WO2018233095A1 - 具有淋巴靶向功能的生物自组装纳米晶注射剂及制备方法 - Google Patents

具有淋巴靶向功能的生物自组装纳米晶注射剂及制备方法 Download PDF

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WO2018233095A1
WO2018233095A1 PCT/CN2017/101801 CN2017101801W WO2018233095A1 WO 2018233095 A1 WO2018233095 A1 WO 2018233095A1 CN 2017101801 W CN2017101801 W CN 2017101801W WO 2018233095 A1 WO2018233095 A1 WO 2018233095A1
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injection
bio
mitoxantrone
assembled
self
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PCT/CN2017/101801
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French (fr)
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刘珺
叶田田
张丹
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沈阳天邦药业有限公司
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Priority to EP17915174.1A priority Critical patent/EP3643299A4/en
Priority to US16/482,054 priority patent/US20200163872A1/en
Publication of WO2018233095A1 publication Critical patent/WO2018233095A1/zh
Priority to PH12019502480A priority patent/PH12019502480A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present application relates to the design and preparation of a new dosage form having a lymphatic targeting function, and provides a prescription composition and a preparation method of a self-assembled nanocrystalline injection.
  • Lymphatic metastasis is one of the important factors affecting the prognosis of malignant tumors.
  • Tumor cells can enter the lymphatic system and diffuse at an early stage.
  • the lymphatic collateral circulation enters the non-affected lymph nodes or lymph nodes away from the primary tumor, forming a so-called jump transfer, which limits the application of surgery.
  • Regional lymph nodes are less sensitive to radiotherapy than primary tumors, and systemic chemotherapy is not effective for metastatic lymph nodes because conventional drugs are not easily transported to lymph nodes. Since the efficacy of treating lymph node metastases directly affects the cure rate and survival rate of patients, the targeting of lymph node metastases is receiving more and more attention. Lymph nodes have become a very promising target organ in cancer chemotherapy.
  • the use of macromolecular substances and particles that are easily phagocytosed by the lymphatic system allows the design of novel drug delivery systems to enable lymphatic targeting.
  • the drug carriers used are emulsions, activated carbon, cyclodextrin, liposomes, microspheres, dendrimers, nanocrystals, etc., and their combination with chemotherapeutic drugs or imaging substances have chemical bonds, emulsification, adsorption, inclusion, and packaging. Sealed.
  • these drug carriers have significant lymphatic targeting properties compared to small molecule drugs, they are also inferior in in vitro storage stability and in vivo dilution stability, and maintain effective drug loading and carrier integrity in physiological environments. Not clear.
  • Nanocrystals also known as nanocrystals or nanosuspensions, are submicron colloidal dispersions prepared by self-assembly or pulverization techniques with a small amount of active agent or polymer as a stabilizer. Between -100nm, therefore also has a good lymphatic targeting function.
  • nanocrystals are directly formed from drugs, and have the advantages of high drug loading, easy industrial production, and relatively low preparation cost. They can also be applied to various drug delivery routes, such as subcutaneous injection and intravenous injection. Oral administration, transdermal administration, and the like.
  • the nanocrystal itself has high activity and is very unstable. When it meets certain activation conditions, it will release excess free energy, and the particles will grow up, which will also lose the characteristics of the nanomaterial.
  • the preparation of drug nanocrystals has two basic processes: bottom-up (in vitro self-assembly technology) and top-down (crushing technology), because there are some insurmountable defects in the in vitro self-assembly technology, such as organic solvent residues, preparation physics. Poor stability, easy to occur in Austrian ripening, etc., so the crushing technology is widely used in the pharmaceutical industry, but due to the large amount of energy generated during the crushing process, crystal transformation may occur.
  • nanocrystals compared with other drug delivery systems, nanocrystals also have poor stability in vitro, stability in vivo is difficult to control, and preparation techniques are affected. The stability of the formulation is difficult and it is difficult to enlarge. Therefore, the preparation of nanocrystals has caused difficulties and hindered clinical application.
  • the above-mentioned novel drug delivery system having a lymphatic targeting function can be selected from the group consisting of tissue interstitial administration, mucosal administration, vascular administration, and digestive administration.
  • interstitial administration is also called interstitial administration.
  • the drug delivery system When the drug delivery system is administered in the subcutaneous, intramuscular, peritumoral, intratumoral and other interstitial tissues, it has both capillary and capillary lymphatic transport and uptake, due to endothelial The intercellular connections are loose, and there are often many open gaps.
  • the drug-loading system can enter the capillary lymphatics through the intercellular space of the capillary lymphatic vessels and the pinocytosis and phagocytosis of the endothelial cells, and then reach the regional lymph nodes through lymphatic drainage. Due to the advantages of this mode of administration in lymphatic targeting, it is the most common mode of administration for drug delivery systems with lymphatic targeting. To date, the FDA has approved three formulations for lymphatic targeting, the sulfur colloid approved in 1974, the isosulfan blue approved in 1981, and the Lymphoseek approved in 2013. Only the nano-carbon suspension injection (Kanalin) of Chongqing Laimei Pharmaceutical Co., Ltd. was approved for listing. The four routes of administration of the four lymphatic targeting functions are selected for intratumoral or peritumoral tissue interstitial administration.
  • Mitoxantrone is a research reagent widely used in molecular biology, pharmacology and other scientific research. It is also an antibiotic-like anticancer drug. Its structure and anticancer effect are similar to those of doxorubicin. It has no amino sugar structure, does not generate free radicals, and inhibits lipid peroxidation. It can kill any cell cycle tumor. Cells, proliferation and non-proliferating cells are inhibited, so their anti-tumor activity is comparable or slightly higher than doxorubicin, significantly higher than cyclophosphamide, fluorouracil, methotrexate, vincristine and cytarabine, anti-cancer Broad spectrum.
  • the mitoxantrone powder and solution have color, and the raw material powder has a purple-brown powder appearance, which is dark blue in aqueous solution and dark purple-red in concentrated sulfuric acid.
  • the marketed mitoxantrone hydrochloride for injection is a solution, and the administration route is intravenous injection, and the indications are mainly for malignant lymphoma, breast cancer and acute leukemia. There are no pharmaceutical preparations of mitoxantrone and its salts that have been marketed and have lymphatic targeting functions.
  • the technical problem solved by the present application is to provide a prescription composition, a preparation method, a administration method and a design principle of a solution-type injection capable of self-assembly into a nanocrystal in a living organism.
  • Bio-assembled nanocrystalline injection with lymphatic targeting function the components thereof contain mitoxantrone or mitoxantrone salt in mass to volume ratio (g/mL): 0.05%-5%, osmotic pressure regulating agent : 0.1-10%, the rest is solvent.
  • it further contains a buffer: 0.01-0.1%, an antioxidant: 0.01-0.1%, an adsorbent: 0.05-1%, and a filler: 0-20%.
  • the concentration of mitoxantrone or mitoxantrone in the injection ranges from 0.1% to 1%.
  • the pH range is from 2.0 to 6.0.
  • the pH ranges from 3.5 to 5.5.
  • the osmotic pressure ranges from 285 to 2317 mmol/Kg.
  • the osmotic pressure ranges from 307 to 1503 mmol/Kg.
  • the mitoxantrone salt is selected from the group consisting of mitoxantrone hydrochloride, mitoxantrone oxalate, mitoxantrone sulfate, mitoxantrone phosphate, mitoxantrone acetate, and mitoxantrone citrate.
  • mitoxantrone hydrochloride mitoxantrone oxalate
  • mitoxantrone sulfate mitoxantrone phosphate
  • mitoxantrone acetate mitoxantrone citrate
  • the bioself-assembled nanocrystals have a particle size ranging from 1 to 1000 nm under physiological conditions.
  • the bio-assembled nanocrystals have a particle size ranging from 10 to 100 nm under physiological conditions.
  • the bio-assembled nanocrystals have a particle size ranging from 30 to 60 nm under physiological conditions.
  • the injection type is divided into a small water needle or a powder injection, the filler in the small water needle is 0, and the filler in the powder injection is greater than 0 and less than or equal to 20%.
  • the osmotic pressure adjusting agent uses sodium chloride, glucose, sorbitol, mannitol, glycerin, PEG, phosphate, citrate, and a mixture of one or more of the above;
  • the buffering agent is one or more of acetic acid, sodium acetate, citric acid, and sodium citrate;
  • the antioxidant is one or more of sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, sodium thiosulfate, and disodium edetate;
  • the filler is monosaccharide glucose, fructose, galactose, ribose or deoxyribose, or saccharose, trehalose, maltose, lactose, or mannitol using polymeric sugars, One or more of sorbitol, lactitol, xylitol, maltitol, erythritol;
  • the adsorbent is activated carbon.
  • the lymph nodes produce a blue color visible to the naked eye.
  • the tumor metastasis lymph nodes shrink or return to normal size.
  • the administration of the injection is a solution type
  • mitoxantrone or a salt thereof is present in a completely dissolved molecular form.
  • the nano-crystals are self-assembled at the injection site.
  • the mode of administration of the lymphatic targeting function is administered by interstitial injection.
  • interstitial injection including subcutaneous interstitial injection, intermuscular injection, intermuscular injection, submucosal injection, intraperitoneal injection, intra-tissue interstitial injection, tissue interstitial injection, or several administrations a combination of ways.
  • a preparation method of a biological self-assembled nanocrystalline injection having a lymphatic targeting function, wherein the injection is a small water needle bio-assembled nanocrystalline injection comprising the following preparation process: weighing an appropriate amount of raw materials, and mixing the cans Add 70% of the water for injection, add the prescription amount of the auxiliary material, stir for about 15 minutes to dissolve, add mitoxantrone or mitoxantrone salt, stir for about 20 minutes to dissolve, put into activated carbon, at 40-60 After stirring for 20 minutes at °C, the concentrated solution was filtered through a 3 ⁇ m stainless steel folding filter and transferred to a dilution tank. The injection water was added to the total amount of the solution, stirred for 10 minutes, and the liquid was separated by two series. The bacteria filter was filtered, and the filter pore size was 0.22 ⁇ m, which was aseptically sealed and sealed.
  • the injection is a powder needle bio-assembled nanocrystalline injection, and comprises the following preparation process: weighing an appropriate amount of raw materials, adding a prescription amount of 70% water for injection to the concentrated tank, adding a prescription amount of the auxiliary material, stirring Dissolve in about 15 minutes, add mitoxantrone or mitoxantrone salt, stir for about 20 minutes to dissolve, put into activated carbon, stir and absorb at 40-60 ° C for 20 minutes, and filter the concentrated solution through 3 ⁇ m stainless steel folding filter.
  • the dried biological self-assembled nanocrystalline injection lyophilized powder can be administered by interstitial injection after hydration shaking, and one or more of water for injection, physiological saline and glucose injection are selected for the hydration medium.
  • the mass percent by volume in this application refers to g/mL.
  • mitoxantrone hydrochloride is selected as an active component of biological self-assembled nanocrystals, and mitoxantrone hydrochloride is present in the form of a well-soluble hydrochloride salt under the acidic conditions formed by the preparation of the product.
  • the pH value of the acidic condition has a key influence on the self-assembled nanocrystal behavior of the injection.
  • the pH range of the injection is between 2.0 and 6.0
  • the interstitial injection is used as the administration mode, and the interstitial part is characterized.
  • the body fluid of pH 7.4 is reduced to mitoxantrone after neutralization of the hydrochloric acid group of mitoxantrone hydrochloride under physiological conditions, thereby reducing the solubility and causing precipitation of nanocrystals.
  • the pH of the injection is less than 2.0, the body fluid of pH 7.4 cannot achieve neutralization, so that the nanocrystals cannot be effectively produced.
  • the pH of the injection When the pH of the injection is greater than 6.0, the mitoxantrone hydrochloride in the injection solution produces an unstable appearance. The quality of the injection. After effective prescription optimization, it is determined that when the pH of the injection is between 3.5 and 5.5, the neutralization of pH 7.4 is the most effective, and a large number of nanocrystals satisfying lymphatic targeting function can be rapidly formed in the interstitial site. .
  • the concentration of the drug significantly affects the amount of formation and the rate of formation of the nanocrystals.
  • the optimum concentration of the present application is in the range of 0.05-5% (g/mL, W: V), which is lower than the concentration range of administration. After the interstitial injection, it is difficult to form nanocrystals with targeting ability under physiological conditions, and the height is high. In the concentration range of administration, it has toxic side effects on the lymphatic system.
  • the circulation rate of the fluid in the interstitial site is slow.
  • the motility of the drug injected into the lymphatic system from the interstitial site mainly comes from the difference of osmotic pressure between the interstitial site and the lymphatic system.
  • the hypotonic injection is not conducive to the drug drainage through the capillary lymphatic space. Lymphatic system, so the isotonic or hyperosmolarity of the injection is beneficial to the lymphatic drainage of the self-assembled nanocrystals.
  • the suitable osmotic pressure range in this application is 285 to 2317 mmol/Kg, and the optimum osmotic pressure range is 307 to 1503 mmol/Kg.
  • the lymphatic drainage force is insufficient, and when the osmotic pressure is greater than 2317 mmol/Kg, The concentration of the osmotic pressure adjusting agent in the injection is too high, resulting in irritation at the injection site.
  • the amount of body fluid in the interstitial injection site is small, the pH value is 7.4, and the circulation speed is slow. It is because of the characteristics of such physiological conditions that it is appropriate.
  • the pH of the injection, the appropriate concentration and the appropriate osmotic pressure, the water solubility of the mitoxantrone hydrochloride is reduced after the hydrochloric acid group is removed, and the nanocrystals having a particle diameter of more than 30 nm and less than 60 nm are self-assembled at the interstitial site, lymph
  • lymph There is a certain overlap gap between the endothelial cells of the tube. Small molecule drugs and nanoparticles with a particle size of less than 20 nm will produce a dispersion effect at the injection site after subcutaneous injection.
  • the particle size ranges from 20 nm to 100 nm and can pass through the lymphatic endothelial cell gap. , produces a good lymphatic targeting effect.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone hydrochloride in the small water needle type biological self-assembled nanocrystal injection prepared by the prescription process was 0.58%, the pH was determined to be 3.5, and the osmotic pressure was 196 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 50 nm under interstitial biological conditions.
  • the level of metastasis after subcutaneous interstitial administration was dark blue visible to the naked eye, and the secondary and tertiary lymph nodes were light blue in the naked eye.
  • the weight and volume of metastatic lymph nodes at all levels decreased significantly or returned to normal and were not significantly toxic to the injection site and lymphocytes.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone hydrochloride in the small water needle type bio-assembled nanocrystalline injection prepared by the prescription process was 0.57%, the pH was determined to be 3.5, and the osmotic pressure was 938 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 50 nm under interstitial biological conditions.
  • the primary, secondary and tertiary lymph nodes metastasized after subcutaneous interstitial administration are dark blue visible to the naked eye.
  • the weight and volume of metastatic lymph nodes at all levels decreased significantly or returned to normal and were not significantly toxic to the injection site and lymphocytes.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone oxalate in the small water needle type self-assembled nanocrystalline injection prepared by the prescription process was 0.056%, the pH was determined to be 5.5, and the osmotic pressure was 1503 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 40 nm under interstitial biological conditions.
  • the primary, secondary and tertiary lymph nodes metastasized after subcutaneous interstitial administration were light blue visible to the naked eye.
  • the weight and volume of metastatic lymph nodes at all levels decreased to some extent and were not significantly toxic to the injection site and lymphocytes.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone sulfate in the small water needle type bio-assembled nanocrystalline injection prepared by the prescription process was 5.44%, and the pH was determined to be 3.5,307 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 60 nm under interstitial biological conditions.
  • the primary, secondary and tertiary lymph nodes metastasized after subcutaneous interstitial administration were dark blue visible to the naked eye.
  • the weight and volume of metastatic lymph nodes at all levels returned to normal size and were significantly toxic to the injection site and lymphocytes.
  • the prescription of powder needle bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the preparation process is as follows:
  • the drug solution is filtered through two serial sterilization filters (pore size: 0.22 ⁇ m), dissolved in mannitol, and lyophilized to remove water to obtain a dried biologically self-assembled nanocrystalline injection lyophilized powder. After shaking, it can be administered by interstitial injection.
  • the powder-needle type self-assembled nanocrystalline injection prepared by the prescription process has a smooth surface without collapse and no shrinkage after freeze-drying.
  • a mitoxantrone hydrochloride injection was formed, wherein the concentration of mitoxantrone hydrochloride was 0.52%, the pH was determined to be 3.5, and the osmotic pressure was 938 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 40 nm under interstitial biological conditions.
  • the metastatic primary, secondary, and tertiary lymph nodes were dark blue visible to the naked eye. The weight and volume of metastatic lymph nodes at all levels were significantly reduced or restored to normal size and were not significant at the injection site and lymphocytes. toxicity.
  • the prescription of powder needle bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the preparation process is as follows:
  • the drug solution is filtered through two serial sterilization filters (pore size: 0.22 ⁇ m), dissolved in sucrose, and lyophilized to remove water, to obtain a dried lyophilized powder for biological self-assembly nanocrystal injection, and hydrated before use. After the interstitial injection can be administered.
  • the powder-needle type self-assembled nanocrystalline injection prepared by the prescription process freezes and shrinks on the surface. After the injection water was hydrated, a mitoxantrone injection was formed, wherein the concentration of mitoxantron phosphate was 0.499%, the pH was determined to be 5.5, and the osmotic pressure was 307 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 55 nm under interstitial biological conditions. After subcutaneous interstitial administration, the metastatic primary, secondary, and tertiary lymph nodes were dark blue visible to the naked eye. The weight and volume of metastatic lymph nodes at all levels were significantly reduced or restored to normal size and were not significant at the injection site and lymphocytes. toxicity.
  • the prescription of powder needle bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the preparation process is as follows:
  • the drug solution is filtered through two serial sterilization filters (pore size: 0.22 ⁇ m), dissolved in trehalose, and then lyophilized to remove water to obtain a dried biologically self-assembled nanocrystalline injection lyophilized powder. After shaking, it can be administered by interstitial injection.
  • the powder-needle type self-assembled nanocrystalline injection prepared by the prescription process freezes and shrinks on the surface. After the injection water was hydrated, the mitoxantrone injection was formed, wherein the concentration of mitoxantrone acetate was 0.52%, the pH was measured to be 3.5, and the osmotic pressure was 1503 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 60 nm under interstitial biological conditions. After subcutaneous interstitial administration, the metastatic primary, secondary, and tertiary lymph nodes were dark blue visible to the naked eye. The weight and volume of metastatic lymph nodes at all levels were significantly reduced or restored to normal size and were not significant at the injection site and lymphocytes. toxicity.
  • 1000 in the present application means that the prepared injection is divided into 1000 parts.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone hydrochloride in the small water needle type self-assembled nanocrystalline injection prepared by the prescription process was 0.01%, the pH was determined to be 6.83, and the osmotic pressure was 196 mmol/Kg. Only the metastatic lymph nodes after subcutaneous interstitial administration are light blue and difficult to distinguish by the naked eye. There was no significant effect on the weight and volume of metastatic lymph nodes at all levels.
  • the prescription of the small water needle type bio-assembled nanocrystalline injection and the dosage of 1000 are as follows:
  • the concentration of mitoxantrone hydrochloride in the small water needle type biological self-assembled nanocrystal injection prepared by the prescription process was 10.34%, the pH value was 1.66, and the osmotic pressure was 196 mmol/Kg.
  • the self-assembled nanocrystals have a particle size of 120 nm under interstitial biological conditions. After subcutaneous interstitial administration, the metastatic primary, secondary, and tertiary lymph nodes are dark blue visible to the naked eye. The weight and volume of metastatic lymph nodes at all levels are significantly reduced or restored to normal size, but there are injection sites and lymphocytes. Significant toxicity.
  • the isosulfan blue solution (IB) was selected as the control solution, and the formulation and preparation process were as follows:
  • liquid medicine qualified for foreign matter inspection is filled with nitrogen and potted, and each branch is 5.1 ⁇ 0.1mL. Sterilize at 121 ° C for 15 min. Leak detection, light inspection, labeling, packaging, that is, the finished product.
  • the mitoxantrone liposome (MIH-LIP) was selected as the control nano preparation, and the formulation and preparation process were as follows:
  • the MIH-LIP was prepared by the thin film method: the soybean lecithin and cholesterol in the urban area were dissolved in 5 mL of anhydrous ether, and the mixture was vacuumed to dryness with anhydrous ether. The appropriate amount of phosphate buffer was added to hydrate to obtain a uniform pale yellow emulsion. Transfer to a polypropylene centrifuge tube, place the tube in an ice bath probe for 5 min (working 50 s, intermittent 10 s). The pale yellow translucent liquid was poured into a weighing mitoxantrone original flask and rotated at 37 ° C to form a uniform blue-black colloidal solution. Store at 4 ° C for later use.
  • MIH-Injection mitoxantrone bioinjection injection
  • transmission electron microscope and other detection methods require dry samples for detection, and can not completely simulate the real self-assembly in the body environment.
  • Morphology so the atomic force microscope was selected to characterize the morphology of self-assembled nanoparticles under physiological conditions. The results showed that MIH can self-assemble into nanoparticles with particle size of about 60 nm under physiological conditions, and the shape is round and uniform, and the particle size is uniform. Consistent. Under acidic conditions, the particle size is about 10 nm.
  • the same concentration of IB solution was placed under physiological conditions, and its particle size and morphology were determined by atomic force microscopy.
  • the results showed that IB under physiological conditions, the particle size of single particles was 10 nm to 20 nm, and the particle size was uniform. The shape is round.
  • the particle size of the control preparation MIH-LIP was measured using a Malvern particle size analyzer. The test results show that the particle size of MIH-LIP is about 140 nm.
  • the pH of the injected solution was adjusted to 1.5, 2.0, 2.5, 3.5, 4.5, 5.5, 6.0, 6.5, 7.0, and the 0.22 ⁇ m filter was used after adjusting the pH for 1 hour. After filtration, the content of the drug was determined. When the pH was between 1.5 and 6.0, the drug content was 98.45 ⁇ 0.61% to 101.72 ⁇ 0.20%. When the pH was 6.5 and 7.0, the drug content showed a significant decrease, respectively. It was 83.76 ⁇ 0.53% and 75.38 ⁇ 0.22%, indicating that the pH of the injection was greater than 6.0, and the drug in the injection showed unstable precipitation.
  • the pH of the injected injection was 1.5, 2.0, 2.5, 3.5, 4.5, 5.5, 6.0, and the mode of administration of interstitial injection was selected.
  • the mice were injected subcutaneously in the foot of Kunming mice. At the preset time point, the mice were sacrificed by cervical dislocation, and the first, second and third grade lymph nodes of the mice were removed, and the staining was observed. When the pH is less than 2.0, only the first stage lymph nodes can be stained.
  • the third-grade lymph nodes can be stained, but the pH is 2.0, 2.5 and 6.0, the third-stage lymph nodes are blue, and the tertiary lymph nodes are between pH 3.5 and 5.5. Both can be dyed and are dark blue.
  • the osmotic pressure values of the respectively adjusted injections were 112,196,285,307,1503,2317,3011mmol/Kg, and the mode of administration of interstitial injection was selected in Kunming.
  • the mouse foot was injected subcutaneously. At the preset time point, the mice were sacrificed by cervical dislocation, and the mouse administration site was taken for H&E staining to evaluate local irritation.
  • the osmotic pressure was 3011 mmol/Kg, and redness and swelling occurred. H&E staining showed local irritation, and other osmotic pressure values did not show significant local irritation.
  • the osmotic pressure values of the respectively adjusted injections were 112,196,285,307,1503,2317,3011mmol/Kg, and the mode of administration of interstitial injection was selected in Kunming.
  • the mouse foot was injected subcutaneously. At the preset time point, the mice were sacrificed by cervical dislocation, and the first, second and third grade lymph nodes were harvested, and the staining was observed. When the osmotic pressure value is less than 285 mmol/Kg, only the primary lymph node can be stained.
  • the tertiary lymph nodes When the osmotic pressure is between 285 and 2317 mmol/Kg, the tertiary lymph nodes can be stained, but when the osmotic pressure is 285 and 2317 mmol/Kg, the third-stage lymph nodes are blue, at pH 307-1503 mmol/Kg. When in between, the tertiary lymph nodes can be stained and dark blue.
  • MIH-Injection subcutaneously inject the mitoxantrone bio-injection injection into the foot of Kunming mice 20 ⁇ L.
  • the mice were sacrificed by cervical dislocation.
  • the first, second and third grade lymph nodes of the mice were harvested, and the staining was observed.
  • the content of MIH in the lymph nodes was determined by HPLC.
  • the results of the trial showed that MIH-Injection was analyzed by the area under the curve (AUC) and the maximum peak concentration (Cmax) after interstitial administration, for primary lymph nodes (PLNS), secondary lymph nodes (ILNS) and tertiary lymph nodes.
  • AUC area under the curve
  • Cmax maximum peak concentration
  • RLNS has a good targeting effect, taking the blood drug content as a reference (there is no detectable drug in the blood), and the targeting efficiency can reach 100%, that is, the particle size after self-assembly by interstitial administration can satisfy all the lymphatics.
  • the system does not enter the blood circulation.
  • PLNs popliteal lymph nodes
  • ILNs iliac lymph nodes
  • RLNs renal lymph nodes
  • IB and MIH-LIP were used as control preparations to explore the effects of different particle size factors on the lymphatic targeting ability of the drug delivery system.
  • Interstitial injections of IB (20 nm), MIH-Injection (60 nm) and MIH-LIP (130 nm) were selected. After two hours of injection, the test results are shown in Table 2 below. It is indicated that the mitoxantrone bio-assembled nanocrystals give a certain particle size of the mitoxantrone hydrochloride drug, which has better lymphatic targeting ability than liposome, and is superior to the lymphatic targeting of common injection. ability.
  • PLNs popliteal lymph nodes
  • ILNs iliac lymph nodes
  • RLNs renal lymph nodes
  • MIH-Injection group MIH-LIP group
  • IB group Three rats in each group were subcutaneously administered to the soles of mice. The mice were sacrificed by cervical dislocation 10 min after administration, and the first, second and third grade lymph nodes were dissected to observe the staining effect. The results of the test showed that the degree of grade III lymph node staining of MIH-Injection was deeper than that of IB group and MIH-LIP group, the effect was good, and the lymph node tracer effect was good. MIH-Injection and MIH-LIP had more advantages of lymph node tracing. The reason may be that the fluidity of the solution is superior to the fluidity of the liposome.
  • Phase III The third cycle of chemotherapy
  • Phase IV The fourth cycle of chemotherapy
  • MPLNs mestastatic popliteal lymph nodes
  • MILNs mestastatic iliac lymph nodes
  • Phase III The third cycle of chemotherapy
  • Phase IV The fourth cycle of chemotherapy
  • MPLNs mestastatic popliteal lymph nodes
  • MILNs mestastatic iliac lymph nodes

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Abstract

一种具有淋巴靶向功能的生物自组装纳米晶注射剂，以及使用生物自组装纳米晶注射剂靶向淋巴***的给药方式。所述注射剂含有0.05％-5％米托蒽醌及其盐类、0.1-10％渗透压调节剂、0.001-0.1％缓冲剂、0.01-0.1％抗氧剂、0.05-1％吸附剂、0％-20％填充剂，给药方式为间质注射，被动靶向淋巴***后***会产生肉眼可见的蓝色。

Description

具有淋巴靶向功能的生物自组装纳米晶注射剂及制备方法 技术领域
本申请涉及具有淋巴靶向功能的新剂型的设计和制备，提供了一种在体自组装纳米晶注射剂的处方组成、制备方法。
背景技术
淋巴道转移是影响恶性肿瘤预后的重要因素之一。肿瘤细胞在早期即可进入淋巴***而扩散,通过***侧支循环进入非所属***或远离原发灶的***，形成所谓跳跃式转移,使外科手术的应用受到限制。区域***对放疗的敏感性低于原发灶，全身化疗对转移***的疗效不佳，原因在于常规剂型的化疗药物不易转运至***。由于治疗***转移灶的疗效直接影响患者的治愈率和生存率，所以针对***转移灶的靶向越来越受到重视。***已成为肿瘤化疗中一个非常有潜力的靶器官。
利用大分子物质和微粒易被淋巴***吞噬的特性设计新型给药***使淋巴靶向得以实现。目前所用药物载体有乳剂、活性炭、环糊精、脂质体、微球、树状大分子、纳米结晶等，它们与化疗药物或成像物质的结合方式有化学键、乳化、吸附、包合、包封等。虽然这些药物载体与小分子药物相比具有明显的淋巴靶向性，但是同时这些载体的体外储存稳定性和体内稀释稳定性较差，生理环境下维持有效的药物包载和载体完整性的能力也不明确。
纳米结晶(nanocrystals)也称为纳米晶或纳米混悬液，是以少量表明活性剂或聚合物为稳定剂，通过自组装或粉碎技术制备的一种亚微胶体分散系，其粒径在1-100nm之间，因此也具有良好淋巴靶向功能。相比于其它给药***，纳米晶体直接由药物形成，具有载药量高、易工业化生产、制备成本相对较低的优势,同时还能应用于多种给药途径，如皮下注射、静脉注射、口服给药、经皮给药等。
纳米晶体本身具有较高的活性，非常不稳定，满足一定激活条件时，就会释放出过剩的自由能，粒子长大，从而也将失去纳米材料所具有的特性。目前制备药物纳米结晶有自下而上(体外自组装技术)和自上而下(破碎技术)两种基本工艺，因为体外自组装技术种类有一些无法克服的缺陷，如有机溶剂残留、制剂物理稳定性差、易发生奥式熟化等，因此破碎技术在医药行业中应用相对广泛，但是由于在破碎过程中会产生大量的能量，可能产生晶型转化现象。因此与其他给药***相比，纳米晶体也具有体外稳定性差、体内稳定性难于控制、制备技术会影 响制剂的稳定性且难于放大的缺陷。因此对纳米晶体的制备产生了困难，阻碍了临床应用。
以上所述的具有淋巴靶向功能的新型给药***，给药方式可以选择组织间隙给药、粘膜给药、血管给药、消化道给药等。其中，组织间隙给药也称为间质给药，当给药***进行皮下、肌内、瘤周、瘤内等组织间隙给药时同时具有毛细血管和毛细***的转运和摄取，由于内皮细胞间连接松散，常有许多开放的间隙存在，载药***可通过毛细***内皮细胞间间隙和内皮细胞的胞饮及吞噬作用进入毛细***内，然后通过淋巴引流到达区域***。由于这种给药方式在淋巴靶向方面的优势，成为了具有淋巴靶向功能的给药***最为常见的给药方式。迄今为止，FDA共批准过三种用于淋巴靶向的制剂，分别为1974年批准的硫胶体、1981年批准的异硫蓝和2013年批准的Lymphoseek。国内仅有重庆莱美药业的纳米炭混悬注射液(卡纳林)批准上市。这四种具有淋巴靶向功能的已上市制剂的给药方式均选择的是瘤内或瘤周组织间隙给药。
米托蒽醌(Mitoxantrone)是广泛应用于分子生物学，药理学等科研方面的科研试剂。同时也是一种抗生素类抗肿瘤药物，其结构及抗癌作用与阿霉素相近，其无氨基糖结构，不产生自由基，且有抑制脂质过氧化作用，可以杀灭任何细胞周期的肿瘤细胞，增殖与非增殖细胞均受到抑制，因此其抗肿瘤活性相当或略高于阿霉素，明显高于环磷酰胺、氟尿嘧啶、甲氨蝶呤、长春新碱和阿糖胞苷，抗癌谱广。米托蒽醌原料药粉末和溶液均具有颜色，原料药粉末外观为紫褐色粉末，于水溶液中呈深蓝色，于浓硫酸中呈深***。已上市的注射用盐酸米托蒽醌为溶液剂，给药途径为静脉注射，适应症为主要用于恶性淋巴瘤、乳腺癌和急性白血病。国内外均无已上市并且具有淋巴靶向功能的米托蒽醌及其盐类的药物制剂上市。
发明内容
本申请多解决的技术问题是提供了一种能够在体生物自组装成纳米晶体的溶液型注射剂的处方组成、制备方法、给药方式和设计原理。
本申请是通过如下技术方案实现的：
一种具有淋巴靶向功能的生物自组装纳米晶注射剂，其组分按照质量体积比(g/mL)含有米托蒽醌或米托蒽醌盐类：0.05％-5％，渗透压调节剂：0.1-10％，其余为溶剂。
可选的，还含有缓冲剂：0.01-0.1％、抗氧剂：0.01-0.1％、吸附剂：0.05-1％，填充剂：0-20％。
可选的，注射剂中米托蒽醌或米托蒽醌盐类浓度范围为0.1％到1％。
pH范围为2.0到6.0。
可选的，pH范围为3.5到5.5。
渗透压范围为285到2317mmol/Kg。
可选的，渗透压范围为307到1503mmol/Kg。
可选的，米托蒽醌盐类采用盐酸米托蒽醌、草酸米托蒽醌、硫酸米托蒽醌、磷酸米托蒽醌、醋酸米托蒽醌、枸橼酸米托蒽醌中的一种或几种。
生物自组装纳米晶在生理条件下粒径范围为1-1000nm。
可选的，生物自组装纳米晶在生理条件下粒径范围为10-100nm。
可选的，生物自组装纳米晶在生理条件下粒径范围为30-60nm。
可选的，注射剂类别分为小水针或粉针剂，小水针中填充剂为0，粉针剂中填充剂为大于0小于等于20％。
可选的，所述渗透压调节剂采用氯化钠、葡萄糖、山梨醇、甘露醇、甘油、PEG、磷酸盐、枸橼酸盐、采用上述一种或者几种物质的混合物；
可选的，所述的缓冲剂采用醋酸、醋酸钠、枸橼酸、枸橼酸钠中的一种或者几种；
可选的，所述的抗氧剂采用亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、依地酸二钠中的一种或者几种；
可选的，所述的填充剂采用单糖类的葡萄糖、果糖、半乳糖、核糖或脱氧核糖、或者采用二糖类的蔗糖、海藻糖、麦芽糖、乳糖、或者采用聚合糖类的甘露醇、山梨醇、乳糖醇、木糖醇、麦芽糖醇、赤藓糖醇中的一种或者几种；
可选的，所述吸附剂为活性炭。
可选的，实现淋巴靶向功能后，***会产生肉眼可见的蓝色。
可选的，实现淋巴靶向功能后，肿瘤转移***缩小或恢复正常大小。
可选的，其中给药注射剂为溶液型，米托蒽醌或其盐类以完全溶解的分子形式存在。
可选的，溶液型注射剂间质给药后，在注射部位自组装成纳米晶体。
可选的，实现淋巴靶向功能给药方式为间质注射给药。
可选的，包括皮下间质注射、肌肉间质注射、肌肉间质注射、粘膜下间质注射、腹腔间质注射、组织内间质注射、组织周间质注射，或者采用上述几种给药方式的组合。
一种具有淋巴靶向功能的生物自组装纳米晶注射剂的制备方法，所述的注射剂为小水针类生物自组装纳米晶注射剂，包含如下制备流程：称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入米托蒽醌或米托蒽醌盐类，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟，浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟，药液经两支串联的除 菌过滤器过滤，过滤器孔径为0.22μm，无菌分装封口。
可选的，所述注射剂为粉针类生物自组装纳米晶注射剂，包含如下制备流程：称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入米托蒽醌或米托蒽醌盐类，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟，浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟，药液经两支串联的除菌过滤器，孔径为0.22μm，过滤，加入甘露醇溶解后，经冷冻干燥去除水分，得到干燥的生物自组装纳米晶注射用冻干粉，使用前水化振摇后可间质注射给药，水化介质选择注射用水、生理盐水、葡萄糖注射液中的一种或者几种。
本申请中的质量体积百分比均指的是g/mL。
本申请选择盐酸米托蒽醌作为生物自组装纳米晶体的活性成分，盐酸米托蒽醌在本品制备形成的酸性条件下以水溶性良好的盐酸盐形式存在。
(1)酸性条件的pH值对注射剂体内自组装纳米晶行为有关键的影响，当注射剂的pH范围在2.0到6.0之间时，以间质注射作为给药方式，***位的特点是存在pH7.4的体液，在生理条件pH环境，盐酸米托蒽醌的盐酸基团被中和后还原成为米托蒽醌，从而溶解度降低产生纳米晶析出的现象。当注射剂的pH值小于2.0，pH7.4的体液不能实现中和作用，从而无法有效产生纳米晶；当注射剂的pH值大于6.0，注射液中盐酸米托蒽醌产生不稳定的析出现象，影响注射剂的质量。经过有效的处方优化，确定当注射剂的pH值范围在3.5到5.5之间时，pH7.4体液的中和作用发挥的最充分，能够在***位快速形成大量满足淋巴靶向功能的纳米晶。
(2)***位体液量少，因此给药浓度显著影响纳米晶的生成量和生成速度。本申请的最适给药浓度范围为0.05-5％(g/mL，W：V)，低于给药浓度范围，间质注射后在生理条件下难于形成具有靶向能力的纳米晶体，高于给药浓度范围，对淋巴***会产生毒副作用。
(3)***位体液循环速度较慢，***位注射的药物进入淋巴***的动力主要来自于***位和淋巴***的渗透压差异，低渗注射剂不利于药物通过毛细***间隙引流至淋巴***，因此注射剂等渗或偏高渗有利于自主装纳米晶的淋巴引流。本申请中适合的渗透压范围为285到2317mmol/Kg，最适的渗透压范围为307到1503mmol/Kg，当渗透压小于285mmol/Kg时，淋巴引流动力不足，当渗透压大于2317mmol/Kg，在注射剂中渗透压调节剂浓度过高，产生注射部位刺激性。
间质注射部位体液量少、pH值7.4、循环速度较慢，正因为这样的生理条件特征，在适当 的注射剂pH值、适当的给药浓度和适当的渗透压，盐酸米托蒽醌脱去盐酸基团后水溶性降低，在***位自组装成为粒径大于30nm、小于60nm的纳米晶体，***内皮细胞间存在一定交叠间隙，小分子药物和粒径小于20nm的纳米粒子，皮下注射后会在注射部位产生弥散效果，粒径在20nm到100nm的范围内，能通过***内皮细胞间隙，产生良好的淋巴靶向效果。
具体实施方式
实施例1
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000001
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入盐酸米托蒽醌，搅拌约20分钟使溶解。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中盐酸米托蒽醌的浓度为0.58％，测定pH值为3.5，渗透压为196mmol/Kg。在间质生物条件下自组装纳米晶的粒径为50nm。皮下间质给药后转移的一级呈肉眼可见的深蓝色，二级和三级***呈肉眼可见浅蓝色。各级转移***的重量和体积显著下降或恢复正常且对注射部位和淋巴细胞没有显著毒性。
实施例2
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000002
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约 15分钟使溶解，再加入盐酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，无菌分装封口。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中盐酸米托蒽醌的浓度为0.57％，测定pH值为3.5，渗透压为938mmol/Kg。在间质生物条件下自组装纳米晶的粒径为50nm。皮下间质给药后转移的一级、二级和三级***呈肉眼可见的深蓝色。各级转移***的重量和体积显著下降或恢复正常且对注射部位和淋巴细胞没有显著毒性。
实施例3
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000003
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入草酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，无菌分装封口。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中草酸米托蒽醌的浓度为0.056％，测定pH值为5.5，渗透压为1503mmol/Kg。在间质生物条件下自组装纳米晶的粒径为40nm。皮下间质给药后转移的一级、二级和三级***呈肉眼可见的浅蓝色，各级转移***的重量和体积有一定的下降且对注射部位和淋巴细胞没有显著毒性。
实施例4
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000004
Figure PCTCN2017101801-appb-000005
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入硫酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，无菌分装封口。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中硫酸米托蒽醌的浓度为5.44％，测定pH值为3.5，307mmol/Kg。在间质生物条件下自组装纳米晶的粒径为60nm。皮下间质给药后转移的一级、二级和三级***呈肉眼可见的深蓝色，各级转移***的重量和体积恢复至正常大小且对注射部位和淋巴细胞有显著毒性。
实施例5
粉针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000006
制备工艺如下：
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入除填充剂以外的处方量的辅料，搅拌约15分钟使溶解，再加入盐酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，加入甘露醇溶解后，经冷冻干燥去除水分，得到干燥的生物自组装纳米晶注射用冻干粉，使用前水 化振摇后可间质注射给药。
由该处方工艺制备的粉针型生物自组装纳米晶注射剂冷冻干燥后表面光滑无塌陷，无皱缩。注射用水水化后形成盐酸米托蒽醌注射液，其中盐酸米托蒽醌的浓度为0.52％，测定pH值为3.5，渗透压为938mmol/Kg。在间质生物条件下自组装纳米晶的粒径为40nm。皮下间质给药后，转移的一级、二级和三级***呈肉眼可见的深蓝色，各级转移***的重量和体积显著减小或恢复至正常大小且对注射部位和淋巴细胞没有显著毒性。
实施例6
粉针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000007
制备工艺如下：
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入除填充剂以外的处方量的辅料，搅拌约15分钟使溶解，再加入磷酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，加入蔗糖溶解后，经冷冻干燥去除水分，得到干燥的生物自组装纳米晶注射用冻干粉，使用前水化振摇后可间质注射给药。
由该处方工艺制备的粉针型生物自组装纳米晶注射剂冷冻干燥后表面出现塌陷和皱缩。注射用水水化后形成磷酸米托蒽醌注射液，其中磷酸米托蒽醌的浓度为0.499％，测定pH值为5.5，渗透压为307mmol/Kg。在间质生物条件下自组装纳米晶的粒径为55nm。皮下间质给药后，转移的一级、二级和三级***呈肉眼可见的深蓝色，各级转移***的重量和体积显著减小或恢复至正常大小且对注射部位和淋巴细胞没有显著毒性。
实施例7
粉针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000008
制备工艺如下：
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入除填充剂以外的处方量的辅料，搅拌约15分钟使溶解，再加入醋酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，加入海藻糖溶解后，经冷冻干燥去除水分，得到干燥的生物自组装纳米晶注射用冻干粉，使用前水化振摇后可间质注射给药。
由该处方工艺制备的粉针型生物自组装纳米晶注射剂冷冻干燥后表面出现塌陷和皱缩。注射用水水化后形成醋酸米托蒽醌注射液，其中醋酸米托蒽醌的浓度为0.52％，测定pH值为3.5，渗透压为1503mmol/Kg。在间质生物条件下自组装纳米晶的粒径为60nm。皮下间质给药后，转移的一级、二级和三级***呈肉眼可见的深蓝色，各级转移***的重量和体积显著减小或恢复至正常大小且对注射部位和淋巴细胞没有显著毒性。
本申请中1000支指的是制备出的注射剂分成1000份。
对比例1
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000009
Figure PCTCN2017101801-appb-000010
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入盐酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，无菌分装封口。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中盐酸米托蒽醌的浓度为0.01％，测定pH值为6.83，渗透压为196mmol/Kg。皮下间质给药后仅转移的一级***呈淡蓝色且肉眼较难分辨。对各级转移***的重量和体积没有显著影响。
对比例2
小水针型生物自组装纳米晶注射剂的处方及1000支的用量如下：
Figure PCTCN2017101801-appb-000011
称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入盐酸米托蒽醌，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟。浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟。药液经两支串联的除菌过滤器(孔径为0.22μm)过滤，无菌分装封口。
由该处方工艺制备的小水针型生物自组装纳米晶注射剂中盐酸米托蒽醌的浓度为10.34％，测定pH值为1.66，渗透压为196mmol/Kg。在间质生物条件下自组装纳米晶的粒径为120nm。皮下间质给药后，转移的一级、二级和三级***呈肉眼可见的深蓝色，各级转移***的重量和体积显著减小或恢复至正常大小，但是对注射部位和淋巴细胞有显著毒性。
对照例
1.对照溶液剂的制备
选择异硫蓝溶液剂(IB)作为对照溶液剂，处方和制备工艺如下：
处方：
Figure PCTCN2017101801-appb-000012
注射用水加至1000mL
制成200支
制备工艺：
称取处方量的磷酸氢二钠、磷酸二氢钾和氯化钠，加入处方量80％的注射用水，搅拌约15min使溶解，再加入处方量IB，搅拌约20min使溶解，投入药用炭，于50℃搅拌吸附20min。浓配液经3μm炭棒过滤后转至稀配罐中，冲洗并合并冲洗液。补加注射用水至稀配罐内药液体积达到规定量后，搅拌约10min。取样检测性状、pH和含量。中间体检验合格的药液经0.22μm微孔滤膜过滤，取样检验可见异物。可见异物检验合格的药液充氮、灌封，每支5.1±0.1mL。121℃灭菌15min。检漏，灯检，贴标，包装，即得成品。
2.对照纳米制剂的制备
选择盐酸米托蒽醌脂质体(MIH-LIP)作为对照纳米制剂，处方和制备工艺如下：
处方：
Figure PCTCN2017101801-appb-000013
制备工艺：
采用薄膜法制备MIH-LIP：分别城区大豆卵磷脂、胆固醇，溶解于5mL无水***中，抽真空旋转至无水***全部去尽，加入适量磷酸盐缓冲液，水化，得到均匀淡黄色乳液，转入聚丙烯离心管，离心管放置在冰浴探头超声，3min(工作50s，间歇10s)。将此淡黄色半透明液体倒入装有已称重的盐酸米托蒽醌原底烧瓶中，37℃旋转至生成均匀的蓝黑色胶体溶液。4℃保存备用。
3.粒径测定
使用生理盐水模拟体内生理环境，将盐酸米托蒽醌生物自组装注射剂(MIH-Injection)置于生理环境中，以粒径为考察指标，检测其生物条件下自组装情况。由于盐酸米托蒽醌(MIH) 比较特殊，传统的粒径检测手段如马尔文粒度仪等不能准确检测出其粒径范围，而透射电镜等检测方法需要干燥的样品来进行检测，均不能完整的模拟在体环境的真实自组装形态，故而选择原子力显微镜，对其生理条件下自组装纳米粒的形态进行表征，结果表明，MIH在生理条件下可自组装成粒径在60nm左右的纳米粒子，形态圆整，分散均匀，粒度一致。而在酸性条件下，粒径约为10nm。
4.IB粒径的测定
按照上述方法，将同等浓度的IB溶液置于生理条件下，原子力显微镜对其粒径及形貌进行测定，试验结果显示，IB在生理条件下，单个粒子的粒径为10nm到20nm，粒度均匀，形貌圆整。
5.MIH-LIP粒径的测定
使用马尔文粒度测定仪，对对照制剂MIH-LIP的粒径进行测定。试验结果显示，MIH-LIP的粒径为140nm左右。
6.pH值考察
6.1.pH值对药物稳定性的影响
以盐酸米托蒽醌作为模型药物，分别调节的注射液的pH值为1.5，2.0，2.5，3.5，4.5，5.5，6.0，6.5，7.0，在调节pH值1小时后利用0.22μm的滤膜过滤后测定药物的含量，当pH值在1.5和6.0之间时，药物含量在98.45±0.61％～101.72±0.20％，当pH值为6.5和7.0使，药物含量出现了一个明显的下降，分别为83.76±0.53％和75.38±0.22％，说明注射液的pH值大于6.0时，注射液中药物出现了不稳定析出的现象。
6.2.pH值对药物靶向能力的影响
以盐酸米托蒽醌作为模型药物，分别调节的注射液的pH值为1.5，2.0，2.5，3.5，4.5，5.5，6.0，选择间质注射的给药方式，于昆明种小鼠脚掌皮下注射，在预设时间点，将小鼠脱颈椎致死，摘取小鼠一、二、三级***，观察其染色情况。当pH值小于2.0时，仅有一级***能够染色。当pH值在2.0～6.0之间时，三级***能染色，但是pH值为2.0，2.5和6.0时，第三级***蓝色较浅，在pH值3.5～5.5之间时，三级***均能染色，且为深蓝色。
7.渗透压考察
7.1.渗透压对注射部位刺激性的影响
以盐酸米托蒽醌作为模型药物，分别调节的注射液的渗透压值分别为112，196，285，307，1503，2317，3011mmol/Kg，选择间质注射的给药方式，于昆明种小鼠脚掌皮下注射，在预设时间点，将小鼠脱颈椎致死，摘取小鼠给药部位进行H&E染色，评价局部刺激性，当注射液 的渗透压是3011mmol/Kg，出现红肿现象，H&E染色结果显示具有局部的刺激性，其他渗透压值没有表现出明显的局部刺激性。
7.2.渗透压对药物靶向能力的影响
以盐酸米托蒽醌作为模型药物，分别调节的注射液的渗透压值分别为112，196，285，307，1503，2317，3011mmol/Kg，选择间质注射的给药方式，于昆明种小鼠脚掌皮下注射，在预设时间点，将小鼠脱颈椎致死，摘取一、二、三级***，观察其染色情况。当渗透压值小于285mmol/Kg时，仅有一级***能够染色。当渗透压值在285～2317mmol/Kg之间时，三级***都能够染色，但是渗透压值为285和2317mmol/Kg时，第三级***蓝色较浅，在pH值307～1503mmol/Kg之间时，三级***均能染色，且为深蓝色。
8.间质给药后的淋巴靶向能力的考察
对这一新型生物体内自组装纳米药物递送体系的淋巴靶向能力进行考察，选择间质注射的给药方式，于昆明种小鼠脚掌皮下注射盐酸米托蒽醌生物自组装注射液(MIH-Injection)20μL。在预设时间点，将小鼠脱颈椎致死，摘取小鼠一、二、三级***，观察其染色情况，并用HPLC对***中MIH的含量进行测定。试验结果表明，MIH-Injection经间质给药后，由药时曲线下面积(AUC)和最大峰浓度(Cmax)可知，对一级***(PLNS)、二级***(ILNS)和三级***(RLNS)靶向效果良好，以血液中药物含量做参考(血液中未检测到药物存在)，靶向效率可达到100％，即通过间质给药自组装后的粒径能满足全部进入淋巴***而不进入血液循环。
表1.MIH-Injection皮下注射后在***的药动学参数
Figure PCTCN2017101801-appb-000014
注释：PLNs：popliteal lymph nodes腘***
ILNs：iliac lymph nodes髂***
RLNs：renal lymph nodes肾***
AUC(药时曲线下面积)
Cmax(最大峰浓度)
同时，分别以IB和MIH-LIP作为对照制剂，探索了不同粒径因素对给药***淋巴靶向能力的影响。选择间质注射IB(20nm)、MIH-Injection(60nm)和MIH-LIP(130nm)，注射两个小时后，试验结果如下表2。说明盐酸米托蒽醌生物自组装纳米晶赋予了盐酸米托蒽醌药物分子一定的粒径，使其具备了优于脂质体的淋巴靶向能力，而优于普通注射液的淋巴靶向能力。
表2.具有不同粒径的制剂的淋巴靶向能力
Figure PCTCN2017101801-appb-000015
注释：PLNs：popliteal lymph nodes腘***
ILNs：iliac lymph nodes髂***
RLNs：renal lymph nodes肾***
9.淋巴靶向速度考察
昆明种小鼠9只，随机分成三组：MIH-Injection组、MIH-LIP组、IB组。每组3只，分别于小鼠脚掌皮下给药。给药后10min将小鼠脱颈椎致死，解剖取出一、二、三级***，观察染色效果。试验结果显示，MIH-Injection的三级***染色程度均深于IB组和MIH-LIP组，效果均良好，***示踪效果良好，MIH-Injection与MIH-LIP比较更具有***示踪优势，探讨其原因可能是溶液的流动性优于脂质体的流动性导致。
10.转移***治疗效果的考察
10.1.局部间质给药对淋巴转移瘤的抑制效果考察
对局部给药方案下，不同疗程一、二级***大小进行比较，分别考察***的质量和体积两个指标。结果见表3，分析该组数据可得出结论，MIH-Injection对淋巴转移癌的抑制效果明显，各级淋巴转移瘤体积和重量出现明显的缩小，并且其疗效与化疗周期有关。MIH-Injection局部给药对转移的二级淋巴瘤的抑制作用与对转移的一级淋巴瘤的抑制作用差异不显著(p>0.05)。
表.3 局部注射组不同疗程***大小的比较
Figure PCTCN2017101801-appb-000016
注：
PhaseⅠ：化疗第一周期
PhaseⅡ：化疗第二周期
PhaseⅢ：化疗第三周期
PhaseⅣ：化疗第四周期
MPLNs：mestastatic popliteal lymph nodes腘***
MILNs：mestastatic iliac lymph nodes髂***
V：体积
m：质量
10.2.局部给药方案与静脉给药方案对淋巴转移瘤的抑制效果的比较
设计不同的给药方案，通过比较局部注射与静脉注射MIH-Injection在不同的给药周期对淋巴瘤的治疗效果。以下为局部给药组和静脉给药组实验数据列表，由结果可知局部给药的治疗方式比较全身静注而言，对淋巴瘤的抑制作用程度大，起效快。
表4.局部注射与静注组不同疗程***大小的比较
Figure PCTCN2017101801-appb-000017
注：
PhaseⅠ：化疗第一周期
PhaseⅡ：化疗第二周期
PhaseⅢ：化疗第三周期
PhaseⅣ：化疗第四周期
MPLNs：mestastatic popliteal lymph nodes腘***
MILNs：mestastatic iliac lymph nodes髂***
V：体积
m：质量
以上所述，仅为本申请较佳的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本申请的保护范围之内。

Claims (21)

  1. 一种具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，其组分按照质量体积比(g/mL)含有以下组分：米托蒽醌或米托蒽醌盐类：0.05％-5％，渗透压调节剂：0.1-10％，其余为溶剂。
  2. 根据权利要求1所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，还含有缓冲剂：0.001-0.1％、抗氧剂：0.01-0.1％、吸附剂：0.05-1％，填充剂：0-20％。
  3. 根据权利要求1-2之一所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，注射剂中米托蒽醌或米托蒽醌盐类浓度范围为0.1％到1％。
  4. 根据权利要求1-2之一所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，pH范围为2.0到6.0。
  5. 根据权利要求4所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，pH范围为3.5到5.5。
  6. 根据权利要求1-2之一所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，渗透压范围为285到2317mmol/Kg。
  7. 根据权利要求4所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，渗透压范围为307到1503mmol/Kg。
  8. 根据权利要求1-5之一所述的生物自组装纳米晶注射剂，其特征在于，米托蒽醌盐类采用盐酸米托蒽醌、草酸米托蒽醌、硫酸米托蒽醌、磷酸米托蒽醌、醋酸米托蒽醌、枸橼酸米托蒽醌中的一种或几种。
  9. 根据权利要求1-6之一所述的生物自组装纳米晶注射剂，其特征在于，生理条件下粒径范围为1-1000nm。
  10. 根据权利要求7所述的生物自组装纳米晶注射剂，其特征在于，生理条件下粒径范围为10-100nm。
  11. 根据权利要求8所述的生物自组装纳米晶注射剂，其特征在于，生理条件下粒径范围为30-60nm。
  12. 根据权利要求1-9之一所述的生物自组装纳米晶注射剂，其特征在于，注射剂类别分为小水针或粉针剂，小水针中填充剂为0，粉针剂中填充剂为大于0小于等于20％。
  13. 根据权利要求1-10之一所述的具有淋巴靶向功能的生物自组装纳米晶注射剂，其特征在于，
    所述渗透压调节剂采用氯化钠、葡萄糖、山梨醇、甘露醇、甘油、PEG、磷酸盐、枸橼酸盐中的一种或者几种；
    所述缓冲剂采用醋酸、醋酸钠、枸橼酸、枸橼酸钠中的一种或者几种；
    所述抗氧剂采用亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、依地酸二钠中的一种或者几种；
    所述填充剂采用单糖类的葡萄糖、果糖、半乳糖、核糖或脱氧核糖、或者采用二糖类的蔗糖、海藻糖、麦芽糖、乳糖、或者采用聚合糖类的甘露醇、山梨醇、乳糖醇、木糖醇、麦芽糖醇、赤藓糖醇中的一种或者几种；
    所述吸附剂为活性炭。
  14. 根据权利要求1-11之一所述的生物自组装纳米晶注射剂，其特征在于，实现淋巴靶向功能后，***会产生肉眼可见的蓝色。
  15. 根据权利要求1-12之一所述的生物自组装纳米晶注射剂，其特征在于，实现淋巴靶向功能后，肿瘤转移***缩小或恢复正常大小。
  16. 根据权利要求1-13之一所述的生物自组装纳米晶注射剂，其特征在于，其中给药注射剂为溶液型，米托蒽醌或其盐类以完全溶解的分子形式存在。
  17. 根据权利要求1-14之一所述的生物自组装纳米晶注射剂，其特征在于，溶液型注射剂间质给药后，在注射部位自组装成纳米晶体。
  18. 根据权利要求1-15之一所述的生物自组装纳米晶注射剂，其特征在于，实现淋巴靶向功能给药方式为间质注射给药。
  19. 根据权利要求16所述的生物自组装纳米晶注射剂，其特征在于，包括皮下间质注射、肌肉间质注射、肌肉间质注射、粘膜下间质注射、腹腔间质注射、组织内间质注射、组织周间质注射，或者采用上述几种给药方式的组合。
  20. 一种权利要1-17之一所述的生物自组装纳米晶注射剂的制备方法，其特征在于，所述注射剂为小水针类生物自组装纳米晶注射剂，包含如下制备流程：称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入米托蒽醌或米托蒽醌盐类，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟，浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟，药液经两支串联的除菌过滤器过滤，过滤器孔径为0.22μm，无菌分装封口。
  21. 根据权利要求1-17之一所述的生物自组装纳米晶注射剂的制备方法，其特征在于，所 述注射剂为粉针类生物自组装纳米晶注射剂，包含如下制备流程：称取适量的原辅料，向浓配罐中加入处方量70％注射用水，加入处方量的辅料，搅拌约15分钟使溶解，再加入米托蒽醌或米托蒽醌盐类，搅拌约20分钟使溶解，投入活性炭，于40-60℃搅拌吸附20分钟，浓配液经3μm不锈钢折叠滤芯过滤后转至稀释罐中，补加注射用水至配液总量，搅拌10分钟，药液经两支串联的除菌过滤器，孔径为0.22μm，过滤，加入甘露醇溶解后，经冷冻干燥去除水分，得到干燥的生物自组装纳米晶注射用冻干粉，使用前水化振摇后可间质注射给药，水化介质选择注射用水、生理盐水、葡萄糖注射液中的一种或者几种。
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