WO2021213488A1 - Method and composition for inhibiting cytokine storm - Google Patents

Abstract

Provided is amniotic fluid and/or an extract thereof from non-human animals, especially birds and non-human mammals, for inhibiting a cytokine storm and preventing and treating cytokine storm syndrome.

Description

Method and composition for inhibiting cytokine storm Technical field

The present invention relates to a method and composition for inhibiting cytokine storm.

Background technique

Cytokine storm syndrome (CSS) 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.

The hallmark of 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-α, IL-1, IL-6, IL -12, IFN-α, IFN-β, IFN-γ, MCP-1 and IL-8, leading to a cytokine storm.

Various infectious and non-communicable diseases have a causal relationship with CSS. Hemophagocytic lymphohistiocytosis (HLH) is a typical inflammatory disease characterized by an uncontrolled cytokine storm. According to its etiology and pathogenesis, HLH can be divided into primary (pHLH) or secondary (sHLH). SHLH associated with autoimmune or autoinflammatory diseases is called macrophage activation syndrome (MAS). After the body is infected with microorganisms, it can also cause the rapid mass production of a variety of cytokines in body fluids, which can cause acute respiratory distress syndrome and multiple organ failure.

Summary of the invention

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.

In one or more embodiments, the agent or drug is a pharmaceutical composition comprising amniotic fluid and/or extracts thereof as described herein and optionally pharmaceutically acceptable excipients.

In one or more embodiments, 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.

In one or more embodiments, 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.

In one or more embodiments, 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.

In one or more embodiments, 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.

In one or more embodiments, the cytokine storm or cytokine storm syndrome is caused by influenza virus and/or coronavirus infection.

In one or more embodiments, the coronavirus is COVID-19.

In one or more embodiments, the cytokine storm or cytokine storm syndrome is caused by SARS, MERS, H5N1 influenza virus or H7N9 influenza virus.

Description of the drawings

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.

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.

Figure 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.

Figure 13: The P8 peak was further separated by C18 AQ on the Hitachi HPLC system, and the cell viability test found that the higher purity P8-2 had cell viability.

Figure 14: HPLC chart of higher purity P8, L-dopa (Log P = 0.05) and Vitamin B12 (Log P = 1.897).

Figure 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.

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.

Figure 17: Immunofluorescence staining of the heart of mice with myocardial infarction (PH3, cTnT, DAPI).

Figure 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.

Figure 20: The area of cardiac fibrosis in mice with myocardial infarction was significantly reduced after amniotic fluid (EE) treatment compared with untreated group (NS).

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. Upper left image: blank group (untreated); upper right image: control group (given LPS); lower left image: treatment group (LPS+20% EE); lower right image: apoptosis results detected by AnnexinV-FITC-PI.

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.

Detailed ways

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a preferred technical solution.

The inventors found that the growth factor group contained in non-human animal amniotic fluid and/or its extract can inhibit cytokine storm. Therefore, 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.

In the present invention, 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. Preferably, 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. For example, when using eggs, 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. For example, when duck eggs are used, duck eggs with embryo age of 8-10 days, especially 8-9 days, may be the best.

Conventional methods can be used to obtain 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.

Here, 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. In certain embodiments, 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. For example, using surgical scissors to cut the abdominal cavity of a mouse 8-14 days pregnant, carefully remove and cut open the uterus, 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.

It should be understood that, if necessary, the 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. It should be understood that all steps of obtaining amniotic fluid need to be carried out under sterile conditions; in addition, 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℃ and used after thawing.

In certain embodiments, the present invention uses an extract of amniotic fluid. Preferably, 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. Preferably, the extract is a neutral fraction. Preferably, 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. For example, 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. Alternatively, 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.

In some embodiments, 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. Specifically, the method may include the following steps:

(1) A neutral fraction with a molecular weight of 150-2000 Daltons is separated from the amniotic fluid; and

(2) Separating from the neutral fraction with a molecular weight of 150-2000 Daltons to obtain a neutral fraction with a molecular weight of 150-1200 Daltons.

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.

In this article, various commercially available 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. Generally, when using a gel chromatography column, you can first _{equilibrate the gel chromatography column with ddH 2} O, and the flow rate can be determined according to the actual situation. For example, in certain embodiments, the flow rate may be 0.5-50 ml/min, such as 1 ml/min. Usually, the ultraviolet absorption is between 200-300nm, such as 280nm. After the UV absorption curve is stable and returns to the baseline, the balance is ended. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation situation. After sample loading, 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.

Herein, methods known in the art can be used to separate charged components from uncharged components. For example, it can be achieved using ion exchange methods. Both anion exchange and cation exchange can be used in the method of the present invention. In certain embodiments, 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. Alternatively, 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.

Generally, when performing ion exchange, the ion exchange column is first equilibrated with a buffer. 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. In certain embodiments, 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. For example, in certain embodiments, the flow rate may be 0.5-50 ml/min, such as 1 ml/min. Usually, after the 280nm UV absorption curve is stable and returns to the baseline, 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.

In 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 .

The main purpose of 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. Here, 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.

Usually, the _{gel column can be equilibrated with ddH 2} O first, and the flow rate can be determined according to the actual situation. For example, in certain embodiments, the flow rate may be 0.5-50 ml/min, such as 1 ml/min. Usually, after the 280nm UV absorption curve is stable and returns to the baseline, the equilibrium is ended. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation situation. After loading the sample, 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. In one or more embodiments, 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.

For similar separation methods, please refer to CN 201810909193.0, the entire content of which is incorporated herein by reference.

In some embodiments, 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. Generally, 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. For example, such 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.

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;

Mobile phase: acetonitrile and ultrapure water;

Elution method: gradient elution, 0-10CV, acetonitrile (A) from 5% to 12%, ddH ₂ O (B) from 95% to 88%;

Flow rate of mobile phase: 10ml/min;

Column temperature: 25.0℃;

Sample flow rate: 1ml/min;

Loading volume: 50ml.

When other chromatographic columns are used, 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.

In certain embodiments, 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. When performing gradient elution for the first reverse 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). As mentioned above, 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.

In the second phase of reverse phase chromatography for gradient elution, 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). In some embodiments, 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%. Preferably, in the second reverse phase chromatographic separation, 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;

Mobile phase: acetonitrile and ultrapure water;

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%;

Flow rate of mobile phase: 0.8ml/min;

Column temperature: 25.0℃;

Loading volume: 20 microliters.

It should be understood that when two reversed-phase chromatographic separations are used, when different chromatographic separation columns are used, different mobile phases, gradient changes and flow rates can be selected according to the composition, performance and specifications of the different separation columns to obtain the best elution effect. . In addition, when different reversed-phase columns, mobile phases, and elution methods are used, the selection of eluent will be different. You can refer to the method of determining eluent described herein to select a suitable eluent as the present invention. Extracts.

In some embodiments, when performing the first reverse phase chromatography separation, the fractions 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.

For the extract obtained from the separation of amniotic fluid by reversed-phase chromatography and the biological activity of the extract, please refer to CN 201910887556.X, the entire content of which is incorporated herein by reference.

It should be understood that in combination with CN 201810911038.2, CN 201810909485.4, CN 201810909193.0 and CN 201910887556.X, it can be seen that the amniotic fluid and extracts from various sources described in the present invention have the same or similar biological activities, and when used in the present invention, all Inhibit cytokine storm, thereby treating or preventing cytokine storm syndrome.

The 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. For example, 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.

Here, 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-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, IFN- γ, 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. In some embodiments, the cytokine storm is caused by a microbial infection. In some embodiments, the cytokine storm of the present invention is caused by viral infection. Viral infections include but are not limited to coronavirus, influenza virus, etc. More specifically, in some embodiments, the cytokine storm of the present invention is caused by COVID-19, SARS, MERS, H5N1 influenza virus or H7N9 influenza virus. In some embodiments, the use of cellular immunotherapy, such as treatment with CAR-T cells, also produces a cytokine storm. In addition, 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-α, IL-1 , IL-6, IL-12, IFN-α, IFN-β, IFN-γ, 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. In some embodiments, the cytokine storm syndrome is caused by a microbial infection. In some embodiments, 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. In some embodiments, the use of cellular immunotherapy, such as treatment with CAR-T cells, can also produce cytokine storm syndrome. As is well known in the art, cytokine storm syndrome includes HLH (hemophagocytic lymphohistiocytosis) and MAS (macrophage activation syndrome). In some embodiments, cytokine storm syndrome refers to cytokine storm syndrome that occurs in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome.

In a particularly preferred embodiment of the present invention, 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.

Therefore, 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. In this context, 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. Herein, the subject or patient generally refers to a mammal, especially a human. Herein, 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.

The 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. In some embodiments, 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.

The pharmaceutical composition containing the amniotic fluid and/or its extracts described herein usually also contains pharmaceutically acceptable excipients. As used herein, "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. In some embodiments, 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. For example, the dosage for humans is usually 1-200ml/time, and it can be injected daily or weekly. In certain embodiments, 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. Preferably, the pharmaceutical composition contains 5-40% (v/v) or 10%-35% of amniotic fluid and/or extracts thereof, preferably 15-30%.

Hereinafter, the present invention will be explained in the form of specific embodiments. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods, reagents, and instruments used in the examples are conventional methods, reagents, and instruments in the art.

Example

Example 1: Materials and methods

1. Material

a) Instruments and tools

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).

b) Reagents and biological materials

Eggs with embryo age of 7 days.

2. Experimental process

Take the egg, tap the flat blunt end placed upwards to break the eggshell, and peel the eggshell to form a hole about 2 cm in diameter. The edge should be as flat as possible. Use tweezers to carefully tear open the shell membrane and vitelline membrane, taking care not to damage the amniotic membrane. Observe the development of embryos, and only embryos that are well-developed and meet the standards of the corresponding stage can be used to extract amniotic fluid.

Pour the amniotic membrane and connected tissues covering the embryo from the shell to the petri dish, pierce the amniotic membrane with a syringe to extract the amniotic fluid. The amniotic fluid is poured into the centrifuge tube in the ice box.

Take out the embryos in the amniotic membrane with tweezers, collect them in a stainless steel sieve placed on ice, homogenize the collected embryos with a blender every hour, package them in a sterile plastic storage tank, and place them at -80°C. In the refrigerator. It can be placed upright after freezing.

^{The amniotic fluid extract collected through the test of the Midland TM} 1800 ultraviolet spectrophotometer. For the standard operating procedure of the photometer, please refer to the user manual.

Balance the centrifuge tube for collecting amniotic fluid extract and centrifuge it in a Zhongjia ^TM KDC-2046 low-speed refrigerated centrifuge at 5°C and 3500 rpm for 20 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.

Example 2: Component detection

In this example, 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 25μl 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:

Time (min)	water(%)	Acetonitrile (%)	Flow rate (ml/min)
0.0	100.0	0.0	0.8
11.0	100.0	0.0	0.8
17.0	95.0	5.0	0.8
30.0	90.0	10.0	0.8
45.0	55.0	45.0	0.8
60.0	0.0	100.0	0.8
70.0	0.0	100.0	0.8

In this example, amniotic fluid with embryo age of 7, 11, and 13 days was tested, and the results are shown in Figures 1-3.

Example 4: Effect on chicken embryo fibroblasts

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).

Take the embryos of 7-day-old eggs, rinse the surface of the embryos with PBS, and suck up the liquid with a pipette. Take out the internal organs of the embryo, and cut the remaining tissues until there are no large particles or clumps visible to the naked eye. Add 1ml of 0.25% pancreatin-EDTA, mix it with the tissue with a pipette tip, and draw the suspension into a 15ml centrifuge tube. Wash the petri dish with 1ml of 0.25% pancreatin-EDTA, and draw the suspension to the same 15ml centrifuge tube. Put the centrifuge tube in a 37°C water bath, after 5-7 minutes of digestion, add 8ml of DMEM medium (containing PBS) to neutralize the trypsin-EDTA. Put the centrifuge tube into the centrifuge and centrifuge for 5-10 seconds. Take out the centrifuge tube and collect the supernatant. The centrifugal supernatant was centrifuged at 2000 rpm for 2 min. Discard the supernatant, add 4ml of DMEM medium, and resuspend the cells with a pipette tip. Inject 1ml of cell suspension into a 10cm cell culture dish, and then add 10ml of DMEM medium. Shake the petri dish in the cross direction at least 20 times in each direction to make the cells evenly distributed. Incubate at 37°C and 5% CO ₂ . When the cells cover 70%-90% of the bottom of the petri dish, the cells are passaged.

Take the petri dish out of the incubator, and collect the original culture medium in a centrifuge tube. Carefully add 5ml of PBS to wash the cells. After that, add 500 μl 0.25% pancreatin-EDTA, put the petri dish into the incubator, and digest for 1 minute. Gently tap the side of the petri dish to speed up the digestion process. When the cell clumps are quickly broken down and most of the cells are floating, quickly add 9.5ml of the recovered original culture medium to neutralize the trypsin-EDTA. Use a pipette to blow the bottom of the petri dish, collect as much cell suspension as possible into a 15ml centrifuge tube, and centrifuge at 2000rpm for 3min. Discard the supernatant, add 4ml of DMEM medium, and resuspend the cells with a pipette tip. Inject 1ml of the cell suspension into 10cm cell culture dishes containing 10ml of fresh medium containing different volume ratios of amniotic fluid. Shake the petri dish in the cross direction at least 20 times in each direction to make the cells evenly distributed, and incubate at 37°C and 5% CO ₂ .

Take good-growing chicken embryo fibroblasts and collect the original culture medium in a centrifuge tube. Carefully add 5ml of PBS to wash the cells. Be careful not to damage the cell layer. Shake gently and pour out the PBS. Add 100 μl of 0.25% trypsin-EDTA to digest for 2-5 minutes (24-well plate), and neutralize with 100 μl of culture medium. Use a pipette tip to make a single cell suspension. Dilute the single cell suspension according to a certain multiple, and add an equal amount of 0.4% trypan blue staining solution for staining. The appropriate dilution multiple is that the number of cells after dilution is between 20-200. Pipette an appropriate amount (15μl) of cell suspension, add samples from the upper and lower edges of the cover glass to the hemocytometer, and count the number of viable cells under the microscope. Calculate the total number of living cells and adjust the cell concentration to 1×10 ⁵ cells/ml. Sampling was taken every 24 hours, and 3 well cells were taken each time, and routine trypsin-EDTA digestion was carried out, single cell suspension was prepared, and counted under microscope. Draw a growth curve with time (day) as the horizontal axis and cell concentration as the vertical axis. Number of cells=total cell count/4×10 ⁴ ×dilution factor, cell concentration=number of cells/ml.

The result is shown in Figure 4. 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

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.

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).

On the first day before the experiment, prepare a 6-well plate, use a marker to draw 5-6 evenly distributed horizontal lines on the back of the 6-well plate with a ruler, and pass through the holes; then draw a vertical vertical line at the center line. Indicates the location of the scratch. ^{About 5×10 5} cells in the logarithmic growth phase are added to each well. In principle, the fusion rate reaches 90% after overnight inoculation.

On the day of the experiment, draw a line along the vertical line of the marker pen perpendicular to the bottom surface of the 6-well plate with the tip of the gun compared to the ruler. Try not to tilt or bend as much as possible. It is best to use the same gun head between different holes, with a width of 1000-2000μm. Wash each well with 2ml PBS 3 times to wash away the cells at the scratches. Add 2ml of culture medium containing different content of EE to each well, and culture it routinely, changing the medium every 48 hours. Time from scratch is 0h, take pictures at fixed points every 24 hours, and measure the cell spacing on both sides of the scratch. Observe the cell growth status in each hole; draw a chart with time (days) as the horizontal axis and the distance of the scratches in each hole as the vertical axis; calculate the healing speed of the scratches in each hole.

The results are shown in Figures 5 and 6. 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.

Example 6

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.

Measure cell viability: After digesting the well-growing AC16, spread it in a 96-well plate, 8000 cells/well, five replicate wells in each group. After culturing for 2 hours in a 5% CO ₂ saturated humidity 37°C incubator, the cells adhere to the wall. After starvation culture with DMEM medium for 24 hours, it was replaced with DMEM and DMEM with 10% FBS, and DMEM medium with 2.5%, 5%, 10% and 20% (volume ratio) mouse EE added respectively. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance value was detected at 450nm in a microplate reader.

The result is shown in Figure 7.

Example 7: Purification of active compounds in amniotic fluid

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.

1. Material

1.1 Purified sample: fresh egg amniotic fluid with embryo age of 7 days, 50ml.

1.2 Main experimental equipment and consumables

1) GE AKTA purifier;

2) Gel column GE Sephacryl S-200;

3) Anion exchange column GEHiPrep Q;

4) GEHiPrep 26/10 Desalting;

5) Gel column GEHiLoad 16/600 Superdex75pg;

6) Superloop 10ml.

2. Method

2.1 Solution preparation

Preparation of Sodium Phosphate Buffer A (50mM Na ₂ HPO ₄ +NaH ₂ PO ₄ , pH 8.0): 46.6ml 1M Na ₂ HPO ₄ and 3.4ml 1M NaH ₂ PO _{4 were} mixed, and ddH ₂ O was added to make the volume 1L.

2.2 Experimental method

2.2.2 Sample processing: 50ml of fresh amniotic fluid, add appropriate amount of hexane, centrifuge at 2500rpm, 4℃ for 20min to obtain the water phase, filter with 0.22μm filter membrane.

2.2.3 Sample purification

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;

Elution: Use degassed ddH ₂ O to elute the crude product at a flow rate of 2 ml/min, and collect fractions in an equal volume, 3 ml/tube. 2 Column volume (240ml) elution;

Repeat the separation and purification 5 times, and mix the parts with the same peak time in each time;

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);

Measure cell viability: After digesting the well-growing AC16, spread it in a 96-well plate, 8000 cells/well, five replicate wells in each group. After culturing for 2 hours in a 5% CO ₂ saturated humidity 37°C incubator, the cells adhere to the wall. After starvation culture with DMEM medium for 24 hours, it was replaced with 10% FBS DMEM, DMEM and medium containing 20% fraction. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance value was detected at 450nm in a microplate reader.

3. Experimental results

The chromatogram of the unbound part separated by gel column GE HiLoad 16/600 Superdex75 pg is shown in Figure 8. The cell viability test traces the biologically active growth factor groups, and the results are shown in Figure 9.

Example 8

1. Experimental materials

(1) Purified sample: fresh egg amniotic fluid with embryo age of 7 days, 400ml.

(2) Main experimental equipment and consumables

GE AKTA purifier; The phase separation column is UniSil 10-100 C18;

Hitachi HPLC system; using LaChrom-C18 AQ separation column;

The elution solvent is degassed chromatography grade acetonitrile and ddH ₂ O.

2. Experimental method

(1) Sample processing: 400ml fresh egg amniotic fluid, add appropriate amount of hexane, centrifuge at 2500rpm, 4℃ for 20 minutes to obtain the water phase, filter with 0.22μm filter membrane.

(2) Sample purification

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;

Gradient elution: 0-10CV, acetonitrile (A) from 5% to 12%, ddH ₂ O (B) from 95% to 88%. Flow rate 10 ml/min, collect fractions in equal volume, 3 ml/tube;

Repeat the separation and purification 20 times, and combine the fractions with the same peak time in each experiment;

Freeze dry each part of the sample.

Test cell viability: After AC16 is digested, it is plated in a 96-well plate, 8000 cells/well, five replicate wells in each group. After culturing for 2 hours in a 5% CO ₂ saturated humidity 37°C incubator, the cells adhere to the wall. After starvation culture with medium DMEM for 24 hours, the medium was replaced with DMEM containing 10% FBS, DMEM and 20% (volume ratio) of different fractions. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance value was detected at 450nm in a microplate reader. Cell viability%=(absorption value of experimental group-absorption value of blank hole)/(absorption value of control group-absorption value of blank hole)×100%.

Step 2: Hitachi C18 reversed phase separation column separation

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 20μl. The detection wavelength of the DAD detector is 250 nm, and the detection time is 0-20 minutes.

Freeze-dry each part of the sample;

Test cell viability: After AC16 is digested, it is plated in a 96-well plate, 8000 cells/well, five replicate wells in each group. After culturing for 2 hours in a 5% CO ₂ saturated humidity 37°C incubator, the cells adhere to the wall. After starvation culture with DMEM medium for 24 hours, it was replaced with DMEM containing 10% FBS, DMEM and medium containing 20% (volume ratio) of different fractions. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance value was detected at 450nm in a microplate reader. Cell viability%=(absorption value of experimental group-absorption value of blank hole)/(absorption value of control group-absorption value of blank hole)×100%.

3. Experimental results

1. The chromatogram of fresh amniotic fluid separated by UniSil 10-100 C18 is shown in Figure 10. The cell viability test results are shown in Figure 12. The results show that the P6, P7, and P8 peaks in Figure 10 have major biological activities in AC16 cells.

2. The HPLC results of P6, P7 and P8 peaks are shown in Figure 11. Compared with the original crude amniotic fluid, the results show that UniSil 10-100 C18 separation has a higher resolution, which is very suitable as the first step of preparation and separation. As shown in Figure 13, the cell viability test found that P8-2 contained in the P8 peak has biological activity in AC16 cells. The above results indicate that the higher purity P8-2 is one of the important compounds in the P8 peak and amniotic fluid that can promote cell proliferation.

3. In order to determine the hydrophobic coefficient of P8 peak, prepare P8 peak, L-dopa and VB12 standard compound solutions with water respectively, and eluted with Agilent SB-Aq column. The specific elution conditions are as follows:

Chromatographic column: Agilent SB-Aq, 4.6 x 250mm, 5micron;

Instrument: Hitachi Primaide type high performance liquid chromatograph;

Parameters: detection wavelength 250nm, column temperature 25℃;

Mobile phase: acetonitrile, water;

Sample processing: each sample is dissolved in water;

Elution:

Time (minutes)	Acetonitrile (%)	water(%)
0	0	100
11	0	100
17	5	95
30	10	90
45	45	55
50	100	0

The result is shown in Figure 14. 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.

Example 9

1. Material

Commonly used 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.

The 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.

Preparation of 0.1mol/L 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.

0.5% Triton X-100 preparation: Triton X-100 stock solution 5ml, 1×PBS 995ml.

2. Test method

(1) Immunofluorescence

(a) Process the cell slide or frozen section according to the experimental requirements, wash with PBS, 5min×3 times.

(b) 0.5% Triton X-100 was permeated at room temperature for 15 minutes, washed with PBS, 5 minutes × 3 times.

(c) Goat serum was blocked at 37°C for 30 minutes.

(d) Discard the serum, dilute the primary antibody in an appropriate proportion, drop the covering tissue, and place it in a humidified chamber overnight at 4°C.

(e) Take out the wet box, reheat at 37°C for 30 minutes, wash the slides or tissue sections with PBS, 5 minutes × 3 times.

(f) Dilute the secondary antibody according to an appropriate ratio, add dropwise to the covering tissue, and incubate at 37°C for 30min to 60min.

(g) Wash the nucleus with PBS 3 times, 5 min each time, and stain the nucleus with DAPI for 10 min.

(h) Wash with PBS 3 times, 5 minutes each time, and then observe and analyze under a fluorescent microscope after mounting the anti-extractable mounting tablets.

(2) H&E dyeing

(a) Slices with a thickness of 4μm, fished at 42℃, copied over at 60℃, and stored at room temperature.

(b) Dewaxing paraffin sections to water: xylene 3 times, 20min each time; gradient alcohol (100%, 95%, 95%, 90%, 80%) hydration, respectively: 2min, 2min, 2min, 1min , 1min, wash with tap water for 5min.

(c) Wash with PBS 3 times, 5 min each time.

(d) Hematoxylin staining for 5 min.

(e) Rinse with tap water for 10 minutes.

(f) Double differentiation with 1% hydrochloric acid and alcohol, and rinse with tap water for 5 minutes.

(g) 1% ammonia water returns to blue for 2 minutes, and rinses with tap water for 5 minutes.

(h) Eosin staining for 1-5 min.

(i) Use 80%, 90%, 95%, 95%, and 100% alcohol for dehydration, respectively, for 1 min, 2 min, 2 min, 2 min, and 2 min.

(j) Xylene is transparent 3 times, 2 min each time.

(k) Seal the film with neutral gum and observe under the microscope.

(3) Masson three-color dyeing

(a) Dewax the paraffin sections to water.

(b) Chromizing treatment (potassium dichromate overnight treatment).

(c) Wash with tap water and distilled water in sequence.

(d) Stain the nucleus with Harris' hematoxylin staining solution or Weigert hematoxylin solution for 1-2 minutes.

(e) Wash thoroughly, if over-stained, it can be differentiated with hydrochloric acid and alcohol for 2-3s.

(f) The ammonia water returns to blue for 2 minutes.

(g) Use Masson Ponceau Acid Redness Solution for 5-10 minutes.

(h) 1% phosphomolybdic acid aqueous solution is differentiated for 3-5 min.

(i) Stain with 1% aniline blue or light green solution for 5 minutes.

(j) The 1% glacial acetic acid aqueous solution is differentiated for a few seconds.

(k) 95% alcohol, anhydrous alcohol, transparent xylene, and sealing with neutral gum.

Results: 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.

(4) Establishment of mouse myocardial infarction model

For 8 weeks, C57BL/6J male 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. Prepare the skin, open the chest between 3-4 intercostals, expose the heart, ligate the left anterior descending branch with 7-0 prolene suture, you will see that the apex of the heart turns white, suture the intercostal space, suture the skin, and disinfect. Turn off the anesthetic and continue to ventilate until the mouse wakes up.

(5) Establishment of mouse heart failure model

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.

(6) Take material, fix and slice

(a) 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.

(b) Frozen sections are used for immunofluorescence = paraffin sections are used for H&E and Masson trichrome staining.

(c) After the specimen is stained with Masson's three-color stain, the size of myocardial infarction is measured with Image J image analysis software. The calculation formula for the area of myocardial infarction is:

Take 5 sections of each specimen and calculate the average value.

3. Statistical analysis

All experimental results are expressed in Mean±SEM. Two-tailed tailed t test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups. P<0.05 is the standard of significant statistical difference. All experimental results are graphed and analyzed using GraphPad Prism 5 (Software, Inc.) and Image J software.

4. Experimental results

(I) 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). In the 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 (LVEF) is a key classic indicator of left ventricular function. 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. In Figure 19, the blue is the infarct fibrosis tissue, and 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.

Only promoting the regeneration of cells in the heart tissue cannot make the area of fibroblasts significantly smaller than the control group. Therefore, in addition to promoting the regeneration of cells in the heart tissue, 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.

(II) 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). In the 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 (LVEF) 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.

Example 10

An experimental large white pig was used to pass the percutaneous arterial catheter (PCI) to the anterior descending coronary artery of the heart for 50 minutes after the inflatable balloon blockage was removed, and a large white pig’s cardiac ischemia reperfusion model was constructed. The chicken EE (1ml/kg) obtained by the method is treated. Determine basic cardiac function before surgery. The results are shown in Figures 21 and 22.

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). Calculating the ΔEF and ΔFS with the difference from the preoperative basic value, it was found that 1 week after EE treatment, the EF and FS decreased significantly compared with the preoperative decrease. 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) .

After the large white pig ischemia-reperfusion (IR) model, the administration group was immediately treated with chicken EE, and the control group was given 5% glucose. Through observation and monitoring video during feeding, it was found that the large white pigs in the control group had less active time and looked tired. One week after the operation, statistics found that the daily activity time of large white pigs in the treatment group was significantly higher than that in the control group (Figure 22, D). Eight weeks after EE treatment, samples were taken and stained with triphenyltetrazolium chloride (TTC) after cardiac slice excision. It was found that the myocardium from the apex to the anterior wall of the control group became thinner, white after staining, and the left ventricle was slightly dilated; the treatment group There was a slight infarction from the apex of the heart to the anterior wall of the left ventricle, and the ventricular wall was not significantly thinned. At the same time, there was an increase in adipose tissue in the heart tissue of the control group (Figure 22, A). Statistics showed that the infarct appeared white area after TTC staining in the treatment group. Significantly lower than the control group (Figure 22, B).

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).

The above results indicate that 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. In addition, 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. Similarly, these results indicate that EE treatment also changed the ratio of macrophage subtypes in the inflammatory response, selectively activated and increased CCR ⁺ and CCR2 ⁺ CX3CR1 ⁺ macrophages, and inhibited the cytokine storm, thereby Suppresses fibrosis.

Example 11

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.

LPS (Lipopolysaccharide, a component of the outer wall of the cell wall of Gram-negative bacteria) can cause inflammation of lung epithelial cells. 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, treat the cells with 10μg/ml lipopolysaccharide LPS (MCE) for 24h, extract RNA for qPCR or digestion for flow analysis.

After LPS treatment for 24 hours, it was digested with 0.25% trypsin, and apoptosis was detected with AnnexinV-FITC-PI. The flow cytometry results are shown in Figure 23. EE reduced Calu-3 cell apoptosis caused by LPS.

The qPCR results are shown in Figure 24. The results showed that EE significantly reduced the expression of inflammatory factors caused by LPS, such as IL-1β, IL-6 and TNF-α, while EE increased the anti-inflammatory factors IFN-γ and IL-10.

The results proved that the embryonic stem cell extract growth factor group reduces the inflammation of lung epithelial cells caused by LPS.

Example 12

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 4μl/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-1β, IL-6, TNF-α 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-γ and IL-10 indicate that EE reduces the inflammation of lung epithelial cells caused by the COVID-19 protein pseudovirus.

Example 13

A mouse pneumonia model was constructed by inhaling LPS. For 8-10 weeks, 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 50μl, 200μg/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 μl of saline through the tail vein, and the treatment group was injected with 100 μl 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.

The results of pulmonary function measurement are shown in Figure 26. The results showed that EE significantly reduced the inspiratory resistance and expiratory resistance of mice with LPS pneumonia, and improved lung compliance.

The survival curve and weight change of the mice were counted, and the results are shown in Figure 27. The results showed that EE significantly improved the survival rate of mice with LPS pneumonia and recovered their body weight faster.

The results of HE staining suggest that EE reduces the pneumonia-induced exudate caused by LPS, especially on the third day when the inflammation is more serious. There were no obvious abnormalities in the organs outside the lungs, and there was no obvious difference between the two groups.

The results of HE staining are shown in Figures 28 and 29. The results showed that EE reduced the pneumonia-induced exudate caused by LPS, especially on the third day when the inflammation was more severe. There were no obvious abnormalities in the organs outside the lungs, and there was no obvious difference between the two groups.

Claims (10)

1. The use 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,

   Wherein, the amniotic fluid is derived from eggs with embryo age of 5-12 days, preferably eggs with embryo age of 6-11 days, more preferably eggs with embryo age of 7-9 days, more preferably eggs with embryo age of 7-8 days , Or from eggs of other avians other than chickens whose developmental period corresponds to the developmental period of the embryonic-age egg; or embryos from rodents with a gestational age of 8-14 days, or The embryos of non-human mammals other than rodents corresponding to the developmental period of rodents aged 8-14 days.
2. The application according to claim 1, wherein the causes of the cytokine storm or cytokine storm syndrome include infectious, acute injury, organ transplantation, rheumatic and neoplastic causes.
3. The application according to claim 1, wherein the cytokine storm or cytokine storm syndrome is caused by viral infection, preferably caused by influenza virus and/or coronavirus infection, or caused by cellular immunotherapy.
4. The application according to claim 3, wherein the coronavirus is COVID-19, SARS, MERS, H5N1 influenza virus or H7N9 influenza virus.
5. The application according to claim 1, wherein the cytokine storm or cytokine storm syndrome is a cytokine that occurs in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome Storm or cytokine storm syndrome.
6. The application according to any one of claims 1 to 5, wherein the active ingredients contained in the extract are not combined with the ion exchange column at pH 5.8-8.0, and the ingredients contained in the extract The molecular weight is in the range of 150-2000 Daltons.
7. The application according to claim 6, characterized in that the active ingredients contained in the extract do not bind to the ion exchange column at pH 7.0-8.0, and the molecular weight of the ingredients contained in the extract is 150-2000 Daltons. Scope.
8. The application according to claim 6, wherein 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 150-1200 Dalton range.
9. The application according to any one of claims 1 to 5, wherein 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 separated by reverse phase chromatography.
10. The application according to claim 1, wherein the amniotic fluid is from 6-11 days old eggs, 8-10 days old duck eggs or 8-14 days pregnant mice, and the cytokine storm or cell Factor storm syndrome is caused by COVID-19 infection.

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