Artificial platelet and use thereof

Abstract

The present invention relates to an artificial platelet comprising: a wound-site targeting module; a fibrinogen-binding module; and a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin. The modules may be mass-produced via a simple production process in the form of a recombinant protein-based fusion protein, and, unlike conventional technologies that assist platelet functions, may replace the core functions of platelets in recognizing a wound and forming a fibrin network at a wound site, and accordingly, hemostasis may be achieved via an independent mechanism regardless of platelet production or destruction, and thus, the artificial platelet may be used as a novel type of artificial platelet or bleeding inhibitor that is not inhibited by an antiplatelet agent.

Description

Artificial Platelets and Their Uses

[0001] The present invention relates to artificial platelets and their uses.

[0002] Blood is a bodily fluid that plays a role in supplying oxygen and nutrients to cells and collecting and transporting carbon dioxide and waste products generated by cellular metabolism. Blood is released outside the blood vessels due to trauma or internal bleeding within the vessels; this process is called hemorrhage. When bleeding occurs, a hemostatic process takes place to prevent blood loss. Hemostatic refers to the process where, when a blood vessel is injured and blood comes into contact with foreign substances, various blood clotting factors present in platelets or plasma undergo complex pathways to form a blood clot, thereby stopping the bleeding at the wound site. The hemostatic process is broadly classified into primary and secondary hemostatics. When bleeding occurs, blood flow is first obstructed by vasoconstriction, and with the help of vWF (von Willebrand factor), platelets attach to the inner wall of the blood vessel and aggregate to form a primary platelet plug; this is called primary hemostatics. At the same time, the successive activation of various types of blood coagulation factors produces insoluble fibrin, and ultimately, a secondary hemostatic plug is formed, leading to secondary hemostasis. The representative mechanism of action of the blood coagulation factors involved in the secondary hemostasis process is that when a blood vessel is damaged, tissue factor (TF), which is present outside the blood vessel, reacts with inactive prothrombin present in the plasma along with Factor VII to convert prothrombin into active thrombin. Subsequently, the active thrombin catalyzes the reaction of hydrolyzing soluble fibrinogen in the blood to convert it into insoluble fibrin, and as this fibrin clumps together to more firmly secure the platelet plug, the hemostasis process that stops bleeding is achieved.

[0003] Hemostasis methods include mechanical, cold / hot, and chemical methods. The most basic mechanical method involves applying direct pressure to the bleeding site using gauze or cotton; for bleeding caused by lacerations or cuts, instruments such as sutures, clamps, and clips can be used to stop the bleeding; and for bone hemorrhages, bone wax can be used. Cold / hot methods include cold compresses, which lower body temperature to restrict blood flow to the bleeding site, and electrocautery, a type of electrosurgical device that uses high-frequency electrical energy to apply heat to tissues to coagulate and stop bleeding. Representative chemical methods include hemostatic agents. Hemostatic agents are broadly classified into astringents, which suppress direct bleeding through an astringent action that forms a film when applied to the skin or mucous membranes; coagulant, which promotes blood coagulation by using various factors involved in the blood coagulation process as drugs when bleeding occurs; antifibrinolytics, which help stop bleeding by preventing the breakdown of fibrin produced during the blood coagulation process; and local hemostatic agents, which help stop bleeding quickly by directly absorbing blood lost due to bleeding and increasing the concentration of blood cells present in the blood.

[0004] The object of the present invention is to provide a fusion protein that can be used as an artificial platelet or a recombinant vector encoding the same.

[0005] In addition, the objective of the present invention is to provide a hemostatic composition.

[0006] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bleeding.

[0007] In addition, the objective of the present invention is to provide a method for preventing or treating bleeding.

[0008] To achieve the above objective, the present invention provides a fusion protein comprising: a wound site targeting module; a fibrinogen binding module; and a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin.

[0009] In addition, the present invention provides a recombinant vector encoding the fusion protein.

[0010] In addition, the present invention provides a hemostatic composition comprising the fusion protein or recombinant vector.

[0011] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of bleeding comprising the fusion protein or recombinant vector.

[0012] In addition, the present invention provides a method for preventing or treating bleeding, comprising the step of administering the above pharmaceutical composition to an individual.

[0013] The present invention relates to an artificial platelet comprising: a wound site targeting module; a fibrinogen binding module; and a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin. The modules can be mass-produced through a simple production process in the form of recombinant protein-based fusion proteins. Unlike conventional technologies that assist platelet function, the modules can replace the core functions of platelets, such as wound recognition and forming a fibrin network at the wound site. Therefore, hemostasis is possible through an independent mechanism unrelated to the production or destruction of platelets, and the artificial platelet can be utilized as a new type of artificial platelet or hemorrhage inhibitor that is not inhibited by antiplatelet agents.

[0014] Figure 1 is a figure showing the peptide structure of the fusion protein (SP-FA-Apple1-linker-vWFA1-Fc) of the present invention.

[0015] FIG. 2 is a schematic diagram illustrating the hemorrhage-inhibiting action of the fusion protein of the present invention.

[0016] Figures 3 and 4 show the purification results of the fusion protein hFA-PT-A1 of the present invention.

[0017] Figure 5 is a figure showing the purification results of the fusion protein mFA-PT-A1 of the present invention.

[0018] Figure 6 is a schematic diagram of the module combination of scFv-based fusion proteins.

[0019] Figure 7 shows the purification results of scFv-based fusion protein #2-6-7.

[0020] Figure 8 is a figure showing the purification results of the fusion protein mFA-PT-A1 of the present invention.

[0021] Figure 9 is a figure showing the collagen, thrombin, and fibrinogen binding ability of the fusion protein hFA-PT-A1 of the present invention.

[0022] Figure 10 is a figure showing the collagen, thrombin, and fibrinogen binding ability of the fusion protein mFA-PT-A1 of the present invention.

[0023] Figure 11 shows the prothrombin and fibrinogen binding ability (binding affinity) of three scFv-based fusion proteins (#10-6-7, #2-6-7, #11-6-7).

[0024] Figure 12 shows the collagen, prothrombin, and fibrinogen binding ability of scFv-based fusion protein #2-6-7.

[0025] Figure 13 shows the collagen, prothrombin, and fibrinogen binding ability of scFv-based fusion protein #17-6-7.

[0026] Figure 14 shows the results of analyzing the artificial platelet effect of the hFA-PT-A1 fusion protein of the present invention in normal platelet mice and thrombocytopenia mice (Anti-CD41) trauma models.

[0027] Figure 15 is a figure confirming the artificial platelet effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention in an animal model of thrombocytopenia in which a liver incision was applied.

[0028] Figure 16 shows the results of confirming the artificial platelet effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention by immunofluorescence staining in an animal model of thrombocytopenia with liver resection.

[0029] Figure 17 is a figure confirming the artificial platelet effect of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein of the present invention on thrombocytopenia through colon occult blood.

[0030] Figure 18 is a figure confirming the artificial platelet effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention after producing a colon occult blood model by applying a chemotherapy to an animal model of thrombocytopenia.

[0031] Figure 19 is a figure confirming the blood half-life of the hFA-PT-A1 fusion protein of the present invention in a mouse.

[0032] Figure 20 is a figure confirming the effect of the mFA-PT-A1 and #17-6-7 fusion proteins of the present invention on the formation of a fibrin network structure.

[0033] Figure 21 is a figure confirming the effect of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutant of the present invention on the formation of a fibrin network structure.

[0034] Figure 22 is a figure confirming the area of ​​fibrin network formation of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutant of the present invention.

[0035] Figures 23 and 24 show the alpha-thrombin (α-Thrombin) binding ability of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutants of the present invention.

[0036] Figure 25 is a figure confirming the change in bleeding amount when the mFA-PT-A1 and #17-6-7 fusion protein mutant of the present invention is applied.

[0037] Figure 26 is a figure showing the results of the wound site targeting analysis of the mFA-PT-A1 and #17-6-7 fusion protein mutants of the present invention.

[0038] Figure 27 is a figure showing the final hemostasis time by tracking the change in the bleeding area at the liver incision site over time when the sodium alginate gel mixed with the mFA-PT-A1 and #17-6-7 fusion proteins of the present invention was applied to an animal model of thrombocytopenia and an animal model with a liver incision.

[0039] Figure 28 is a figure confirming the artificial platelet effect when a sodium alginate gel mixed with the mFA-PT-A1 and #17-6-7 fusion proteins of the present invention is applied to an animal model of thrombocytopenia and an animal model with liver resection.

[0040] Figure 29 is a figure showing the degree of accumulation of artificial platelets in each organ by detecting the fluorescence signal of the NIR750 dye in each organ after injecting the mFA-PT-A1 and #17-6-7 fusion proteins of the present invention into a mouse labeled with NIR750 dye and extracting the heart, lungs, liver, and kidneys after 24 or 48 hours.

[0041] Figure 31 is a figure showing whether microthrombi were formed in lung tissue by injecting the mFA-PT-A1, #17-6-7 fusion protein, mucin, and Fc control of the present invention into the tail vein of a mouse.

[0042] Figure 32 is a figure showing the number and distribution of thrombi in lung tissue using frozen thin sections and immunofluorescence staining techniques after injecting the mFA-PT-A1, #17-6-7 fusion protein, mucin, and Fc control of the present invention into the tail vein of a mouse.

[0043] Figure 33 is a figure showing the analysis of microthrombi formation and immunological changes in a mouse model when the mFA-PT-A1, #17-6-7 fusion protein and Fc control of the present invention were administered multiple times by injecting them into the tail vein a total of three times at 4-day intervals.

[0044] Figure 34 is a figure showing the trend of immune cell differentiation and the ratio of T cells, B cells, and inflammatory cells to total immune cells in a mouse model when the mFA-PT-A1, #17-6-7 fusion protein and Fc control of the present invention were administered multiple times by injecting them into the tail vein a total of three times at 4-day intervals.

[0045] Figure 35 is a figure showing the increase or decrease in immune cells in the glomeruli of the kidneys of a mouse model using a histosaturation technique when the mFA-PT-A1, #17-6-7 fusion protein and Fc control of the present invention were administered a total of three times by injecting them into the tail vein at 4-day intervals.

[0046] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0047] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0048] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention.

[0049] Throughout this specification, not only are conventional one- and three-character codes for naturally occurring amino acids used, but generally accepted three-character codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc., are also used. Additionally, amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB nomenclature as follows:

[0050] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

[0051]

[0052] In one aspect, the present invention relates to a fusion protein comprising (1) a wound site targeting module; (2) a fibrinogen binding module; and (3) a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin.

[0053] In one embodiment, the wound site targeting module may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

[0054] In one embodiment, the fibrinogen binding module may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 4 to 6.

[0055] In one embodiment, a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 7 to 12.

[0056] In one embodiment, the fusion protein may further include a signal peptide (SP), which may be included at the N-terminus of the fusion protein for the production of the fusion protein in mammalian cells, and the signal peptide may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 13 to 15.

[0057] In one embodiment, the fusion protein may additionally include an Fc domain, which may be included at the C-terminus of the fusion protein for the stability or multimer formation of the fusion protein, and the Fc domain may include the amino acid sequence of SEQ ID NO. 16.

[0058] In one embodiment, the fusion protein may further include a linker, the linker may be located between a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin and prothrombin and a wound site targeting module, and may include the amino acid sequence of GSGSG (SEQ ID NO. 24).

[0059] In one embodiment, the fusion protein is combined in the order of a fibrinogen binding module; a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin; and a wound site targeting module,

[0060] It may be combined in the order of a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin; a wound site targeting module; and a fibrinogen binding module, but is not limited thereto.

[0061] In one embodiment, the fusion protein may further comprise a labeling module and may comprise one or more selected from the group consisting of chromogenic enzymes, radioisotopes, chromophores, luminescent substances, and fluorescent substances, and the fluorescent substance may be a cyanine (Cy) series, rhodamine series, Alexa series, BODIPY series, or ROX series fluorescent substance, and rhodamine, cyanine 3, cyanine 5, pyrene, cyanine 2, green fluorescent protein (GFP;Green Fluorescent Protein), red fluorescent protein (RFP), Calcein, Fluorescein Isothiocyanate (FITC), Alexa 488, 6-Carboxy-Fluorescein (FAM), 2',4',5',7'-Tetrachloro-6-Carboxy-4,7-Dichlorofluorescein (HEX), 2',7'-Dichloro-6-Carboxy-4,7-Dichlorofluorescein (TET), Fluorescein Chlorotriazinyl, Fluorescein, Oregon Green, Magnesium Green, Calcium Green, 6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein (JOE), It may be tetramethylrhodamine, tetramethyl-rhodamine isothiocyanate (TRITC), carboxytetramethylrhodamine (TAMRA), rhodamine phalloidin, pyronin Y, lissamine, ROX (X-rhodamine), calcium crimson, Texas Red, Nile Red, malachite green, thiadicarbocyanine, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), or To1-biotin.

[0062] In one embodiment, the fusion protein may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 17 to 22.

[0063] In one embodiment, the fusion protein may include a variant thereof or an analog thereof.

[0064] In the present invention, the terms "peptide," "polypeptide," and "protein" may be used interchangeably.

[0065] The term "peptide" as used in the present invention refers to an amino acid polymer that may include not only natural amino acids but also non-protein amino acids as components.

[0066] As used in the present invention, the term "variant" refers to a corresponding amino acid sequence comprising at least one amino acid difference (substitution, insertion, or deletion) when compared to a reference material. In certain embodiments, the "variant" has high amino acid sequence homology and / or conservative amino acid substitution, deletion, and / or insertion when compared to the reference sequence.

[0067] As used in the present invention, the term "analogous" may include an analog in which one or more other functional groups are substituted for the side chain or alpha-amino acid backbone of an amino acid. Examples of side chain or backbone modified peptide analogs include, but are not limited to, hydroxyproline in which a pyrrolidine ring is substituted with a hydroxyl group, or N-methylglycine "peptoid." Types of protein analogs are known in the art.

[0068] The peptide / protein variant according to the present invention is interpreted to include variants in which an amino acid residue is conservatively substituted at a specific amino acid residue position.

[0069] In the present invention, "conservative substitution" means a modification of a variant comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of biological or biochemical function of the peptide / protein variant. "Conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. A class of amino acid residues having a similar side chain is defined in the art and is well known. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0070] The fusion protein according to the present invention may be produced by a standard synthesis method, a recombinant expression system, or any other method in the art. Accordingly, the fusion protein according to the present invention may be synthesized by a number of methods, including, for example, a method comprising the following:

[0071] (a) a method for synthesizing a protein stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and separating and purifying the final protein product; or

[0072] (b) a method of expressing a nucleic acid construct encoding a protein in a host cell and recovering the expression product from a host cell culture; or

[0073] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a protein and recovering the expression product; or

[0074] A method for obtaining a protein fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a protein, and recovering the protein.

[0075] In one aspect, the present invention relates to a nucleic acid encoding a fusion protein of the present invention or a recombinant vector comprising said nucleic acid.

[0076] In one embodiment, the recombinant vector may include the nucleotide sequence of SEQ ID NO. 23.

[0077] In one aspect, the present invention relates to a hemostatic composition comprising a fusion protein of the present invention, a variant thereof or an analog thereof, or a nucleic acid encoding the same or a recombinant vector comprising said nucleic acid.

[0078] In one embodiment, the hemostatic composition may be an artificial platelet.

[0079] In one embodiment, the hemostatic composition comprises collagen, gelatin, cellulose, chitosan, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, agarose, alginate, polysaccharide, starch, carboxymethyl cellulose, calcium salt, thrombin, prothrombin, prethrombin, fibrinogen, fibrin, fibronectin, heparinase, factor X / Xa, factor VII / VIIa, factor IX / IXa, factor XI / XIa, factor XII / XIIa, tissue factor, It may additionally include batroxobin, ancrod, ecarin, von Willebrand factor, albumin, platelet surface glycoprotein, vasopressin, vasopressin analog, epinephrine, selectin, coagulant-promoting toxin, plasminogen activator inhibitor, platelet activator, synthetic peptide having hemostatic activity, chlorhexidine gluconate (CHG), triclosan, silver, a diluent, a saline solution, or a combination thereof.

[0080] In one embodiment, the hemostatic composition may be in the form of powder, nanofiber, paste, gel, foam, liquid, or spray.

[0081] In the present invention, the term "hydrogel" refers to a hydrophilic network structure material in which polymer chains form a three-dimensional structure. Through a sol-gel phase transition, a liquid in which water is used as a dispersion medium solidifies, loses its fluidity, and forms a porous structure. Hydrogels become transparent as they swell through the absorption of hydrophilic components, including water, in an aqueous solution, and also possess suitable mechanical properties. This degree of swelling and the mechanical properties of the polymer depend mainly on the properties of the polymer network structure. Polymers are primarily used as materials to produce such hydrogels, and these polymers are classified into natural polymers and synthetic polymers depending on the origin of the material.

[0082] In the present invention, the term "biodegradable polymer" refers to a polymer that spontaneously and gradually degrades in vivo after a certain period and possesses one or more characteristics among biocompatibility, hemoaffinity, anti-calcification properties, cellular nutrient component, and intercellular matrix formation ability. The types of such biodegradable polymers are not limited to, but include fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone) (PL), polyanhydride, polyorthoester, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), and poly-N-isopropylacrylamide (PNIPAAm). Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, copolymers thereof, mixtures thereof, etc. are included.

[0083] In one aspect, the present invention relates to a hemostatic material in which the hemostatic composition of the present invention is covalently fixed to a non-colloidal porous dressing material.

[0084] In one embodiment, the hemostatic material may be a sponge, fabric, non-woven fabric, pre-formed shape, particulate material, granular material, or sheet.

[0085] In one aspect, the present invention relates to artificial blood comprising a fusion protein of the present invention.

[0086] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of bleeding comprising a fusion protein of the present invention, a variant thereof or an analog thereof, or a nucleic acid encoding said fusion protein or a recombinant vector comprising said nucleic acid.

[0087] In one embodiment, the bleeding may be internal bleeding, bleeding due to trauma, excessive bleeding, occult blood, bleeding caused by an anticoagulant, antithrombotic agent or antiplatelet agent, bleeding due to a blood coagulation disorder-related disease, or bleeding due to a platelet-related disease.

[0088] In one embodiment, the platelet-related disease may be thrombocytopenia, immune thrombocytopenia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, Bernard-Soulier Syndrome, Glanzmann's thrombasthenia, or chemotherapy-induced thrombocytopenia.

[0089] In one embodiment, the disease associated with the blood coagulation disorder may be an acquired coagulation factor deficiency or a congenital coagulation factor deficiency.

[0090] In one embodiment, the composition of the present invention may additionally include a known hemorrhage treatment agent in addition to the active ingredient and may be used in combination with other known treatments for the treatment of these diseases.

[0091] As used in the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of bleeding by administering the pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of bleeding by administering the composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases to which the composition of the present invention is effective, and to judge the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0092] In the present invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating bleeding, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0093] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, type of bleeding, cause of bleeding, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0094] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0095] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, and an oral or injectable formulation is more preferred.

[0096] As used in the present invention, the term "administration" means providing a specific substance to an individual or patient by any appropriate method. Depending on the intended method, it may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally. The dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectables can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), non-aqueous solvents, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0097] As used in the present invention, the term "individual" refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, in which the hemorrhage has occurred or may occur, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual. The pharmaceutical composition of the present invention may be administered in conjunction with existing therapeutic agents.

[0098] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0099] In one aspect, the present invention relates to a food composition for the prevention or improvement of bleeding comprising genetically modified stem cells or kidney organoids differentiated therefrom, or culture medium thereof or exosomes isolated therefrom.

[0100] In one embodiment, the food-grade acceptable salt may include a salt derived from a food-grade acceptable organic acid, inorganic acid, or base.

[0101] When the composition of the present invention is used as a food composition, the compound may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The composition may include a food-grade additive that is food-gradely acceptable in addition to the active ingredient, and the amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0102] As used in the present invention, the term "food" refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone a certain degree of processing to become edible, and in a conventional sense includes health functional foods, beverages, food additives, beverage additives, etc.

[0103] The term "food auxiliary additive" as used in the present invention refers to a component that can be added to food as an auxiliary component, and is added to manufacture health functional foods of each formulation, and can be appropriately selected and used by a person skilled in the art. Examples of food auxiliary additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food auxiliary additives of the present invention are not limited by the above examples.

[0104] The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the ordinary technical field, and during such manufacturing, raw materials and ingredients commonly added in the ordinary technical field may be added. Furthermore, the formulation of the health functional food may also be manufactured without restriction as long as it is a formulation recognized as a health functional food. The health functional food of the present invention may be consumed as an adjuvant to enhance the effect of a hemorrhage treatment drug.

[0105] In addition, there are no restrictions on the types of health foods to which the composition of the present invention can be used. Furthermore, the composition of the present invention may be prepared by mixing other appropriate auxiliary ingredients and known additives that may be contained in health functional foods, depending on the choice of a person skilled in the art. Examples of foods to which it can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it may be prepared by adding it to juices, teas, jellies, and juices prepared using the extract according to the present invention as the main ingredient.

[0106] As used in the present invention, the term "improvement" refers to any act of reducing parameters related to a target disease, such as the degree of symptoms, by administering a composition according to the present invention.

[0107] In one aspect, the present invention relates to a method for preventing or treating bleeding, comprising the step of administering a pharmaceutical composition for preventing or treating bleeding to an individual.

[0108] The term "individual" as used in this invention refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and includes humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0109] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0110] Example 1. Preparation of artificial platelets

[0111] 1-1. Preparation of FA-PT-A1 Fusion Protein

[0112] 1-1-1. Design of FA-PT-A1 Fusion Protein

[0113] A fusion protein was designed comprising a wound site targeting module containing an A1 domain of vWF (vWFA1, A1), a peptide having the ability to bind to collagen exposed at the wound site (human or mouse vWF A1 domain: SEQ ID NO. 1 or 2), a fibrinogen binding module containing a fibrinogen-specific binding peptide (FA) (human or mouse fibrinogen binding peptide: SEQ ID NO. 4 or 5), and a thrombin binding and activation module containing an Apple1 domain (PT), a thrombin / prethrombin / prothrombin-specific binding peptide (human or mouse Apple1 domain: SEQ ID NO. 7 or 8). In addition, for production in mammalian cells, a signal peptide (SP) (SP of hFA-PT-A1: SEQ ID NO. 13 or SP of mFA-PT-A1: SEQ ID NO. 14) was added to the N-terminus of the fusion protein, and a Fc domain (SEQ ID NO. 16) was added to the C-terminus for purification, stability, and multimer formation to design a fusion protein (hFA-PT-A1 or mFA-PT-A1) (Fig. 1). The A1 of the fusion protein designed in this way specifically binds to collagen exposed at the wound site, and fibrinogen specifically binds to the FA of the fusion protein to localize fibrinogen at the wound site; thrombin binds to the PT of the fusion protein or activates prethrombin into thrombin, and the activated thrombin cleaves the N-terminus of fibrinogen to convert it into fibrin, and the GPR (Gly-Pro-Arg) sequence exposed by the cleavage binds to other fibrinogen / fibrin to form a polymer. At this time, since the artificial platelet exists as a dimer through the Fc site, it performs a cross-linking role between fibrinogen polymers, thereby promoting the formation and stabilization of the fibrinogen network structure (Fig. 2).

[0114]

[0115] 1-1-2. Expression and Purification of hFA-PT-A1 / mFA-PT-A1 Fusion Protein

[0116] DNA encoding the fusion protein designed in Example 1-1-1 above was expressed in mammalian cells and then purified. Specifically, the corresponding fusion protein DNA was transduced into 1 L of culture medium of CHO-S or HEK293-F cell lines using linear PEI and DMSO, and after 24 hours, tryptone was added and cultured for 1 week. Subsequently, the fusion protein in the culture medium was purified by protein A-based affinity chromatography.

[0117] As a result, the purification of the fusion protein hFA-PT-A1 was successfully carried out, and a high-purity fusion protein was obtained in a yield of 7.53 µg per 1 ml of mammalian cell culture medium (Figs. 3 and 4). In addition, the purification of mFA-PT-A1 targeting mice was successfully carried out, and a high-purity fusion protein was obtained in a yield of 4 µg per 1 ml of mammalian cell culture medium (Fig. 5).

[0118]

[0119] 1-2. Preparation of scFv Fusion Proteins

[0120] 1-2-1. Derivation of scFv Fusion Protein

[0121] After screening scFvs that can be used as wound site targeting modules, fibrinogen binding modules, and thrombin binding and activation modules using phage-display library screening, fusion proteins were created by combining each scFv capable of collagen binding, fibrinogen binding, and thrombin binding, and scFv-based fusion proteins were prepared by fusing the Fc domain to the C-terminus.

[0122] As a result of phage-display library screening, collagen-binding scFv was selected as clone #6 (SEQ No. 3), fibrinogen-binding scFv was selected as clone #7 (SEQ No. 6), and prothrombin-binding scFv was selected as four clones (#2, #10, #11 and #17) (SEQ Nos. 9 to 12), and scFv-based fusion proteins #2-6-7, #10-6-7, #11-6-7 and #17-6-7 were derived by combining them (Fig. 6).

[0123]

[0124] 1-2-2. Expression and Purification of scFv Fusion Protein

[0125] DNA encoding scFv-based fusion proteins #2-6-7, #10-6-7, #11-6-7, and #17-6-7, as well as fusion proteins containing an Fc domain at the C-terminus and a TST tag, was prepared and expressed in mammalian cells for purification. Specifically, the corresponding fusion protein DNA was transfected into 1 L of CHO-S or HEK293-F cell culture medium using linear PEI and DMSO, and after 24 hours, tryptone was added and cultured for one week. Subsequently, the fusion proteins in the culture medium were purified using protein A-based affinity chromatography.

[0126] As a result, the purification of scFv-based fusion proteins #2-6-7, #10-6-7, #11-6-7, and #17-6-7 was successfully carried out, and in particular, high-purity fusion protein #2-6-7 was obtained in a yield of 1 µg per 1 ml of mammalian cell culture medium (Fig. 7). In addition, the purification of #17-6-7 targeting mice was successfully carried out, and high-purity fusion protein #17-6-7 was obtained in a yield of 8.2 µg per 1 ml of mammalian cell culture medium (Fig. 8).

[0127]

[0128] Example 2. Analysis of the binding ability of artificial platelets

[0129] 2-1. Analysis of Collagen, Thrombin, and Fibrinogen Binding Ability of FA-PT-A1 Fusion Protein

[0130] The collagen binding ability, thrombin binding ability, and thrombin activity-dependent fibrinogen binding ability of the hFA-PT-A1 fusion protein and mFA-PT-A1 fusion protein purified in Example 1-1 above were analyzed by ELISA. Specifically, three target proteins, prothrombin, collagen, and fibrinogen were each adsorbed onto a 96-well immunoplate and then blocked with an albumin (BSA) solution. FA-PT-A1 fusion protein solutions diluted to different concentrations were added to the corresponding wells to induce binding. After the washing process, the dissociation constant of the FA-PT-A1 fusion protein was calculated by measuring the absorbance at a wavelength of 450 nm resulting from the reaction between the bound FA-PT-A1 fusion protein and protein A, which specifically binds to the human-derived fc domain, and the horseradish peroxidase fused thereto and 3,3',5,5'-tetramethylbenzidine (TMB). For fibrinogen binding ability, the FA-PT-A1 fusion protein was mixed with 1 U / ml of alpha-thrombin (α-thrombin), activated at room temperature for 30 minutes, and then used for ELISA analysis.

[0131] As a result, the FA-PT-A1 fusion protein was found to have binding affinities of 88.3 nM, 13.4 nM, and 223 nM for prothrombin, collagen, and fibrinogen, respectively, confirming that it possesses collagen binding ability, thrombin binding ability, and thrombin activity-dependent fibrinogen binding ability (Fig. 9). In addition, the mFA-PT-A1 fusion protein was found to have binding affinities of 275 pM, 12 pM, and 3.24 nM for prothrombin, collagen, and fibrinogen, respectively, confirming that it possesses collagen binding ability, thrombin binding ability, and thrombin activity-dependent fibrinogen binding ability (Fig. 10).

[0132]

[0133] 2-2. Analysis of Collagen, Thrombin, and Fibrinogen Binding Ability of scFv Fusion Proteins

[0134] The binding ability of the scFv-based fusion proteins #2-6-7, #10-6-7, and #11-6-7 purified in Examples 1-2 above to prothrombin, collagen, and fibrinogen was analyzed by ELISA. Specifically, the three target proteins, prothrombin, collagen, and fibrinogen were each adsorbed onto a 96-well immunoplate and then blocked with albumin (BSA) solution. After adding scFv protein solutions diluted to different concentrations to the corresponding wells to induce binding, the absorbance at a wavelength of 450 nm resulting from the reaction of the bound scFv protein with a human-derived Fc domain-specific antibody or a 2B8-specific antibody fused with horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB) was measured to calculate the dissociation constant of the FA-PT-A1 fusion protein.

[0135] As a result, all three fusion proteins were found to have concentration-dependent binding abilities to prothrombin, collagen, and fibrinogen, with the #2-6-7 fusion protein exhibiting the highest binding affinity for prothrombin (Fig. 11). In particular, the binding affinities of the #2-6-7 fusion protein to prothrombin, collagen, and fibrinogen were confirmed to be 35 nM, 17 nM, and 22 nM, respectively (Fig. 12). Additionally, the binding affinities of the #17-6-7 fusion protein to prothrombin, collagen, and fibrinogen were confirmed to be 25 pM, 19 pM, and 31 pM, respectively (Fig. 13).

[0136]

[0137] Example 3. Analysis of the hemostatic effect of artificial platelets

[0138] 3-1. Hemostatic effect on trauma

[0139] The artificial platelet effect of the FA-PT-A1 fusion protein of the present invention was confirmed in normal platelet mice and thrombocytopenia mice (Anti-CD41) trauma models. Specifically, normal mice and mice in which thrombocytopenia was induced by subcutaneous injection of anti-CD41 24 hours prior to the experiment were each injected with the FA-PT-A1 fusion protein at a dose of 5 mg / kg via the tail vein. One hour later, a 2 mm deep wound was made in the tail vein using a #10 surgical knife, and the incision site was completely immersed in physiological saline maintained at 37°C to collect the lost blood. Subsequently, the collected blood was centrifuged to separate the red blood cell layer, and the total blood loss and bleeding time were analyzed by quantifying hemoglobin (Hb) using Drabkin's reagent.

[0140] As a result, when the FA-PT-A1 fusion protein of the present invention was injected into the tail vein at a dose of 5 mg / kg in a normal platelet mouse group, the average hemorrhage volume decreased by 68.6% from 34,156 Hb nmol to 10,705 Hb nmol compared to the group injected with PBS, and the average hemorrhage time decreased by 22.8% from 92 seconds to 71 seconds. In a model in which thrombocytopenia was induced by anti-CD41, when the FA-PT-A1 fusion protein was injected into the tail vein at a dose of 5 mg / kg, the average hemorrhage volume decreased by 98.6% from 97,801 Hb nmol to 1,376 Hb nmol compared to the group injected with PBS, and the average hemorrhage time decreased by 83.7% from 540 seconds to 88 seconds (Fig. 14).

[0141]

[0142] 3-2. Hemostatic effect on an animal model of thrombocytopenia

[0143] 3-2-1. Hepatectomy Model

[0144] The artificial platelet effect of the FA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention was confirmed in a liver laceration model of a thrombocytopenia animal model. Specifically, FA-PT-A1 fusion protein and #17-6-7 fusion protein were injected into thrombocytopenia mice, in which thrombocytopenia was induced by subcutaneous injection of anti-CD41 24 hours prior to the experiment, via the tail vein at a dose of 5 mg / kg. One hour after the tail vein injection, the mice were placed under inhalation anesthesia, the peritoneum was incised to expose the left lobe of the liver, and a partial incision measuring 3 mm in length and 2 mm in depth was performed using a surgical knife #10. Using sterile gauze weighed in advance, blood loss from the left lobe incision site of the liver was absorbed at intervals of 10, 20, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, 480, 540, and 600 seconds to measure the amount of bleeding over time.

[0145] As a result, compared to the control group, FA-PT-A1 fusion protein and #17-6-7 fusion protein each showed statistical significance in inhibiting hemorrhage volume, and when comparing the total hemorrhage volume at 600 seconds, the average hemorrhage volumes of the control group, human immunoglobulin Fc domain injection / anti-CD41 injection group, FA-PT-A1 fusion protein injection / anti-CD41 injection group, and #17-6-7 fusion protein injection / anti-CD41 injection group were 0.2839, 0.8893, 0.5376, and 0.5003 (mg / g mice), respectively, confirming that FA-PT-A1 fusion protein and #17-6-7 fusion protein reduced the average hemorrhage volume by 41% compared to the control group (Fig. 15). In addition, immunofluorescence staining results showed that a fibrin (green) network was normally formed at the site of the left liver lobe incision in mice with normal platelet counts. Within the group injected subcutaneously with anti-CD41, it was observed that the group of mice injected with human immunoglobulin via tail vein did not form a fibrin (green) network, while the group injected with FA-PT-A1 fusion protein and #17-6-7 fusion protein via tail vein showed the formation of a fibrin (green) network similar to the normal platelet mouse group (Fig. 16). Simultaneously, in the group injected with FA-PT-A1 fusion protein and #17-6-7 fusion protein via tail vein, an anti-human immunoglobulin Fc domain (red) signal was observed at the same location as the fibrin (green) network, confirming that FA-PT-A1 fusion protein and #17-6-7 fusion protein were induced to the site of the left liver lobe incision in mice during the hemorrhage process (Fig. 16).

[0146]

[0147] 3-2-2. Colonic Occult Blood Model

[0148] The artificial platelet effects of the FA-PT-A1 fusion protein and #2-6-7 fusion protein of the present invention on thrombocytopenia were confirmed through colonic occult blood. Specifically, anti-CD41 was subcutaneously injected at 0.75 mg / kg, followed by tail vein injection of the FA-PT-A1 fusion protein and #2-6-7 fusion protein of the present invention and a human immunoglobulin Fc domain as a control at 5 mg / kg, respectively, and colonic occult blood and platelet counts were analyzed one day later. For colonic occult blood, fecal samples were collected 24 hours after intravenous injection of anti-CD41 and fusion proteins. The fecal samples were dissolved in a 0.53 M potassium hydroxide (KOH) solution at 37°C for 30 minutes, then centrifuged to collect the supernatant. 5 μl of the supernatant separated in a 96-well plate was mixed with 95 μl of a luminol solution for hemoglobin detection (0.53 M potassium hydroxide (KOH), 1.5% hydrogen peroxide (H2O2), 0.04% luminol), and the chemiluminescence intensity at a wavelength of 425 nm was measured. The measured chemiluminescence intensity was used to obtain standard curves for human hemoglobin concentration ranges of 0, 8, and 80 μg / ml, and then used to backcalculate the hemoglobin concentration of each fecal sample.

[0149] As a result, it was confirmed that the FA-PT-A1 fusion protein and #2-6-7 fusion protein of the present invention reduced average colonic occult blood by approximately 27.5% and 33.7%, respectively. In addition, when the average platelet count in the blood of the same groups was examined, the platelet counts for the human immunoglobulin Fc domain administration / no anti-CD41 administration group, human immunoglobulin Fc domain administration / anti-CD41 administration group, FA-PT-A1 fusion protein administration / anti-CD41 administration group, and #2-6-7 fusion protein administration / anti-CD41 administration group were 711.1, 32.0, 40.0, and 46.0 x10^3 / μl, respectively, confirming that thrombocytopenia induced by anti-CD41 infusion was induced normally (Fig. 17).

[0150]

[0151] 3-2-3. Chemotherapy-induced colonic occult blood model

[0152] After constructing an occult blood model by applying chemotherapy to an animal model of thrombocytopenia, the artificial platelet effects of the FA-PT-A1 fusion protein and #2-6-7 fusion protein of the present invention were confirmed. Specifically, the anticancer drug carboplatin was injected intraperitoneally at 50 mg / kg for 3 days, and on the 3rd day of the carboplatin intraperitoneal injection, anti-CD41 was injected subcutaneously at 0.75 mg / kg to induce thrombocytopenia in the mice. After 24 hours, 5 mg / kg of the FA-PT-A1 fusion protein or #2-6-7 fusion protein was injected into the tail vein, and the average occult blood and average platelet counts were analyzed after 24 hours.

[0153] As a result, the average colonic occult blood volumes of the human immunoglobulin Fc domain administration / anti-CD41 non-administration group, human immunoglobulin Fc domain administration / anti-CD41 administration group, FA-PT-A1 fusion protein administration / anti-CD41 administration group, and #2-6-7 fusion protein administration / anti-CD41 administration group were 4, 353, 212, and 145 Hb nmol, respectively, and the colonic occult blood volume reduction effects of FA-PT-A1 fusion protein and #2-6-7 fusion protein were confirmed to be 40% and 59%, respectively. In addition, the average platelet count in the blood of the same group was examined, and the platelet counts of the human immunoglobulin Fc domain administration / anti-CD41 non-administration group, human immunoglobulin Fc domain administration / anti-CD41 administration group, FA-PT-A1 fusion protein administration / anti-CD41 administration group, and #2-6-7 fusion protein administration / anti-CD41 administration group were 589, 49.0, 11.3, and 98.33 x10^3 / μl, respectively, confirming that thrombocytopenia induced by anti-CD41 infusion was normal (Fig. 18).

[0154]

[0155] Example 4. Analysis of artificial platelet stability

[0156] To analyze the blood half-life of the FA-PT-A1 fusion protein of the present invention, the fusion protein FA-PT-A1 was injected via the tail vein at a dose of 5 mg / kg. Subsequently, 30 μl of blood was collected from the jugular vein of mice at intervals of 1, 6, 12, 24, 36, 48, and 72 hours, and the plasma was separated by centrifugation at 17,000 xg to exclude blood cells. ELISA analysis was performed on the separated plasma, and it was confirmed that the blood concentration of the fusion protein FA-PT-A1 decreased to 338 nM, which is half of the initial average blood concentration of 675 nM for 5 mg / kg of fusion protein FA-PT-A1, after 41 hours. Through this, it was confirmed that the blood half-life of the fusion protein FA-PT-A1 in mice is 41 hours (Fig. 19).

[0157]

[0158] Example 5. Analysis of the effect on the formation of fibrin network structure of artificial platelets

[0159] To analyze the effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention on the fibrin network structure formation process, 1 U / ml of activated thrombin and 20 nM of mFA-PT-A1 fusion protein and #17-6-7 fusion protein were mixed with 5 mg / ml of fibrinogen solution (10% FITC fluorescently labeled fibrinogen, 90% fibrinogen solution) on a microslide for microscopy. The polymerization reaction of fibrin was induced at room temperature for 2 hours, after which 1 mM of the thrombin inhibitor Dabigatran was added to stop the polymerization reaction. Subsequently, the fibrin network structure was analyzed using a laser scanning microscope at 630x magnification for fluorescence imaging. As a result, it was confirmed that a fibrin network structure of higher density was formed in a sample mixed with mFA-PT-A1 fusion protein and #17-6-7 fusion protein at a concentration of 20 nM (Fig. 20).

[0160]

[0161] Example 6. Analysis of the effect of artificial platelet mutants on fibrin network structure formation

[0162] To analyze the effect of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutants (ΔFg, ΔPT, ΔCol) of the present invention on the formation of a fibrin network structure, fusion protein mutants at concentrations of 3.2 nM and 20 nM, respectively, and 1 U / ml of activated thrombin were mixed with a fibrinogen solution (10% FITC fluorescently labeled fibrinogen, 90% fibrinogen solution) at a concentration of 5 mg / ml on a microslide for microscopy. After inducing the polymerization reaction of fibrin at room temperature for 2 hours, the polymerization reaction was stopped by adding 1 mM of the thrombin inhibitor Dabigatran. Subsequently, the fibrin network structure was analyzed by fluorescence imaging at 630x magnification using a laser scanning microscope. As a result, the fusion protein mutants corresponding to Full length hFA-PT-A1 fusion protein, Full length #2-6-7 fusion protein, hFA-PT-A1_ΔPT, hFA-PT-A1_ΔCol, #2-6-7_ΔPT, and #2-6-7_ΔCol showed increases in the area of ​​the fibrin network structure of 227%, 277%, 157%, 156%, 468%, and 243%, respectively, compared to the control group. In the case of the fusion protein mutant corresponding to ΔFg, hFA-PT-A1_ΔFg, and #2-6-7_ΔFg, the density of the fibrin network structure was -33% and +33%, respectively, compared to the control group, confirming that the change in the area of ​​the fibrin network structure was not significant compared to mutants with fibrinogen binding ability. (Figs. 21, 22)

[0163]

[0164] Example 7. Analysis of Alpha-Thrombin (α-Thrombin) and Collagen Binding Ability of Artificial Platelet Mutants

[0165] To analyze the binding ability of the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutants (ΔFg, ΔPT, ΔCol) of the present invention to activated alpha-thrombin (α-Thrombin), the hFA-PT-A1 fusion protein and #2-6-7 fusion protein mutants (ΔFg, ΔPT, ΔCol) were pre-adsorbed at a concentration of 100 μg / ml onto a collagen type I and III coated - clear bottom 96-well plate. After binding at concentration ranges of 0, 8, 20, 50, 125, 313, and 781 nM for 2 hours at room temperature, 1 U / ml of activated thrombin was mixed in to induce additional binding with the fusion protein for 1 hour at room temperature. Afterward, all wells were washed and Z-GGR-AMC, a substrate of activated alpha-thrombin, was mixed in. The concentration of AMC, the reaction product of alpha-thrombin - Z-GGR-AMC, was measured at 1-minute intervals for 50 minutes using a spectrophotometer set to 37°C. As a result, for the hFA-PT-A1_ΔFg and hFA-PT-A1_ΔCol fusion protein pro-thrombin binding mutants corresponding to the concentration range of 781 nM, the AMC concentration at 2900 seconds decreased slightly to -19% and -25%, respectively, compared to the full-length fusion proteins, but it was confirmed that they possessed pro-thrombin binding ability similar to the full-length fusion proteins. In addition, by observing that no AMC fluorescence was detected for hFA-PT-A1_ΔPT, hFA-PT-A1_ΔCol, #2-6-7__ΔPT, and #2-6-7_ΔCol corresponding to the concentration range of 781 nM, it was confirmed that the prothrombin-binding and collagen-binding mutants lacked prothrombin-binding and collagen-binding abilities, respectively. (Figs. 23 and 24)

[0166]

[0167] Example 8. Analysis of Wound Site Targeting by Artificial Platelet Collagen Binding Ability Mutant (ΔCol)

[0168] The artificial platelet effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein mutant (ΔCol) of the present invention was confirmed in a liver laceration model of a thrombocytopenia animal model. Specifically, the mFA-PT-A1 fusion protein and the #17-6-7 fusion protein collagen binding ability mutant (ΔCol) were injected into thrombocytopenia mice, in which thrombocytopenia was induced by subcutaneous injection of anti-CD41 24 hours prior to the experiment, via the tail vein at a dose of 5 mg / kg. One hour after the tail vein injection, the mice were placed under inhalation anesthesia, the peritoneum was incised to expose the left lobe of the liver, and a partial incision measuring 3 mm in length and 2 mm in depth was performed using a surgical knife #10. Using sterile gauze weighed in advance, blood loss from the left lobe incision site of the liver was absorbed at intervals of 10, 20, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, 480, 540, and 600 seconds to measure the amount of bleeding over time.

[0169] As a result, it was confirmed that compared to the control group, the mFA-PT-A1 fusion protein collagen binding mutant did not show statistical significance in inhibiting hemorrhage, while the #17-6-7 fusion protein collagen binding mutant showed statistical significance in inhibiting hemorrhage. When comparing the total hemorrhage at 600 seconds, the average hemorrhage for the control group, human immunoglobulin Fc domain injection / anti-CD41 injection group, mFA-PT-A1 fusion protein collagen binding mutant injection / anti-CD41 injection group, and #17-6-7 fusion protein collagen binding mutant injection / anti-CD41 injection group was 0.2696, 1.073, 0.9630, and 0.7534 (mg / g mouse), respectively, confirming that the mFA-PT-A1 fusion protein collagen binding mutant and the #17-6-7 fusion protein collagen binding mutant reduced the average hemorrhage by 10% and 29%, respectively, compared to the control group (Fig. 25). In addition, immunofluorescence staining results showed that a fibrin (green) network was normally formed at the site of the left liver lobe incision in mice with normal platelet counts. It was observed that among the group injected subcutaneously with anti-CD41, the group injected with human immunoglobulin via tail vein did not form a fibrin (green) network, and it was confirmed that the formation of the fibrin (green) network was inhibited in the group injected with the mFA-PT-A1 fusion protein and #17-6-7 fusion protein collagen-binding mutant via tail vein, similar to the group injected with human immunoglobulin via tail vein. At the same time, in the group injected with the mFA-PT-A1 fusion protein and #17-6-7 fusion protein via tail vein, no anti-human immunoglobulin Fc domain (red) signal was observed at the same location as the fibrin (green) network, confirming that the mFA-PT-A1 and #17-6-7 fusion protein collagen-binding mutants were not induced to the site of the left liver lobe incision in mice during the hemorrhage process (Fig. 26).

[0170]

[0171] Example 9. Analysis of the hemorrhage reduction effect by applying artificial platelets to the wound site

[0172] The artificial platelet effect of the mFA-PT-A1 fusion protein and #17-6-7 fusion protein of the present invention, mixed into a sodium alginate gel, was confirmed in a liver laceration model of thrombocytopenia. Specifically, thrombocytopenia mice were prepared by inducing thrombocytopenia in normal mice by subcutaneously injecting anti-CD41 24 hours prior to the experiment. After inhalation anesthesia, the peritoneum was incised to expose the left lobe of the liver, and a partial incision measuring 3 mm in length and 2 mm in depth was performed using a #10 surgical knife. For local treatment, FA-PT-A1 or T17-C6-F7 was mixed with 1% (w / v) sodium alginate to adjust the final concentration to 500 nM. Immediately after inducing liver laceration, 100 μL of the pre-mixed hemostatic hydrogel was applied directly to the wound site. To initiate gelation, 4 μL of 50 mM calcium chloride was added and mixed with sodium alginate gel. The liver laceration surgery procedure was videotaped, and time-lapse images were extracted at 30-second intervals (Fig. 27). The time-lapse images were analyzed using image analysis software to track changes in the bleeding area over time and determine the final hemostasis time, thereby statistically analyzing the hemostatic effect on the Fc control group, mFA-PT-A1, and #17-6-7 livers (Fig. 28).

[0173]

[0174] Example 10. Analysis of organ-specific distribution of artificial platelets after injection into an animal model

[0175] The mFA-PT-A1 fusion protein, #17-6-7 fusion protein, and Fc control of the present invention were labeled with the fluorescent dye NIR750. NIR750-labeled-Fc control, NIR750-labeled-mFA-PT-A1, and NIR750-labeled-#17-6-7 were administered to mice via the tail vein at a concentration of 5 mg / kg, and in vivo circulation was induced for 24 and 48 hours. Major organs such as the liver, lungs, heart, and kidneys were excised at predetermined time points and scanned using a biofluorescence analyzer (Fig. 29). Three mice were assigned to each group, and the organ accumulation patterns for each sample were confirmed using representative images. Additionally, the fluorescence intensity accumulated in each excised organ was measured and normalized to the size of the organ to perform statistical quantitative analysis, thereby verifying the in vivo distribution characteristics for each sample (Fig. 30).

[0176]

[0177] Example 11. Analysis of safety and immune response after injection of artificial platelets in an animal model

[0178] The mFA-PT-A1 fusion protein (5 mg / kg), #17-6-7 fusion protein (5 mg / kg), Fc control (5 mg / kg), and mucin (0.5 mg / kg) of the present invention were administered via the tail vein of mice 30 minutes prior to euthanasia. 30 minutes after tail vein administration, lung tissues from mice administered the mFA-PT-A1 fusion protein, #17-6-7 fusion protein, Fc control, and mucin were surgically excised and fixed with 4% paraformaldehyde. The lungs were inflated by injecting a 50% OCT compound (Tissue-Tek) mixed with phosphate-buffered saline (PBS), followed by dehydration in 30% sucrose and embedding in the OCT compound, which was then stored at -80°C. Subsequently, 10 μm thick frozen sections were prepared using a freeze-threader (CM-1850, Leica), and the tissue sections were stained with an anti-CD41 antibody (Fig. 31). As a result of analyzing images obtained using a confocal microscope to measure the CD41-positive area and the number of microthrombi, it was verified that the CD41-positive area and the number of microthrombi in the Fc control group, mFA-PT-A1 fusion protein, and #17-6-7 fusion protein groups were statistically significantly reduced compared to the mucin group, while the mFA-PT-A1 fusion protein and #17-6-7 fusion protein groups did not show a statistically significant difference compared to the Fc control group (Fig. 32).In addition, to verify the safety of multiple intravenous administration of mFA-PT-A1 fusion protein and #17-6-7 fusion protein, mFA-PT-A1 fusion protein (5 mg / kg), #17-6-7 fusion protein (5 mg / kg), and Fc control group (5 mg / kg) were injected into mice via the tail vein at 4-day intervals for a total of 3 times. Blood was collected via the jugular vein on days 2 and 10 of the experiment to track immune cell differentiation and changes in T cells, B cells, and inflammatory cells relative to total immune cells. On day 11 of the experiment, mice were euthanized, and the increase or decrease in immune cells in the glomeruli of the kidneys was verified using histosaturation techniques. As a result of tracking the differentiation and increase / decrease trends of immune cells through blood collection, it was verified that the differentiation ratios and increase / decrease trends of T cells, B cells, and inflammatory cells did not show a statistically significant difference in the mFA-PT-A1 fusion protein and #17-6-7 fusion protein groups compared to the Fc control group (Fig. 34). In addition, microscopic image analysis verified that there was no significant change in the increase or decrease of immune cells in the glomeruli within the kidneys in the mFA-PT-A1 fusion protein and #17-6-7 fusion protein groups compared to the Fc control group (Fig. 35).

Claims

1. (1) Wound site targeting module; (2) Fibrinogen binding module; and (3) A fusion protein comprising a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin and prothrombin.

2. In Paragraph 1, A wound site targeting module is a fusion protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3.

3. In Paragraph 1, The above fibrinogen binding module is a fusion protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 4 to 6.

4. In Paragraph 1, A fusion protein comprising a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin, wherein the binding or activation module comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs 7 to 12.

5. In Paragraph 1, A fusion protein additionally containing a signal peptide (SP).

6. In Paragraph 1, A fusion protein that additionally contains an Fc domain.

7. In Paragraph 1, A fusion protein that additionally includes a linker.

8. In Paragraph 1, The above fusion protein is combined in the order of a fibrinogen binding module; a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin; and a wound site targeting module, A fusion protein that is combined in the order of a binding or activation module for one or more selected from the group consisting of thrombin, prethrombin, and prothrombin; a wound site targeting module; and a fibrinogen binding module.

9. A recombinant vector encoding the fusion protein of claim 1.

10. A hemostatic composition comprising the fusion protein of claim 1 or the recombinant vector of claim 9.

11. In Paragraph 10, Collagen, Gelatin, Cellulose, Chitosan, Matrigel, Methacrylated Gelatin (GelMA), Hyaluronic acid, Agarose, Alginate, Polysaccharide, Starch, Carboxymethyl Cellulose, Calcium Salt, Thrombin, Prothrombin, Prethrombin, Fibrinogen, Fibrin, Fibronectin, Heparinase, Factor X / Xa, Factor VII / VIIa, Factor IX / IXa, Factor XI / XIa, Factor XII / XIIa, Tissue Factor, Batroxobin, A hemostatic composition further comprising ancrod, ecarin, von Willebrand factor, albumin, platelet surface glycoprotein, vasopressin, vasopressin analog, epinephrine, selectin, coagulant-promoting toxin, plasminogen activator inhibitor, platelet activator, synthetic peptide having hemostatic activity, chlorhexidine gluconate (CHG), triclosan, a diluent, a saline solution, or a combination thereof.

12. In Paragraph 10, The above composition is a hemostatic composition in the form of powder, nanofiber, paste, gel, foam, liquid, or spray.

13. A pharmaceutical composition for the prevention or treatment of bleeding comprising the fusion protein of claim 1 or the recombinant vector of claim 9.

14. In Paragraph 13, A pharmaceutical composition for the prevention or treatment of bleeding, wherein the above bleeding is internal bleeding, bleeding caused by trauma, excessive bleeding, occult blood, bleeding caused by anticoagulants, antithrombotic agents or antiplatelet agents, bleeding caused by blood coagulation disorder-related diseases, or bleeding caused by platelet-related diseases.

15. In Paragraph 14, A pharmaceutical composition for the prevention or treatment of bleeding in which platelet-related diseases are thrombocytopenia, immune thrombocytopenia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, Bernard-Soulier syndrome, Glanzmann's thrombasthenia, or chemotherapy-induced thrombocytopenia.

16. In Paragraph 14, A pharmaceutical composition for the prevention or treatment of bleeding, wherein the disease associated with the above-mentioned blood coagulation disorder is an acquired coagulation factor deficiency or a congenital coagulation factor deficiency.

17. A method for preventing or treating bleeding comprising the step of administering the pharmaceutical composition of claim 13 to an individual.

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