Use of non-human mammalian amniotic fluid or extract thereof in treatment or prevention of heart failure

Abstract

The present invention relates to the use of non-human mammalian amniotic fluid or an extract thereof in the treatment or prevention of heart failure. Specifically, the present invention relates to the use of amniotic fluid and / or an extract thereof in the preparation of a drug for reducing or ameliorating atrial fibrillation, increasing six-minute walking distance, increasing upright sitting duration, prolonging sleep duration, ameliorating the edema of the whole body or lower limbs (especially the legs), ameliorating chest distress, increasing ejection fraction, reducing heart rate variability, reducing the N-terminal pro-brain natriuretic peptide, reducing the level of pro-inflammatory or cytotoxic factors, and / or improving the level of anti-inflammatory or cytoprotective factors; wherein the amniotic fluid is derived from chicken eggs at an embryonic age of 5-12 days, or from eggs of avian species other than chickens at a developmental stage corresponding to the developmental stage of the chicken eggs at the embryonic age; or from embryos of rodents at a gestational age of 8-14 days, or from embryos of non-human mammals other than rodents at a developmental stage corresponding to the developmental stage of the rodents at a gestational age of 8-14 days.

Description

Application of amniotic fluid or its extracts from non-human mammals in the treatment or prevention of heart failure Technical Field

[0001] This invention relates to the use of amniotic fluid from non-human mammals or its extracts in the treatment or prevention of heart failure. Background Technology

[0002] Heart failure patients are typically classified according to the severity of their condition, with the New York Heart Association (NYHA) functional classification system being the primary method currently used.

[0003] Grade I: Patients have heart disease, but their daily activity levels are not restricted, and general activities do not cause symptoms of heart failure such as fatigue or shortness of breath. Patients can perform daily household chores, take walks, climb stairs, etc., without experiencing significant fatigue or shortness of breath. These patients usually have relatively mild conditions, and their cardiac function is still adequately able to meet their body's daily needs.

[0004] Grade II: Mild limitation of physical activity, no symptoms at rest, but symptoms of heart failure such as fatigue and shortness of breath may occur with normal activity. Patients can perform mild physical activities, such as walking slowly for a distance or doing some simple housework, but will feel short of breath and fatigue when the activity level is slightly higher. Cardiac function is impaired to some extent, and appropriate care should be taken during daily activities to avoid overexertion.

[0005] Grade III: Physical activity is significantly limited; even activities below normal limits can cause symptoms of heart failure such as fatigue and shortness of breath. Patients can only perform very light activities, such as walking slowly indoors; even a few steps or slight activity will cause significant shortness of breath and palpitations. Cardiac function is severely impaired, significantly impacting the patient's daily life and requiring more rest and treatment.

[0006] Grade IV: Unable to engage in any physical activity; heart failure symptoms are present even at rest and worsen with activity. Patients may experience shortness of breath, weakness, and edema even at rest, and these symptoms will escalate rapidly with slight activity. The condition is very serious, requiring long-term bed rest and aggressive treatment and care. Summary of the Invention

[0007] The first invention provides the use of amniotic fluid and / or its extracts in the preparation of a medicament for treating heart failure, or in the preparation of a medicament for improving cardiac function; wherein the amniotic fluid is derived from an egg with an embryonic age of 5-12 days, preferably an egg with an embryonic age of 6-11 days, more preferably an egg with an embryonic age of 7-9 days, even more preferably an egg with an embryonic age of 7-8 days, or from an egg of a poultry other than a chicken whose developmental stage corresponds to the developmental stage of the egg with the embryonic age; or from an embryo of a rodent with a gestational age of 8-14 days, or from an embryo of a non-human mammal other than a rodent whose developmental stage corresponds to the developmental stage of a rodent with a gestational age of 8-14 days.

[0008] A second aspect of the present invention provides the use of amniotic fluid and / or its extracts in the preparation of one or more of the following medicaments: reducing atrial fibrillation in the recipient, increasing the recipient's six-minute walking distance, increasing the recipient's upright sitting time, prolonging the recipient's sleep time, improving general or lower limb edema in the recipient, and improving the recipient's chest tightness; wherein the amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, even more preferably eggs with an embryonic age of 7-8 days, or from eggs of poultry other than chickens whose developmental stage corresponds to the developmental stage of the eggs with the embryonic age; or from rodent embryos with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents with a gestational age of 8-14 days.

[0009] A third aspect of the present invention provides the use of amniotic fluid and / or its extracts in the preparation of one or more of the following formulations: (1) reducing heart rate variability in the recipient, (2) increasing ejection fraction in the recipient, (3) reducing N-terminal pro-brain natriuretic peptide in the recipient, (4) reducing the level of pro-inflammatory or cytotoxic factors in the recipient, and (5) increasing the level of anti-inflammatory or cytoprotective factors in the recipient; wherein the amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of poultry other than chickens whose developmental stage corresponds to the developmental stage of the eggs with the embryonic age; or from the embryos of rodents with a gestational age of 8-14 days, or from the embryos of other non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents with a gestational age of 8-14 days.

[0010] In one or more embodiments, the inflammatory or cytotoxic factor is selected from one or more of CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL17, MMP1, MMP2, MMP7, MMP9, MMP12, IL-1α, IL-1β, IL-6, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD31, CD62p, SYK, KMT5A, CNBP, IFN-γ, TNF-α, and VEGF-A.

[0011] In one or more embodiments, the amniotic fluid and its extracts reduce the levels of pro-inflammatory or cytotoxic factors via signaling pathways. In some embodiments, the signaling pathways include, but are not limited to, one or more of the following: chemokine family signaling pathways, matrix metalloproteinase family signaling pathways, interleukin family signaling pathways, CD antigen family signaling pathways, atherosclerosis-related signaling pathways, JAK / STAT signaling pathways, Th1 signaling pathways, and angiogenesis-related VEGF-A signaling pathways.

[0012] In one or more embodiments, the anti-inflammatory or cytoprotective factor is selected from one or more of IL-4, IL-10, iNOS, and NQO1.

[0013] In one or more embodiments, the amniotic fluid and its extracts enhance the levels of anti-inflammatory or cytoprotective factors in the recipient through signaling pathways. In some embodiments, the signaling pathways include, but are not limited to, cytokine-inducible signaling pathways and / or the antioxidant NQO1 signaling pathway.

[0014] A fourth aspect of the present invention provides the use of amniotic fluid and / or extracts thereof in the preparation of formulations for the treatment and / or prevention of diseases mediated by one or more of the following signaling pathways: (a) chemokine family signaling pathways, (b) matrix metalloproteinase family signaling pathways, (c) interleukin family signaling pathways, (d) CD antigen family signaling pathways, (e) atherosclerosis-associated signaling pathways, (f) JAK / STAT signaling pathways, (g) Th1 signaling pathways, (h) angiogenesis VEGF-A signaling pathways, (i) cytokine-inducible signaling pathways, and (j) antioxidant NQO1 signaling pathways.

[0015] In one or more embodiments, diseases mediated by chemokine family signaling pathways include, but are not limited to, diseases mediated by one or more chemokines of CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, and CCL17.

[0016] In one or more embodiments, diseases mediated by matrix metalloproteinase family signaling pathways include, but are not limited to, diseases mediated by one or more matrix metalloproteinases of MMP1, MMP2, MMP7, MMP9, and MMP12.

[0017] In one or more embodiments, diseases mediated by interleukin family signaling pathways include, but are not limited to, diseases mediated by one or more cytokine families such as the IL-1 family, IL-6 family, IL-10 family, IL-12 family, and IL-17 family.

[0018] In one or more embodiments, the CD antigen family includes, but is not limited to, CD31 and / or CD62p.

[0019] In one or more embodiments, diseases mediated by atherosclerosis-related signaling pathways include, but are not limited to, diseases mediated by the SYK signaling pathway and / or diseases mediated by the KMT5A signaling pathway.

[0020] In one or more embodiments, diseases mediated by the JAK / STAT signaling pathway include, but are not limited to, diseases mediated by IFN-γ cytokines.

[0021] In one or more embodiments, diseases mediated by the Th1 signaling pathway include, but are not limited to, diseases mediated by the TNF-α cytokine.

[0022] In one or more embodiments, diseases mediated by cytokine-inducible signaling pathways include, but are not limited to, diseases mediated by iNOS cytokines.

[0023] In one or more embodiments, the drug or formulation is administered to a mammal, particularly a human. In one or more embodiments, the subject suffers from heart failure, including but not limited to NYHA class I, II, III, or IV heart failure.

[0024] In one or more embodiments, the total atrial fibrillation time of the subject is higher than the control level, where the control level is the total atrial fibrillation time of a healthy control.

[0025] In one or more embodiments, the proportion of atrial fibrillation in the subjects is higher than the control level, where the control level is the proportion of atrial fibrillation in healthy controls.

[0026] In one or more embodiments, the subject has a higher atrial fibrillation frequency than the control level, where the control level is the atrial fibrillation frequency of a healthy control.

[0027] In one or more embodiments, the subject's six-minute walking distance is lower than the control level, which is the six-minute walking distance of a healthy control.

[0028] In one or more embodiments, the degree of edema in the whole body or lower limbs of the subject is higher than the control level, where the control level is the whole body or lower limbs of a healthy control.

[0029] In one or more embodiments, the ejection fraction of the subject is lower than the control level, which is the ejection fraction of a healthy control.

[0030] In one or more embodiments, the reduction of heart rate variability includes increasing the standard deviation normal-to-normal interval (SDNN), increasing the standard deviation mean normal-to-normal interval (SDANN), increasing the standard deviation normal-to-normal interval of the square root mean (rMSDD), and / or increasing the percentage of normal-to-normal interval differences greater than 50 milliseconds (pNN50). In one or more embodiments, the subject's SDNN is lower than the control level, which is the SDNN of a healthy control. In one or more embodiments, the subject's SDANN is lower than the control level, which is the SDANN of a healthy control. In one or more embodiments, the subject's rMSDD is lower than the control level, which is the rMSDD of a healthy control. In one or more embodiments, the subject's pNN50 is lower than the control level, which is the pNN50 of a healthy control.

[0031] In one or more embodiments, the N-terminal pro-brain natriuretic peptide (NTNP) level of the subject is higher than the control level, which is the N-terminal NTNP level of a healthy control.

[0032] In one or more embodiments, the object has a higher level of inflammatory or cytotoxic factors than a control level, wherein the control level is the level of inflammatory or cytotoxic factors in a healthy control.

[0033] In one or more embodiments, the object has a lower level of anti-inflammatory or cytoprotective factors than a control level, wherein the control level is the level of anti-inflammatory or cytoprotective factors in a healthy control.

[0034] In one or more embodiments, the dosage of the drug or preparation is 0.5 to 2.5 ml / kg / day, for example 0.5 to 1.5 ml / kg / day or 0.8 to 1.2 ml / kg / day.

[0035] In one or more embodiments, the drug or formulation is administered once every half day, daily, every two days, every three days, every four days, every five days, every six days, or every seven days, or once every half month, once a month, once every three months, once every six months, or once a year.

[0036] In one or more embodiments, the drug or preparation is administered twice daily, for a course of treatment every 7 days, or once every seven days.

[0037] In one or more embodiments, the amniotic fluid is derived from chicken eggs, duck eggs, goose eggs, or combinations thereof.

[0038] In one or more embodiments, the amniotic fluid is derived from a rodent that is 10-14 days pregnant.

[0039] In one or more embodiments, the drug or preparation contains cryopreserved amniotic fluid and / or its extracts, or is a lyophilized reagent of the amniotic fluid and / or its extracts.

[0040] In one or more embodiments, the drug or preparation is an infusion solution. In one or more embodiments, the drug or preparation may contain saline for injection, water for injection, or glucose injection. Attached Figure Description

[0041] Figure 1 shows the results of patient M002 before and after medication. The first row from top to bottom shows the results before medication, and the second row shows the results after the third day of medication.

[0042] Figure 2 shows the CT curves of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients M002–M004. Day 30 data for patient M002 were obtained from a laboratory report submitted on September 20, 2024, at Huashan Hospital (Hongqiao Campus), affiliated with Fudan University, after patient discharge. The pro-BNP level in M002 on Day 30 was 66.18 pg / mL (reference range <300 pg / mL), and NT-proBNP was not measured. Patients S001–S002 and M001 were all healthy subjects with N-terminal pro-brain natriuretic peptide (NT-proBNP) levels within the normal range; therefore, S001–S002 and M001 are not included in the figure.

[0043] Figure 3 shows the ejection fraction (LVEF) percentage (%)-T curve for patients M002-M004. S001-S002 and M001 were all healthy subjects with ejection fraction (LVEF) percentages (%) within the normal range, therefore S001-S002 and M001 are not included in the figure.

[0044] Figure 4 shows the flow cytometry results of subject S001 (Day 0, Day 1).

[0045] Figure 5 shows the flow cytometry results of subject S001 (Day 3, Day 7).

[0046] Figure 6 shows the flow cytometry results of subject S002 (Day 0, Day 1).

[0047] Figure 7 shows the flow cytometry results of subject S002 (Day 3, Day 7).

[0048] Figure 8 shows the flow cytometry results of the M001 subjects (Day 0, Day 1, Day 3).

[0049] Figure 9 shows the flow cytometry results of the M001 subjects (Day 7, Day 10, Day 14).

[0050] Figure 10 shows the flow cytometry results of subjects M002 (Day 0, Day 1, Day 3).

[0051] Figure 11 shows the flow cytometry results of subjects M002 (Day 7, Day 10, Day 14).

[0052] Figure 12 shows the flow cytometry results of subjects M003 (Day 0, Day 1, Day 3).

[0053] Figure 13 shows the flow cytometry results of subjects M003 (Day 7, Day 10, Day 14).

[0054] Figure 14 shows the flow cytometry results of subjects M004 (Day 0, Day 1, Day 3).

[0055] Figure 15 shows the flow cytometry results of subjects M004 (Day 7, Day 10, Day 14).

[0056] Figure 16 shows the CT curves of cytokines (IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-11) for six subjects, S001-S002 and M001-M004.

[0057] Figure 17 shows the CT curves of cytokines (IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27) for six subjects, S001-S002 and M001-M004.

[0058] Figure 18 shows the CT curves of cytokines (CD31, CD61, CD62p, CD68, CD206, CCL2, CCL5, CCL17) for six subjects, S001-S002 and M001-M004.

[0059] Figure 19 shows the CT curves of cytokines (CXCL4, CXCL9, CXCL10, and CXCL11) for six subjects, S001-S002 and M001-M004.

[0060] Figure 20 shows the CT curves of cytokines (MMP-1, MMP-2, MMP-7, MMP-9, MMP-12) for six subjects, S001-S002 and M001-M004.

[0061] Figure 21 shows the CT curves of cytokines (LPS, TGF-β1, TNF-α, IFN-γ) in six subjects, S001-S002 and M001-M004.

[0062] Figure 22 shows the CT curves of cytokines (Arg-1, iNOS, VEGF-A, NF-κB p65) for six subjects, S001-S002 and M001-M004.

[0063] Figure 23 shows the CT curves of cytokines (TLR4, HO1, NQO1, Nrf2) for six subjects, S001-S002 and M001-M004.

[0064] Figure 24 shows the CT curves of cytokines (Syk, KMT5A, CNBP) for six subjects, S001-S002 and M001-M004. Detailed Implementation

[0065] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0066] Amniotic fluid and / or its extracts

[0067] In this article, amniotic fluid may be derived from poultry eggs or non-human mammals. Poultry eggs refer to the eggs of poultry. Preferred poultry are domestic fowl, such as chickens, ducks, and geese. Preferably, the present invention uses poultry eggs with an embryonic age of 5-20 days, more preferably 6-15 days. It should be understood that the suitable embryonic age may differ for different poultry eggs. For example, when using chicken eggs, it is preferred to use eggs with an embryonic age of 5-12 days, more preferably eggs with an embryonic age of 6-11 days, even more preferably eggs with an embryonic age of 7-9 days, and even more preferably eggs with an embryonic age of 7-8 days. When using eggs from other poultry species, eggs whose developmental stage corresponds to the developmental stage of the aforementioned embryonic ages can be used. For example, when using duck eggs, duck eggs with an embryonic age of 8-10 days, especially 8-9 days, may be best.

[0068] In some embodiments, the eggs are eggs of poultry, especially eggs with an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, and more preferably 7-8 days, particularly chicken eggs.

[0069] Amniotic fluid from poultry eggs can be obtained using conventional methods. For example, the blunt end of an egg of the appropriate embryonic age can be tapped to crack the shell, creating an opening approximately 2 cm in diameter. Then, the shell membrane and yolk membrane are carefully peeled away with tweezers, taking care not to damage the amnion. The amnion and associated tissues surrounding the embryo are poured from the shell into a petri dish. Amniotic fluid is then extracted by inserting a syringe into the amnion until it adheres tightly to the embryo, thus obtaining the amniotic fluid used in this invention.

[0070] In this invention, amniotic fluid may also be derived from non-human mammals, especially rodents, such as mice. Other non-human mammals may be common livestock, such as cattle, sheep, dogs, cats, and pigs. In some embodiments, the amniotic fluid is derived from rodent embryos with a gestational age of 8-14 days, or from the embryos of non-human mammals whose developmental stage corresponds to that of rodents with a gestational age of 8-14 days. Amniotic fluid can be obtained using conventional methods. For example, the abdominal cavity of a mouse gestating at 8-14 days is cut open with surgical scissors, the uterus is carefully removed and cut open, and amniotic fluid is extracted by inserting a syringe into the amnion until the amnion is tightly attached to the embryo, thereby obtaining the amniotic fluid used in this invention.

[0071] It should be understood that, if necessary, the amniotic fluid may be centrifuged to separate any impurities that may be present, such as egg yolk, to obtain the purest amniotic fluid possible. The supernatant obtained after centrifugation is the amniotic fluid used in this invention. It should be understood that all steps in obtaining the amniotic fluid must be performed under sterile conditions; furthermore, "amniotic fluid" as used herein refers to "pure" amniotic fluid, i.e., amniotic fluid isolated from avian eggs or non-human mammalian embryos that does not contain other components found in avian eggs or non-human mammalian embryos and is not contaminated by exogenous substances. Pure amniotic fluid can be stored in a freezer at -60°C or below and thawed before use.

[0072] This document provides a pharmaceutical composition containing amniotic fluid and / or its extracts as described herein, particularly amniotic fluid and / or its extracts from poultry eggs, more preferably amniotic fluid and / or its extracts from eggs with an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, and more preferably 7-8 days. The pharmaceutical composition may be amniotic fluid and / or its extracts cryopreserved at -60°C or below, or a lyophilized reagent thereof, such as lyophilized amniotic fluid and / or its extracts. The pharmaceutical composition may also contain other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection, or glucose injection.

[0073] This study found that amniotic fluid and its extracts can treat heart failure or improve cardiac function. Specifically, they reduced atrial fibrillation, increased six-minute walking distance, increased upright sitting time, prolonged sleep time, improved generalized or lower limb (especially leg) edema, reduced chest tightness, decreased heart rate variability, increased ejection fraction, decreased N-terminal pro-brain natriuretic peptide levels, decreased levels of pro-inflammatory or cytotoxic factors, and increased levels of anti-inflammatory or cytoprotective factors.

[0074] Pharmaceutical Uses

[0075] Therefore, this invention provides the use of amniotic fluid and / or its extracts in the preparation of one or more of the following pharmaceutical preparations: reducing or improving atrial fibrillation in the recipient, increasing the recipient's six-minute walking distance, increasing the recipient's upright sitting time, prolonging the recipient's sleep time, improving generalized or lower limb (especially leg) edema in the recipient, and improving the recipient's chest tightness. In some embodiments, one or more of the above applications are achieved by reducing the recipient's heart rate variability, increasing the recipient's ejection fraction, reducing the recipient's N-terminal pro-brain natriuretic peptide, reducing the recipient's levels of pro-inflammatory or cytotoxic factors, and / or increasing the recipient's levels of anti-inflammatory or cytoprotective factors.

[0076] In some embodiments, the present invention also provides the use of amniotic fluid and / or its extracts in the preparation of one or more formulations that: reduce heart rate variability in the recipient, increase ejection fraction in the recipient, reduce N-terminal pro-brain natriuretic peptide in the recipient, reduce levels of pro-inflammatory or cytotoxic factors in the recipient, and increase levels of anti-inflammatory or cytoprotective factors in the recipient.

[0077] In this article, pro-inflammatory or cytotoxic factors include, but are not limited to, one or more of CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL17, MMP1, MMP2, MMP7, MMP9, MMP12, IL-1α, IL-1β, IL-6, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD31, CD62p, SYK, KMT5A, CNBP, IFN-γ, TNF-α, and VEGF-A. In some embodiments, the amniotic fluid and its extracts reduce the levels of pro-inflammatory or cytotoxic factors through different signaling pathways. In some embodiments, the signaling pathways include, but are not limited to, one or more of the following: chemokine family signaling pathways, matrix metalloproteinase family signaling pathways, interleukin family signaling pathways, CD antigen family signaling pathways, atherosclerosis-related signaling pathways, JAK / STAT signaling pathways, Th1 signaling pathways, and angiogenesis VEGF-A signaling pathways.

[0078] In this document, anti-inflammatory or cytoprotective factors include, but are not limited to, one or more of IL-4, IL-10, iNOS, and NQO1. In some embodiments, the amniotic fluid and its extracts enhance the levels of anti-inflammatory or cytoprotective factors through different signaling pathways. In some embodiments, the signaling pathways include, but are not limited to, cytokine-inducible signaling pathways and / or the antioxidant NQO1 signaling pathway.

[0079] The amniotic fluid and / or its extracts described herein can be used directly for the purposes described herein, administered to the desired subjects. Administration may be parenteral, such as intravenous or intracardiac injection. In some embodiments, a therapeutically effective amount of amniotic fluid and / or its extracts may be mixed with an appropriate amount of saline for injection, water for injection, or glucose injection, and then administered, for example, by intravenous infusion or intracardiac injection.

[0080] The dosage and frequency of administration are determined by healthcare professionals based on the specific condition, the patient's age, and gender. Generally, for the treatment of a specific disease, the therapeutically effective dose refers to a dosage sufficient to improve or alleviate disease-related symptoms in some way. Such a dose may be administered as a single dose or as part of an effective treatment regimen. The dosage may cure the disease, but administration is usually intended to improve its symptoms. Repeated administration is generally required to achieve the desired symptom improvement. For example, the typical dosage for humans is 0.5–2.5 ml / kg / day, such as 0.5–1.5 ml / kg / day or 0.8–1.2 ml / kg / day, which can be divided into two doses per day, with an interval of approximately 4 hours between doses. The dosage for an adult can be 1–80 ml / dose, such as 1–35 ml / dose or 1–70 ml / dose, administered daily or weekly by injection at a rate of approximately 35 ml / h, such as 10–20 ml / h or 10–70 ml / h, with each administration lasting longer than 1 hour. In some implementations, the dosing frequency may be once daily, once every two days, once every three days, once every four days, once every five days, or once every six days, or once every two weeks, monthly, once every three months, once every four months, once every five months, once every six months, or annually. In an exemplary implementation, the dosing frequency is twice daily, or once weekly, with each week constituting one course of treatment.

[0081] Disease treatment methods

[0082] This document also provides a method for treating heart failure, and a method for improving heart failure in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the amniotic fluid and / or its extract, or a pharmaceutical composition containing said amniotic fluid and / or its extract, as described herein. In particular, the step of using amniotic fluid from poultry eggs and / or its extract, or a pharmaceutical composition or preparation containing said amniotic fluid from poultry eggs and / or its extract. Preferably, the poultry eggs are as defined herein or as described in any embodiment herein.

[0083] This document also provides a method for reducing or improving atrial fibrillation, increasing six-minute walking distance, increasing upright sitting time, prolonging sleep time, improving generalized or lower limb (especially leg) edema, improving chest tightness, increasing ejection fraction, reducing heart rate variability, decreasing N-terminal pro-brain natriuretic peptide, decreasing levels of pro-inflammatory or cytotoxic factors, and / or increasing levels of anti-inflammatory or cytoprotective factors, the method comprising the step of administering a therapeutically effective amount of the amniotic fluid and / or its extracts, or a pharmaceutical composition containing said amniotic fluid and / or its extracts, as described herein, to the desired subject. Particularly, the step of administering amniotic fluid from poultry eggs and / or its extracts, or a pharmaceutical composition or preparation containing said amniotic fluid from poultry eggs and / or its extracts. Preferably, the poultry eggs are as defined herein or as described in any embodiment herein.

[0084] Subject / Patient

[0085] In this document, "subject," "patient," or "application subject" refers to the intended recipient of the amniotic fluid and / or its extracts, drugs, formulations, or pharmaceutical compositions described herein, and generally refers to an animal, such as a mammal, particularly a human. In a preferred embodiment, the subject suffers from heart failure, including but not limited to NYHA class I, II, III, or IV heart failure. In an exemplary embodiment, the subject suffers from class II, III, or IV heart failure.

[0086] In this document, reducing or improving atrial fibrillation (in the subjects) includes reducing the total duration of atrial fibrillation, reducing the proportion of atrial fibrillation, and / or reducing the number of atrial fibrillation episodes. In some embodiments, the total duration of atrial fibrillation in the subjects is higher than the control level, for example, 1000–7000 minutes, 1200–1500 minutes, or 6000–7000 minutes, where the control level is the total duration of atrial fibrillation in healthy controls (typically 0 minutes). In some embodiments, the proportion of atrial fibrillation in the subjects is higher than the control level, for example, 10–100%, 55–60%, 60–75%, 65–75%, 10.0–15.0%, or 10.5–11.0%, where the control level is the proportion of atrial fibrillation in healthy controls (typically 0%). In some embodiments, the number of atrial fibrillation episodes in the subjects is higher than the control level, for example, 1–5 times, 3–5 times, or 4–6 times, where the control level is the number of atrial fibrillation episodes in healthy controls (typically 0 times).

[0087] In some implementations, the subject's six-minute walking distance is lower than the control level, for example, 200–410 meters, 240–300 meters, 250–350 meters, or 300–380 meters, where the control level is the six-minute walking distance of a healthy control (typically more than 500 meters).

[0088] In some implementations, the subject's upright sitting time is lower than the control level, which is the upright sitting time of a healthy control.

[0089] In some implementations, the subjects' sleep duration is lower than the control level, which is the sleep duration of healthy controls (typically around 8 hours).

[0090] In some embodiments, the degree of edema in the whole body or lower limbs (especially the legs) of the subject is higher than the control level, which is the whole body or lower limbs (especially the legs) of a healthy control.

[0091] In some embodiments, the ejection fraction of the subject is lower than the control level, for example, 20-60% or 30-40%, where the control level is the ejection fraction of a healthy control (typically higher than 60%).

[0092] In this paper, reducing heart rate variability includes increasing the standard deviation normal-to-normal interval (SDNN), increasing the standard deviation mean normal-to-normal interval (SDANN), increasing the square root mean standard deviation normal-to-normal interval (rMSDD), and / or increasing the percentage of normal-to-normal intervals differing by more than 50 milliseconds (pNN50). In some embodiments, the subject's SDNN is lower than the control level, for example, 70–90 ms or 75–80 ms, where the control level is the SDNN of healthy controls (typically 100–150 ms). In some embodiments, the subject's SDANN is lower than the control level, for example, 70–79 ms or 70–75 ms, where the control level is the SDANN of healthy controls (typically 80–140 ms). In some embodiments, the subject's rMSDD is lower than the control level, for example, 10–25 ms or 10–26 ms, where the control level is the rMSDD of healthy controls (typically 15–45 ms). In some embodiments, the pNN50 of the object is lower than the control level, for example, 3-10% or 3-5%, where the control level is the pNN50 of a healthy control (typically 1-12%).

[0093] In some embodiments, the N-terminal pro-brain natriuretic peptide (PTP) level of the subject is higher than the control level, for example, 300–6000 pg / mL or 1000–5000 pg / mL, where the control level is the N-terminal PTP level of a healthy control (typically less than 300 mg / mL).

[0094] In some embodiments, the levels of inflammatory or cytotoxic factors in the subject are higher than those in a control, where the control level is the level of inflammatory or cytotoxic factors in a healthy control.

[0095] In some embodiments, the CXCL4 level of the subjects is 480–620 ng / mL, 500–610 ng / mL, or 530–580 ng / mL. In some embodiments, the CXCL4 level of the healthy controls is 300–550 ng / mL, 320–450 ng / mL, or 400–480 ng / mL.

[0096] In some embodiments, the CXCL9 level of the subjects is 2500–3000 pg / mL or 2700–3000 pg / mL. In some embodiments, the CXCL9 level of the healthy controls is 2000–3200 pg / mL, 1500–2500 pg / mL, or 2900–3000 pg / mL.

[0097] In some embodiments, the CXCL10 level of the subjects is 200–250 pg / mL or 210–240 pg / mL. In some embodiments, the CXCL10 level of the healthy controls is 200–220 pg / mL or 150–220 pg / mL.

[0098] In some embodiments, the CXCL11 level of the subjects is 3500–4300 pg / mL, 3700–400 pg / mL, or 3700–3800 pg / mL. In some embodiments, the CXCL11 level of the healthy controls is 2500–3500 pg / mL, 3700–4200 pg / mL, or 3900–4100 pg / mL.

[0099] In some embodiments, the CCL2 level of the subjects is 90–120 ng / mL, 100–110 ng / mL, or 100–120 ng / mL. In some embodiments, the CCL2 level of the healthy controls is 90–115 ng / mL or 100–110 ng / mL.

[0100] In some embodiments, the CCL5 level of the subjects is 75–95 ng / mL, 80–90 ng / mL, or 88–92 ng / mL. In some embodiments, the CCL5 level of the healthy controls is 85–95 ng / mL, 90–96 ng / mL, or 89–96 ng / mL.

[0101] In some embodiments, the CCL17 level of the subjects is 2400–3200 pg / mL, 2700–3000 pg / mL, or 2900–3000 pg / mL. In some embodiments, the CCL17 level of the healthy controls is 2500–3500 pg / mL, 2800–3400 pg / mL, or 2700–3000 pg / mL.

[0102] In some embodiments, the MMP1 level of the subjects is 7.5–12.0 ng / mL, 10.0–11.0 ng / mL, or 9.0–10.5 ng / mL. In some embodiments, the MMP1 level of the healthy controls is 9–12 ng / mL or 10–11 ng / mL.

[0103] In some embodiments, the MMP2 level of the subjects is 430–520 ng / mL, 490–510 ng / mL, or 430–500 ng / mL. In some embodiments, the MMP2 level of the healthy controls is 350–480 ng / mL, 400–420 ng / mL, or 360–400 ng / mL.

[0104] In some embodiments, the MMP7 level of the subjects is 25–39 ng / mL, 32–36 ng / mL, or 29–34 ng / mL. In some embodiments, the MMP7 level of the healthy controls is 25–45 ng / mL, 35–38 ng / mL, or 35–40 ng / mL.

[0105] In some embodiments, the MMP9 level of the subjects is 35–55 ng / mL, 40–45 ng / mL, or 45–55 ng / mL. In some embodiments, the MMP9 level of the healthy controls is 34–55 ng / mL, 45–50 ng / mL, or 50–55 ng / mL.

[0106] In some embodiments, the MMP12 level of the subjects is 8.5–13.5 ng / mL, 9.5–11.9 ng / mL, or 13.0–13.5 ng / mL. In some embodiments, the MMP12 level of the healthy controls is 8–14 ng / mL, 10–13 ng / mL, or 9–11 ng / mL.

[0107] In some embodiments, the IL-1α level of the subjects is 210–290 pg / mL, 200–290 pg / mL, or 170–300 pg / mL. In some embodiments, the IL-1α level of the healthy controls is 190–230 pg / mL or 200–220 pg / mL.

[0108] In some embodiments, the IL-1β level of the subjects is 100–120 pg / mL, 110–120 pg / mL, or 90–100 pg / mL. In some embodiments, the IL-1β level of the healthy controls is 115–125 pg / mL or 115–130 pg / mL.

[0109] In some embodiments, the IL-6 level of the subjects is 50–65 pg / mL, 52–60 pg / mL, or 62–63 pg / mL. In some embodiments, the IL-6 level of the healthy controls is 45–55 pg / mL or 49–50 pg / mL.

[0110] In some embodiments, the L-12 level of the subjects is 40–60 pg / mL, 50–60 pg / mL, or 40–50 pg / mL. In some embodiments, the L-12 level of the healthy controls is 40–55 pg / mL or 40–50 pg / mL.

[0111] In some embodiments, the IL-13 level of the subjects is 55–68 pg / mL, 50–60 pg / mL, or 65–68 pg / mL. In some embodiments, the IL-13 level of the healthy controls is 50–60 pg / mL or 53–58 pg / mL.

[0112] In some embodiments, the IL-17α level of the subjects is 40–50 pg / mL, 43–49 pg / mL, or 48–49 pg / mL. In some embodiments, the IL-17α level of the healthy controls is 40–48 pg / mL or 42–46 pg / mL.

[0113] In some embodiments, the IL-18 level of the subjects is 350–380 pg / mL, 360–380 pg / mL, or 350–370 pg / mL. In some embodiments, the IL-18 level of the healthy controls is 380–430 pg / mL, 380–420 pg / mL, or 420–430 pg / mL.

[0114] In some embodiments, the IL-23 level of the subjects is 440–600 pg / mL, 500–580 pg / mL, or 440–500 pg / mL. In some embodiments, the IL-23 level of the healthy controls is 440–555 pg / mL or 550–580 pg / mL.

[0115] In some embodiments, the IL-27 level of the subjects is 100–120 pg / mL or 105–110 pg / mL. In some embodiments, the IL-27 level of the healthy controls is 95–130 pg / mL or 120–130 pg / mL.

[0116] In some embodiments, the CD31 level of the subjects is 120–165 ng / mL, 120–161 ng / mL, or 140–145 ng / mL. In some embodiments, the CD31 level of the healthy controls is 140–165 ng / mL, 150–150 ng / mL, or 145–150 ng / mL.

[0117] In some embodiments, the CD62p level of the subjects is 30–45 ng / mL or 35–41 ng / mL. In some embodiments, the CD62p level of the healthy controls is 40–43 ng / mL or 40–45 ng / mL.

[0118] In some embodiments, the SYK level of the subjects is 900–1090 pg / mL, 900–1000 pg / mL, or 1005–1086 pg / mL. In some embodiments, the SYK level of the healthy controls is 1000–1100 pg / mL or 1030–1075 pg / mL.

[0119] In some embodiments, the KMT5A level of the subjects is 17–20 ng / mL or 18–20 ng / mL. In some embodiments, the KMT5A level of the healthy controls is 15–25 ng / mL, 18–25 ng / mL, or 15–20 ng / mL.

[0120] In some embodiments, the CNBP level of the subjects is 19–25 ng / mL or 20–22 ng / mL. In some embodiments, the CNBP level of the healthy controls is 15–21 ng / mL, 18–21 ng / mL, or 15–18 ng / mL.

[0121] In some embodiments, the IFN-γ level of the subjects is 800–1070 pg / mL or 810–950 pg / mL. In some embodiments, the IFN-γ level of the healthy controls is 750–1100 pg / mL or 790–920 pg / mL.

[0122] In some embodiments, the TNF-α level of the subjects is 100–125 pg / mL, 105–110 pg / mL, or 102–122 pg / mL. In some embodiments, the TNF-α level of the healthy controls is 95–105 pg / mL or 98–100 pg / mL.

[0123] In some embodiments, the VEGF-A level of the subjects is 650–730 pg / mL, 670–700 pg / mL, or 700–725 pg / mL. In some embodiments, the VEGF-A level of the healthy controls is 640–750 pg / mL or 700–730 pg / mL.

[0124] In some embodiments, the level of anti-inflammatory or cytoprotective factors in the subject is lower than the control level, which is the level of anti-inflammatory or cytoprotective factors in healthy controls.

[0125] In some embodiments, the IL-4 level of the subjects is 16–30 pg / mL, 15–50 pg / mL, or 16–23 pg / mL. In some embodiments, the IL-4 level of the healthy controls is 15–25 pg / mL or 25–22 pg / mL.

[0126] In some embodiments, the IL-10 level of the subjects is 450–720 pg / mL, 650–720 pg / mL, or 450–665 pg / mL. In some embodiments, the IL-10 level of the healthy controls is 450–730 pg / mL, 600–730 pg / mL, or 450–600 pg / mL.

[0127] In some embodiments, the iNOS level of the subjects is 0.70–2.10 ng / mL, 0.70–1.60 ng / mL, or 1.60–2.10 ng / mL. In some embodiments, the iNOS level of the healthy controls is 0.40–0.50 ng / mL or 0.46–0.47 ng / mL.

[0128] In some embodiments, the NQO1 level of the subject is 6.0–7.0 ng / mL, 6.0–9.0 ng / mL, or 6.5–7.0 ng / mL. In some embodiments, the NQO1 level of the healthy control is 3.5–6.0 ng / mL or 5.5–6.0 ng / mL.

[0129] The present invention has the following beneficial effects:

[0130] The pharmaceutical compositions of the present invention can treat heart failure or improve cardiac function, such as reducing atrial fibrillation, increasing six-minute walking distance, increasing upright sitting time, prolonging sleep time, improving leg edema, improving chest tightness, increasing ejection fraction, reducing heart rate variability, reducing N-terminal pro-brain natriuretic peptide, reducing the level of pro-inflammatory or cytotoxic factors, and increasing the level of anti-inflammatory or cytoprotective factors.

[0131] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0132] Experimental materials

[0133] a) Instruments and tools

[0134] Microcomputer-controlled fully automatic incubator (Zhengda) TM ZF880), clean petri dishes, 1.0ml syringe (Jiangxi Hongda) TM ), tweezers sterilized with 70% alcohol, stainless steel sieve, sterile centrifuge tubes ( #SCT-50ML-25-S) and low-speed refrigerated centrifuge (Zhongjia KDC-2046).

[0135] b) Reagents and biological materials

[0136] Eggs with an embryo age of 7 days.

[0137] Extraction Experiment Procedure

[0138] Take an egg and tap the flat, blunt end facing upwards to crack the shell. Peel the shell open to create a slit about 2 cm in diameter, ensuring the edges are as smooth as possible. Carefully tear apart the shell membrane and yolk membrane with tweezers, being careful not to damage the amnion. Observe the embryonic development; only embryos that are well-developed and meet the standards for the corresponding stage can be used for amniocentesis.

[0139] Pour the amnion and attached tissues surrounding the embryo from the shell into a culture dish. Use a syringe to puncture the amnion and extract amniotic fluid, with the bevel of the syringe facing away from the embryo, until the amnion is in close contact with the embryo. Then, inject the clear, colorless, and foreign matter-free amniotic fluid into a centrifuge tube in an ice box.

[0140] The embryos are removed from the amnion using tweezers and collected in a stainless steel sieve placed on ice. Every hour, the collected embryos are homogenized using a blender, sealed in sterile plastic storage containers, and placed at an angle in a -80°C freezer. After freezing, they can be stored upright.

[0141] Through Meptop TMAmniotic fluid extracts collected by the 1800 UV spectrophotometer are tested. For the standard operating procedure of the spectrophotometer, please refer to the user manual. Qualified extracts can be used for mixing and balancing.

[0142] After balancing the centrifuge tubes used to collect amniotic fluid extract, use Zhongjia... TM Centrifuge the KDC-2046 low-speed refrigerated centrifuge at 5°C and 3500 rpm for 21 minutes (see the user manual for standard centrifuge operating procedures). Decant the supernatant into a clean plastic storage container and store it at -80°C. Reserve 5 ml of each batch for subsequent testing.

[0143] All steps are performed under sterile conditions.

[0144] Experimental Design

[0145] Test drug name (code): EE001; Specification: 35ml / bag; Batch number: MP231201; The infusion pump was set to infusion rate of 35ml / h, infusion time of 40min, and infusion interval of ≥4 hours.

[0146] Treatment duration: Single-dose treatment lasts for 1 day, administered once daily for 7 days, followed by observation. Multiple-dose treatment lasts for 1 week, administered once daily for 7 consecutive days, followed by observation for 7 days. Safety, tolerability, and preliminary efficacy were evaluated at 1, 3, and 7 days after administration. This drug is the amniotic fluid extract described in this article, i.e., amniotic fluid aspiration fluid.

[0147] Efficacy evaluation criteria:

[0148] (1) Efficacy evaluation indicators (mainly): changes in cardiac ejection fraction (color echocardiography), and the influence of exercise;

[0149] (2) Efficacy evaluation indicators (minor): BNP level, changes in myocardial enzyme profile; changes in serum cytokine levels; changes in serum regulatory T cells (CD4+). + CD8 + Subgroup and Treg (e.g.) detection;

[0150] (3) Efficacy assessment intervals: at baseline (D-7 to D-1), on day 1, day 3, and day 7 after a single dose; and on day 1, day 3, day 7, day 10, and day 14 after multiple doses.

[0151] Safety indicators:

[0152] Throughout the trial, safety was evaluated by monitoring and observing vital signs, physical examinations, adverse events, and laboratory test results. The severity of adverse events was primarily determined according to the CTCAE v5.0 standard.

[0153] The inspection items include:

[0154] 1) Hematological examinations: complete blood count, coagulation function, glycated hemoglobin, etc.;

[0155] 2) Liver and kidney function related tests: blood biochemistry, urinalysis;

[0156] 3) Cardiac-related examinations: electrocardiogram (and QTcF calculation), color Doppler echocardiography;

[0157] 4) Cytokine levels; Cytokines include CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL217, MMP1, MMP2, MMP7, MMP9, MMP12, CNBP, IL-1α, IL-1β, IL-4, IL-10, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD61, CD31, CD62p, SYK, KMT5A, TNF-α, VEGF-A, Nrf2, iNOS, NQO1, CNBP, IFN-γ, TGF-β, TLR4, HO1, NF-κB, CD206, CD68, IL-6, IL-11, LPS, and Arg-1.

[0158] 5) Other examinations: Pregnancy check for women of childbearing age, HBV, HCV, HIV tests, etc.

[0159] Single-dose administration: Two healthy subjects, male or female, were enrolled. All healthy subjects were in normal condition and received the drug twice daily via intravenous infusion (approximately 1 ml / kg / day, based on the actual dosage in Table 3). An intravenous infusion pump was used with an infusion rate set at 35 ml / h per bag, and the infusion time was 40 minutes. After each infusion, the second bag was administered at an interval of ≥4 hours. Safety, tolerability, and preliminary efficacy were evaluated at 1, 3, and 7 days post-administration. The single-dose administration procedure and assessment indicators are shown in Table 1 below.

[0160] Table 1

[0161] After a single dose, a multiple-dose safety observation trial was conducted: The dose was 1 ml / kg / day (administered twice intravenously, once in the morning and once in the afternoon, for a total dose of 1 ml / kg / day, using an intravenous infusion pump set to an infusion rate of 35 ml / h per bag, with an infusion time of 40 minutes; after each infusion, the second bag was administered at an interval of ≥4 hours), for 7 consecutive days. Safety, tolerability, and preliminary efficacy were evaluated at 1, 3, 7, 10, and 14 days after the first dose. Three experimental groups were established: Group A (1 healthy subject); Group B (1 subject with mild (Class I) heart failure); and Group C (2 subjects with moderate or severe (Class II-IV) heart failure). Subjects in each group could be male or female. Groups B and C were only conducted after safety was assessed in Group A. The procedure and monitoring indicators for multiple dosing are shown in Table 2 below.

[0162] Table 2

[0163] Inclusion criteria for participants: male and female participants aged 18 to 75 years (inclusive); body mass index (BMI) between 19 and 28 kg / m². 2 (Including upper and lower limits); During the study period (from signing the informed consent form to the last follow-up) and within 90 days after the last dose of medication, subjects are prohibited from donating sperm, eggs, etc., and there is no possibility of pregnancy (or causing a sexual partner to become pregnant), childbirth, or breastfeeding; No history of major diseases, and physical examination, vital signs, and laboratory test results are normal during the screening period, or although they exceed the normal reference range, they are deemed by the researcher to have no clinical significance; Able to communicate normally with clinical staff and able to comply with the requirements of this study; Sign the informed consent form to indicate willingness to participate in this study.

[0164] Exclusion criteria for subjects: Medical conditions that may interfere with drug absorption, distribution, metabolism, or excretion, or may affect study protocol adherence; history of hemophilia or coagulation disorders; history of endocrine, nervous, hematological, immunological (including personal or family history of hereditary immunodeficiency), psychiatric, metabolic disorders, lymphoproliferative disorders, or serious or opportunistic infections (including herpes, tuberculosis) that the investigator considers currently clinically significant; allergic constitution, especially to eggs or chicken; participation in other clinical studies within 90 days prior to the first use of the study product; discontinuation of other prescription or over-the-counter medications that the investigator considers to affect the evaluation results of this study for less than 14 days or 5 half-lives of the drug (whichever is longer) prior to the first use of the study product; history of malignant tumors within the past 5 years; blood donation or significant blood loss (>400 ml) within 90 days prior to the first use of the study product. L); those who have undergone major surgery (as determined by the investigator based on past medical history) or suffered major trauma within 6 months prior to the first use of the study product; those whose vital signs are abnormal and deemed clinically significant by the investigator; those with clinical or laboratory evidence showing one of the following: hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), or syphilis carrier / infected; those with a history of severe kidney disease; those with difficulty obtaining venous blood or a known history of multiple instances of fainting during injections or bleeding; those with a history of regular alcohol consumption within 6 months prior to screening, exceeding 14 drinks / week for men (1 drink = 5 ounces of wine, 12 ounces of beer, or 1.5 ounces of spirits) or exceeding the alcohol test limit during the screening period; those who have consumed any alcoholic beverages within 48 hours prior to the first use of the study product, or those who do not agree to avoid consuming any alcoholic beverages during the trial; and those with poor compliance who are unwilling to follow the study requirements.

[0165] The basic information and medication details of the subjects are shown in the table below.

[0166] Table 3: Basic Information of Subjects

[0167] During the trial, the underlying medications for patient M002 remained unchanged. The underlying medications for patient M002 were as follows: antihypertensive: amlodipine besylate tablets, bisoprolol fumarate tablets, candesartan tablets; uric acid-lowering: benzbromarone tablets; back pain: etorizine acetate tablets, nimesulide dispersible tablets; coronary artery disease: isosorbide dinitrate tablets (discontinued as directed by the physician after day 14; no discomfort was reported on days 30, 51, and 134); and adjuvant medications: vitamin C tablets, vitamin B2 tablets, and coenzyme Q10 soft capsules.

[0168] During the trial, the underlying disease medications for patients M003 remained unchanged. These included: hypoglycemic agents: acarbose tablets, dapagliflozin tablets; diuretics: furosemide tablets, spironolactone tablets; anticoagulants: rivaroxaban tablets; heart failure medications: minotrol succinate extended-release tablets, digoxin tablets, sacubitril / valsartan sodium tablets; antiarrhythmic agents: Wenxin granules; and other medications: rabeprazole sodium enteric-coated tablets, potassium chloride extended-release tablets.

[0169] During the trial, the underlying medications for patient M004 remained unchanged. The underlying medications for patient M004 were: diuretic: spironolactone tablets, anticoagulant: rivaroxaban tablets, and heart failure: minotropin extended-release tablets and veliciguat tablets.

[0170] After a single intravenous (IV) administration (EE001) to healthy subjects in S001 and S002, the subjects were observed for 7 days. After multiple IV administrations (EE001) to healthy subjects in M001, patients in M002, patients in M003, and patients in M004, the subjects were observed for 7 days. The results of complete blood count, blood biochemistry, urinalysis, coagulation function, electrocardiogram, echocardiography, and six-minute walk test were measured.

[0171] The results showed that single-dose (S001-S002) and multiple-dose (M001-M004) administrations did not significantly alter blood routine, urine routine, coagulation parameters, myocardial enzyme profile, or T lymphocyte subsets. No adverse reactions or toxic reactions were observed, and EE001 had no effect. Therefore, EE001 injection has good safety; no adverse reactions or serious adverse reactions occurred in any subjects during single-dose and multiple-dose administrations, and the subjects tolerated it well.

[0172] As shown in Figure 2, for heart failure patients, the N-terminal pro-brain natriuretic peptide (NTNP) level increased after the first dose in patient M002, then gradually decreased, and after Day 7, the NTNP level was significantly lower than before the medication. The NTNP level in patient M003 was higher before the medication, decreased after the medication, and then increased again, but was generally close to the level before the medication, with no significant difference. The NTNP level in patient M004 increased first after the medication and then decreased.

[0173] Table 4: Partial echocardiographic results of 6 subjects (S001-S002, M001-M004)

[0174] As can be seen from Figure 3 and the table above, the ejection fractions of heart failure patients M002 and M003 were significantly increased compared with those before medication.

[0175] Table 5: Results of the six-minute walk of 6 subjects (S001-S002, M001-M004)

[0176] As shown in the table above, for healthy subjects (S001, S002, and M001), there were no significant differences in the six-minute walk test results before and after single and multiple administrations, and no adverse reactions or toxicities were observed. For patients with heart failure (M003 and M004), the six-minute walk distance was significantly increased.

[0177] Table 6: Flow cytometry results (CD3+) of 6 subjects (S001-S002, M001-M004) + CD3 + +CD8 + CD3 + +CD4 + Treg cell)

[0178] As shown in Figures 4 to 15 and the table above, for healthy subjects, there were no significant differences before and after IV administration in both single-dose and multiple-dose groups. For heart failure patients (M002, M003, and M004), Treg cells increased compared to before medication, and no adverse reactions or toxic reactions were observed.

[0179] As shown in Figures 16 to 24, among the 44 cytokines detected, for factors with pro-inflammatory or cytotoxic effects (CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL17, MMP1, MMP2, MMP7, MMP9, MMP12, IL-1α, IL-1β, IL-6, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD31, CD62p, SYK, KMT5A, CNBP, IFN-γ, TNF-α, VEGF-A), 6 subjects showed a significant decreasing trend after drug administration; for factors with anti-inflammatory or cytoprotective effects (IL-4, IL-10, iNOS, NQO1), 6 subjects showed a significant decreasing and increasing trend after drug administration.

[0180] Table 7: 24-hour Holter monitoring of patients with multiple drug administrations in M002

[0181] Figure 1 shows the leg swelling of patient M002 before medication (first row from top to bottom) and after medication (second row from top to bottom) (Day 3). The patient did not use diuretics throughout the entire medication course. Patient M002's chief complaint was significant improvement in symptoms such as chest tightness and bilateral leg edema, as well as mobility, compared to before medication. Specifically, this included:

[0182] 1. After taking the medication (Day 1), the patient reported significant relief from chest tightness after the second dose on the afternoon of the first day.

[0183] 2. After taking the medication (Day 2), the swelling in both legs subsided significantly on the second day; on the second day, one could sit upright (for about 30 minutes), whereas before taking the medication, one could only lie on one's side, prone, or lean against something.

[0184] 3. After taking the medication (Day 5), I went to bed late at night (1-2 am) and felt quite excited, like I had taken ginseng.

[0185] Furthermore, 127 days after starting medication (EE001), patient M002 reported: a significantly improved heart condition, feeling no longer experiencing heart disease, and excellent sleep quality; swelling in the legs had subsided, and shortness of breath was relieved both day and night; the patient could sit, stand, and perform household chores; the right coronary artery showed no plaque or significant stenosis, with only calcified plaques and slight stenosis visible in the proximal segment of the left descending artery, while other lumens were normal; the electrocardiogram was normal; the medication was not only effective for the heart but also had restorative effects on other bodily functions. Medication usage: Cardiac medications have been discontinued. Antihypertensive medication is now only bisoprolol fumarate tablets; the other two medications have been discontinued. Back pain medication has been discontinued. Coenzyme Q10 has been discontinued. Isosorbide dinitrate tablets (discontinued after day 14 as directed by the doctor) have been discontinued for over four months.

[0186] Table 8: 24-hour Holter ECG results of M003 patients after multiple drug administrations

[0187] Table 9: Results of 24-hour Holter monitoring in patients with multiple drug administrations (M004) Note 1: On Day 4, sinus, junctional, and atrial fibrillation heart rates alternated throughout the day, with unclear boundaries. Note 2: During monitoring on Day 4, the patient was allergic to the Holter probe, so this monitoring was not performed on Day 7. Among them, patient M004 developed atrial flutter on Day 14, with the following parameters: atrial flutter heartbeats: 118653, atrial flutter duration: 1351 minutes, atrial flutter rate: 99.7%, number of atrial flutters: 1.

[0188] As can be seen, the 24-hour ECG showed significant improvement in atrial fibrillation in patient M002 compared to before medication, and all indicators of heart rate variability were significantly improved compared to before medication. Furthermore, the same results were observed during a 30-day follow-up of patient M002. The 24-hour ECG also showed significant improvement in atrial fibrillation in patient M003 compared to before medication. The same results were observed during a 30-day follow-up of patient M004. Patient M004 also reported no recurrence of atrial fibrillation after 14 days of medication and felt more energetic while walking.

[0189] Therefore, EE001 injection has a certain effect on improving ejection fraction in some patients with heart failure (e.g., patients M002 and M003), significantly improving various indicators of heart rate variability (e.g., patient M002), and 24-hour Holter monitoring shows a significant elimination effect on atrial fibrillation in patients (e.g., patients M002, M003, and M004), a certain improvement in six-minute walking distance (e.g., patients M003 and M004), and a reduction in the level of N-terminal pro-brain natriuretic peptide (NTP), a heart failure-specific marker (e.g., patient M002). According to patients' reports after medication, EE001 injection can improve the quality of life of heart failure patients to a certain extent, such as increasing the time patients can sit upright, prolonging nighttime sleep, relieving chest tightness, and reducing symptoms such as generalized or lower limb edema (e.g., patient M002); no atrial fibrillation occurred during the medication period, and walking distance and endurance were improved (e.g., patient M004).

[0190] Meanwhile, by monitoring the levels of a total of 44 cytokines in subjects before and after medication, it was found that after administration of EE001 injection, it could significantly reduce the levels of pro-inflammatory or cytotoxic factors, such as CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL17, MMP1, MMP2, MMP7, MMP9, MMP12, IL-1α, IL-1β, IL-6, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD31, CD62p, SYK, KMT5A, CNBP, IFN-γ, TNF-α, and VEGF-A; and significantly increase the levels of anti-inflammatory or cytoprotective factors, such as IL-4, IL-10, iNOS, and NQO1. The decreasing trend in the levels of pro-inflammatory or cytotoxic factors and the increasing trend in the levels of anti-inflammatory or cytoprotective factors became more pronounced with increasing dosing frequency; for example, multiple administrations were more effective than single administrations. In summary, EE001 can inhibit the levels of pro-inflammatory or cytotoxic factors and increase the levels of anti-inflammatory or cytoprotective factors in vivo. The optimal indications and efficacy of EE001 require further clinical validation.

[0191] In summary, EE001 injection is safe and did not produce any adverse reactions in the subjects. It exhibits significant efficacy, enhancing basic cardiac function in patients with heart failure or insufficiency; significantly eliminating atrial fibrillation; increasing cardiac ejection fraction; improving basic motor function; and significantly inhibiting pro-inflammatory cytokines and increasing anti-inflammatory cytokines in the subjects. These effects are similar to the comprehensive effects of stem cells, demonstrating therapeutic efficacy and long-term effectiveness. Further research is needed to determine its optimal indications and clinical efficacy.

Claims

1. The use of amniotic fluid and / or its extracts in the preparation of one or more of the following drugs: reducing atrial fibrillation in drug recipients, increasing six-minute walking distance in drug recipients, increasing upright sitting time in drug recipients, prolonging sleep time in drug recipients, improving generalized or lower limb edema in drug recipients, and improving chest tightness in drug recipients; in, The amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, even more preferably eggs with an embryonic age of 7-8 days, or from eggs of poultry other than chickens whose developmental stage corresponds to the developmental stage of the eggs with the embryonic age; or from rodent embryos with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents with a gestational age of 8-14 days.

2. The use of amniotic fluid and / or its extracts in the preparation of one or more of the following formulations: (1) reducing heart rate variability in the subject of formulation, (2) increasing ejection fraction in the subject of formulation, (3) reducing N-terminal pro-brain natriuretic peptide in the subject of formulation, (4) reducing the level of pro-inflammatory or cytotoxic factors in the subject of formulation, and (5) increasing the level of anti-inflammatory or cytoprotective factors in the subject of formulation. in, The amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, even more preferably eggs with an embryonic age of 7-8 days, or from eggs of poultry other than chickens whose developmental stage corresponds to the developmental stage of the eggs with the embryonic age; or from the embryos of rodents with a gestational age of 8-14 days, or from the embryos of other non-human mammals other than rodents whose developmental stage corresponds to the developmental stage of rodents with a gestational age of 8-14 days. Preferably, the amniotic fluid and / or its extracts reduce the levels of pro-inflammatory or cytotoxic factors in the treatment recipient through one or more of the following signaling pathways: chemokine family signaling pathway, matrix metalloproteinase family signaling pathway, interleukin family signaling pathway, CD antigen family signaling pathway, atherosclerosis-related signaling pathway, JAK / STAT signaling pathway, Th1 signaling pathway, and angiogenesis VEGF-A signaling pathway. Preferably, the amniotic fluid and / or its extracts enhance the anti-inflammatory or cytoprotective factor levels in the treatment recipient through cytokine-inducible signaling pathways and / or the antioxidant NQO1 signaling pathway.

3. The application as described in claim 2, characterized in that, The inflammatory or cytotoxic factor is selected from one or more of CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, CCL17, MMP1, MMP2, MMP7, MMP9, MMP12, IL-1α, IL-1β, IL-6, IL-12, IL-13, IL-17α, IL-18, IL-23, IL-27, CD31, CD62p, SYK, KMT5A, CNBP, IFN-γ, TNF-α, and VEGF-A, and / or The anti-inflammatory or cytoprotective factor is selected from one or more of IL-4, IL-10, iNOS, and NQO1.

4. Use of amniotic fluid and / or its extracts in the preparation of formulations for the treatment and / or prevention of diseases mediated by one or more of the following signaling pathways: (a) Chemokine family signaling pathways (b) Matrix metalloproteinase family signaling pathway (c) Interleukin family signaling pathways (d) CD antigen family signaling pathway, (e) Atherosclerosis-related signaling pathways, (f) JAK / STAT signaling pathway, (g)Th1 signaling pathway, (h) VEGF-A signaling pathway in angiogenesis (i) Cytokine-induced signaling pathways, and (j) The NQO1 signaling pathway of antioxidant factor; Preferably, diseases mediated by chemokine family signaling pathways include diseases mediated by one or more chemokines selected from CXCL4, CXCL9, CXCL10, CXCL11, CCL2, CCL5, and CCL17. Preferably, diseases mediated by matrix metalloproteinase family signaling pathways include diseases mediated by one or more matrix metalloproteinases among MMP1, MMP2, MMP7, MMP9, and MMP12. Preferably, diseases mediated by the interleukin family signaling pathway include diseases mediated by one or more cytokine families such as the IL-1 family, IL-6 family, IL-10 family, IL-12 family, and IL-17 family; Preferably, the CD antigen family includes CD31 and / or CD62p; Preferably, diseases mediated by atherosclerosis-related signaling pathways include diseases mediated by the SYK signaling pathway and / or diseases mediated by the KMT5A signaling pathway; Preferably, diseases mediated by the JAK / STAT signaling pathway include diseases mediated by IFN-γ cytokines; Preferably, diseases mediated by the Th1 signaling pathway include diseases mediated by TNF-α cytokines; Preferably, diseases mediated by cytokine-induced signaling pathways include diseases mediated by iNOS cytokines.

5. The application as described in any one of claims 1-4, characterized in that, The dosage of the drug or preparation is 0.5 to 2.5 ml / kg / day, for example, 0.5 to 1.5 ml / kg / day or 0.8 to 1.2 ml / kg / day.

6. The application as described in any one of claims 1-5, characterized in that, The drug or preparation is administered once every half day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days, or once every half month, once a month, once every three months, once every six months, or once a year. Preferably, the drug or preparation is administered twice a day, for a course of treatment every seven days, or once every seven days.

7. The application as described in any one of claims 1-6, characterized in that, The amniotic fluid is derived from chicken eggs, duck eggs, goose eggs, or a combination thereof; and / or The amniotic fluid comes from rodents that are 10-14 days pregnant.

8. The application as described in any one of claims 1-7, characterized in that, The drug or formulation is administered to mammals, especially humans; preferably, the recipient suffers from heart failure, including but not limited to NYHA class I, II, III, or IV heart failure; and / or The total duration of atrial fibrillation in the subjects was higher than the control level, where the control level was the total duration of atrial fibrillation in healthy controls; and / or The proportion of atrial fibrillation in the subjects was higher than that in the control group, where the control group was the proportion of atrial fibrillation in healthy controls; and / or The subjects had a higher atrial fibrillation frequency than the control group, where the control group was the atrial fibrillation frequency in healthy controls; and / or The subjects' six-minute walking distance was lower than the control level, which was the six-minute walking distance of healthy controls; and / or The degree of edema in the subjects' whole body or lower limbs was higher than the control level, where the control level was the whole body or lower limbs of healthy controls; and / or The ejection fraction of the subject was lower than the control level, where the control level was the ejection fraction of a healthy control; and / or The reduction of heart rate variability includes increasing the standard deviation normal to normal interval (SDNN), increasing the standard deviation mean normal to normal interval (SDANN), increasing the standard deviation normal to normal interval of the square root mean (rMSDD), and / or increasing the percentage of normal to normal intervals with a difference greater than 50 milliseconds (pNN50); preferably, the SDNN of the subject is lower than the control level, and the control level is the SDNN of a healthy control; preferably, the SDANN of the subject is lower than the control level, and the control level is the SDANN of a healthy control; preferably, the rMSDD of the subject is lower than the control level, and the control level is the rMSDD of a healthy control; preferably, the pNN50 of the subject is lower than the control level, and the control level is the pNN50 of a healthy control; and / or The levels of N-terminal pro-brain natriuretic peptide (PTP) in the subjects were higher than those in the control group, where the control group was the N-terminal PTP level in healthy controls; and / or The levels of inflammatory or cytotoxic factors in the subject were higher than those in the control group, where the control group was the level of inflammatory or cytotoxic factors in healthy controls; and / or The level of anti-inflammatory or cytoprotective factors in the subject was lower than that in the control group, where the control group was the level of anti-inflammatory or cytoprotective factors in healthy controls.

9. The application as described in any one of claims 1-8, characterized in that, The drug or preparation contains frozen amniotic fluid and / or its extracts, or is a lyophilized reagent of the amniotic fluid and / or its extracts.

10. The application as described in any one of claims 1-9, characterized in that, The drug or preparation is an infusion solution.

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