Pharmaceutical composition for preventing and treating necrotizing enterocolitis comprising extracellular vesicles derived from thrombin-treated mesenchymal stem cells

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating necrotizing enterocolitis, comprising extracellular vesicles derived from thrombin-treated mesenchymal stem cells. The pharmaceutical composition of the present invention can be expected to prevent and treat necrotizing enterocolitis, including neonatal necrotizing enterocolitis (NEC).

Description

Pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin.

[0002] Necrotizing enterocolitis (NEC) is a common, intractable disease that primarily affects premature infants. It is characterized by the necrosis of the small and large intestine, leading to the development of necrosis. It occurs in approximately 10% of extremely premature infants, and even with treatment, the mortality rate reaches 25%, making it a serious and intractable condition. However, the cause of the disease, as well as effective prevention and treatment methods, remain elusive. While the cause is currently believed to be immaturity, little research has been conducted on a suitable disease model for developing therapeutics.

[0003] All cells must exchange information with their surroundings and other cells in order to survive. To exchange information, cells secrete various substances outside the cells. In addition to soluble factors such as cytokines, hormones, and neurotransmitters, extracellular vesicles, also known as exosomes, have recently been attracting attention as a new type of intercellular information exchanger.

[0004] Extracellular vesicles (EVs) are microscopic particles ranging from several nm to several μm in size secreted from cells or cells into which genetic material has been introduced, and are surrounded by a lipid bilayer. Recently, various studies have been conducted on the important functions of extracellular vesicles.

[0005] Secretion of extracellular vesicles is an evolutionarily conserved phenomenon across all living systems, from bacteria to archaea to eukaryotes.

[0006] In particular, extracellular vesicles, which contain DNA, RNA, functional proteins, and antigens, are used as a new method of intracellular communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Because they are fundamentally cell-derived, extracellular vesicles are biocompatible, unlike other nanoparticles. Furthermore, they can be loaded with or labeled with drugs or biologically active ingredients inside or on their surface. Therefore, ongoing attempts are being made to use them as drug delivery vehicles or raw materials for cosmetics and pharmaceuticals.

[0007] However, development is difficult due to uneconomical productivity (high cost, low yield) and lack of efficient therapeutic agent loading technology, and it is difficult to diversify the administration route of extracellular vesicles, and especially, it is difficult to ensure the stability and safety of the substance when administered orally.

[0008] Viruses, lipid nanoparticles, and virus-like particles (VLPs) have been developed as delivery vehicles for gene therapy. However, existing virus-based delivery systems have side effects, such as reduced efficacy due to immune responses to various viruses. Lipid nanoparticles, as synthetic materials, can cause immune responses with repeated administration. Furthermore, naked gene delivery systems have very low delivery efficiency, limiting their use as therapeutics.

[0009] To solve the above problems, the present inventors developed a pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, which comprises extracellular vesicles derived from mesenchymal stem cells treated with thrombin, for the treatment of necrotizing enterocolitis.

[0010] To improve these problems, the inventors conducted extensive research and found that:

[0011] A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin was designed.

[0012] Accordingly, the present invention aims to provide a pharmaceutical composition for preventing and treating necrotizing enterocolitis, which comprises extracellular vesicles derived from mesenchymal stem cells treated with thrombin.

[0013] In order to achieve the above purpose, the present invention

[0014] A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis is provided, comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin.

[0015] In one embodiment of the present invention, the necrotizing enterocolitis may be necrotizing enterocolitis (NEC).

[0016] In another embodiment of the present invention, the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0017] In another embodiment of the present invention, the composition may further comprise a pharmaceutically acceptable carrier or excipient.

[0018] In another embodiment of the present invention, the composition may be formulated for intravenous or intraperitoneal administration.

[0019] In another embodiment of the present invention, the composition can reduce inflammatory cytokines IL-1α (Interleukin-1α) and TNF-α (Tumor Necrosis Factor-α).

[0020] In another embodiment of the present invention, the composition can increase the tight junction protein ZO-1 (Zonula Occluden protein 1).

[0021] In another embodiment of the present invention, the composition can increase the tight junction protein CLDN1 (Claudin-1).

[0022] In another embodiment of the present invention, the composition can restore diseased tissue to normal tissue.

[0023] In another embodiment of the present invention, the composition can restore mucin secretory cells and intestinal stem cells.

[0024] In another embodiment of the present invention, the recovery of the intestinal stem cells may be achieved by increasing the expression of one or more selected from the group consisting of OLFM4 (Olfactomedin 4) and LGR5 (Leucine-rich repeat-containing G-protein-coupled receptor 5).

[0025] In addition, the present invention provides a use of extracellular vesicles derived from mesenchymal stem cells treated with thrombin for the prevention or treatment of necrotizing enterocolitis.

[0026] In addition, the present invention provides a method for preventing or treating necrotizing enterocolitis, comprising a step of administering extracellular vesicles derived from mesenchymal stem cells treated with thrombin to a subject in need thereof.

[0027] By means of a pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin of the present invention, prevention and treatment of necrotizing enterocolitis, including necrotizing enterocolitis (NEC), can be expected.

[0028] Figure 1 shows the results of comparing and confirming the anti-inflammatory effect of thMSC-EVs by measuring the degree of reduction in the level of inflammatory cytokines by treating thMSC-EVs and naive MSC-EVs in an LPS-induced macrophage model.

[0029] Figure 2 is a schematic diagram illustrating the process of establishing a triple hit enteritis model of NEC using the IEC-6 mouse intestinal epithelial cell line.

[0030] Figure 3 shows the results of confirming the therapeutic effect of thMSC-EVs on necrotizing enterocolitis by measuring cell viability and the expression level of ZO-1, a tight junction protein, by treating thMSC-EVs in a triple hit enterocolitis model.

[0031] Figure 4 shows the results of visually confirming the therapeutic effect of thMSC-EVs in an organoid disease model through morphological observation of organoids after treating thMSC-EVs in organoids derived from neonatal necrotizing enterocolitis tissue.

[0032] Figure 5 shows the results of confirming the therapeutic effect of thMSC-EVs in an organoid disease model at the molecular level by measuring the relative expression level of the mRNA of Cldn1, a tight junction protein, after treating thMSC-EVs in organoids derived from neonatal necrotizing enterocolitis tissue.

[0033] Figure 6 is a graph measuring clinical disease scores, tissue damage, apoptosis, expression levels of intestinal epithelial markers (OLFM4) and stem cell markers (LGR5) after treatment with thMSC-EVs in a neonatal necrotizing enterocolitis mouse model.

[0034] Hereinafter, the present invention will be described in detail.

[0035] The present invention provides a pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin.

[0036] The above term "thrombin" is a proteolytic enzyme involved in blood coagulation, which changes fibrinogen into fibrin when blood vessels are damaged or bleeding occurs, thereby coagulating the blood.

[0037] The above term "mesenchymal stem cell (MSC)" refers to stem cells that exist in cartilage, bone tissue, adipose tissue, and the stroma of bone marrow, differentiated from the mesoderm formed by the division of a fertilized egg. In this case, "mesenchymal stem cell" may be a mesenchymal stem cell derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, placenta, and other tissues, but is not limited thereto.

[0038] The above term "extracellular vesicle (EV)" refers to a substance surrounded by a lipid bilayer and is a microscopic particle ranging from several nm to several μm in size secreted from a cell or a cell into which genetic material has been introduced. Recently, various studies have been conducted on the important functions of extracellular vesicles.

[0039] Secretion of extracellular vesicles is an evolutionarily conserved phenomenon across all living systems, from bacteria to archaea to eukaryotes.

[0040] In particular, extracellular vesicles, which contain DNA, RNA, functional proteins, and antigens, are used as a new method of intracellular communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Because they are fundamentally cell-derived, extracellular vesicles are biocompatible, unlike other nanoparticles. Furthermore, they can be loaded with or labeled with drugs or biologically active ingredients inside or on their surface. Therefore, ongoing attempts are being made to use them as drug delivery vehicles or raw materials for cosmetics and pharmaceuticals.

[0041] The above term “prevention” may mean any act of inhibiting or delaying the onset of necrotizing enterocolitis by administering the pharmaceutical composition according to the present invention.

[0042] The term "treatment" above may mean any action that improves or beneficially changes the symptoms of necrotizing enterocolitis in an individual by administering a pharmaceutical composition according to the present invention.

[0043] In one embodiment of the present invention, the necrotizing enterocolitis may be necrotizing enterocolitis (NEC).

[0044] The term "necrotizing enterocolitis (NEC)" refers to a disease that primarily occurs in premature infants, characterized by necrosis of the small and large intestine mucosa. It is a representative intractable disease. It occurs in approximately 10% of extremely premature infants, and even with treatment, it is a severe and intractable disease with a mortality rate of as high as 25%.

[0045] In another embodiment of the present invention, the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0046] The above term "umbilical cord" refers to the baby's umbilical cord.

[0047] The term "umbilical cord blood" refers to blood collected from the umbilical vein, which connects the placenta and the fetus. Umbilical cord blood is a natural byproduct of childbirth and is much easier to collect than bone marrow and other mesenchymal tissues, which require multiple surgeries. Furthermore, compared to bone marrow transplants, the cord blood storage industry is active and the infrastructure is already in place, making it easier to find donors. Furthermore, cord blood-derived cells do not express HLA-DR (class II), the most important cause of rejection in tissue and organ transplants. Therefore, they can minimize or eliminate immune responses, such as rejection, that have plagued conventional transplants.

[0048] The term "bone marrow" refers to a flexible tissue located in the inner space of the bone, and is also a hematopoietic organ that produces most of the blood in adults. Bone marrow is divided into red bone marrow and white bone marrow according to the ratio of its constituent cells. Red bone marrow is composed mostly of hematopoietic cells, and white bone marrow is composed mostly of adipose tissue.

[0049] The term "Fat" is one of the three major nutrients, along with carbohydrates and proteins. Fat is mainly composed of carbon and hydrogen atoms, so it is hydrophobic, soluble in organic solvents, and insoluble in water. Fat is one of the types of lipids, along with phospholipids and cholesterol.

[0050] The term "muscle" refers to both tendons and flesh, and is the organ responsible for animal movement. Functionally, it is divided into voluntary skeletal muscle and involuntary visceral muscle, and structurally, it is divided into striated and smooth muscle. Muscle is divided into three types: skeletal muscle, cardiac muscle, and smooth muscle, based on morphology, cell signaling pathways, the way contractile force changes, contraction patterns (rhythmic or differential), and the role of the nervous system in muscle function.

[0051] The term "nerve" refers to the organ that allows organisms to sense and respond to their surrounding environment and stimuli. It constitutes the nervous system, particularly the peripheral nervous system, and consists of multiple axons of nerve cells bundled together. Because a single nerve primarily consists of axons originating from adjacent nerve cells, it often transmits only specific information.

[0052] The term "skin" refers to the largest tissue in the integumentary system, composed of numerous epithelial tissues that protect the muscles and organs within the body. When exposed to the external environment, the skin plays a crucial role in protecting the body from pathogens. Other key functions of the skin include insulation, temperature regulation, sensory functions, and the synthesis of vitamin D and the protection of vitamin B folates.

[0053] The term "amnion" refers to the membrane that covers the embryo, covering its outer surface within the uterus. The amniotic sac is filled with a fluid called amniotic fluid, which expands the amniotic sac to form a sac called the amniotic sac, providing a protective environment for the embryo as it grows.

[0054] The term "placenta" refers to a reproductive organ in mammals that transfers maternal nutrients to the fetus and fetal waste products to the mother during development. The placenta is formed through the differentiation and division of embryonic trophectoderm implanted in the maternal endometrium, the subsequent differentiation of endometrial basal cells and vascular cells (endocytes), and the growth of spiral arteries.

[0055] In another embodiment of the present invention, the composition may further comprise a pharmaceutically acceptable carrier or excipient.

[0056] The above term "pharmaceutically acceptable carrier" is one commonly used in formulations, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other conventional additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated using the methods disclosed in Remington's literature.

[0057] The term "pharmaceutically acceptable excipient" above refers to any substance that is used as a carrier or medium for delivery of the pharmaceutical composition according to the present invention, or added to the formulation to improve handling or storage properties, or to facilitate the manufacture of the dosage unit formulation into articles such as capsules or tablets suitable for oral administration. Excipients may include, but are not limited to, diluents, disintegrants, binders, adhesives, wetting agents, lubricants, glidants, fragrances, surfactants, and inclusion compounds.

[0058] In another embodiment of the present invention, the composition may be formulated for intravenous or intraperitoneal administration.

[0059] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dosage level can be determined according to factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0060] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs.

[0061] In another embodiment of the present invention, the composition can reduce inflammatory cytokines IL-1α (Interleukin-1α) and TNF-α (Tumor Necrosis Factor-α).

[0062] The term "cytokine" refers to a relatively small immune protein found in the blood. It is a broad, loosely packed protein secreted by immune cells and plays a crucial role in cell signaling. After being secreted, cytokines can affect other cells or the cells themselves.

[0063] In another embodiment of the present invention, the composition can increase the tight junction protein ZO-1 (Zonula Occluden protein 1).

[0064] The term "tight junction" refers to one of the intercellular junctions observed in the human epidermis. Located in the intercellular spaces of the epidermal granular cell layer, it connects adjacent cells and performs various biological functions, including a barrier function that regulates the movement of electrolytes and water. Occludin, a representative protein component of these tight junctions, is also known to have a high correlation in its expression level with the epidermal biological functionality of the tight junction.

[0065] In another embodiment of the present invention, the composition can increase the tight junction protein CLDN1 (Claudin-1).

[0066] In another embodiment of the present invention, the composition can restore diseased tissue to normal tissue.

[0067] In another embodiment of the present invention, the composition can restore mucin secretory cells and intestinal stem cells.

[0068] In another embodiment of the present invention, the recovery of the intestinal stem cells may be achieved by increasing the expression of one or more selected from the group consisting of OLFM4 (Olfactomedin 4) and LGR5 (Leucine-rich repeat-containing G-protein-coupled receptor 5).

[0069] In addition, the present invention provides a use of extracellular vesicles derived from mesenchymal stem cells treated with thrombin for the prevention or treatment of necrotizing enterocolitis.

[0070] In addition, the present invention provides a method for preventing or treating necrotizing enterocolitis, comprising a step of administering extracellular vesicles derived from mesenchymal stem cells treated with thrombin to a subject in need thereof.

[0071]

[0072] To facilitate understanding of the present invention, the following examples will be described in more detail. However, these examples are intended only to exemplify the content of the present invention and are not intended to limit the scope of the present invention. These examples are provided to more fully explain the present invention to those with average knowledge in the technical field to which the invention pertains.

[0073]

[0074] [Example]

[0075] Example 1. Comparison of the anti-inflammatory effects of thMSC-EVs and naive MSC-EVs.

[0076] Peritoneal macrophages were isolated from mouse pups, and primary cultured peritoneal macrophages were treated with Lipopolysaccharide (LPS) in serum-free culture medium. LPS-induced peritoneal macrophages were co-cultured with Dulbecco's Phosphate-Buffered Saline (DPBS), thrombin-treated Mesenchymal Stem Cell-derived Extracellular Vesicles (thMSC-EVs), or naïve MSC-EVs for 24 h. The levels of inflammatory cytokines Interleukin-1α (IL-1α) and Tumor Necrosis Factor-α (TNF-α) in the cell culture medium were measured using a commercial ELISA kit according to the manufacturer's recommendations.

[0077] As a result, as shown in Fig. 1, when peritoneal macrophages were activated with LPS, the levels of inflammatory cytokines IL-1α and TNF-α were significantly increased compared to the normal control group (Normal ctrl), confirming that the activation of peritoneal macrophages is associated with inflammatory bowel disease. In addition, in the LPS-induced macrophage model, when extracellular vesicles derived from untreated mesenchymal stem cells (naive MSC-EVs) and extracellular vesicles derived from thrombin-treated mesenchymal stem cells (thMSC-EVs) were treated, the levels of inflammatory cytokines were significantly reduced.

[0078] Among them, it was confirmed that the effect of reducing the level of inflammatory cytokines when treated with extracellular vesicles derived from thrombin-treated mesenchymal stem cells (thMSC-EVs) was significantly superior to the effect of reducing the level of inflammatory cytokines when treated with extracellular vesicles derived from untreated mesenchymal stem cells (naive MSC-EVs).

[0079] These results confirmed that the anti-inflammatory effect of extracellular vesicles derived from thrombin-treated mesenchymal stem cells (thMSC-EVs) was superior to that of extracellular vesicles derived from untreated mesenchymal stem cells (naive MSC-EVs).

[0080]

[0081] Example 2. Confirmation of the therapeutic effect of thMSC-EVs on necrotizing enterocolitis (NEC, triple hit model)

[0082] Necrotizing enterocolitis (NEC) is a serious, multifactorial disease primarily affecting premature infants. As shown in Figure 2, a triple-hit NEC enteritis model was established by exposing the IEC-6 rat intestinal epithelial cell line to three conditions: hyperosmotic pressure, oxygen-glucose deprivation, and hypothermic stress.

[0083] These IEC-6 cells were co-cultured with extracellular vesicles derived from mesenchymal stem cells (thMSC-EVs) treated with Dulbecco's Phosphate-Buffered Saline (DPBS) or thrombin as a control. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, and the expression level of the tight junction protein ZO-1 (Zonula Occluden protein 1) was quantified by normalization to GAPDH, a loading control.

[0084] As a result, as shown in Fig. 3, compared to the general enteritis control group (CON), the cell viability and the expression level of ZO-1, a tight junction protein, of the triple hit enteritis in vitro model (Triple Hit) decreased, but the cell viability and the expression level of ZO-1, a tight junction protein, of the group treated with extracellular vesicles derived from mesenchymal stem cells (thMSC-EVs) treated with thrombin (Triple Hit + EV) of the triple hit enteritis in vitro model significantly increased.

[0085] Among the components of the intestinal barrier, tight junctions are one of the dynamic structures that regulate the permeability of the interstitial pathway, and considering that patients with inflammatory bowel disease (IBD) such as Crohn's disease have destroyed tight junction structures, which increases the permeability, these results confirm that thrombin-treated mesenchymal stem cell-derived extracellular vesicles (thMSC-EVs) have an excellent intestinal inflammation treatment effect through increased expression levels of ZO-1, a tight junction protein.

[0086]

[0087] Example 3. Evaluation of efficacy in an organoid model of neonatal necrotizing enterocolitis.

[0088] Organoids were generated from necrotizing enterocolitis tissue obtained during intestinal resection in premature infants. These organoids were co-cultured with thrombin-treated mesenchymal stem cell-derived extracellular vesicles (thMSC-EVs) at a 1:10 dilution. The normal and abnormal morphologies of the organoids were observed microscopically. The relative mRNA levels of Cldn1 (Claudin-1), a tight junction protein, were compared in organoids derived from untreated or EV-treated intestinal tissue.

[0089] As a result, as shown in Fig. 4, it was confirmed that the number of organoids forming a morphology close to normal (recovering to normal state) significantly increased in the group (EV) treated with extracellular vesicles derived from mesenchymal stem cells (thMSC-EVs) treated with thrombin compared to the untreated group (NT).

[0090] In addition, as shown in Fig. 5, it was confirmed that the expression level of Cldn1, which is important for cell survival and tissue composition, significantly increased in the group treated with extracellular vesicles derived from thrombin-treated mesenchymal stem cells (thMSC-EVs).

[0091] These results morphologically and molecularly confirm that thrombin-treated mesenchymal stem cell-derived extracellular vesicles (thMSC-EVs) have an excellent therapeutic effect on neonatal necrotizing enterocolitis in an organoid disease model.

[0092]

[0093] Example 4. Tissue protective effect of th.MSC-EVs in an in vivo NEC model.

[0094] Postnatal day 4 (P4) outbred mouse pups from the Institute of Cancer Research (ICR) strain were used as an in vivo NEC model, weighing 2.5–3 g. A total of 60 g of lipopolysaccharide (LPS) was orally administered to the pups using a 10 μL pipette. Hyperosmolar formula was prepared by adding powdered formula to Esbilac liquid puppy milk replacer. 100 μL of hyperosmolar formula per 3 g of body weight was gavage-fed to the pups using a 1-French G polyurethane catheter. Mice fed formula were exposed to 4°C for 10 minutes to induce hypothermia, and then exposed to 5% O2 and 95% N2 gas in a chamber for 20 minutes to induce hypoxia. Then, for 3 consecutive days immediately after hypoxic exposure, 100 μL of phosphate-buffered saline (PBS) or 2 × 10 10 Dogs were administered intraperitoneally (IP) with thMSC-EVs. Three cycles of gavage, hypoxia, and hypothermia were performed daily at 4-hour intervals. Clinical disease scores were measured before sacrifice. The obtained intestinal tissue was embedded in paraffin, sectioned, and stained to assess apoptosis, goblet cells, and the amount of intestinal stem cells.

[0095] As a result, as shown in Fig. 6, compared to the NEC control group, the clinical sickness score, tissue disease score (Histological Damage Score), and number of tissue cell death (TUNEL (+) cells) of animals in the extracellular vesicle-treated group (NEC+thEV) derived from mesenchymal stem cells treated with thrombin were significantly reduced, and it was confirmed that the cells that secrete mucin, which acts as the first defense barrier of the intestinal epithelium, and the intestinal stem cell markers OLFM4 (Olfactomedin 4) and LGR5 (Leucine-rich repeat-containing G-protein-coupled receptor 5) involved in the regeneration of the intestinal epithelium were significantly increased.

[0096] These results suggest that thrombin-treated mesenchymal stem cell-derived extracellular vesicles (thMSC-EVs) have the effect of promoting protection and regeneration of intestinal tissue.

[0097]

[0098] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, comprising extracellular vesicles derived from mesenchymal stem cells treated with thrombin.

2. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the necrotizing enterocolitis is neonatal necrotizing enterocolitis (NEC).

3. In claim 1, A pharmaceutical composition for preventing and treating necrotizing enterocolitis, characterized in that the mesenchymal stem cells are derived from at least one tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

4. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition further comprises a pharmaceutically acceptable carrier or excipient.

5. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition is formulated for intravenous or intraperitoneal administration.

6. In claim 1, The above composition is a pharmaceutical composition for preventing and treating necrotizing enterocolitis, characterized in that it reduces inflammatory cytokines IL-1α (Interleukin-1α) and TNF-α (Tumor Necrosis Factor-α).

7. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition increases the tight junction protein ZO-1 (Zonula Occluden protein 1).

8. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition increases the tight junction protein CLDN1 (Claudin-1).

9. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition restores diseased tissue to normal tissue.

10. In claim 1, A pharmaceutical composition for the prevention and treatment of necrotizing enterocolitis, characterized in that the composition restores mucin secretory cells and intestinal stem cells.

11. In claim 10, A pharmaceutical composition for preventing and treating necrotizing enterocolitis, characterized in that the recovery of the intestinal stem cells is characterized by increased expression of one or more selected from the group consisting of OLFM4 (Olfactomedin 4) and LGR5 (Leucine-rich repeat-containing G-protein-coupled receptor 5).

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