治疗终点的抑瘤率。
组别 溶媒对照组 RT R837 RT+R837
原位瘤 0 71.15％ 38.73％ 96.44％
远端瘤 0 29.67％ 17.23％ 68.14％
综上，微米级咪喹莫特混悬液制剂可以和外放射治疗联合，增强体内抗肿瘤免疫反应，尤其是放大了放疗中的远端效应，抑制远端肿瘤的生长。
实施例E2：微米级咪喹莫特混悬液制剂联合酒精消融的治疗实验。
酒精消融是肿瘤局部化学消融疗法的一种，通过将无水乙醇注射到肿瘤内部，使肿瘤组织凝固坏死，从而达到治疗的目的。但是单纯通过注射酒精或盐酸等化学消融手段，在不影响周边正常组织的剂量下，很难做到肿瘤的完全清除。本实施例中，将微米级咪喹莫特混悬液制剂联合酒精消融疗法，证明本发明所述微米级咪喹莫特混悬液制剂联合化学疗法的抗肿瘤效果。
首先建立小鼠皮下肿瘤模型。具体地，将肿瘤细胞接种至小鼠背部，待肿瘤体积生长至100mm 3时，将小鼠随机分为5组，每组5只小鼠，分组情况如下：
Control：空白对照组；
R837：瘤周注射微米级咪喹莫特混悬液；
ETOH：瘤内注射无水乙醇；
ETOH+R837(25)：瘤周注射25μL微米级咪喹莫特混悬液，瘤内注射无水乙醇；
ETOH+R837(50)：瘤周注射50μL咪喹莫特混悬液，瘤内注射无水乙醇。
其中微米级咪喹莫特混悬液的浓度为12mg/mL，无水乙醇的注射剂量为30μL。联合治疗组中，先将微米级咪喹莫特混悬液制剂注射在肿瘤周围的皮下部位，间隔10分钟左右，再将无水乙醇通过瘤内注射的方式进行给药。监测小鼠肿瘤体积变化，制作肿瘤生长曲线，结果如图14所示。
在实验中发现，仅施用无水乙醇组的小鼠，其肿瘤中心部位出现肿瘤纤维化并结痂，但由于外围肿瘤组织未被完全消除，后逐渐发展，外径继续增加，导致该组别肿瘤体积的数值与对照组差异不大，因此肿瘤生长曲线中，酒精消融组与空白对照组的肿瘤生长曲线几乎重合。除该特殊情况外，可从图14中看出，联合施用微米级咪喹莫特混悬剂和酒精消融组别的小鼠肿瘤生长被明显抑制，施用不同剂量的微米级咪喹莫特，均能提高酒精消融的疗效，达 到更好的肿瘤治疗效果，抑制肿瘤生长，说明微米级咪喹莫特混悬剂能够增强肿瘤的酒精消融治疗效果。
实施例F 咪喹莫特混悬液可帮助实现铂类化药的缓释，增强免疫原性细胞死亡引起的抗肿瘤免疫反应的效果
实施例F1：验证奥沙利铂与咪喹莫特混悬液混合使用，对奥沙利铂的缓释效果
在小鼠肿瘤背部接种结肠癌(CT26)肿瘤细胞，建立小鼠皮下肿瘤模型，待接种部位形成肿块约一周，小鼠随机分为两组，分别为：
OXA：奥沙利铂溶液
OXA-R837：与微米级咪喹莫特混悬液混合后的奥沙利铂溶液
对应地，向每组小鼠瘤内注射奥沙利铂溶液或混合有微米级咪喹莫特混悬液制剂的奥沙利铂溶液，然后在不同时间点(10min、30min、1h、3h、6h、9h、12h、24h、48h、72h)取小鼠血液样本，并于终点处死小鼠，获得主要器官及肿瘤，用电感耦合等离子质谱(ICP-MS)检测血液样本和脏器中铂离子的相对含量并制作统计图。结果如图15、图16所示。
图15是奥沙利铂的生物分布情况，较游离的奥沙利铂相比，与咪喹莫特微米级混悬液预混后进行给药，能够显著增加铂药在肿瘤部位的滞留量，注射药物72h后，混合注射组中，小鼠肿瘤部位的铂含量是游离组中的数十倍，说明咪喹莫特微米级混悬液能够增加铂类化药在肿瘤部位的滞留，减缓奥沙利铂的释放。
图16是奥沙利铂在血液中的浓度随时间变化的情况。与游离的奥沙利铂药物溶液相比，混合了微米级咪喹莫特混悬液的奥沙利铂，缓释效果更明显，具体表现在，达峰浓度更低、达峰时间更迟，在血液中存在的时间更长。具体的达峰时间(Tmax)、达峰浓度(Cmax)及曲线下面积(AUC)情况如表14所示，与微米级咪喹莫特混悬液混合后，延长了奥沙利铂在循环系统中的暴露时间，增加了奥沙利铂的生物利用度。
表14奥沙利铂药代动力学数据
组别 T max(h) C max(ng/g) AUC all
OXA 3 638.685 7850.866
OXA+R837 6 565.728 10438.525
实施例F2：奥沙利铂与微米级咪喹莫特混悬液制剂联合使用增效抗肿瘤效果
在小鼠背部左右两侧分别接种不同数量(左侧是右侧接种量的1/5，)结肠癌(CT26)肿瘤细胞，建立小鼠皮下双边肿瘤模型，右侧视为原位肿瘤，左侧视为远端肿瘤。待原位肿瘤大小约100mm 3，将小鼠随机分为4组，分别进行治疗。分组情况如下：
VEHICAL：溶媒组，注射生理盐水30μL
OXA：注射奥沙利铂溶液30μL
R837：注射微米级咪喹莫特混悬液30μL
OXA+R837：注射微米级咪喹莫特混悬液和奥沙利铂混合后的混悬液30μL
在治疗第一天进行原位肿瘤的瘤内注射给药，其中，奥沙利铂的浓度为4mg/mL，咪喹莫特的浓度为6mg/mL，给药后记录小鼠肿瘤体积并制作肿瘤生长曲线，结果如图17(原位肿瘤生长曲线)、图18(远端肿瘤生长曲线)所示。图17为小鼠原位肿瘤生长曲线，经过计算，治疗组的抑瘤率如表15所示。
表15治疗后第15天不同组别的抑瘤率
组别 VEHICAL OXA R837 OXA+R837
原位瘤 0 69.56％ 43.94％ 86.26％
远端瘤 0 41.87％ 22.94％ 69.95％
根据金氏公式q＝E(A+B)/(EA+EB-EA*EB)计算药物协同作用，其中E(A+B)为联合治疗组的抑瘤率，EA和EB分别为两种组分单独使用时的抑瘤率，当q≥1时，说明两种成分具有协同效果。计算得到原位瘤抑瘤率的q约为1.1，远端瘤的q约为1.27，说明微米级咪喹莫特混悬液具有协同增效奥沙利铂化疗的效果。
同时，奥沙利铂能够引起肿瘤免疫原性死亡，咪喹莫特的加入会增强抗肿瘤免疫效果，引起全身抗肿瘤免疫反应，从而抑制远端肿瘤生长，与单独使用每一种组分相比，奥沙利铂和咪喹莫特同时进行瘤内注射后能够有效抑制远端肿瘤的生长，图18中的联合治疗组小鼠肿瘤生长最慢且组内差异越小。
实施例G 咪喹莫特微米级混悬液制剂对蒽环类化药缓释作用的考察
实施例G1：咪喹莫特微米级混悬液制剂与多柔比星(DOX)混合后，多柔比星的体外释放实验。
分组及样品制备：
1)DOX：多柔比星的水溶液，浓度为3mg/mL；
2)DOX+R837：用1mL咪喹莫特微米级混悬液制剂(含卵磷脂且已灭菌完成的样品，其中咪喹莫特的浓度为12mg/mL)溶解3mg多柔比星。
具体实验步骤：将配制好的各组溶液分别加入透析袋(截留分子量为3500D)中，置于500mL的PBS溶液中透析，在不同时间点检测透析液中药物浓度，检测手段为检测药物所在波段的紫外吸光度，计算药物含量，并与起始药物含量做比值，制作不同样品透析液的药物相对含量变化曲线，结果如图19所示。其中微米级咪喹莫特混悬液与多柔比星混合后，其药物释放曲线趋势明显减缓，说明微米级咪喹莫特混悬液能够显著降低药物的释放速率，达到缓释的效果。体外模拟释放实验能够在一定程度上反应药物在体内的行为，说明微米级咪喹莫特混悬液制剂与多柔比星预混后注射，能够减缓药物在原位注射后的释放速率，能够延长化疗药物在肿瘤部位的滞留时间、增加药物在肿瘤部位的滞留量，从而加强药物在肿瘤部位的作用效果、降低药物对系统的毒副作用。
实施例G2：改变咪喹莫特微米级混悬液制剂和多柔比星的混合比例，验证缓释效果。
分组及样品制备：
1)DOX：多柔比星的水溶液，浓度为3mg/mL；
2)DOX+R837：用微米级咪喹莫特混悬液制剂溶解多柔比星，咪喹莫特混悬液制剂的浓度为12mg/mL，多柔比星的终浓度为3mg/mL；
3)DOX+R837(1/3)：用微米级咪喹莫特混悬液制剂溶解多柔比星，咪喹莫特混悬液制剂的浓度为4mg/mL，多柔比星的终浓度为3mg/mL。
用与实施例G1相同的检测方法和数据处理方式绘制三组的药物释放曲线，结果如图20所示。降低了咪喹莫特微米级混悬液的用量之后，药物的释放速率有所增加，但相较于单纯的多柔比星的水溶液，释放速率仍然减缓，进一步说明微米级咪喹莫特混悬液制剂能够减缓多柔比星的释放，该缓释效果与两者的比例相关，微米级咪喹莫特混悬液制剂的浓度越高，缓释效果越好，多柔比星与咪喹莫特的浓度比例为1：1～1：18。
实施例G3：表柔比星(EPI)与微米级咪喹莫特混悬液制剂混合后的体外释放实验。
分组及样品制备：
1)EPI：表柔比星的水溶液，浓度为2mg/mL；
2)EPI+R837：用微米级咪喹莫特混悬液制剂溶解表柔比星，表柔比星的终浓度为2mg/mL；
与实施例G1相同，将样品至于透析袋(截留分子量为3500D)中，缓释体系为500mL的PBS溶液，检测不同时间点的药物释放量，计算释放百分比并绘制药物释放曲线，结果如 图21所示。结果显示，在体外释放实验的早期(6h之前)，不同组别的药物释放趋势近似，但随着时间的延长，与微米级咪喹莫特混悬液制剂混合的表柔比星释放减缓。
与多柔比星的缓释效果相比，表柔比星的缓释效果略有降低，发明人推测认为，混合液中的蒽环类化药可能与咪喹莫特微米颗粒之间形成了一定的π-πstacking作用力，而多柔比星和表柔比星互为异构体，从分析结构上看，多柔比星容易和咪喹莫特形成更稳定的π-πstacking作用力，因此会表现出更强的缓释作用。
当药物与混悬剂的相对浓度过高时，多余的药物处于游离的状态，该部分药物会在短时间内快速释放，而通过与咪喹莫特微米颗粒之间的π-π相互作用稳定的化药，其药物代谢的行为变化与微米级咪喹莫特颗粒相关联，瘤内注射混合液后，化药与咪喹莫特微米级颗粒的药代动力学相似，滞留增强，释放变缓。
实施例G4：磷脂酰甘油作为助悬剂得到的微米级咪喹莫特混悬液制剂对表柔比星的缓释作用
分组及样品制备：
EPI：表柔比星的水溶液，浓度为2mg/mL；
EPI+R837(0.25PG)：用磷脂酰甘油助悬得到的微米级咪喹莫特混悬液制剂溶解表柔比星，其中磷脂酰甘油和咪喹莫特的质量比为0.25：1，表柔比星的终浓度为2mg/mL；
EPI+R837(3PG)：用磷脂酰甘油助悬得到的微米级咪喹莫特混悬液制剂溶解表柔比星，其中磷脂酰甘油和咪喹莫特的质量比为3：1，表柔比星的终浓度为2mg/mL。
同实施例G1相似的办法，用透析实验检测药物的释放情况，结果如图22所示，微米级咪喹莫特混悬液制剂溶解的表柔比星，药物溶出速率明显放缓，24小时药物释放量，仅为对照组的1/4左右，增加磷脂酰甘油的比例，药物的释放更加缓慢，磷脂酰甘油表面的负电荷与表柔比星表面的正电荷形成了静电作用力，因此表现出更强的缓释效果。
对所公开的实施例的上述说明，使得本技术领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对于本领域技术人员而言将是显而易见的。本发明不会被限制于本文所示的这些实施例，只需要符合与本文所公开的原理与特点一致即可。

Claims (45)

  1. 一种自缓释免疫佐剂混悬液，其由脂溶性免疫佐剂和表面活性剂组成，余量为分散介质，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散介质中形成混悬液。
  2. 根据权利要求1所述的自缓释免疫佐剂混悬液，其中所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
  3. 根据权利要求1-2中任一项所述的自缓释免疫佐剂混悬液，其中所述脂溶性免疫佐剂颗粒为粒径为0.5-5微米的核壳复合颗粒。
  4. 根据权利要求1-3中任一项所述的自缓释免疫佐剂混悬液，其中所述脂溶性免疫佐剂核壳复合微米颗粒的粒径为1-2微米。
  5. 根据权利要求1-4中任一项所述的自缓释免疫佐剂混悬液，其中所述脂溶性免疫佐剂为咪喹莫特微米颗粒。
  6. 根据权利要求5所述的自缓释免疫佐剂混悬液，其中所述咪喹莫特微米颗粒的平均粒径为0.5～5微米。
  7. 根据权利要求1-6中任一项所述的自缓释免疫佐剂混悬液，其中所述表面活性剂为含有高级脂肪酸链的表面活性剂。
  8. 根据权利要求7所述的自缓释免疫佐剂混悬液，其中所述含有高级脂肪酸链的表面活性剂包括阴离子型表面活性剂。
  9. 根据权利要求8所述的自缓释免疫佐剂混悬液，其中所述阴离子型表面活性剂包括油酸钠、十二烷基硫酸钠、硬脂酸钠、N-月桂酰肌胺酸钠、椰油酰基甲基牛磺酸钠、N-月桂酰基谷胺酸钠、月桂醇聚氧乙烯醚羧酸钠、十二烷基磷酸酯的至少一种。
  10. 根据权利要求7所述的自缓释免疫佐剂混悬液，其中所述含有高级脂肪酸链的表面活性剂包括两亲性离子型表面活性剂。
  11. 根据权利要求7所述的自缓释免疫佐剂混悬液，其中所述含有高级脂肪酸链的表面活性剂包括磷脂类离子型表面活性剂。
  12. 根据权利要求11所述的自缓释免疫佐剂混悬液，其中所述磷脂类离子型表面活性剂包括卵磷脂、大豆磷脂、磷脂酰甘油、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇中的至少一种。
  13. 根据权利要求1-6中任一项所述的自缓释免疫佐剂混悬液，其中所述表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。
  14. 根据权利要求13所述的自缓释免疫佐剂混悬液，其中所述表面活性剂包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338，泊洛沙姆407中的至少一种。
  15. 根据权利要求1-6中任一项所述的自缓释免疫佐剂混悬液，其中所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链。
  16. 根据权利要求15所述的自缓释免疫佐剂混悬液，其中所述表面活性剂包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000，卵磷脂中的至少一种。
  17. 根据权利要求1-6中任一项所述的自缓释免疫佐剂混悬液，其中所述表面活性剂为两种亲水亲油平衡值不同的表面活性剂的混合物。
  18. 根据权利要求1-12、16-17中任一项所述的自缓释免疫佐剂混悬液，其包括咪喹莫特混悬液制剂，所述咪喹莫特混悬液制剂包括：咪喹莫特微米颗粒、含有高级脂肪酸链的表面活性剂和分散介质。
  19. 根据权利要求18所述的自缓释免疫佐剂混悬液，其中所述咪喹莫特微米颗粒的浓度为1～18mg/mL，所述含有高级脂肪酸链的表面活性剂与咪喹莫特微米颗粒的质量比为0.025～3：1。
  20. 根据权利要求19所述的自缓释免疫佐剂混悬液，其中所述含有高级脂肪酸链的表面活性剂与咪喹莫特微米颗粒的质量比为0.1～1：1。
  21. 一种自缓释免疫佐剂混悬液的制备方法，其包括如下步骤：
    S1：将表面活性剂和脂溶性免疫佐剂分散在同一分散体系中，搅拌得到混悬液；
    S2：对上述制得的混悬液进行均质处理/进行高剪切工艺处理；
    S3：对均质后/高剪切工艺处理后的混悬液进行灭菌处理。
  22. 根据权利要求21所述的制备方法，其包括如下步骤：
    S1：将脂溶性免疫佐剂通过气流粉碎工艺形成初级微米级粉体；
    S2：向S1步骤获得的脂溶性免疫佐剂初级微米级粉体中按照脂溶性免疫佐剂：表面活性剂质量比(1：0.025～5)加入表面活性剂的水溶液，进行高压均质工艺处理，处理结束后取出匀浆；或
    S2’：向S1步骤获得的脂溶性免疫佐剂微米粉体中按脂溶性免疫佐剂：表面活性剂质量比(1：0.025～5)加入表面活性剂的水溶液，进行高剪切工艺处理，处理结束后取出匀浆；
    S3：高压灭菌处理。
  23. 根据权利要求22所述的制备方法，其中步骤S2中所述的表面活性剂包括两种溶解度不 同表面活性剂。
  24. 根据权利要求22所述的制备方法，其中所述高压灭菌处理的条件为105℃～150℃下处理10-20分钟。
  25. 一种咪喹莫特混悬液制剂的制备方法，其包括如下步骤：
    S1：将含有高级脂肪酸链的表面活性剂和咪喹莫特微米颗粒分散在同一分散体系中，搅拌得到混悬液；
    S2：将上述制得的混悬液进行均质处理；
    S3：对均质后的混悬液进行灌装，封闭后进行高温高压灭菌。
  26. 根据权利要求25所述的制备方法，其中所述高温高压灭菌的条件为：110～145℃，5～30min。
  27. 权利要求21-26中任一项所述的制备方法获得的自缓释免疫佐剂混悬液在制备肿瘤辅助治疗药物中的应用。
  28. 一种自缓释免疫佐剂组合物，其包括第一组合物和第二组合物；所述第一组合物由脂溶性免疫佐剂和表面活性剂组成，余量为分散剂，所述表面活性剂包覆所述脂溶性免疫佐剂形成微米级颗粒，并分散在所述分散剂中形成混悬液；所述第二组合物包括易溶性海藻酸盐和保护填充剂，形成的冻干粉。
  29. 根据权利要求28所述的自缓释免疫佐剂组合物，其中所述脂溶性免疫佐剂包括咪喹莫特(R837)、雷西莫特(R848)或吡喃葡糖苷脂质A(MPLA)中的至少一种。
  30. 根据权利要求28所述的自缓释免疫佐剂组合物，其中所述表面活性剂的疏水结构部分含不少于20个的氧丙烯基单元。
  31. 根据权利要求30所述的自缓释免疫佐剂组合物，其中所述表面活性剂包括泊洛沙姆188，泊洛沙姆237，泊洛沙姆338和泊洛沙姆407中的至少一种。
  32. 根据权利要求28所述的自缓释免疫佐剂组合物，其中所述表面活性剂的疏水结构部分含总数不少于15个碳原子的一条或多条碳氢链。
  33. 根据权利要求32所述的自缓释免疫佐剂组合物，其中所述表面活性剂包括倍半油酸山梨坦，大豆磷脂，单硬脂酸甘油酯，聚山梨酯40，聚山梨酯60，聚山梨酯65，聚山梨酯80，聚山梨酯85，硬脂山梨坦(司盘60)，硬脂酸盐，维生素E聚琥珀酸乙二醇酯，聚氧乙烯烷基醚，硬脂酸聚氧乙烯酯，硬脂酸聚烃氧(40)酯，蔗糖硬脂酸酯，聚氧乙烯蓖麻油衍生物，聚西托醇1000和卵磷脂中的至少一种。
  34. 根据权利要求28所述的自缓释免疫佐剂组合物，其中所述表面活性剂为两种亲水亲油平衡值不同的表面活性剂的混合物。
  35. 根据权利要求28-34中任一项所述的自缓释免疫佐剂组合物，其中所述分散剂为水或生理盐水。
  36. 根据权利要求28-35中任一项所述的自缓释免疫佐剂组合物，其中所述保护填充剂为甘露醇或乳糖。
  37. 根据权利要求28-36中任一项所述的自缓释免疫佐剂组合物，所述第二组合物还包括pH调节剂。
  38. 权利要求1-20中任一项所述的自缓释免疫佐剂混悬液或权利要求28-37中任一项所述的自缓释免疫佐剂组合物在制备抗肿瘤联合免疫治疗制剂中的应用。
  39. 根据权利要求38所述的应用，其中所述自缓释免疫佐剂包含咪喹莫特混悬液制剂。
  40. 根据权利要求39所述的应用，其中所述咪喹莫特混悬液制剂与铂类化药预混，协助铂类化药的缓释。
  41. 根据权利要求39所述的应用，其中所述咪喹莫特混悬液制剂与蒽环类化药预混，协助蒽环类化药的缓释。
  42. 权利要求1-20中任一项所述的自缓释免疫佐剂混悬液或权利要求28-37中任一项所述的自缓释免疫佐剂组合物在制备放疗增敏剂中的应用。
  43. 权利要求1-20中任一项所述的自缓释免疫佐剂混悬液或权利要求28-37中任一项所述的自缓释免疫佐剂组合物在制备化疗增敏剂中的应用。
  44. 权利要求1-20中任一项所述的自缓释免疫佐剂混悬液或权利要求28-37中任一项所述的自缓释免疫佐剂组合物在制备热疗增敏剂中的应用。
  45. 权利要求1-20中任一项所述的自缓释免疫佐剂混悬液或权利要求28-37中任一项所述的自缓释免疫佐剂组合物在制备酒精消融增敏剂中的应用。
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