WO2021110073A1 - 含有榄香烯的药物组合物及其制备方法和用途 - Google Patents

含有榄香烯的药物组合物及其制备方法和用途 Download PDF

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WO2021110073A1
WO2021110073A1 PCT/CN2020/133470 CN2020133470W WO2021110073A1 WO 2021110073 A1 WO2021110073 A1 WO 2021110073A1 CN 2020133470 W CN2020133470 W CN 2020133470W WO 2021110073 A1 WO2021110073 A1 WO 2021110073A1
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Prior art keywords
cancer
pharmaceutical composition
elemene
oil
composition according
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PCT/CN2020/133470
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English (en)
French (fr)
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龚涛
柯潇
郑强
叶亮
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成都康弘药业集团股份有限公司
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Priority to JP2022533393A priority Critical patent/JP2023504821A/ja
Priority to US17/781,611 priority patent/US20230355544A1/en
Priority to EP20895953.6A priority patent/EP4070786A4/en
Publication of WO2021110073A1 publication Critical patent/WO2021110073A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • 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
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin

Definitions

  • the present invention relates to the field of pharmaceutical preparations, in particular to a pharmaceutical composition containing elemene and a preparation method and application thereof.
  • Elemene is a volatile oily compound extracted from plants such as Curcuma Wenyujin (Curcuma Wenyujin Y.H. Chen et C. ling) and Citronella (Cymbopoqon citratus ((DC.)) Stapt). The compound presents a light yellow or yellow clear liquid with a pungent fennel smell. Elemene currently used clinically is a mixture containing ⁇ -elemene, ⁇ -elemene, ⁇ -elemene and ⁇ -elemene. It is widely used in malignant pleural effusion, lung cancer, gastrointestinal tumors and Other superficial tumors.
  • Elemene is almost insoluble in water and highly fat-soluble, especially in organic solvents such as petroleum ether and diethyl ether. Its molecular formula is C 15 H 24 and consists only of hydrocarbons. Due to its unique chemical structure and physical and chemical properties, there are great difficulties and obstacles when preparing pharmaceutical preparations. In order to increase the solubility of elemene, castor oil polyhydroxyoxygen is added to the market prescription in the process of making it into injection. Excipients such as esters cause serious hemolysis and irritation.
  • This application provides a pharmaceutical composition comprising elemene, injection oil and a protein carrier, which can effectively reduce one or more side effects of the pharmaceutical composition administered to the human body.
  • the protein carrier used in the pharmaceutical composition includes a protein, and any suitable protein can be used.
  • suitable proteins include but are not limited to albumin, immunoglobulins, including but not limited to IgA, lipoprotein, apolipoprotein B, ⁇ -acid glycoprotein, ⁇ -2-macroglobulin, thyroglobulin, transferrin Protein, fibronectin, factor VII, factor VIII, factor IX, factor X and the like.
  • the protein carrier can be of natural origin or synthetically prepared.
  • the protein carrier is a non-blood protein, such as casein, alpha-lactalbumin, and beta-lactoglobulin.
  • the protein carrier includes albumin, such as human serum albumin (HSA), bovine serum albumin, and the like.
  • Human serum albumin is a highly soluble globulin, Mr65K, composed of 585 amino acids.
  • HSA is the most abundant protein in plasma and constitutes 70-80% of the colloidal osmotic pressure of human plasma.
  • the "injectable oil” used in the pharmaceutical composition includes, but is not limited to, ethyl oleate, benzyl benzoate, medium chain triglycerides (MCT) and vegetable oils, of which vegetable oils One or more selected from soybean oil, safflower oil, cottonseed oil, corn oil, sunflower oil, peanut oil, and olive oil. In one or more embodiments of the present application, the injectable oil is soybean oil.
  • the pharmaceutical composition may be made into particles, wherein the particle size of the particles is less than 180 nm, for example, about 70-170 nm, preferably about 70-150 nm.
  • the composition includes nanoparticles of any shape (for example, spherical or non-spherical shape), and in some embodiments, the average or median diameter of the particles is no greater than about 180 nm. In some embodiments, the average or median diameter of the particles is about 50 to about 180 nm. In some embodiments, the average or median diameter of the particles is about 60 to about 180 nm. In some embodiments, the average or median diameter of the particles is about 70 to about 170 nm. In some embodiments, the average or median diameter of the particles is from about 70 to about -170 nm. In some embodiments, the average or median diameter of the particles is between about 70 to about 150 nm.
  • any shape for example, spherical or non-spherical shape
  • the average or median diameter of the particles is no greater than about 180 nm. In some embodiments, the average or median diameter of the particles is about 50 to about 180 nm. In some embodiments, the average or median diameter of the particles is about 60 to about 180
  • the average or median diameter of the particles is about 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, or 150 nm.
  • the particles are sterile and filterable.
  • the elemene in the pharmaceutical composition is selected from one or more of ⁇ -elemene, ⁇ -elemene, ⁇ -elemene and ⁇ -elemene.
  • the elemene in the pharmaceutical composition is ⁇ -elemene.
  • the ratio of the albumin carrier, elemene, and injection oil in the pharmaceutical composition is the weight ratio of the feed.
  • the weight ratio of the protein carrier to elemene in the pharmaceutical composition is 0.520; for example, 0.5-10.
  • the weight ratio of the injection oil to elemene in the pharmaceutical composition is 0.3510; for example, 0.5-10.
  • the pharmaceutical composition further contains a lyoprotectant.
  • the lyophilization protective agent is selected from one or more of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, and dextran, preferably trehalose or sucrose.
  • the ratio of the weight of the lyoprotectant to the volume of the solution of the pharmaceutical composition is 3:100-20:100g/mL, preferably 5:100-10:100g/mL mL.
  • the pharmaceutical composition further contains one or more of isotonic regulator, antioxidant, preservative, and pH regulator.
  • the isotonic adjusting agent is one or more of glycerol, sorbitol, mannitol or glucose;
  • the pH adjusting agent is one or more of sodium hydroxide, sodium citrate, citric acid, phosphoric acid, acetic acid or hydrochloric acid.
  • the preservative is one or more of alkyl hydroxyphenyl esters, benzoic acid, sodium benzoate, sorbic acid, chloretidine acetate, and benzalkonium bromide;
  • the antioxidant is sodium sulfite, sodium bisulfite , Sodium metabisulfite, sodium thiosulfate, ascorbic acid, tert-butyl p-hydroxyanisole, 2,6-di-tert-butylated hydroxytoluene, one or more of vitamin E.
  • the pharmaceutical composition can be made into particles, wherein the particle size of the particles is less than 180 nm; for example, about 70-150 nm.
  • the weight ratio of the soybean oil to elemene in the pharmaceutical composition is about 0.35-3, and the weight ratio of the human albumin to elemene is about
  • the weight ratio of the soybean oil to elemene is about 0.5-3; the weight ratio of the human albumin to elemene is about 0.5-2.5; or the weight ratio of the pharmaceutical composition
  • the weight ratio of soybean oil to elemene is about 0.35-1.5, and the weight ratio of human albumin to elemene is about 1.5-10.
  • the weight ratio of soybean oil to elemene is about 0.5, and the weight ratio of human serum albumin to elemene is about 1.5.
  • the weight ratio of soybean oil to elemene is about 0.75, and the weight ratio of human serum albumin to elemene is about 5.
  • the weight ratio of soybean oil to elemene is about 1.5, and the weight ratio of human serum albumin to elemene is about 3-5.
  • a pharmaceutical composition which contains an organic solvent during the preparation process.
  • the organic solvent is selected from chloroform, dichloromethane, tert-butanol, isopropanol, ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, One or more of methylpyrrolidone.
  • the fat-soluble organic solvent is a mixed solvent of dichloromethane and ethanol.
  • the volume ratio of methylene chloride to ethanol is 1:1-8, preferably 1:4.
  • the pharmaceutical composition can be prepared into particles, the particle size of which is about 70-150 nm.
  • the weight ratio of the soybean oil and elemene in the pharmaceutical composition is about 2.4-10, and the weight ratio of the human albumin to elemene is about 0.5-10; preferably, the weight ratio of the pharmaceutical composition is about 0.5-10.
  • the weight ratio of soybean oil to elemene is about 2.4, and the weight ratio of human albumin to elemene is about 3.
  • One or more embodiments of the present application provide a method for preparing the pharmaceutical composition of the present application, which includes the following steps:
  • step (3) Homogenizing the emulsion described in step (2) under high pressure, and optionally evaporating the emulsion under reduced pressure to remove the organic solvent to obtain a nanoparticle solution.
  • One or more embodiments of the present application provide a method for preparing the pharmaceutical composition of the present application, and the process includes the following steps:
  • step (3) The emulsion described in step (3) is homogenized under high pressure to obtain an albumin nanoparticle solution.
  • step (2) in step (2), a shear or ultrasonic method is used for mixing, and in step (3), a high-pressure homogenization or micro-jet homogenization method is used for homogenization.
  • step (3) may also include flushing the homogenizer pipeline with an appropriate amount of trehalose aqueous solution, replacing the remaining homogeneous solution in the homogenizer, and then using an appropriate amount of superfluous solution. Rinse the homogenizer pipeline with pure water to replace the residual trehalose aqueous solution, combine it with the homogenizer, and filter it after mixing.
  • the shearing can be done with a mixer.
  • the rotation speed of shearing with the mixer is 5000 rpm-10000 rpm.
  • the shear time of the mixer is 1 min-20 min, preferably, for example, 2 min-10 min or, more preferably, 1 min-10 min.
  • the shearing with a mixer is preferably multiple shearing, preferably, for example, more than 2 times.
  • the shearing is performed at 5000 rpm for 1 min for the first time and shearing for 5 minutes at 10,000 rpm for the second time.
  • the high-pressure homogeneous pressure is 200-1600 bar, preferably 250-1550 bar, for example.
  • the high-pressure homogenization time is 1-10 min, for example, preferably 2-10 min, such as further preferably 5-8 min.
  • the high-pressure homogenization is preferably performed multiple times, preferably, for example, more than 2 times.
  • the pressure of the homogenization of the microjet is 20000 psi.
  • the time for the homogenization of the microjet is 30 minutes.
  • One or more embodiments of the present application also provide a preparation method of the pharmaceutical composition of the present application, and an organic solvent is used in the preparation process.
  • the preparation method of the pharmaceutical composition of the present application includes the following steps:
  • step (4) The emulsion described in step (4) is evaporated under reduced pressure to remove the organic solvent to obtain an albumin nanoparticle solution.
  • the organic solvent in step (1) is selected from chloroform, dichloromethane, tert-butanol, isopropanol, ethyl acetate, ethanol, tetrahydrofuran, dioxane, One or more of acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide, and methyl pyrrolidone; for example, a mixed solvent of dichloromethane and ethanol, the volume ratio of ethanol to dichloromethane in the mixed solvent is 1:1-8, such as 1:4.
  • a shearing or ultrasonic method can be used for mixing, and the step (4) can be homogenized by a high pressure or microjet method.
  • the shearing can be done with a mixer.
  • the rotation speed of shearing with the mixer is 5000 rpm-10000 rpm.
  • the shear time of the mixer is 1 min-20 min, for example, 2 min-10 min.
  • the shearing with a mixer is preferably multiple shearing, for example, more than 2 times.
  • the shearing is performed at 5000 rpm for 1 min for the first time and shearing for 5 minutes at 10,000 rpm for the second time.
  • the high-pressure homogeneous pressure is 200-1600 bar, such as 250-1550 bar.
  • the time for high-pressure homogenization is 1-10 min, such as 2-10 min, such as 5-8 min more preferably.
  • the high-pressure homogenization is preferably performed multiple times, for example, more than 2 times.
  • the pressure of the homogenization of the microjet is 20000 psi.
  • the time for the homogenization of the microjet is 30 minutes.
  • the preparation method of the pharmaceutical composition of the present application further includes the step of freeze-drying the obtained albumin nanoparticle solution.
  • One or more embodiments of the present application provide the use of the pharmaceutical composition of the present application in drugs for preventing or treating cancer.
  • the cancers involved in the present application include adrenocortical carcinoma, unexplained medullary metaplasia, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, and basal cell carcinoma , Cholangiocarcinoma, bladder cancer, bone cancer, glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, neuroectodermal tumor , Visual pathway and hypothalamic glioma and malignant glioma, bronchial adenoma, carcinoid tumor, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disease, endometrial Cancer, ependymoma, necessarily tumor family, eye cancer, gallbladder cancer, gastric cancer
  • the cancers involved in this application include lung cancer, liver cancer, esophageal cancer, nasopharyngeal cancer, brain tumors, bone metastases, gastric cancer, intestinal cancer, uterine cancer, cervical cancer, and reproductive cancer.
  • brain tumors include gliomas, brainstem gliomas, astrocytomas of the cerebellum or brain, malignant gliomas, ependymomas, oligodendrocytes Glioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, visual pathway and hypothalamic glioma or malignant glioma; the astrocytoma of the cerebellum or brain is fibrous Like cell astrocytoma or diffuse astrocytoma or anaplastic (malignant) astrocytoma.
  • the pharmaceutical composition of the present application can be used in combination with radiotherapy and chemotherapy for malignant tumors such as lung cancer, liver cancer, esophageal cancer, nasopharyngeal cancer, brain tumors, and bone metastases to enhance the therapeutic effect It reduces the side effects of radiotherapy and chemotherapy, and can also be used for intervention, intracavitary chemotherapy and the treatment of cancerous pleural and ascites.
  • malignant tumors such as lung cancer, liver cancer, esophageal cancer, nasopharyngeal cancer, brain tumors, and bone metastases.
  • the compound of the present application can be used in combination with other drugs, the other drugs include anticancer drugs, and the anticancer drugs include temozolomide, paclitaxel, docetaxel, and taxane.
  • the other drugs include anticancer drugs, and the anticancer drugs include temozolomide, paclitaxel, docetaxel, and taxane.
  • Gemcitabine anti-VEGF drugs, PD-1 antibody drugs; wherein the anti-VEGF drugs include AVASTIN, ranibizumab, aflibercept or conbercept; wherein the PD-1 antibody drugs include nivolumab, Pabo Livizumab, Teriplizumab, Sintilizumab, Cimiprizumab, Atezolizumab, Avitizumab, or Duvalizumab.
  • One or more embodiments of the present application provide a method for treating human diseases, including administering a therapeutically effective amount of the pharmaceutical composition of the present application.
  • the pharmaceutical composition is administered parenterally, by inhalation, intraperitoneal, intravesical, intramuscular, intravenous, intratracheal, subcutaneous, intraocular, intrathecal, transdermal, rectal or vaginal administration .
  • the pharmaceutical composition of the present application can be made into a pharmaceutical preparation suitable for intravenous administration, a pharmaceutical preparation for parenteral administration, a pharmaceutical preparation for gastrointestinal administration, and aerosol.
  • a pharmaceutical preparation suitable for intravenous administration a pharmaceutical preparation for parenteral administration
  • a pharmaceutical preparation for gastrointestinal administration a pharmaceutical preparation for gastrointestinal administration
  • aerosol a pharmaceutical preparation suitable for aerosol.
  • Pharmaceutical preparations for vaginal administration or other suitable pharmaceutical preparations which are solid preparations, liquid preparations or gas preparations.
  • the pharmaceutical composition of the present application can be used as a liquid preparation for injection, which comprises elemene and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier includes an albumin carrier and Injection oil, wherein the composition can be made into particles, wherein the particle size of the particles is 70-150nm, and the weight ratio of albumin carrier to elemene in the composition is about It is 0.5-20, and the weight ratio of the injection oil to elemene is about 0.35-10.
  • the liquid formulation in the present application may be a stable aqueous suspension reconstituted from sterile lyophilized powder.
  • One or more embodiments of the present application provide a sealed container containing the pharmaceutical composition of the present application, and the sealed container may be a unit-dose container or a multi-dose container.
  • the sealed container may be a pre-filled syringe.
  • the pharmaceutical composition is a liquid composition or a dry composition.
  • the pharmaceutical composition is freeze-dried and sterile.
  • the nanoformulations or nanoparticles described herein may be in the form of a dry formulation (such as a lyophilized composition) or suspended in a biocompatible medium.
  • Suitable biocompatible media include, but are not limited to, water, aqueous buffered media, saline, buffered saline, optionally buffered amino acid solutions, optionally buffered protein solutions, optionally buffered sugar solutions, optionally buffered vitamin solutions, Optional buffered synthetic polymer solution, lipid-containing emulsion, etc.
  • the "individual” as used herein is a mammal, including but not limited to primates, humans, cows, horses, felines, canines, or rodents.
  • treatment is a method of obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, any one or more of the following: reducing one or more symptoms produced by the disease, reducing the severity of the disease, stabilizing the disease (for example, preventing or delaying the deterioration of the disease) ), prevent or delay the spread of the disease (such as metastasis), prevent or delay the occurrence or recurrence of the disease, delay or slow the progression of the disease, improve the disease state, provide relief (whether partial or full) of the disease, reduce one or more The dosage of other drugs necessary to treat the disease, delay the progression of the disease, increase the quality of life and/or prolong survival.
  • the composition reduces one or more cancer-related symptoms compared to corresponding symptoms in the same subject prior to treatment or compared to corresponding symptoms in other subjects who did not receive the composition.
  • the severity of each symptom is at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • Treatment also includes the reduction of the pathological consequences of cancer. The methods of the invention take into account any one or more of these therapeutic aspects.
  • pharmaceutically acceptable or “pharmacologically compatible” means a substance that is not biological or otherwise undesired, for example, without causing any significant undesired biological effects or in combination with the composition.
  • the substance can be incorporated into the pharmaceutical composition administered to the patient if any other ingredients contained in it do not interact in a harmful way.
  • the pharmaceutically acceptable carrier or excipient preferably meets the standards required by toxicology and production testing.
  • Preparations for oral administration may be (a) liquid solutions, such as effective amounts of active ingredients dissolved in diluents such as water, saline or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of solids or particles Active ingredient in the form of (c) a suspension in a suitable liquid, and (d) a suitable emulsion.
  • liquid solutions such as effective amounts of active ingredients dissolved in diluents such as water, saline or orange juice
  • capsules, sachets or tablets each containing a predetermined amount of solids or particles Active ingredient in the form of (c) a suspension in a suitable liquid, and (d) a suitable emulsion.
  • the tablet form may include lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearin One or more of acid and other excipients, coloring agents, diluents, buffers, wetting agents, preservatives, flavoring agents and pharmacologically compatible excipients.
  • the lozenge form may contain the active ingredients in flavors, usually sucrose and gum arabic or tragacanth, and lozenges containing the active ingredients in an inert base, such as gelatin and glycerin, or sucrose and gum arabic, emulsions, gels, etc. In addition to the active ingredient, it also contains excipients known in the art.
  • Preparations for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, and may include suspensions.
  • the preparation can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored under freeze-drying (lyophilization) conditions immediately before use, requiring only the addition of sterile liquid excipients, such as Water for injection.
  • Extemporaneous injection solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules and tablets.
  • the aerosol contains the pharmaceutical composition of the present invention, which includes aqueous and non-aqueous, isotonic sterile solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes, and may contain suspending agents, enhancers
  • aqueous and non-aqueous sterile suspensions of solvents, thickeners, stabilizers and preservatives, alone or in combination with other suitable components, can be made into aerosol preparations for administration by inhalation.
  • These aerosol formulations can be placed in pressurized acceptable propellants, such as difluoromethane, propane, nitrogen, and the like. They can also be formulated as drugs in non-pressure formulations, for example in a nebulizer or nebulizer.
  • suppositories can be prepared by using various bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the preparations for vaginal administration can be presented as vaginal suppositories, tampons, creams, gels, pastes, foams or spray formulations, which, in addition to the active ingredients, also contain suitable carriers known in the art.
  • Figure 1 shows the distribution of hydrated particle size of ⁇ -elemene albumin nanoparticles
  • Figure 2 shows the potential diagram of ⁇ -elemene albumin nanoparticles
  • Figure 3 shows a transmission electron microscope image of ⁇ -elemene albumin nanoparticles
  • Figure 4 shows the investigation of particle size stability of different freeze-dried samples after reconstitution
  • Figure 5 shows the investigation of the stability of the content of different freeze-dried samples
  • Figure 6 shows the survival period of each group of tumor-bearing mice
  • FIG. 7 shows the results of the hemolytic test
  • Figure 8 shows the tumor growth curve of the U87R1 mouse orthotopic tumor model
  • Figure 9 shows the survival curve of the U87R1 mouse orthotopic tumor model.
  • the soybean oil involved in the present invention is derived from soybean oil (SIO, injection grade, Lot19C4520/Lot18C4816); the human serum albumin (HSA) involved in the present invention is derived from human serum albumin (20%, Baxter, LotA1U133A) or (20%, Grifols) , LotALAFD03042).
  • the commonly used detection methods of the present invention include particle size and particle size distribution, content determination, hemolysis experiment, etc., and the details are as follows:
  • the particle size and particle size distribution were measured with a particle size analyzer (MALVERN ZETASIZER NANO ZS90). Take 25 ⁇ L of sample, add 1ml of ultrapure water to dilute and test, detection angle: 90°; dispersion medium: water; temperature 25°C.
  • MALVERN ZETASIZER NANO ZS90 a particle size analyzer
  • the particle size is the Z-average value (average particle size of the sample) measured by the particle size analyzer, and the particle size distribution is the PDI value measured by the particle size analyzer.
  • test solution Take 1 bottle of this product, remove the cap, accurately weigh it (m 1 ), add an appropriate amount of water (make the ratio of API to water 20mg:1ml), accurately weigh it (m 2 ), and shake
  • To reconstitute accurately weigh about 500mg of the reconstituted solution (approximately equivalent to API 10mg) (m 3 ), put it in a 50ml measuring flask, first add about 1.5ml of water, and then slowly add acetonitrile to 80% of the full scale, ultrasonic Leave it to cool for 10 minutes, add acetonitrile to dilute to the mark, shake well, and filter with a 0.45 ⁇ m nylon filter. After using the reconstituted solution, clean the vial with water and wipe dry, and accurately weigh the empty vial (m 4 ).
  • Preparation of blood cell suspension Take a few milliliters of blood from rabbits (male New Zealand rabbits, reared for 1 week in laboratory, weighing 2.5kg-3kg, blood taken from the heart), put them into a beaker, stir the blood with a glass rod or bamboo stick, and remove Fibrinogen makes defibrinated blood. Add about 10 times the amount of 0.9% sodium chloride solution, mix well, centrifuge at 1500 rpm/min for 15 minutes, discard the supernatant, and then wash the precipitated red blood cells with 0.9% sodium chloride solution 2-3 times as described above to reach the supernatant It does not show red. The obtained red blood cells were prepared into a 2% blood cell suspension with 0.9% sodium chloride solution for use.
  • Sample preparation According to the content of the sample, take an appropriate amount and dilute it with physiological saline to obtain a 1 mg/mL sample (actual content).
  • Drug group Add 2.5 mL of 2% blood cell suspension, 2.2 mL of normal saline, and 0.3 mL of drugs to the test tube.
  • Positive group Add 2.5 mL of 2% blood cell suspension and 2.5 mL of distilled water to the test tube.
  • Negative group add 2.5 mL of 2% blood cell suspension and 2.5 mL of normal saline to the test tube.
  • OD value determination draw 100uL of the upper layer into a 96-well plate, and measure the OD value at a wavelength of 540nm.
  • Hemolysis value calculation method (sample OD value-negative OD value)/(positive OD value-negative OD value).
  • the preparation of ⁇ -elemene albumin nanoparticles includes the following steps: 1) Weigh 200 mg of elemene and 600 mg of soybean oil, and dissolve them in 2 mL of dichloromethane to obtain an oil phase. 2) Take 650 mg of human serum albumin and add it to water for injection to dissolve to obtain a 3.6% (g/ml) human serum albumin aqueous solution by mass volume percentage to obtain the water phase. 3) Under the action of water bath ultrasound, the oil phase is added dropwise to the water phase, and the probe is ultrasonicated for 8 minutes (250W) to make colostrum.
  • the freeze-drying parameters are: pre-freezing at -45°C for 5 hours; turn on the vacuum pump to keep the vacuum at about 10Pa, program the temperature to increase, dry at -40°C for 4 hours, dry at -30°C for 10 hours, and then in turn at -20°C, -10°C, 0°C, Dry at 5°C and 10°C for 4 hours respectively.
  • the freeze-dried samples prepared above were reconstituted with pure water, and the following methods were used to investigate the particle size, potential and microscopic morphology of the nanoparticles.
  • the Malvern particle size analyzer Before the measurement, preheat the Malvern particle size analyzer for 30 minutes, take 50 ⁇ L of the elemene albumin nanoparticle aqueous solution, dilute to 1 mL with water, shake slightly (to avoid bubbles), and place it in a Malvern Nano-ZS90 particle size dish or a potential dish Measure particle size and Zeta potential.
  • the measurement temperature is 25°C
  • the equilibration time is 120s, and the rest of the items retain the default values of the instrument
  • the Malvern V2.2 workstation is used for data acquisition and processing.
  • the obtained elemene albumin nanoparticle size diagram and zeta potential are shown in Figure 1 and Figure 2. It can be seen from Figure 1 and Figure 2 that the particle size of elemene albumin nanoparticles is about 110 nm, the PDI is 0.127, and the potential is about -18 mV.
  • the pipette sucks the elemene albumin nanoparticle aqueous solution and drops it on the copper net. After it evaporates naturally, the 1% phosphotungstic acid solution is dropped on the copper net by the same method, and it evaporates naturally. Take a few drops of pure water and carefully rinse the surface of the copper net. After being evaporated, the morphology of the nanoparticles was observed in the transmission electron microscope as shown in Figure 3.
  • the nanoparticles are spherical, regular in shape, relatively uniform in particle size, and the particle diameter is about 100nm.
  • Elemene albumin nanoparticles were prepared according to the method of Test Example 1, wherein the injection oil was prepared with the same amount of soybean oil, corn oil, tea oil, and medium chain triglyceride (MCT), and different injection oils were used as raw materials.
  • MCT medium chain triglyceride
  • the particle size of the prepared nanoparticles was measured by a laser particle size analyzer. After lyophilization, the drug content of each group of preparations before and after lyophilization was measured, and the drug loss rate during the lyophilization process was calculated.
  • the preparation of soybean oil-free elemene albumin nanoparticles includes the following steps: 1) Weigh 200 mg of elemene and dissolve it in 2 mL of dichloromethane to obtain an oil phase. 2) Take 650 mg of human serum albumin and add it to water for injection to dissolve to obtain a 3.6% (g/ml) human serum albumin aqueous solution by mass volume percentage to obtain the water phase. 3) Under the action of water bath ultrasound, the oil phase is added dropwise to the water phase, and the probe is ultrasonicated for 8 minutes (250W) to make colostrum. 4) Transfer the obtained colostrum to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1300 bar, and the homogenization times are 9 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered to sterilize the system to obtain an aqueous solution of elemene albumin nanoparticles.
  • the volume of water for injection is adjusted to 10 mg/ml.
  • 5) Add 3% sucrose to the obtained elemene albumin nanoparticles, and freeze-dry according to the freeze-drying parameters of Test Example 1. The drug content before and after the freeze-drying of a certain volume of the preparation was measured respectively, and the drug loss rate during the freeze-drying process was calculated.
  • Table 1 The drug loss rate of different injection oil preparation samples after freeze-drying
  • Instrument model MALVERN ZETASIZER NANO ZS90, detection angle: 90°; detection mode: automatic; detection times: 3; detection type: nanoparticles; refractive index of the sample to be tested: 1.340; dispersion medium: water; temperature 25°C ; Viscosity: 0.8872cp; Dispersion medium refractive index: 1.330; Equilibration time: 120s; Particle size dish model: DTS0012.
  • the results are shown in Figure 4 and Figure 5.
  • the particle size of the preparation without soybean oil added during the preparation process is larger than 200nm.
  • the particle size of the sample reconstituted gradually increases, and the drug content is Declining trend.
  • the particle size stability is better, and the stability of the main drug content is gradually enhanced.
  • the mass ratio of soybean oil/elemene is 1-10
  • the prepared nanoparticle freeze-dried sample does not aggregate within 30 days at room temperature
  • the particle size is 100-150nm
  • the PDI is less than 0.3
  • ⁇ -elemene is mainly The content of the medicine has little change.
  • the amount of elemene is 1 part and the amount of soybean oil is less than 1 part, the stability of the prepared nanoparticles is not good.
  • Tumor modeling Trypsinize C6 cells that are well-grown and in log phase, centrifuge, discard the upper layer of waste liquid, wash once with PBS, centrifuge to discard the upper layer of waste liquid, and resuspend in PBS to make the final C6 cell density approximately It is 1 ⁇ 106 pcs/5 ⁇ L.
  • Healthy male Kunming mice 28-30g were anesthetized by intraperitoneal injection of an appropriate amount of 4% chloral hydrate, shaved the top of the head, and smeared with 75% ethanol for disinfection. Use sterilized ophthalmic scissors to cut along the sagittal midline of the head.
  • micro-injection needle to aspirate 5 ⁇ L of the above-mentioned C6 cells with a density of 1 ⁇ 106 cells/5 ⁇ L, along the vertical direction of the small hole, insert the needle 4mm, retract the needle 1mm, leave the cell space, slowly inject the cell suspension into the brain, microneedle Stay in the small hole for 5 minutes and then take it out. Suture the wound with surgical thread, and finally smear the suture surface with a cotton swab moistened with double resistance.
  • mice On the 10th day after tumor inoculation, the tumor-bearing mice were divided into normal saline group (NS), commercially available elemene liposomes (Lip, Dalian Jingang, approval number: National Medicine Zhunzi H10960114), prepared by Test Example 1. Elemene albumin nanoparticles (NPs), 12 mice per group, with a dosage of 30 mg/kg, were given once every 2 days for a total of 4 administrations. The survival time of each mouse was recorded and the survival curve was made.
  • NS normal saline group
  • NPs Elemene albumin nanoparticles
  • the survival time test results are shown in Figure 6.
  • the results of the experiment show that C6 glioma-bearing mice given elemene albumin nanoparticles show a longer survival time.
  • the average survival time of the physiological group was 21 days
  • the average survival time of the elemene liposome group was 28 days
  • the average survival time of the elemene albumin nanoparticle group was 41 days. Therefore, the elemene albumin nanoparticles prepared in the experiment have the effect of prolonging the survival period of mice bearing C6 glioma.
  • the New Zealand rabbits male, laboratory-suitable breeding for 1 week, weight 2.5kg-3kg were divided into commercially available elemene injection (Ailineng, CSPC Yuanda Pharmaceutical Co., Ltd., batch number 17050302) and preparation of test example 1.
  • the two preparations were respectively formulated into a medicinal solution with a concentration of 6 mg/mL with glucose solution and administered by intravenous drip.
  • venous blood was drawn from the ear vein into a centrifuge tube coated with sodium heparin, centrifuged at 3500r/min for 15 minutes, and the supernatant was taken to observe the color.
  • the organic solvent is a mixed solvent of 26.67 ml of dichloromethane and 6.67 ml of absolute ethanol.
  • Measure 50ml (10g) of 20% human serum albumin add an appropriate amount of ultrapure water to dilute evenly, and dilute to 333ml to obtain a 3% (g/ml) human serum albumin aqueous solution, that is, the water phase.
  • the obtained colostrum was transferred to a microfludizer (Microfludizer, M-110-EH) for high-pressure homogenization at a homogenization pressure of 20,000 psi and homogenization for 30 minutes.
  • the organic solvent-containing emulsion is passed through a thin film evaporator (Shanghai Deda Tianyi) to remove the organic solvent.
  • the parameters are: temperature 30°C, vacuum degree 4000pa, liquid feeding speed 100rpm, scraper speed 50rpm. Filter through 0.45 ⁇ m and 0.22 ⁇ m PVDF flat filters, make the volume constant, and add the freeze-dried protectant sucrose 3% (g/ml).
  • the homogenized solution after adding the freeze-dried protective agent is freeze-dried. After the freeze-drying is completed, the freeze-dried powder is re-dissolved with sterilized ultrapure water, and the particle size is 78.34nm and the PDI is 0.207.
  • Sample 3 (elemene injection (trade name: Ailineng)): CSPC Yuanda (Dalian) Pharmaceutical Co., Ltd., batch number: 19061502; particle size detected by particle size analyzer is 14.74nm PDI: 0.035
  • Kunming mice Male, SPF grade, body weight 20-25g were used for the experiment, and the drug was administered through the tail vein: the dose was 50mg/kg; the brain tissue samples were collected at different time periods and frozen at -20°C in time. Used for detection of drug content in brain tissue. Three samples, 3 Kunming mice of each formulation at each time point, a total of 45 mice, the eyeballs were removed at 5 min, 15 min, 30 min, 1 h, 2 h after administration, and blood was collected, brain tissue was collected, and the drug content in the brain tissue was detected. GC-MS was used to quantitatively analyze ⁇ -elemene in Kunming mouse plasma and brain tissue biological samples. The half-life and brain tissue exposure in brain tissue were studied in Table 2.
  • the homogenized solution after adding trehalose was freeze-dried. After the freeze-drying was completed, the freeze-dried powder was re-dissolved with sterilized ultrapure water, and the particle size was determined to be 144.07nm with a particle size analyzer and the PDI was 0.123. The loss rate of elemene content before and after freeze-drying was 1.27%.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 250-300bar, homogenization is 2min, and 1350-1400bar is homogenization for 24min.
  • measure 150ml of the homogenized solution with a graduated cylinder add 15g of trehalose, shake up to dissolve, and filter with a 0.45 ⁇ m microporous membrane to obtain the elemene albumin nanoparticle solution.
  • the particle size measured by the particle size meter is 132.4nm, and the PDI is 0.133.
  • the homogenized solution after adding trehalose is freeze-dried.
  • the freeze-dried powder is re-dissolved in sterilized ultrapure water, and the particle size is 123.68nm and PDI 0.144 with a particle size analyzer.
  • the loss rate of elemene content after freeze-drying was 1.58%.
  • the lyophilized powder was re-dissolved in sterilized ultrapure water, and the particle size was determined to be 181.56 nm with a particle size analyzer, and the PDI was 0.251.
  • the loss rate of elemene content after lyophilization was 16.47%.
  • filter with a 0.22 ⁇ m filter membrane measure 50ml 2% HSA solution (including HSA 1g) and add it to the filtered homogeneous solution, homogenize 1 cycle at 140bar, discharge the homogeneous solution, and measure with a measuring cylinder Take 40ml, add 4g trehalose, shake up to dissolve, filter with 0.45 ⁇ m microporous membrane to obtain elemene albumin nanoparticle solution.
  • the particle size measured by the particle size meter is 93.11nm, and the PDI is 0.131.
  • the homogenized solution after adding trehalose was lyophilized, and after lyophilization was completed, the lyophilized powder was re-dissolved in sterilized ultrapure water, and the particle size was 811.70 nm and the PDI was 0.313 by a particle size analyzer.
  • the loss rate of elemene content after lyophilization was 82.64%.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 116.4 nm, and the PDI is 0.118.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 105.3 nm, and the PDI is 0.116.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 20 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle diameter was 121.3 nm, and the PDI was 0.133.
  • the obtained colostrum was transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure was 1400 bar, and the homogenization times were 20 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid medicine after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous filter membrane to obtain an aqueous solution of elemene albumin nanoparticles.
  • Add 3% sucrose for freeze-drying After freeze-drying is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 112.6nm and the PDI was 0.101.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 140.3 nm, and the PDI is 0.164.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 125.3 nm, and the PDI is 0.148.
  • the obtained colostrum was transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure was 1000 bar, and the homogenization times were 20 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 119.3 nm, and the PDI is 0.143.
  • the prepared nanoparticle solution is configured to have a drug concentration of 10 mg/ml, and an appropriate amount of freeze-dried protective agent is added.
  • the amount of the freeze-dried protective agent is 3% (g/ml) and freeze-dried. After lyophilization, it was reconstituted with water for injection and the particle size was measured. The results are shown in the table below.
  • the experimental results show that during the freeze-drying process of elemene albumin nanoparticles, adding various freeze-dried protective agents can obtain freeze-dried powders with loose structure and uniform texture, which can be reconstituted with water, and the particle size after reconstitution does not exceed 200nm.
  • sucrose is used as a freeze-dried protective agent, and the size of the nanoparticles obtained from the sample reconstitution after freeze-drying is the smallest.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1400 bar, and the homogenization times are 10 times.
  • the organic solvent is removed by rotary evaporation, the 0.22 ⁇ m microporous filter membrane is filtered and sterilized, the dilution configuration is 15mg/ml, and 3% sucrose is added, lyophilized, capped, and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection, and the particle size was determined. The measured particle size is 103.5 nm, and the PDI is 0.123.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 20 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid medicine after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous filter membrane to obtain an aqueous solution of elemene albumin nanoparticles.
  • the dilution configuration is that the drug concentration is 10mg/ml, 3% sucrose is added for lyophilization, and after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 115.2nm and the PDI was 0.102.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 20 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid medicine after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous filter membrane to obtain an aqueous solution of elemene albumin nanoparticles.
  • the dilution configuration is the drug concentration of 10mg/ml, and 3% glucose is added for freeze-drying. After the freeze-drying is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 109.6nm and the PDI was 0.113.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 20 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid medicine after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous filter membrane to obtain an aqueous solution of elemene albumin nanoparticles.
  • Add 3% trehalose for lyophilization press the lid after lyophilization, and store at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 112.6nm and the PDI was 0.101.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous membrane.
  • the dilution configuration is that the drug concentration is 10mg/ml, 3% sucrose is added for lyophilization, and after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection. The measured particle size was 128.1nm and the PDI was 0.132.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered through a 0.22 ⁇ m microporous membrane to sterilize.
  • the dilution configuration is that the drug concentration is 5mg/ml, 3% sucrose is added for lyophilization, and after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection. The measured particle size was 116.8nm and the PDI was 0.113.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1400 bar, and the homogenization times are 10 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous membrane.
  • the dilution configuration is that the drug concentration is 1mg/ml, and 3% sucrose is added for lyophilization.
  • the lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 121.8nm and the PDI was 0.111.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1400 bar, and the homogenization times are 10 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous membrane.
  • the dilution configuration is that the drug concentration is 10mg/ml, 3% sucrose is added for lyophilization, and after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 121.1nm and the PDI was 0.109.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous membrane.
  • the dilution configuration is that the drug concentration is 12mg/ml, 3% sucrose is added for lyophilization, after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection. The measured particle size was 109.7nm and the PDI was 0.105.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1400 bar, and the homogenization times are 10 times.
  • the organic solvent is removed by rotary evaporation.
  • the liquid after rotary steaming is filtered and sterilized through a 0.22 ⁇ m microporous membrane.
  • the dilution configuration is that the drug concentration is 10mg/ml, 3% sucrose is added for lyophilization, and after lyophilization is completed, it is capped and stored at 4°C.
  • the lyophilized sample was reconstituted with water for injection.
  • the measured particle size was 112.7nm and the PDI was 0.112.
  • the obtained colostrum is transferred to a high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 1000 bar, and the homogenization times are 16 times.
  • the organic solvent is removed by rotary evaporation, and the 0.22 ⁇ m microporous filter membrane is filtered and sterilized to obtain an aqueous solution of elemene albumin nanoparticles.
  • the measured particle size is 126.1 nm, and the PDI is 0.122.
  • the two phases are mixed uniformly, and the resulting solution is transferred to a high-pressure homogenizer for high-pressure homogenization, 400-500bar 4-6 times, 1300-1400bar 8-10 times, after homogenization is completed, it is removed by rotary evaporation Organic solvent, microporous filter membrane to filter and sterilize, and let stand for a while to remove bubbles in the preparation to obtain elemene albumin nanoparticle solution.
  • the measured nanoparticle size is 99.4nm, and the PDI is 0.096.
  • the obtained elemene albumin nanoparticle solution was fixed to a concentration of 10 mg/ml, 3% sucrose was added, lyophilized, and reconstituted with ultrapure water, and the particle size was measured to be 101.6 nm and the PDI was 0.113.
  • colostrum is transferred to a high-pressure homogenizer (ATS, AH-1500) for high-pressure homogenization, the homogenization pressure is 250-300bar, homogenization is 2min, and 1450-1550bar is homogenization for 6min.
  • ATS high-pressure homogenizer
  • the obtained colostrum is transferred to a high-pressure homogenizer (ATS, AH-1500) for high-pressure homogenization, the homogenization pressure is 250-300bar, homogenization is 2min, and 1450-1550bar is homogenization for 6min.
  • ATS high-pressure homogenizer
  • AH-1500 high-pressure homogenizer
  • measure the total volume of the homogenized solution with a graduated cylinder add trehalose at a 10% mass/volume ratio (g/ml), shake up to dissolve, filter with a 0.45 ⁇ m microporous filter membrane, and obtain elemene albumin Nanoparticle solution.
  • the particle size and PDI were detected, and the hemolysis value was determined. The specific results are shown in Table 6.
  • the obtained colostrum was transferred to a microfludizer (Microfludizer, M-110-EH) for homogenization at a homogenization pressure of 20,000 psi and homogenization for 30 minutes. Filter through 0.45 ⁇ m and 0.22 ⁇ m PVDF flat filters, and add the corresponding different freeze-dried protective agents (g/ml) in Table 7 to constant volume. The homogenized solution after adding the freeze-dried protective agent was freeze-dried. After the freeze-drying was completed, the freeze-dried powder was re-dissolved with sterilized ultrapure water to detect the particle size, PDI and hemolysis value. The specific results are shown in Table 7.
  • the organic solvent is a mixed solvent of 26.67 ml of dichloromethane and 6.67 ml of absolute ethanol.
  • Measure 50ml (10g) of 20% human serum albumin (HSA) add an appropriate amount of ultrapure water to dilute evenly, and obtain an aqueous solution of human serum albumin with a mass volume percentage of 3% (g/ml), that is, the water phase.
  • the obtained colostrum was transferred to a microfludizer (Microfludizer, M-110-EH) for high-pressure homogenization at a homogenization pressure of 20,000 psi and homogenization for 30 minutes.
  • the organic solvent-containing emulsion is passed through a thin film evaporator to remove the organic solvent.
  • the parameters are: temperature 30°C, vacuum degree 4000pa, liquid feeding speed 100rpm, scraper speed 50rpm. Filter through 0.45 ⁇ m and 0.22 ⁇ m PVDF flat filters, and add lyoprotectant 10% (g/ml) trehalose to constant volume.
  • the homogenized solution after adding the freeze-dried protective agent is freeze-dried, and after the freeze-drying is completed, the freeze-dried powder is re-dissolved in sterilized ultrapure water.
  • the detected particle size was 76.28, PDI was 0.254, and hemolysis value was 16.3.
  • the organic solvent is a mixed solvent of 1 ml of ethanol and 4 ml of dichloromethane, and the oil phase is obtained.
  • HSA human serum albumin
  • the obtained colostrum is transferred to a high-pressure homogenizer (ATS, AH-1500) for high-pressure homogenization, the homogenization pressure is 250-300 bar, homogenization is 2 min, and the homogenization is 1450-1550 bar for 6 min.
  • the organic solvent was removed with a rotary evaporator at 35° C., 150 rpm, and a vacuum degree of 30 mbar. After completion, measure the total volume of the homogenized liquid with a graduated cylinder, add trehalose at 10% mass/volume ratio (g/ml), shake it to dissolve, filter with a 0.45 ⁇ m microporous filter membrane to obtain elemene albumin nanoparticles Solution.
  • the prepared samples were put into a 30°C oven, and samples were taken at 0h, 24h, and 48h, and the particle size, PDI and ⁇ -elemene content were determined. The measurement results are shown in Table 9.
  • HSA human serum albumin
  • the obtained colostrum is transferred to a high-pressure homogenizer (ATS, AH-1500) for high-pressure homogenization, the homogenization pressure is 250-300bar, homogenization is 2min, and 1350-1450bar is homogenization for 6min.
  • ATS high-pressure homogenizer
  • AH-1500 high-pressure homogenizer
  • the prepared samples were put into a 30°C constant temperature box, and samples were taken at 0h, 24h, and 48h, and the particle size, PDI and content were determined. The measurement results are shown in Table 11.
  • the obtained liquid is transferred to a high-pressure homogenizer (ATS, AH-1500) for high-pressure homogenization, the homogenization pressure is 250-300 bar, homogenization is 2 minutes, and 1500-1550 bar is homogenized for 6 minutes, and the homogenized liquid is collected.
  • the trehalose aqueous solution was replaced, combined with the homogenized solution, mixed well, and filtered with a 0.22 ⁇ m sterile filter membrane.
  • the samples were divided into EP tubes, stored in a refrigerator at 2-8°C, and samples were taken on the 0th day and 2 months to detect particle size and PDI. The results are shown in Table 12.
  • mice 50 nude mice were anesthetized with 2.5% isoflurane, a small hole ( ⁇ 0.5 mm) was drilled in the skull of the nude mouse, and the cells (5x10 4 cells) expressing the luminescent reporter gene U87MGR1 human glioma cells) were injected into the left forehead (2.5mm lateral and 0.5mm anterior bregma, depth 2.5mm).
  • In vivo bioluminescence imaging assay method nude mice were injected with D-luciferin solution (120mg/kg) subcutaneously. 10 minutes after the injection of fluorescein, within the exposure time of 1-5 minutes, use dark multi-color illumination to capture the light image of the animal (for Gluc bioluminescence imaging, a single intravenous injection of coelenterazine (20mg/kg, total volume is 150 ⁇ l)).
  • the luciferase activity in the tumor is obtained by recording photon counts over 5 minutes with a CCD camera without illumination.
  • Use Perkin Elmer's Living image software 4.3.1 to quantitatively analyze the image intensity.
  • the tumor volume is measured once a week.
  • the animals will be weighed weekly to monitor their health.
  • the administration was discontinued for two weeks, followed by observation for three weeks.
  • Graphpad software was used to record the survival and tumor volume of each group of mice, and a Kaplan-Meier chart was generated.
  • Elemene injection (trade name: Ailineng): CSPC Yuanda (Dalian) Pharmaceutical Co., Ltd. (batch number: 17050302)
  • Avery Energy Take 7.5mL of 20mg/mL commercially available Avery Energy and dilute it with 17.5mL of normal saline into a sample solution of 25ml 6mg/mL.
  • ⁇ -elemene micelle preparation take 5.91mL of 25.41mg/mL ⁇ -elemene micelle preparation, dilute it with 19.09mL of normal saline to 25ml-6mg/mL sample solution.
  • ⁇ -elemene albumin preparations Take 12 beta-elemene albumin preparations, add 2.01 mL of normal saline to each, and mix with 24 ml 6 mg/mL sample solution.
  • mice Take male KM mice, 20-25g/mouse, and randomly divide them into 13 groups according to their body weight, each with 7 rats. Groups are grouped according to the established dosage according to body weight, and the dosage of each sample is set: 100mg/kg, 130mg/kg, 170mg/kg, 220mg/kg, and a blank group is set up. The mice were injected into the tail vein, and the administration site, post-administration reaction and death were observed after the administration. The results of the study are shown in Table 15.

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Abstract

涉及药物组合物，其包含榄香烯、蛋白载体和注射用油，其具有较好的安全性和稳定性。

Description

含有榄香烯的药物组合物及其制备方法和用途 技术领域
本发明涉及药物制剂领域，具体而言，涉及含有榄香烯的药物组合物及其制备方法和用途。
背景技术
榄香烯是从植物温郁金(Curcuma Wenyujin Y.H.Chen et C.ling)、香茅(Cymbopoqon citratus((DC.))Stapt)等植物中提取的挥发性油状化合物。该化合物呈现淡黄色或黄色的澄明液体，有辛辣的茴香气味。目前临床上所用榄香烯为混合物，含有α-榄香烯、β-榄香烯、δ-榄香烯和γ-榄香烯，广泛用于恶性胸膜腔积液、肺癌、消化道肿瘤以及其他浅表性肿瘤。由于目前上市的榄香烯以及制剂中含有蓖麻油聚羟氧酯等辅料具有较大的刺激性，因此上市药物用药后大部分病人会有较严重的静脉炎、发热、局部疼痛、过敏反应或者轻度消化道反应，以及发热等不良反应。在实际临床用药过程中由于其静脉炎的发生率过高，且复发率更高，对于静脉滴注的速度要求很高，要严格控制，造成滴注时间很长。
榄香烯在水中几乎不溶，脂溶性很强，尤其在石油醚、乙醚等有机溶剂中溶解性很好，其分子式为C 15H 24，仅由碳氢化合物组成。由于其独特的化学结构和理化性质，制成药物制剂时存在很大的难度和障碍，为了增加榄香烯的溶解度，将其制成注射液的过程中，上市处方添加了蓖麻油聚羟氧酯等辅料，造成其溶血严重、刺激性大的问题。另外，虽然紫杉醇白蛋白纳米制剂的上市，为解决临床副反应提供了一种新的药物制剂形式，但是常温下为油状的榄香烯的化学结构及其理化性质决定将其制备成白蛋白制剂存在诸多困难，因此，目前并未有相应研究和开发。
发明内容
本申请提供了一种药物组合物，其包含榄香烯、注射用油和蛋白载体，其能有效地减少药物组合物给药到人体内的一种或多种副作用。
在本申请的一个或多个实施方式中，药物组合物中所用的蛋白载体包括蛋白质，可以使用任何合适的蛋白质。合适的蛋白质的实例包括但不限于白蛋白、免疫球蛋白，包括但不限于IgA、脂蛋白、载脂蛋白B、α-酸性糖蛋白、β-2-巨球蛋白，甲状腺球蛋白、转铁蛋白、纤连蛋白、因子VⅡ、因子VIII、因子IX、因子X和类似物。蛋白载体可以是天然来源或是合成制备的。在一些实施方式中，蛋白载体是非血液蛋白质，如酪蛋白、α-乳清蛋白和β-乳球蛋白。在一些实施方式中，蛋白载体包括白蛋白，如人血清白蛋白(HSA)，牛血清白蛋白等。人血清白蛋白是高度可溶性球蛋白，Mr65K，由585个氨基酸组成。HSA是血浆中最丰富的蛋白质，并且构成人血浆胶体渗透压的70-80％。
在本申请的一个或多个实施方式中，药物组合物中所用“可注射用油”包括但不限于油酸乙酯、苯甲酸苄酯、中链甘油三酯(MCT)和植物油，其中植物油选自大豆油、红花油、棉籽油、玉米油、向日葵油、花生油、橄榄油的一种或多种。在本申请的一个或多个实施方式中，可注射用油为大豆油。
在本申请的一个或多个实施方式中，药物组合物可以被制成颗粒，其中所述颗粒的粒径小于180nm，例如约为70-170nm，优选约为70-150nm。
在本申请的一个或多个实施方式中，组合物包含任何形状(例如，球形或非球形形状)的纳米颗粒，在一些实施方式中，颗粒的平均或中间直径不大于约180nm。在一些实施方式中，颗粒的平均或中间直径为约50至约180nm。在一些实施方式中，颗粒的平均或中间直径为约60至约180nm。在一些实施方式中，颗粒的平均或中间直径为约70至约170nm。在一些实施方式中，颗粒的平均或中间直径为约70至约-170nm。在一些实施方式中，颗粒的平均或中间直径为约70至约150nm之间。在一些实施方式中，颗粒的平均或中间直径为约60nm、70nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm或150nm。在一些实施方式中，颗粒是无菌、可过滤的。
在本申请的一个或多个实施方式中，药物组合物中的榄香烯选自α-榄香烯、β-榄香烯、γ-榄香烯以及δ-榄香烯的一种或多种。在本申请的一个或多个实施方式中，药物组合物中的榄香烯为β-榄香烯。
在本申请的一个或多个实施方式中，药物组合物中所述白蛋白载体、榄香烯和注射用油的比例为投料的重量比。
在本申请的一个或多个实施方式中，药物组合物中所述蛋白载体与榄香烯的重量比为0.520；例如0.5-10。
在本申请的一个或多个实施方式中，药物组合物中所述注射用油与榄香烯的重量比为0.3510；例如0.5-10。
在本申请的一个或多个实施方式中，药物组合物还含有冻干保护剂。所述冻干保护剂选自葡萄糖、蔗糖、麦芽糖、乳糖、甘露糖、海藻糖、甘氨酸、右旋糖酐中的一种或几种，优选海藻糖或蔗糖。在一个或多个实施方式中，其中所述冻干保护剂的重量与所述药物组合物的溶液的体积之比为3:100-20:100g/mL，优选5:100-10:100g/mL。
在本申请的一个或多个实施方式中，药物组合物还含有等渗调节剂、抗氧化剂、防腐剂、pH调节剂中的一种或几种。所述等渗调节剂为甘油、山梨醇、甘露醇或葡萄糖的一种或多种；所述pH调节剂为氢氧化钠、柠檬酸钠、柠檬酸、磷酸、醋酸或盐酸的一种或多种；所述防腐剂为羟苯烷基酯类、苯甲酸、苯甲酸钠、山梨酸、醋酸氯乙定、苯扎溴胺的一种或多种；所述抗氧化剂为亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、抗坏血酸、叔丁基对羟基茴香醚、2,6-二叔丁基化羟基甲苯、维生素E的一种或多种。
在本申请的一个或多个实施方式中，还提供了一种药物组合物在制备过程中不使用有机溶剂。
在本申请的一个或多个实施方式中，药物组合物可以制成颗粒，其中所述颗粒的粒径小于180nm；例如约为70-150nm。
在本申请的一个或多个实施方式中，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.35-3，所述人血白蛋白与榄香烯的重量比约为0.5-20；优选所述大豆油与榄香烯 的重量比约为0.5-3；所述人血白蛋白与榄香烯的重量比约为0.5-2.5；或者所述药物组合物中所述大豆油与榄香烯的重量比约为0.35-1.5，所述人血白蛋白与榄香烯的重量比约为1.5-10。
在本申请的一个或多个实施方式中，大豆油与榄香烯的重量比约为0.5，人血清白蛋白与榄香烯的重量比约为1.5。
在本申请的一个或多个实施方式中，大豆油与榄香烯的重量比约为0.75，人血清白蛋白与榄香烯的重量比约为5。
在本申请的一个或多个实施方式中，大豆油与榄香烯的重量比约为1.5，人血清白蛋白与榄香烯的重量比约为3-5。
在本申请的一个或多个实施方式中，还提供了一种药物组合物，其在制备过程中含有有机溶剂。其中所述有机溶剂选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或几种。其中所述脂溶性有机溶剂为二氯甲烷与乙醇的混合溶剂。
其中所述二氯甲烷与乙醇的体积比1:1-8，优选1:4。
所述药物组合物可以制备成颗粒，所述颗粒的粒径约为70-150nm。
所述药物组合物中所述大豆油和榄香烯的重量比约为2.4-10，所述人血白蛋白和榄香烯的重量比约为0.5-10；优选所述药物组合物中所述大豆油和榄香烯的重量比约为2.4，所述人血白蛋白和榄香烯的重量比约为3。
本申请的一个或多个实施方式提供了本发明申请的药物组合物的制备方法，其包括以下步骤：
(1)将榄香烯与注射用油混合均匀，任选地加入有机溶剂，得到榄香烯和注射用油的溶液；将蛋白载体溶解于水，得到蛋白载体溶液；
(2)将步骤(1)中获得的两份溶液混合形成乳液；
(3)将步骤(2)中所述的乳液高压均质，任选地将所述乳液减压蒸发除去有机溶剂，得到纳米粒溶液。
本申请的一个或多个实施方式提供了本发明申请的药物组合物的制备方法，过程其包括以下步骤：
(1)取榄香烯和注射用油混合均匀；
(2)取白蛋白，溶解于水中；
(3)将两者混合形成乳液；
(4)将步骤(3)中所述的乳液高压均质，得到白蛋白纳米粒溶液。
在本申请的一个或多个实施方式中，其中步骤(2)中采用剪切或超声的方法进行混合，步骤(3)中采用高压均质或微射流均质的方法进行均质。
在本申请的一个或多个实施方式中，步骤(3)中还可包括用适量海藻糖水溶液冲洗均质机管路，将残留在均质机内的均质液置换出来，再用适量超纯水冲洗均质机管路，将残 留的海藻糖水溶液置换出来，与均质液合并，混匀后再过滤。
在本申请的一个或多个实施方式中，其中剪切可以用混合机剪切。
在本申请的一个或多个实施方式中，其中用混合机剪切的转速为5000rpm-10000rpm。
在本申请的一个或多个实施方式中，其中混合机的剪切时间为1min-20min，优选例如2min-10min或，更优选1min-10min。
在本申请的一个或多个实施方式中，其中用混合机剪切优选多次剪切，优选例如大于2次。
在本申请的一个或多个实施方式中，优选第一次5000rpm剪切1min，第二次10000rpm剪切5min。
在本申请的一个或多个实施方式中，其中高压均质的压力200-1600bar，优选例如250-1550bar。
在本申请的一个或多个实施方式中，其中高压均质的时间为1-10min，例如优选2-10min，比如进一步优选5-8min。
在本申请的一个或多个实施方式中，其中高压均质优选进行多次高压均质，优选例如大于2次。
在本申请的一个或多个实施方式中，其中微射流均质的压力为20000psi。
在本申请的一个或多个实施方式中，其中微射流均质的时间为30min。
本申请的一个或多个实施方式还提供了本申请的所述药物组合物的制备方法，在制备过程中使用有机溶剂。
在本申请的一个或多个实施方式中，本申请药物组合物的制备方法包括以下步骤：
(1)取榄香烯和注射用油溶于脂溶性有机溶剂；
(2)取白蛋白，溶解于水中；
(3)将步骤(1)和(2)中得到的溶液混合，形成乳液；
(4)将步骤(3)中所述的乳液进行均质；
(5)将步骤(4)中所述的乳液减压蒸发除去有机溶剂，得到白蛋白纳米粒溶液。
在本申请的一个或多个实施方式中，步骤(1)中所述有机溶剂选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或几种；例如，二氯甲烷与乙醇的混合溶剂，混合溶剂中乙醇与二氯甲烷的体积比为1:1-8，例如1:4。
在本申请的一个或多个实施方式中，其中所述步骤(3)中可以采用剪切或超声的方法进行混合，其中所述步骤(4)可以采用高压或微射流的方法进行均质。
在本申请的一个或多个实施方式中，其中剪切可以用混合机剪切。
在本申请的一个或多个实施方式中，其中用混合机剪切的转速为5000rpm-10000rpm。
在本申请的一个或多个实施方式中，其中混合机的剪切时间为1min-20min，例如2min-10min。
在本申请的一个或多个实施方式中，其中用混合机剪切优选多次剪切，例如大于2次。
在本申请的一个或多个实施方式中，优选第一次5000rpm剪切1min，第二次10000rpm剪切5min。
在本申请的一个或多个实施方式中，其中高压均质的压力200-1600bar，例如250-1550bar。
在本申请的一个或多个实施方式中，其中高压均质的时间为1-10min，例如2-10min，比如进一步优选5-8min。
在本申请的一个或多个实施方式中，其中高压均质优选进行多次高压均质，例如大于2次。
在本申请的一个或多个实施方式中，其中微射流均质的压力为20000psi。
在本申请的一个或多个实施方式中，其中微射流均质的时间为30min。
在本申请的一个或多个实施方式中，本申请的药物组合物的制备方法过程还包括将得到的白蛋白纳米粒溶液冻干的步骤。
本申请的一个或多个实施方式提供了本申请的药物组合物在预防或治疗癌症的药物中的用途。
在本申请的一个或多个实施方式中，本申请所涉及的癌症包含肾上腺皮质癌、原因不明的髓样化生、AIDS相关的癌、肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、神经胶质瘤、室管膜瘤、少突神经胶质瘤、脑膜瘤、颅咽管瘤、成血管细胞瘤、成神经管细胞瘤、神经外胚层肿瘤、视觉通路和下丘脑神经胶质瘤和恶性胶质瘤、支气管腺瘤、类癌瘤、中枢神经系统淋巴瘤、子宫颈癌、结肠癌、结肠直肠癌、慢性骨髓增生性疾病、子宫内膜癌、室管膜瘤、尤文肿瘤家族、眼癌、胆囊癌、胃癌、胃肠道类癌瘤、胃肠道间质瘤、生殖细胞癌瘤、妊娠滋养层肿瘤、头颈癌、肝癌、喉癌、白血病、唇及口腔癌、肺癌、淋巴瘤、成神经管细胞瘤、黑色素瘤、间皮瘤、转移性颈部鳞状细胞癌、多发性内分泌瘤形成综合征、骨髓增生异常综合征、骨髓增生异常/骨髓增生性疾病、鼻腔和鼻窦癌、成神经细胞瘤、神经内分泌癌、口咽癌、鼻咽癌、脑肿瘤、骨转移癌、肠癌、食道癌、卵巢癌、胰腺癌、皮肤癌、甲状旁腺癌、阴茎癌、腹膜癌、咽癌、嗜铬细胞瘤、成松果体细胞瘤和幕上原始神经外胚层肿瘤、垂体瘤、胸膜肺的胚细胞瘤、淋巴瘤、原发性中枢神经系统淋巴瘤、肺淋巴管肌瘤病、直肠癌、肾癌、肾盂及输尿管癌、横纹肌肉瘤、唾腺癌、小肠癌、鳞状上皮细胞癌、睾丸癌、咽喉癌、胸腺瘤及胸腺癌、甲状腺癌、尿道癌、阴道癌。
在本申请的一个或多个实施方式中，本申请所涉及的癌症包含肺癌、肝癌、食道癌、鼻咽癌、脑肿瘤、骨转移癌、胃癌、肠癌、子宫癌、子宫颈癌、生殖细胞癌瘤、子宫内膜癌、妊娠滋养层肿瘤、乳腺癌、皮肤癌、淋巴癌、白血病、恶性黑色素瘤；
在本申请的一个或多个实施方式中，脑肿瘤包含神经胶质瘤、脑干神经胶质瘤、小脑或大脑的星形细胞瘤、恶性神经胶质瘤、室管膜瘤、少突神经胶质瘤、脑膜瘤、颅咽管瘤、 成血管细胞瘤、成神经管细胞瘤、视觉通路和下丘脑神经胶质瘤或恶性胶质瘤；所述小脑或大脑的星形细胞瘤为纤维状细胞星形细胞瘤或弥漫星形细胞瘤或间变性(恶性)星形细胞瘤。
在本申请的一个或多个实施方式中，本申请的药物组合物可以合并用于对肺癌、肝癌、食道癌、鼻咽癌、脑瘤、骨转移癌等恶性肿瘤的放疗、化疗以增强疗效，降低放疗和化疗的毒副作用，还可用于介入、腔内化疗及癌性胸腹水的治疗。
在本申请的一个或多个实施方式中，本申请的化合物可以与其它药物联用，所述其它药物包含抗癌药物，所述抗癌药物包含替莫唑胺、紫杉醇、多西他赛、紫杉烷、吉西他滨、抗VEGF药物、PD-1抗体药物；其中抗VEGF药物包含AVASTIN、雷珠单抗、阿柏西普或康柏西普；其中PD-1抗体药物包含纳武利尤单抗、帕博丽珠单抗、特瑞普利单抗、信迪利单抗、西米普利单抗、阿特珠单抗、阿维单抗或度伐利尤单抗。
本申请的一个或多个实施方式提供了治疗人类疾病的方法，包括给予治疗有效量的本申请的药物组合物。所述药物组合物的给药方式为通过非肠道、通过吸入、腹腔内、膀胱内、肌肉内、静脉内、气管内、皮下、眼内、鞘内、透皮给药、直肠或阴道内。
在本申请的一个或多个实施方式中，本申请的药物组合物可以制成适于静脉给药的药物制剂、胃肠外给药的药物制剂、胃肠给药的药物制剂、气雾剂、阴道给药的药物制剂或其它合适的药物制剂，所述药物制剂为固体制剂、液体制剂或气体制剂。
在本申请的一个或多个实施方式中，本申请的药物组合物可作为注射用液体制剂，其包含榄香烯和药学上可接受的载体，其中药学上可接受的载体包括白蛋白载体和注射用油，其中组合物可以制成颗粒，其中所述颗粒的粒径70-150nm，所述组合物中白蛋白载体与榄香烯的重量比为白蛋白载体和榄香烯的重量比约为0.5-20，所述注射用油和榄香烯的比例重量比约为0.35-10。
在本申请的一个或多个实施方式中，本申请中的液体制剂可以为由无菌冻干粉重建的稳定的水悬浮液。
本申请的一个或多个实施方式提供包含本申请的药物组合物的密封容器，密封容器可以为单位剂量容器或多剂量容器。密封容器可以为预填充注射器。药物组合物为液体组合物或干燥组合物。药物组合物为冻干的、无菌的。
在本申请的一个或多个实施方式中，本文所述的纳米制剂或纳米颗粒可以以干燥制剂形式存在(如冻干组合物)或悬浮于生物相容性介质中。合适的生物相容性介质包括但不限于水、含水缓冲介质、盐水、缓冲盐水、任选缓冲的氨基酸溶液、任选缓冲的蛋白质溶液、任选缓冲的糖溶液、任选缓冲的维生素溶液、任选缓冲的合成聚合物溶液、含脂类乳剂等。
除非另有清楚地指出，在本文中使用的“个体”是哺乳动物，包括但不限于灵长类动物、人、牛、马、猫科动物、犬科动物或啮齿动物。
如在本文所使用，“治疗”是获得包括临床结果在内的有益或所需的结果的方法。对于 本发明目的，有益或所需临床结果包括但不限于下列任一或多个：减少由疾病产生的一种或多种症状的、减轻疾病程度、稳定疾病(例如预防或延迟了疾病的恶化)、预防或延迟疾病播散(例如转移)、预防或延迟疾病发生或复发、延迟或减慢疾病进展、改善疾病状态、提供疾病的缓和(不论是部分或全部的)、减少一种或多种治疗疾病必需的其它药物的剂量、延迟疾病进展、增加生活质量和/或延长生存。在一些实施方式中，与在治疗之前相同受实验者中的相应症状相比或与没有接受组合物的其它受实验者中的相应症状相比，组合物减少了与癌症相关的一种或多种症状的严重性，为至少大约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％中的任一种。“治疗”也包括癌症的病理后果减轻。本发明方法考虑了这些治疗方面中的任一或多种。
如在本文所使用的，“药学上可接受的”或“药理学相容的”意思是不是生物学的或另外不想要的物质，例如在没有引起任何显著不想要的生物效果或与组合物中包含的任何其它成分没有以有害方式相互作用的情况下，该物质可以被掺入给予患者的药物组合物中。药学上可接受的载体或赋形剂优选满足毒物学和生产测试要求的标准。
在本文提及“大约”某个值或参数包括(并描述)涉及该值或参数本身的实施方式。例如，提及“大约X”的描述包括对“X”的描述。
本发明药物组合物有多种合适的制剂。下面的制剂和方法仅仅是示例性的，并不是限制性的。
口服给药的制剂可由(a)液体溶液，例如有效量的溶解在稀释剂如水，盐水或橙汁中的活性成分，(b)胶囊，香囊或片剂，每个含有预定量的固体或颗粒形式的活性成分，(c)在适当液体中的悬浮液，和(d)适当的乳剂。片剂形式可包括乳糖、甘露醇、玉米淀粉、马铃薯淀粉、微晶纤维素、阿拉伯胶、明胶、胶态二氧化硅、交联羧甲基纤维素钠、滑石、硬脂酸镁、硬脂酸和其它赋形剂、着色剂、稀释剂、缓冲剂、润湿剂、防腐剂、调味剂和药理学相容的赋形剂中的一种或多种。含片形式可包含香料中的活性成分，通常是蔗糖和阿拉伯胶或黄蓍胶，以及在惰性基质中包含活性成分的锭剂，例如明胶和甘油，或蔗糖和阿拉伯胶，乳剂，凝胶等，除了活性成分外，还包含本领域已知的赋形剂。
胃肠外给药的制剂包括水性和非水性，等渗无菌注射溶液，其可包含抗氧化剂、缓冲剂、抑菌剂和使制剂与预期接受者的血液等渗的溶质，以及可包括悬浮剂、增溶剂、增稠剂、稳定剂和防腐剂的含水和非水无菌悬浮液。所述制剂可存在于单位剂量或多剂量的密封容器中，例如安瓿和小瓶中，并且可在使用前立即在冷冻干燥(冻干)条件下储存，仅需要加入无菌液体赋形剂，例如水，用于注射。可由前述类型的无菌粉末，颗粒和片剂制备临时注射溶液和悬浮液。
气雾剂包含本发明的药物组合物，所述药物组合物包括水性和非水性，等渗无菌溶液，其可包含抗氧化剂、缓冲剂、抑菌剂和溶质，以及可包含悬浮剂、增溶剂、增稠剂、稳定剂和防腐剂的水性和非水性无菌悬浮液，其单独或与其它合适的组分组合，可制成气雾剂制剂，通过吸入给药。这些气溶胶制剂可被置于加压的可接受的推进剂中，例如二氟甲烷、 丙烷、氮气等。它们也可以配制成非压力制剂的药物，例如在喷雾器或雾化器中。
其它合适的制剂也是可能的，例如栓剂可以通过使用各种碱例如乳化碱或水溶性碱来制备。阴道给药的制剂可呈现为阴道栓剂、棉塞、乳膏、凝胶、糊剂、泡沫剂或喷雾制剂，除了活性成分外，还含有本领域已知的合适的载体。
说明书附图
图1表示β-榄香烯白蛋白纳米粒水合粒径分布图；
图2表示β-榄香烯白蛋白纳米粒电位图；
图3表示β-榄香烯白蛋白纳米粒透射电镜图；
图4表示不同冻干样品复溶后粒径稳定性考察；
图5表示不同冻干样品放置含量稳定性考察；
图6表示各组荷瘤小鼠生存期；
图7表示溶血性实验结果；
图8表示U87R1小鼠原位瘤模型的肿瘤生长曲线；
图9表示U87R1小鼠原位瘤模型的生存曲线。
具体实施方式
以下实施例是对本发明的进一步说明，并非本发明范围的限制。下面参考实施例进一步详细阐述本发明，但是本领域技术人员应当理解，本发明并不限于这些实施例以及使用的制备方法。而且，本领域技术人员根据本发明的描述可以对本发明进行等同替换、组合、改良或修饰，但这些都将包括在本发明的范围内。
本发明涉及的大豆油来自大豆油(SIO，注射级，Lot19C4520/Lot18C4816)；本发明涉及的人血清白蛋白(HSA)来自人血清白蛋白(20％，Baxter，LotA1U133A)或(20％，Grifols，LotALAFD03042)。
本发明常用检测方法含有粒径及粒径分布、含量测定、溶血实验等，具体如下：
粒径和粒径分布
用粒度仪(MALVERN ZETASIZER NANO ZS90)测定粒径和粒径分布。取25μL样品，加入1ml超纯水稀释后进行检测，检测角：90°；分散介质：水；温度25℃。
其中粒径为粒度仪测出的Z-average值(样品粒径平均值)，其中粒径分布为粒度仪测出的PDI值。
含量测定
照高效液相色谱法(中国药典2015年版四部通则0512)测定。
色谱柱：
Figure PCTCN2020133470-appb-000001
IG(4.6×150mm，5μm)，乙腈-水(55：45)为流动相，流速1.0ml/min，进样量20μL，柱温为30℃，检测波长为210nm。
对照品溶液(0.2mg/ml)的制备(外标法)：精密称取β-榄香烯对照品约10mg至已 加入少量乙腈的50ml量瓶中，加乙腈溶解并稀释至刻度，摇匀，即得。
供试品溶液的制备：取本品1瓶去盖，精密称定(m 1)，加入适量水(使API与水的比例为20mg：1ml)后，精密称定(m 2)，振摇使复溶，精密称取复溶溶液约500mg(约相当于API 10mg)(m 3)，置50ml量瓶中，先加入约1.5ml的水，再缓慢加入乙腈至满量程的80％，超声10min，放冷，再加乙腈稀释至刻度，摇匀，用孔径0.45μm的尼龙滤头过滤即得。复溶溶液使用完毕后，用水清洗西林瓶并擦干，精密称定空瓶重量(m 4)。
精密量取20μl，注入液相色谱仪，记录色谱图。按外标法以峰面积计算供试品中β-榄香烯(C 15H 24)的含量。
计算公式：
Figure PCTCN2020133470-appb-000002
式中
Ax：供试品溶液中API峰面积
A R：对照品的峰面积
C R：对照品的浓度
溶血值的测定方法
血细胞悬液制备：取家兔(雄性新西兰兔、实验室适用性饲养1周、体重2.5kg-3kg，心脏取血)血数毫升，放入烧杯中，用玻璃棒或竹签搅动血液，除去纤维蛋白原，使成脱纤血液。加入0.9％氯化钠溶液约10倍量，混匀，1500rpm/min离心15min，弃去上清液，沉淀的红细胞再用0.9％氯化钠溶液按上述方法洗涤2-3次，至上清液不显红色为止。将所得红细胞用0.9％氯化钠溶液配成2％的血细胞悬液，备用。
样品制备：根据样品含量，取适量用生理盐水依次稀释获得1mg/mL样品(实际含量)。
反应观察：按下列方式加入各药物，混匀后，立即置于37℃水浴锅中进行温育，3h后观察溶血情况。
药物组：试管中依次加入2.5mL 2％的血细胞悬液、2.2mL生理盐水、0.3mL药物。
阳性组：试管中依次加入2.5mL 2％的血细胞悬液、2.5mL蒸馏水。
阴性组：试管中依次加入2.5mL 2％的血细胞悬液、2.5mL生理盐水。
OD值测定：吸取上层液100uL至96孔板中，540nm波长下测定OD值。
溶血值的计算方式：(样品OD值-阴性OD值)/(阳性OD值-阴性OD值)。
试验例1
制备β-榄香烯白蛋白纳米粒，包括如下步骤：1)称取200mg榄香烯和600mg大豆油，溶于2mL二氯甲烷中，得到油相。2)取人血清白蛋白650mg加入注射用水溶解，得质量体积百分比为3.6％(g/ml)人血清白蛋白水溶液，即得到水相。3)在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。4)将所得初乳转移到高压均质仪中进行高压均质，均质压力为1300bar，均质次数9次。均质完成后的体系通过 旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液，注射用水定容至10mg/ml，5)往所得榄香烯白蛋白纳米粒中加入3％蔗糖，冻干。冻干参数为：-45℃预冻5h；开启真空泵保持真空度为10Pa左右，程序升温，-40℃干燥4h，-30℃干燥10h，随后依次于-20℃、-10℃、0℃、5℃、10℃分别干燥4h。将上述制备的冻干样品采用纯水复溶，采用如下方法对纳米粒粒径、电位和微观形态进行考察。
测量前马尔文粒度仪预热30min，取榄香烯白蛋白纳米粒水溶液50μL，加水稀释至1mL，轻微震荡摇匀(避免产生气泡)，置于马尔文Nano-ZS90粒径皿或电位皿中测量粒径及Zeta电位。测量温度为25℃，平衡时间120s，其余各项保留仪器默认值，以Malvern V2.2工作站进行数据采集处理。所得的榄香烯白蛋白纳米粒粒径图，zeta电位，见图1、图2。由图1和图2可见，榄香烯白蛋白纳米粒的粒径约为110nm左右，PDI为0.127，电位约为-18mV。
移液枪吸取榄香烯白蛋白纳米粒水溶液，滴于铜网上，自然挥干后同法将1％磷钨酸溶液滴于铜网上，自然挥干，吸取纯净水数滴小心冲洗铜网表面，挥干后于透射电镜观察形态如图3，纳米粒呈圆球形，形状规整，粒径较为均匀，微粒直径为100nm左右。
试验例2
按照试验例1的方法制备榄香烯白蛋白纳米粒，其中注射用油分别采用等量大豆油、玉米油、茶油、中链甘油三酯(MCT)，制备出以不同注射用油为原料的榄香烯白蛋白纳米粒，采用激光粒度仪测定制备的纳米粒的粒径，冻干后，分别测定各组制剂冻干前后的药物含量，计算冻干过程中药物的损失率。
制备不含大豆油的榄香烯白蛋白纳米粒，包括如下步骤：1)称取200mg榄香烯溶于2mL二氯甲烷中，得到油相。2)取人血清白蛋白650mg加入注射用水溶解，得质量体积百分比为3.6％(g/ml)人血清白蛋白水溶液，即得到水相。3)在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。4)将所得初乳转移到高压均质仪中进行高压均质，均质压力为1300bar，均质次数9次。均质完成后的体系通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液，注射用水定容至10mg/ml。5)往所得榄香烯白蛋白纳米粒中加入3％蔗糖，按试验例1冻干参数冻干。分别测定一定体积制剂冻干前后的药物含量，计算冻干过程中药物的损失率。
另按照上述方法制备仅是不含冻干保护剂3％蔗糖的大豆油、玉米油、茶油、中链脂肪酸甘油酯组，分别测定一定体积制剂冻干前后的药物含量，计算冻干过程中药物的损失率。所述结果如表1所示。
表1不同注射用油制备样品冷冻干燥后药物损失率
Figure PCTCN2020133470-appb-000003
实验表明，将注射用油与榄香烯混合后制备成白蛋白纳米粒较不加注射用油制备的纳米粒冻干损失率均有不同程度降低。
试验例3
按照试验例1的方法，调整大豆油用量，分别按照β-榄香烯/大豆油质量比为5:1，1:1，1:3，1:5，1:10，1:20和不加注射用油投料，制备榄香烯白蛋白纳米粒。
采用激光粒度仪测定制备的纳米粒的粒径，加入3％蔗糖冻干后，将冻干样品放置于室温干燥器内，定期取出样品，复溶，测定30d内冻干样品粒径和主药含量的变化情况。具体检测参数：仪器型号：MALVERN ZETASIZER NANO ZS90，检测角：90°；检测模式：自动；检测次数：3；检测类型：纳米粒；待测样品折射率：1.340；分散介质：水；温度25℃；黏度：0.8872cp；分散介质折射率：1.330；平衡时间：120s；粒径皿型号：DTS0012。
结果如图4和图5所示，制备过程中不加大豆油的制剂冻干后粒径大于200nm，随着存储时间延长，样品复溶后的粒径呈逐渐增大的趋势，药物含量呈不断降低的趋势。逐渐增加制剂中大豆油的比例，冻干后样品复溶的粒径均小于200nm，粒径稳定性较好，主药含量稳定性也逐渐增强。其中，当大豆油/榄香烯的质量比在1-10时，制备的纳米粒冻干样品室温放置30d内无聚集现象，粒径为100-150nm，PDI小于0.3，β-榄香烯主药含量变化小。当榄香烯1份，大豆油的用量小于1份时，制备的纳米粒稳定性欠佳。
试验例4
榄香烯白蛋白纳米粒对小鼠脑胶质瘤的治疗效果考察，实验方法如下所述。
肿瘤建模：将生长良好且处于对数期的C6细胞用胰酶消化，离心，弃去上层废液，用PBS洗一次，离心弃去上层废液，PBS重悬，使最终C6细胞密度约为1×106个/5μL。健康雄性昆明小鼠(28-30g)，腹腔注射适量4％水合氯醛使其麻醉，将头顶的毛发剃除后，75％乙醇涂抹消毒，用消毒的眼科剪沿头部矢状中线开一小口，用沾有10％双氧水的棉签快速去除组织膜，分离出颅骨，再用沾有PBS的棉签清洗双氧水；将小鼠的头部固定于脑立体定位仪上，定位距离前囟点右1.8mm，后0.6mm的位置，用2mL注射器的针头在定位点钻一个小孔，不得过深以免出血。用微量进样针吸取上述密度为1×106个/5μL的C6细胞5μL，沿小孔垂直方向，进针4mm，退针1mm，留出细胞空间，缓慢将细胞悬液注入脑内，微针在小孔位置停留5min后取出。用手术线将伤口缝合，最后用沾有双抗的棉签涂抹缝合表面。
接种肿瘤后第10天，将荷瘤小鼠分为生理盐水组(NS)、市售榄香烯脂质体(Lip，大连金港，批准文号：国药准字H10960114)、试验例1制备的榄香烯白蛋白纳米粒(NPs)，每组12只，给药剂量为30mg/kg，每2天给一次，共给药4次，记录每只小鼠存活时间，制作生存曲线。
生存期考查结果如图6所示，实验的结果表明给予榄香烯白蛋白纳米粒的荷C6胶质瘤 小鼠，表现出更长的生存期。生理组的平均生存期为21天，榄香烯脂质体组的平均生存时间为28天，榄香烯白蛋白纳米粒组的平均生存期为41天。因此，实验所制备的榄香烯白蛋白纳米粒具有延长荷C6胶质瘤小鼠生存期的作用。
试验例5
溶血实验：
将新西兰兔(雄性，实验室适用性饲养1周，体重2.5kg-3kg)分为市售榄香烯注射液(艾利能，石药集团远大制药有限公司，批号17050302)和试验例1制备的榄香烯白蛋白纳米粒(NPs)两个组，每组2只，给药剂量为50mg/kg。用葡萄糖溶液分别将两种制剂配制成浓度为6mg/mL的药液，静脉滴注给药。
给药完成后10-15min内耳缘静脉抽取1ml左右静脉血于涂有肝素钠的离心管中，3500r/min离心15min，取上清液观察颜色。
所得血浆颜色如图7所示，该结果表明给予榄香烯白蛋白纳米粒的新西兰兔未表现出溶血现象，刺激性小，具有较高的安全性。
试验例6
1.1实验样品的制备
样品1
称取3.33gβ-榄香烯、8g大豆油，混匀，得到油相。量取20％人血清白蛋白50ml(10g)，加入适量超纯水稀释均匀，定容至333ml，得质量体积百分比为3％(g/ml)的人血清白蛋白水溶液，即水相。将水相倒入油相中，用混合机(FLUKO，FA30/30F)10000rpm剪切10min，形成初乳。将所得初乳转移到微射流均质机(Microfludizer，M-110-EH)中进行均质，均质压力20000psi，均质30min。经0.45μm和0.22μm PVDF平板过滤器进行过滤，定容，加入冻干保护剂蔗糖3％(g/ml)，将加入冻干保护剂后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，用粒径仪检测粒径为175.97nm，PDI为0.188。
样品2
称取3.33gβ-榄香烯、8g大豆油，溶于有机溶剂中，混匀，得到油相。所述有机溶剂为26.67ml二氯甲烷与6.67ml无水乙醇的混合溶剂。量取20％人血清白蛋白50ml(10g)，加入适量超纯水稀释均匀，定容至333ml，得质量体积百分比为3％(g/ml)的人血清白蛋白水溶液，即水相。将水相溶液倒入油相中，用混合机(FLUKO，FA30/30F)10000rpm剪切10min，形成初乳。将所得初乳转移到微射流均质机(Microfludizer，M-110-EH)中进行高压均质，均质压力20000psi，均质30min。均质完成后含有机溶剂的乳液通过薄膜蒸发仪(上海德大天一)除去有机溶剂，参数为：温度30℃，真空度4000pa，进液速度100rpm，刮板速度50rpm。经0.45μm和0.22μm PVDF平板过滤器进行过滤，定容，加入冻干保护剂蔗糖3％(g/ml)。将加入冻干保护剂后的均质液进行冻干，完成冻干后，用灭菌超纯水 复溶冻干粉，用粒径仪检测粒径为78.34nm，PDI为0.207。
样品3(榄香烯注射液(商品名：艾利能)):石药集团远大(大连)制药有限公司，批号：19061502；用粒径仪检测粒径为14.74nm PDI：0.035
1.2榄香烯白蛋白纳米粒的PK和小鼠脑组织分布
采用昆明小鼠(雄性，SPF级，体重20-25g)进行实验，通过尾静脉给药：给药剂量50mg/kg；脑组织样品于不同时间段采集好后，及时于-20℃冻存，用于脑组织中药物含量检测。三个样品，每种剂型每个时间点3只昆明小鼠，共计45只，给药后于5min、15min、30min、1h、2h摘除眼球取血，收集脑组织，检测脑组织中药物含量。采用GC-MS进行昆明小鼠血浆和脑组织生物样品中β-榄香烯的定量分析，其中脑组织中半衰期及脑组织暴露量的研究结果见表2。
表2半衰期及脑组织暴露量研究结果
  样品1 样品2 样品3
粒径(nm) 175.97 78.34 14.7
脑组织中半衰期(min) 41.8 57.5 33.6
脑部药物暴露量(mg/L*min) 2808.7 3008.6 2812.7
试验例7
样品4的制备及稳定性考察
称取1gβ-榄香烯、3g大豆油，混匀，得到油相。量取20％人血清白蛋白15ml(3g)，加入85mL超纯水稀释均匀，得质量体积百分比为3％(g/ml)的人血清白蛋白水溶液，即水相。将水相溶液倒入油相中，用混合机5000rpm剪切1min，10000rpm剪切5min，形成初乳。将所得初乳转移到高压均质机中进行高压均质，均质压力250-300bar，均质2min，1350-1400bar均质9min。均质完成后，用量筒测量均质液总体积，约为50ml，加入5g海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。用粒径仪测得粒径为141.0nm，PDI为0.162。将加入海藻糖后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，用粒径仪检测粒径为144.07nm，PDI为0.123。冻干前后榄香烯含量损失率为1.27％。
样品5的制备及稳定性考察
称取4gβ-榄香烯、2g大豆油，混匀，得到油相。量取20％人血清白蛋白30ml(6g)，加入170mL超纯水稀释均匀，得质量体积百分比为3％(g/ml)的人血清白蛋白水溶液，即水相。将水相溶液倒入油相中，用混合机5000rpm剪切1min，10000rpm剪切10min，形成初乳。将所得初乳转移到高压均质机中进行高压均质，均质压力250-300bar，均质2min，1350-1400bar均质24min。均质完成后，用量筒量取150ml均质液，加入15g海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。用粒径仪测得粒径为 132.4nm，PDI为0.133。将加入海藻糖后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，用粒径仪检测粒径为123.68nm，PDI 0.144。冻干后榄香烯含量损失率为1.58％。
样品6的制备及稳定性考察
称取1gβ-榄香烯、2g蛋黄卵磷脂，加入50ml超纯水，用混合机5000rpm剪切1min，10000rpm剪切5min，再将粘附于瓶壁上的凝脂刮下至溶液中，再15000rpm剪切3min，至未见明显未分散磷脂，得到初乳。将初乳转移到高压均质机中进行高压均质，均质压力250-300bar，均质2min，1300-1350bar均质12min。完成均质后用0.45μm滤膜过滤，量取50ml 2％的HSA溶液(含HSA 1g)加入到过滤后的均质液中，280bar均质1个循环，将均质液放出，用量筒量取40ml，加入4g海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得白蛋白纳米粒溶液。用粒径仪测得粒径为150.86nm，PDI 0.114。将加入海藻糖后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，用粒径仪检测粒径为181.56nm，PDI为0.251。冻干后榄香烯含量损失率为16.47％。
样品7的制备及稳定性考察
称取1gβ-榄香烯、2g蛋黄卵磷脂、0.5g TPGS，加入50ml超纯水，用混合机5000rpm剪切1min，10000rpm剪切5min，再将粘附于瓶壁上的凝脂刮下至溶液中，再15000rpm剪切3min，至未见明显未分散磷脂，得到初乳。将初乳转移到高压均质机中进行高压均质，均质压力250-300bar，均质2min，1300-1350bar均质12min。完成均质后用0.22μm滤膜过滤，量取50ml 2％的HSA溶液(含HSA 1g)加入到过滤后的均质液中，140bar均质1个循环，将均质液放出，用量筒量取40ml，加入4g海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。用粒径仪测得粒径为93.11nm，PDI 0.131。将加入海藻糖后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，用粒径仪检测粒径为811.70nm，PDI为0.313。冻干后榄香烯含量损失率为82.64％。
实施例1
称取200mgβ-榄香烯和600mg大豆油，溶于2mL有机溶剂中，涡旋混匀，得到油相。所述的有机溶剂是二氯甲烷、或乙酸乙酯或二氯甲烷与乙醇的混合。取人血清白蛋白650mg加入18mL注射用水溶解，得质量体积百分比为3.6％(g/ml)的人血清白蛋白水溶液，即得到水相。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后再通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。
表3有机溶剂单因素考查结果
编号 有机溶剂 粒径(d.nm) PDI
1 无有机溶剂 142.1 0.094
2 乙酸乙酯 122.0 0.164
3 二氯甲烷 108.8 0.129
4 二氯甲烷/乙醇(4:1) 98.5 0.130
实验结果表明，制备过程中无论是否将β-榄香烯和大豆油溶于有机溶剂中均可制备得到粒径适宜的β-榄香烯白蛋白制剂，多种有机溶剂中，二氯甲烷或二氯甲烷与乙醇的混合溶剂制备得到的纳米粒粒径更小。
实施例2
称取200mgβ-榄香烯和600mg大豆油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，2mL)中，涡旋混匀，得到油相体积为2mL。取人血清白蛋白650mg采用注射用水溶解，得质量体积百分比为3.6％(g/ml)人血清白蛋白水溶液，再加入100mg聚乙二醇-12-羟基硬脂酸酯即得到水相。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后再通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒径为116.4nm，PDI为0.118。
实施例3
称取200mgβ-榄香烯和400mg中长链脂肪酸甘油酯，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，2mL)中，涡旋混匀，得到油相体积为2mL。取人血清白蛋白900mg加入18mL注射用水溶解，得质量体积百分比(g/ml)为5.0％人血清白蛋白水溶液，即得到水相。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250w)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒径为105.3nm，PDI为0.116。
实施例4
称取800mgβ-榄香烯和2400mg芝麻油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，10mL)中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水100mL溶解，得质量体积百分比(g/ml)为4％人血清白蛋白水溶液，既得到水相。将油相逐滴滴加到水相中，采用组织捣碎机10000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数20次。均质完成后再通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒 径为121.3nm，PDI为0.133。
实施例5
称取800mgβ-榄香烯和2400mg橄榄油，先后投入二氯甲烷/乙醇混合溶剂(二氯甲烷：乙醇＝4:1，5mL)中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比为4％(g/ml)人血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，采用组织捣碎机12000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数20次。将均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为112.6nm，PDI为0.101。
实施例6
称取200mgβ-榄香烯和600mg大豆油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，10mL)中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比(g/ml)为10％人血清白蛋白水溶液，即得到水相。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒径为140.3nm，PDI为0.164。
实施例7
称取200mgβ-榄香烯和1000mg大豆油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，5mL)中，涡旋混匀，得到油相。取人血清白蛋白650mg加入注射用水溶解，得质量体积百分比(g/ml)为3.6％人血清白蛋白水溶液，即得到水相。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒径为125.3nm，PDI为0.148。
实施例8
称取800mgβ-榄香烯和2400mg大豆油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，10mL)中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比(g/ml)为4％人血清白蛋白水溶液，既得到水相。将油相逐滴滴加到水相中，采用组织捣碎机10000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪 中进行高压均质，均质压力为1000bar，均质次数20次。均质完成后通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。测得粒径为119.3nm，PDI为0.143。取制备的纳米粒溶液配置为药物浓度10mg/ml，加入适量冻干保护剂，冻干保护剂用量为3％(g/ml)，冻干。冻干后加入注射用水复溶，测定粒径。结果如下表所示。
表4不同冻干保护剂冻干后榄香烯白蛋白纳米粒的粒径
冻干保护剂 无 葡萄糖 蔗糖 海藻糖 甘露醇 乳糖 麦芽糖
粒径(nm) 187.4 160.7 153.5 167.4 172.1 166.3 164.5
PDI 0.233 0.165 0.125 0.167 0.133 0.178 0.196
实验结果表明，榄香烯白蛋白纳米粒冻干过程中，加入各种冻干保护剂均可以得到结构疏松，质地均匀的冻干粉末，加水均可复溶，复溶后粒径均不超过200nm。其中，蔗糖作为冻干保护剂冻干后样品复溶所得的纳米粒粒径最小。
实施例9
称取500mgβ-榄香烯和1200mg中长链脂肪酸甘油酯，溶于二氯甲烷与乙醇(二氯甲烷：乙醇＝4:1，5mL)的混合溶剂中，涡旋混匀，得到油相。取人血清白蛋白1500mg加入50mL注射用水溶解，得质量体积百分比(g/ml)为3％人血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成再通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，稀释配置为药物浓度15mg/ml，加入3％蔗糖，冻干，压盖，4℃保存。取冻干后样品加入注射用水复溶，测定粒径。测得粒径为103.5nm，PDI为0.123。
实施例10
称取800mgβ-榄香烯和2400mg芝麻油，先后投入二氯甲烷/异丙醇(二氯甲烷：异丙醇＝4:1，5mL)混合溶剂中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比为4％(g/ml)人血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，采用组织捣碎机10000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数20次。将均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。稀释配置为药物浓度10mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为115.2nm，PDI为0.102。
实施例11
称取800mgβ-榄香烯和2400mg橄榄油，先后投入二氯甲烷/叔丁醇混合溶剂(二氯甲烷：叔丁醇＝4:1，5mL)中，涡旋混匀，得到油相。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比为4％(g/ml)人血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，采用组织捣碎机10000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数20次。将均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。稀释配置为药物浓度10mg/ml，加入3％葡萄糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为109.6nm，PDI为0.113。
实施例12
称取800mgβ-榄香烯和2400mg芝麻油，先后投入二氯甲烷/四氢呋喃混合溶剂(二氯甲烷：四氢呋喃＝4:1，5mL)中，涡旋混匀，得到油相体积为5mL。取人血清白蛋白4000mg加入注射用水溶解，得质量体积百分比为4％(g/ml)人血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，采用组织捣碎机10000rpm，剪切10min，制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数20次。将均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌，即得榄香烯白蛋白纳米粒的水溶液。加入3％海藻糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为112.6nm，PDI为0.101。
实施例13
称取500mgβ-榄香烯和1200mg大豆油，先后投入异丙醇(5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比为3％(g/ml)牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌。稀释配置为药物浓度10mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为128.1nm，PDI为0.132。
实施例14
称取500mgβ-榄香烯和1200mg大豆油，先后投入叔丁醇(5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比为3％(g/ml)牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤 除菌。稀释配置为药物浓度5mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为116.8nm，PDI为0.113。
实施例15
称取500mgβ-榄香烯和1200mg芝麻油，先后投入二氯甲烷/异丙醇混合溶剂(二氯甲烷：异丙醇＝4:1，5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比为3％(g/ml)牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌。稀释配置为药物浓度1mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为121.8nm，PDI为0.111。
实施例16
称取500mgβ-榄香烯和1200mg大豆油，先后投入二氯甲烷/叔丁醇混合溶剂(二氯甲烷：叔丁醇＝4:1，5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比为3％(g/ml)牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌。稀释配置为药物浓度10mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为121.1nm，PDI为0.109。
实施例17
称取500mgβ-榄香烯和1200mg橄榄油，先后投入二氯甲烷/乙醇混合溶剂(二氯甲烷：乙醇＝4:1，5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比(g/ml)为3％牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌。稀释配置为药物浓度12mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为109.7nm，PDI为0.105。
实施例18
称取500mgβ-榄香烯和1200mg大豆油，先后投入二氯甲烷/四氢呋喃混合溶剂(二氯 甲烷：四氢呋喃＝4:1，5mL)中，涡旋混匀，得到油相。取牛血清白蛋白1500mg加入注射用水溶解，得质量体积百分比(g/ml)为3％牛血清白蛋白水溶液，得到水相。将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1400bar，均质次数10次。均质完成后的体系通过旋转蒸发除去有机溶剂。旋蒸后的药液经过0.22μm微孔滤膜过滤除菌。稀释配置为药物浓度10mg/ml，加入3％蔗糖进行冻干，冻干完成后进行压盖，4℃保存。取冻干后样品加入注射用水复溶，测得粒径为112.7nm，PDI为0.112。
实施例19
称取200mg榄香烯和600mg大豆油，溶于二氯甲烷与乙醇的混合溶剂(二氯甲烷：乙醇＝4:1，2mL)中，涡旋混匀，得到油相。取人血清白蛋白650mg采用18mL0.5％氯化钠溶液溶解，得质量体积百分比(g/ml)为3.6％人血清白蛋白溶液。在水浴超声作用下，将油相逐滴滴加到水相中，探头超声8min(250W)制成初乳。将所得初乳转移到高压均质仪中进行高压均质，均质压力为1000bar，均质次数16次。均质完成后的体系通过旋转蒸发除去有机溶剂，0.22μm微孔滤膜过滤除菌，既得榄香烯白蛋白纳米粒的水溶液。测得粒径为126.1nm，PDI为0.122。
实施例20
称取β-榄香烯500mg和大豆油1500mg，溶于5mL二氯甲烷/乙醇(二氯甲烷：乙醇＝8:1)混合溶剂中，得到油相，称取人血清白蛋白2500mg溶解于45mL水中，即得到水相。在超声作用下，使两相混合均匀，将所得溶液转移到高压均质仪进行高压均质，400-500bar 4-6次，1300-1400bar 8-10次，均质完成后再通过旋转蒸发除去有机溶剂，微孔滤膜过滤除菌，静置片刻以除去制剂中的气泡，即得榄香烯白蛋白纳米粒溶液。测定纳米粒粒径为99.4nm，PDI为0.096。将所得的榄香烯白蛋白纳米粒溶液定容至浓度为10mg/ml，加入3％蔗糖，冻干，加入超纯水复溶后测得粒径为101.6nm，PDI为0.113。
实施例21
按照表5所列组分量称取相应的β-榄香烯、大豆油、MCT(中链甘油三酯)和表面活性剂(HS-15，即15-羟基硬脂酸聚乙二醇酯；吐温80；泊洛沙姆P188)混匀，得到油相。量取20％人血清白蛋白(按质量折算如表5所示)，加入超纯水稀释到总体积50ml，混合均匀，得到人血清白蛋白水溶液，即水相。在水相溶液中加入适量的表面活性剂，倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳。将所得初乳转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1450-1550bar均质6min。均质完成后，用量筒测量均质液总体积，按10％(g/ml)加入海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。检测粒 径、PDI，并测定溶血值，具体结果见表5。
表5
Figure PCTCN2020133470-appb-000004
实施例22
按照表6所列组分量称取β-榄香烯、大豆油混匀，得到油相。量取人血清白蛋白(质量折算如表6所示)，加入超纯水稀释到总体积50ml，混合均匀，得到人血清白蛋白水溶液，即水相。水相倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳。将所得初乳转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1450-1550bar均质6min。均质完成后，用量筒测量均质液总体积，按10％质量/体积比(g/ml)加入海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。检测粒径、PDI，并测定溶血值，具体结果见表6。
表6
Figure PCTCN2020133470-appb-000005
实施例23
称取3.33gβ-榄香烯、8g大豆油，混匀，得到油相。量取20％人血清白蛋白(HSA)50ml(10g)，加入适量超纯水稀释均匀，得质量体积百分比(g/ml)为3％的人血清白蛋白水溶液，即水相。将水相溶液倒入油相中，用混合机(FLUKO，FA30/30F)10000rpm剪切10min，形成初乳。将所得初乳转移到微射流均质机(Microfludizer，M-110-EH)中进行均质，均质压力20000psi，均质30min。经0.45μm和0.22μm PVDF平板过滤器进行过滤，定容，分别加入表7中对应的不同的冻干保护剂(g/ml)。将加入冻干保护剂后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉，检测粒径、PDI和溶血值，具体结果见表7。
表7
Figure PCTCN2020133470-appb-000006
实施例24
称取3.33gβ-榄香烯、8g大豆油，溶于有机溶剂中，混匀，得到油相。所述有机溶剂为26.67ml二氯甲烷与6.67ml无水乙醇的混合溶剂。量取20％人血清白蛋白(HSA)50ml(10g)，加入适量超纯水稀释均匀，得质量体积百分比为3％(g/ml)的人血清白蛋白水溶液，即水相。将水相溶液倒入油相中，用混合机(FLUKO，FA30/30F)10000rpm剪切10min，形成初乳。将所得初乳转移到微射流均质机(Microfludizer，M-110-EH)中进行高压均质，均质压力20000psi，均质30min。均质完成后含有机溶剂的乳液通过薄膜蒸发仪除去有机溶剂，参数为：温度30℃，真空度4000pa，进液速度100rpm，刮板速度50rpm。经0.45μm和0.22μm PVDF平板过滤器进行过滤，定容，加入冻干保护剂10％(g/ml)海藻糖。将加入冻干保护剂后的均质液进行冻干，完成冻干后，用灭菌超纯水复溶冻干粉。检测粒径为76.28、PDI为0.254和溶血值为16.3。
实施例25
如表8所列组分量称取β-榄香烯、大豆油，溶于有机溶剂中，所述有机溶剂为1ml乙醇与4ml二氯甲烷的混合溶剂混匀，得到油相。量取适量的20％的人血清白蛋白(HSA)(折合重量如表8所示)，加入适量的超纯水稀释至总体积50ml，即水相。将水相溶液倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳。将所得初乳转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar， 均质2min，1450-1550bar均质6min。用旋转蒸发仪35℃，150rpm，真空度30mbar除去有机溶剂。完成后，用量筒测量均质液总体积，按10％质量/体积比(g/ml)加入海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。
表8
Figure PCTCN2020133470-appb-000007
将制备好的样品放入30℃烘箱，于0h、24h、48h取样，进行粒径、PDI和β-榄香烯含量的测定，测量结果如表9所示。
表9
Figure PCTCN2020133470-appb-000008
实施例26
按照表10所列组分称取β-榄香烯、大豆油，得到油相。量取20％人血清白蛋白(HSA)适量(折合质量如表10所示)，加入适量超纯水稀释至总体积50ml，即水相。将水相溶液倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初 乳。将所得初乳转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1350-1450bar均质6min。完成后，用量筒测量均质液总体积，按质量/体积比(g/ml)加入表10所示的海藻糖，摇匀溶解，用0.45μm微孔滤膜过滤，即得榄香烯白蛋白纳米粒溶液。
表10
Figure PCTCN2020133470-appb-000009
将制备好的样品放入30℃恒温箱，于0h、24h、48h取样，进行粒径、PDI和含量的测定，测量结果如表11所示。
表11
Figure PCTCN2020133470-appb-000010
Figure PCTCN2020133470-appb-000011
实施例27
称取2gβ-榄香烯、1.5g大豆油，混匀，得到油相。量取20％人血清白蛋白45ml(9g)，即水相。将水相溶液倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳。将所得初乳转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1450-1550bar均质6min，收集均质液。用海藻糖水溶液(25ml水+10g海藻糖)冲洗均质机管路，将残留在均质机内的均质液置换出来，再用30ml水超纯水冲洗均质机管路，将残留的海藻糖水溶液置换出来，与均质液合并，混匀，用0.22μm无菌滤膜过滤。用粒径仪检测得到粒径106.4nm，PDI为0.08。
实施例28
表13
Figure PCTCN2020133470-appb-000012
称取表13所示的β-榄香烯、大豆油，混匀，得到油相。量取20％人血清白蛋白(50ml，10g)，即水相。将水相溶液倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳，将所得液体转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1500-1550bar均质6min，称取海藻糖10g，加入纯水溶 解至50ml(其中5号样品是加入海藻糖15g，加入纯水溶解至105ml)，将此海藻糖水溶液加入均质机中冲洗均质机管路，将残留在均质机内的均质液置换出来，与均质液合并，混匀，用0.45μm无菌滤膜过滤。测试各样品的溶血值。并将样品分装至EP管中，存放于30℃恒温箱，于第0h、24h、48h取样进行粒径、PDI和β-榄香烯含量的测定，结果见表14。
表14
Figure PCTCN2020133470-appb-000013
实施例29
称取2gβ-榄香烯、1.5g大豆油，混匀，得到油相。量取20％人血清白蛋白50ml(10g)，即水相。将水相溶液倒入油相中，用混合机(IKA，T25)5000rpm剪切1min，10000rpm剪切5min，形成初乳，并用5ml注射用水清洗剪切机头，与初乳合并。将所得液体转移到高压均质机(ATS，AH-1500)中进行高压均质，均质压力250-300bar，均质2min，1500-1550bar均质6min，收集均质液。用22.5ml海藻糖水溶液(含海藻糖5g)冲洗均质机管路，将残留在均质机内的均质液置换出来，再用22.5ml超纯水冲洗均质机管路，将残留的海藻糖水溶液置换出来，与均质液合并，混匀，用0.22μm无菌滤膜过滤。将样品分装至EP管中，存放于2-8℃冰箱，于第0天和2个月分别取样检测粒径、PDI，结果见表12。
表12
时间 粒径 PDI
第0天 87.93 0.107
2个月 89.39 0.119
实施例30药效研究
样品的制备：
称取β-榄香烯4.5g，大豆油10.8g，将二者混合均匀，得到油相。量取乙醇4ml，二氯甲烷16ml，混合均匀得有机相A。将油相加有机相定溶至36ml，得有机相B。
称取人血清白蛋白13.5g加至405ml纯化水中，搅拌溶解，将有机相B加入到水相中，使用高速剪切机A档搅拌2min，然后B档搅拌1min，得到初乳A，初乳A分3份，用细胞破碎仪250w 8min得初乳B。将初乳B加入高压均质机中，先200bar 3个循环，再1300bar 5个循环，得白蛋白纳米粒。将上述纳米粒于37℃下减压浓缩1h，加入3％蔗糖12.30g，0.22μm过滤，进行冻干。粒径81.03nm、PDI为0.174。
实验方法：
人源脑肿瘤异种移植小鼠模型的建立：用2.5％异氟烷麻醉50只裸鼠，在裸鼠头骨上钻一个小洞(<0.5毫米)，将表达发光报告基因的细胞(5x10 4个U87MGR1人脑胶质瘤细胞)注射入左前额(2.5mm横向和0.5mm前囟前，深度2.5mm)。
活体生物发光成像测定方法：裸鼠皮下单次注射D-荧光素溶液(120mg/kg)。注射荧光素10分钟后，在1-5分钟的曝光时间内，使用暗多色照明拍摄动物的光图像(对于Gluc的生物发光成像，单次静脉注射腔肠素(20mg/kg，总体积为150微升))。肿瘤内的荧光素酶活性是在没有照明的情况下，用CCD摄像机记录超过5分钟的光子计数来获得的。使用Perkin Elmer公司的Living image软件4.3.1定量分析图像强度。
裸鼠接种1周后，进行活体生物发光成像评估肿瘤体积，选取32只肿瘤体积相近的裸鼠随机分组，8只小鼠(雌雄各半)/组，尾静脉注射给药3周，3次/周：
1)模型组(control)(PBS配制0.1％DMSO)，n＝8；
2)50mg/kg替莫唑胺(TMZ)，n＝8；
3)50mg/kg艾利能(ELE mic)，n＝8；
4)50mg/kg(Alb)，n＝8；
每周检测肿瘤体积一次。
在实验过程中，动物将每周称重以监测其健康状况。
给药两周停药，随后观察三周。使用Graphpad软件记录各组小鼠的存活情况以及肿瘤体积，生成Kaplan-Meier图。小鼠原位瘤模型的肿瘤生长曲线见图8(使用Alb制剂处理荷瘤裸鼠，3次/周×2周(n＝8，雌雄各半)；与替莫唑胺(TMZ)相比较，p<0.01)，小鼠原位瘤模型的生存曲线见图9(使用Alb处理荷瘤裸鼠，3次/周×2周(n＝8，雌雄各半)；与替莫唑胺(TMZ)相比较，p<0.01)。
实施例31安全性研究
样品的制备：
榄香烯注射液(商品名：艾利能)：石药集团远大(大连)制药有限公司(批号：17050302)
β-榄香烯胶束制剂：
称取RH40 17.08g，60℃加热溶解，加入2mlβ-榄香烯，60℃下加热搅拌5min，称取丙二醇14.40g，纯化水38.72g，混匀后60℃加热10min。将丙二醇水溶液加入至含β-榄香烯的RH40溶液中，60℃水浴搅拌30min，即得β-榄香烯胶束。将上述胶束于0.22μm过滤，分装成5ml/支。
β-榄香烯白蛋白制剂：
称取β-榄香烯2.5001g，大豆油6.001g，将二者混合均匀，备用。量取乙醇2ml，二氯甲烷8ml，混合均匀备用。将油相用有机相定溶至12ml备用。
称取人血清白蛋白4.5015g加至135ml纯化水中，搅拌溶解，将有机相加入到水相中，使用高速剪切机A档搅拌2min，然后B档搅拌0.5min，得到初乳A，初乳A分3份，用细胞破碎仪250w 8min得初乳B。将初乳B加入高压均质机中，先200bar 3个循环，再1300-1400bar 5个循环，得白蛋白纳米粒。将上述纳米粒于37℃下减压浓缩1h，加入蔗糖1.20g，0.22μm过滤，进行冻干。
溶液配置：
使用生理盐水，将三种制剂分别配置成6mg/mL的溶液。
艾利能：取20mg/mL的市售艾利能7.5mL，用17.5mL生理盐水稀释成25ml 6mg/mL的样品溶液。
β-榄香烯胶束制剂：取25.41mg/mL的β-榄香烯胶束制剂5.91mL，用19.09mL的生理盐水，稀释成25ml 6mg/mL的样品溶液。
β-榄香烯白蛋白制剂：取12支β-榄香烯白蛋白制剂，每支加入2.01mL生理盐水，混合既得24ml 6mg/mL的样品溶液。
试验方法：
取雄性KM小鼠，20-25g/只，按体重随机分为13组，每组7只。根据体重按照既定给药剂量分组，每个样品剂量设定：100mg/kg、130mg/kg、170mg/kg、220mg/kg，另设一组空白组。对小鼠进行尾静脉注射，给药完成后观察给药部位、给药后反应和死亡情况。研究结果见表15。
表15不同样品的安全性研究
Figure PCTCN2020133470-appb-000014
Figure PCTCN2020133470-appb-000015

Claims (68)

  1. 药物组合物，其包含榄香烯、注射用油和蛋白载体。
  2. 根据权利要求1所述的药物组合物，其中所述药物组合物为颗粒状，所述颗粒的粒径为小于180nm，优选50-180nm。
  3. 根据权利要求2所述的药物组合物，其中所述颗粒的粒径约为70-170nm。
  4. 根据权利要求3所述的药物组合物，其中所述颗粒的粒径约为70-150nm。
  5. 根据权利要求1所述的药物组合物，其中所述榄香烯选自α-榄香烯、β-榄香烯、γ-榄香烯以及δ-榄香烯的一种或多种。
  6. 根据权利要求5所述的药物组合物，其中所述榄香烯为β-榄香烯。
  7. 根据权利要求1所述的药物组合物，其中所述蛋白载体为白蛋白，所述白蛋白选自人血清蛋白或牛血清蛋白的一种或两种。
  8. 根据权利要求7所述的药物组合物，其中所述蛋白载体为人血清蛋白。
  9. 根据权利要求1所述的药物组合物，其中所述白蛋白载体与榄香烯的重量比为0.5-20。
  10. 根据权利要求9所述的药物组合物，其中所述白蛋白载体与榄香烯的重量比为0.5-10。
  11. 根据权利要求1所述的药物组合物，其中所述注射用油与榄香烯的重量比为0.35-10。
  12. 根据权利要求11所述的药物组合物，其中所述注射用油与榄香烯的重量比为0.5-10。
  13. 根据权利要求1-12中任一项所述的药物组合物，其中所述注射用油为大豆油、红花油、棉籽油、玉米油、向日葵油、花生油、橄榄油、芝麻油、茶油、中长链脂肪酸甘油酯或中链甘油三酯的一种或多种。
  14. 根据权利要求1-13中任一项所述的药物组合物，其还包含冻干保护剂。
  15. 根据权利要求14所述的药物组合物，其中所述冻干保护剂选自葡萄糖、蔗糖、麦芽糖、乳糖、甘露糖、海藻糖、甘氨酸、右旋糖酐中的一种或几种。
  16. 根据权利要求15所述的药物组合物，其中所述冻干保护剂为海藻糖或蔗糖。
  17. 根据权利要求14-16所述的药物组合物，其中所述冻干保护剂的重量与所述药物组合物的溶液的体积之比为3:100-20:100g/mL。
  18. 根据权利要求1-16中任一项所述的药物组合物，其还包含等渗调节剂、抗氧化剂、防腐剂、pH调节剂中的一种或几种。
  19. 根据权利要求18所述的药物组合物，其中所述等渗调节剂为甘油、山梨醇、甘露醇或葡萄糖的一种或多种。
  20. 根据权利要求18所述的药物组合物，其中所述pH调节剂为氢氧化钠、柠檬酸钠、柠檬酸、磷酸、醋酸或盐酸的一种或多种。
  21. 根据权利要求18所述的药物组合物，其中所述防腐剂选自羟苯烷基酯类、苯甲酸、苯甲酸钠、山梨酸、醋酸氯乙定、苯扎溴胺的一种或多种。
  22. 根据权利要求18所述的药物组合物，其中所述抗氧化剂选自亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、抗坏血酸、叔丁基对羟基茴香醚、2,6-二叔丁基化羟基甲苯、维生素E的一种或多种。
  23. 根据权利要求1-22所述的药物组合物，其在制备过程中不含有有机溶剂。
  24. 根据权利要求23所述的药物组合物，其中所述药物组合物被制成颗粒，其中所述颗粒的粒径小于180nm。
  25. 根据权利要求24所述的药物组合物，其中所述颗粒的粒径约为70-150nm。
  26. 根据权利要求23-25所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.35-3；所述人血白蛋白与榄香烯的重量比约为0.5-20。
  27. 根据权利要求26所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.5-3；所述人血白蛋白与榄香烯的重量比约为0.5-2.5。
  28. 根据权利要求26所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.35-1.5，优选0.35-0.75；所述人血白蛋白与榄香烯的重量比约为1.5-10。
  29. 根据权利要求28所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.5；所述人血白蛋白与榄香烯的重量比约为1.5。
  30. 根据权利要求28所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为0.75；所述人血白蛋白与榄香烯的重量比约为5。
  31. 根据权利要求28所述的药物组合物，其中所述药物组合物中所述大豆油与榄香烯的重量比约为1.5；所述人血白蛋白与榄香烯的重量比约为3-5。
  32. 根据权利要求1-22所述的药物组合物，其在制备过程中含有有机溶剂。
  33. 根据权利要求32所述的药物组合物，其中所述有机溶剂选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或几种。
  34. 根据权利要求33所述的药物组合物，其中所述有机溶剂为二氯甲烷与乙醇的混合溶剂。
  35. 根据权利要求34所述的药物组合物，其中所述二氯甲烷与乙醇的体积比1:1-8。
  36. 根据权利要求35所述的药物组合物，其中所述二氯甲烷与乙醇的体积比1:4。
  37. 根据权利要求33-36任一项所述的药物组合物，其特征在于所述颗粒的粒径约为70-150nm。
  38. 根据权利要求37所述的药物组合物，其特征在于所述药物组合物中所述大豆油和榄香烯的重量比约为2.4-10；所述人血白蛋白和榄香烯的重量比约为0.5-10。
  39. 根据权利要求38所述的药物组合物，其特征在于所述药物组合物中所述大豆油和榄香烯的重量比约为2.4；所述人血白蛋白和榄香烯的重量比约为3。
  40. 制备权利要求1-39中任一项所述的药物组合物的方法，其包括以下步骤：
    (1)将榄香烯与注射用油混合均匀，任选地加入有机溶剂，得到榄香烯和注射用油的溶液；将蛋白载体溶解于水，得到蛋白载体溶液；
    (2)将步骤(1)中获得的两份溶液混合形成乳液；
    (3)将步骤(2)中所述的乳液高压均质，任选地将所述乳液减压蒸发除去有机溶剂，得到纳米溶液。
  41. 根据权利要求40所述的方法，其中步骤(1)使用有机溶剂，优选将榄香烯溶解于有机溶剂中再和注射用油混合。
  42. 根据权利要求41所述的方法，其中所述注射用油为大豆油，所述白蛋白载体为人血白蛋白，所述大豆油与榄香烯的重量比约为2.4-10；所述人血白蛋白与榄香烯的重量比约为0.5-10。
  43. 根据权利要求40所述的方法，其还包括将得到的蛋白载体纳米粒溶液冻干的步骤。
  44. 根据权利要求40所述的方法，其中在所述步骤(2)中采用剪切或超声的方法进行混合，步骤(3)中采用高压均质或微射流均质的方法进行均质。
  45. 权利要求1-39中任一项所述的药物组合物在制备用于预防或治疗癌症的药物中的用途。
  46. 根据权利要求45所述的用途，其中述癌症为肾上腺皮质癌、原因不明的髓样化生、AIDS相关的癌、肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、神经胶质瘤、室管膜瘤、少突神经胶质瘤、脑膜瘤、颅咽管瘤、成血管细胞瘤、成神经管细胞瘤、神经外胚层肿瘤、视觉通路和下丘脑神经胶质瘤和恶性胶质瘤、乳腺癌、支气管腺瘤、类癌瘤、中枢神经系统淋巴瘤、子宫颈癌、结肠癌、结肠直肠癌、慢性骨髓增生性疾病、子宫内膜癌、室管膜瘤、食管癌、尤文肿瘤家族、眼癌、胆囊癌、胃肠道类癌瘤、胃肠道间质瘤、生殖细胞癌瘤、妊娠滋养层肿瘤、头颈癌、肝癌、喉癌、白血病、唇及口腔癌、肺癌、淋巴瘤、成神经管细胞瘤、黑素瘤、间皮瘤、转移性颈部鳞状细胞癌、多发性内分泌瘤形成综合征、骨髓增生异常综合征、骨髓增生异常/骨髓增生性疾病、鼻腔和鼻窦癌、鼻咽癌、成神经细胞瘤、神经内分泌癌、口咽癌、脑肿瘤、骨转移癌、胃癌、肠癌、食道癌、卵巢癌、胰腺癌、乳腺癌、皮肤癌、甲状旁腺癌、阴茎癌、腹膜癌、咽癌、嗜铬细胞瘤、成松果体细胞瘤和幕上原始神经外胚层肿瘤、垂体瘤、胸膜肺的胚细胞瘤、淋巴瘤、原发性中枢神经系统淋巴瘤、肺淋巴管肌瘤病、直肠癌、肾癌、肾盂及输尿管癌、横纹肌肉瘤、唾腺癌、皮肤癌、小肠癌、鳞状上皮细胞癌、睾丸癌、咽喉癌、胸腺瘤及胸腺癌、甲状腺癌、尿道癌、或阴道癌。
  47. 根据权利要求46所述的用途，其中所述癌症为肺癌、肝癌、食道癌、鼻咽癌、脑肿瘤，骨转移癌、胃癌、肠癌、子宫癌、子宫颈癌、生殖细胞癌瘤、子宫内膜癌、妊娠滋养层肿瘤、乳腺癌、皮肤癌、淋巴癌、白血病、或恶性黑色素瘤。
  48. 根据权利要求47所述的用途，其中所述癌症为脑肿瘤，优选为神经胶质瘤、脑干 神经胶质瘤、小脑或大脑的星形细胞瘤、恶性神经胶质瘤、室管膜瘤、少突神经胶质瘤、脑膜瘤、颅咽管瘤、成血管细胞瘤、成神经管细胞瘤、视觉通路和下丘脑神经胶质瘤或恶性胶质瘤。
  49. 根据权利要求48所述的用途，其中所述小脑或大脑的星形细胞瘤为纤维状细胞星形细胞瘤或弥漫星形细胞瘤或间变性(恶性)星形细胞瘤。
  50. 治疗人类疾病的方法，包括向由此需要的对象给予治疗有效量的权利要求1-37中任一项所述的药物组合物。
  51. 根据权利要求50所述的方法，其中所述药物组合物的给药方式为通过非肠道、通过吸入，腹腔内、膀胱内，肌肉内，静脉内、气管内，皮下，眼内，鞘内、透皮给药、直肠或阴道内。
  52. 根据权利要求51所述的方法，其中所述给药方式为静脉内给药。
  53. 药物制剂，其包含权利要求1-39中任一项所述的药物组合物。
  54. 根据权利要求53所述的药物制剂，其中所述药物制剂为静脉给药的药物制剂、胃肠外给药的药物制剂、胃肠给药的药物制剂、呼吸道给药的药物制剂、阴道给药的药物制剂或其它合适的药物制剂；所述呼吸道给药的药物制剂为气雾剂。
  55. 根据权利要求53所述的药物制剂，其中所述药物制剂为固体制剂、液体制剂或气体制剂。
  56. 根据权利要求53所述的药物制剂，其中所述液体制剂为注射用液体制剂，其包含榄香烯和药学上可接受的载体，其中所述药学上可接受的载体包括白蛋白和大豆油。
  57. 根据权利要求55所述的药物制剂，其为颗粒剂，所述颗粒剂的粒径70-150nm，其中所述组合物中白蛋白与榄香烯的重量比为为0.5-20，所述注射用油与榄香烯的重量比为0.35-10。
  58. 根据权利要求55所述的药物制剂，其中所述液体制剂是由无菌冻干粉重建的稳定的水悬浮液。
  59. 根据权利要求53所述的药物制剂，其还包含其它药物，所述其它药物包括抗癌药物。
  60. 根据权利要求59所述的药物制剂，所述抗癌药物包括替莫唑胺、紫杉醇、多西他赛、紫杉烷、吉西他滨、抗VEGF药物或PD-1抗体药物。
  61. 根据权利要求60所述的药物制剂，其中抗VEGF药物包括AVASTIN、雷珠单抗、阿柏西普或康柏西普。
  62. 根据权利要求56所述的药物制剂，其中所述PD-1抗体药物包含纳武利尤单抗、帕博丽珠单抗、特瑞普利单抗、信迪利单抗、西米普利单抗、阿特珠单抗、阿维单抗或度伐利尤单抗。
  63. 密封容器，其包含权利要求1-39中任一项所述的药物组合物或权利要求53-62中任一项所述的药物制剂。
  64. 根据权利要求63所述的密封容器，其中所述密封容器为单位剂量容器或多剂量容器。
  65. 根据权利要求63所述的密封容器，其中所述药物组合物为液体组合物或干燥固体组合物。
  66. 根据权利要求63所述的密封容器，其中所述药物组合物是冻干的。
  67. 根据权利要求63所述的密封容器，其中所述药物组合物是无菌的。
  68. 根据权利要求63所述的密封容器，其中所述密封容器为预填充注射器。
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