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WO2021213488A1 - Procédé et composition permettant d'inhiber un orage cytokinique - Google Patents

Procédé et composition permettant d'inhiber un orage cytokinique Download PDF

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Publication number
WO2021213488A1
WO2021213488A1 PCT/CN2021/089156 CN2021089156W WO2021213488A1 WO 2021213488 A1 WO2021213488 A1 WO 2021213488A1 CN 2021089156 W CN2021089156 W CN 2021089156W WO 2021213488 A1 WO2021213488 A1 WO 2021213488A1
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Prior art keywords
amniotic fluid
days
extract
cytokine storm
eggs
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Chinese (zh)
Inventor
钱进
孙宁
崔白苹
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Zhejiang Hygeiancells Biomedical Co Ltd
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Zhejiang Hygeiancells Biomedical Co Ltd
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    • 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
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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

Definitions

  • the present invention relates to a method and composition for inhibiting cytokine storm.
  • Cytokine storm syndrome is a serious life-threatening disease. Its clinical features are systemic inflammation, methemorrhaginemia, hemodynamic instability, and multiple organ failure (MOF). If left untreated, it may lead to death, which is an important factor in the dangerous clinical manifestations of H7N9, H5N1, and SARS. Studies have shown that it occurs in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome.
  • CSS is an uncontrolled and dysfunctional immune response, involving the continuous activation and expansion of lymphocytes and macrophages, which secrete large amounts of cytokines such as TNF- ⁇ , IL-1, IL-6, IL -12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1 and IL-8, leading to a cytokine storm.
  • cytokines such as TNF- ⁇ , IL-1, IL-6, IL -12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1 and IL-8
  • HHLH Hemophagocytic lymphohistiocytosis
  • pHLH primary
  • sHLH secondary
  • SHLH associated with autoimmune or autoinflammatory diseases is called macrophage activation syndrome (MAS).
  • MAS macrophage activation syndrome
  • the present invention provides the application of amniotic fluid and/or extracts thereof in the preparation of reagents for inhibiting cytokine storm or drugs for treating or preventing cytokine storm syndrome, wherein the amniotic fluid is derived from eggs with embryonic age of 5-12 days, preferably Eggs with an embryo age of 6-11 days, more preferably eggs with an embryo age of 7-9 days, more preferably eggs with an embryo age of 7-8 days, or from the developmental stage and the developmental stage of the eggs of the embryonic age Corresponding eggs of avian species other than chickens; or embryos from rodents with a gestational age of 8-14 days, or rodents whose developmental period corresponds to that of rodents with a gestational age of 8-14 days Embryos of non-human mammals other than animals.
  • the agent or drug is a pharmaceutical composition comprising amniotic fluid and/or extracts thereof as described herein and optionally pharmaceutically acceptable excipients.
  • the active ingredient contained in the extract is not combined with the ion exchange column at pH 5.8-8.0, and the molecular weight of the ingredient contained in the extract is in the range of 150-2000 Daltons Inside.
  • the active ingredients contained in the extract are not bound to the ion exchange column at pH 7.0-8.0, and the molecular weight of the ingredients contained in the extract is in the range of 150-2000 Daltons, but not Limited to this range.
  • the active ingredients contained in the extract are not combined with the ion exchange column at pH 7.0-8.0, and the molecular weight of the ingredients contained in the extract is in the range of 150-1200 Daltons Inside.
  • the octanol/water partition coefficient Log P of the active ingredient contained in the extract is in the range of 0.05-1.897, preferably between 0.3-1.5; preferably, the extract is reversed Obtained by phase chromatography.
  • the cytokine storm or cytokine storm syndrome is caused by influenza virus and/or coronavirus infection.
  • the coronavirus is COVID-19.
  • the cytokine storm or cytokine storm syndrome is caused by SARS, MERS, H5N1 influenza virus or H7N9 influenza virus.
  • Figure 1 HPLC detection results of amniotic fluid from eggs with embryo age of 7 days.
  • Figure 2 HPLC detection results of amniotic fluid from eggs with embryo age of 11 days.
  • Figure 3 HPLC detection result of amniotic fluid from eggs with embryo age of 13 days.
  • Figure 4 Growth curve of chicken embryo fibroblasts under different culture conditions.
  • Figure 5 The effect of amniotic fluid from eggs on the growth viability and migration ability of human umbilical vein endothelial cells (HUVEC). Among them, the abscissa represents the culture medium, and the ordinate represents the OD450 value.
  • HAVEC umbilical vein endothelial cells
  • Figure 6 The effect of amniotic fluid from duck eggs on the growth viability and migration ability of chicken embryonic fibroblasts. Among them, the abscissa represents the culture medium, and the ordinate represents the OD450 value.
  • Figure 7 The effect of amniotic fluid from mice on the growth vigor of AC16 cells.
  • Figure 8 GE HiLoad 16/600 Superdex75pg separation chromatogram.
  • FIG. 9 Cell viability detection gel column GE HiLoad 16/600 Superdex75pg separation fraction.
  • the abscissa represents the culture medium, where FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; “EE” represents freeze-dried amniotic fluid; S-200B represents the fraction of peak B; QUNBOUND represents the unbound fraction of the anion column; 3-1 to 3-6 respectively represent the middle volume fraction 1-6 in the third step purification.
  • Figure 10 Chromatogram of fresh egg amniotic fluid separated by UniSil 10-100 C18.
  • Figure 11 HPLC chart of peaks P6, P7, and P8 of the components of amniotic fluid extract.
  • Figure 12 Cell viability detection revealed that the P6, P7, and P8 peaks in Figure 4 have biological activity.
  • FIG. 15 Ejection fraction of mice with myocardial infarction.
  • the ejection fraction and the left ventricular short axis shortening rate of mice can be measured by cardiac ultrasound. It can be seen from the figure that the treatment of amniotic fluid (EE) significantly improved the ejection fraction of mice with myocardial infarction, and the cardiac function was significantly improved.
  • EE amniotic fluid
  • Figure 16 Short-axis shortening rate of left ventricle in mice with myocardial infarction.
  • the ejection fraction and the left ventricular short axis shortening rate of mice can be measured by cardiac ultrasound. It can be seen from the figure that the treatment of amniotic fluid (EE) significantly increased the short-axis shortening rate of the left ventricle in mice with myocardial infarction, and the cardiac function was significantly improved.
  • EE amniotic fluid
  • FIG. 17 Immunofluorescence staining of the heart of mice with myocardial infarction (PH3, cTnT, DAPI).
  • FIG. 18 Immunofluorescence staining of the heart of mice with myocardial infarction (AuroraB, cTnT, DAPI). It can be seen from the figure that the PH3-positive and AuroraB-positive cells in the hearts of the mice in the treatment group increased significantly, indicating that EE treatment significantly triggered the regeneration of heart cells in the mice with myocardial infarction.
  • Figure 19 Masson trichrome staining of the heart of mice with myocardial infarction. It can be seen from the figure that the mice with myocardial infarction have severe fibrosis, and the left ventricular wall is significantly thinned. After amniotic fluid (EE) treatment, the left ventricular wall thinning is not obvious, and the fibrosis is significantly reduced.
  • EE amniotic fluid
  • FIG. 20 The area of cardiac fibrosis in mice with myocardial infarction was significantly reduced after amniotic fluid (EE) treatment compared with untreated group (NS).
  • EE amniotic fluid
  • Figure 21 EE improves heart function in pigs with myocardial infarction and reduces left ventricular remodeling.
  • Figure 22 EE reduces the area of heart infarction in IR pigs and prolongs the activity time.
  • Figure 23 Flow cytometry results of the control group and the treatment group after 24 hours of LPS treatment of human alveolar epithelial cells Calu-3.
  • Figure 24 The qPCR results of the control group and the treatment group after LPS treatment of human alveolar epithelial cells Calu-3 for 24 hours.
  • Figure 25 The qPCR results of the control group and the treatment group after the treatment of human alveolar epithelial cells Calu-3 with COVID-19 protein pseudovirus for 24 hours.
  • Figure 26 Results of pulmonary function measurement of the control group and the treatment group in the mouse pneumonia model constructed by LPS.
  • Figure 27 Survival curves and body weight changes of the control group and the treatment group in the mouse pneumonia model constructed by LPS.
  • Figure 28 HE staining results of lung tissues of control group (NS) and treatment group (EE) animals in the mouse pneumonia model constructed by LPS.
  • Figure 29 HE staining results of liver (columns 1-3) and kidney (columns 4-6) in the control group (NS) and the treatment group (EE) in the mouse pneumonia model constructed by LPS.
  • this article relates to the use of amniotic fluid and/or extracts thereof to inhibit cytokine storm, or to treat or prevent cytokine storm syndrome.
  • amniotic fluid can be derived from poultry eggs and non-human mammals.
  • Poultry eggs refer to poultry eggs.
  • the preferred birds are poultry, such as chickens, ducks and geese.
  • the present invention uses poultry eggs with embryo age of 5-20 days, preferably 6-15 days. It should be understood that for different eggs, the appropriate embryo age may not be the same.
  • eggs with embryo age of 5-12 days are preferably used, eggs with embryo age of 6-11 days are more preferably used, eggs with embryo age of 7-9 days are more preferably used, and embryo age is more preferably used.
  • Eggs for 7-8 days When using eggs of other avians, eggs whose developmental stage corresponds to the developmental stage of the eggs of the embryonic age mentioned above can be used.
  • duck eggs with embryo age of 8-10 days, especially 8-9 days may be the best.
  • amniotic fluid from poultry eggs For example, the blunt end of an egg of the corresponding embryonic age can be knocked to break the eggshell, and the eggshell can be peeled to form a hole with a diameter of about 2 cm. Then use tweezers to carefully tear open the shell membrane and vitelline membrane, taking care not to damage the amniotic membrane. Pour the amniotic membrane and connected tissues covering the embryo from the shell to a petri dish, and pierce the amniotic membrane with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.
  • amniotic fluid can also be derived from non-human mammals, especially rodents, such as from mice.
  • Other non-human mammals may be common domestic animals, such as cattle, sheep, dogs, cats, pigs, and so on.
  • the amniotic fluid is derived from embryos of rodents with a gestational age of 8-14 days, or from non-humans whose developmental period corresponds to that of rodents with a gestational age of 8-14 days. The embryo of a mammal. Conventional methods can be used to obtain amniotic fluid.
  • amniotic fluid can be centrifuged to separate possible impurities, such as egg yolk, to obtain pure amniotic fluid as much as possible.
  • the supernatant obtained after centrifugation is the amniotic fluid used in the present invention.
  • the "amniotic fluid” referred to herein shall refer to "pure" amniotic fluid, that is, the amniotic fluid isolated from poultry eggs or non-human mammalian embryos does not contain Amniotic fluid that contains other components in poultry eggs or embryos of non-human mammals, and is not contaminated by foreign substances. Pure amniotic fluid can be stored in the refrigerator below -60°C and used after thawing.
  • the present invention uses an extract of amniotic fluid.
  • the active ingredient contained in the extract does not bind to the ion exchange column between pH 5.8 and 8.0, and more preferably does not bind to the ion exchange column between pH 7.0 and 8.0.
  • the extract is a neutral fraction.
  • the molecular weight of the components contained in the extract is in the range of 150-2000 Daltons, but is not limited to this range; preferably, it is in the range of 150-1200 Daltons.
  • the neutral fraction with a molecular weight of 150-2000 Daltons, preferably 150-1200 Daltons can be separated from the amniotic fluid, thereby obtaining the extract.
  • Gel columns and ion exchange columns well known in the art can be used to implement the method herein.
  • a well-known gel chromatography column (various gel chromatography columns as described below) can be used to separate a fraction with a molecular weight of 150-2000 Daltons from the amniotic fluid, and then use an ion exchange method (such as using the following The ion exchange column) separates the neutral fraction from the fraction.
  • the neutral fraction can be separated from the amniotic fluid by an ion exchange method (such as the ion exchange column described below), and then a gel chromatography column (various gel chromatography columns as described below) can be used to separate the neutral fraction from the amniotic fluid. ) Separate the neutral fraction with a molecular weight in the range of 150-2000 Daltons.
  • a neutral fraction with a molecular weight of 150-2000 daltons can be separated from the amniotic fluid first, and then a fraction with a molecular weight in the range of 150-1200 daltons can be separated therefrom.
  • the method may include the following steps:
  • a neutral fraction with a molecular weight of 150-2000 Daltons is separated from the amniotic fluid.
  • Step (1) can be achieved by using gel chromatography and ion exchange methods.
  • a gel chromatography column is used to separate the components of the amniotic fluid with a molecular weight of 150-2000 Daltons, and an uncharged (neutral) fraction can be obtained by ion exchange.
  • gel chromatography columns can be used to implement gel chromatography.
  • Such gel chromatography columns include but are not limited to Sephacryl S-100, Sephacryl S-200, Sephacryl S-300, Sephacryl S. -400, Superose 12, Superose 6, Superdex 12 and Superdex 6, etc. It should be understood that any other gel chromatography packing with a separation range of 100-10000 Daltons can also be used.
  • the flow rate may be 0.5-50 ml/min, such as 1 ml/min.
  • the ultraviolet absorption is between 200-300nm, such as 280nm.
  • the sample can be loaded.
  • the sample flow rate is determined according to the actual preparation situation.
  • the crude product can be eluted with degassed ddH 2 O, and the fractions with molecular weight between 150-2000 Daltons can be collected. If necessary, the separation by gel chromatography can be repeated several times, and the fractions with the same peak time during each separation can be mixed.
  • anion exchange and cation exchange can be used in the method of the present invention.
  • an anion exchange method is used herein.
  • Commercially available anion exchange columns can be used, including but not limited to GE's DEAE Sepharose, ANX Sepharose, Q Sepharose, Capto DEAE, Capto Q, Mono Q, Mini Q, etc. It should be understood that other brands of anion exchange packing may also be used.
  • commercially available cation exchange columns can also be used, including but not limited to CM Sepharose, SP Sepharose, Capto S, Mono S, Mini S, etc.
  • the buffer may be a conventional buffer in the art, for example, a phosphate buffer, especially a sodium phosphate buffer may be used.
  • the pH of the buffer can be determined according to the ion exchange column used. For example, when using an anion exchange column, you can use a buffer with a pH of 7.5 to 8.5, preferably 7.5 to 8.0 to balance the anion exchange column; when using a cation exchange column, you can use a buffer with a pH of 5.8 to 7.0, preferably 5.8 to 6.5 for balance Cation exchange column.
  • the sodium phosphate buffer contains Na 2 HPO 4 and NaH 2 PO 4 and has a pH of about 5.8 or 8.0.
  • the present invention preferably uses an anion exchange column for separation.
  • the flow rate can be determined according to the actual situation.
  • the flow rate may be 0.5-50 ml/min, such as 1 ml/min.
  • the equilibrium is ended. After the equilibration is over, you can load the sample and collect the outflow part (that is, the part that is not bound to the column). The sample flow rate is determined according to the actual preparation situation.
  • step (1) gel chromatography can be performed first to separate the fraction with a molecular weight of 150-2000 Daltons, and then ion exchange can be performed to separate the neutral fraction; alternatively, ion exchange can be performed first to separate the fraction The neutral fraction in the amniotic fluid, and then gel chromatography is used to separate the active ingredients in the neutral fraction with a molecular weight in the range of 150-2000 Daltons to obtain a neutral fraction with a molecular weight between 150-2000 Daltons .
  • step (2) is to further separate the neutral fraction obtained in step (1) to obtain active ingredients with a molecular weight in the range of 150-1200 Daltons.
  • commercially available gel chromatography columns can be used to separate fractions with a molecular weight in the range of 150-1200 Daltons.
  • Suitable gel chromatography columns include, but are not limited to, HiLoad Superdex 16/600 Superdex 75pg, Superdex Peptide, Superdex 200 and Superdex 30 from GE. It should be understood that other brands of gel chromatography packing with a separation range of 500-10000 Daltons can also be used.
  • the gel column can be equilibrated with ddH 2 O first, and the flow rate can be determined according to the actual situation.
  • the flow rate may be 0.5-50 ml/min, such as 1 ml/min.
  • the sample can be loaded. The sample flow rate is determined according to the actual preparation situation.
  • the crude product can be eluted with degassed ddH 2 O, and the fractions are collected to obtain the fraction with the molecular weight of the components in the range of 150-1200 Daltons, which is the extract described herein.
  • the amniotic fluid and/or its extract of the present invention or the composite dressing prepared from it as the main raw material is called DWS.
  • the extract obtained by the above method is formulated into a solution with a pH of 5.8-8.0 and passed through a variety of ion exchange columns (including DEAE Sepharose, Q Sepharose, Mono Q, CM Sepharose, SP Sepharose and Mono S). None of the active ingredients are combined with these ion exchange columns.
  • the octanol/water partition coefficient Log P of the active ingredient contained in the extract of the present invention is in the range of 0.05-1.897, preferably between 0.3-1.5; preferably, the extract is separated by reversed-phase chromatography get.
  • the stationary phase of reversed-phase chromatographic columns usually uses silica gel as a carrier, and a layer of non-polar molecules is bonded on the surface.
  • the bonded non-polar group can be selected from C18 alkyl, C8 alkyl, phenyl, C4 alkyl, etc. and their derivatives.
  • the present invention preferably uses a C18 reverse phase chromatographic column, that is, a reverse phase chromatographic column bonded with a C18 alkyl group.
  • the present invention can be implemented using reversed-phase chromatography columns known in the art.
  • reversed-phase chromatography columns can be obtained from commercially available sources, including UniSil 10-100 C18, LaChrom-C18, Inertsil ODS, Zorbax ODS, ACE C18, SunFire C18, Symmetry C18, Hypersil GOLD C18, Luna C18, Hypersil BDS C18, Hypersil ODS C18, SyncronisaQ C18 and Syncronis C18, etc.
  • the mobile phase of reversed-phase chromatography is a certain proportion of water and an organic solvent that is miscible with water.
  • the organic solvent can be selected from methanol, acetonitrile, ethanol, tetrahydrofuran, isopropanol, dioxane, acetone, etc., preferably methanol and acetonitrile are used.
  • the organic solvent used is a chromatographic grade organic solvent, and the water is 100% ultrapure water.
  • the mobile phase Before implementing reversed-phase chromatography, the mobile phase can be used to equilibrate the reversed-phase chromatography column. After the absorption curve stabilizes and returns to the baseline, the balance can be stopped.
  • the sample can be loaded in a conventional way, and the flow rate of the sample can be determined according to the actual production situation, such as the material, specifications and flow of the column used. After sample loading, gradient elution can be performed.
  • the concentration of the organic solvent in the mobile phase may vary slightly depending on the type of organic solvent, which can be easily determined by those skilled in the art. In some embodiments, the percentage gradient of organic solvent in the mobile phase of the present invention can be from 5% to 12% (volume percentage), and the gradient of water can be from 95% to 88% (volume percentage).
  • the flow rate of the mobile phase can also be determined according to the actual production situation. Choose a fraction with an elution volume between 51-731 ml, preferably a fraction with an elution volume between 250-504 ml, more preferably an elution volume between 278-353 ml and/or between 354-429 ml And/or the fraction between 430-504 ml, or the similar fraction obtained by other similar chromatographic columns in the same proportion, is the extract described herein.
  • the elution volume is determined as follows:
  • Reverse phase separation column C18 reverse phase separation column
  • Elution method gradient elution, 0-10CV, acetonitrile (A) from 5% to 12%, ddH 2 O (B) from 95% to 88%;
  • the elution volume can be selected according to the above method; in other words, when other chromatographic columns are used, the selected elution volume should correspond to the elution volume determined by the above method.
  • two reverse phase chromatographic separations can be performed.
  • the resolution of the first reversed phase chromatographic separation may be lower than that of the second reversed phase chromatographic separation.
  • the percentage gradient of the polar organic solvent can vary from 5% to 12% (volume percentage), and the percentage gradient of water can vary from 95% to 88% (volume percentage).
  • the first reversed-phase chromatographic separation when performing the first reversed-phase chromatographic separation, take the fraction with an elution volume between 51-731 ml, preferably the fraction with an elution volume between 250-504 ml, and more preferably with an elution volume of 278-353 ml between and/or between 354-429 milliliters and/or between 430-504 milliliters, or other similar chromatographic columns with similar fractions obtained in the same proportion, the second reverse phase chromatographic separation is performed. Due to different loading sample volumes and/or different column volumes of reversed-phase separation columns, the elution volume will be different accordingly.
  • the gradient of organic solvent can vary from 0% to 7% (volume percentage), and the gradient of water can vary from 100% to 93% (volume percentage).
  • the organic solvent when performing gradient elution in the second reverse phase chromatographic separation, in the first 0-3 minutes, the organic solvent changes from 0% to 5.5%, and the ultrapure water changes from 100% to 94.5%; In 3-50 minutes, the organic solvent changes gradually from 5.5% to 7%, and the ultrapure water changes gradually from 94.5% to 93%.
  • the eluate with an elution time between 11-12.5 minutes and the eluate with an elution time between 13-14 minutes are taken. The elution time is determined as follows:
  • Reverse phase separation column C18 reverse phase separation column
  • Elution method gradient elution, 0-3 minutes, acetonitrile from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile from 5.5% to 7%, The ultrapure water changes gradually from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%;
  • Loading volume 20 microliters.
  • the fractions when performing the first reverse phase chromatography separation, are collected, and then the activity of each fraction to promote the proliferation of cells (such as the human cardiomyocyte cell line AC16) is tested using conventional techniques in the art. Afterwards, the fractions with the activity of promoting cell proliferation were further separated by reversed-phase chromatography. After the second reverse phase chromatography separation, the collected fractions can be tested for cell proliferation activity to obtain fractions with cell proliferation activity.
  • the fraction can be a mixture of different components.
  • amniotic fluid and/or extracts thereof described herein can be used as active ingredients of medicines for in vivo administration to a subject in need.
  • an effective amount of the amniotic fluid and/or extracts thereof described herein, or a pharmaceutical composition containing the amniotic fluid and/or extracts thereof can be administered to a subject in need.
  • the animal may be a mammal, especially a human.
  • the cytokine storm of the present invention refers to a variety of cytokines in body fluids (human or animal body) such as TNF- ⁇ , IL-1, IL-6, IL-12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1 and IL-8 are rapidly and massively produced.
  • the causes of cytokine storms include infectiousness, acute injury, organ transplantation, rheumatic and neoplastic causes.
  • the cytokine storm is caused by a microbial infection.
  • the cytokine storm of the present invention is caused by viral infection. Viral infections include but are not limited to coronavirus, influenza virus, etc.
  • the cytokine storm of the present invention is caused by COVID-19, SARS, MERS, H5N1 influenza virus or H7N9 influenza virus.
  • the use of cellular immunotherapy such as treatment with CAR-T cells, also produces a cytokine storm.
  • studies have shown that cytokine storms occur in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome.
  • the cytokine storm syndrome described in the present invention is an uncontrolled and dysfunctional immune response in a subject, the continuous activation and expansion of lymphocytes and macrophages, and the secretion of large amounts of cytokines such as TNF- ⁇ , IL-1 , IL-6, IL-12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1 and IL-8, leading to systemic inflammation, hyperferritinemia, hemodynamic instability and multiple organ failure (MOF).
  • the causes of cytokine storm syndrome include infectiousness, acute injury, organ transplantation, rheumatic and neoplastic causes.
  • the cytokine storm syndrome is caused by a microbial infection.
  • the cytokine storm syndrome of the present invention is caused by viral infection, especially influenza virus and/or coronavirus infection. More specifically, in some embodiments, the cytokine storm syndrome of the present invention is caused by COVID-19, SARS, MERS, H5N1 influenza virus or H7N9 influenza virus.
  • the use of cellular immunotherapy, such as treatment with CAR-T cells can also produce cytokine storm syndrome.
  • cytokine storm syndrome includes HLH (hemophagocytic lymphohistiocytosis) and MAS (macrophage activation syndrome).
  • cytokine storm syndrome refers to cytokine storm syndrome that occurs in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome.
  • amniotic fluid is used, especially the amniotic fluid of poultry eggs described herein, and more preferably amniotic fluid of eggs is used to inhibit cytokine storm, or treat or prevent cytokine storm syndrome.
  • the present invention provides a method for inhibiting cytokine storm, or treating or preventing cytokine storm syndrome, which method comprises administering to a subject in need an effective amount of the amniotic fluid and/or extract thereof of the present invention, or containing all The steps of the pharmaceutical composition of amniotic fluid and/or its extract are described. Also provided is the application of amniotic fluid and/or its extracts in the preparation of reagents for inhibiting cytokine storm or drugs for treating or preventing cytokine storm syndrome, as well as reagents for inhibiting cytokine storm or treating or preventing cytokine storm Syndrome of amniotic fluid and/or its extract or pharmaceutical composition as described herein.
  • an effective amount refers to a dose that can treat, prevent, alleviate and/or alleviate a disease or condition in a subject.
  • the therapeutically effective dose can be determined according to factors such as the patient's age, gender, the disease and its severity, and other physical conditions of the patient.
  • the subject or patient generally refers to a mammal, especially a human.
  • treatment and prevention have well-known meanings in the art, and "inhibiting" cytokine storm refers to preventing the occurrence of cytokine storm or reducing its severity.
  • amniotic fluid and/or its extract described herein can be used directly or used in the methods and uses described herein to give a subject in need.
  • the mode of administration may be parenteral administration, such as intravenous injection.
  • a therapeutically effective amount of amniotic fluid and/or its extract can be mixed with an appropriate amount of normal saline for injection, water for injection or glucose injection, and then administered by, for example, intravenous infusion.
  • composition containing the amniotic fluid and/or its extracts described herein usually also contains pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients refer to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: antibiotics, moisturizers Agents, pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavor enhancers, Wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.
  • pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, or other suitable non-active ingredients used in combination with pharmacological compounds. Active ingredient.
  • the dosage and frequency of administration can be determined by the medical staff according to the specific condition, the age and gender of the patient. Generally, for the treatment of a specific disease, a therapeutically effective amount refers to an amount sufficient to ameliorate or alleviate the symptoms associated with the disease in some way. Such a dose can be administered as a single dose, or it can be administered according to an effective treatment regimen. The dosage may cure the disease, but the administration is usually to improve the symptoms of the disease. Generally, repeated administration is required to achieve the desired symptom improvement.
  • the dosage for humans is usually 1-200ml/time, and it can be injected daily or weekly.
  • the frequency of administration may be multiple times a day, twice a day, every two days, every three days, every four days, every five days, or every six days, or once every half month , Or once a month.
  • This document also provides a pharmaceutical composition, which contains the amniotic fluid and/or extracts thereof, especially amniotic fluid and/or extracts from poultry eggs, more preferably the embryo age is 5-12 Days, more preferably 6-11 days, more preferably 6-9 days, more preferably 7-8 days of amniotic fluid from eggs and/or extracts thereof.
  • the pharmaceutical composition may be cryopreservation of amniotic fluid and/or its extract or its lyophilized reagent, such as freeze-dried amniotic fluid and/or its extract, below -60°C.
  • the pharmaceutical composition may also contain other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection, or glucose injection.
  • the pharmaceutical composition contains 5-40% (v/v) or 10%-35% of amniotic fluid and/or extracts thereof, preferably 15-30%.
  • Microcomputer automatic incubator (Zhengda TM ZF880), clean petri dish, 1.0ml syringe (Jiangxi Hongda TM ), 70% alcohol-sterilized forceps, stainless steel sieve, sterile centrifuge tube ( #SCT-50ML-25-S) and low-speed refrigerated centrifuge (Zhongjia KDC-2046).
  • amniotic fluid extract collected through the test of the Midland TM 1800 ultraviolet spectrophotometer.
  • the standard operating procedure of the photometer please refer to the user manual.
  • a Hitachi Primaide-type high performance liquid chromatograph was used to detect the amniotic fluid components of eggs of different embryonic ages. Perform detection according to the operating instructions of the chromatograph. Wherein, before the start of the detection, wash with 100% acetonitrile for 30 minutes, the flow rate time is 0.8ml/min, and then equilibrate with water for 30 minutes, the flow rate 0.8ml/min time. Take 25 ⁇ l sample and eliminate air bubbles, click the "data acquisition" button of the software that comes with the chromatograph, select "method 2", click "single analysis start” at the bottom of the screen, and start to inject the sample when the system displays "waiting for injection”. The injection should be rapid, and the valve should be switched after the injection.
  • the method 2 is as follows:
  • amniotic fluid with embryo age of 7, 11, and 13 days was tested, and the results are shown in Figures 1-3.
  • This example tests the effect of the egg amniotic fluid (EE) of Example 1 on the growth of chicken embryonic fibroblasts under different culture conditions.
  • the composition of the DMEM medium used in this example is as follows: #Cat. 11960077, add 1% L-glutamine ( #G0200) and 5% FBS ( #Cat.10099141)), 0.25% pancreatin-EDTA (Hangzhou Keyi Biological TM #CY003), PBS (BI TM #02-024-1ACS), 0.4% trypan blue stain (BBI TM #72-57- 1).
  • Figure 4 shows that after 96 hours of co-cultivation, the number of chicken embryo fibroblasts in the experimental group with EE was significantly higher than the number of cells in the control without EE.
  • Example 5 Cell viability and migration ability in amniotic fluid extract
  • Example 2 The same method as in Example 1 was used to obtain the amniotic fluid of duck eggs with an embryonic age of 8 days.
  • the scratch test was used to test the effect of egg amniotic fluid on chicken embryo fibroblasts and duck egg amniotic fluid on the growth viability and migration ability of human umbilical vein endothelial cells (HUVEC).
  • Duck egg amniotic fluid was obtained from duck eggs of 8 days embryonic age, and obtained by the method of Example 1.
  • Chicken embryo fibroblasts were obtained using the method described in Example 4, and human umbilical vein endothelial cells were obtained from commercial sources.
  • composition of the DMEM medium used in this example is as follows: #Cat.11960077, add 1% L-glutamine ( #G0200) and 5% FBS ( #Cat.10099141)), 0.25% pancreatin-EDTA (Hangzhou Keyi Biological TM #CY003), PBS (BI TM #02-024-1ACS), 0.4% trypan blue stain (BBI TM #72-57- 1).
  • Figure 5 shows the effect of amniotic fluid from eggs on the growth viability and migration ability of human umbilical vein endothelial cells (HUVEC). The addition of 5% (volume ratio) of amniotic fluid obviously promotes the healing of HUVEC.
  • Figure 6 shows the effect of amniotic fluid from duck eggs on the growth vigor and migration ability of chicken embryonic fibroblasts. The addition of amniotic fluid also shows a very obvious promotion effect on the healing of chicken embryonic fibroblasts.
  • HUVEC human umbilical vein endothelial cells
  • Example 1 Refer to the method described in Example 1 to obtain the amniotic fluid of the 13-14 days gestational age of the mice, balance the centrifuge tube collecting the amniotic fluid extract and use the Zhongjia TM KDC-2046 low-speed refrigerated centrifuge at 5°C, 3500rpm for 21 minutes (See the manual for the standard operating procedure of the centrifuge). The supernatant was decanted and transferred to a clean plastic storage tank and stored in a refrigerator at -80°C. Reserve 5ml samples for subsequent testing in each batch. All steps are performed under sterile conditions.
  • the purpose of this embodiment is to gradually purify the biologically active compounds in chicken embryo amniotic fluid through analytical column gel column Sephacryl S-200, anion exchange column HiPrep Q, desalting column HiPrep 26/10 Desalting, HiLoad 16/600 Superdex 75 pg.
  • Step 1 Gel column GE Sephacryl S-200
  • ddH 2 O balance gel column flow rate 2ml/min, until the 280nm UV absorption curve is stable and returns to the baseline;
  • Sample loading flow rate 1ml/min, sample loading volume 10ml;
  • Step 2 Anion exchange column GE HiPrep Q
  • Sodium phosphate buffer A (50mM Na 2 HPO 4 +NaH 2 PO 4 , pH 8.0) equilibrate the anion exchange column: flow rate 2ml/min, until the 280nm UV absorption curve is stable and returns to the baseline;
  • Sample loading Take the biologically active part after the first step of purification, use the pump to load the sample flow rate 1.5ml/min, the sample volume 250ml, and at the same time collect the unbound part of the anion column in an equal volume, 2ml/tube;
  • Desalting replace the bound and unbound fractions in the ion column with GE HiPrep 26/10 Desalting into degassed ddH 2 O, and collect the desalted fractions;
  • Step 3 Gel column GE HiLoad 16/600 Superdex75pg
  • ddH 2 O balance gel column flow rate 1ml/min, until the 280nm UV absorption curve is stable and returns to the baseline;
  • Sample loading flow rate 1ml/min, sample loading volume 10ml;
  • Elution Elute the sample with degassed ddH 2 O, flow rate 1ml/min, collect fractions in equal volume, 2ml/tube. Elution 1.5 column volume (240ml);
  • phase separation column is UniSil 10-100 C18;
  • the elution solvent is degassed chromatography grade acetonitrile and ddH 2 O.
  • Sample processing 400ml fresh egg amniotic fluid, add appropriate amount of hexane, centrifuge at 2500rpm, 4°C for 20 minutes to obtain the water phase, filter with 0.22 ⁇ m filter membrane.
  • the first step using acetonitrile (A) and ddH 2 O (B) as mobile phases, UniSil 10-100 C18 reverse phase separation column;
  • Equilibrate the reversed phase column equilibrate the reversed phase column with 5% acetonitrile (A) as the mobile phase at a flow rate of 10ml/min until the 280nm UV absorption curve is stable and returns to the baseline;
  • Sample loading flow rate 1ml/min, sample loading volume is 50ml;
  • the 8th peak of the primary active fraction separated by AKTA was further separated and purified by HPLC (Hitachi).
  • the mobile phase is acetonitrile and ultrapure water.
  • Use gradient elution, the specific parameters are as follows: 0-3 minutes, acetonitrile changes from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile changes from 5.5% to 7 %, the ultrapure water changes gradually from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%.
  • the flow rate of the mobile phase was 0.8mL/min, the column temperature was 25.0°C, and the injection volume was 20 ⁇ l.
  • the detection wavelength of the DAD detector is 250 nm, and the detection time is 0-20 minutes.
  • Figure 14 shows that the hydrophobicity of P8 is between L-dopa and VB12. Since the octanol/water partition coefficient Log P of L-dopa is 0.05, and the octanol/water partition coefficient Log P of VB12 is 1.897, the octanol/water partition coefficient Log P of P8 is between 0.05 and 1.897. It is preferably between 0.1 and 1.897, more preferably between 0.5 and 1.897 or between 0.5 and 1.5.
  • reagents such as sodium hydroxide, sodium chloride, potassium chloride, hydrated sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen carbonate, sodium carbonate, magnesium chloride, acetone, concentrated sulfuric acid, concentrated hydrochloric acid, xylene, absolute ethanol, Paraffin wax and sucrose were purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium lauryl sulfate and ethylenediamine tetraacid were purchased from Sigma in the United States; Triton X-100 and heparin were purchased from Beijing Dingguo Company; Tween-20 was purchased from American Thermo Fisher Company; Chloral hydrate was purchased from Beijing Soleibao Technology Co., Ltd.; Paraformaldehyde and Masson Masson Tricolor Staining Kit were purchased from Google Biotechnology Co., Ltd.; OCT embedding agent was purchased from Japan Sakura Company; Anti-fluorescence extract The anti-blocking tablets were purchased from the Vector company of the United States.
  • Rabbit anti-human/mouse Aurora B antibody was purchased from Sigma Aldrich, USA; rabbit anti-human/mouse phosphorylated histone H3 polyclonal antibody was purchased from Merck Millipore, Germany; rabbit anti-human/mouse cTnT polyclonal antibody was purchased from Abcam, UK; Alexa Fluor 594-labeled goat anti-rabbit IgG, Alexa Fluor 488-labeled goat anti-rabbit IgG, Alexa Fluor 594-labeled goat anti-mouse IgG, and Alexa Fluor 488-labeled goat anti-mouse IgG were purchased from Life Technologies, USA; DAPI was purchased from Sigma Aldrich, USA; goats The serum working solution was purchased from Wuhan Boster Biological Engineering Co., Ltd.
  • Trizol was purchased from Invitrogen, USA; Adriamycin hydrochloride was purchased from Shanghai Shenggong Biological Engineering Co., Ltd.
  • the experimental animals were male C57BL/6J mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Leica Dmi8 fluorescence microscope and Leica IM50 image acquisition system were purchased from Leica, Germany; the small animal ultrasound system was purchased from VisualSonics, Canada.
  • phosphate buffer (1 ⁇ PBS): NaCl 8.0g, KCl 0.2g, Na 2 PO 4 ⁇ H 2 O 3.58g, KH 2 PO 4 0.24, adjust the pH to 7.4, deionized water Dilute to 1000ml, autoclave and store at 4°C.
  • Triton X-100 preparation Triton X-100 stock solution 5ml, 1 ⁇ PBS 995ml.
  • the collagen fibers, mucus, and cartilage were blue (for example, the light green liquid stained green), the cytoplasm, muscle, cellulose, and glial were red, and the nucleus was black and blue.
  • mice were anesthetized with isoflurane in the induction box, the frequency of the ventilator was 115 times/min, the respiratory ratio was 1:1, and the tidal volume was 1.5ml.
  • a 20g indwelling needle plastic tube was used to intubate the trachea through the mouth, connected to a small animal ventilator, and continued anesthesia with pure oxygen containing 2.5% isoflurane.
  • mice C57BL/6J male mice were injected with doxorubicin (5mg/kg) once in 7 days at 8 weeks. After a total of four injections, the mice would have heart failure, which was verified by echocardiography.
  • mice After treatment for 1 week and 8 weeks after surgery, the mice were killed by intraperitoneal injection of 10% chloral hydrate (200 mg/kg), and the heart was taken out. The liver and kidney were taken for 1 week, OCT embedding or paraffin embedding.
  • mice myocardial infarction model Establish a mouse myocardial infarction model with reference to the method described in (4) above.
  • the established mouse myocardial infarction models were divided into control group (NS) and chicken embryo amniotic fluid (EE) treatment group (6 in each group).
  • EE treatment group 100 microliters of the EE prepared in Example 1 was injected through the tail vein every two days, and on the 21st day of the third week, a total of 10 injections were made.
  • the control group was injected with 100 microliters of normal saline 10 times in the same manner.
  • LVEF Left ventricular ejection fraction
  • the increase of left ventricular ejection fraction indicates that the cardiac function of mice after myocardial infarction can be improved.
  • the ejection fraction of mice was calculated by cardiac ultrasound, and the results are shown in Figure 15. It can be seen from Figure 15 that by the 3rd week, the treatment of EE significantly increased the left ventricular ejection fraction of the mice with myocardial infarction, indicating that the treatment of EE significantly improved the cardiac function of the mice after myocardial infarction.
  • the left ventricular short axis shortening rate (LVFS) of each group of mice was calculated by echocardiography, and the results are shown in Figure 16. It can be seen from Figure 16 that by the 3rd week, the EE treatment significantly increased the LVFS of the mice with myocardial infarction, that is, improved the cardiac function of the mice after myocardial infarction.
  • PH3 staining is an indicator of cell regeneration in the heart.
  • the mice in each group after 21 days of treatment were sacrificed, frozen sections of myocardial tissue were prepared, and PH3 staining was performed according to the method described in point (1) above.
  • the results are shown in FIG. 17. It can be clearly seen from Fig. 17 that the PH3 staining positive (green fluorescent dots, indicated by the arrow) cells in the heart tissue of the mice in the EE treatment group increased significantly, indicating that the EE treatment promoted the regeneration of cells in the heart tissue.
  • AuroraB staining is an indicator for judging the regeneration of cells in the heart. AuroraB staining was performed according to the method described in point (1) above, and the results are shown in Figure 18. It can be clearly seen from Figure 18 that the AuroraB staining positive (green fluorescent dots, arrows point) cells in the heart tissues of the mice in the EE treatment group Significant increase, indicating that the treatment of EE promoted the regeneration of cells in the heart tissue.
  • Masson staining is a classic method for judging cardiac infarct tissue and fibrous tissue.
  • the mice in each group after 21 days of treatment were sacrificed, and paraffin sections of myocardial tissue were prepared and stained according to the aforementioned point (3).
  • the results are shown in FIG. 19.
  • the blue is the infarct fibrosis tissue
  • the red is the muscle tissue. From the figure, it can be seen that the mice with myocardial infarction have severe fibrosis, and the fibrosis is significantly reduced after EE treatment; it indicates that EE treatment prevents small Fibrosis after myocardial infarction in rats.
  • EE treatment also changed the ratio of macrophage subtypes in the inflammatory response, selectively activated and increased CCR + and CCR2 + CX3CR1 + macrophages, and inhibited Cytokine storm, thereby inhibiting cardiomyocyte death and fibrosis.
  • mice Refer to the above method (5) to construct a mouse heart failure model.
  • the established mouse heart failure model was divided into a control group and a chicken embryo extract (EE) treatment group (6 mice in each group).
  • EE treatment group 100 microliters of the EE prepared in Example 1 was injected through the tail vein every two days, and on the 21st day of the third week, a total of 10 injections were made.
  • the control group was injected with 100 microliters of normal saline 10 times in the same manner.
  • Left ventricular ejection fraction is a key classic indicator of left ventricular function.
  • the increase of left ventricular ejection fraction indicates that the cardiac function of mice after heart failure can be improved.
  • the ejection fraction of mice was measured by echocardiography, and the results are shown in Figure 20. It can be seen from Figure 20 that by the 3rd week, the treatment of EE significantly increased the left ventricular ejection fraction of the mice with heart failure, indicating that the treatment of EE significantly improved the heart function of the mice with heart failure. The area of left ventricular fibrosis was significantly reduced.
  • Figure 21 shows that chicken EE treatment of myocardial infarction large white pigs can increase the left ventricular ejection fraction and short axis shortening rate of myocardial infarct large white pigs.
  • the heart function of the large white pigs in the control group after the operation shows a gradual decline, while the left ventricular function of the EE treatment group
  • the ventricular function recovered to a certain extent, and the EF and FS at 2 weeks, 4 weeks and 8 weeks after the operation were significantly higher than those of the control group ( Figure 21, A and C).
  • the decrease in the treatment group was significant at 2, 4, and 8 weeks. Lower than the control group ( Figure 21, B and D).
  • the stroke volume of the treatment group was significantly higher than that of the control group during 1-8 weeks after surgery (Figure 21, E).
  • the volume and diameter of the control left ventricular end-systole have an upward trend, and the treatment group is lower than the control group ( Figure 21, F and I), indicating that EE improves the left ventricular contractility.
  • the end-diastolic volume and diameter of the control left ventricle showed a rising trend, and the drug group showed a trend of rising first and then falling ( Figure 21, G and H), indicating that EE reversed part of the ventricular remodeling caused by myocardial infarction (MI) .
  • MI myocardial infarction
  • the left ventricular anterior wall tissue of the infarct area was taken for Masson's trichrome staining, and it was found that the control group showed transmural infarction and the ventricular wall became thin; the EE treatment group had cardiac fibrosis interspersed in the myocardial space, and the ventricular wall was not significantly thinned (Figure 22). , C).
  • EE can significantly increase the left ventricular ejection fraction and stroke volume of ischemia-reperfusion large white pigs, reduce the left ventricular remodeling caused by myocardial infarction, reduce the pulmonary congestion of ischemia-reperfusion large pigs, and increase the perfusion rate. Daily activity volume.
  • the results of TTC staining showed that the area of cardiac infarction in the EE treatment group was significantly lower than that in the control group; the results of tissue Masson staining showed that the anterior wall of the left heart of large white pigs in the control group had transmural infarction, and the area of fibrosis was significantly higher than that in the EE treatment group; Fluorescence staining results show that EE can increase the angiogenesis in the infarcted area of large white pigs.
  • This example tested the use of chicken embryo amniotic fluid (EE) obtained in Example 1 to repair lung and extrapulmonary organ damage caused by pneumonia.
  • EE chicken embryo amniotic fluid
  • LPS Lipopolysaccharide, a component of the outer wall of the cell wall of Gram-negative bacteria
  • Cultivate human alveolar epithelial cells Calu-3 in a 6-well cell culture plate (culture medium is DMEM+10% FBS+0.1mg/ml penicillin/streptomycin), use LPS to construct a cell pneumonia model when the cell density reaches about 40-50%
  • the control group culture medium is the culture medium DMEM+10% (volume ratio, the same below) FBS+0.1mg/ml penicillin/streptomycin
  • the treatment tissue culture medium is the culture medium DMEM+10% FBS+20% (volume For comparison, the same below) EE+0.1mg/ml penicillin/streptomycin
  • MCE lipopolysaccharide LPS
  • Culture human alveolar epithelial cells Calu-3 in a 6-well plate (culture medium is DMEM+10%FBS+0.1mg/ml penicillin/streptomycin), the cell density is about 40-50%, use 4 ⁇ l/ml COVID-19 Protein pseudovirus (Shanghai Yisheng Biotechnology Co., Ltd.) was treated for 24 hours, and 20% EE was added to the treatment tissue culture solution.
  • culture medium is DMEM+10%FBS+0.1mg/ml penicillin/streptomycin
  • the cell density is about 40-50%
  • use 4 ⁇ l/ml COVID-19 Protein pseudovirus (Shanghai Yisheng Biotechnology Co., Ltd.) was treated for 24 hours, and 20% EE was added to the treatment tissue culture solution.
  • the qPCR results are shown in Figure 25.
  • the results showed that EE significantly reduced the expression of inflammatory factors IL-1 ⁇ , IL-6, TNF- ⁇ and leukocyte chemotactic factors Il-23, CXCL-5, IL-17a caused by COVID-19 protein pseudovirus, while EE increased
  • the high anti-inflammatory factors IFN- ⁇ and IL-10 indicate that EE reduces the inflammation of lung epithelial cells caused by the COVID-19 protein pseudovirus.
  • a mouse pneumonia model was constructed by inhaling LPS.
  • C57BL/6J mice were anesthetized with 0.3% sodium pentobarbital solution 0.3ml/20g, a 20G indwelling needle was intubated through the oral cavity, and 50 ⁇ l, 200 ⁇ g/ml LPS was dripped into the intubation. Liquid, the mouse slowly inhales the LPS as it breathes, then remove the cannula, and place the mouse on a 37°C hot plate until the mouse wakes up.
  • the mice were randomly divided into 2 groups, the control group was injected with 100 ⁇ l of saline through the tail vein, and the treatment group was injected with 100 ⁇ l of EE through the tail vein. The mice are weighed daily. Pulmonary function was measured and samples were taken at 1 day, 3 days and 7 days after the model to observe the condition of lung tissue and its extrapulmonary organs.

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Abstract

L'invention concerne du liquide amniotique et/ou un extrait de celui-ci issu d'animaux non humains, en particulier d'oiseaux et de mammifères non humains, pour inhiber un orage cytokinique et prévenir et traiter un syndrome d'orage cytokinique.
PCT/CN2021/089156 2020-04-23 2021-04-23 Procédé et composition permettant d'inhiber un orage cytokinique Ceased WO2021213488A1 (fr)

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WO2015120077A1 (fr) * 2014-02-04 2015-08-13 Gonzalez Jose Javier Lopez Cellules souches mésenchymateuses adultes biologiquement optimisées
CN110857434A (zh) * 2018-08-10 2020-03-03 浙江楚沅生物科技有限公司 促进细胞生长和组织修复的方法及组合物

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WO2015120077A1 (fr) * 2014-02-04 2015-08-13 Gonzalez Jose Javier Lopez Cellules souches mésenchymateuses adultes biologiquement optimisées
CN110857434A (zh) * 2018-08-10 2020-03-03 浙江楚沅生物科技有限公司 促进细胞生长和组织修复的方法及组合物

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EP4424317A4 (fr) * 2021-10-29 2025-08-06 Anhui Hygeiancells Biomedical Co Ltd Médicament pour la prévention ou le traitement de l'entérite et du cancer intestinal

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