WO2021056815A1 - 一种化疗免疫组合药物及其制备方法 - Google Patents

一种化疗免疫组合药物及其制备方法 Download PDF

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WO2021056815A1
WO2021056815A1 PCT/CN2019/122492 CN2019122492W WO2021056815A1 WO 2021056815 A1 WO2021056815 A1 WO 2021056815A1 CN 2019122492 W CN2019122492 W CN 2019122492W WO 2021056815 A1 WO2021056815 A1 WO 2021056815A1
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mixture
imiquimod
immune
sodium alginate
chemotherapeutic
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English (en)
French (fr)
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刘庄
许欢
陶惠泉
巢宇
赵琪
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苏州百迈生物医药有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/555Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/66Phosphorus compounds
    • A61K31/675Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of drugs for the treatment of tumors, in particular to a drug composition for chemotherapy and immunity combined therapy, as well as a preparation method and application.
  • Chemotherapy is currently one of the three main treatment methods for clinical treatment of tumors. Most cancer patients need to receive a certain degree of chemotherapy. For those tumors that are prone to metastasis or have metastasized, chemotherapy is the main treatment method. However, traditional chemotherapeutics also damage normal organs, and the commonly used clinical chemotherapy modes are systemic administration, which is not very selective for the lesions, and the side effects of chemotherapy are very large.
  • the immune checkpoint blockade represents the encouraging achievements of tumor immunotherapy in recent years, this therapy still has important limitations, including low clinical response rate (about 20%) and non-specific immune response. Side effects etc.
  • the current low clinical response rate of clinical immune checkpoint blocking therapy means that most patients do not respond to this expensive therapy.
  • the technical problem to be solved by the present invention is to provide a new type of chemotherapeutic immune drug composition, which can produce a synergistic anti-cancer effect, and reduce side effects, reduce the probability of cancer metastasis, and reduce the probability of cancer recurrence. It can effectively kill tumors in situ while inhibiting the immune response, reducing the growth of distant metastatic tumors and the probability of tumor recurrence. At the same time, the production process is optimized and the product stability is good.
  • the present invention provides the following technical solutions:
  • a chemotherapeutic and immune combination drug which contains a chemotherapeutic drug that can cause immunogenic death and an immune adjuvant, characterized in that: the chemotherapy and immune combination drug comprises a first mixture and a second mixture, and the first mixture contains an immune adjuvant
  • the second mixture contains a chemotherapeutic agent that can cause immunogenic death; the chemotherapeutic agent that can cause immunogenic death is oxaliplatin, and the immunological adjuvant is imiquimod R837, which also includes Poloxamer 188 and sodium alginate ALG;
  • the first mixture is that the imiquimod R837 and poloxamer 188 are mixed and ball milled to obtain a uniformly dispersed imiquimod emulsion, and the imiquimod particles have a particle size of 0.5-3 microns.
  • the special emulsion is sterilized by high temperature, humidity and heat;
  • the second mixture is that the sodium alginate ALG and oxaliplatin are stirred and mixed with water, and filtered through a micron filter membrane to sterilize the mixture to prepare a mixture;
  • the first mixture and the second mixture are mixed to form a chemotherapy and immune combination drug.
  • a chemotherapeutic immune combination drug which contains a chemotherapeutic drug that can cause immunogenic death and an immune adjuvant:
  • the chemotherapy and immune combination drug includes a first mixture and a second mixture, the first mixture contains an immune adjuvant, and the The second mixture contains a chemotherapeutic agent that can cause immunogenic death;
  • the chemotherapeutic agent that can cause immunogenic death is oxaliplatin, the immunological adjuvant is imiquimod R837, and also includes poloxamer 188 and sodium alginate ALG;
  • the first mixture is that the imiquimod R837 and poloxamer 188 are mixed and ball milled to obtain a uniformly dispersed imiquimod emulsion.
  • the imiquimod particles have a particle size of 0.5-3 microns.
  • Mott emulsion, oxaliplatin and water are mixed and stirred evenly and sterilized by high temperature and humidity;
  • the second mixture is that the sodium alginate is mixed with water, and then filtered and sterilized by a micron filter membrane to prepare a mixture;
  • the first mixture and the second mixture are mixed to form a chemotherapy and immune combination drug.
  • the mass ratio of imiquimod R837 and poloxamer 188 is 1: (0.1-5), and the high temperature sterilization is 105° C. to 150 °C humid heat sterilization for 10-15 minutes;
  • the second mixture is filtered and sterilized through a 0.22 micron filter membrane, and lyophilized to form a lyophilized powder.
  • the mass ratio of imiquimod R837 and poloxamer 188 is 1: (0.1-5), and the high temperature sterilization is 105° C. to 150 °C humid heat sterilization for 10-15 minutes;
  • the second mixture is filtered and sterilized through a 0.22 micron filter membrane, and lyophilized to form a lyophilized powder.
  • a chemotherapeutic immune combination drug which contains a chemotherapeutic drug capable of causing immunogenic death and an immune adjuvant
  • the chemotherapy and immune combination drug comprises a first mixture and a second mixture, the first mixture contains the immune adjuvant, the The second mixture contains chemotherapeutics that can cause immunogenic death;
  • the first mixture is that the immune adjuvant is imiquimod, imiquimod and surfactant are mixed and ball milled to obtain a uniformly dispersed imiquimod emulsion, and the imiquimod particles are 0.5-300 microns Particle size, imiquimod emulsion is sterilized by high temperature, humid heat, and the surfactant is poloxamer 407, or polysorbate 80 (Tween 80), or polyethylene glycol-12-hydroxystearate (Solutol HS 15), or egg yolk lecithin, or polyoxyethylene (35) castor oil, or vitamin E succinate polyethylene glycol ester, or one or more of sodium hydroxymethyl cellulose;
  • the second mixture is a mixture of sodium alginate and oxaliplatin, which is stirred and mixed with water, and filtered through a micron filter membrane to sterilize the mixture;
  • the first mixture and the second mixture are mixed to form a chemotherapy and immune combination drug.
  • the sodium alginate is replaced with chitosan, or fibrinogen, or alginate, or hyaluronic acid;
  • the imiquimod R837 is replaced with imidazoquinoline, or glucopyranoside lipid
  • oxaliplatin Oxa is replaced with anthracyclines, or cyclophosphamide, or bortezomib, or gemcitabine, or pentafluorouracil, or toxin.
  • a chemotherapeutic and immune combination drug which contains a chemotherapeutic drug that can cause immunogenic death and an immune adjuvant, characterized in that: the chemotherapy and immune combination drug comprises a first mixture and a second mixture, and the first mixture contains an immune adjuvant The second mixture contains a chemotherapeutic drug that can cause immunogenic death;
  • the first mixture is that the imiquimod R837 and the surfactant are mixed and ball milled to obtain a uniformly dispersed imiquimod emulsion.
  • the imiquimod particles have a particle size of 0.5-3 microns.
  • the emulsion is mixed with oxaliplatin and water and stirred evenly and then sterilized by high temperature and humidity;
  • the surfactant is poloxamer 407, or polysorbate 80 (Tween 80), or polyethylene glycol-12-hydroxystearate (Solutol HS 15), or egg yolk lecithin, or polyoxyethylene One or more of ethylene (35) castor oil, or vitamin E succinate polyethylene glycol ester, or sodium hydroxymethyl cellulose;
  • the second mixture is that the sodium alginate is mixed with water, and then filtered and sterilized by a micron filter membrane to prepare a mixture;
  • the first mixture and the second mixture are mixed to form a chemotherapy and immune combination drug.
  • the sodium alginate can be replaced with chitosan, or fibrinogen, or alginate, or hyaluronic acid;
  • the imiquimod R837 can be replaced with imidazoquinoline, or glucopyranoside lipid;
  • the oxaliplatin Oxa can be replaced with anthracyclines, or cyclophosphamide, or bortezomib, or gemcitabine, or pentafluorouracil, or toxin.
  • a method for preparing a chemotherapy and immune combination drug which is characterized in that it comprises the following steps:
  • the first step Weigh imiquimod R837 and surfactant poloxamer 188 according to the ratio 1: (0.1-5), add water ball mill for 2-3 hours, take out the homogenate after completion, add water, stir and mix, 105 °C ⁇ 150°C damp heat sterilization for 10-15 minutes;
  • Step 2 Weigh sodium alginate ALG and oxaliplatin, add water, stir, filter and sterilize the obtained solution through a micron filter membrane; after pre-cooling, perform freeze-drying;
  • the third step Add the second lyophilized powder of the mixture to the first solution of the mixture, shake and mix well, and then inject.
  • a chemotherapeutic and immune combination drug for the treatment of colon cancer tumors which contains a chemotherapeutic drug and an immune adjuvant that can cause immunogenic death, and is characterized in that: the chemotherapy and immune combination drug comprises a first mixture and a second mixture.
  • One mixture contains an immunological adjuvant, and the second mixture contains a chemotherapeutic drug that can cause immunogenic death;
  • the first mixture is that the immune adjuvant is imiquimod, imiquimod and surfactant are mixed and ball milled to obtain a uniformly dispersed imiquimod emulsion, and the imiquimod particles are 0.5-3 microns Particle size, imiquimod emulsion is sterilized by high temperature, humid heat, and the surfactant is poloxamer 407, or polysorbate 80 (Tween 80), or polyethylene glycol-12-hydroxystearate (Solutol HS 15), or egg yolk lecithin, or polyoxyethylene (35) castor oil, or vitamin E succinate polyethylene glycol ester, or one or more of sodium hydroxymethyl cellulose;
  • the second mixture is a mixture of sodium alginate and oxaliplatin, which is stirred and mixed with water, and filtered through a micron filter membrane to sterilize the mixture;
  • the first mixture and the second mixture are mixed to form a chemotherapy and immune combination drug.
  • the present invention also provides an in-situ gel-forming chemotherapeutic immunotherapy biopolymer pharmaceutical composition, which contains: the first component is alginate, which can form a porous gel with calcium ions in the body,
  • the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate;
  • the second component is chemotherapeutics that can cause immunogenic death
  • the third group of components are immune adjuvants.
  • the immune adjuvant is imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and requine One or more of mods.
  • the second type of chemotherapeutics that can cause immunogenic death are anthracyclines such as adriamycin and epirubicin
  • anthracyclines such as adriamycin and epirubicin
  • vitamins, mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil, and toxins such as maytansine.
  • in-situ gel-forming chemotherapy and immune combined therapy biopolymer pharmaceutical composition it also includes a fourth type of component immune checkpoint inhibitor or IDO inhibitor, the fourth type of component immune checkpoint inhibitor
  • Antibodies usually include anti-CTLA-4, anti-PD-1 and anti-PD-L1
  • small molecule inhibitors usually include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS -230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1, peptide inhibitors include DPPA-1;
  • the IDO inhibitors include small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole.
  • the first type of component is sodium alginate
  • the second type of component is doxorubicin hydrochloride
  • the third The class component is imiquimod
  • the mass ratio of sodium alginate, adriamycin hydrochloride and imiquimod is 50-800 to 1-100 to 1-100.
  • the mass ratio of sodium alginate, adriamycin hydrochloride and imiquimod is 200-400 to 10 to 75 to 10 to 75.
  • the sodium alginate concentration is more than 5 mg/ml.
  • a method for preparing a biopolymer pharmaceutical composition for in-situ gel-forming chemotherapy and immunotherapy comprising:
  • the first type of component is sodium alginate
  • the second type of component is oxaliplatin
  • the third The class component is imiquimod hydrochloride
  • the mass ratio of sodium alginate, oxaliplatin and imiquimod is 50-800 to 1-75 to 1-100.
  • the mass ratio of sodium alginate, oxaliplatin and imiquimod is 200-400 to 10 to 75 to 10 to 75.
  • the method for preparing the in-situ gel-forming chemotherapeutic immune combined therapy biopolymer pharmaceutical composition includes:
  • the first type component is sodium alginate
  • the second type component is pentafluorouracil
  • the third type group Divided into imiquimod hydrochloride
  • the first type component is sodium alginate
  • the second type component is cyclophosphamide
  • the third type component is imiquimod hydrochloride.
  • the first type of component is sodium alginate; the second type of component is doxorubicin hydrochloride or oxaliplatin; and third The first component is imiquimod hydrochloride; the fourth component is anti-PDL1 antibody.
  • the first type of component is potassium alginate or ammonium alginate
  • the second type of component is doxorubicin hydrochloride or oxaliplatin
  • the third component is imiquimod hydrochloride
  • the fourth component is anti-PDL1 antibody.
  • An in-situ gel-forming chemotherapeutic immune combined therapy biopolymer pharmaceutical composition consisting of: the first component is alginate, said alginate can form a porous gel with calcium ions in the body, said seaweed
  • the acid salt is one or more of sodium alginate, potassium alginate and ammonium alginate;
  • the second type of component is a chemotherapeutic agent that can cause immunogenic death;
  • the third type of component is an immune adjuvant.
  • An in-situ gel-forming chemotherapeutic immune combined therapy biopolymer pharmaceutical composition consisting of: the first component is alginate, said alginate can form a porous gel with calcium ions in the body, said seaweed
  • the acid salt is one or more of sodium alginate, potassium alginate and ammonium alginate;
  • the second type of chemotherapeutics that can cause immunogenic death are anthracyclines such as adriamycin and epirubicin , Mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and one or more of toxins such as maytansine;
  • the third component is an immune adjuvant, the immune The adjuvant is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod.
  • An in-situ gel-forming chemotherapeutic immune combined therapy biopolymer pharmaceutical composition consisting of: the first component is alginate, said alginate can form a porous gel with calcium ions in the body, said seaweed
  • the acid salt is one or more of sodium alginate, potassium alginate and ammonium alginate;
  • the second type of chemotherapeutics that can cause immunogenic death are anthracyclines such as adriamycin and epirubicin , Mitoxantrone, oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and one or more of toxins such as maytansine;
  • the third component is an immune adjuvant, the immune The adjuvant is one or more of imiquimod (R837), CpG oligonucleotide, monophosphoryl lipid A and resiquimod;
  • the fourth type of component immune checkpoint inhibitors or IDO inhibitors the fourth type of component immune checkpoint inhibitor antibodies usually have anti-CTLA-4, anti-PD-1 and anti-PD-L1, small molecules Inhibitors usually include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1, peptides Class inhibitors include DPPA-1;
  • the IDO inhibitors include BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole small molecules.
  • An in-situ gel-forming, chemotherapy and immune combined therapy biopolymer pharmaceutical composition consisting of: the first type of component is sodium alginate, the second type of component is doxorubicin hydrochloride; the third type of component is imiquimol Particularly, the mass ratio of the sodium alginate, adriamycin hydrochloride and imiquimod is 50-800 to 1-100 to 1-100.
  • the present invention provides a series of pharmaceutical compositions.
  • this composition system there are mainly four types of components.
  • the first type of components can be combined with other types of components according to the actual situation, including:
  • the first type of component sodium alginate excipients, which can produce gel with calcium ion plasma in the human body or animal body;
  • the second component chemotherapeutics that cause immunogenic death
  • the third component immune adjuvant
  • the fourth component immune checkpoint inhibitors or IDO inhibitors.
  • the first type of component excipients, usually sodium alginate, potassium alginate, ammonium alginate, etc. These polysaccharides will cross-link each other to form gels when they encounter divalent ions such as calcium ions, so when the drug is wrapped in it Later, the formed gel can effectively slow the release of the drugs in it, thereby enhancing the efficacy and reducing the side effects.
  • Sodium alginate is a natural polysaccharide, which has the stability, solubility, viscosity and safety required for pharmaceutical excipients.
  • Sodium alginate has been widely used in the food industry and medicine.
  • Sodium alginate is the most widely used water-soluble alginate.
  • Sodium alginate can quickly undergo ion exchange when encountering calcium ions to form a gel, and there are sufficient calcium ions in the human body or animal body, so it can form gel in situ in the body.
  • the two alginates, potassium alginate and ammonium alginate although the cations contained in them are different from sodium alginate, they can also be cross-linked with calcium ions to form a porous gel, thereby acting as a slow-release drug.
  • sodium alginate is usually extracted from seaweed, so sodium alginate is a better choice.
  • the second component chemotherapeutics that can cause immunogenic death
  • anthracyclines such as doxorubicin, epirubicin, mitoxantrone, etc.
  • oxaliplatin such as doxorubicin
  • cyclophosphamide such as cyclophosphamide
  • bortezo Rice gemcitabine
  • pentafluorouracil and toxins such as maytansine and so on.
  • These drugs have been clinically approved, and recent studies have shown that these drugs can cause the immunogenic death of cancer cells.
  • These dead cancer cells express calreticulin, which is easily recognized and taken up by immune cells, especially antigen-presenting cells. , To help immune cells recognize tumor cells and cause an effective anti-tumor immune response.
  • immune adjuvants are non-specific immune proliferatives, which refer to auxiliary substances that can enhance the body's immune response to antigens or change the type of immune response together with antigens or pre-injected into the body.
  • immune adjuvants There are many types of immune adjuvants, and there is no unified classification method.
  • Freund's adjuvant and cytokine adjuvant are more commonly used.
  • the immunobiological effects of immune adjuvants are to enhance immunogenicity, enhance antibody titer, change the type of antibody production, cause or enhance delayed hypersensitivity reactions, but the specific mechanism of immune adjuvants is not yet fully understood. Different adjuvants The mechanism of action is also different.
  • TLR Toll-like receptors
  • immunomodulators include immune checkpoint inhibitors or IDO inhibitors.
  • Immune checkpoint inhibitors include antibody inhibitors or small molecule inhibitors.
  • Antibody inhibitors usually include anti-CTLA-4, anti-PD-1 and anti-PD-L1, and small molecule inhibitors usually include CA-170. , PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 and JQ1.
  • Peptide inhibitors include DPPA-1.
  • IDO inhibitors include small molecules such as BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat and 4-phenylimidazole, which can inhibit IDO enzymes and enhance the effect of antigen presenting cells. Because tumor cells deceive the immune system and escape the immune response, these antibodies are needed to suppress the immune response that protects the tumor, so that immune cells can better kill tumor cells.
  • the first type of component excipients can also be referred to as component one; the second type of component ICD chemotherapeutic drugs can also be referred to as component two; the third type of component immune adjuvant can also be referred to as component three; the fourth type of component immune Checkpoint inhibitors can also be referred to as component four.
  • Freeze-dried powder is a sterile powder injection prepared by freezing the medicinal solution into a solid in a sterile environment, and vacuuming the water to sublimate and dry it.
  • the preparation process of the mixed medicinal solution of component one, component two, component three and component four and freeze-dried preparation is as follows.
  • This patent mainly relates to four types of raw material ingredients: the first type of component excipient sodium alginate (solid powder), the second type of component ICD type chemotherapeutics (solid powder), the third type of component immune adjuvant (solid powder) , The fourth component of immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies, commercial products for clinical use, with freeze-dried powder or injection as raw materials).
  • Preparation scheme 1 Mix the solid powders of component one excipient, component two ICD chemotherapeutics, and component three immune adjuvant in a certain proportion and put them into a large beaker, add deionized water (or physiological saline, or phosphate buffer solution) ), at room temperature 25 degrees Celsius, stir with a stirring blade at a speed of 50 to 500 revolutions per minute until the solution is clear and transparent; if necessary, formulate component four immune checkpoint inhibitors into injections according to the product instructions, and add them to the above mixed solution; After the above solution is evenly stirred, take out a separate bottle and freeze-dry it. After the lyophilized powder is reconstituted, there should be no turbidity and flocculent precipitation.
  • Preparation scheme 2 Weigh component 1, component 2, and component 3 of the target mass, respectively, add deionized water (or physiological saline, or phosphate buffer solution) to prepare three separate solutions; Prepare the injection solution in four parts according to the product instructions; mix the above-mentioned solutions in a suitable volume ratio, stir with a stirring blade at a rotation speed of 50 to 500 revolutions per minute at 25 degrees Celsius at room temperature until the solution is uniform and not turbid, take out the divided bottle and freeze-dry. After the lyophilized powder is reconstituted, there should be no turbidity and flocculent precipitation. There is little difference between Option 1 and Option 2.
  • deionized water or physiological saline, or phosphate buffer solution
  • Preparation scheme 3 Weigh component 1, component 2, and component 3 of the target mass, respectively, add deionized water (or physiological saline, or phosphate buffer solution) to prepare three independent solutions; According to the product instructions, it is divided into four to prepare injections; for ICD drugs containing hydrochloride, the solutions of component 2, component 3, and component 4 need to be stirred and mixed first, and during the stirring process (25 degrees Celsius, the stirring paddle is used Rotation speed 50 to 300 revolutions per minute) slowly drop the solution of component 1 until the whole solution is evenly stirred.
  • deionized water or physiological saline, or phosphate buffer solution
  • Usage plan 1 After re-dissolving the lyophilized powder injection of the composition of the above four types of components in physiological saline, the composition solution is directly injected into the tumor site of the patient through clinical interventional administration and direct puncture administration. The multi-point injection method is adopted to ensure that the composition solution evenly fills the entire tumor. After the composition is injected into the tumor, firstly, the first type of component alginate will form a gel when it encounters calcium ions. The first type of component will quickly gel after encountering calcium ions in the tissue.
  • a porous network cross-linked structure is formed, so that the other three types of components mixed in the alginate can be slowly released, thereby enhancing its effect and reducing toxic and side effects;
  • the second type of component ICD chemotherapeutics can not only kill effectively Tumor cells can also cause immunogenic death, produce tumor-related antigens, and activate tumor-specific immune responses; again, the third type of immune adjuvant enhances the ability of antigen-presenting cells to further amplify the corresponding immune response;
  • the use of the fourth component of immune checkpoint inhibitors or IDO inhibitors prevents the metastatic tumor from escaping the immune response, so that immunotherapy can kill the tumor more effectively, thereby inhibiting tumor metastasis and recurrence.
  • usage plan 2 After re-dissolving the lyophilized powder injection of the composition of the first, second, and third components in physiological saline, the composition solution is directly injected into the patient through clinical interventional administration and direct puncture administration At the tumor site, multi-point injection is used during injection to ensure that the composition solution evenly fills the entire tumor.
  • This treatment method is recommended to be used in combination with the fourth component of immune checkpoint inhibitors: the combination plan includes the addition of immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1 Or anti-PD-L1 antibody) or IDO inhibitor, one-time intratumor local injection; it can also be an intravenous injection of immune checkpoint inhibitor after local treatment of a mixture of one, two, and three components. (Reference example in embodiment twelve)
  • Use plan three the composition of four components is sprayed on the wound, and then the calcium ion solution is sprayed to form a gel
  • the tumor patient has a normal surgical resection of the lesion, considering the problem that the surgical resection cannot completely remove the tumor cells in the lesion, it can be
  • the freeze-dried powder injections of the above four types of components are reconstituted with physiological saline, and then sprayed on the wound site after surgical resection with a syringe or spray bottle, and then an appropriate amount of calcium chloride solution can be sprayed on the site to make it gel.
  • the wound is sutured. This program helps to eliminate the remaining cancer cells, and can inhibit tumor metastasis and recurrence. (Example Sixteen)
  • Usage plan four (the composition of three components is sprayed on the wound, and then the calcium ion solution is sprayed to form a gel + the fourth component is combined for use): after the normal surgical resection of the lesion site, the surgical resection is considered incomplete
  • the freeze-dried powder injections of the first, second, and third components can be reconstituted with normal saline, and then sprayed on the wound site after surgical resection with a syringe or spray bottle. Spray an appropriate amount of calcium chloride solution on the site to make it gel, and finally suture the wound. This program helps to eliminate the remaining cancer cells, and can inhibit tumor metastasis and recurrence.
  • the combination plan includes the addition of immune checkpoint inhibitors (anti-CTLA- 4. Anti-PD-1 or anti-PD-L1 antibody) or IDO inhibitor, one-time intratumor local injection; it can also be intravenously administered after local treatment of a mixture of one, two, and three components Inject immune checkpoint inhibitors.
  • Alginate is a natural polysaccharide, which is safe and non-toxic, has good biocompatibility, and can be degraded. It is a good biological material.
  • the commonly used method is to combine alginate and calcium ions in vitro to form a gel implantable material. This method of use not only limits its application in the body, it often requires surgery or intervention, which is difficult to operate. The damage to the patient is large, and it is not conducive to combined drug treatment. Therefore, we inject alginate into the tumor, use the calcium ions in the tumor tissue to make the sodium alginate gel in situ in the tumor, and use the formed cross-linked structure to slowly release the drug mixed in the alginate , The sustained-release effect is better.
  • This technology has broad application prospects. It can be directly injected with a syringe for the treatment of tumors, with simple operation and low invasiveness. It can also be sprayed on the wound site after the operation with a sprayer, and combined with the operation to clean the residual cancer cells, it is expected to be targeted at different Patients are given personalized treatment, and the cost is low.
  • the third type of immune adjuvant mentioned in the technical scheme has not been clinically used to directly treat tumors.
  • These small molecule immunomodulators themselves do not have antiviral and antitumor effects, and are often used as auxiliary adjuvants of vaccines to enhance the immunogenicity of antigens.
  • Imiquimod R873
  • the technology of this patent uses ICD chemotherapeutics and immune adjuvants to be injected together.
  • ICD drugs kill tumors to produce tumor antigens, antigens and adjuvants act like tumor vaccines, which can not only inhibit metastasis, but also prevent tumors. relapse.
  • the technology of this patent creates a new strategy for the direct treatment of tumors with immune adjuvants and chemotherapeutic drugs.
  • the pharmaceutical composition formed by mixing the relevant components of this patent can produce a distinctive and unexpected synergistic anti-cancer effect, and can reduce the side effects of conventional treatments, reduce the probability of cancer metastasis, and reduce the probability of cancer recurrence, providing a Efficient tumor-specific immunotherapy can effectively kill tumors in situ while suppressing the immune response, reducing the growth of distant metastatic tumors and the probability of tumor recurrence, which can help patients under the premise of relatively controlling costs Extend the life cycle and improve the quality of life.
  • the related technical solutions in this patent can solve the problem of imiquimod insoluble in water, the problem of stability of imiquimod after sterilization, and the problem of sterilization of sodium alginate.
  • sodium alginate and imiquimod R837 cannot be mixed together.
  • Sodium alginate ALG needs to be filtered and sterilized, but R837 particles cannot be filtered (the minimum particle diameter after ball milling is 500nm, and filter sterilization requires 220nm filter membrane, so it cannot pass); on the other hand, R837 needs to be sterilized by moist heat, and sodium alginate ALG will degrade at high temperature. Both R837 and ALG cannot be sterilized together.
  • Sodium alginate is a relatively special natural biopolymer material.
  • Poloxamer 188 is particularly preferred. Without the addition of poloxamer 188, after the R837 ball mill emulsion is sterilized by moist heat at 121°C, it will cause the emulsion to become unstable and produce obvious precipitation and particles, and the water dispersibility is greatly reduced. Poloxamer 188 can greatly help R837 Ensure water dispersibility and stability after sterilization.
  • the combined use of sodium alginate, chemotherapeutics, and immune adjuvants can achieve a relatively excellent therapeutic effect.
  • sodium alginate, chemotherapeutics and immune adjuvants, and then Adding poloxamer 188 to form a composition can produce better therapeutic effects without the need to combine PD-1, and the effect is better, but the treatment cost is lower. See Figure 30 and Figure 31 for specific experimental data. .
  • Fig. 1 is the preparation process of the lyophilized powder injection of the composition of sodium alginate and imiquimod hydrochloride in Example 1, and the instructions for use thereof.
  • Fig. 2 is a scanning electron microscope picture of the composition of sodium alginate and imiquimod hydrochloride in Example 1 after the lyophilized powder injection was reconstituted into a gel.
  • Figure 3 is the release curve and data statistics of imiquimod drug at different concentrations of sodium alginate in Example 1.
  • Fig. 4 is the release curve and data statistics of imiquimod drug at different concentrations of imiquimod in Example 1.
  • Figure 5 is a scanning electron microscope picture of the lyophilized powder injection of the composition of sodium alginate and CpG oligonucleotide in Example 2 after being reconstituted into a gel.
  • Fig. 6 is the release curve and data statistics of CpG drug at different concentrations of sodium alginate in Example 2.
  • Fig. 7 is a CpG drug release curve and data statistics under different CpG concentrations in Example 2.
  • Fig. 8 is a scanning electron microscope picture of the composition of sodium alginate and adriamycin hydrochloride in Example 3 after the freeze-dried powder injection was reconstituted into a gel.
  • Figure 9 is the release curve and data statistics of doxorubicin hydrochloride drug at different concentrations of sodium alginate in Example 3.
  • Fig. 10 is the release curve and data statistics of doxorubicin hydrochloride at different concentrations of doxorubicin hydrochloride in Example 3.
  • Fig. 11 is a scanning electron microscope picture of the composition of sodium alginate and oxaliplatin in Example 4 after the freeze-dried powder injection was reconstituted into a gel.
  • Figure 12 is the oxaliplatin drug release curve and data statistics at different concentrations of sodium alginate in Example 4.
  • Figure 13 is the oxaliplatin drug release curve and data statistics when the concentration of oxaliplatin in Example 4 is different.
  • Figure 14 is a scanning electron microscope picture of the sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride freeze-dried powder injection in Example 5 after being reconstituted into a gel.
  • Fig. 15 shows the rheological performance test of sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride freeze-dried powder injection after reconstitution in Example 5.
  • Figure 16 is a scanning electron microscope picture of the lyophilized powder injection of sodium alginate, oxaliplatin and imiquimod hydrochloride in Example 6 after being reconstituted into a gel.
  • Fig. 17 is a scanning electron microscope picture of sodium alginate, adriamycin hydrochloride, imiquimod hydrochloride, and anti-PDL1 antibody freeze-dried powder injection in Example 9 after being reconstituted into a gel.
  • Figure 18 shows the detection of antibody activity after reconstitution of sodium alginate, adriamycin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody freeze-dried powder injection in Example 9.
  • Figure 19 shows the tumor growth curve and data statistics of the combination of sodium alginate and imiquimod hydrochloride in Example 13 combined with radiofrequency ablation therapy and anti-PDL1 antibody therapy on a mouse colon cancer tumor model.
  • Figure 20 shows the tumor growth curve and data statistics of the combination of sodium alginate and imiquimod hydrochloride in Example 13 in combination with HIFU treatment and anti-PDL1 antibody treatment on a mouse colon cancer tumor model.
  • Figure 21 shows the growth curve and data statistics of the second planting tumor caused by the combination of sodium alginate and imiquimod hydrochloride in Example 13 in combination with HIFU treatment and anti-PDL1 antibody treatment on a mouse colon cancer tumor model .
  • Figure 22 shows the tumor growth curve and data statistics of the lyophilized powder of the composition of sodium alginate and oxaliplatin in Example 14 after treatment of colon cancer in mice.
  • Fig. 23 shows the body weight curve and data statistics of mice treated with colon cancer of the lyophilized powder of the composition of sodium alginate and oxaliplatin in Example 14.
  • Figure 24 is an in situ tumor growth curve after treatment with sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in a mouse bilateral tumor model in Example 15.
  • Figure 25 shows the growth curve and data statistics of the distal tumor after treatment with sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in a mouse bilateral tumor model in Example 15.
  • Fig. 26 shows the tumor growth curve and data statistics of sodium alginate, oxaliplatin, imiquimod hydrochloride and anti-PDL1 antibody in the mouse bilateral tumor model after being cured and re-inoculated with the tumor in Example 15.
  • Figure 27 shows the fluorescence imaging data of mice after treatment with sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride, and anti-PDL1 antibody on a mouse orthotopic breast cancer tumor model in Example 16.
  • Figure 28 shows the tumor growth curve and data statistics after treatment with sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody in the mouse brain cancer model in Example 17.
  • Figure 29 shows the fluorescence imaging data of mice after treatment with sodium alginate, doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody on the mouse tumor surgical resection model in Example 18.
  • Figure 30 shows the tumor growth of different treatment modalities by direct drug injection to a larger tumor (initial volume> 120 cubic millimeters) in Example 19.
  • Figure 31 shows the growth of the contralateral small tumor (initial volume ⁇ 50 mm) in Example 19 without direct injection of drugs and different treatment methods.
  • Example 1 Preparation and use of lyophilized powder injection of sodium alginate (first component) and imiquimod (third component) hydrochloride composition
  • Step 1 Preparation of Imiquimod (the third component) hydrochloride.
  • the purpose of this step is to change the water-insoluble imiquimod into the water-soluble hydrochloride form.
  • the lyophilization time is long enough to ensure the complete removal of hydrochloric acid residues.
  • Step 2 Preparation of the lyophilized powder injection of the composition of sodium alginate (the first component) and imiquimod (the third component) hydrochloride can adopt the following three methods.
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate and 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and dissolve in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50 ⁇ 300 rpm to After the solution is clear and transparent, the temperature is maintained at 20-40 degrees Celsius and the pH is ⁇ 6.5. The solution is freeze-dried to obtain the composition freeze-dried powder injection.
  • Method 2 Weigh 10 to 80 mg of sodium alginate and dissolve in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50 to 300 revolutions per minute until the solution is clear and transparent, freeze-dry to obtain a freeze-dried powder injection, and then mix with 0.1 ⁇ 10 mg of imiquimod hydrochloride freeze-dried powder is uniformly mixed by shaking and mixing the solid to obtain a composition freeze-dried powder injection.
  • Method 3 Dissolve 0.1-10 mg of imiquimod hydrochloride lyophilized powder in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, and then add 10 to sodium alginate. 80 mg is dissolved in the aqueous phase solution, and the continuously stirred imiquimod hydrochloride solution is dripped at a volume ratio of 1 to 20 to ensure that the mixture is clear and transparent without flocculent precipitation. After all the sodium alginate solution has been added, the mixed solution is taken out and freeze-dried to obtain a composition freeze-dried powder injection.
  • Figure 1 shows the preparation process of the lyophilized powder injection of the composition of sodium alginate (the first component) and imiquimod (the third component) hydrochloride, and the instructions for use thereof.
  • Fig. 2 is a scanning electron microscope picture of a freeze-dried powder injection of a composition prepared by the method described in Fig. 1 after gelling. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Step 3 Release curve of imiquimod in the lyophilized powder injection of the composition of sodium alginate (the first component) and imiquimod (the third component) hydrochloride.
  • the drug sustained-release carrier means that the drug slowly enters the blood to reduce the blood drug concentration, and the preparation of sustained-release long-acting drugs that can slowly release the drug components is often very necessary in the treatment.
  • the drug release curve refers to the release of the encapsulated drug after we simulate the composition in vitro to form a gel.
  • the following is the release curve obtained by fixing the dosage of imiquimod and changing the dosage of sodium alginate.
  • Preparation of sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection, wherein the sodium alginate concentration is 1, 10, 20, 40 and 80 mg, Imiquimod is 2 mg.
  • the lyophilized powder injection of the composition is re-dissolved in 1 ml of aqueous solution and shaken until it is clear and transparent.
  • 200 ⁇ l of 5 mg per ml of calcium chloride solution is added to make it into a gel, and chlorine is added.
  • the reason for calcification is to simulate the situation that the composition is injected into the tumor in vitro to meet the gelation of calcium ions and then the sustained release of the drug, and the gel is soaked in 1 ml of phosphate buffer solution and stirred. 0.5, 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of imiquimod.
  • Figure 3 shows the drug release curve and statistical table of imiquimod at different concentrations of sodium alginate. It can be seen from the figure that when the concentration of sodium alginate is 5 mg/ml and above, imiquimod has an obvious sustained release Therefore, the concentration of sodium alginate in the composition is preferably 5 mg/ml to 80 mg/ml. When the concentration of sodium alginate is 10 mg/ml, it has been optimized. When the concentration of sodium alginate is 20 mg/ml, it basically reaches the peak value. When the concentration is increased again, the effect is not improved significantly.
  • the following is the release curve obtained by fixing the dosage of sodium alginate and changing the dosage of imiquimod.
  • Preparation of sodium alginate (first component) and imiquimod (third component) hydrochloride composition freeze-dried powder injection, wherein the concentration of imiquimod is 1, 2.5, 5, 7.5 and 10 mg (Maximum solubility).
  • Sodium alginate is 20 mg.
  • the lyophilized powder injection of the composition is re-dissolved in 1 ml of aqueous solution and shaken until it is clear and transparent, and then 5 mg per ml of calcium chloride solution is added to make it gel, and The colloid was immersed in 1 ml of phosphate buffer solution and stirred, and the drug content in the phosphate buffer solution was determined on the 0th, 0.25, 0.5, 1, 2, 4, and 8 days as the release of imiquimod.
  • Figure 4 shows the imiquimod drug release curve and statistical table at different concentrations of imiquimod. It can be seen from the figure that when the concentration of imiquimod is higher than 7.5 mg/ml, the composition has obvious gel formation Rapid drug release, and subsequent release is faster than the low concentration, but the sustained release effect is still obvious, so the concentration of imiquimod in the composition freeze-dried powder injection is selected to be 0.1-10 mg/ml. This shows that imiquimod is effective regardless of the concentration. Although the high concentration will release quickly, it also has an obvious slow-release effect.
  • the preferred mass ratio of sodium alginate and imiquimod hydrochloride obtained through the above experiment is 50-800 to 1-100, and the more preferred mass ratio is 200-400 to 10-75.
  • Example 2 Lyophilized powder injection of the composition of sodium alginate (the first component) and CpG oligonucleotide (the third component)
  • Step 1 Preparation of freeze-dried powder injection of the composition of sodium alginate and CpG oligonucleotide
  • Figure 5 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is reconstituted into a gel. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Step 2 CpG release curve in the freeze-dried powder injection of the composition of sodium alginate and CpG oligonucleotide
  • the following is the release curve obtained by fixing the dosage of CpG oligonucleotide and changing the dosage of sodium alginate.
  • composition freeze-dried powder injection wherein the sodium alginate concentration is 1, 10, 20, and 40 mg, and the CpG oligonucleotide is 0.2 mg, and the composition freeze-dried powder injection is reconstituted separately Dissolve in 1 ml of aqueous solution and shake until it is clear and transparent, then add 200 ⁇ l of 5 mg per ml of calcium chloride solution to make it gel, and soak the gel in 1 ml of phosphate buffer solution and stir. , 0.5, 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of CpG oligonucleotides.
  • Figure 6 shows the release curve of CpG drug at different concentrations of sodium alginate. It can be seen from the figure that when the concentration of sodium alginate is 20 mg or more, CpG oligonucleotides have an obvious slow-release phenomenon, so the composition freezes
  • the concentration of sodium alginate in the dry powder injection is selected from 5 mg/ml to 80 mg/ml. When the concentration of sodium alginate is 1 mg/ml, the effect is not obvious. When the concentration of sodium alginate is 10 mg/ml, the effect has been optimized. When the concentration of sodium alginate is 20 mg/ml, it basically reaches the peak value, and the concentration is increased. When, the effect is not obvious anymore.
  • the following is the release curve obtained by fixing the amount of sodium alginate and changing the amount of CpG oligonucleotide.
  • Preparation of sodium alginate and CpG oligonucleotide composition freeze-dried powder injection, wherein the CpG oligonucleotide concentration is 0.1, 0.25, 0.5, 1, and 2 mg, sodium alginate is 20 mg, and the composition is freeze-dried powder injection Re-dissolve in 1 ml of the aqueous solution and shake until it is clear and transparent, then add 5 mg per ml of calcium chloride solution to make it gel, and soak the gel in 1 ml of phosphate buffer solution and stir. 0.5, 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of CpG oligonucleotides.
  • Figure 7 shows the release curve of CpG drug at different concentrations of CpG oligonucleotides. From Figure 8 it can be seen that when the concentration of CpG oligonucleotides is higher than 1 mg/ml, there is a relatively obvious acute release, and the subsequent release rate There is not much change. For cost considerations, the price of CpG oligonucleotides is almost RMB 10,000/mg. Therefore, the concentration of CpG oligonucleotides in the composition freeze-dried powder injection is selected to be 0.1-2 mg/ml, preferably The concentration of CpG oligonucleotide is selected to be 0.1-0.5 mg/ml.
  • the optimal mass ratio of sodium alginate and CpG oligonucleotide obtained through the above experiment is 50-800 to 1-20, and the more preferable mass ratio is 200-400 to 1-20.
  • Example 3 Lyophilized powder injection of the composition of sodium alginate (the first component) and doxorubicin hydrochloride (the second component)
  • Step 1 Preparation of freeze-dried powder injection of the composition of sodium alginate and adriamycin hydrochloride:
  • Method 1 Weigh 20 ⁇ 80 mg of sodium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride and dissolve in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50 to 300 revolutions per minute until the solution is clear and transparent, and then The solution is freeze-dried to obtain the composition freeze-dried powder injection.
  • Method 2 Dissolve 0.1-10 mg of adriamycin hydrochloride in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent, and then dissolve 10 to 80 mg of sodium alginate In the aqueous solution, drip the constantly stirred adriamycin hydrochloride solution at a volume ratio of 1 to 20 to ensure that the mixture is clear and transparent without flocculent precipitation. After all the sodium alginate solution has been added, the mixed solution is taken out and freeze-dried to obtain a composition freeze-dried powder injection.
  • Figure 8 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is reconstituted into a gel. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Step 2 Release curve of adriamycin in the freeze-dried powder injection of the composition of sodium alginate and adriamycin hydrochloride
  • composition freeze-dried powder injection wherein the sodium alginate concentration is 1, 10, 20, and 40 mg, and adriamycin hydrochloride is 2 mg, and the composition freeze-dried powder injection is reconstituted separately 1 ml of aqueous solution and shake until it is clear and transparent, then add 200 ⁇ l of 5 mg per ml of calcium chloride solution to make it gel, and soak the gel in 1 ml of phosphate buffer solution and stir. 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of doxorubicin hydrochloride.
  • Figure 9 shows the release curve of doxorubicin hydrochloride at different concentrations of sodium alginate. It can be seen from the figure that when the concentration of sodium alginate is 10 mg or more, doxorubicin hydrochloride has an obvious slow-release phenomenon, so the combination
  • the concentration of sodium alginate in the freeze-dried powder injection is preferably 5 mg/ml to 80 mg/ml.
  • adriamycin hydrochloride composition freeze-dried powder injection, wherein the concentration of adriamycin hydrochloride is 1, 2.5, 5, 7.5 and 10 mg (maximum solubility), sodium alginate is 20 mg, and the composition is lyophilized
  • the powder injections were re-dissolved in 1 ml of aqueous solution and shaken until clear and transparent, and then 5 mg per ml of calcium chloride solution was added to make it gel, and the gel was immersed in 1 ml of phosphate buffer solution and stirred. 0.25, 0.5, 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of doxorubicin hydrochloride.
  • Figure 10 shows the release curve of doxorubicin hydrochloride at different concentrations of doxorubicin hydrochloride. It can be seen from the figure that when the concentration of doxorubicin hydrochloride is higher than 7.5 mg, there is a relatively obvious rapid release, and the subsequent release is also lower than that The concentration should be fast, but the sustained-release effect is still obvious, so the concentration of adriamycin in the composition freeze-dried powder injection is selected to be 0.1-10 mg/ml.
  • the preferred mass ratio of sodium alginate and adriamycin hydrochloride obtained through the above experiment is 50-800 to 1-100, and the more preferred mass ratio is 200-400 to 10-75.
  • Example 4 Lyophilized powder injection of the composition of sodium alginate (the first component) and oxaliplatin (the second component)
  • Step 1 Preparation of freeze-dried powder injection of the composition of sodium alginate and oxaliplatin
  • Figure 11 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is reconstituted into a gel. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Step 2 Release curve of oxaliplatin in the lyophilized powder injection of the composition of sodium alginate and oxaliplatin
  • Preparation of sodium alginate and oxaliplatin composition freeze-dried powder injection wherein the sodium alginate concentration is 1, 10, 20 and 40 mg, oxaliplatin is 2 mg, and the composition freeze-dried powder injection is re-dissolved separately 1 ml of aqueous solution and shake until it is clear and transparent, then add 200 ⁇ l of 5 mg per ml of calcium chloride solution to make it gel, and soak the gel in 1 ml of phosphate buffer solution and stir. 1,2,4,8 days to determine the content of the drug in the phosphate buffer solution is the release of oxaliplatin.
  • Figure 12 shows the oxaliplatin drug release curve at different concentrations of sodium alginate. It can be seen from the figure that when the concentration of sodium alginate is 10 mg or more, oxaliplatin has an obvious slow-release phenomenon, so the combination
  • the concentration of sodium alginate in the freeze-dried powder injection is selected from 5 mg/ml to 80 mg/ml.
  • Preparation of sodium alginate and oxaliplatin composition freeze-dried powder injection wherein the oxaliplatin concentration is 1, 2.5, 5 and 7.5 mg (maximum solubility), sodium alginate is 20 mg, the composition is freeze-dried powder injection Re-dissolve in 1 ml of the aqueous solution and shake until it is clear and transparent, then add 5 mg per ml of calcium chloride solution to make it gel, and soak the gel in 1 ml of phosphate buffer solution and stir. 0.5, 1, 2, 4, 8 days to determine the content of the drug in the phosphate buffer solution is the release of oxaliplatin.
  • Figure 13 shows the oxaliplatin drug release curve at different oxaliplatin concentrations. It can be seen from the figure that when the oxaliplatin concentration is higher than 7.5 mg, there is a relatively obvious acute release and a relatively obvious acute release And the subsequent release is faster than the low concentration, but the sustained release effect is still obvious, so the concentration of oxaliplatin in the composition freeze-dried powder injection is selected to be 0.1-7.5 mg/ml.
  • the preferred mass ratio of sodium alginate and oxaliplatin obtained through the above experiment is 50-800 to 1-75, and the more preferred mass ratio is 200-400 to 10-75.
  • Example 5 Sodium alginate (first component), doxorubicin hydrochloride (second component) and imiquimod (third component) hydrochloride freeze-dried powder injection
  • Step 1 Preparation of sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride freeze-dried powder injection
  • Method one (dissolve components one, two and three in the aqueous solution, stir, and then freeze-dry the mixed solution): Weigh 10 to 80 mg of sodium alginate (the first component) and imiquimod (the third Class component) 0.1-10 mg of hydrochloride lyophilized powder and 0.1-10 mg of doxorubicin hydrochloride (the second component) are dissolved in 1 ml of aqueous solution, using a stirring paddle at 50-300 revolutions per minute Stir at a speed until the solution is clear and transparent, and then freeze-dry the solution to obtain a composition freeze-dried powder injection.
  • Method two (components one and two are dissolved in the water phase solution, the mixed solution is lyophilized to obtain the lyophilized powder after stirring, and the lyophilized powder is mixed with the lyophilized powder of component three):
  • Weigh the sodium alginate (the first component ) 10 ⁇ 80 mg and 0.1 ⁇ 10 mg of doxorubicin hydrochloride (the second component) are dissolved in 1 ml of aqueous solution, stirred with a stirring paddle at a speed of 50 to 300 revolutions per minute until the solution is clear and transparent, and then lyophilized
  • the freeze-dried powder is obtained, and 0.1-10 mg of imiquimod (the third component) hydrochloride freeze-dried powder is uniformly mixed by shaking the solid and the solid to obtain the composition freeze-dried powder injection.
  • Method three (components two and three are dissolved in the aqueous solution, and a clear solution is obtained after stirring.
  • the solution of component one is dropped into the aforementioned mixture at a ratio of 1:20, and then the final mixture is lyophilized to obtain a lyophilized solution.
  • Powder Dissolve 0.1-10 mg doxorubicin hydrochloride and 0.1-10 mg imiquimod hydrochloride in 1 ml of aqueous solution, and stir until the solution is reached with a stirring paddle at a speed of 50-300 revolutions per minute.
  • the preferred mass ratio of the first component, the second component and the third component in the composition is 50-800 to 1-100 to 1-100, and the more preferred mass ratio is 200 to 400 to 10. ⁇ 75 to 10 ⁇ 75. .
  • Figure 14 is a scanning electron microscope picture of the composition after the freeze-dried powder injection is reconstituted into a gel. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Step 2 Determination of rheological properties of sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride freeze-dried powder injection after reconstitution
  • Figure 15 shows the rheological properties of different concentrations of sodium alginate, adriamycin and imiquimod composition lyophilized powder after reconstitution after exposure to calcium ions. It can be seen from the figure that when the concentration of sodium alginate is 1 mg/ml, its storage modulus is smaller than the loss modulus, showing fluid behavior. When the concentration of sodium alginate reaches more than 10 mg/ml, its storage modulus The amount is greater than the loss modulus, showing gel behavior, which proves that sodium alginate will form a colloid when it encounters calcium ions above 10 mg/ml.
  • Example 6 Sodium alginate (first component), oxaliplatin (second component) and imiquimod hydrochloride (third component) freeze-dried powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 7.5 mg of oxaliplatin, and dissolve them in 1 ml of aqueous solution. After stirring at a speed of ⁇ 300 revolutions per minute until the solution is clear and transparent, the solution is freeze-dried to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 to 80 mg of sodium alginate and 0.1 to 7.5 mg of oxaliplatin to 1 ml of aqueous solution, stir with a stirring blade at a speed of 50 to 300 revolutions per minute until the solution is clear and transparent, and then freeze-dried. The dry powder is then mixed with 0.1-10 mg of imiquimod hydrochloride lyophilized powder through solid-solid shaking to obtain a composition lyophilized powder injection.
  • Method 3 Dissolve 0.1-10 mg of oxaliplatin and 0.1-10 mg of imiquimod hydrochloride in 1 ml of aqueous solution, stir with a stirring paddle at a speed of 50-300 rpm until the solution is clear and transparent , And then dissolve 20-80 mg of sodium alginate in 1 ml of aqueous solution, and drop it into the continuously stirred mixed solution at a volume ratio of 1 to 20 to ensure that the mixed solution is clear and transparent without flocculent precipitation. After all the sodium alginate solution has been added, the mixed solution is taken out and freeze-dried to obtain a composition freeze-dried powder injection.
  • the mass ratio of the first type component, the second type component and the third type component in the composition is 50-800 to 1 to 75 to 1 to 100, and the more preferred mass ratio is 200 to 400 to 10 to 75 to 10 ⁇ 75. .
  • Figure 16 is a scanning electron microscope picture of the freeze-dried powder injection of the composition after gelling. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Example 7 Sodium alginate (first component), pentafluorouracil (second component) and imiquimod hydrochloride (third component) freeze-dried powder injection
  • Method 1 Weigh 10 to 80 mg of sodium alginate, 1 to 5 mg of pentafluorouracil and 0.1 to 10 mg of imiquimod hydrochloride, and dissolve in 1 ml of 2 mg per ml of sodium hydroxide solution, and shake it to the solution After being clear and transparent, the solution is freeze-dried to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of sodium alginate and 1 ⁇ 5 mg of pentafluorouracil, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then freeze-dry to obtain freeze-dried powder and 0.1 ⁇ 10 mg of imiquimod hydrochloride The freeze-dried powder is uniformly mixed by shaking to obtain the composition freeze-dried powder injection.
  • Example 8 Sodium alginate (first component), cyclophosphamide (second component) and imiquimod hydrochloride (third component) lyophilized powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate, 1 ⁇ 5 mg of cyclophosphamide and 0.1 ⁇ 10 mg of imiquimod hydrochloride, and dissolve in 1 ml of aqueous solution. Shake well until the solution is clear and transparent. Freeze-drying to obtain the composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of sodium alginate and 1 ⁇ 5 mg of cyclophosphamide, dissolve in 1 ml of aqueous solution, shake until the solution is clear and transparent, then freeze-dry to obtain freeze-dried powder and 0.1 ⁇ 10 mg of imiquimod hydrochloric acid The freeze-dried salt powder is uniformly mixed by shaking to obtain the composition freeze-dried powder injection.
  • Example 9 Sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) Component) Lyophilized powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, and dissolve in 1 ml of aqueous solution, and shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of anti-PDL1 solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of sodium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of anti-PDL1 solution, mix well and freeze-dried. The dry powder and 0.1-10 mg of imiquimod hydrochloride lyophilized powder are uniformly mixed by shaking to obtain the composition lyophilized powder injection.
  • Figure 17 is a scanning electron microscope picture of the freeze-dried powder injection of the composition after gelling. It can be seen from the figure that the composition still has good gel forming ability after freeze-drying and reconstitution, and it can be seen from the electron microscope pictures that there are many micron-level pores after gelling, which is important to the sustained release of drugs.
  • Figure 18 shows the activity test of antibody anti-PDL1 after lyophilization. From the experimental results, it can be seen that the peak value of anti-PDL1 antibody binding to cell surface PDL1 antibody flow cytometry after lyophilization is consistent with the peak value of pure anti-PDL1 antibody. It shows that lyophilization does not affect the activity of anti-PDL1 antibody.
  • Example 10 Sodium alginate (first component), oxaliplatin (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth component) Component) Lyophilized powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 7.5 mg of oxaliplatin, and dissolve in 1 ml of aqueous solution, and shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of anti-PDL1 solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of sodium alginate and 0.1 ⁇ 10 mg of oxaliplatin, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of anti-PDL1 solution, mix well and freeze-dried. The dry powder and 0.1-10 mg of imiquimod hydrochloride lyophilized powder are uniformly mixed by shaking to obtain the composition lyophilized powder injection.
  • Example 11 Other alginates (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component), and anti-PDL1 antibody ( The fourth component) freeze-dried powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of potassium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride and dissolve in 1 ml of aqueous solution, and shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of anti-PDL1 solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of potassium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of anti-PDL1 solution, mix uniformly and lyophilize. The freeze-dried powder is then mixed with 0.1-10 mg of imiquimod hydrochloride freeze-dried powder and mixed uniformly by solid-solid shaking to obtain a composition freeze-dried powder injection.
  • Method 1 Weigh 10 ⁇ 80 mg of ammonium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride and dissolve in 1 ml of aqueous solution, shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of anti-PDL1 solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of ammonium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of anti-PDL1 solution, mix well, and freeze-dried. The dry powder and 0.1-10 mg of imiquimod hydrochloride lyophilized powder are uniformly mixed by shaking to obtain the composition lyophilized powder injection.
  • Alginates of different cations can form a good composition with the other three components, and still have the ability to form a gel and release slowly.
  • Example 12 Alginate (the first component) and doxorubicin hydrochloride (the second component) and imiquimod hydrochloride (the third component) and IDO inhibitor 4-phenyl Imidazole (fourth component) freeze-dried powder injection
  • Method 1 Weigh 10 ⁇ 80 mg of sodium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, and dissolve in 1 ml of aqueous solution, and shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of 4-phenylimidazole solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of sodium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of 4-phenylimidazole solution and mix well.
  • the freeze-dried powder is obtained by drying, and the freeze-dried powder is mixed with 0.1-10 mg of imiquimod hydrochloride by solid-solid shaking to obtain a composition freeze-dried powder injection.
  • Method 1 Weigh 10 ⁇ 80 mg of potassium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride and dissolve in 1 ml of aqueous solution, and shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of 4-phenylimidazole solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of potassium alginate and 0.1 ⁇ 10 mg of doxorubicin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of 4-phenylimidazole solution, mix well and freeze dry The obtained freeze-dried powder and 0.1-10 mg of imiquimod hydrochloride freeze-dried powder are uniformly mixed by shaking to obtain a composition freeze-dried powder injection.
  • Method 1 Weigh 10 ⁇ 80 mg of ammonium alginate, 0.1 ⁇ 10 mg of imiquimod hydrochloride lyophilized powder and 0.1 ⁇ 10 mg of doxorubicin hydrochloride and dissolve in 1 ml of aqueous solution, shake well until the solution is clear After being transparent, 100 micrograms to 5 mg of 4-phenylimidazole solution are added and mixed uniformly, and the solution is lyophilized to obtain a composition freeze-dried powder injection.
  • Method 2 Weigh 10 ⁇ 80 mg of ammonium alginate and 0.1 ⁇ 10 mg of adriamycin hydrochloride, dissolve in 1 ml of aqueous solution and shake until the solution is clear and transparent, then add 100 ⁇ g ⁇ 5 mg of 4-phenylimidazole solution, mix well and freeze dry The obtained freeze-dried powder and 0.1-10 mg of imiquimod hydrochloride freeze-dried powder are uniformly mixed by shaking to obtain a composition freeze-dried powder injection.
  • Example 13 Study on the curative effect of the composition of sodium alginate (the first component) and imiquimod hydrochloride (the third component) on a colon cancer model
  • Step 1 Sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection combined with radiofrequency ablation therapy and immune checkpoint suppression therapy anti-PDL1 antibody efficacy the study.
  • mice Mouse colon cancer tumors were planted on the left and right ends of the back of the mice (the left side is regarded as the in situ tumor, the right side is regarded as the distal tumor), and the tumor-bearing mice were divided into four groups, each with 5 mice for treatment experiments.
  • Group 1 Radiofrequency ablation treatment of the left orthotopic tumor alone (reference example);
  • the second group After radiofrequency ablation of the left tumor in situ, anti-pdl1 antibody was injected into the tail vein (reference example);
  • the third group Intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) on the left side of the tumor, followed by radiofrequency ablation therapy;
  • the fourth group Intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) on the left side of the tumor, followed by radiofrequency ablation therapy plus tail vein injection of anti-PDL1 antibody therapy.
  • Step 2 Sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection combined with high-energy focused ultrasound knife (HIFU) and immune checkpoint suppression therapy anti- Efficacy study of PDL1 antibody.
  • HIFU high-energy focused ultrasound knife
  • mice Mouse colon cancer tumors were planted on the left and right ends of the back of the mice (the left side is regarded as the in situ tumor, the right side is regarded as the distal tumor), and the tumor-bearing mice were divided into four groups, each with 5 mice for treatment experiments.
  • the first group the left orthotopic tumor is treated with HIFU alone (reference example);
  • the second group After HIFU treatment of the left orthotopic tumor, anti-pdl1 antibody was injected into the tail vein (reference example);
  • the third group HIFU treatment after intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) on the left side of the tumor;
  • the fourth group Intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) on the left side of the tumor, followed by HIFU treatment plus tail vein injection of anti-PDL1 antibody treatment.
  • Step 3 Sodium alginate (first component) and imiquimod hydrochloride (third component) composition freeze-dried powder injection combined with high-energy focused ultrasound knife (HIFU) and immune checkpoint suppression therapy anti- PDL1 antibody causes immune memory effect research.
  • HIFU high-energy focused ultrasound knife
  • the colon cancer-bearing mice were divided into six groups, 5 mice in each group.
  • the first group the normal saline group
  • the second group tail vein anti-PDL1 antibody treatment (reference example);
  • the third group HIFU treatment alone (reference example);
  • the fourth group tail vein anti-pdl1 antibody treatment after HIFU treatment (reference example);
  • the fifth group HIFU treatment after intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1);
  • the sixth group intratumoral injection of sodium alginate and imiquimod hydrochloride composition freeze-dried powder injection (Example 1) after HIFU treatment and tail vein anti-PDL1 antibody treatment.
  • mice in the fifth and sixth groups were significantly slower than that of the control group, and was significantly inhibited.
  • the tumor growth of the sixth group of mice was slower than that of the fifth group, and some of them were no longer able to grow. Grow a tumor.
  • Example 14 Study on the curative effect of sodium alginate (first component) and oxaliplatin (second component) composition freeze-dried powder injection on colon cancer model
  • the colon cancer-bearing mice were divided into 6 groups, with 5 mice in each group for treatment experiments.
  • mice were injected with saline intratumor respectively (reference example);
  • the second group Oxaliplatin (1.5 mg per kilogram of body weight) (reference example of single chemotherapeutic agent);
  • the third group intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (0.375 mg/kg body weight) (Example 4);
  • the fourth group intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (0.75 mg/kg body weight) (Example 4);
  • the fifth group intratumoral injection of oxaliplatin and sodium alginate composition freeze-dried powder injection (1.5 mg/kg body weight) (Example 4);
  • the sixth group oxaliplatin (3 mg/kg body weight) was injected into the tail vein (a reference case with a single chemotherapeutic agent).
  • mice in the tail vein injection of oxaliplatin group showed a significant weight loss in the first four days, indicating that the intravenous injection has certain toxic and side effects, while the intratumoral injection showed no obvious toxic side effect.
  • the side effects of intratumoral administration using the composition of the patent technical scheme are lower than that of intravenous administration.
  • Example 15 Sodium alginate (first component), oxaliplatin (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth A study on the efficacy of freeze-dried powder injection on a bilateral tumor (one tumor on the left and right sides) model.
  • mice colon cancer tumors were planted on the left and right ends of the back of the mice (the left side is regarded as the in situ tumor, the right side is regarded as the distal tumor), and the tumor-bearing mice are divided into 7 groups, each group has 6 mice for combined immunization experiment.
  • mice were injected with saline intratumor respectively (reference example);
  • the second group Oxaliplatin, Imiquimod and anti-PDL1 composite solution (reference example);
  • the third group sodium alginate and oxaliplatin composition freeze-dried powder injection (Example 4) combined with intravenous injection of anti-PDL1 (reference example);
  • the fourth group intratumoral injection of sodium alginate, oxaliplatin and anti-PDL1 composition freeze-dried powder injection (Example 4);
  • the fifth group intratumoral injection of sodium alginate, oxaliplatin and imiquimod composition lyophilized powder injection (Example 6);
  • the sixth group intratumoral injection of sodium alginate, oxaliplatin and imiquimod anti-PDL1 composition freeze-dried powder injection (Example 10);
  • the seventh group sodium alginate, oxaliplatin and imiquimod composition freeze-dried powder injection (Example 6) combined with anti-PDL1 intravenous injection (reference example).
  • Example 16 Sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth The study on the efficacy of freeze-dried powder injection in breast cancer metastasis model
  • mice with 4T1 breast cancer tumors in situ on the breast pads were divided into 6 groups, with 6 mice in each group for treatment experiments on metastatic tumor models.
  • mice were injected with saline intratumor respectively (reference example);
  • the second group doxorubicin and imiquimod hydrochloride and anti-PDL1 antibody (reference example);
  • the third group intratumoral injection of sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride composition freeze-dried powder injection (Example 5);
  • the fourth group intratumoral injection of sodium alginate, adriamycin hydrochloride and anti-PDL1 antibody composition freeze-dried powder injection (Example 5);
  • the fifth group intratumoral injection of sodium alginate, adriamycin, imiquimod and anti-PDL1 antibody composition freeze-dried powder injection (Example 9);
  • the sixth group sodium alginate, adriamycin and imiquimod hydrochloride composition freeze-dried powder injection (Example 5) combined with intravenous injection of anti-PDL1 antibody (reference example).
  • Example 17 Sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth (Class-component) Freeze-dried Powder Injection on the Efficacy of Mouse Brain Cancer
  • mice The first group: intracranial injection of normal saline in mice (reference example);
  • mice were intraperitoneally injected with temotomil (reference example);
  • mice were injected intracranially with imiquimod hydrochloride, anti-PDL1 antibody and sodium alginate composition (reference example);
  • mice were injected intracranially with a freeze-dried powder injection of a composition of sodium alginate and adriamycin (Example 3);
  • mice were injected intracranially with doxorubicin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody composition (reference example);
  • mice were injected intracranially with sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride composition freeze-dried powder injection (Example 5);
  • mice were injected intracranially with a combination of sodium alginate, adriamycin hydrochloride and anti-PDL1 antibody (reference example);
  • mice are injected intracranially with sodium alginate, adriamycin hydrochloride, imiquimod hydrochloride and anti-PDL1 antibody composition freeze-dried powder injection (Example 9);
  • mice were treated with intracranial injection of sodium alginate, adriamycin hydrochloride and imiquimod hydrochloride freeze-dried powder injection (reference example).
  • Figure 28 is the mortality curve of mice. It can be seen from the figure that the survival time of mice in the eighth and ninth groups is twice as long as that of the control group, indicating that the treatment effect is better.
  • Example 18 Sodium alginate (first component), doxorubicin hydrochloride (second component), imiquimod hydrochloride (third component) and anti-PDL1 antibody (fourth (Class component) freeze-dried powder injection in the mouse model after tumor resection
  • mice The subcutaneous breast cancer mice were randomly divided into three groups, and six mice in each group were treated with sodium alginate, adriamycin, imiquimod, and anti-PDL1 antibody composite gel treatment experiment. After the mice were surgically removed most of the subcutaneous tumors (removal of the mouse subcutaneous tumors to preserve their adjacent skin and muscle).
  • the first group is not processed (reference example);
  • the second group of simple surgery (reference example).
  • the treatment effect is judged by observing the metastasis and recurrence of the tumor after surgery, and the conclusion is drawn by small animal bioluminescence imaging. It can be seen from Figure 29 that the tumors of the third group of mice in the key group have a good effect of inhibiting metastasis and recurrence, which proves that our sodium alginate, doxorubicin, imiquimod and anti-PDL1 antibody composite gel The role of.
  • Example 19 The pharmaceutical composition of this example can be prepared in two dosage forms.
  • the first dosage form is as follows:
  • Component 1 according to the ratio of imiquimod R837: surfactant poloxamer 188: (0.1-5). Weigh imiquimod R837 and surfactant poloxamer 188. Preferably 1g R837, add appropriate amount of Poloxamer 188 (0.15g, 0.3g, 0.5g, 1g, 2g, 3g, 4g, 5g), add 10ml water ball mill for 3 hours, after the end, take out 9ml homogenate and transfer to 250ml In the beaker, add 141ml of first-grade water, stir at 500rpm for 1 hour and mix well. Use a syringe to suck the suspension and fill it into 10ml vials of 5ml each, totaling 30 vials. Cover the rubber cover and seal it with the aluminum cover, then sterilize it with moist heat at 121°C for 12 minutes;
  • Component 2 Prepare 0.1-5% sodium alginate and 0.1-1% oxaliplatin solution, preferably weigh out sodium alginate ALG (3g, or 1.5g), appropriate amount of oxaliplatin (159mg, 300mg, 450mg) ,90mg), add 300ml first-grade water, stir in a 500ml beaker for 2-6 hours (25°C-40°C, 200-850rpm, seal the bottle with plastic wrap). The obtained solution was filtered through a 0.22 micron filter membrane to sterilize. Fill a 20ml vial with a 60ml syringe, 10ml per bottle, 30 bottles in total. After being pre-cooled in a refrigerator at -80°C for 30 minutes, it was freeze-dried for 30 hours. After freeze-drying, cover the rubber cover and seal the aluminum cover.
  • the second dosage form is as follows:
  • Component 1 According to the ratio of imiquimod R837:surfactant poloxamer 188 to 1:(0.1-5), weigh R837 and surfactant, and add 0.1-1% oxaliplatin.
  • Component 2 Prepare a 0.1-5% solution of sodium alginate. Preferably, weigh 3g or 1.5g of sodium alginate ALG, add 300ml of first-grade water, and stir in a 500ml beaker for 2-6 hours (25°C-40°C, 200-850rpm, seal the bottle with plastic wrap). The obtained solution was filtered through a 0.22 micron filter membrane to sterilize. Fill a 20ml vial with a 60ml syringe, 10ml per bottle, 30 bottles in total. After pre-cooling in the refrigerator at -80°C for 30 minutes, freeze-dry for 30 hours. After freeze-drying, cover the rubber cover and seal the aluminum cover.
  • the specific effects of this embodiment for treatment are as follows.
  • the first dosage form is used, and the second dosage form has similar therapeutic effects as the first dosage form.
  • mice Plant mouse colon cancer tumors on the left and right ends of the back of the mouse (the left side is regarded as the tumor in situ, the right side is regarded as the distal tumor), and the tumor-bearing mice are divided into 5 groups, with 6 mice in each group as a combination Immunity treatment experiment.
  • mice were injected with saline intratumor respectively (reference example);
  • the second group intratumoral injection of oxaliplatin + poloxamer 188 dispersed imiquimod particles;
  • the third group intratumoral injection of oxaliplatin + sodium alginate;
  • the fourth group intratumoral injection of Poloxamer 188 dispersed imiquimod particles + sodium alginate;
  • the fifth group intratumoral injection of oxaliplatin + poloxamer 188 dispersed imiquimod particles + sodium alginate;
  • Poloxamer P188 ball mill is added to R837 ball mill emulsion.
  • Poloxamer 188 is a new type of polymer nonionic surfactant. It has many uses including: as emulsifier, stabilizer and solubilizer. Further enhance the water dispersibility and stability of R837 ball mill emulsion.
  • solubilizing pharmaceutical excipients used include: poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), egg yolk Phospholipids, polyoxyethylene (35) castor oil, vitamin E polyethylene glycol succinate, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 800, or one of sodium hydroxymethyl cellulose Or multiple.
  • polysorbate 80 polyethylene glycol-12-hydroxystearate
  • Solutol HS 15 polyethylene glycol-12-hydroxystearate
  • egg yolk lecithin polyoxyethylene
  • polyoxyethylene 35
  • castor oil vitamin E succinate polyethylene glycol ester, or sodium hydroxymethyl cellulose have a good solubilizing effect on R837.
  • polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 800 have general solubilization effects.
  • Polysorbate 80 Teween 80
  • Polyoxyethylene castor oil as a non-ionic surfactant, although it can increase the water solubility of most insoluble drugs, it will cause the release of histamine and cause a variety of toxic and side effects, such as severe allergic reactions, toxic kidney damage, Neurotoxicity, cardiovascular toxicity, etc.
  • Solutol HS 15 has the most toxic and side effects for muscle stimulation and hemolysis experiments, while polyethylene glycol 200 has the least side effects.
  • Polyethylene glycol is a stable hydrophilic substance, non-toxic, non-irritating, solubilizing, increasing stability and prolonging effect on many drugs.
  • Poloxamer 188 is a series of multi-purpose pharmaceutical excipients. It is 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. It has been used clinically as an emulsifier and solubilizer for intravenous administration.
  • Poloxamer 188 can help R837 ensure water dispersibility and stability after sterilization.
  • Table 1 Water dispersibility of ball milled imiquimod R837 before and after adding surfactant
  • Surfactant a Long-term stability after autoclaving 0.15:1 A large number of granular aggregates appear 0.3:1 A large number of granular aggregates appear 0.5:1 A large number of granular aggregates appear 1:1 A large number of granular aggregates appear 2:1 A large number of granular aggregates appear 3:1 Evenly dispersed and no granular aggregates appear 4:1 Evenly dispersed and no granular aggregates appear
  • Applicable surfactants include: poloxamer 188, poloxamer 407, polysorbate 80 (Tween 80), polyethylene glycol-12-hydroxystearate (Solutol HS 15), poly Oxyethylene (35) castor oil, vitamin E succinate polyethylene glycol ester, sodium hydroxymethyl cellulose.
  • Surfactant b Long-term stability after autoclaving 0.15:1 A large number of granular aggregates appear 0.3:1 A large number of granular aggregates appear 0.5:1 A large number of granular aggregates appear 1:1 Evenly dispersed and no granular aggregates appear 2:1 Evenly dispersed and no granular aggregates appear 3:1 Evenly dispersed and no granular aggregates appear 4:1 Evenly dispersed and no granular aggregates appear 5:1 Evenly dispersed and no granular aggregates appear
  • Applicable surfactants include: egg yolk lecithin.
  • Surfactant c R837 Long-term stability after autoclaving 0.15:1 Large flocculent aggregates 0.3:1 Large flocculent aggregates 0.5:1 A large number of flocculent aggregates appear 1:1 A large number of flocculent aggregates appear 2:1 Large flocculent aggregates 3:1 A large number of flocculent aggregates appear 4:1 A large number of flocculent aggregates appear 5:1 A large number of flocculent aggregates appear
  • Applicable surfactants include: polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 800.
  • Poloxamer 188 itself is viscous, the concentration is too high, the viscosity is very large, and to avoid introducing impurities.
  • Sodium alginate is a relatively special natural biopolymer material. Because it decomposes at high temperatures, it cannot be sterilized by traditional high temperature and humid heat. This patent uses filter sterilization to retain the properties of sodium alginate. In addition, from the perspective of sterilization, sodium alginate and imiquimod R837 cannot be mixed together. Sodium alginate ALG needs to be filtered and sterilized, but R837 particles cannot be filtered (the minimum particle diameter after ball milling is 500nm, and the filter kills Bacteria need a 220nm filter membrane, so they cannot pass); on the other hand, R837 needs moist heat sterilization, and sodium alginate ALG will degrade at high temperature. Therefore, both R837 and ALG cannot be sterilized together.
  • Imiquimod R837 needs to be made into ball-milled granular formulations is that if R837 is formulated into hydrochloride, there is a contraindication with OXA, and OXA will react with chloride ions to inactivate; secondly, the hydrochloride of R837 will increase The viscosity of sodium alginate ALG is not convenient to use.

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Abstract

本发明公开了一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;所述能引起免疫原性死亡的化疗药为奥沙利铂,所述免疫佐剂为咪喹莫特R837,还包括有泊洛沙姆188和海藻酸钠ALG;第一混合物为,所述咪喹莫特R837与泊洛沙姆188混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌;第二混合物为,所述海藻酸钠ALG与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;第一混合物与第二混合物混合构成化疗免疫组合药物。本发明要解决的技术问题是提供一种新型化疗免疫药物组合物,提供新的能够产生协同抗癌作用,并且降低副作用,降低癌症转移概率,降低癌症复发概率的抗癌药物组合物,可以在有效杀灭原位肿瘤的同时通过免疫反应抑制、降低远端转移肿瘤的生长和肿瘤复发的概率,同时生产工艺较为优化,产品稳定性好。

Description

一种化疗免疫组合药物及其制备方法 技术领域
本发明涉及治疗肿瘤药物领域,尤其涉及化疗免疫联合治疗药物组合物,以及制备方法、应用。
背景技术
化疗是目前临床治疗肿瘤的三种主要治疗方法之一,大部分的癌症患者都需要接受一定程度的化疗,对那些有转移倾向的或者已经转移的肿瘤,化疗更是主要的治疗手段。然而,传统化疗药对正常器官也有损伤,而且临床常用的化疗模式都是全身性给药,对病变部位并没有很好地选择性,化疗毒副作用非常大。
尽管以免疫检查点阻断为代表肿瘤免疫治疗近年来取得了令人鼓舞的成就,这一疗法还存在重要的局限性,包括临床响应率低(20%左右)、非特异性免疫反应带来的副作用等。特别是目前临床免疫检查点阻断疗法的临床响应率低,意味着大部分患者对于这一代价昂贵的疗法是没有响应的。为了进一步提升肿瘤治疗的疗效和响应率,有必要改善现有疗法的用药途径,并发展针对肿瘤的化疗-免疫联合治疗实现协同效应。例如,需要考虑如何更好的将ICD类化疗药物对肿瘤细胞的杀伤局限在肿瘤原位,避免对整个身体造成损害;如何更好的放大癌细胞死亡后其肿瘤相关抗原的免疫原性以获得更强的肿瘤特异性免疫反应;如何更有效结合免疫检查点抑制剂(如CTLA-4、PD-1/PD-L1抗体)或IDO抑制剂的作用从而进一步通过调节免疫平衡增强针对肿瘤的特异性免疫反应等一系列难题。这方面的新技术开发对癌症高发、而抗癌原研药研发较为落后的中国更是具有重要的现实意义。此外,如何能在局部治疗的同时又抑制肿瘤转移并预防其复发,一直是困扰全球的难题。
同时,相关药物生产的操作性,以及药物产品的灭菌和后续稳定性,也都是难题。
发明内容
本发明要解决的技术问题是提供一种新型化疗免疫药物组合物,提供新的 能够产生协同抗癌作用,并且降低副作用,降低癌症转移概率,降低癌症复发概率的抗癌药物组合物,可以在有效杀灭原位肿瘤的同时通过免疫反应抑制、降低远端转移肿瘤的生长和肿瘤复发的概率,同时生产工艺较为优化,产品稳定性好。
为解决相关技术问题,本发明提供了如下技术方案:
一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;所述能引起免疫原性死亡的化疗药为奥沙利铂,所述免疫佐剂为咪喹莫特R837,还包括有泊洛沙姆188和海藻酸钠ALG;
第一混合物为,所述咪喹莫特R837与泊洛沙姆188混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌;
第二混合物为,所述海藻酸钠ALG与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
第一混合物与第二混合物混合构成化疗免疫组合药物。
一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;所述能引起免疫原性死亡的化疗药为奥沙利铂,所述免疫佐剂为咪喹莫特R837,还包括有泊洛沙姆188和海藻酸钠ALG;
第一混合物为,所述咪喹莫特R837与泊洛沙姆188混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,将咪喹莫特乳液与奥沙利铂及水混合搅匀经过高温湿热灭菌;
第二混合物为所述海藻酸钠与水混合,然后经过微米滤膜过滤除菌制成混合物;
第一混合物与第二混合物混合构成化疗免疫组合药物。
作为所述的化疗免疫组合药物的一种优选方案:所述咪喹莫特R837与泊洛沙姆188的质量配比为1:(0.1-5),所述高温灭菌为105℃~150℃湿热灭菌10-15分钟;
所述第二混合物经0.22微米滤膜过滤除菌,并经冻干后制成冻干粉。
作为所述的化疗免疫组合药物的一种优选方案:所述咪喹莫特R837与泊洛沙姆188的质量配比为1:(0.1-5),所述高温灭菌为105℃~150℃湿热灭菌10-15分钟;
所述第二混合物经0.22微米滤膜过滤除菌,并经冻干后制成冻干粉。
一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
第一混合物为,所述免疫佐剂为咪喹莫特,咪喹莫特与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-300微米粒径,咪喹莫特乳液经过高温湿热灭菌,所述表面活性剂为泊洛沙姆407,或聚山梨酯80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油,或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
第二混合物为,海藻酸钠与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
第一混合物与第二混合物混合构成化疗免疫组合药物。
作为所述的化疗免疫组合药物的一种优选方案:所述海藻酸钠替换为壳聚糖、或纤维蛋白原、或藻酸盐、或透明质酸;
所述咪喹莫特R837替换为咪唑喹啉,或吡喃葡糖苷脂质;
所述奥沙利铂Oxa替换为蒽环类药物、或环磷酰胺、或硼替佐米、或吉西他滨、或五氟尿嘧啶、或毒素。
一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
第一混合物为,所述咪喹莫特R837与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,将咪喹莫特乳液与奥沙利铂及水混合搅匀经过高温湿热灭菌;
所述表面活性剂为泊洛沙姆407,或聚山梨酯80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油, 或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
第二混合物为所述海藻酸钠与水混合,然后经过微米滤膜过滤除菌制成混合物;
第一混合物与第二混合物混合构成化疗免疫组合药物。
作为所述的化疗免疫组合药物的一种优选方案:所述海藻酸钠能替换为壳聚糖、或纤维蛋白原、或藻酸盐、或透明质酸;
所述咪喹莫特R837能替换为咪唑喹啉,或吡喃葡糖苷脂质;
所述奥沙利铂Oxa能替换为蒽环类药物、或环磷酰胺、或硼替佐米、或吉西他滨、或五氟尿嘧啶、或毒素。
一种化疗免疫组合药物的制备方法,其特征在于包括如下步骤:
第一步:按比例1:(0.1-5)称取咪喹莫特R837和表面活性剂泊洛沙姆188,加水球磨2-3小时,结束后取出匀浆,加水,搅拌混匀,105℃~150℃湿热灭菌10-15分钟;
第二步:称取海藻酸钠ALG与奥沙利铂,加水,搅拌,将获得的溶液经微米滤膜过滤除菌;预冷后,进行冻干;
第三步:使用时将混合物二冻干粉加入混合物一溶液中,充分震荡混匀溶解后注射。
一种治疗结肠癌肿瘤的化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
第一混合物为,所述免疫佐剂为咪喹莫特,咪喹莫特与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌,所述表面活性剂为泊洛沙姆407,或聚山梨酯80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油,或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
第二混合物为,海藻酸钠与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
第一混合物与第二混合物混合构成化疗免疫组合药物。
本发明还提供一种原位成胶化疗免疫联合治疗生物高分子药物组合物,其含有:第一类组分为海藻酸盐,所述海藻酸盐能与体内的钙离子形成多孔凝胶,所述的海藻酸盐为海藻酸钠、海藻酸钾和海藻酸铵中的一种或多种;
第二类组分为能引起免疫原性死亡的化疗药;
第三类组分为免疫佐剂。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述免疫佐剂为咪喹莫特(R837)、CpG寡核苷酸、单磷酰脂质A和瑞喹莫德中的一种或多种。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述第二类组分能引起免疫原性死亡的化疗药为蒽环类药物如阿霉素,表阿霉素,米托蒽醌,奥沙利铂,环磷酰胺,硼替佐米,吉西他滨,五氟尿嘧啶和毒素如美登素中的一种或多种。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:还包括有第四类组分免疫检查点抑制剂或IDO抑制剂,所述第四类组分免疫检查点抑制剂抗体通常有anti-CTLA-4、anti-PD-1和anti-PD-L1,小分子抑制剂类通常有CA-170、PM-327、BMS-8、BMS-37、BMS-202、BMS-230、BMS242、BMS-1001、BMS-1166、BMS-1001、BMS-1166和JQ1,肽类抑制剂有DPPA-1;
所述IDO抑制剂包括BMS-986205、IDO inhibitor 1、NLG919,NLG8189,PF-06840003,Epacadostat和4-苯基咪唑等小分子。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述第一类组分为海藻酸钠,所述第二类组分为盐酸阿霉素;所述第三类组分为咪喹莫特,所述海藻酸钠,盐酸阿霉素及咪喹莫特的质量比为50~800比1~100比1~100。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述海藻酸钠,盐酸阿霉素及咪喹莫特的质量比为200~400比10~75比10~75。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述海藻酸钠浓度为5毫克每毫升以上。
制备原位成胶化疗免疫联合治疗生物高分子药物组合物的方法,所述方法包括:
将海藻酸钠和咪喹莫特盐酸盐冻干粉和盐酸阿霉素溶于水相溶液中,搅拌至溶液澄清透明,后将溶液冻干得到组合物冻干粉;
或者,将海藻酸钠和盐酸阿霉素溶于水相溶液,搅拌至溶液澄清透明,后冻干得到冻干粉,与咪喹莫特盐酸盐冻干粉通过固体与固体震荡混合均匀得到组合物冻干粉;
或者,将阿霉素盐酸盐和咪喹莫特盐酸盐溶于水相溶液中,搅拌至溶液澄清透明,后将海藻酸钠水相溶液中,滴入不断搅拌的混合溶液,保证混合液澄清透明没有絮状沉淀,将混合液取出冻干得到组合物冻干粉。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述第一类组分为海藻酸钠,所述第二类组分为奥沙利铂;所述第三类组分为咪喹莫特盐酸盐,所述海藻酸钠,奥沙利铂及咪喹莫特的质量比为50~800比1~75比1~100。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述海藻酸钠,奥沙利铂及咪喹莫特的质量比为200~400比10~75比10~75。
制备所述的原位成胶化疗免疫联合治疗生物高分子药物组合物的方法,所述方法包括:
将海藻酸钠和咪喹莫特盐酸盐冻干粉和奥沙利铂溶于水相溶液中,搅拌至溶液澄清透明后将溶液冻干得到组合物冻干粉;
或者,将海藻酸钠和奥沙利铂溶于水相溶液搅拌至溶液澄清透明后冻干得到冻干粉,然后再与咪喹莫特盐酸盐冻干粉通过固固震荡混合均匀得到组合物冻干粉。
或者,将奥沙利铂和咪喹莫特盐酸盐溶于水相溶液中,搅拌至溶液澄清透明,后将海藻酸钠溶于水相溶液中,滴入不断搅拌的混合溶液,保证混合液澄清透明没有絮状沉淀,将混合液取出冻干得到组合物冻干粉。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:所述第一类组分为海藻酸钠,所述第二类组分为五氟尿嘧啶;所述第三类组分为咪喹莫特盐酸盐;
或者,所述第一类组分为海藻酸钠,所述第二类组分为环磷酰胺;所述第三类组分为咪喹莫特盐酸盐。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案: 第一类组分为海藻酸钠;第二类组分为盐酸阿霉素或奥沙利铂;和第三类组分为咪喹莫特盐酸盐;第四类组分为anti-PDL1抗体。
作为原位成胶化疗免疫联合治疗生物高分子药物组合物的一种优选方案:第一类组分为海藻酸钾或海藻酸铵;第二类组分为盐酸阿霉素或奥沙利铂;和第三类组分为咪喹莫特盐酸盐;第四类组分为anti-PDL1抗体。
一种原位成胶化疗免疫联合治疗生物高分子药物组合物,组成为:第一类组分为海藻酸盐,所述海藻酸盐能与体内的钙离子形成多孔凝胶,所述的海藻酸盐为海藻酸钠、海藻酸钾和海藻酸铵中的一种或多种;第二类组分为能引起免疫原性死亡的化疗药;第三类组分为免疫佐剂。
一种原位成胶化疗免疫联合治疗生物高分子药物组合物,组成为:第一类组分为海藻酸盐,所述海藻酸盐能与体内的钙离子形成多孔凝胶,所述的海藻酸盐为海藻酸钠、海藻酸钾和海藻酸铵中的一种或多种;第二类组分能引起免疫原性死亡的化疗药为蒽环类药物如阿霉素,表阿霉素,米托蒽醌,奥沙利铂,环磷酰胺,硼替佐米,吉西他滨,五氟尿嘧啶和毒素如美登素中的一种或多种;第三类组分为免疫佐剂,所述免疫佐剂为咪喹莫特(R837)、CpG寡核苷酸、单磷酰脂质A和瑞喹莫德中的一种或多种。
一种原位成胶化疗免疫联合治疗生物高分子药物组合物,组成为:第一类组分为海藻酸盐,所述海藻酸盐能与体内的钙离子形成多孔凝胶,所述的海藻酸盐为海藻酸钠、海藻酸钾和海藻酸铵中的一种或多种;第二类组分能引起免疫原性死亡的化疗药为蒽环类药物如阿霉素,表阿霉素,米托蒽醌,奥沙利铂,环磷酰胺,硼替佐米,吉西他滨,五氟尿嘧啶和毒素如美登素中的一种或多种;第三类组分为免疫佐剂,所述免疫佐剂为咪喹莫特(R837)、CpG寡核苷酸、单磷酰脂质A和瑞喹莫德中的一种或多种;
第四类组分免疫检查点抑制剂或IDO抑制剂,所述第四类组分免疫检查点抑制剂抗体通常有anti-CTLA-4、anti-PD-1和anti-PD-L1,小分子抑制剂类通常有CA-170、PM-327、BMS-8、BMS-37、BMS-202、BMS-230、BMS242、BMS-1001、BMS-1166、BMS-1001、BMS-1166和JQ1,肽类抑制剂有DPPA-1;
所述IDO抑制剂包括BMS-986205、IDO inhibitor 1、NLG919,NLG8189,PF-06840003,Epacadostat和4-苯基咪唑小分子。
一种原位成胶化疗免疫联合治疗生物高分子药物组合物,组成为:第一类 组分为海藻酸钠,第二类组分为盐酸阿霉素;第三类组分为咪喹莫特,所述海藻酸钠,盐酸阿霉素及咪喹莫特的质量比为50~800比1~100比1~100。
本发明提供了一系列药物组合物。在这个组合物体系中,主要有四类组分,可以由第一类组分与其他几类组分根据实际情况进行不同组合,包括:
第一类组分:海藻酸钠类辅料,能与人体或动物体内钙离子等离子产生凝胶;
第二类组分:引起免疫原性死亡的化疗药;
第三类组分:免疫佐剂;
第四类组分:免疫检查点抑制剂或IDO抑制剂。
第一类组分,辅料,通常有海藻酸钠、海藻酸钾和海藻酸铵等,这类多糖在遇到钙离子等二价离子后会相互交联形成凝胶,因此当药物被包裹其中后,形成的凝胶能有效地缓释其中的药物,从而增强疗效,减弱副作用。
海藻酸钠是一种天然多糖,具有药物制剂辅料所需的稳定性、溶解性、粘性和安全性。海藻酸钠已经在食品工业和医药领域得到了广泛应用。海藻酸钠是应用最广泛的水溶性海藻酸盐。海藻酸钠遇到钙离子可迅速发生离子交换,生成凝胶,而人体或动物体内有充足的钙离子,因此,可以体内原位成胶。
而海藻酸钾和海藻酸铵两种海藻酸盐,虽然所含阳离子与海藻酸钠不同,但同样可以与钙离子交联形成多孔的凝胶,从而起到缓释药物的作用。目前海藻中提取的通常是海藻酸钠,所以海藻酸钠是更优选择。
第二类组分,能引起免疫原性死亡的化疗药,有蒽环类药物如阿霉素,表阿霉素,米托蒽醌等,还有奥沙利铂,环磷酰胺,硼替佐米,吉西他滨,五氟尿嘧啶和毒素如美登素等等。这些药物都已经被临床批准,并且近年来研究表明,这些药物会引起癌细胞免疫原性死亡,这些死亡的癌细胞会表达容易被免疫细胞尤其是抗原提呈细胞识别并摄取的钙网织蛋白,帮助免疫细胞识别肿瘤细胞,引起有效的抗肿瘤免疫反应。
第三类组分,免疫佐剂,简称佐剂,即非特异性免疫增生剂,指那些同抗原一起或预先注入机体内能增强机体对抗原的免疫应答能力或改变免疫应答类型的辅助物质。免疫佐剂种类很多,目前尚无统一的分类方法,应用比较多的是福氏佐剂和细胞因子佐剂。免疫佐剂的免疫生物学作用是增强免疫原性、增强抗体的滴度、改变抗体产生的类型、引起或增强迟发超敏反应,但免疫佐剂的 具体作用机制尚未完全明了,不同佐剂作用的机制也不尽相同。通常有咪喹莫特(R837)、CpG寡核苷酸、单磷酰脂质A和瑞喹莫德等等,他们都是Toll样受体(Toll-like receptors简称TLR)的激动剂,能够帮助抗原提呈细胞提呈抗原,因此免疫佐剂可以将化疗产生的肿瘤相关抗原更好地提呈给T细胞,从而放大免疫反应。
第四类组分,免疫调控剂包括免疫检查点抑制剂或IDO抑制剂。免疫检查点抑制剂包括抗体类抑制剂或小分子抑制剂,抗体类抑制剂通常有anti-CTLA-4、anti-PD-1和anti-PD-L1,小分子抑制剂类通常有CA-170、PM-327、BMS-8、BMS-37、BMS-202、BMS-230、BMS242、BMS-1001、BMS-1166、BMS-1001、BMS-1166和JQ1,肽类抑制剂有DPPA-1。IDO抑制剂包括BMS-986205、IDO inhibitor 1、NLG919,NLG8189,PF-06840003,Epacadostat和4-苯基咪唑等小分子,能够抑制IDO酶从而增强抗原提呈细胞的作用。由于肿瘤细胞会欺骗免疫系统,逃逸免疫反应,所以需要这些抗体来抑制保护肿瘤的免疫反应,使得免疫细胞能更好地杀伤肿瘤细胞。
第一类组分辅料也可简称组分一;第二类组分ICD类化疗药物也可简称组分二;第三类组分免疫佐剂也可简称组分三;第四类组分免疫检查点抑制剂也可简称组分四。
冻干粉是在无菌环境下将药液冷冻成固态,抽真空将水分升华干燥而成的无菌粉注射剂。
组分一、组分二、组分三与组分四的混合药液与冻干制剂的制备过程如下。
本专利主要涉及到四类原料成分:第一类组分辅料海藻酸钠(固体粉末),第二类组分ICD类化疗药物(固体粉末),第三类组分免疫佐剂(固体粉末),第四类组分免疫检查点抑制剂(anti-CTLA-4、anti-PD-1或anti-PD-L1抗体,临床使用的商业化产品,以冻干粉或注射液形式为原料)。
制备方案一:将组分一辅料、组分二ICD类化疗药物、组分三免疫佐剂的固体粉末按一定比例混和后入大烧杯中,加入去离子水(或生理盐水、或磷酸缓冲溶液),在室温25摄氏度用搅拌桨以转速50到500转每分搅拌至溶液澄清透明;根据需要,将组分四免疫检查点抑制剂根据产品说明书配制成注射液,加入上述混和溶液中;将上述溶液搅拌均匀后,取出分瓶冻干。冻干粉复溶后,不能出现浑浊和絮状沉淀。
制备方案二:分别称取目标质量的组分一、组分二、组分三,分别加入去离子水(或生理盐水、或磷酸缓冲溶液)配制为三份独立的溶液;根据需要,将组分四根据产品说明书配制成注射液;将上述溶液以合适的体积比混合,室温25摄氏度用搅拌桨以转速50到500转每分搅拌至溶液均匀不浑浊,取出分瓶冻干。冻干粉复溶后,不能出现浑浊和絮状沉淀。方案一和方案二差别不大。
制备方案三:分别称取目标质量的组分一、组分二、组分三,分别加入去离子水(或生理盐水、或磷酸缓冲溶液)配制为三份独立的溶液;根据需要,将组分四根据产品说明书配制成注射液;对于含盐酸盐的ICD药物,需要先将组分二、组分三、组分四的溶液搅拌混合,并在搅拌过程中(25摄氏度,搅拌桨以转速50到300转每分)将组分一溶液缓慢滴入,至整个溶液搅拌均匀,相比于方案一和方案二,当组分二或组分三为盐酸盐类药物时,考虑到pH值对溶液稳定性的影响,需按该方案制备组合物,使得制备的组合物混合溶液没有絮状沉淀的现象,保证混合均匀,溶液澄清透明随后取出分瓶冻干。冻干粉复溶后,不会出现浑浊和絮状沉淀。
四类组分的混合药液与冻干制剂的使用方案说明。
使用方案一:将上述四类组分的组合物冻干粉针剂通过生理盐水复溶后,通过临床介入给药和直接穿刺给药的方式,将组合物溶液直接注射到患者肿瘤部位,注射时采用多点注射的方式,保证组合物溶液均匀充满整个肿瘤。在组合物注射入肿瘤后,首先,利用第一类组分海藻酸盐遇到钙离子会形成凝胶的特性,第一类组分在遇到组织内的钙离子后会快速凝胶化,形成多孔的网状交联结构,使得混合在海藻酸盐里的其他三类组分能够得到缓慢释放,从而增强其效果降低毒副作用;其次,第二类组分ICD化疗药不仅能有效地杀伤肿瘤细胞而且能使其产生免疫原性死亡,产生肿瘤相关抗原,激活肿瘤特异的免疫反应;再次,第三类组分免疫佐剂增强了抗原提呈细胞的能力进一步放大了相应的免疫反应;最后,利用第四类组分免疫检查点抑制剂或IDO抑制剂使得转移的肿瘤不能逃逸免疫反应,使得免疫治疗能更有效地杀伤肿瘤,从而抑制肿瘤的转移和复发。(实施例十二)
使用方案二:将上述第一、二、三类组分的组合物冻干粉针剂通过生理盐水复溶后,通过临床介入给药和直接穿刺给药的方式,将组合物溶液直接注射到患者肿瘤部位,注射时采用多点注射的方式,保证组合物溶液均匀充满整个 肿瘤。该治疗方式推荐与第四类组分免疫检查点抑制剂联用:联用的方案包括在注射液中根据病人的个体情况加入免疫检查点抑制剂(anti-CTLA-4、anti-PD-1或anti-PD-L1抗体)或IDO抑制剂,一次性瘤内局部注射;也可以是在一、二、三类组分混和液注射的局部治疗后,静脉给药注射免疫检查点抑制剂。(实施例十二中参照例)
使用方案三(四类组分的组合物喷涂创口,然后喷涂钙离子溶液形成凝胶):肿瘤患者在正常手术切除病灶部位后,考虑到手术切除不能完全清除病灶部位的肿瘤细胞的问题,可以将上述四类组分的冻干粉针剂通过生理盐水复溶,然后用注射器或者喷瓶喷在手术切除后的创口部位,随后可以在该部位喷洒适量的氯化钙溶液使其凝胶化,最后在将创口缝合。该方案有助于消灭残存的癌细胞,并且能抑制肿瘤转移和复发。(实施例十六)
使用方案四(三类组分的组合物喷涂创口,然后喷涂钙离子溶液形成凝胶+再加第四类组分联合使用):肿瘤患者在正常手术切除病灶部位后,考虑到手术切除不能完全清除病灶部位的肿瘤细胞的问题,可以将上述第一、二、三类组分的冻干粉针剂通过生理盐水复溶,然后用注射器或者喷瓶喷在手术切除后的创口部位,随后可以在该部位喷洒适量的氯化钙溶液使其凝胶化,最后在将创口缝合。该方案有助于消灭残存的癌细胞,并且能抑制肿瘤转移和复发。该治疗方式推荐在治疗后与第四类组分免疫检查点抑制剂或IDO抑制剂联用:联用的方案包括在注射液中根据病人的个体情况加入免疫检查点抑制剂(anti-CTLA-4、anti-PD-1或anti-PD-L1抗体)或IDO抑制剂,一次性瘤内局部注射;也可以是在一、二、三类组分混和液注射的局部治疗后,静脉给药注射免疫检查点抑制剂。
采用本专利的技术方案,会具有如下的有益技术效果:
一:海藻酸盐是一种天然多糖,其本身安全无毒,生物相容性好,可以降解,是很好的生物材料。然而在医药领域,常用的使用方式是将海藻酸盐和钙离子在体外结合形成凝胶植入式材料,这样的使用方式既限制了其在体内的应用,往往需要配合手术或者介入,操作难度大,对患者造成的损伤大,又不利于联合药物治疗。因此,我们采用将海藻酸盐注射到肿瘤内部,利用肿瘤组织内自有的钙离子使海藻酸钠在肿瘤原位成胶,利用形成的交联网状结构缓释混合在海藻酸盐中的药物,缓释效果更好。该技术有广阔的使用前景,既可以使 用注射器直接注射用于治疗肿瘤,操作简单,侵入性小;又可以使用喷雾器在手术后对伤口部位喷洒,配合手术清扫残留的癌细胞,有望针对不同的病人做个性化的治疗,且成本较低。
二:临床常规的化疗大多是静脉给药或是灌注式给药,该治疗方式没有很好的选择性和靶向性,对病灶和正常组织都有损伤,副作用大,患者会承受极大的生理和心理的伤害。并且,常规的化疗需要维持一定的血药浓度,使用剂量大,给药次数多,不仅大大增加了副作用,而且提高了给药的成本。我们采用直接肿瘤内给药的策略并配合凝胶缓释技术,使得化疗药更长时间停留在病灶部位,最大的发挥药物的作用,大大降低了药物对正常组织的损害。直接瘤内给药和缓释使得病灶内的有效药物浓度能长时间的保持在很高的范围,在保证药效的同时可以减少给药的次数,进一步降低副作用和成本。
三:该技术方案中提到的第三类组分免疫佐剂,在临床上还没有用来直接治疗肿瘤的先例。这些小分子免疫调节剂其本身并不具有抗病毒和抗肿瘤的效果,往往只是作为疫苗的辅助佐剂增强抗原的免疫原性。例如咪喹莫特(R873),通常是作为软膏制剂用来治疗成人外生殖器和肛周尖锐湿疣的药物,还没有在临床肿瘤治疗上使用。本专利的技术采用ICD化疗药和免疫佐剂一同注射的方式,在ICD药物杀伤肿瘤产生肿瘤抗原的同时,抗原和佐剂起到了类似肿瘤疫苗的作用,不仅可以抑制转移瘤,而且还能预防肿瘤复发。本专利的技术开创了免疫佐剂配合化疗药物直接治疗肿瘤的新策略。
四:目前,不管是科研前线还是临床上,免疫检查点抑制疗法都饱受关注。然而,尽管这类抗体在一些病人身上起到了奇迹般的效果,但是其有效性并不是百分之百。也就是说,针对不同的适应症不同的病人,检查点抑制剂的效果有待进一步研究。目前的研究表明,肿瘤可以分为热肿瘤和冷肿瘤,对于那些突变多,抗原表达高的肿瘤,往往检查点抑制疗法的效果显著。我们的方案,通过化疗杀死肿瘤提供抗原,又配合佐剂放大其免疫反应,意味着对大多数的肿瘤,本专利的方案都可以把其变成热肿瘤,大大提高检查点抑制剂的有效性。
五:本专利相关组分混合而成的药物组合物能够产生与众不同、意想不到的协同抗癌作用,并且能够降低常规治疗的副作用,降低癌症转移概率,降低癌症复发概率,提供了一种高效的肿瘤特异性免疫治疗方案,可以在有效杀灭原位肿瘤的同时通过免疫反应抑制、降低远端转移肿瘤的生长和肿瘤复发的概 率,能够在相对控制成本的前提下,有助于患者延长生存周期,提高生活质量。
六:进一步的,本专利中相关技术方案能够解决咪喹莫特难溶于水的问题、咪喹莫特灭菌后稳定性的问题,以及海藻酸钠的灭菌问题。从灭菌的角度考虑,不能把海藻酸钠和咪喹莫特R837配在一起,海藻酸钠ALG需要过滤灭菌,但R837颗粒无法过滤(球磨后颗粒直径最低为500nm,而过滤灭菌需要220nm的滤膜,因此不能通过);另一方面,R837需要湿热灭菌,而海藻酸钠ALG高温会降解。R837和ALG二者不能在一起灭菌。海藻酸钠是比较特殊的天然生物高分子材料,由于其在高温下会分解,因此无法采用传统的高温湿热灭菌,在本专利采用过滤灭菌,保留了海藻酸钠的性质。本专利相关实施例的剂型以及制备方法,解决了相关技术问题。同时,选择性增加了表面活性剂,尤其优选泊洛沙姆188。在不加入泊洛沙姆188的情况下,R837球磨乳液在121℃湿热灭菌后,会导致乳液不稳定产生明显的沉淀和颗粒,水分散性大打折扣,泊洛沙姆188可以大大帮助R837在灭菌后保证水分散性和稳定性。
根据本专利的技术方案,其中海藻酸钠、化疗药、以及免疫佐剂联合使用PD-1可以取得比较优异的治疗效果,但是,进一步优化的,海藻酸钠、化疗药与免疫佐剂,再加上泊洛沙姆188形成组合物,则不需要联合PD-1就可以产生更好的治疗效果,效果更好,但是治疗成本更低,参见图30、图31,可以得知具体实验数据。
附图说明
图1是实施例一中海藻酸钠与咪喹莫特盐酸盐组合物冻干粉针剂的制备流程,以及其使用说明。
图2是实施例一中海藻酸钠与咪喹莫特盐酸盐组合物冻干粉针剂复溶成胶后的扫描电镜图片。
图3是实施例一中海藻酸钠浓度不同时的咪喹莫特药物释放曲线及数据统计。
图4是实施例一中咪喹莫特浓度不同时的咪喹莫特药物释放曲线及数据统计。
图5是实施例二中海藻酸钠与CpG寡核苷酸组合物冻干粉针剂复溶成胶后的扫描电镜图片。
图6是实施例二中海藻酸钠浓度不同时的CpG药物释放曲线及数据统计。
图7是实施例二中CpG浓度不同时的CpG药物释放曲线及数据统计。
图8是实施例三中海藻酸钠与盐酸阿霉素组合物冻干粉针剂复溶成胶后的扫描电镜图片。
图9是实施例三中海藻酸钠浓度不同时的盐酸阿霉素药物释放曲线及数据统计。
图10是实施例三中盐酸阿霉素浓度不同时的盐酸阿霉素药物释放曲线及数据统计。
图11是实施例四中海藻酸钠与奥沙利铂组合物冻干粉针剂复溶成胶后的扫描电镜图片。
图12是实施例四中海藻酸钠浓度不同时的奥沙利铂药物释放曲线及数据统计。
图13是实施例四中奥沙利铂浓度不同时的奥沙利铂药物释放曲线及数据统计。
图14是实施例五中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂复溶成胶后的扫描电镜图片。
图15为实施例五中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂复溶后流变性能测试。
图16为实施例六中海藻酸钠与奥沙利铂和咪喹莫特盐酸盐冻干粉针剂复溶成胶后的扫描电镜图片。
图17为实施例九中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体冻干粉针剂复溶成胶后的扫描电镜图片。
图18为实施例九中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体冻干粉针剂复溶后抗体活性检测。
图19为实施例十三中海藻酸钠和咪喹莫特盐酸盐组合物联合射频消融治疗和anti-PDL1抗体治疗在小鼠结肠癌肿瘤模型上肿瘤生长曲线及数据统计。
图20为实施例十三中海藻酸钠和咪喹莫特盐酸盐组合物联合HIFU治疗和anti-PDL1抗体治疗在小鼠结肠癌肿瘤模型上肿瘤生长曲线及数据统计。
图21为实施例十三中海藻酸钠和咪喹莫特盐酸盐组合物联合HIFU治疗和anti-PDL1抗体治疗在小鼠结肠癌肿瘤模型上引起第二次种植肿瘤的生长曲线及 数据统计。
图22为实施例十四中海藻酸钠与奥沙利铂组合物冻干粉小鼠结肠癌治疗后肿瘤生长曲线及数据统计。
图23为实施例十四中海藻酸钠与奥沙利铂组合物冻干粉小鼠结肠癌治疗后小鼠体重曲线及数据统计。
图24为实施例十五中海藻酸钠与奥沙利铂和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠双边肿瘤模型上治疗后原位肿瘤生长曲线。
图25为实施例十五中海藻酸钠与奥沙利铂和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠双边肿瘤模型上治疗后远端肿瘤生长曲线及数据统计。
图26为实施例十五中海藻酸钠与奥沙利铂和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠双边肿瘤模型上治愈后再次接种肿瘤后的肿瘤生长曲线及数据统计。
图27为实施例十六中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠原位乳腺癌肿瘤模型上治疗后小鼠荧光成像的数据。
图28为实施例十七中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠脑癌模型上治疗后肿瘤生长曲线及数据统计。
图29为实施例十八中海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体在小鼠肿瘤手术切除模型上治疗后小鼠荧光成像的数据。
图30为实施例十九中对较大肿瘤(起始体积>120立方毫米)直接药物注射,不同治疗方式的肿瘤生长情况。
图31为实施例十九中对侧小肿瘤(起始体积<50立方毫米)无药物直接注射,不同治疗方式的肿瘤生长情况。
具体实施方式
实施例一:海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻干粉针剂的制备以及使用
步骤一:咪喹莫特(第三类组分)盐酸盐的制备。称取咪喹莫特50~100毫克于50毫升玻璃混和容器中,向其中加入1毫升1M稀盐酸,待白色粉末状的咪喹莫特充分溶解至无色透明后加入去离子水稀释,使得咪喹莫特的终浓度为2.5~5毫克每毫升。将溶液冻干,获得咪喹莫特盐酸盐冻干粉。该步骤 目的是使得不溶于水的咪喹莫特变成溶于水的盐酸盐形式。需足够长的冻干时间已确保盐酸残留的完全去除。
步骤二:海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻干粉针剂的制备,可以采用如下三种方法。
方法一:称取海藻酸钠10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明后,温度保持在20~40摄氏度,pH在~6.5。将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克溶于1毫升水相溶液,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明后,冻干得到冻干粉针剂,然后与0.1~10毫克咪喹莫特盐酸盐冻干粉通过固体与固体震荡混合均匀得到组合物冻干粉针剂。
方法三将0.1~10毫克咪喹莫特盐酸盐冻干粉溶于1毫升水相溶液,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将海藻酸钠10~80毫克溶于水相溶液中,以体积比1比20滴入不断搅拌的咪喹莫特盐酸盐溶液,保证混合液澄清透明没有絮状沉淀。待海藻酸钠溶液全部加完后,将混合液取出冻干得到组合物冻干粉针剂。
图1为海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻干粉针剂的制备流程,以及其使用说明。
图2为图1所述方法制备的一种组合物冻干粉针剂成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
步骤三:海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻干粉针剂中咪喹莫特释放曲线。药物缓释载体是指让药物缓慢进入血液,减小血液药物浓度,制备能够缓慢释放药物成分的缓释性长效药品在治疗中经常是非常需要的。药物释放曲线指的是我们在体外模拟组合物成胶后,其中包裹药物的释放情况。
如下为固定咪喹莫特用量,改变海藻酸钠用量,得到的释放曲线。
制备海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻 干粉针剂,其中海藻酸钠浓度为1、10、20、40和80毫克,咪喹莫特为2毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入200微升5毫克每毫升氯化钙溶液使其成胶,加入氯化钙的原因是要在体外模拟组合物注射入肿瘤后遇到钙离子的成胶后缓释药物的情况,并将该胶体浸泡浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为咪喹莫特的释放。
图3为海藻酸钠浓度不同时的咪喹莫特药物释放曲线及统计表格,从图中可知,在海藻酸钠浓度为5毫克每毫升及其以上时,咪喹莫特有一个明显的缓释现象,所以组合物中海藻酸钠的浓度优选为5毫克每毫升到80毫克每毫升。海藻酸钠浓度为10毫克每毫升时,已经比较优化了,海藻酸钠浓度为20毫克每毫升时基本达到峰值,浓度再提高时,效果提升不明显了。
如下为固定海藻酸钠用量,改变咪喹莫特用量,得到的释放曲线。
制备海藻酸钠(第一类组分)与咪喹莫特(第三类组分)盐酸盐组合物冻干粉针剂,其中咪喹莫特浓度为1、2.5、5、7.5和10毫克(最大溶解度),海藻酸钠为20毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为咪喹莫特的释放。
图4为咪喹莫特浓度不同时的咪喹莫特药物释放曲线及统计表格,从图中可知,在咪喹莫特浓度高于7.5毫克每毫升时,组合物在成胶时有明显的快速的药物释放,并且后续的释放也比低浓度时要快,然而缓释作用依然明显,所以组合物冻干粉针剂中咪喹莫特的浓度选为0.1~10毫克每毫升。这表明,不管咪喹莫特浓度高低,都是有效的,高浓度虽然释放会快,但是也有明显的缓释作用。
通过上述实验得到海藻酸钠与咪喹莫特盐酸盐的优选质量配比为50~800比1~100,更优选的质量配比为200~400比10~75。
实施例二:海藻酸钠(第一类组分)与CpG寡核苷酸(第三类组分)组合物冻干粉针剂
步骤一:海藻酸钠与CpG寡核苷酸组合物冻干粉针剂的制备
称取海藻酸钠10~80毫克和CpG寡核苷酸0.1~5毫克溶于1毫升水相溶 液中,充分震荡至溶液澄清透明后将溶液冻干得到组合物冻干粉针剂。
图5为该组合物冻干粉针剂复溶成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
步骤二:海藻酸钠与CpG寡核苷酸组合物冻干粉针剂中CpG释放曲线
如下为固定CpG寡核苷酸用量,改变海藻酸钠用量,得到的释放曲线。
制备海藻酸钠与CpG寡核苷酸组合物冻干粉针剂,其中海藻酸钠浓度为1、10、20和40毫克,CpG寡核苷酸为0.2毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入200微升5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为CpG寡核苷酸的释放。
图6为海藻酸钠浓度不同时的CpG药物释放曲线,从图中可知,在海藻酸钠浓度为20毫克及其以上时,CpG寡核苷酸有一个明显的缓释现象,所以组合物冻干粉针剂中海藻酸钠的浓度选为5毫克每毫升到80毫克每毫升。海藻酸钠浓度为1毫克每毫升时,效果不太明显,海藻酸钠浓度为10毫克每毫升时,效果已经比较优化了,海藻酸钠浓度为20毫克每毫升时基本达到峰值,浓度再提高时,效果提升不明显了。
如下为固定海藻酸钠用量,改变CpG寡核苷酸用量,得到的释放曲线。
制备海藻酸钠与CpG寡核苷酸组合物冻干粉针剂,其中CpG寡核苷酸浓度为0.1、0.25、0.5、1和2毫克,海藻酸钠为20毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为CpG寡核苷酸的释放。
图7为CpG寡核苷酸浓度不同时的CpG药物释放曲线,从图8中可知,CpG寡核苷酸浓度高于1毫克每毫升时,出现了比较明显的急性释放,而后续的释放速度并没有太大变化,出于成本考虑,CpG寡核苷酸的价格差不多1万人民币/毫克,所以组合物冻干粉针剂中CpG寡核苷酸的浓度选为0.1~2毫克每毫升,优选为CpG寡核苷酸的浓度选为0.1~0.5毫克每毫升。
通过上述实验得到海藻酸钠与CpG寡核苷酸的最佳质量配比为50~800比1~20,更优选的质量配比为200~400比1~20。
实施例三:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)组合物冻干粉针剂
步骤一:海藻酸钠与盐酸阿霉素组合物冻干粉针剂的制备:
方法一:称取海藻酸钠20~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将溶液冻干得到组合物冻干粉针剂。
方法二:将0.1~10毫克阿霉素盐酸盐溶于1毫升水相溶液,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将海藻酸钠10~80毫克溶于水相溶液中,以体积比1比20滴入不断搅拌的阿霉素盐酸盐溶液,保证混合液澄清透明没有絮状沉淀。待海藻酸钠溶液全部加完后,将混合液取出冻干得到组合物冻干粉针剂。
图8为该组合物冻干粉针剂复溶成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
步骤二:海藻酸钠与盐酸阿霉素组合物冻干粉针剂中阿霉素释放曲线
制备海藻酸钠与盐酸阿霉素组合物冻干粉针剂,其中海藻酸钠浓度为1、10、20和40毫克,盐酸阿霉素为2毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入200微升5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为盐酸阿霉素的释放。
图9为海藻酸钠浓度不同时的盐酸阿霉素药物释放曲线,从图中可知,在海藻酸钠浓度为10毫克及其以上时,盐酸阿霉素有一个明显的缓释现象,所以组合物冻干粉针剂中海藻酸钠的浓度优选为5毫克每毫升到80毫克每毫升。
制备海藻酸钠与盐酸阿霉素组合物冻干粉针剂,其中盐酸阿霉素浓度为1、2.5、5、7.5和10毫克(最大溶解度),海藻酸钠为20毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为盐酸阿霉素的释放。
图10为盐酸阿霉素浓度不同时的盐酸阿霉素药物释放曲线,从图中可知,盐酸阿霉素浓度高于7.5毫克时,出现了比较明显的快速释放,并且后续的释放也比低浓度时要快,然而缓释作用依然明显,所以组合物冻干粉针剂中阿霉素的浓度选为0.1~10毫克每毫升。
通过上述实验得到海藻酸钠与盐酸阿霉素的优选质量配比为50~800比1~100,更优选的质量配比为200~400比10~75。
实施例四:海藻酸钠(第一类组分)与奥沙利铂(第二类组分)组合物冻干粉针剂
步骤一:海藻酸钠与奥沙利铂组合物冻干粉针剂的制备
称取海藻酸钠10~80毫克和奥沙利铂1~7.5毫克溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将溶液冻干得到组合物冻干粉针剂。
图11为该组合物冻干粉针剂复溶成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
步骤二:海藻酸钠与奥沙利铂组合物冻干粉针剂中奥沙利铂释放曲线
制备海藻酸钠与奥沙利铂组合物冻干粉针剂,其中海藻酸钠浓度为1、10、20和40毫克,奥沙利铂为2毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入200微升5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为奥沙利铂的释放。
图12为海藻酸钠浓度不同时的奥沙利铂药物释放曲线,从图中可知,在海藻酸钠浓度为10毫克及其以上时,奥沙利铂有一个明显的缓释现象,所以组合物冻干粉针剂中海藻酸钠的浓度选为5毫克每毫升到80毫克每毫升。
制备海藻酸钠与奥沙利铂组合物冻干粉针剂,其中奥沙利铂浓度为1、2.5、5和7.5毫克(最大溶解度),海藻酸钠为20毫克,将组合物冻干粉针剂分别复溶于1毫升水相溶液并震荡至澄清透明,后加入5毫克每毫升氯化钙溶液使其成胶,并将该胶体浸泡在1毫升磷酸缓冲溶液中搅拌,在第0、0.25、0.5、1、2、4、8天测定磷酸缓冲溶液中药物的含量既为奥沙利铂的释放。
图13为奥沙利铂浓度不同时的奥沙利铂药物释放曲线,从图中可知,奥 沙利铂浓度高于7.5毫克时,出现了比较明显的急性释放,出现了比较明显的急性释放,并且后续的释放也比低浓度时要快,然而缓释作用依然明显,所以组合物冻干粉针剂中奥沙利铂的浓度选为0.1~7.5毫克每毫升。
通过上述实验得到海藻酸钠与奥沙利铂的优选质量配比为50~800比1~75,更优选的质量配比为200~400比10~75。
实施例五:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特(第三类组分)盐酸盐冻干粉针剂
步骤一:海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂的制备
方法一(组分一、二、三溶于水相溶液中,搅拌,然后混合溶液冻干):称取海藻酸钠10~80毫克(第一类组分)和咪喹莫特(第三类组分)盐酸盐冻干粉0.1~10毫克和盐酸阿霉素(第二类组分)0.1~10毫克溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将溶液冻干得到组合物冻干粉针剂。
方法二(组分一、二溶于水相溶液中,搅拌后混合溶液冻干得到冻干粉,与组分三的冻干粉固固混合):称取海藻酸钠(第一类组分)10~80毫克和盐酸阿霉素(第二类组分)0.1~10毫克溶于1毫升水相溶液,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后冻干得到冻干粉,与0.1~10毫克咪喹莫特(第三类组分)盐酸盐冻干粉通过固体与固体震荡混合均匀得到组合物冻干粉针剂。
方法三(组分二、三溶于水相溶液中,搅拌后得到澄清溶液,将组分一的溶液按1:20滴入前述混合液中,然后将最终的混合液冻干后得到冻干粉):将0.1~10毫克阿霉素盐酸盐和0.1~10毫克咪喹莫特盐酸盐溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将海藻酸钠20~80毫克溶于1毫升水相溶液中,以体积比1比20滴入不断搅拌的混合溶液,保证混合液澄清透明没有絮状沉淀。待海藻酸钠溶液全部加完后,将混合液取出冻干得到组合物冻干粉针剂。
该组合物中第一类组分、第二类组分和第三类组分的优选质量比为50~800比1~100比1~100,更优选的质量配比为200~400比10~75比10~75。。
图14为该组合物冻干粉针剂复溶成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看 出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
步骤二:海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂复溶后流变性质的测定
将海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂溶解于1毫升磷酸缓冲溶液溶液中。分别检测20微升1,5,10和20毫克每毫升海藻酸钠的组合物与20微升10毫克每毫升的钙离子溶液混合后的流变力学性质。
图15为不同浓度海藻酸钠和阿霉素与咪喹莫特组合物冻干粉针剂复溶后在接触钙离子后的流变力学性质。从图中可以看出,当海藻酸钠浓度为1毫克每毫升时,其存储模量小于损耗模量,表现出流体的行为,当海藻酸钠浓度达到10毫克每毫升以上时,其存储模量大于损耗模量,表现出凝胶的行为,证明海藻酸钠在10毫克每毫升以上时遇到钙离子会形成胶体。
实施例六:海藻酸钠(第一类组分)与奥沙利铂(第二类组分)和咪喹莫特盐酸盐(第三类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和奥沙利铂0.1~7.5毫克溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明后将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和奥沙利铂0.1~7.5毫克溶于1毫升水相溶液用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明后冻干得到冻干粉,然后再与0.1~10毫克咪喹莫特盐酸盐冻干粉通过固固震荡混合均匀得到组合物冻干粉针剂。
方法三:将0.1~10毫克奥沙利铂和0.1~10毫克咪喹莫特盐酸盐溶于1毫升水相溶液中,用搅拌桨以50~300转每分的速度搅拌至溶液澄清透明,后将海藻酸钠20~80毫克溶于1毫升水相溶液中,以体积比1比20滴入不断搅拌的混合溶液,保证混合液澄清透明没有絮状沉淀。待海藻酸钠溶液全部加完后,将混合液取出冻干得到组合物冻干粉针剂。
该组合物中第一类组分、第二类组分和第三类组分的质量比为50~800比1~75比1~100,更优选的质量配比为200~400比10~75比10~75。。
图16为该组合物冻干粉针剂成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
实施例七:海藻酸钠(第一类组分)与五氟尿嘧啶(第二类组分)和咪喹莫特盐酸盐(第三类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和五氟尿嘧啶1~5毫克和咪喹莫特盐酸盐0.1~10毫克溶于1毫升2毫克每毫升的氢氧化钠溶液中,充分震荡至溶液澄清透明后将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和五氟尿嘧啶1~5毫克溶于1毫升水相溶液震荡至溶液澄清透明后冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
实施例八:海藻酸钠(第一类组分)与环磷酰胺(第二类组分)和咪喹莫特盐酸盐(第三类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和环磷酰胺1~5毫克和咪喹莫特盐酸盐0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和环磷酰胺1~5毫克溶于1毫升水相溶液震荡至溶液澄清透明后冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
实施例九:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
图17为该组合物冻干粉针剂成胶后的扫描电镜图片。从图中可以看出,组合物在冻干复溶后仍具备不错的成胶能力,并且从电镜图片中可以看出其成胶后有很多微米级别的孔道,对药物缓释有重要帮助。
图18为抗体anti-PDL1冻干后的活性测试,从实验结果可以看出,冻干后anti-PDL1抗体结合细胞表面PDL1抗体流式所跑出的峰值和单纯的anti-PDL1抗体峰值一致,说明冻干并不影响anti-PDL1抗体的活性。
实施例十:海藻酸钠(第一类组分)与奥沙利铂(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和奥沙利铂0.1~7.5毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和奥沙利铂0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
实施例十一:其他海藻酸盐(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及以及anti-PDL1抗体(第四类组分)冻干粉针剂
制备:海藻酸钾(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及以及anti-PDL1抗体(第四类组分)冻干粉针剂
方法一:称取海藻酸钾10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钾10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明,后加入100微克~5毫克anti-PDL1溶液混合均匀冻干得到冻干粉,然后与0.1~10毫克咪喹莫特盐酸盐冻干粉,通过固固震荡混合均匀得到组合物冻干粉针剂。
制备:海藻酸铵(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及以及anti-PDL1抗体(第四类组分)冻干粉针剂
方法一:称取海藻酸铵10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸铵10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明后加入100微克~5毫克anti-PDL1溶液混合均匀冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
不同阳离子的海藻酸盐都能与其他三类组分构成良好的组合物,并仍具备成胶缓释的能力。
实施例十二:海藻酸盐(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及IDO抑制剂4-苯基咪唑(第四类组分)冻干粉针剂
制备:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及4-苯基咪唑(第四类组分)冻干粉针剂
方法一:称取海藻酸钠10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克4-苯基咪唑溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钠10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明,后加入100微克~5毫克4-苯基咪唑溶液混合均匀冻干得到冻干粉,然后与0.1~10毫克咪喹莫特盐酸盐冻干粉,通过固固震荡混合均匀得到组合物冻干粉针剂。
制备:海藻酸钾(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及以及4-苯基咪唑(第四类组分)冻干粉针剂
方法一:称取海藻酸钾10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克4-苯基咪唑溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸钾10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升 水相溶液震荡至溶液澄清透明后加入100微克~5毫克4-苯基咪唑溶液混合均匀冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
制备:海藻酸铵(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及以及4-苯基咪唑(第四类组分)冻干粉针剂
方法一:称取海藻酸铵10~80毫克和咪喹莫特盐酸盐冻干粉0.1~10毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液中,充分震荡至溶液澄清透明后加入100微克~5毫克4-苯基咪唑溶液混合均匀将溶液冻干得到组合物冻干粉针剂。
方法二:称取海藻酸铵10~80毫克和盐酸阿霉素0.1~10毫克溶于1毫升水相溶液震荡至溶液澄清透明后加入100微克~5毫克4-苯基咪唑溶液混合均匀冻干得到冻干粉与0.1~10毫克咪喹莫特盐酸盐冻干粉通过震荡混合均匀得到组合物冻干粉针剂。
如下为不同组合物的协同增效治疗效果相关实验及数据统计。
实施例十三:海藻酸钠(第一类组分)和咪喹莫特盐酸盐(第三类组分)组合物冻干粉针剂在结肠癌模型上的疗效研究
步骤一:海藻酸钠(第一类组分)和咪喹莫特盐酸盐(第三类组分)组合物冻干粉针剂联合射频消融治疗和免疫检查点抑制疗法anti-PDL1抗体的疗效研究。
在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(左边视为原位肿瘤,右边视为远端肿瘤),并将荷瘤小鼠分为四组,每组5只做治疗实验。
第一组:左边原位肿瘤单独射频消融治疗(参照例);
第二组:左边原位肿瘤射频消融治疗后尾静脉注射anti-pdl1抗体(参照例);
第三组:左边原位肿瘤瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后射频消融治疗;
第四组:左边原位肿瘤瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后射频消融治疗加尾静脉注射anti-PDL1抗体治疗。
小鼠在经过不同的治疗后每隔两天用游标卡尺测量其右边远端肿瘤的长和宽,肿瘤的体积为(长乘以(宽的平方))除以2。实验结果表明,小鼠左 边的原位肿瘤都被射频消融治疗所消灭,而第四组小鼠的右边远端肿瘤得到了明显的抑制,说明了海藻酸钠和咪喹莫特盐酸盐组合物在射频消融治疗后能更好地激起抗肿瘤免疫反应,并且和anti-PDL1抗体有很好的协同效果。(图19)
步骤二:海藻酸钠(第一类组分)和咪喹莫特盐酸盐(第三类组分)组合物冻干粉针剂联合高能聚焦超声刀(HIFU)和免疫检查点抑制疗法anti-PDL1抗体的疗效研究。
在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(左边视为原位肿瘤,右边视为远端肿瘤),并将荷瘤小鼠分为四组,每组5只做治疗实验。
第一组:左边原位肿瘤单独HIFU治疗(参照例);
第二组:左边原位肿瘤HIFU治疗后尾静脉注射anti-pdl1抗体(参照例);
第三组:左边原位肿瘤瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后HIFU治疗;
第四组:左边原位肿瘤瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后HIFU治疗加尾静脉注射anti-PDL1抗体治疗。
小鼠在经过不同的治疗后每隔两天用游标卡尺测量其右边远端肿瘤的长和宽,肿瘤的体积为(长乘以(宽的平方))除以2。实验结果表明,小鼠左边的原位肿瘤都被HIFU治疗所消灭,而第四组小鼠的右边远端肿瘤得到了明显的抑制,说明了海藻酸钠和咪喹莫特盐酸盐组合物在HIFU治疗后能更好地激起抗肿瘤免疫反应,并且和anti-PDL1抗体有很好的协同效果。(图20)
步骤三:海藻酸钠(第一类组分)和咪喹莫特盐酸盐(第三类组分)组合物冻干粉针剂联合高能聚焦超声刀(HIFU)和免疫检查点抑制疗法anti-PDL1抗体引起免疫记忆效果研究。
将结肠癌荷瘤小鼠分为六组,每组5只。
第一组:生理盐水组;
第二组:尾静脉anti-PDL1抗体治疗(参照例);
第三组:单独HIFU治疗(参照例);
第四组:HIFU治疗后尾静脉anti-pdl1抗体治疗(参照例);
第五组:瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后HIFU治疗;
第六组:瘤内注射海藻酸钠和咪喹莫特盐酸盐组合物冻干粉针剂(实施例一)后HIFU治疗并尾静脉anti-PDL1抗体治疗。
在小鼠肿瘤被HIFU治疗所消除40天后,在这些经过不同治疗的小鼠身上再次种植结肠癌肿瘤细胞,用游标卡尺测量其右边远端肿瘤的长和宽,肿瘤的体积为(长乘以(宽的平方))除以2。实验结果表明,第五组和第六组小鼠再次种植的肿瘤生长明显比对照组要慢,受到显著抑制,第六组小鼠的肿瘤生长比第五组还要慢甚至有部分不再能长出肿瘤。说明海藻酸钠(第一类组分)和咪喹莫特盐酸盐(第三类组分)组合物冻干粉针剂联合高能聚焦超声刀(HIFU)和免疫检查点抑制疗法anti-PDL1抗体能显著引起小鼠的免疫记忆从而预防肿瘤复发。(图21)
实施例十四:海藻酸钠(第一类组分)和奥沙利铂(第二类组分)组合物冻干粉针剂在结肠癌模型上的疗效研究
将结肠癌荷瘤小鼠分为6组,每组5只做治疗实验。
第一组:小鼠分别瘤内注射生理盐水(参照例);
第二组:奥沙利铂(1.5毫克每千克体重)(单独化疗药参照例);
第三组:瘤内注射奥沙利铂与海藻酸钠组合物冻干粉针剂(0.375毫克每千克体重)(实施例四);
第四组:瘤内注射奥沙利铂与海藻酸钠组合物冻干粉针剂(0.75毫克每千克体重)(实施例四);
第五组:瘤内注射奥沙利铂与海藻酸钠组合物冻干粉针剂(1.5毫克每千克体重)(实施例四);
第六组:尾静脉注射奥沙利铂(3毫克每千克体重)(单独化疗药参照例)。
在瘤内注射后,每隔两天用游标卡尺测量肿瘤的长和宽,肿瘤的体积为(长乘以(宽的平方))除以2。从肿瘤生长曲线(图22)可以看出,瘤内注射海藻酸钠和奥沙利铂的组合物冻干粉针剂在0.75毫克千克体重的剂量时,其治疗效果已经超过了尾静脉注射3毫克每千克体重和单纯药物注射1.5毫克每千克体重;而瘤内注射海藻酸钠和奥沙利铂的组合物冻干粉针剂在1.5毫克千克体重的计量时,其肿瘤生长受到了明显的抑制,疗效显著。从小鼠的体重(图23)可以看出,尾静脉注射奥沙利铂组小鼠在前四天体重出现明显下降,说明静脉注射存在一定的毒副作用,而瘤内注射则没有表现出明显 的毒副作用。明显看出采用本专利技术方案的组合物进行瘤内给药副作用比静脉给药低。
实施例十五:海藻酸钠(第一类组分)与奥沙利铂(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂在双边肿瘤(左右两边各有一个肿瘤)模型上的疗效研究。
在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(左边视为原位肿瘤,右边视为远端肿瘤),并将荷瘤小鼠分为7组,每组6只做联合免疫的治疗实验。
第一组:小鼠分别瘤内注射生理盐水(参照例);
第二组:奥沙利铂和咪喹莫特与anti-PDL1复合溶液(参照例);
第三组:海藻酸钠与奥沙利铂组合物冻干粉针剂(实施例四)联合anti-PDL1静脉注射(参照例);
第四组:瘤内注射海藻酸钠与奥沙利铂和anti-PDL1组合物冻干粉针剂(实施例四);
第五组:瘤内注射海藻酸钠与奥沙利铂和咪喹莫特组合物冻干粉针剂(实施例六);
第六组:瘤内注射海藻酸钠与奥沙利铂和咪喹莫特anti-PDL1组合物冻干粉针剂(实施例十);
第七组:海藻酸钠与奥沙利铂和咪喹莫特组合物冻干粉针剂(实施例六)联合anti-PDL1静脉注射(参照例)。
对左侧原位肿瘤进行注射在瘤内注射原位肿瘤后,对右侧远端肿瘤不进行注射,每隔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。从原位肿瘤生长曲线和远端肿瘤生长曲线(图24和图25)可以看出,第6组和第7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。在第6组和第7组小鼠存活两个月后,再次种植结肠癌细胞,发现肿瘤生长受到明显的抑制,说明有效的防止了肿瘤的复发(图26)。
实施例十六:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂在乳腺癌转移瘤模型上的疗效研究
将乳房垫原位肿瘤4T1乳腺癌的小鼠分为6组,每组6只做转移瘤模型的治疗实验。
第一组:小鼠分别瘤内注射生理盐水(参照例);
第二组:阿霉素和咪喹莫特盐酸盐与anti-PDL1抗体(参照例);
第三组:瘤内注射海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐组合物冻干粉针剂(实施例五);
第四组:瘤内注射海藻酸钠与盐酸阿霉素和anti-PDL1抗体组合物冻干粉针剂(实施例五);
第五组:瘤内注射海藻酸钠与阿霉素和咪喹莫特以及anti-PDL1抗体组合物冻干粉针剂(实施例九);
第六组:海藻酸钠与阿霉素和咪喹莫特盐酸盐组合物冻干粉针剂(实施例五)联合anti-PDL1抗体静脉注射(参照例)。
第一组对照组直接通过手术去除小鼠肿瘤。小鼠在第十五天进行治疗,并每隔五天拍摄小动物活体荧光成像。从实验结果可以看出,第五组和第六组都有很好的治疗效果。(图27)
实施例十七:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂在小鼠脑癌上的疗效探究
将脑癌小鼠分为九组,每组六只做脑癌治疗实验。
第一组:小鼠颅内注射生理盐水(参照例);
第二组:小鼠腹腔注射替莫咗咪(参照例);
第三组:小鼠颅内注射咪喹莫特盐酸盐和anti-PDL1抗体以及海藻酸钠组合物(参照例);
第四组:小鼠颅内注射海藻酸钠与阿霉素组合物冻干粉针剂(实施例三);
第五组:小鼠颅内注射盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体组合物(参照例);
第六组:小鼠颅内注射海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐组合物冻干粉针剂(实施例五);
第七组:小鼠颅内注射海藻酸钠与盐酸阿霉素和anti-PDL1抗体组合物(参照例);
第八组:小鼠颅内注射海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐以及anti-PDL1抗体组合物冻干粉针剂(实施例九);
第九组:小鼠颅内注射海藻酸钠与盐酸阿霉素和咪喹莫特盐酸盐冻干粉针剂联用anti-PDL1抗体治疗(参照例)。
观察小鼠死亡情况。图28为小鼠的死亡率曲线,从图中可以看出,第八组和第九组小鼠存活时间比对照组长一倍,表明其治疗效果更佳。
实施例十八:海藻酸钠(第一类组分)与盐酸阿霉素(第二类组分)和咪喹莫特盐酸盐(第三类组分)以及anti-PDL1抗体(第四类组分)冻干粉针剂在小鼠肿瘤手术切除后模型上疗效研究
将皮下乳腺癌小鼠随机分为三组,每组六只做海藻酸钠与阿霉素和咪喹莫特以及anti-PDL1抗体复合凝胶治疗实验。小鼠在手术切除大部分皮下肿瘤(切除小鼠皮下肿瘤保留其癌旁皮肤和肌肉)后。
第一组不处理(参照例);
第二组单纯手术(参照例);
第三组手术后在创口部位涂抹海藻酸钠与阿霉素和咪喹莫特以及anti-PDL1抗体复合凝胶(实施例九)。
通过观察手术后肿瘤的转移和复发情况来判断治疗效果,通过小动物生物自发光成像来得出结论。从图29中可知,关键组第三组小鼠的肿瘤得到很好地抑制转移和复发的效果,证明了我们的海藻酸钠与阿霉素和咪喹莫特以及anti-PDL1抗体复合凝胶的作用。
实施例十九:本实施例的药物组合物可以采用两种剂型制备。
第一种剂型如下:
组分一,按咪喹莫特R837:表面活性剂泊洛沙姆188的比例1:(0.1-5)。称取咪喹莫特R837和表面活性剂泊洛沙姆188。优选1g R837,加入适量的泊洛沙姆188(0.15g,0.3g,0.5g,1g,2g,3g,4g,5g),加10ml水球磨3小时,结束后,取出匀浆9ml移入到250ml烧杯中,加入141ml一级水,500rpm搅拌1小时混匀。以注射器吸取混悬液灌装到10ml西林瓶中,每瓶5ml,共30瓶。盖上橡胶盖,并以铝盖封好后,121℃湿热灭菌12 分钟;
组分二:配制海藻酸钠0.1-5%,奥沙利铂0.1-1%溶液,优选的称取海藻酸钠ALG(3g,或1.5g),奥沙利铂适量(159mg,300mg,450mg,90mg),加入300ml一级水,于500ml烧杯中搅拌2-6小时(25℃-40℃,200-850rpm,以保鲜膜封住瓶口)。将获得的溶液经0.22微米滤膜过滤除菌。以60ml注射器灌装到20ml西林瓶,每瓶10ml,共30瓶。经过-80℃冰箱预冷30分钟后,冻干30小时。冻干后盖上橡胶盖,封上铝盖。
使用时,将组分二冻干粉加入到组分一溶液中,通过震荡的方式充分混匀后注射。
第二种剂型如下:
组分一:按咪喹莫特R837:表面活性剂泊洛沙姆188的比例为1:(0.1-5)称取R837和表面活性剂,加入0.1-1%的奥沙利铂。优选1g咪喹莫特R837,加入适量的泊洛沙姆188(0.15g,0.3g,0.5g,1g,2g,3g,4g,5g),加10ml水球磨3小时,结束后,取出匀浆9ml移入到250ml烧杯中,加入141ml一级水,奥沙利铂适量(159mg,300mg,450mg,900mg),100-500rpm搅拌0.5-3小时混匀。以50ml注射器吸取混悬液灌装到10ml西林瓶中,每瓶5ml,共30瓶。盖上橡胶盖,并以铝盖封好后,121℃湿热灭菌12分钟;
组分二:配制海藻酸钠0.1-5%溶液。优选的,称取海藻酸钠ALG3g或1.5g,加入300ml一级水,于500ml烧杯中搅拌2-6小时(25℃-40℃,200-850rpm,以保鲜膜封住瓶口)。将获得的溶液经0.22微米滤膜过滤除菌。以60ml注射器灌装到20ml西林瓶,每瓶10ml,共30瓶。-80℃冰箱预冷30分钟后,冻干30小时。冻干后盖上橡胶盖,封上铝盖。
使用时,将组分二冻干粉加入到组分一溶液中,通过震荡的方式充分混匀后注射。
本实施例用于治疗的具体效果如下,采用第一种剂型,第二种剂型与第一种剂型的治疗效果差不多。
实验方法:在小鼠背部左右两端分别种植小鼠结肠癌肿瘤(左边视为原位肿瘤,右边视为远端肿瘤),并将荷瘤小鼠分为5组,每组6只做联合免疫的治疗实验。
第一组:小鼠分别瘤内注射生理盐水(参照例);
第二组:瘤内注射奥沙利铂+泊洛沙姆188分散的咪喹莫特颗粒;
第三组:瘤内注射奥沙利铂+海藻酸钠;
第四组:瘤内注射泊洛沙姆188分散的咪喹莫特颗粒+海藻酸钠;
第五组:瘤内注射奥沙利铂+泊洛沙姆188分散的咪喹莫特颗粒+海藻酸钠;
对左侧原位肿瘤进行注射,在瘤内注射原位肿瘤后,对右侧远端肿瘤不进行注射,每隔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和远端肿瘤生长曲线(图30和图31)可以看出,第5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,更为重要的是,这是在没有联合anti-PDL1抗体的前提下就获得了良好的治疗效果,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。
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,采用咪喹莫特R837球磨法,将不溶于水的R837粉末在水中球磨后使得粉末越磨越细,使得其拥有良好的水分散性。
在R837球磨乳液中加入泊洛沙姆P188球磨,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:作乳化剂,稳定剂和增溶剂,可以进一步增强R837球磨乳液的水分散性和稳定性。
所用增溶性药用辅料包括:泊洛沙姆188,泊洛沙姆407,聚山梨酯80(吐温80),聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),蛋黄卵磷脂,聚氧乙烯(35)蓖麻油,维生素E琥珀酸聚乙二醇酯,聚乙二醇200,聚乙二醇400,聚乙二醇800,或羟甲基纤维素钠中的一种或多种。其中泊洛沙姆188,泊洛沙姆407,聚山梨酯80(吐温80),聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),蛋黄卵磷脂,聚氧乙烯(35)蓖麻油,维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种对R837都有很好的增溶效果。
但是,聚乙二醇200,聚乙二醇400,聚乙二醇800增溶效果一般。且近年来,不良反应病例报道逐渐增多。聚山梨酯80(吐温80)作为其常用增溶剂辅料,成为了不良反应致因的研究焦点。聚氧乙烯蓖麻油作为非离子表面活性剂,虽能增加大多数难溶性药物的水溶性,但会导致组织胺的释放,引起多种毒副反应,如严重的过敏反应、中毒性肾损伤、神经毒性、心脏血管毒性等。根据文献报道,对肌肉的刺激及溶血实验,Solutol HS 15的毒副作用最大,而聚乙二醇200的毒副作用最小。聚乙二醇是稳定的亲水性物质,无毒、无刺激性,对很多药物有增溶、增加稳定性和延效作用。
泊洛沙姆188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,目前已作为静脉给药的乳化剂和增溶剂应用于临床。
进一步的实验发现,在不加入泊洛沙姆188的情况下,R837球磨乳液在121℃湿热灭菌后,会导致乳液不稳定产生明显的沉淀和颗粒,水分散性大打折扣。泊洛沙姆188可以帮助R837在灭菌后保证水分散性和稳定性。
表格1:加入表面活性剂前后球磨咪喹莫特R837的水分散性
Figure PCTCN2019122492-appb-000001
表格2:添加不同表面活性剂球磨的R837高压灭菌后的再分散性
Figure PCTCN2019122492-appb-000002
表格3:加入不同比例表面活性剂的球磨R837高压灭菌后的长期稳定性
表面活性剂a:R837 高压灭菌后的长期稳定性
0.15:1 出现大量颗粒状聚集体
0.3:1 出现大量颗粒状聚集体
0.5:1 出现大量颗粒状聚集体
1:1 出现大量颗粒状聚集体
2:1 出现大量颗粒状聚集体
3:1 均匀分散且未出现颗粒状聚集体
4:1 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
a:适用的表面活性剂包括:泊洛沙姆188,泊洛沙姆407,聚山梨酯80(吐温80),聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),聚氧乙烯(35)蓖麻油,维生素E琥珀酸聚乙二醇酯,羟甲基纤维素钠。
表面活性剂b:R837 高压灭菌后的长期稳定性
0.15:1 出现大量颗粒状聚集体
0.3:1 出现大量颗粒状聚集体
0.5:1 出现大量颗粒状聚集体
1:1 均匀分散且未出现颗粒状聚集体
2:1 均匀分散且未出现颗粒状聚集体
3:1 均匀分散且未出现颗粒状聚集体
4:1 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
b:适用的表面活性剂包括:蛋黄卵磷脂。
表面活性剂c:R837 高压灭菌后的长期稳定性
0.15:1 出现大量絮状聚集体
0.3:1 出现大量絮状聚集体
0.5:1 出现大量絮状聚集体
1:1 出现大量絮状聚集体
2:1 出现大量絮状聚集体
3:1 出现大量絮状聚集体
4:1 出现大量絮状聚集体
5:1 出现大量絮状聚集体
c:适用的表面活性剂包括:聚乙二醇200,聚乙二醇400,聚乙二醇800。
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,且避免引入杂质。
海藻酸钠是比较特殊的天然生物高分子材料,由于其在高温下会分解,因此无法采用传统的高温湿热灭菌,本专利采用过滤灭菌,保留了海藻酸钠的性质。另外,从灭菌的角度考虑,不能把海藻酸钠和咪喹莫特R837配在一起,海藻酸钠ALG需要过滤灭菌,但R837颗粒无法过滤(球磨后颗粒直径最低为500nm,而过滤灭菌需要220nm的滤膜,因此不能通过);另一方面,R837需要湿热灭菌,而海藻酸钠ALG高温会降解。因此,R837和ALG二者不能在一起灭菌。
另外,之所以咪喹莫特R837要做成球磨颗粒剂型,是因为R837如果配成盐酸盐,与OXA存在配伍禁忌,OXA会与氯离子反应失活;其次,R837的盐酸盐会增加海藻酸钠ALG的粘度,不方便使用。
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。本发明将不会被限制于本文所示的这些实施例,而是只需要符合与本文所公开的原理与特点一致即可。

Claims (10)

  1. 一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;所述能引起免疫原性死亡的化疗药为奥沙利铂,所述免疫佐剂为咪喹莫特R837,还包括有泊洛沙姆188和海藻酸钠ALG;
    第一混合物为,所述咪喹莫特R837与泊洛沙姆188混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌;
    第二混合物为,所述海藻酸钠ALG与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
    第一混合物与第二混合物混合构成化疗免疫组合药物。
  2. 一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;所述能引起免疫原性死亡的化疗药为奥沙利铂,所述免疫佐剂为咪喹莫特R837,还包括有泊洛沙姆188和海藻酸钠ALG;
    第一混合物为,所述咪喹莫特R837与泊洛沙姆188混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,将咪喹莫特乳液与奥沙利铂及水混合搅匀经过高温湿热灭菌;
    第二混合物为所述海藻酸钠与水混合,然后经过微米滤膜过滤除菌制成混合物;
    第一混合物与第二混合物混合构成化疗免疫组合药物。
  3. 根据权利要求1所述的化疗免疫组合药物,其特征在于:所述咪喹莫特R837与泊洛沙姆188的质量配比为1:(0.1-5),所述高温灭菌为105℃~150℃湿热灭菌10-15分钟;
    所述第二混合物经0.22微米滤膜过滤除菌,并经冻干后制成冻干粉。
  4. 根据权利要求2所述的化疗免疫组合药物,其特征在于:所述咪喹莫特R837与泊洛沙姆188的质量配比为1:(0.1-5),所述高温灭菌为105℃~150℃湿热灭菌10-15分钟;
    所述第二混合物经0.22微米滤膜过滤除菌,并经冻干后制成冻干粉。
  5. 一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
    第一混合物为,所述免疫佐剂为咪喹莫特,咪喹莫特与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌,所述表面活性剂为泊洛沙姆407,或聚山梨酯80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油,或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
    第二混合物为,海藻酸钠与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
    第一混合物与第二混合物混合构成化疗免疫组合药物。
  6. 根据权利要求5所述的化疗免疫组合药物,其特征在于:所述海藻酸钠替换为壳聚糖、或纤维蛋白原、或藻酸盐、或透明质酸;
    所述咪喹莫特R837替换为咪唑喹啉,或吡喃葡糖苷脂质;
    所述奥沙利铂Oxa替换为蒽环类药物、或环磷酰胺、或硼替佐米、或吉西他滨、或五氟尿嘧啶、或毒素。
  7. 一种化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
    第一混合物为,所述咪喹莫特R837与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,将咪喹莫特乳液与奥 沙利铂及水混合搅匀经过高温湿热灭菌;
    所述表面活性剂为泊洛沙姆407,或聚山梨酯80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油,或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
    第二混合物为所述海藻酸钠与水混合,然后经过微米滤膜过滤除菌制成混合物;
    第一混合物与第二混合物混合构成化疗免疫组合药物。
  8. 根据权利要求7所述的化疗免疫组合药物,其特征在于:所述海藻酸钠能替换为壳聚糖、或纤维蛋白原、或藻酸盐、或透明质酸;
    所述咪喹莫特R837能替换为咪唑喹啉,或吡喃葡糖苷脂质;
    所述奥沙利铂Oxa能替换为蒽环类药物、或环磷酰胺、或硼替佐米、或吉西他滨、或五氟尿嘧啶、或毒素。
  9. 一种化疗免疫组合药物的制备方法,其特征在于包括如下步骤:
    第一步:按比例1:(0.1-5)称取咪喹莫特R837和表面活性剂泊洛沙姆188,加水球磨2-3小时,结束后取出匀浆,加水,搅拌混匀,105℃~150℃湿热灭菌10-15分钟;
    第二步:称取海藻酸钠ALG与奥沙利铂,加水,搅拌,将获得的溶液经微米滤膜过滤除菌;预冷后,进行冻干;
    第三步:将第二步中的冻干粉加入到第一步的混合物溶液中,充分震荡混合均匀。
  10. 一种治疗结肠癌肿瘤的化疗免疫组合药物,其含有能引起免疫原性死亡的化疗药和免疫佐剂,其特征在于:所述化疗免疫组合药物包括第一混合物与第二混合物,所述第一混合物含有免疫佐剂,所述第二混合物含有能引起免疫原性死亡的化疗药;
    第一混合物为,所述免疫佐剂为咪喹莫特,咪喹莫特与表面活性剂混合球磨制成得到均匀分散的咪喹莫特乳液,所述咪喹莫特颗粒为0.5-3微米粒径,咪喹莫特乳液经过高温湿热灭菌,所述表面活性剂为泊洛沙姆407,或聚山梨酯 80(吐温80),或聚乙二醇-12-羟基硬脂酸酯(Solutol HS 15),或蛋黄卵磷脂,或聚氧乙烯(35)蓖麻油,或维生素E琥珀酸聚乙二醇酯,或羟甲基纤维素钠中的一种或多种;
    第二混合物为,海藻酸钠与奥沙利铂与水搅拌混合,经过微米滤膜过滤除菌制成混合物;
    第一混合物与第二混合物混合构成化疗免疫组合药物。
PCT/CN2019/122492 2019-09-26 2020-01-07 一种化疗免疫组合药物及其制备方法 WO2021056815A1 (zh)

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