WO2023066044A1 - 外泌体冻干粉针剂或外泌体液体制剂的保护剂 - Google Patents

外泌体冻干粉针剂或外泌体液体制剂的保护剂 Download PDF

Info

Publication number
WO2023066044A1
WO2023066044A1 PCT/CN2022/124068 CN2022124068W WO2023066044A1 WO 2023066044 A1 WO2023066044 A1 WO 2023066044A1 CN 2022124068 W CN2022124068 W CN 2022124068W WO 2023066044 A1 WO2023066044 A1 WO 2023066044A1
Authority
WO
WIPO (PCT)
Prior art keywords
exosome
exosomes
freeze
sorbitol
mannitol
Prior art date
Application number
PCT/CN2022/124068
Other languages
English (en)
French (fr)
Inventor
王伊
徐铭枝
董亚南
周伟
Original Assignee
谛邈生物科技(北京)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 谛邈生物科技(北京)有限公司 filed Critical 谛邈生物科技(北京)有限公司
Publication of WO2023066044A1 publication Critical patent/WO2023066044A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of pharmaceutical preparations, in particular to a protective agent for exosome freeze-dried powder injection or exosome liquid preparation.
  • exosomes In the study of exosomes, there are still few studies on the preparation of exosomes. The preparations used clinically are mainly liquid preparations. Only a few scientific studies have mentioned lyophilized preparations of exosomes. For the clinical application of exosomes, there is insufficient research on formulation development. Through the study of various preparation excipients, we have determined the excipient formulations of milk-derived exosomes and cell-derived exosome liquid preparations, freeze-dried powder injection preparations, and sublingual tablets.
  • exosome lyophilized and liquid preparations derived from milk exosomes and cell-derived exosomes, and excipient formulations for sublingual tablets is a necessary step for exosome-based innovative drugs to move from research laboratories to clinical applications, and is of great importance. practical significance.
  • the present invention provides exosome freeze-dried powder injection or exosome liquid formulation of auxiliary materials.
  • the present invention solves the problem of fragmentation and aggregation of exosomes derived from milk and exosomes derived from cells under freeze-dried and frozen storage conditions respectively, and determines liquid preparations, freeze-dried preparations and sublingual preparations of exosomes from different sources.
  • the present invention provides the application of one or more of mannitol, sorbitol, sucrose, arginine or glycine as a protective agent in the preparation of exosome freeze-dried powder injection or exosome liquid preparation.
  • the exosomes include milk-derived exosomes or cell-derived exosomes.
  • the content of mannitol is 1% ⁇ 10%, 1% ⁇ 6% or 1% ⁇ 12%; the content of sorbitol is 1% ⁇ 15%, 1% ⁇ 10% or 3%-15%; the content of sucrose is 1%-10%, 6%-25% or 2.5%-35%; the content of arginine is 0.01M-0.5M; the content of glycine is 0.01M-1M.
  • the present invention also provides a protective agent for preparing exosome freeze-dried powder injection or exosome liquid preparation, including one or more of mannitol, sorbitol, sucrose, arginine or glycine.
  • the content of mannitol is 1%-10%, 1%-6% or 1%-12%; the content of sorbitol is 1%-15%, 1% % ⁇ 10% or 3% ⁇ 15%; the content of sucrose is 1% ⁇ 10%, 6% ⁇ 25% or 2.5% ⁇ 35%; the content of arginine is 0.01M ⁇ 0.5M; the content of glycine is 0.01 M ⁇ 1M.
  • the exosome freeze-dried powder injection is milk exosome freeze-dried powder injection, which is characterized in that it includes a combination of 1%-10% mannitol and 1%-15% sorbitol or a combination of 1% to 10% sucrose and 1% to 15% sorbitol.
  • the exosome liquid preparation is a milk exosome liquid preparation, including 6% to 25% sucrose, 1% to 10% sorbitol, 0.01M to 0.5M arginine and A composition of 0.01M-1M glycine.
  • the exosome liquid preparation is a cell-derived exosome freeze-dried powder injection, comprising a composition of 1%-6% mannitol and 3%-15% sorbitol.
  • the exosome liquid preparation is a liquid preparation of cell-derived exosomes, comprising a composition of 2.5% to 35% sucrose and 1% to 12% mannitol, wherein sucrose and /or Mannitol can be replaced by sorbitol.
  • the present invention also provides the application of the protective agent in the preparation of exosome freeze-dried powder injection or exosome liquid preparation.
  • the present invention also provides exosome freeze-dried powder injection or exosome liquid preparation, including exosome and the protective agent.
  • the present invention has determined milk-derived exosomes and cell-derived exosome liquid preparations, freeze-dried powder injection preparations, and auxiliary material formulas for sublingual tablets through research on various preparation auxiliary materials (selected disintegrating agent: microcrystalline fiber
  • selected disintegrating agent microcrystalline fiber
  • selected fillers microcrystalline cellulose, microcrystalline cellulose-mannitol, microcrystalline cellulose-micronized silica gel, lactose, starch, modified starch, mannitol, sorbitol, xylitol, erythritol Thucose, trehalose, pregelatinized starch, powdered sugar, glucose, dextrin, calcium sulfate, any one or a mixture of two or more
  • selected flavoring agents stevio
  • the optimal formula is a combination of 1%-10% mannitol and 1%-15% sorbitol, in which mannitol can be replaced by sucrose;
  • the optimal formula is 6%-25% sucrose, 1%-10% sorbitol, 0.01M-0.5M arginine, 0.01M-1M glycine, which have the same effect on milk exosomes. have obvious protective effects;
  • the optimal formula is 1%-6% mannitol, and 3%-15% sorbitol has a protective effect;
  • the optimal formula is 2.5%-35% sucrose and 1%-12% mannitol, which has the best effect on sample preservation. Among them, sucrose and mannitol can be replaced by sorbitol.
  • the optimum formula is for every 1000 exosome sublingual tablets, the content of the main drug is 10 5 -10 13 particles, and the content of croscarmellose sodium is 1-50 g (cross-linked sodium cellulose can be used) Polyvinylpyrrolidone, microcrystalline cellulose substitute), starch content is 20-400g (can be replaced by xylitol, lactose, mannitol); aspartame 0-50g (can be replaced by powdered sugar), pure water 5-100g (can be replaced by starch slurry), talcum powder 1g-10g (can be replaced by magnesium stearate).
  • Fig. 1 shows the slow freezing result of the freezing box; wherein, Fig. 1 (A) is the sample particle number; Fig. 1 (B) shows the purity;
  • Fig. 2 shows liquid nitrogen quick freezing result; Wherein, Fig. 2 (A) is sample particle number; Fig. 2 (B) shows purity;
  • Figure 3 shows the preservation effect of different concentrations of sucrose and sorbitol on the cryopreservation of milk exosomes; wherein, Figure 3 (A) shows the purity; Figure 3 (B) shows the number of sample particles;
  • Figure 4 shows the protective effect of different excipient combinations on the cryopreservation of milk exosomes; wherein, Figure 4(A) shows the purity; Figure 4(B) shows the number of sample particles;
  • Figure 5 shows the protective effects of different cryoprotectants on cell-derived exosomes under cryopreservation conditions at -80°C; among them, Figure 5(A) shows the purity; Figure 5(B) shows the number of sample particles;
  • Figure 6 shows the preservation effect of different exosome combinations under freeze-drying conditions; wherein, Figure 6(A) shows the purity; Figure 6(B) shows the number of sample particles;
  • Figure 7 shows the preservation effect of exosomes after further optimization for 3% mannitol+5% sorbitol conditions under freeze-drying conditions; wherein, Figure 7 (A) shows the purity; Figure 7 (B) shows the number of sample particles; 7(C) shows the retention map of frozen samples;
  • Figure 8 shows the protective effect of the combination of lyoprotectants on cell-derived exosomes under freeze-drying conditions; wherein, Figure 8(A) shows the purity; Figure 8(B) shows the number of sample particles;
  • Fig. 9 shows a comparison chart of purity and particle number between the existing preservation technology (PBS) of milk exosome liquid preparation and the technology of the present invention (5% sorbitol);
  • Fig. 10 shows the comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of milk exosome freeze-dried powder injection;
  • Fig. 11 shows the comparison chart of the purity and the number of particles between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of the exosome liquid preparation;
  • Fig. 12 shows the comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of the cell exosome freeze-dried powder injection;
  • Figure 13 shows a pharmacokinetic profile
  • the invention discloses a protective agent for exosome freeze-dried powder injection or exosome liquid preparation.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. What needs to be pointed out in particular is that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • exosomes used in clinical research are solution preparations stored at -80°C.
  • Preparations stored at low temperature have disadvantages such as troublesome transportation, high cost, and high requirements for storage equipment.
  • storing exosomes at low temperature without adding excipients will cause exosomes to rupture and aggregate.
  • the present invention solves the problem of fragmentation and aggregation of exosomes derived from milk and exosomes derived from cells under freeze-dried and frozen storage conditions respectively, and determines liquid preparations and freeze-dried preparations of exosomes from different sources.
  • Optimal storage method for tablets are Optimal storage method for tablets.
  • the raw materials and reagents used in the exosome freeze-dried powder injection or the protective agent of the exosome liquid preparation provided by the present invention can be purchased from the market.
  • Exosomes have a lipid bilayer structure similar to the cell membrane structure.
  • the most commonly used freezing method for cells is gradient cooling.
  • exosomes are derived from cell secretion, and cell secreted proteins are usually frozen by liquid nitrogen quick freezing. During the cryopreservation process, exosomes will aggregate and rupture.
  • Sucrose, arginine, glycine, and urea are commonly used excipients, which are helpful for drug dispersion.
  • DMSO is a commonly used cryopreservation solution for cells, which can prevent cell rupture.
  • group A was frozen at -80°C in a cell freezing box, and group B was quickly frozen with liquid nitrogen, and stored at -80°C for 2 days
  • Nanoflow NanoFCM detects the particle number and purity of the sample
  • Exosomes have a lipid bilayer structure similar to the cell membrane structure. Exosomes from different sources have different optimal storage conditions. Different excipient ingredients and different concentrations have different protective effects on samples.
  • Nanoflow NanoFCM detects the particle number and purity of the sample
  • sucrose Compared with other concentrations of sucrose, 25% sucrose has the best protective effect on milk exosomes 2) Compared with other concentrations of sorbitol, 5% sorbitol has the best protective effect on milk exosomes
  • Exosomes have a lipid bilayer structure similar to the cell membrane structure. Exosomes from different sources have different optimal storage conditions.
  • Cell-derived exosomes Cell supernatants were filtered sequentially with 0.2 ⁇ m filter membranes. The initially purified sample is first filtered by TFF, and then purified by core700 to obtain cellular exosomes.
  • Nanoflow NanoFCM detects the particle number and purity of the sample.
  • Exosomes have a lipid bilayer structure similar to the cell membrane structure. Exosomes from different sources have different optimal storage conditions.
  • Nanoflow NanoFCM detects the particle number and purity of the sample.
  • Exosomes have a lipid bilayer structure similar to the cell membrane structure. Exosomes from different sources have different optimal storage conditions.
  • Nanoflow NanoFCM detects the particle number and purity of the sample.
  • Step 1 Freeze-dry the main ingredient and sieve it to 80-120 mesh;
  • Step 2 Dry the filler, disintegrant, flavoring agent, and lubricant, pulverize, and sieve with 80-120 mesh for pretreatment;
  • Step 3 Mix the pretreated exosome freeze-dried powder, filler, flavoring agent and disintegrant evenly;
  • Step 4 Add the binder to the above-mentioned material mixed well to make a soft material, granulate, and dry;
  • Step 5 add lubricant to the dry granules and mix them evenly, and then compress them into tablets to obtain the finished product.
  • Obtained sublingual tablet carries out animal efficacy evaluation:
  • exosome preservation and preparations for clinical research are mainly liquid preparations, without freeze-dried agents and sublingual tablets, and liquid preparations are directly frozen without adding excipients, which will cause exosomes to aggregate, rupture, and affect Efficacy of exosomes. And there is no preparation process for milk exosomes and cell-derived exosomes.
  • the present invention solves the liquid preparations of milk exosomes and cell-derived exosomes, the preparation process development of freeze-dried powder injections and sublingual tablets, and develops a new type of exosome preparations.
  • the comparative data shall be reviewed by the examiner:
  • Control group PBS was used as cryopreservation agent
  • Method 1 Add 500uL 10% sorbitol to 500uL fresh milk exosome sample, add 500uLPBS to 500uL sample of the control group, freeze it in liquid nitrogen, and store it at -80°C; when the sample is tested, melt the sample at 4°C and centrifuge at 12000rpm for 10min , the supernatant was taken, and the concentration of the sample was detected by NanoFCM. After the sample was digested with 1% Triton, the purity of the sample was detected and calculated by NanoFCM.
  • Method 2 Add 500uL 12% mannitol to 500uL fresh cell exosome samples, add 500uLPBS to 500uL samples of the control group, freeze them in liquid nitrogen, and store them at -80°C; when testing samples, melt the samples at 4°C and centrifuge at 12000rpm for 10min , the supernatant was taken, and the concentration of the sample was detected by NanoFCM. After the sample was digested with 1% Triton, the purity of the sample was detected and calculated by NanoFCM.
  • Method 3 Add 500uL 12% mannitol to 500uL fresh milk or cell exosome samples, add 500uLPBS to 500uL samples of the control group, freeze in liquid nitrogen, store at -80°C after quick freezing in liquid nitrogen, and freeze dry in a freeze dryer ;
  • sample detection after the samples of the experimental group and the control group were redissolved with 1mL H 2 O, centrifuged at 12000rpm for 10min, the supernatant was taken, and the concentration of the sample was detected by NanoFCM. After the sample was digested with 1%Trtion, the purity of the sample was detected and calculated by NanoFCM .
  • Fig. 9 shows a comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (5% sorbitol) of milk exosome liquid preparation.
  • the sample was added with 5% sorbitol as a protective agent, and the purity and number of particles of the sample were significantly higher than that of the PBS group, indicating that 5% sorbitol can effectively protect the structural integrity and dispersibility of the sample during cryopreservation.
  • Fig. 10 shows a comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of milk exosome freeze-dried powder injection. Adding 6% mannitol as an excipient in the sample, the purity and number of particles of the sample were significantly higher than that of the PBS group, indicating that 6% sorbitol can effectively protect the structural integrity and dispersibility of the sample during freeze-drying.
  • PBS existing preservation technology
  • 6% mannitol the technology of the present invention
  • Fig. 11 shows the comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of the exosome liquid preparation.
  • PBS existing preservation technology
  • 6% mannitol 6% mannitol
  • the sample was added with 6% sorbitol as a protective agent, and the purity and particle number of the sample were significantly higher than that of the PBS group, indicating that 6% sorbitol can effectively protect the structural integrity and dispersion of the sample during cryopreservation.
  • Fig. 12 shows the comparison chart of purity and particle number between the existing preservation technology (PBS) and the technology of the present invention (6% mannitol) of cell exosome freeze-dried powder injection.
  • PBS existing preservation technology
  • 6% mannitol 6% mannitol
  • the sample was added with 6% sorbitol as a protective agent, and the purity and particle number of the sample were significantly higher than that of the PBS group, indicating that 6% sorbitol can effectively protect the structural integrity and dispersion of the sample during cryopreservation.
  • 5% sorbitol has obvious protective effect on milk exosome liquid preparation
  • 6% mannitol has obvious protective effect on milk exosome freeze-dried powder injection, cell exosome freeze-dried powder injection or liquid preparation.
  • the protective effect can maintain the stability of exosomes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明涉及药物制剂领域，特别涉及外泌体冻干粉针剂或外泌体液体制剂的保护剂。本发明通过多种制剂辅料的研究，确定了牛奶来源外泌体和细胞来源外泌体液体制剂和冻干粉针制剂，舌下片剂的辅料配方。

Description

外泌体冻干粉针剂或外泌体液体制剂的保护剂
本申请要求于2021年10月20日提交中国专利局、申请号为202111222193.1、发明名称为“外泌体冻干粉针剂或外泌体液体制剂的保护剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物制剂领域，特别涉及外泌体冻干粉针剂或外泌体液体制剂的保护剂。
背景技术
外泌体的研究中，外泌体的制剂研究尚少。临床所用的制剂以液体制剂为主。只有少量科研研究中提到外泌体的冻干制剂。对于外泌体的临床应用，制剂开发研究不足。我们通过多种制剂辅料的研究，确定了牛奶来源外泌体和细胞来源外泌体液体制剂和冻干粉针制剂，舌下片剂的辅料配方。
表1
Figure PCTCN2022124068-appb-000001
Figure PCTCN2022124068-appb-000002
目前，临床研究所用到外泌体多数是-80℃储存的溶液制剂。低温储存的制剂存在运输麻烦，成本高，保存设备要求高等缺点。而且在不添加辅料条件下，低温储存外泌体，会造成外泌体破裂，聚集。
因此，提供开发牛奶外泌体和细胞来源的外泌体冻干和液体制剂，舌下片剂的辅料配方，是基于外泌体的创新药从科研实验室走向临床应用的必要步骤，具有重要的现实意义。
发明内容
有鉴于此，本发明提供了外泌体冻干粉针剂或外泌体液体制剂的辅料配方。本发明解决了牛奶来源外泌体和细胞来源外泌体分别在冻干和冻存条件下，外泌体破碎和聚集问题，确定了不同来源外泌体的液体制剂、冻干制剂和舌下片剂的最适保存方法。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了甘露醇、山梨醇、蔗糖、精氨酸或甘氨酸中的一种或多种作为保护剂在制备外泌体冻干粉针剂或外泌体液体制剂中的应用。
在本发明的一些具体实施方案中，所述外泌体包括牛奶来源外泌体或细胞来源外泌体。
在本发明的一些具体实施方案中，甘露醇的含量为1％～10％、1％～6％或1％～12％；山梨醇的含量为1％～15％、1％～10％或3％～15％；蔗糖的含量为1％～10％、6％～25％或2.5％～35％；精氨酸的含量为0.01M～0.5M；甘 氨酸的含量为0.01M～1M。
本发明还提供了用于制备外泌体冻干粉针剂或外泌体液体制剂的保护剂，包括甘露醇、山梨醇、蔗糖、精氨酸或甘氨酸中的一种或多种。
在本发明的一些具体实施方案中，所述保护中，甘露醇的含量为1％～10％、1％～6％或1％～12％；山梨醇的含量为1％～15％、1％～10％或3％～15％；蔗糖的含量为1％～10％、6％～25％或2.5％～35％；精氨酸的含量为0.01M～0.5M；甘氨酸的含量为0.01M～1M。
在本发明的一些具体实施方案中，所述外泌体冻干粉针剂为牛奶外泌体冻干粉针剂，其特征在于，包括1％～10％甘露醇和1％～15％山梨醇的组合物，或1％～10％蔗糖和1％～15％山梨醇的组合物。
在本发明的一些具体实施方案中，所述外泌体液体制剂为牛奶外泌体液体制剂，包括6％～25％蔗糖，1％～10％山梨醇，0.01M～0.5M精氨酸和0.01M～1M甘氨酸的组合物。
在本发明的一些具体实施方案中，所述外泌体液体制剂为细胞来源外泌体冻干粉针剂，包括1％～6％的甘露醇和3％～15％山梨醇的组合物。
在本发明的一些具体实施方案中，所述外泌体液体制剂为细胞来源外泌体的液体制剂，包括2.5％～35％蔗糖和1％～12％甘露醇的组合物，其中，蔗糖和/或甘露醇可经山梨醇替代。
本发明还提供了所述保护剂在制备外泌体冻干粉针剂或外泌体液体制剂中的应用。
本发明还提供了外泌体冻干粉针剂或外泌体液体制剂，包括外泌体和所述的保护剂。
本发明通过多种制剂辅料的研究，确定了牛奶来源外泌体和细胞来源外泌体液体制剂和冻干粉针制剂，舌下片剂的辅料配方(所选用的崩解剂：微晶纤维素，交联羧甲基纤维素钠，交联聚乙烯吡咯烷酮，羧甲基淀粉钠，低取代羟丙基纤维素，淀粉，吐温，十二烷基硫酸钠中的仍一种或两种以上的混合物；所选用的填充剂：微晶纤维素，微晶纤维素-甘露醇，微晶纤维素-微粉硅胶，乳糖，淀粉，改性淀粉，甘露醇，山梨醇，木糖醇，赤藓糖，海藻糖，预胶化淀粉，糖粉，葡萄糖，糊精，硫酸钙中的任一种 或两种以上的混合物；所选用矫味剂：甜菊甙，糖粉，甘草甜素，阿司帕坦，三氯蔗糖，甜蜜素，索马甜，糖精等一种或两种以上的混合物；所选用的粘合剂：纯化水，淀粉浆，羟丙基甲基纤维素，聚乙烯吡咯烷酮，卡波姆，糊精，明胶浆，阿拉伯胶浆，海藻酸钠和糖浆中的一种或两种以上的混合物；所用润滑剂：硬脂酸，硬脂酸镁，硬脂酸钙，微粉硅胶，滑石粉，氢化植物油，聚乙二醇4000，聚乙二醇6000，十二烷基硫酸钠，十二万级硫酸镁，富马酸硬质酸钠中的任一种或两种以上的混合物)。为外泌体的临床应用提供了制剂工艺。
1)对于牛奶外泌体冻干粉针剂，最优配方是1％-10％甘露醇与1％-15％山梨醇组合，其中甘露醇可用蔗糖替代；
2)对于牛奶外泌体液体制剂，最优配方是6％-25％蔗糖，1％-10％山梨醇，0.01M-0.5M精氨酸，0.01M-1M甘氨酸，对牛奶外泌体均有明显的保护效果；
3)对于细胞来源外泌体冻干粉针剂，最优配方是1％-6％的甘露醇，3％-15％山梨醇具有保护效果；
4)对于细胞来源外泌体的液体制剂，最优配方2.5％-35％蔗糖与1％-12％甘露醇，对样品保存效果最好，其中，蔗糖，甘露醇可用山梨醇替代。
对于舌下片，最优配方每1000片外泌体舌下片中，主药的含量为10 5-10 13颗粒数，交联羧甲基纤维素钠的含量为1-50g(可用交联聚乙烯吡咯烷酮，微晶纤维素替代)，淀粉的含量为20-400g(可用木糖醇，乳糖，甘露醇替代)；阿司帕坦0-50g(可用糖粉替代)，纯水5-100g(可用淀粉浆替代)，滑石粉1g-10g(可用硬脂酸镁替代)。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示冻存盒慢冻结果；其中，图1(A)是样品颗粒数；图1(B)示纯度；
图2示液氮速冻结果；其中，图2(A)是样品颗粒数；图2(B)示纯度；
图3示不同浓度蔗糖和山梨醇对牛奶外泌体冻存的保存效果；其中，图3(A)示纯度；图3(B)示样品颗粒数；
图4示不同辅料组合，对牛奶外泌体冻存的保护效果；其中，图4(A)示纯度；图4(B)示样品颗粒数；
图5示-80℃冻存条件下，不同冻存保护剂对细胞来源外泌体的保护效果；其中，图5(A)示纯度；图5(B)示样品颗粒数；
图6示冻干条件下，不同辅料组合对外泌体的保存效果；其中，图6(A)示纯度；图6(B)示样品颗粒数；
图7示冻干条件下，针对3％甘露醇+5％山梨醇条件进一步优化后对外泌体的保存效果；其中，图7(A)示纯度；图7(B)示样品颗粒数；图7(C)示冻样品留存图；
图8示冻干条件下，冻干保护剂的组合对细胞来源外泌体的保护效果；其中，图8(A)示纯度；图8(B)示样品颗粒数；
图9示牛奶外泌体液体制剂现有保存技术(PBS)与本发明技术(5％山梨醇)，纯度与颗粒数对比图；
图10示牛奶外泌体冻干粉针剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图；
图11示细胞外泌体液体剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图；
图12示细胞外泌体冻干粉针剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图；
图13示药代动力学曲线图。
具体实施方式
本发明公开了外泌体冻干粉针剂或外泌体液体制剂的保护剂，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视 为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
临床研究所用到外泌体多数是-80℃储存的溶液制剂。低温储存的制剂存在运输麻烦，成本高，保存设备要求高等缺点。而且在不添加赋形剂条件下，低温储存外泌体，会造成外泌体破裂，聚集。本发明解决了牛奶来源外泌体和细胞来源外泌体分别在冻干和冻存条件下，外泌体破碎和聚集问题，确定了不同来源外泌体的液体制剂和冻干制剂，舌下片剂的最适保存方法。
本发明提供的外泌体冻干粉针剂或外泌体液体制剂的保护剂所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 冻存方法对外泌体的影响
原理：外泌体是具有和细胞膜结构类似的脂质双分子层结构。细胞常用的冻存方式是梯度降温。但是外泌体源于细胞分泌，细胞分泌蛋白通常采用液氮速冻的方法进行冻存。外泌体在冻存过程时，会出现聚集，破裂。蔗糖，精氨酸，甘氨酸，尿素是常用的赋形剂，有助于药物分散剂，DMSO是细胞常用的冻存液，能防止细胞破裂。
操作步骤：
1)牛奶外泌体的制备：3M硫酸铵沉淀牛奶，室温进行沉淀，静置1.5h，沉淀后的牛奶样品6000rpm，离心20min，接着将上清依次用0.8μm，0.45μm和0.2μm的滤膜进行过滤。初纯的样品采用TFF初滤，再用core700进行精纯，获得牛奶外泌体
2)用PBS分别配制4M、2M、1M尿素，50％、25％、12.5％蔗糖，1M、0.5M、0.25M甘氨酸，0.2M、0.1M、0.05M精氨酸储液。20％、2％DMSO。按下述表格加入外泌体和各试剂，各2份(A组和B组)
表2
Figure PCTCN2022124068-appb-000003
Figure PCTCN2022124068-appb-000004
3)样品混合均匀后，A组用细胞冻存盒-80℃冻存，B组用液氮速冻后，放入-80℃保存2天
4)冻存的样品从-80℃取出，25℃水浴快速溶解
5)12000g离心10min，将上清转移至新的1.5mL离心管中
6)纳米流式NanoFCM检测样品颗粒数和纯度
实验结果：
1)冻存盒慢冻，结果如图1所示：
表3
Figure PCTCN2022124068-appb-000005
Figure PCTCN2022124068-appb-000006
表4
Figure PCTCN2022124068-appb-000007
2)液氮速冻，结果如图2所示：
表5
Figure PCTCN2022124068-appb-000008
表6
Figure PCTCN2022124068-appb-000009
Figure PCTCN2022124068-appb-000010
实验结论：
1)对于冻存盒慢冻方法保存的样品，所有辅料均不能很好的保持外泌体结构完整性和分散性，所有样品的颗粒数下降了一个数量级，纯度下降了至少50％。综合分析，0.25M甘氨酸的保存最好，效率是45.1％
2)对于液氮速冻方法保存的样品，所有辅料的抗聚集效果均不佳，所有样品的颗粒数均下降一个数量级。25％精氨酸能较好的保持外泌体的结构完整性，保存效率是82.8％。
3)综合对比冻存盒慢冻和液氮速冻的方法，虽然各条件下，最佳的保存辅料不一样，但是，从外泌体结构完整性的保存效率上看，液氮速冻的方法更适合外泌体的保存。
实施例2 -80℃冻存条件下，牛奶来源外泌体最优保存方法
原理：外泌体是具有和细胞膜结构类似的脂质双分子层结构。不同来源的外泌体，最适储存条件不同。不同的辅料成分，不同浓度，对样品的保护效果不同。
实验步骤
1)牛奶外泌体的制备：3M硫酸铵沉淀牛奶，室温进行沉淀，静置1.5h，沉淀后的牛奶样品6000rpm，离心20min，接着将上清依次用0.8μm，0.45μm和0.2μm的滤膜进行过滤。初纯的样品采用TFF初滤，再用core700进行精纯，获得牛奶外泌体
2)用PBS配制50％，25％，12.5％蔗糖储液，40％，20％，10％山梨醇储液，0.4M精氨酸，2M甘氨酸
3)按下述表格配制溶液
表7
Figure PCTCN2022124068-appb-000011
表8
Figure PCTCN2022124068-appb-000012
4)样品混合均匀后，用液氮速冻后，放入-80℃保存2天
5)冻存的样品从-80℃取出，25℃水浴快速溶解
6)12000g离心10min，将上清转移至新的1.5mL离心管中
7)纳米流式NanoFCM检测样品颗粒数和纯度
实验结果：
1)不同浓度蔗糖和山梨醇对牛奶外泌体冻存的保存效果，如图3所示：
表9 样品纯度
Figure PCTCN2022124068-appb-000013
表10 样品颗粒数
Figure PCTCN2022124068-appb-000014
2)不同辅料组合，对牛奶外泌体冻存的保护效果，如图4所示：
表11 样品纯度
  新鲜样品 25％蔗糖 5％山梨醇 0.1M精氨酸 0.25M甘氨酸
PBS 96.3％ 92.7％ 91.74％ 92.28％ 83.83％
25％蔗糖   \ 12.82％ 82.2％ 56.02％
5％山梨醇   12.82％ \ 0％ 60.11％
0.1M精氨酸   82.2％ 0％ \ 0％
0.25M甘氨酸   56.02％ 60.11％ 0％ \
表12 样品颗粒数
  新鲜样品 25％蔗糖 5％山梨醇 0.1M精氨酸 0.25M甘氨酸
PBS 3.52×10 9 3.37×10 9 6.58×10 9 4.59×10 9 5.41×10 9
25％蔗糖   \ 3.7×10 9 0.297×10 9 3.06×10 9
5％山梨醇   3.7×10 9 \ 3.32×10 9 5.25×10 9
0.1M精氨酸   0.297×10 9 3.32×10 9 \ 2.37×10 9
0.25M甘氨酸   3.06 5.25 2.37 \
实验结论：
1)相对于其他浓度的蔗糖，25％蔗糖对牛奶外泌体的保护效果最好2)相对于其他浓度的山梨醇，5％山梨醇对牛奶外泌体的保护效果最好
3)25％蔗糖，5％山梨醇，0.1M精氨酸，0.25M甘氨酸，单独添加时，对牛奶外泌体均有明显的保护效果。但将这些辅料进行混合时，对样品的保护效果，从颗粒数和纯度上看，并没有优于单独使用这些辅料。
实施例3 -80℃冻存条件下，细胞来源外泌体最优保存方法
原理：外泌体是具有和细胞膜结构类似的脂质双分子层结构。不同来源的外泌体，最适储存条件不同。
实验步骤：
1)细胞来源外泌体：细胞上清依次用0.2μm的滤膜进行过滤。初纯的样品采用TFF初滤，再用core700进行精纯，获得细胞外泌体。
2)用PBS配制50％蔗糖，40％山梨醇，15％甘露醇，按下述表格配制溶液。
表13
Figure PCTCN2022124068-appb-000015
3)样品混合均匀后，用液氮速冻后，放入-80℃保存2天；
4)冻存的样品从-80℃取出，25℃水浴快速溶解；
5)12000g离心10min，将上清转移至新的1.5mL离心管中；
6)纳米流式NanoFCM检测样品颗粒数和纯度。
实验结果，如图5所示：
表14 纯度
甘露醇 Fresh PBS 25％蔗糖 5％山梨醇
0 84.73％ 68.98％ 0％ 0％
3％   0％ 0％ 36.45％
6％   77.21％ 0％ 30.38％
表15 颗粒数
甘露醇 Fresh PBS×10 9 25％蔗糖×10 9 5％山梨醇×10 9
0 1.56 0.978 0.438 0.0571
3％   0.257 0.0663 0.529
6％   0.645 0.706 0.525
实验结论：从结果上看，在冻存条件下，6％的甘露醇，外泌体纯度最高，颗粒数也较好的保存，因此6％的甘露醇对细胞来源外泌体的保护效果最好。
实施例4 冻干条件下，牛奶来源外泌体最优保存方法
原理：外泌体是具有和细胞膜结构类似的脂质双分子层结构。不同来源的外泌体，最适储存条件不同。
实验步骤：
1)牛奶外泌体的制备：3M硫酸铵沉淀牛奶，室温进行沉淀，静置1.5h，沉淀后的牛奶样品6000rpm，离心20min，接着将上清依次用0.8μm，0.45μm和0.2μm的滤膜进行过滤。初纯的样品采用TFF初滤，再用core700进行精纯，获得牛奶外泌体
2)用PBS配制50％蔗糖，40％山梨醇，15％，10％甘露醇，按下述表格配制溶液
表16
Figure PCTCN2022124068-appb-000016
表17
Figure PCTCN2022124068-appb-000017
Figure PCTCN2022124068-appb-000018
3)样品混合均匀后，用液氮速冻后，放入冻干机中，冻24h；
4)冻干样品用100μLPBS重溶；
5)12000g离心10min，将上清转移至新的1.5mL离心管中；
6)纳米流式NanoFCM检测样品颗粒数和纯度。
实验结果：
1)不同辅料组合对外泌体的保存效果，如图6所示：
表18 颗粒数
甘露醇浓度 Fresh×10 9 PBS×10 9 25％蔗糖×10 9 5％蔗糖×10 9
0 5.04 1.78 2.17 0.938
3％   0.479 1.98 1.82
6％   1.35 1.97 2.05
表19 纯度
甘露醇浓度 Fresh PBS×10 9 25％蔗糖 5％蔗糖
0 46.76％ 0％ 77.54％  
3％   0％ 59.2％ 100％
6％   100％ 65.53％ 100％
2)针对3％甘露醇+5％山梨醇条件进一步优化，结果如图7所示：
表20 纯度
Figure PCTCN2022124068-appb-000019
Figure PCTCN2022124068-appb-000020
表21 颗粒数×10 9
Figure PCTCN2022124068-appb-000021
实验结论：
1)从结果上看，3％甘露醇与5％山梨醇组合，对外泌体的保存效果更好。
2)在针对3％甘露醇与5％山梨醇组合优化实验中，综合纯度和颗粒数，同时考虑高浓度辅料对冻干样品的影响，辅料越多，冻干剂体积越大，会增加溶解难度。因此，3％山梨醇和2％甘露醇的保存效果较优。
实施例5
1.冻干条件下，细胞来源外泌体最优保存方法
原理：外泌体是具有和细胞膜结构类似的脂质双分子层结构。不同来源的外泌体，最适储存条件不同。
实验步骤：
1)细胞来源外泌体的制备：细胞上清经0.22μm滤膜过滤后，采用TFF初滤，再用core700进行精纯，获得细胞外泌体；
2)用PBS配制50％蔗糖，40％山梨醇，15％，10％甘露醇，按下述表格配制溶液。
表22
Figure PCTCN2022124068-appb-000022
3)样品混合均匀后，用液氮速冻后，放入冻干机中，冻24h；
4)冻干样品用100μLPBS重溶；
5)12000g离心10min，将上清转移至新的1.5mL离心管中；
6)纳米流式NanoFCM检测样品颗粒数和纯度。
实验结果如图8所示：
表23 细胞来源外泌体冻干制剂颗粒数
甘露醇浓度 新鲜样品×10 9 PBS×10 9 25％蔗糖×10 9 5％蔗糖×10 9
0 1.56 1.06 1.87 0.637
3％   0.522 1.48 1.23
6％   0.815 1.28 1.16
表24 细胞来源外泌体冻干制剂纯度
甘露醇浓度 Fresh PBS 25％蔗糖 5％蔗糖
0 84.73％ 0％ 52.56％ 20.57％
3％   28.94％ 55.51％ 7.9％
6％   69.18％ 84.18％ 59.49％
实验结论：25％蔗糖与6％甘露醇组合，对样品保存效果最好。
实施例6
外泌体舌下片的最优辅料配方
制备步骤如下：
第一步：将所述的主药冷冻干燥，过筛80-120目；
第二步：将填充剂，崩解剂，矫味剂，润滑剂烘干，粉碎，过筛80-120目预处理；
第三步：将预处理的外泌体冻干粉，填充剂，矫味剂和崩解剂混合均匀；
第四步：将粘合剂加入混匀的上述无聊中，制成软材，制粒，干燥；
第五步：干颗粒加入润滑剂混匀，再进行压片得到成品。
实施例：将主药外泌体加入赋形剂，冷冻干燥后，与填充剂淀粉，崩解剂羧甲基纤维素钠，润滑剂滑石粉进行烘干，粉碎，过筛混合，制成软材，用20目网筛制粒，并风干至水分≤3％，再加入润滑剂，进行总混，最后压片得到外泌体舌下片。
获得的舌下片进行动物药效评价：
1)主药在制备过程中，采用PKH67进行荧光标记。舌下片的制备同上述步骤一致步骤五
2)将制备好的荧光标记的舌下片给予小鼠，检测血清中的外泌体颗粒数，并进行药代动力学分析，结果如表25、图13所示。
表25 药代动力学参数
Figure PCTCN2022124068-appb-000023
Figure PCTCN2022124068-appb-000024
对比例
现有临床研究用的外泌体保存及制剂以液体制剂为主，没有冻干剂和舌下片，并且液体制剂在不添加辅料时，直接冻存，会造成外泌体聚集，破裂，影响外泌体的疗效。并且没有牛奶外泌体和细胞来源外泌体的制剂工艺。本发明解决了牛奶外泌体和细胞来源外泌体的液体制剂，冻干粉针剂和舌下片剂的制剂工艺开发，开发了新型的外泌体制剂。
对比数据请审查员审核：
对照组：采用PBS作为冻存剂；
实验组1：采用5％山梨醇作为冻存保护剂；
实验组2：采用6％甘露醇作为冻干赋形剂；
实验方法：
方法一：在500uL新鲜牛奶外泌体样品中，加入500uL 10％山梨醇，对照组500uL样品中加入500uLPBS，液氮速冻后，-80℃保存；样品检测时，4℃溶化样品，12000rpm离心10min，取上清，NanoFCM检测样品浓度，样品用1％Trtion消化后，NanoFCM检测并计算获得样品纯度。
方法二：在500uL新鲜细胞外泌体样品中，加入500uL 12％甘露醇，对照组500uL样品中加入500uLPBS，液氮速冻后，-80℃保存；样品检测时，4℃溶化样品，12000rpm离心10min，取上清，NanoFCM检测样品浓度，样品用1％Trtion消化后，NanoFCM检测并计算获得样品纯度。
方法三：在500uL新鲜牛奶或细胞外泌体样品中，加入500uL 12％甘露醇，对照组500uL样品中加入500uLPBS，液氮速冻后，-80℃保存液氮速冻后，冻干机中冻干；样品检测时，实验组和对照组的样品均用1mL H 2O复溶后，12000rpm离心10min，取上清，NanoFCM检测样品浓 度，样品用1％Trtion消化后，NanoFCM检测并计算获得样品纯度。
实验结果：
图9示牛奶外泌体液体制剂现有保存技术(PBS)与本发明技术(5％山梨醇)，纯度与颗粒数对比图。样品添加5％山梨醇作为保护剂，样品的纯度和颗粒数明显高于PBS组，说明5％山梨醇能够有效保护样品冻存时的结构完整性和分散性。
图10示牛奶外泌体冻干粉针剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图。样品中添加6％甘露醇作为赋形剂，样品的纯度和颗粒数明显高于PBS组，说明6％山梨醇能够有效保护样品冻干时的结构完整性和分散性。
图11示细胞外泌体液体剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图。样品添加6％山梨醇作为保护剂，样品的纯度和颗粒数明显高于PBS组，说明6％山梨醇能够有效保护样品冻存时的结构完整性和分散性。
图12示细胞外泌体冻干粉针剂制剂现有保存技术(PBS)与本发明技术(6％甘露醇)，纯度与颗粒数对比图。样品添加6％山梨醇作为保护剂，样品的纯度和颗粒数明显高于PBS组，说明6％山梨醇能够有效保护样品冻存时的结构完整性和分散性。
综上，5％山梨醇对牛奶外泌体液体制剂具有明显的保护效果；6％的甘露醇对牛奶外泌体冻干粉针剂，细胞外泌体的冻干粉针剂或液体制剂具有明显的保护效果，均能保持外泌体的稳定性。
以上对本发明所提供的外泌体冻干粉针剂或外泌体液体制剂的保护剂进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 甘露醇、山梨醇、蔗糖、精氨酸或甘氨酸中的一种或多种作为保护剂在制备外泌体冻干粉针剂或外泌体液体制剂中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述外泌体包括牛奶来源外泌体或细胞来源外泌体。
  3. 如权利要求1或2所述的应用，其特征在于，甘露醇的含量为1％～10％、1％～6％或1％～12％；山梨醇的含量为1％～15％、1％～10％或3％～15％；蔗糖的含量为1％～10％、6％～25％或2.5％～35％；精氨酸的含量为0.01M～0.5M；甘氨酸的含量为0.01M～1M。
  4. 用于制备外泌体冻干粉针剂或外泌体液体制剂的保护剂，其特征在于，包括甘露醇、山梨醇、蔗糖、精氨酸或甘氨酸中的一种或多种。
  5. 如权利要求4所述的保护剂，其特征在于，甘露醇的含量为1％～10％、1％～6％或1％～12％；山梨醇的含量为1％～15％、1％～10％或3％～15％；蔗糖的含量为1％～10％、6％～25％或2.5％～35％；精氨酸的含量为0.01M～0.5M；甘氨酸的含量为0.01M～1M。
  6. 如权利要求4或5所述的保护剂，其特征在于，所述外泌体冻干粉针剂为牛奶外泌体冻干粉针剂，其特征在于，包括1％～10％甘露醇和1％～15％山梨醇的组合物，或1％～10％蔗糖和1％～15％山梨醇的组合物。
  7. 如权利要求4或5所述的保护剂，其特征在于，所述外泌体液体制剂为牛奶外泌体液体制剂，包括6％～25％蔗糖，1％～10％山梨醇，0.01M～0.5M精氨酸和0.01M～1M甘氨酸的组合物。
  8. 如权利要求4或5所述的保护剂，其特征在于，所述外泌体液体制剂为细胞来源外泌体冻干粉针剂，包括1％～6％的甘露醇和3％～15％山梨醇的组合物。
  9. 如权利要求4或5所述的保护剂，其特征在于，所述外泌体液体制剂为细胞来源外泌体的液体制剂，包括2.5％～35％蔗糖和1％～12％甘露醇的组合物，其中，蔗糖和/或甘露醇可经山梨醇替代。
  10. 外泌体冻干粉针剂或外泌体液体制剂，其特征在于，包括外泌体 和如权利要求5～9任一项所述的保护剂。
PCT/CN2022/124068 2021-10-20 2022-10-09 外泌体冻干粉针剂或外泌体液体制剂的保护剂 WO2023066044A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111222193.1 2021-10-20
CN202111222193.1A CN115990261B (zh) 2021-10-20 2021-10-20 外泌体冻干粉针剂或外泌体液体制剂的保护剂

Publications (1)

Publication Number Publication Date
WO2023066044A1 true WO2023066044A1 (zh) 2023-04-27

Family

ID=85990877

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/124068 WO2023066044A1 (zh) 2021-10-20 2022-10-09 外泌体冻干粉针剂或外泌体液体制剂的保护剂

Country Status (2)

Country Link
CN (1) CN115990261B (zh)
WO (1) WO2023066044A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115990261A (zh) * 2021-10-20 2023-04-21 谛邈生物科技(北京)有限公司 外泌体冻干粉针剂或外泌体液体制剂的保护剂
CN117598291A (zh) * 2024-01-15 2024-02-27 北京恩康医药有限公司 一种外泌体保护液及其应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107006452A (zh) * 2017-04-20 2017-08-04 深圳市赛欧细胞生物科技有限公司 一种人脐带间充质干细胞源外泌体冻干保存方法及其应用
WO2018078524A1 (en) * 2016-10-28 2018-05-03 Pharmaexceed S.R.L. Process for isolating and lyophilizing extracellular vesicles
CN108721200A (zh) * 2018-06-07 2018-11-02 武汉赛云博生物科技有限公司 一种人间充质干细胞来源的外泌体美容制剂的制备方法及应用
CN111759863A (zh) * 2020-07-23 2020-10-13 青岛利福特生物医学研究所 一种含有间充质干细胞外泌体的冻干粉胶囊及其制备方法
US20200329697A1 (en) * 2018-07-28 2020-10-22 Exocobio Inc. Method for lyophilizing exosome
WO2021062057A1 (en) * 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Exogenous loading of exosomes via lyophilization
CN113018245A (zh) * 2021-03-15 2021-06-25 广州赛莱拉生物基因工程有限公司 一种牛胎盘细胞外泌体冻干粉及其制备方法
CN113171332A (zh) * 2021-03-18 2021-07-27 深圳玄鸟生物科技有限公司 一种外泌体冻干粉、其制备方法及护肤品

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MA45481A (fr) * 2015-06-10 2018-04-18 Univ Texas Utilisation d'exosomes pour le traitement de maladies
KR102394636B1 (ko) * 2015-06-30 2022-05-09 (주)아모레퍼시픽 피부 미백용 조성물 및 피부 미백 효능 물질을 스크리닝하는 방법
CN110087658A (zh) * 2016-10-12 2019-08-02 新加坡科技研究局 一种用于冻干外泌体的方法
CN107245472B (zh) * 2017-06-08 2021-08-06 北京智能宝生物科技有限公司 一种人间充质干细胞外泌体冻干粉的制备方法、使用方法
CN110897993A (zh) * 2018-09-14 2020-03-24 陕西博鸿生物科技集团有限公司 一种外泌体冻干粉的制备方法及其应用
CN110559254A (zh) * 2019-10-20 2019-12-13 张新梅 一种外泌体组合物、其制备方法及应用
CN112205389A (zh) * 2020-10-23 2021-01-12 广州佳为生物科技有限公司 一种外泌体冻干保护剂和冻干粉复苏液及其应用
CN112514892B (zh) * 2020-12-25 2022-06-14 广州赛莱拉干细胞科技股份有限公司 一种外泌体冻存保护液及其制备方法
CN115990261B (zh) * 2021-10-20 2024-01-05 光武惠文生物科技(北京)有限公司 外泌体冻干粉针剂或外泌体液体制剂的保护剂

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018078524A1 (en) * 2016-10-28 2018-05-03 Pharmaexceed S.R.L. Process for isolating and lyophilizing extracellular vesicles
CN107006452A (zh) * 2017-04-20 2017-08-04 深圳市赛欧细胞生物科技有限公司 一种人脐带间充质干细胞源外泌体冻干保存方法及其应用
CN108721200A (zh) * 2018-06-07 2018-11-02 武汉赛云博生物科技有限公司 一种人间充质干细胞来源的外泌体美容制剂的制备方法及应用
US20200329697A1 (en) * 2018-07-28 2020-10-22 Exocobio Inc. Method for lyophilizing exosome
WO2021062057A1 (en) * 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Exogenous loading of exosomes via lyophilization
CN111759863A (zh) * 2020-07-23 2020-10-13 青岛利福特生物医学研究所 一种含有间充质干细胞外泌体的冻干粉胶囊及其制备方法
CN113018245A (zh) * 2021-03-15 2021-06-25 广州赛莱拉生物基因工程有限公司 一种牛胎盘细胞外泌体冻干粉及其制备方法
CN113171332A (zh) * 2021-03-18 2021-07-27 深圳玄鸟生物科技有限公司 一种外泌体冻干粉、其制备方法及护肤品

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115990261A (zh) * 2021-10-20 2023-04-21 谛邈生物科技(北京)有限公司 外泌体冻干粉针剂或外泌体液体制剂的保护剂
CN115990261B (zh) * 2021-10-20 2024-01-05 光武惠文生物科技(北京)有限公司 外泌体冻干粉针剂或外泌体液体制剂的保护剂
CN117598291A (zh) * 2024-01-15 2024-02-27 北京恩康医药有限公司 一种外泌体保护液及其应用
CN117598291B (zh) * 2024-01-15 2024-04-09 北京恩康医药有限公司 一种外泌体保护液及其应用

Also Published As

Publication number Publication date
CN115990261B (zh) 2024-01-05
CN115990261A (zh) 2023-04-21

Similar Documents

Publication Publication Date Title
WO2023066044A1 (zh) 外泌体冻干粉针剂或外泌体液体制剂的保护剂
CA2707484C (en) Improved formulations and methods for lyophilization and lyophilates provided thereby
Aboelwafa et al. Optimization and in vivo pharmacokinetic study of a novel controlled release venlafaxine hydrochloride three-layer tablet
BRPI0609071A2 (pt) composicão estável de ciclosporina nanoparticulada, método para preparar uma ciclosporina nanoparticulada, uso da composição de ciclosporina nanoparticulada estável, e, composição de liberação controlada
JPH09504429A (ja) 組換えウイルスの保存方法
EP2995307B1 (en) Oral pharmaceutical composition for increasing hypoxia tolerance
JP2010264249A (ja) 改善された再生能を有する凍結乾燥体
US10973766B2 (en) Oral pharmaceutical dosage form for the delivery of a peptide and/or protein
CN104107173A (zh) 一种罗氟司特片剂及其制备方法
WO2024103667A1 (zh) 一种冻干保护剂及粪菌的冻干工艺
CN101874789B (zh) 含有兰索拉唑的冻干粉针
CN111773191A (zh) 含有盐酸去氧肾上腺素的复方固体制剂及其制备方法
CN103055305B (zh) 一种注射用含细胞色素c药物组合物的冻干制剂、制备方法
CN112336692A (zh) 一种叶酸片及其制备方法
CN112294770A (zh) 单硝酸异山梨酯复方制剂及其应用和制备方法
Eriksson et al. Investigations into the stabilisation of drugs by sugar glasses: II: Delivery of an inulin-stabilised alkaline phosphatase in the intestinal lumen via the oral route
CN104069074B (zh) 一种注射用奥拉西坦及其制备方法
US4778816A (en) Anti-asthmatic composition and process for preparing the same
CN101502641B (zh) 一种注射给药的环孢素药物组合物
CN102988402A (zh) 一种含有环磷腺苷化合物药物组合物及其制备方法
WO2003030866A1 (de) Stabile galenische gefriergetrocknete arzneimittelzubereitung von rekombinanten carbohydratbindenden polypeptiden
CN1596893A (zh) 注射用西眯替丁冻干制剂及其制备方法
CN110790675A (zh) 一种秋水仙碱化合物及其制备方法、制剂、应用与药物组合物
CN104910147A (zh) 阿哌沙班晶体及其制备方法
CN114177149B (zh) 一种负载20(s)-原人参二醇的固体分散体及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22882654

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE