Treatment

Abstract

To provide a pharmaceutical composition that treats a mammalian subject by a therapeutic approach based on a cell enhanced in order to promote repair, regeneration and / or restoration of tissue property and neuron property.SOLUTION: A pharmaceutical composition for reduction or inhibition of T cell proliferation in a mammalian subject, and / or for increasing phagocytosis of a macrophage, where the pharmaceutical composition includes a mammalian amnion exosome, and one or more pharmaceutically acceptable carriers, fillers and / or diluents.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Application data) This application is incorporated herein by reference in its entirety, and is hereby incorporated by reference in its entirety. Australian Provisional Patent Application No. 2015902 filed on June 12, 2015, entitled 214 and an Australian provisional patent filed on April 12, 2016 entitled "Method of Treatment" This application relates to and claims priority from patent application number 2016901349. do.

[0002] (Technical field) The present disclosure generally relates to potent anti-inflammatory drugs for promoting tissue and neuronal repair, regeneration and / or recovery. Methods of treatment in mammalian subjects with optimized cell-based therapeutic approaches - Patents.com Drugs and methods for making drugs useful in treating mammalian subjects are also encompassed by the present disclosure. [Background technology]

[0003] Bibliographic details of the publications referred to by the authors in this specification are given at the end of this description. They are collected in alphabetical order.

[0004] Any reference to any prior art in this specification does not necessarily mean that this prior art is a common Nothing in this document shall be construed as an admission or suggestion that any of the foregoing forms part of the general public's knowledge. should not be interpreted.

[0005] Modern medicine has made great strides with the identification of biological agents such as chemotherapy and antibiotics. However, many drugs have multiple functions, and some may have off-target physiological effects. Cell-based therapies have been proposed as the next pillar of modern medicine (Fishb ack et al. (2013), Sci. Transl. Med. 5:179, ps7).

[0006] One of the rate-limiting factors for cell-based therapies is the possibility of inconsistent product quality. Mesenchymal stem cells (MSCs) have been well documented in the literature, e.g. While the strain has been extensively characterized and has achieved clinical trial quality, it generally requires serial passage for use. This has detrimental effects on when and how the cells can be used. It can and often does have an impact.

[0007] One approach to counter this challenge is to use preserved mesenchymal progenitor cells. This at least avoids the delay between collection and treatment. introduces donor-to-donor variability in cell potency and does not address the adverse effects of serial passaging.

[0008] An interim measure to address this issue is to have a "master" cell bank. , which is the number of passages a cell can undergo before senescence and epigenic / karyotypic changes occur. However, this does not overcome the inevitable problem that there are limitations to 2011), Aging, (Albany NY), 3:873-888). After repeated medication There is also a risk of immune rejection.

[0009] As a case in point, with significant improvements in obstetric surveillance and neonatal care, a large number of premature babies are being born. One particularly debilitating condition is hypertension, resulting in an increased prevalence of "diseases of prematurity." Bronchopulmonary dysplasia (BPD) is an incurable chronic lung disease in premature infants. BPD is characterized by abnormalities and arrest of blood flow and destruction of pulmonary capillary structures. BPD is associated with neonatal morbidity and mortality. Survivors of BPD are at serious risk for obstructive pulmonary disease in early adulthood. (Doyle et al., (2006), Paediatrics, 118:108-113) and common chronic health disorders and cognitive decline (Lodha et al. (2014), PLoSONE There is also a serious risk of BPD for the reasons outlined above. While these have been proposed as potential cell-based therapies, they have demonstrated widely differing functional efficacy. There may be many cells with this condition, which is separate from causing emotional stress. This may delay other treatment options.

[0010] The problem of damage to pulmonary capillary structures is not limited to premature human infants. The horse racing industry, in particular, has been plagued by exercise-induced pulmonary hemorrhage. Some jurisdictions are facing the problem of emergency hemorrhage (EIPH). For example, a horse that exhibits more than two nosebleeds after a race is banned from further racing for life. This results in devastating economic losses. Therapeutic approaches may raise ethical issues regarding performance enhancement. In any event, such an approach would reproduce capillary rupture. The possibility is low.

[0011] The beneficial effects of human amniotic epithelial cells (hAECs) have been documented (e.g., Ho dges et al. (2012), Am. J. Obstet. Gynerol. 206:448 e8-448e15;Murphy et al. (2012), Cell Transplant ,I:1477-1492;Vosdoganes et al.(2013),Cytother apy,15:1021-1029;Yawno et al.(2013),Dev.Neuro sci.35:272-282). However, its mechanism of action needs to be determined. There is a gender.

[0012] Therefore, the problem of cell-based therapy needs to be addressed and alternative strategies are required. It is clear that:

[0013] According to the present invention, the vesicular medium for cell communication is mammalian amniotic epithelium. It has been identified that amniotic exosomes are released from amniotic endothelial cells (AECs). The vesicles called extracellular vesicles are nanometer-sized extracellular vesicles (50-60%) derived from late endosomes. 100 nm) and is released from the cell surface.

[0014] Taught herein are improved forms of mammalian amniotic epithelial cell-based therapy. The improvement is due to the release of steroids by epithelial cells and the activation of endogenous repair mechanisms. This involves the use of nano-sized amniotic exosomes, which exert a restorative effect. It acts directly on immune cells, in particular by reducing T cell proliferation and increasing macrophage phagocytosis. Increases phagocytosis, activates stem cells, and inhibits collagen production in activated fibroblasts It is shown herein that amniotic exosomes contain proteins (e.g., proteins) and genetic molecules (e.g., miRNA, mRNA, and non-coding RNA) The present invention relates to the release of exosomal cargo profiles in the form of It has been proposed in.

[0015] Exosome biogenesis involves the inward budding of the limiting membrane of late endosomes into intraluminal vesicles The late endosome then fuses with the plasma membrane to release the exosome. Once secreted, exosomes are absorbed by target cells located in close proximity to the parent cell. Mechanistically, exosomes can be internalized by the endothelium or can travel distally via the circulation. They act as complex vectors containing the parent cell's material, which are then delivered to the target cell. It may contain proteins and genetic material to be transferred.

[0016] Thus, the present invention is based on the development of an enhanced approach to cell-based therapy. The present disclosure provides immunomodulatory, pro-regenerative and restorative agents released from mammalian amniotic epithelial cells. The present invention teaches the use of amniotic exosomes to have a therapeutic effect on T cell proliferation. and enhance macrophage phagocytosis, and promote beneficial proteome molecules and miRNAs. Activating endogenous stem cells through the release of genetic molecules such as mRNA and non-coding RNA Amniotic exosomes are used herein to promote wound healing. Promotes tissue repair, regeneration and recovery, including the maintenance of cells, and combats the effects of neurodegeneration and injury. It has been proposed that these drugs induce neuroprotection, including improving neuronal function and promoting repair and neuroregeneration. Amniotic exosomes have also been shown to promote collagen production in activated fibroblasts. Exosomes also improve kidney, liver, pancreas, heart, and lung damage. as well as the treatment of fibrotic conditions in these organs (e.g., liver or lung fibrosis). including, for example, ischemia-reperfusion injury or organ damage. Disease or adverse events in the systemic vasculature It has been proposed that exosomes promote repair and regeneration following myelination. It is also useful in promoting the development of demyelinating diseases such as multiple sclerosis. It has been proposed to be useful in the treatment of disorders.

[0017] Amniotic exosomes have beneficial effects not only in humans but also in non-human mammals Thus, the present invention extends to human and animal applications. AECs derived from human subjects are not intended to be limiting. In the book it is called "hAEC".

[0018] Examples of animal applications include the prevention of exercise-induced pulmonary hemorrhage in racing animals, including horses, racing dogs, and camels. It is treatment.

[0019] Amniotic exosomes are produced by culturing mammalian amniotic epithelial cells in a bioreactor and using conditioned medium. Amniotic exosomes can be produced in large quantities by isolating them from the culture medium. The cells can be maintained as an immortalized cell line. Amniotic exosomes are required The compound can be isolated when heated or stored in a lyophilized state.

[0020] An innovative feature of the present invention is the ability to selectively bind mammalian amniotic epithelial cells to amniotic exosomes. There is no need to identify a compatible donor. Exosomes do not induce harmful immune responses. Rather, the donor must be aware of the stage of pregnancy and / or the health of the newborn or full-term baby. However, in one embodiment, the amniotic exosome is selected based on other characteristics such as The cells were derived from human AECs from patients at the end of their pregnancy. tissue or neuronal repair, regeneration and / or regeneration for various physiological conditions such as They produce amniotic exosomes, which are at least good at promoting recovery. Therefore, aspects of the present invention provide a method for treating a desired condition without the drawbacks of cell-based therapy. Donor selection to identify amniotic epithelial cells that produce amniotic exosomes useful for This can lead to the generation of a bank of amniotic epithelial cells. Or, specific batches of cells can be selected based on condition.

[0021] Pharmaceutical compositions comprising amniotic exosomes, therapeutic kits comprising amniotic exosomes, and / or Reagents and bioreactors for screening suitable donor or amniotic epithelial cell lines Kits are also encompassed by the teachings of the present disclosure.

[0022] Taught herein are enhanced or modified cell-based therapies. Thus, what is made possible herein is the use of amniotic epithelial cells to generate a mammalian, including human, subject. Improved cell-based therapy protocols for treating cancer, the improvement comprising immortalizing The amniotic exosomes were isolated from the amniotic epithelial cells and used to promote remyelination. administered systemically or locally to a subject in need of tissue or neuronal repair, regeneration, and / or restoration. This includes providing.

[0023] Some drawings contain representations or objects in color. Color photographs are available upon request. They are available from the patent owner or from the appropriate patent office, where a fee is charged. may be imposed. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows (A) an electron micrograph of amniotic exosomes showing the typical cup-shaped morphology and diameter of approximately 100 nm; (B) a photograph depicting the expression of Alix and TSG101, markers of exosome biogenesis. Alix and TSG101 are biomarkers for exosomes. [Figure 2] FIG. 2 shows graphs demonstrating that (A) amniotic exosomes inhibit T cell proliferation similarly to hAEC-conditioned medium; and (B) amniotic exosomes enhance macrophage phagocytosis (n=3). [Figure 3] FIG. 3 is a graph showing that amniotic exosomes improve tissue:airspace ratio in a mouse model of bronchopulmonary dysplasia (BPD). [Figure 4] FIG. 4 is a schematic representation of the experimental timelines used in Example 3, showing intra-amniotic LPS injection at E16, exosome / cell injection at day 4 of life, and sacrifice time points (marked with crosses). [Figure 5] FIG. 5 shows graphs demonstrating that (A) term exosomes are more immunosuppressive than preterm exosomes; (B) term exosomes are better able to enhance macrophage phagocytosis as demonstrated by pHRodo labeling (n=3 donors per group). [Figure 6] Figure 6 shows a graph demonstrating that amniotic exosomes reverse established lung inflammation and fibrosis in a bleomycin-induced pulmonary fibrosis mouse model (A); (B) 6-8 month old female C57BL6 mice. 7 days after bleomycin challenge, 10 μg or 50 μg of exosomes derived from full-term pregnant hAECs were administered intranasally. [Figure 7] Figure 7 shows a graph demonstrating that amniotic exosomes reverse the activation of human primary lung fibroblasts in vitro. When cultured in the presence of 5 mg / mL transforming growth factor β, exosomes reduced alpha-smooth muscle actin protein levels within 24 hours. [Figure 8A]Figure 8A is a schematic diagram showing that amniotic exosomes contain miRNAs that target the cytokine-cytokine receptor signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 8B] Figure 8B is a schematic diagram showing that amniotic exosomes contain miRNAs that target the cytokine-cytokine receptor signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 8C] Figure 8C is a schematic diagram showing that amniotic exosomes contain miRNAs that target the cytokine-cytokine receptor signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 9A] Figure 9A is a schematic diagram showing that amniotic exosomes contain miRNAs that target the Wnt signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 9B] Figure 9B is a schematic diagram showing that amniotic exosomes contain miRNAs that target the Wnt signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 10] 10 is a schematic diagram showing that amniotic exosomes contain miRNAs that target the PI3K-Akt signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 11] 11 is a schematic diagram showing that amniotic exosomes contain miRNAs that target the TGFβ signaling pathway. Yellow boxes indicate targets by one or more miRNAs. [Figure 12] Figure 12 shows (A) a graph and (B, C) photographs showing the lung regenerative effect of amniotic exosomes containing tissue airspace ratio (%) among healthy control hAECs, full-term exosomes, and pre-term exosomes. "Full-term" exosomes are exosomes isolated from hAECs at the end of pregnancy. "Pre-term" exosomes are isolated prior to full term pregnancy. [Figure 13]Figure 13 (A-C) is a photographic representation showing that amniotic exosomes induce regeneration in the lung in a manner similar to hAECs. Dark staining is evidence of elastin-positive leading edges. [Figure 14] Figure 14 is a graph showing that amniotic exosomes, but not fibroblast exosomes, induce a resident stem cell response in the lung that is significantly greater than the response induced by hAECs. [Figure 15] FIG. 15 shows graphs demonstrating that amniotic exosomes were anti-fibrotic in the liver, as demonstrated using Sirius red staining of CCL4+ exosomes versus CCL4+ saline in liver tissue sections (A) and immunohistochemistry with alpha-smooth muscle activin (α-SMA). [Figure 16] FIG. 16 is a graph showing differences at the proteome level between exosomes derived from full-term versus pre-term hAECs. [Figure 17] Figure 17 is a graph showing a comparison of cellular components between hAECs and whole mesenchymal stem cells (MSCs). Dark gray: whole exosomes from hAECs. Light gray: whole exosomes from MSCs (Anderson et al. (2016), Stem cells, http: / / doi.ory / 10.1002 / stem.2298). [Figure 18] Figure 18 is a graph showing a comparison of biological processes between hAECs and whole MSCs (Anderson et al. (2016), supra). Dark grey: whole exosomes from hAECs. Light grey: whole exosomes from MSCs (Anderson et al. (2016), supra). DETAILED DESCRIPTION OF THE INVENTION

[0025] Throughout this specification, unless the context otherwise requires, the word "comprise" Or, for example, "comprises" or "comprising" )" refers to a stated element or integer or method step, or an element or integer or Although the inclusion of a group of method steps is implied, any element or integer or method step or element or integer or It will be understood that a " " does not imply the exclusion of groups of method steps.

[0026] As used herein, the singular forms "a," "an," and "the" are used where the context clearly indicates. Plural aspects are included unless otherwise indicated. Thus, for example, a reference to "(a) a disease or condition" "(an) exosome" encompasses a single disease or condition as well as two or more diseases or conditions; a reference to an exosome includes two or more exosomes as well as a single exosome; and the like. Enabling aspects are encompassed by the term "the present invention." A "disease" or "condition" is a "disorder." All such embodiments are possible within the broad scope of the present invention. Any such variants and derivatives are encompassed by the "forms" of the present invention.

[0027] The present disclosure is within the context of repair, regeneration and restoration of cells, tissues, neural pathways and endocrine pathways. and enhanced cells that facilitate the treatment of mammalian subjects for a number of diseases and conditions commonly encountered in the The present disclosure teaches a method for the treatment of amniotic epithelial cells (AECs) using a method for the treatment of amniotic epithelial cells (AECs) in mammals. Amniotic membrane exosomes isolated from conditioned medium exert beneficial immunomodulatory physiological and biochemical properties. The present invention teaches that mammalian amniotic exosomes have a specific biological profile. reduces T cell proliferation, enhances macrophage phagocytosis, and promotes the proliferation of proteome molecules and m Endogenous stem cells via the release of genetic molecules such as iRNA, mRNA, and non-coding RNA. They activate cells, exerting effects on immune cells. They also stimulate activated fibroblasts. They also suppress collagen production in cells. Importantly, amniotic exosomes are immunogenic. Therefore, allogeneic amniotic exosomes were used. It can be used.

[0028] Amniotic exosomes also reverse established lung inflammation and pulmonary fibrosis It also reverses the activation of primary lung fibroblasts. It also reverses the activation of, for example, liver, pancreas, heart and It also applies to fibrosis in other organs, such as the kidney. Tokine receptor signaling pathway, Wnt signaling pathway, PI3K-Akt signaling transduction pathways, TGFβ signaling pathways, and a diverse range of physiological and neurological processes miRNA, mRNA and non-coding RNA targeting signaling pathways involved in Contains A.

[0029] The present specification describes mammalian amniotic exosomes that can be used to treat cells, tissues including organs, neural pathways, and the entire body. Inducing repair, regeneration and restoration of vascular components and promoting wound healing The present invention teaches that amniotic exosomes promote repair, regeneration, and recovery of the brain and spinal cord, and that they are Promoting repair of degenerative conditions, inducing recovery of organ damage following trauma, disease or drug abuse Promoting repair after other damage to the brain, such as stroke or traumatic brain injury It is proposed herein that exosomes can be used to treat demyelinating diseases such as multiple sclerosis, ophthalmopathy, and the like. Neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis, and adrenal glands Treatment of demyelinating diseases, conditions, or disorders such as leukodystrophy and adrenomyeloneuropathy In one embodiment, amniotic exosomes are administered to the lungs. It promotes repair of damage, which is important in the treatment of bronchopulmonary dysplasia (BPD) in human neonates. This is important for example in horses, racing dogs (e.g. greyhounds) and rabbits. It also has animal applications in the treatment of exercise-induced pulmonary hemorrhage (EIPH) in racing animals such as do.

[0030] Thus, the invention made available herein is a method of treating a mammalian subject, the method comprising: The method comprises the steps of: (a) providing mammalian amniotic epithelial cells derived from a donor mammal of the same species as the mammalian sheep; This includes systemic or local administration of membrane exosomes.

[0031] Reference to a "mammalian subject" includes a mammal in need of treatment. In the present specification, the mammalian subject is a human. The term "AEC" means "amniotic epithelial cells." Human When derived from a mammalian cell line, the AECs are referred to as "hAECs."

[0032] Thus, the present specification provides guidance in methods of treating human subjects, which methods include administering to human subjects Systemic or local administration of human amniotic exosomes derived from human allogeneic amniotic epithelial cells derived from nurses Includes giving.

[0033] In another embodiment, the mammalian subject is, for example, a horse, cow, sheep, goat, pig, Non-human animals such as, but not limited to, alpacas, llamas, dogs, cats or camels It is a mammal.

[0034] In one embodiment, the mammalian subject is in need of treatment. The term "treatment" refers to the treatment of cells, tissues, and repair, regeneration, or promotion of regeneration of physiological pathways, including neuronal and endocrine pathways; and / or Examples include recovery from ischemia-reperfusion injury or stroke, internal and superficial wounds, ulcers and Organs containing circulatory vessels such as capillaries, arteries, and veins containing such blood vessels, followed by scarring , neurodegenerative conditions, and injuries to the brain and spinal cord (including traumatic brain injury and spinal cord injury) The present invention includes, but is not limited to, repair, regeneration and / or restoration of exosomal tissue. Thesomes also have utility in treating fibrotic diseases, conditions, or disorders of the lung, liver, heart, kidney, and pancreas. Exosomes have also been proposed for the treatment of organ fibrosis, e.g., multiple sclerosis. optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis, and adrenoleukodystrophy for use in the treatment of demyelinating diseases, conditions or disorders such as myelomeningocele and adrenal neuropathy Amniotic exosomes are also contemplated for clinical applications to treat diseases or conditions, as well as for the treatment of skin diseases. It is useful in cosmetic preparations to promote skin renewal or scar or wound healing.

[0035] While it is not intended that the present invention be limited to any one theory or mode of action, In mammals, amniotic exosomes are small molecules for communication from amniotic epithelial cells. The proteome represents the cellular medium and provides a mixture of beneficial molecules to promote repair, regeneration, and recovery. It is proposed herein to release proteome molecules and genetic molecules. It has also been suggested that the molecular profile differs depending on the gestational stage of the donor from which the amniotic epithelial cells are obtained. Thus, the present disclosure provides methods for the preparation of immortalized embryos derived from various donors at various stages of gestation. The present invention teaches the creation of a bank of mammalian amniotic epithelial cells. The epithelial cells are then used for therapeutic purposes. Based on the disease or condition in the subject to be treated and the protein produced by the amniotic exosomes, The present specification provides a method for selecting a gene from a bank based on its molecular and genetic molecular profile. Depending on the disease or condition being treated, a particular proteomic and / or genetic profile It is taught that amniotic exosomes having the following structure may be preferred.

[0036] Accordingly, another aspect taught herein is a method of treating a mammalian subject, the method comprising: The law is (i) Identifying donors, if necessary; and (ii) The proteome and / or structure of amniotic exosomes produced by epithelial cells in culture The method involves the transfer of the chromosome from a specific donor or immortalized amniotic membrane from a donor based on the genetic profile. Selecting skin cells; (iii) generating conditioned medium from the selected immortalized amniotic epithelial cells; and (iv) isolating amniotic exosomes from the conditioned medium; and (v) administering the amniotic exosomes systemically or locally to a mammalian subject.

[0037] In one embodiment, the mammalian subject is a human subject. Another aspect is a method of treating a human subject, the method comprising: (i) Identifying donors, if necessary; and (ii) The proteome and / or structure of amniotic exosomes produced by epithelial cells in culture The method involves the transfer of the chromosome from a specific donor or immortalized amniotic membrane from a donor based on the genetic profile. Selecting skin cells; (iii) generating conditioned medium from the selected immortalized amniotic epithelial cells; and (iv) isolating amniotic exosomes from the conditioned medium; and (v) administering the amniotic exosomes systemically or locally to a human subject.

[0038] In another embodiment, mammalian amniotic exosomes are isolated and their proteome profiles are analyzed. Selected mammalian amniotic exosomes with predetermined profile and genetic profile A bank of amniotic exosomes is then generated based on their profile. are selected for use in medical treatment.

[0039] Accordingly, this document provides guidance on methods for treating mammalian subjects, which methods include: , (i) Identifying donors, if necessary; and (ii) Proteomic and / or genetic profiles of agents released by exosomes Selecting amniotic exosomes from a specific donor or donors based on their profile and, (iii) systemically or locally administering the amniotic exosomes to a mammalian subject.

[0040] In one embodiment, the mammalian subject is a human.

[0041] Accordingly, taught herein is a method of treating a human subject, the method comprising: (i) Identifying donors, if necessary; and (ii) Proteomic and / or genetic profiles of agents released by exosomes Selecting amniotic exosomes from a specific donor or donors based on their profile and, (iii) administering the amniotic exosomes systemically or locally to a human subject.

[0042] When used in therapeutics, amniotic exosomes can be used to treat drugs, agents, therapeutic agents, cell-based It may also be referred to as an agent, active ingredient, etc. derived from a therapy. References to "therapy" include clinical This includes both medical and cosmetic treatments.

[0043] Further taught herein is a method for cellular or neuronal repair in a mammalian subject. , a method of inducing regeneration and / or recovery, the method comprising: The delivery of allogeneic amniotic exosomes to a mammalian subject for a time and under conditions sufficient to induce This includes internal or topical administration.

[0044] In one embodiment, the subject herein is a method for cellular or neuronal repair in a human subject, Methods for inducing regeneration and / or recovery are made possible, which methods involve cellular or neuronal repair. Systemic administration of allogeneic amniotic exosomes to human subjects for a time and under conditions sufficient to induce remission. or topical administration.

[0045] In a further embodiment, contemplated herein is the administration of cellular or neural Mammalian amniotic membrane extract in the manufacture of a medicament for neuronal repair, regeneration and / or restoration The use of isosomes.

[0046] In one embodiment, the mammal is a human.

[0047] Thus, the present specification further provides a method for the treatment of cellular or neuronal repair in a human subject. The present specification further teaches the use of human amniotic exosomes in the manufacture of a drug. For example, demyelinating diseases, conditions, or disorders, including, but not limited to, multiple sclerosis. The present invention teaches the use of human amniotic exosomes in the manufacture of a medicament for the treatment of

[0048] Taught herein is an isolated sample of amniotic exosomes derived from amniotic epithelial cells. This includes human amniotic exosomes derived from amniotic epithelial cells. These include isolated samples of cells. The present invention therefore proposes the use of amniotic exosomes in mammalian subjects. COMPOSITION COMPRISING SELECTED ALLOGENIC MAMMALIAN AMNIOTIC EXOSOMES FOR USE IN TREATING AN INFECTIOUS SUBJECT - Patent application The pharmaceutical composition may further comprise one or more cosmetically acceptable carriers, excipients and / or diluents. Includes.

[0049] In one embodiment, the mammalian subject is a human subject.

[0050] Accordingly, the present invention provides human allogeneic amniotic exosomes for use in treating a human subject. The present invention teaches a pharmaceutical composition comprising: and / or a diluent.

[0051] Additionally, the composition includes a human allogeneic amniotic membrane exosome for use in treating a human subject. The cosmetic composition may further comprise one or more pharmaceutically acceptable salts. The term "carrier," "excipient," and / or "diluent" includes carriers, excipients, and / or diluents that may be present in the formulation.

[0052] Pharmaceutically acceptable carriers include, for example, bioactive compounds that act to stabilize amniotic exosomes. Physiologically acceptable compounds can be included. For example, carbohydrates such as glucose, sucrose or dextran, e.g., ascorbic acid, antioxidants such as acetic acid or glutathione, chelating agents, low molecular weight proteins, or water or excipients, including saline, or other stabilizers and / or buffers. Surfactants are used to stabilize, enhance, or reduce the absorption of amniotic exosomes. Pharmaceutically acceptable carriers and formulations (including liposome carriers) are known to those skilled in the art and are well described in the scientific and patent literature. For example, Remington's Pharmaceutical Sciences (19 90), 18th Edition, Mack Publishing Company, Easton, (See 'Remington's').

[0053] Other physiologically acceptable compounds include compounds that inhibit microbial growth or activity in amniotic exosome preparations. Preservatives that are useful for preventing the formation of granules include preservatives. Those skilled in the art will recognize that pharmaceutically acceptable compounds, including physiologically acceptable compounds, are well known. The choice of carrier to be used will depend, for example, on the route of administration of the amniotic exosomes of the present invention and the nature of the exosomes. The specific physiological or biochemical properties of the proteins and nucleic acids produced by This will give you a full understanding of the

[0054] Administration of amniotic exosomes in the form of a pharmaceutical composition can be by any convenient means known to those skilled in the art. The route of administration may be any route depending on the disease or condition or site of injury. intratracheally, nasopharynx, intravenously, intraperitoneally, intrathoracically, subcutaneously, intracranially, intradermally Intramuscular, intraocular, intrathecal, intracerebral, intranasal, intrarectal, topical administration, These include, but are not limited to, adhesive patches, dressings, and implants. Amniotic exosomes can be sprayed onto a subject with severe burns, for example.

[0055] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions.

[0056] Sterile injection solutions in the form of dispersions are generally prepared by dissolving amniotic exosomes in a sterile medium. It is prepared by incorporating various sterilized active ingredients.

[0057] For parenteral administration, amniotic exosomes are formulated with a pharmaceutical carrier and administered as a suspension. Examples of suitable carriers are water, saline, dextrose solution, fructose solution, and the like. The carrier may be a solution of ethanol, or an oil of animal, vegetable, or synthetic origin. For example, it may contain a preservative, a buffer, etc. In some cases, they may be formulated in cerebrospinal fluid.

[0058] For transmucosal or transdermal administration, the barrier to be permeated must be overcome in order to deliver the agent. Any suitable penetrant can be used. Such penetrants are generally known in the art. For transmucosal administration, for example, bile salts and fusidic acid derivatives. Surface active agents can be used to enhance penetration. Transmucosal administration can be through nasal sprays or suppositories. For example, Sayani and Chien (1996), Crit. Rev.Ther.Drug Carrier Syst.13:85-184.

[0059] The amniotic exosomes of the present invention can deliver exosomes internally over time. The exosomal membranes may also be administered by sustained delivery or sustained release mechanisms, such as amniotic exosomal membranes. Biodegradable microspheres or capsules or other biodegradable materials capable of sustained delivery of the drug. Polymeric structures can be included in the formulations of the present invention (see, for example, Putney and Burk e,(1998), Nat.Biotech.16:153-157).

[0060] In preparing the pharmaceutical compositions of the present invention, various formulation techniques may be used and manipulated. The in vivo distribution can be altered. Many methods for altering in vivo distribution are known to those skilled in the art. Examples of such methods include, for example, the use of proteins, lipids (e.g., liposomes), charcoal, etc. Protection of exosomes in vesicles composed of materials such as hydrates or synthetic polymers has been demonstrated. For a general discussion of pharmacokinetics, see, for example, Remington's Please refer to.

[0061] The pharmaceutical composition of the present invention can be administered in various unit dosage forms depending on the method of administration. Such dosages are usually advisory in nature and are adjusted according to the specific therapeutic context. The amount of amniotic exosomes adequate to achieve this is defined as an "effective amount." The dosage schedule and effective amount, i.e., "dosing regimen," for a disease or condition stage, severity of the disease or condition, the general state of the patient's health, the patient's physical condition, age, medications This will depend on a variety of factors, including the formulation and concentration, or choice of amniotic exosomes. In formulating a dosage regimen for a patient, the mode of administration is also taken into consideration. The rate of clearance of the drug composition must also be taken into consideration. Egleton and Davis, (1997), Peptides, 18 :1431-1439;Langer,(1990),Science,249:152 See, e.g., 7-1533. In one embodiment, between 0.05 μg and 100 μg increments. amniotic exosomes will be administered. This includes 0.1 μg to 50 μg and 0.1 μg to 2 0 μg and any amount therebetween.

[0062] According to these methods, amniotic exosomes or pharmaceutical compositions comprising same are administered to one or more other It may also be co-administered in combination with an agent. References herein to "co-administered" , simultaneous administration via the same or different routes in the same formulation or in two different formulations; or sequential administration by the same or different routes. The reference to refers to a time difference of seconds, minutes, hours or days between the administration of amniotic exosomes and another agent. Co-administration may occur in any order. Examples of agents that may be co-administered include Generally, the choice of another agent is based on the disease or condition being treated. become.

[0063] Alternatively, targeting therapies can be used to target cells with targeting molecules such as antibodies or cell-specific ligands. The amniotic exosomes can be delivered to a cell type or location in the body using a dispensing system. For example, targeting to promote localized treatment at the site of need may be beneficial for various reasons. This may be desirable for some reasons.

[0064] Further taught herein is the production of amniotic exosomes. This may be in the form of a bioreactor, a batch reactor, or a continuous flow reactor. Generally, amniotic epithelial cells are immortalized and cultured in a bioreactor. The resulting conditioned medium is then collected and used to inoculate the amniotic membrane exosomes. The compound may be isolated and formulated for immediate use or may be stored, e.g., lyophilized, for later use. Preserved by drying.

[0065] Kits are also contemplated herein. Kits may be for therapeutic or diagnostic purposes. Therapeutic kits may include a selected batch of lyophilized amniotic exosomes and one or more and / or other pharmaceutically acceptable carriers, excipients and / or diluents, and / or other active agents. The diagnostic kit may also be used to determine the proteomic profile or may include reagents for determining the genetic profile. [Example]

[0066] Embodiments taught herein will now be further described by the following non-limiting examples.

[0067] Example 1: Production of amniotic exosomes We developed a protocol to isolate amniotic exosomes (Figure 1). This is the first description of axosomes and validation of their biological activity. The cells were cultured in serum-free medium (Ultraculture media, Lonza) for 96 hours. Afterwards, cells were removed and exosomes were isolated via serial ultracentrifugation at 110,000 g. Process conditioned medium for approximately 100% of hAECs per million, regardless of gestational age. 1.5-2 μg of purified exosomes are consistently produced. This can be scaled up in bioreactor-type cultures without the need for further purification.

[0068] The ability of amniotic exosomes to exert a similar effect was investigated. Dose-effect (0.1 μg vs. 1 μg) inhibited T cell proliferation to the same extent as hAEC-conditioned medium Depletion of exosomes from hAEC conditioned medium (ExDCM) abolished this effect. This suggests that amniotic exosomes are a key mediator of T cell suppression. This indicates that amniotic exosomes directly enhance the phagocytic activity of macrophages. These findings suggest that the immunomodulatory effects of hAEC conditioned medium are due to exosomes. This shows that this is largely due to

[0069] Example 2: Activity of amniotic exosomes We determined whether amniotic exosomes are functional in vivo. Amniotic exosome 1 An aliquot of 1 μg was injected intravenously into BPD mice on postnatal day 4, and tissue analysis was performed on postnatal day 14. The ratio of alveolar to airspace was evaluated. Amniotic exosomes were effective in restoring alveolar simplification. Amniotic membrane exosomes were effective in resolving inflammation in BPD mice while inhibiting alveolar mononucleosis (Fig. 3). By reducing pulmonary fibrosis and mobilizing endogenous stem cells, they may prevent deleterious changes to lung structure. It plays a major role in preventing or reversing the process.

[0070] In summary, the data demonstrate that amniotic exosomes mediate host immune events and immune responses in a manner similar to parental cells. Amniotic membrane exosomes regulate lung repair and promote the in vivo regeneration of hAECs. It has been proposed that amniotic exosomes can recapitulate the functions of amniotic membrane exosomes. By characterizing the cargoes of these molecules, we can use them to explore a wide range of immunomodulatory and It can also have the effect of promoting recovery.

[0071] Using a mouse model of BPD, neonatal administration of amniotic exosomes treated with hAECs resulted in the development of Restoring lung structure in BPD mice to levels comparable to those of non-BPD animals and promoting the proliferation of lung stem cells It is possible to activate the microenvironment and regulate inflammation, which may contribute to long-term physiological It has further been determined that this treatment will result in improvements in beneficial outcomes (e.g., pulmonary hypertension and pulmonary function). The proteome and mRNA / miRNA content of amniotic exosomes were analyzed. Identify specific pathways involved in hAEC-mediated repair.

[0072] Example 3: Restoration effect of amniotic exosomes in BPD mice The data demonstrate that amniotic exosomes have immunomodulatory and pro-regenerative effects in vitro and in vivo. This shows that amniotic exosomes play a key role in cell crosstalk during repair. To understand how this influences the circulation of hAECs in animal models of BPD, To determine whether amniotic exosomes alone are sufficient to summarize the therapeutic effects, Two doses (1 μg and 10 μg) of amniotic exosomes were administered to the optimized dose of EC. The effects are compared using fibroblasts and fibroblast exosomes as controls.

[0073] We use a mouse model of BPD that identifies two major contributors to human BPD: Combined perinatal inflammation and postnatal hyperoxia) for lung repair at term and term pregnancy To evaluate the effects of amniotic exosomes in rodents on complex diseases such as BPD. While there are limitations to the modeling, this model is amenable to detailed molecular analysis. Similar studies will provide insight into dose-effect assessments and long-term studies investigating adolescent and adult outcomes. Briefly, microfabricated glass needles (inner diameter: 70-80 μm) are used. Using a microinjector (IM-300, Narashige), mouse embryos at E16 were Inject 0.2 µg of lipopolysaccharide (LPS) in 5 µL of saline into each amniotic sac in the rabbit. Once born, the newborn pups and nursing mothers were placed in a hyperoxic (65% oxygen) chamber or Change nursing mothers every 48 hours to prevent oxygen toxicity. This combination of prenatal inflammation and postnatal hyperoxia causes lung damage similar to human BPD. (Vosdoganes et al. (2013), Cytotherapy, 15:102 1-1029; Nold et al. Proc. Natl. Acad. Sci. USA. 110: 14384-14389). Treatment is given on postnatal day 4. Intra-amniotic LPS injection at E16. , Experimental timeline showing exosome / cell injection and sacrifice time points at postnatal day 4 ( ×) is shown in Figure 4.

[0074] The same equipment as described for intra-amniotic injection and a wider glass needle (inner diameter: 100- Inject exosomes or cells intravenously via the superficial temporal vein using a 120 μm catheter. The final injection volume was 10 μL, which was well tolerated by 4-day-old mice. Mouse pups were cultured at postnatal days 7 and 14 to assess pulmonary stem cell recruitment and lung repair. After weaning, two cohorts of animals were then transferred to room air and aged 4 weeks and 12 months. At 10 weeks of age, long-term outcomes, such as pulmonary hypertension and circulatory To assess the effects of neonatal treatments on cardiovascular and respiratory function.

[0075] hAECs are isolated from human pregnant women at term (37-40 weeks). hAECs derived from six donors were pooled equally and used for all animal experiments. Animals receiving hAECs are cultured at 100,000 cells on postnatal day 4 to provide a homogenous population. For amniotic exosomes, one of the pooled hAECs was injected. The portion was cultured in culture medium (10 million cells per 25 mL, Ultraculture media, The cells are then placed in a conditioned medium (Lonza) for 96 hours. Exosomes are then isolated from the conditioned medium. The exosomal properties of the pellet were examined by analyzing exosome markers (TSG101 and Alix) and size and differentiation were confirmed by electron microscopy. The exosomes were resuspended in saline and administered at either 1 μg or 10 μg on postnatal day 4. It is administered at either dose.

[0076] Human lung fibroblasts do not support lung repair and are suitable as a control cell type (Mood ley et al. (2010), Am.J.Respir.Crit.Care Medi.: 643-651). Human lung fibroblasts obtained using the same culture protocol as above and fibroblast exosomes. Fibroblasts were administered at the same dose as hAECs. Fibroblast exosomes were administered at a high dose (10 μg). The experimental groups are listed in Table 1. do.

[0077] [Table 1]

[0078] Immune changes Lungs were harvested and analyzed as previously described (Nold et al. (2013) supra; Tan et al. (2015) supra). ), Stem Cell Res. Ther. 6:8) for flow cytometry. The CD45+ fraction was selected and stained for a combination of surface markers and intracellular cytokines. Using a combination of T cells (CD3, CD4, CD25, IFNγ, IL-4, IL17A, F oxP3), macrophages (CD11b, F4 / 80, CD86, MHCII), neutrophils Number of lymphocytes (CD11c, Ly6G), B cells (B220) and NK cells (NK1.1), Table Changes in the expression and activation status of the bronchial alveolar lavage fluid were collected and analyzed as previously described. (Nold et al. (2013) supra) Cytokine changes are measured using a CT scan (D Systems).

[0079] Mobilization of lung stem / progenitor cells CD45- / CD31- / Sca-1+ / EpCam+ (Lee et al. (2014), C BAS was performed by flow sorting based on the criteria of This measures changes to the CD4 C population of cells derived from the immune cell studies described above. Use the 5+ fraction. AT2 is CD31- / Sca1- / autofluorescence 高 Flow sorting of Single-cell digital PCR (Fluidigm, qdPCR 3 7K) to determine differences in transcriptional profiles. Cell lysis, RNA isolation, and pre-amplification and a 96-well microfluidic plate (C1 single cell) where conversion to cDNA will occur. Single flow-sorted samples were collected using the Autoprep System (Fluidigm). The cells are captured. The sample is then loaded onto a microfluidic card for digital PCR. Analyze the data using the NGuLAR v2.0 analysis toolset. BASC and AT Since the microenvironment activation pathway of 2 has not been fully described, customized 48:4 The 8deltaGene assay identified the recently described BMP1 / NFATc1 / thrombin gene. Stem cell pluripotency, activation, and motility, including the bospondin-1 axis (Lee et al. (2014) supra) Covers personnel and differentiation.

[0080] Simplification of alveoli Quantitative image analysis was performed to measure tissue:airspace ratios and to determine the proportion of alveolar units across all experimental groups. Determine the degree of purification.

[0081] Activation of the host stem cell microenvironment Immunohistochemical staining (SPC+CC10+) was performed for BASC in the terminal bronchioles. The activation state of the pulmonary stem cell microenvironment (Lee et al. (2014) supra) is determined.

[0082] The goal is to determine whether changes to lung structure and recruitment of endogenous pulmonary stem cells result in long-term improvements in lung function and secondary outcomes. The goal is to find out whether this extends to a reduction in subsequent complications.

[0083] Physiological testing Rescued adolescent (4-week-old) and young adult (10-week-old) mice were tested for pulmonary function and cardiac function. An ultrasound examination is performed.

[0084] Echocardiography Mice were anesthetized with 3% isoflurane, followed by 1-2% isoflurane, at 350-450 bpm. Vevo 2100 Ultrasound (Monash Bioimaging) and The lung was measured along the left parasternal long axis plane angled anteriorly using a 40 MHz linear transducer. PW Doppler measurement of arterial acceleration time was performed. M-mode was applied along the right parasternal long axis. The thickness of the right ventricular wall was measured by measuring the thickness of the right ventricular wall. These include in-line ultrasonic nebulizers, ventilators, and connected Connected to a pressure transducer (FlexiVent, SCIREQ, Montreal, Canada) The tracheotomy was performed with an 18G cannula connected to the trachea. Airway resistance and compliance increased gradually. by exposing mice to methacholine (1–30 mg / mL, 3 min per cycle) Evaluate by obtaining forced expiratory volume, vital capacity, and deep expiratory volume. Obtain unrestricted whole-body plethysmography. Unlike recording methods, this does not require training of the animal and allows for short interruptions in ventilation. and then performs a measurement operation during which a predefined pressure or volume waveform is measured. This can lead to problems in plethysmography such as excessive dead space and measurement inaccuracies. Overcome traditional challenges you face.

[0085] Amniotic membrane exosomes induce macrophage polarization in BPD mice. beneficial effects in its ability to induce the expression of g and reduce the activation of neutrophils and dendritic cells It has been proposed that immunological changes may be due to the effects of 1 μg of control hAECs. It has been proposed that a 10 μg dose of amniotic exosomes may be more significant. Thus, reversal of alveolar simplification was observed in animals receiving high doses of amniotic exosomes. This can translate into improvements in long-term physiological outcomes, resulting in dose-dependent, right-handed There will be a reduction in ventricular wall thickening, improvement in pulmonary hypertension and restoration of normal lung function. No changes are expected when C or amniotic exosomes are administered to healthy mice. Fibroblast exosomes have effects on immune cells, lung repair, or long-term physiological outcomes. It is not proposed that

[0086] Example 4: Unique mediators in amniotic exosomes The gestational age of the hAEC donor may have a significant impact on its recovery potential (L Im et al. (2013), Placenta, 34:486-492). Comparisons will be made between cargoes in exosomes recovered from pre- and post-transplant hAECs. Amniotic exosomes derived from term and preterm donors were administered, and alveolar simplification was observed. Amniotic exosomes derived from full-term donors were only restored in animals that received them. Therefore, the ability to activate immunoregulatory and regenerative pathways is important for amniotic exosomes prior to term pregnancy. The initial presence / absence proteome analysis shows that the expression of β-glucan is significantly impaired in the β-glucan-containing cytosol. When performed with exosome cargo, it was shown to be effective for term and preterm donors. 242 and 21 unique proteins were identified, respectively. Using this method, full-term amniotic exosomes have been shown to be effective in wound healing, apoptosis, vascular development, acute inflammation, and and epithelial cell development-related mediators of cell signaling. It can be seen.

[0087] For proteome analysis, amniotic exosomes (term and preterm; per group) were used. In-solution trypsin digestion of the samples (n = 10) was performed, followed by liquid chromatography and absolute Mass spectrometry for quantitative quantification (WEHI Proteomics Laboratory) , Melbourne, Australia). Data was collected using Nano ACQUITY A Nano-ESI source (Proxeo) was coupled to a UPLC system (Waters). Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer Adapted for n) The peak list is obtained for each LC-MS / MS run on a single MASCOT. The proteins are fused and searched against the human Ref-Seq protein database (1% False discovery rate). Pipeline Pilot (Accelrys) and Spotfire Analyze quantitative proteomics data using TIBCO. Wilcoxon Signed-rank tests are used to assess differences in abundance. We classify proteins based on their function and subcellular localization, and identify proteins involved in wound healing, cell survival, and immune regulation. Identify genes involved in node.

[0088] For nucleic acid analysis, we use Massive Analysis of cDNA Ends (MACE). Digital gene expression profiling was performed using GenXpro GmbH. This allows for the identification of, for example, receptors and transcription factors that are typically missing from microarrays. Approximately 20 times deeper than conventional RNASeq (1-20 copies per million transcripts) MACE allows the capture and quantification of transcripts from exosomes. It is optimized for sequencing of RNA and miRNA, and each cDNA molecule is tagged. It combines the benefits of qPCR and RNASeq by Identify alternative polyadenylation sequences that affect iRNA interactions and therefore transcript stability Determine gender and biological relatedness. Gene Ontology enrichment analysis for pairwise comparisons and and gene set enrichment analysis.

[0089] There are unique molecular signatures between term and preterm amniotic exosomes, which These are associated with pro-restorative and regenerative effects.

[0090] Example 5: Pro-regenerative effect The pro-regenerative effects of amniotic exosomes were demonstrated in a neonatal mouse model of bronchopulmonary dysplasia. Following administration of exosomes derived from amniotic tissue at term or preterm, alveolar pull The term "BPD" refers to the bronchopulmonary dysplasia mouse model. It means deru animals.

[0091] Example 6: Mechanism of action of exosomes Human exosomes derived from full-term pregnancy were cultured in human amniotic epithelial cells for their ability to induce lung regeneration. The results are shown in Figures 13A-C. As can be seen in the darkly stained (elastin-positive) tip in Figure 13, the secondary septal ridge (se ptal crests).

[0092] In addition, Figure 14 shows that amniotic exosomes induce a response of endogenous stem cells in the lung. In fact, amniotic exosomes were more than two-fold more effective than hAECs.

[0093] It was also observed that amniotic exosomes directly stimulated enhanced proliferation of exogenous lung stem cells. This was shown to be able to increase the number of alveoli and airways exposed to exosomes compared to controls. Occurred in bronchial and mixed lung tissue.

[0094] Example 7: Exosomes are anti-fibrotic in the liver Adult mice aged 8-12 weeks were cultured using intraperitoneal injections of carbon tetrachloride three times a week for 12 weeks. Liver fibrosis was induced using a steroid injection. At week 8, exosomes (1 μg) were injected twice a week. Shown in Figures 15A and B. Sirius Red assay and alpha-smooth muscle actin (SMA) expression The assay was used to determine the biological fibrotic properties of αSMA. αSMA expression was determined using standard assays, and positive results for Sirius Red and αSMA were obtained. The area of ​​inflammation was measured per field of view. The inflammatory macrophage protein CCL4 was used. CCL4+ exosomes significantly increased per field compared to CCL4+ saline controls. This resulted in fewer fibrotic cells (Fig. 15A and B).

[0095] Example 8: Proteomic cargo Figure 16 shows the proteome integration between term exosomes and preterm hAECs. The results show that the loads of term hAECs are similar compared to preterm hAECs. There was even greater variation among the proteome cargo. The proteins examined are listed in Table 2a and A comparison of useful cellular components between hAECs and total MSCs is shown in Figure 17. Figure 18 also compares the biological processes between hAECs and total MSCs.

[0096] [Table 2a]

[0097] [Table 2b]

[0098] Example 9: Exosomes promote myelination Examining amniotic exosomes in animal models of multiple sclerosis. Exosomes promote remyelination. and promotes the development of multiple sclerosis, as well as optic neuritis, Devic's disease, transverse myelitis, acute myelitis, Disseminated encephalomyelitis, and other conditions such as adrenoleukodystrophy and adrenomyeloneuropathy It is expected to be useful in treating the condition.

[0099] Example 10: Activity of exosomes Exosomes isolated from conditioned medium of human amniotic epithelial cells exert immunomodulatory and regenerative effects Amniotic exosomes contain high levels of HLA-G (among other factors) do.

[0100] The immunosuppressive efficacy of amniotic exosomes corresponds to the gestational age of the donor. In response to the donor potential associated with the This has been previously published.

[0101] Amniotic membrane exosomes reverse lung injury in a neonatal mouse model of bronchopulmonary dysplasia. Intravenously injected exosomes significantly improved the tissue:airspace ratio compared to saline. Consistent with our in vitro findings, full-term amniotic exosomes were associated with BPD. It was superior to pre-term exosomes in its ability to reduce lung injury. The results are shown in Figure 5. Amniotic exosomes have been proposed to be useful for the treatment of pulmonary fibrosis and fibrosis in other organs. It is proposed.

[0102] Amniotic exosomes were established in a mouse model of bleomycin-induced pulmonary fibrosis. Amniotic exosomes reversed lung inflammation and fibrosis 7 days after bleomycin challenge. Intravenous administration significantly increased the proportion of activated myofibroblasts (α-smooth muscle actin positive) in the lungs. This was consistent with a reduction in collagen deposition in the lungs. The results are shown in Figure 6. vinegar.

[0103] Amniotic membrane exosomes directly reverse the activation of primary human lung fibroblasts in vitro. 5 When cultured in the presence of 100 ng / mL transforming growth factor β, amniotic exosomes exhibited a The results are shown in Figure 7.

[0104] As shown in Figure 8, amniotic exosomes are involved in cytokine-cytokine receptor signaling. Contains miRNAs that target a pathway, where a yellow box indicates one or more miRNAs. Indicates the target by.

[0105] As shown in Figure 9, amniotic exosomes express miRNPs that target the Wnt signaling pathway. A, where yellow boxes indicate targets by one or more miRNAs.

[0106] As shown in Figure 10, amniotic exosomes target the PI3K-Akt signaling pathway. The yellow boxes indicate targets by one or more miRNAs. vinegar.

[0107] As shown in Figure 11, amniotic exosomes target the TGFβ signaling pathway. Contains RNA, where yellow boxes indicate targets by one or more miRNAs.

[0108] Amniotic exosomes are at least as effective as AECs, such as hAECs. , which induces cellular and molecular repair mechanisms in various physiological and neural processes. It is clear that they have great capabilities.

[0109] Those skilled in the art will appreciate that the present disclosure described herein may be modified in ways other than those specifically described. It will be fully understood that the present disclosure is susceptible to such changes and modifications. It should be understood that all of the above is intended to be illustrative and not restrictive. individually or collectively enabling the steps, features, compositions and compounds described or shown; and Any combination and combination of any two or more of the steps or features or compositions or compounds Makes everything possible.

[0110] Literature information Anderson et al. (2016) Stem Cells. http: / / doi. org / 10.1002 / stem.2298 Doyle et al. (2006) Paediatrics 118: 108-113 Egleton and Davis (1997) Peptides 18:1431-1 439 Fishback et al. (2013) Sci Transl Med 5:179 ps 7) Hodges et al. (2012) Am J Obstet Gynerol 206: 448e8-448e15 Langer (1990) Science 249:1527-1533 Lee et al. (2014) Cell 156:440-455 Lim et al. (2013) Placenta 34: 486-492 Lodha et al. (2014) PLoS ONE: e90843 Putney and Burke (1998) Nat Biotech 16:153- 157 Remington's Pharmaceutical Sciences (199 0), 18th Edition, Mack Publishing Compan y, Easton, PA. Moodley et al. (2010) Am J Respir Crit Care Me di:643-651 Murphy et al. (2012) Cell Transplant 21:1477-1 492 Nold et al. Proc. Natl Acad. Sci USA 110:14384 -14389 Schellenberg et al. (2011) Aging (Albany NY) 3 :873-888 Sayani and Chien (1996) Crit Rev Ther Drug Carrier Syst 13:85-184 Tan et al. (2015) Stem Cell Res Ther 6:8 Vosdoganes et al. (2013) Cytotherapy 15:1021-1 029 Yawno et al. (2013) Dev Neurosci 35:272-282

Claims

1. 1. A pharmaceutical composition for reducing or inhibiting T cell proliferation and / or increasing macrophage phagocytosis in a mammalian subject, comprising:

1. A pharmaceutical composition comprising mammalian amniotic exosomes, and one or more pharmaceutically acceptable carriers, excipients and / or diluents.

2. The pharmaceutical composition of claim 1, wherein the mammalian amniotic exosomes are human amniotic exosomes.

3. The pharmaceutical composition of claim 1 or 2, wherein the mammalian amniotic exosomes are derived from allogeneic mammalian amniotic epithelial cells derived from a donor mammal of the same species.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the mammalian subject to be treated is a human.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the mammalian subject to be treated is a non-human mammal.

6. The pharmaceutical composition of claims 1 to 3, wherein the mammalian subject to be treated is a racing animal selected from the group consisting of horses, dogs and camels.

7. 6. The pharmaceutical composition of claim 4 or 5, wherein the subject is in need of cellular or neuronal repair, regeneration or restoration of the central nervous system, peripheral nervous system, or systemic vasculature, or wound healing.

8. 6. The pharmaceutical composition of claim 4 or 5, wherein the subject has a neurodegenerative disease or condition.

9. 9. The pharmaceutical composition of claim 8, wherein the neurodegenerative disease or condition is a demyelinating disease.

10. 10. The pharmaceutical composition according to claim 9, wherein the demyelinating disease is multiple sclerosis, optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis, adrenoleukodystrophy or adrenomyeloneuropathy.

11. 9. The pharmaceutical composition of claim 8, wherein the neurodegenerative disease or condition is selected from the group consisting of motor neuron disease, stroke, spinal cord injury, traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the amniotic exosomes contain miRNAs that target cytokine-cytokine receptor, Wnt, PI3K-Akt, and TGFβ signaling pathways.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the amniotic exosomes are derived from a bank of immortalized mammalian amniotic epithelial cell lines.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the amniotic exosomes are selected from a bank of lyophilized amniotic exosomes derived from immortalized mammalian amniotic exosomes.

15. 16. Use of mammalian amniotic exosomes in the manufacture of a medicament for reducing or inhibiting T cell proliferation and / or increasing macrophage phagocytosis in a mammalian subject.

16. The use of claim 15, wherein the mammalian amniotic exosomes are human amniotic exosomes.

17. The use of claim 15, wherein the subject has a neurodegenerative disease or condition.

18. The use described in claim 17, wherein the neurodegenerative disease or neurodegenerative condition is a demyelinating disease.

19. The use of claim 15, wherein the subject is in need of cellular or neuronal repair, regeneration or restoration of the central nervous system, peripheral nervous system, or systemic vasculature, or wound healing.

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