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WO2023096330A1 - Composition permettant de favoriser l'angiogenèse comprenant des protéines de fusion de thymosine bêta-4 - Google Patents

Composition permettant de favoriser l'angiogenèse comprenant des protéines de fusion de thymosine bêta-4 Download PDF

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Publication number
WO2023096330A1
WO2023096330A1 PCT/KR2022/018574 KR2022018574W WO2023096330A1 WO 2023096330 A1 WO2023096330 A1 WO 2023096330A1 KR 2022018574 W KR2022018574 W KR 2022018574W WO 2023096330 A1 WO2023096330 A1 WO 2023096330A1
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Prior art keywords
angiogenesis
cell
thymosin
protein
composition
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Korean (ko)
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김정석
백병하
윤석환
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Union Korea Life Sciences Inc
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Union Korea Life Sciences Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2292—Thymosin; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • composition for promoting angiogenesis comprising a fusion protein of thymosin beta 4.
  • TAT peptide known as a protein transducing domain (PTD) has been developed as a carrier peptide for moving a target protein into cells.
  • PTD protein transducing domain
  • composition for promoting angiogenesis comprising a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to a thymosin beta 4 (T ⁇ 4) protein.
  • compositions for preventing or treating angiogenesis-dependent diseases comprising a fusion protein in which a cell-permeable peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein.
  • Another aspect is a method for promoting angiogenesis comprising administering to a subject in need thereof a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein provides
  • Another aspect is prevention or angiogenesis-dependent disease comprising the step of administering to a subject in need thereof a fusion protein in which a cell-permeable peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein provides a way to treat
  • Another aspect provides the use of a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein for the manufacture of a medicament for preventing or treating angiogenesis-dependent diseases. .
  • composition for promoting angiogenesis comprising a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to a thymosin beta 4 (T ⁇ 4) protein.
  • cell penetrating peptide may refer to a carrier capable of transporting a target protein to the cytoplasm by penetrating a cell membrane regardless of molecular size or property.
  • cell permeability may refer to the ability or property of a peptide to penetrate a cell membrane and penetrate into a cell.
  • the cell-penetrating peptide may be an improved TAT peptide (SEQ ID NO: 1, YGRKKRRRQRRR), and the improved TAT peptide means that one more R is added compared to the wild-type TAT peptide (YGRKKRRQRRR).
  • T ⁇ 4 The “thymosin beta 4 (T ⁇ 4)” was first discovered as an extract from the thymus, and is a relatively small protein composed of 43 amino acids with a molecular weight of 5KDa and present in almost all cells except red blood cells.
  • T ⁇ 4 is originally actin As a protein that regulates G-actin, it is known to inhibit the synthesis of actin polymers by binding to G-actin, induce endothelial cell differentiation and migration, and angiogenesis.
  • angiogenesis As long as it plays a role in coordinating and exhibits efficacy in promoting regeneration and growth of damaged tissue through induction of angiogenesis, cell proliferation, promotion of cell migration, inhibition of cell death, etc., its amino acid sequence is not particularly limited. An amino acid sequence may be used, a mutated amino acid sequence thereof may be used, or a partial fragment thereof may be used.
  • the T ⁇ 4 may be a peptide represented by the amino acid sequence of SEQ ID NO: 2.
  • fusion protein refers to a protein artificially synthesized so that a protein or peptide binds to another heterologous protein or peptide, and may specifically include the cell-permeable peptide and the T ⁇ 4. More specifically, the cell-permeable peptide may be a peptide composed of the amino acid sequence of SEQ ID NO: 1, and the T ⁇ 4 may be a peptide composed of SEQ ID NO: 2.
  • the cell-permeable peptide may be bound to the N-terminus of thymosin beta 4.
  • the fusion protein may include a peptide having a sequence differing in at least one amino acid residue from the amino acid sequence of each domain included therein.
  • Amino acid exchanges in peptides that do not entirely alter the activity of the molecule are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • proteins with increased structural stability against heat or pH or increased protein activity may be included due to mutation or modification in the amino acid sequence.
  • the fusion protein or proteins constituting the fusion protein are prepared by a chemical protein synthesis method known in the art, or a gene encoding the fusion protein is amplified by PCR (polymerase chain reaction) or synthesized by a known method It can be prepared by cloning into an expression vector and expressing it.
  • the fusion protein of the present invention may include a linker peptide between the cell-penetrating peptide and the T ⁇ 4.
  • the cell-penetrating peptide may be directly linked to the N-terminus of T ⁇ 4 through a covalent bond or indirectly through a linker.
  • the linker may be specifically linked using amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, and arginic acid.
  • the fusion protein of the present invention may be a peptide composed of the amino acid sequence of SEQ ID NO: 3.
  • angiogenesis refers to the process of new blood vessel formation, i.e., the development of new blood vessels into cells, tissues or organs.
  • Receptors present in vascular endothelial cells existing in a resting state in existing blood vessels are activated, 2) activated vascular endothelial cells begin to secrete proteolytic enzymes that degrade the surrounding basement membrane and extracellular matrix, and 3) vascular endothelium This can be achieved by cells escaping from the walls of existing blood vessels and migrating and proliferating toward tissues that secrete angiogenic factors.
  • the use for promoting angiogenesis may be one or more uses selected from the group consisting of skin plate regeneration, artificial skin transplantation, and transplantation blood vessel production.
  • the "artificial skin” refers to a coating material used to prevent leakage of body fluids and prevent bacterial invasion for the purpose of protecting the film, and may be made of collagen, chitin, or synthetic polymers in addition to dried pork skin.
  • Another aspect provides a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, comprising a fusion protein in which a cell-permeable peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein.
  • T ⁇ 4 thymosin beta 4
  • the cell-penetrating peptide, thymosin beta4 protein, fusion protein, and angiogenesis are as described above.
  • angiogenesis-dependent disease may refer to a disease accompanied by symptoms in which blood supply is not smooth or angiogenesis is not performed properly, that is, a disease caused by insufficient angiogenesis.
  • the type of disease that can be prevented or treated by the angiogenesis promoting effect of the fusion protein according to one aspect is not particularly limited, but in one embodiment, the angiogenesis-dependent disease is ischemic disease, wound, burn, psoriasis, It may be any one selected from the group consisting of chronic ulcer, cardiovascular hemorrhage, cerebral hemorrhage, bedsore, diabetes, retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, diabetic foot ulcer, and pulmonary hypertension.
  • the ischemic disease is composed of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, angina pectoris, myocardial infarction, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, ischemic stroke, cerebrovascular dementia, brain trauma and neonatal hypoxia It may be any one selected from the group.
  • wound refers to a state in which a living body is damaged, and refers to a pathological condition in which tissues constituting the internal or external surface of a living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organs, or vascular tissue, are divided or destroyed.
  • the wound is a non-healing traumatic wound, destruction of tissue by irradiation, abrasion, bone gangrene, laceration, avulsion, penetrating wound, gunshot wound, wound, Chronic wounds, cuts, frostbite, bruises (contusion or bruise), skin ulcers, skin dryness, skin keratosis, cracks, bursts, dermatitis, pain due to dermatophytosis, surgical wounds, vascular disease wounds, wounds such as corneal wounds, bedsores, It may be any one selected from the group consisting of conditions related to diabetes and poor circulation, such as sciatica and diabetic skin erosion, chronic ulcers, suture sites after plastic surgery, spinal injuries, gynecological wounds, chemical wounds, and acne, and the subject Damage to any part of may include, but is not limited to.
  • tissue regeneration refers to a process of tissue recovery from damage caused by external and internal causes. Damage caused by external causes includes ultraviolet rays, external contaminants, wounds, trauma, and the like, and the internal causes Damage caused by stress or the like.
  • diabetes foot ulcer is the most common complication of diabetes and occurs in more than 50% of diabetic patients. It refers to a complication in which microvessels and nerve cells in the leg area die as some intracellular functions are damaged by the high concentration of blood sugar present in the blood vessels of diabetic patients.
  • the "diabetic retinopathy” is a disease of the capillary wall caused by diabetes (thickening of the basement membrane, reduction of parietal cells, excessive proliferation of endothelial cells), narrowing of the capillaries and eventually occlusion, and complications caused by circulatory disorders of the retinal microvessels. it means.
  • the improved TAT-thymosin ⁇ 4 fusion protein can pass through the cell membrane very effectively, does not lose the original angiogenic activity of thymosin ⁇ 4, and has an angiogenic effect similar to or better than VEGF known to have a great angiogenic effect. Since it can inhibit cell death and induce tissue regeneration, it can be usefully used for the regeneration of blood vessels and other damaged tissues or the treatment of ischemic diseases.
  • prevention may refer to any action that inhibits or delays angiogenesis-dependent diseases by administration of the pharmaceutical composition.
  • treatment refers to any action that improves or benefits the symptoms of a disease by administration of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention is formulated in a form suitable for the intended route of administration.
  • routes of administration include parenteral administration, such as intravenous, intramuscular, intradermal, subcutaneous, oral, intranasal (eg, inhalation), transdermal (eg, topical), via mucous membranes, and through the rectum.
  • the composition is formulated to form a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, nasal or topical administration.
  • compositions suitable for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may further include a solubilizing agent and a local anesthetic, such as ergotamine, to reduce pain at the site of the injection.
  • composition of the present invention is administered topically, the composition may be formulated as an ointment, cream, skin patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form known to those skilled in the art.
  • Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, powders, brighteners, ointments, and the like, where necessary, auxiliaries for sterilization or for changing their properties, such as osmotic pressure (such as preservatives, stabilizers, Wetting agents, buffers or salts).
  • suitable topical dosage forms include aerosols, wherein the solid or liquid carrier in combination with the active ingredient is preferably packaged as a mixture or in a squeeze bottle containing a volatile pressure (such as a compressed gas such as Freon).
  • a volatile pressure such as a compressed gas such as Freon
  • humectants or humectants may be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are known in the art.
  • the composition may be formulated in the form of an aerosol, spray, mist or drops.
  • the prophylactic or therapeutic agent used in the present invention is aerosolized by a nebulizer or package pressure using a suitable propellant (eg, dichloro difluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). It can be easily delivered in a form.
  • a suitable propellant eg, dichloro difluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a measured amount.
  • Capsules and cartridges eg composed of gelatin
  • for use in inhalers or inhalers may be prepared to contain a powder mixture comprising the compound and a suitable powder material such as lactose or starch.
  • the composition may be formulated in forms such as tablets, capsules, kits, soft capsules, solutions, suspensions, and the like.
  • Tablets or capsules may contain pharmaceutically acceptable excipients such as binders (such as pregelatinized starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc or silica); disintegrants (such as potato starch or Explotab); or with a humectant (such as sodium lauryl sulfate). Tablets may be coated by methods known in the art.
  • a liquid formulation suitable for oral administration may be in the form of a solution, syrup or suspension, or a dry form that can be dissolved by water in another suitable solvent before administration, but is not limited thereto.
  • Such liquid preparations may contain pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, cellulose derivatives or hardened edible fats); emulsifying agents (such as lecithin or gum arabic); non-aqueous media (such as almond oil, oil esters, ethanol or fractionated vegetable oil); and a preservative (such as methyl or propyl p-hydroxybenzoate or sorbic acid).
  • Such preparations may further include buffer salts, flavoring agents, coloring agents and sweetening agents.
  • Preparations for oral administration can be suitably formulated to form sustained release, controlled release or continuous release prophylactic or therapeutic agents.
  • the methods of the present invention further include formulating a composition for parenteral administration by injection (eg, injection or continuous infusion).
  • Preparations for injection may be in unit dosage form (eg, in ampoules or multi-unit containers) with an additional preservative.
  • the composition may be in the form of an oil or such as a suspension, solution or emulsion in an oily or aqueous medium, and may contain adjuvants such as suspending agents, stabilizing agents and/or dispersing agents.
  • the active ingredient may be in powder form and dissolved in a suitable medium (eg, sterile, heat-free water).
  • suitable media for the parenteral dosage forms of the present invention are known to those skilled in the art.
  • suitable media for parenteral dosage forms include, but are not limited to, US Pharmacopoeia water for injection;
  • Aqueous media includes but is not limited to Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection and Lactated Ringer's Injection;
  • Water miscible media include, but are not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and methyl benzoate.
  • administering may be used interchangeably and refer to a method or route that results in at least partial localization of a composition to a desired site according to one embodiment.
  • disposition of a composition according to one embodiment into a subject by Administration of the fusion protein or at least a portion of the fusion protein component of a composition according to one embodiment can be performed by any suitable route to a desired location in a living subject.
  • the fusion protein is 1 to 50 ug / ml, for example, 1 to 50 ug / ml, 1 to 40 ug / ml, 1 to 30 ug / ml, 1 to 30 ug / ml, based on the total volume of the composition 20 ug/ml, 1 to 15 ug/ml, 1 to 13 ug/ml, 1 to 10 ug/ml, 1 to 8 ug/ml, 2 to 50 ug/ml, 2 to 40 ug/ml, 2 to 30 ug/ml, 2 to 20 ug/ml, 2 to 15 ug/ml, 2 to 13 ug/ml, 2 to 10 ug/ml, 2 to 8 ug/ml, 4 to 50 ug/ml, 4 to 40 ug /ml, 4 to 30 ug/ml, 4 to 20 ug/ml, 4 to 40 ug /
  • the pharmaceutical composition may be administered in a pharmaceutically effective amount, and the pharmaceutically effective amount may vary depending on the application site, treatment frequency, treatment time, dosage form, patient condition, type of adjuvant, and the like.
  • the dosage of the pharmaceutical composition depends on various factors such as administration route, patient's age, sex, weight, patient's severity, wound type, application area, number of treatments, treatment time, dosage form, patient's condition, type of adjuvant, etc. In light of this, the effective amount should not be construed as limiting the scope of the present invention in any way.
  • Another aspect is a method for promoting angiogenesis comprising administering to a subject in need thereof a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein provides
  • T ⁇ 4 thymosin beta 4
  • Examples of the subject include humans and mammals, and specifically include humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, and the like.
  • the subject may be a subject in need of angiogenesis promotion.
  • Another aspect is prevention or angiogenesis-dependent disease comprising the step of administering to a subject in need thereof a fusion protein in which a cell-permeable peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein provides a way to treat
  • the cell permeable peptide, thymosin beta4 protein, fusion protein, subject, administration, angiogenesis, angiogenesis-dependent disease, prevention, and treatment are as described above.
  • Another aspect provides the use of a fusion protein in which a cell penetrating peptide having the amino acid sequence of SEQ ID NO: 1 is linked to thymosin beta 4 (T ⁇ 4) protein for the manufacture of a medicament for preventing or treating angiogenesis-dependent diseases.
  • T ⁇ 4 thymosin beta 4
  • the cell-permeable peptide, thymosin beta 4 protein, fusion protein, angiogenesis, angiogenesis-dependent disease, prevention and treatment are as described above.
  • the thymosin beta 4 fusion protein can pass through cell membranes very effectively, induce angiogenesis, inhibit apoptosis, and smoothly induce wound healing, thereby regenerating blood vessels and other damaged tissues or ischemic diseases.
  • it can be usefully used as a therapeutic agent for various angiogenesis-dependent diseases including diabetic foot ulcers.
  • FIG. 1 shows the results of immunofluorescence staining using the thymosin ⁇ 4 antibody in order to compare and evaluate whether the improved TAT-thymosin ⁇ 4 was introduced into cells with the control thymosin ⁇ 4.
  • FIG. 2 is a result of evaluating the angiogenesis ability by performing a HUVEC tube formation test after treating the HUVEC cell line with the improved TAT-thymosin ⁇ 4 at various concentrations. As a result of confirming the degree of formation of tubes;
  • Figure 2b is a graph quantifying the results of Figure 2a.
  • Figure 3 shows the result of evaluating the migration ability and the degree of proliferation of cells over time through microscopic observation and ImageJ software after treatment with the improved TAT-thymosin ⁇ 4 in various concentrations in fibroblast cell lines.
  • Figure 4 shows a three-layered cell model composed of keratinocytes, fibroblasts, and epithelial cells manufactured to have a structure similar to that of skin, and cell punch used for mesenchymal stem cells as a positive control group.
  • Figure 4a is a result of observing the degree of reduction of the damaged area for each cell under a microscope
  • Figure 4b is a graph quantifying the results of Figure 4a.
  • Figure 6 is a result of measuring the relative amounts of VEGFR2, p-VEGFR2, ang1, ang2, which are proteins involved in the VEGFR2 signaling pathway, by treating HUVEC cell lines with improved TAT-thymosin ⁇ 4 at various concentrations and then performing Western blotting,
  • Figure 6a is a band image;
  • 6B is a graph in which band density is measured and quantified.
  • FIG. 7 shows that HUVEC cell lines were treated with the improved TAT-thymosin ⁇ 4 together with inhibitors of the VEGFR2 signaling pathway (bevacizubam; B, sorafenib; S), and then Western blotting was performed to detect VEGFA, p-, which are proteins involved in the VEGFR2 signaling pathway.
  • VEGFR2 signaling pathway bevacizubam; B, sorafenib; S
  • Figure 7a is a band image confirming the amount of protein secreted out of the cell
  • Figure 7b is a band image confirming the amount of non-secreted intracellular protein
  • 7c is a graph in which band density is measured and quantified.
  • FIG. 8 is a schematic diagram showing intracellular molecular biological signaling pathways of angiogenesis by the improved TAT-thymosin ⁇ 4 derived from the protein analysis results.
  • Figure 9 is a result of measuring the amount of VEGFR2 apoptosis-related proteins cleaved PARP, claeaved caspase3, and Procaspase 3 by performing Western blot after treatment with the improved TAT-thymosin ⁇ 4 in HUVEC cell lines by concentration
  • Figure 9a is a band image
  • 9B is a graph in which band density is measured and quantified.
  • FIG. 10 analyzes the angiogenesis-related mechanism between thymosin ⁇ 4 (TMSB4X) and VEGF (VEGFA) using a string database capable of analyzing protein-protein interactions, and shows the association as a medium confidence score.
  • FIG. 10A is a result of pictorial representation of the correlation between a median reliability index of 0.400 or more and 20 or less;
  • Figure 10b is a result drawn by culling a protein with a high correlation by culling a high index among them.
  • a DNA fragment in which a cell-permeable peptide and thymosin beta 4 were fused was obtained through PCR using primers, and inserted into pBluescript Sk2+ to prepare a plasmid expression vector.
  • the vector prepared above was transformed into E. coli (RBC Bioscience, New Taipei City, Taipei), and after confirming that the DNA was inserted well, the gene sequence was analyzed, and the expressed fusion protein was recovered and the amino acid sequence was analyzed. Results were shown in SEQ ID NO: 3 in the table below.
  • the culture medium was filtered and applied to a Sepharose FF column to finally produce an improved TAT-T ⁇ 4 fusion protein (SEQ ID NO: 3).
  • the production proceeded through a fermenter, and 1 mM IPTG was treated for efficient expression of the fusion protein.
  • 12g of wet cells were suspended in 60ml lysis buffer (Lysis buffer, 30mM Sodium phosphate, 0.5% Triton X-100, 0.1M NaCl, 5mM EDTA, pH 7.0).
  • the suspension is treated with a sonicator (SONOSMASHER from UL SSO HI-TECH, Korea) to disrupt the cells, and then centrifuged (9,000 rpm, 50 min., Mega21R from Hanil, KOREA) to recover the supernatant, which is a water-soluble cell extract.
  • SONOSMASHER from UL SSO HI-TECH, Korea
  • Example 1 In order to confirm whether the improved TAT-thymosin ⁇ 4 prepared in Example 1 was introduced into cells, immunofluorescence staining was performed using a thymosin ⁇ 4 antibody to confirm whether thymosin ⁇ 4 was located in the cytoplasm.
  • human vascular endothelial cells (HUVEC, 1x104 cells/well) were cultured in a 24 well plate and treated with improved TAT-thymosin ⁇ 4 or thymosin ⁇ 4 at a concentration of 10 ⁇ g/mL for 1 hour.
  • the culture medium was removed, washed with PBS, and fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) at room temperature for 20 minutes.
  • Fixed cells were permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 10 minutes.
  • the cells were washed twice with PBS, blocked with a blocking solution (DAKO North America Inc., Carpinteria, CA, USA) for 1 hour at room temperature, and incubated with the primary antibody for 2 hours at room temperature. Thereafter, the cells were washed twice with PBS and incubated with the fluorescently labeled secondary antibody for 1 hour at room temperature.
  • the antibody used at this time was as follows: Anti-thymosin ⁇ 4 (ab14335, Abcam, Cambridge, MA, USA). Secondary antibodies used were as follows: Alexa Fluor 488-conjugated anti-rabbit IgG to thymosin ⁇ 4 (1:200, Invitrogen, Carlsbad, CA, USA). After all treatments, immunofluorescence images were obtained using a confocal microscope (Carl Zeiss LSM 880, Oberkochen, Germany).
  • FIG. 1 shows the results of immunofluorescence staining using the thymosin ⁇ 4 antibody in order to compare and evaluate whether the improved TAT-thymosin ⁇ 4 was introduced into cells with the control thymosin ⁇ 4.
  • Matrigel was dispensed into a 24-well plate at a concentration of 20 ug/ml and coated for 30 minutes in a 37° C. incubator.
  • 4 Huvec (human umbilical vein endothelial cells) cell lines, 7.5x10 were dispensed in a 24-well plate, treated with improved TAT-thymosin ⁇ 4 at various concentrations, and then cultured in a 37°C incubator for 4 hours
  • calcein AM staining was performed and observed using a confocal microscope.
  • the tube formation ability was evaluated through ImageJ software to determine the degree of tube formation appearing when the obtained cell image was graphically processed.
  • VEGF known to have a great angiogenic effect was used.
  • FIG. 2 is a result of evaluating the angiogenesis ability by performing a HUVEC tube formation test after treating the HUVEC cell line with the improved TAT-thymosin ⁇ 4 at various concentrations. As a result of confirming the degree of formation of tubes;
  • Figure 2b is a graph quantifying the results of Figure 2a.
  • the improved TAT-thymosin ⁇ 4 showed excellent tube forming ability at a concentration of 4 ug / ml to 13 ug / ml, in particular, when treated at a concentration of 10 ug / ml, It was confirmed that the tube formation ability of vascular endothelial progenitor cells was increased to a similar extent to that of VEGF, a positive control group. This means that the improved TAT-thymosin ⁇ 4 treatment can promote the angiogenic ability of vascular endothelial progenitor cells, thereby inducing angiogenesis.
  • Example 1 In order to confirm whether the improved TAT-thymosin ⁇ 4 prepared in Example 1 can induce cell regeneration and recovery by promoting the migration and growth of fibroblasts, a scratch wound healing assay was performed to determine cell migration. ability was evaluated.
  • Figure 3 shows the result of evaluating the migration ability and the degree of proliferation of cells over time through microscopic observation and ImageJ software after treatment with the improved TAT-thymosin ⁇ 4 in various concentrations in fibroblast cell lines.
  • Poly-L-lysine 50 ug/mL was coated on the surface of a 60 mm cell culture dish and a transwell before cell inoculation in a 37° C. incubator for 30 minutes. Thereafter, skin epithelial cells were placed in the lowermost petri dish, fibroblasts were placed in the middle transwell, and keratinocytes were placed in the uppermost transwell at a concentration of 4 ⁇ 10 5 cells/well, respectively, and cultured for 24 hours. After confirming that the cells of each layer had proliferated into a monolayer, a cell punch was used to pierce the cell center, and improved TAT-thymosin ⁇ 4 was added for each concentration and cultured for 24 hours. The reduction of the damaged area for each cell (by skin layer) and the degree of cell proliferation were observed under a microscope and analyzed through ImageJ software.
  • Figure 4 shows a three-layered cell model composed of keratinocytes, fibroblasts, and epithelial cells manufactured to have a structure similar to that of skin, and cell punch used for mesenchymal stem cells as a positive control group.
  • Figure 4a is a result of observing the degree of reduction of the damaged area for each cell under a microscope
  • Figure 4b is a graph quantifying the results of Figure 4a.
  • the hemoglobin of the VEGF-administered group which is a positive control group, was measured at 5.25 ⁇ 0.64 ug/mg, and the hemoglobin of the TAT-thymosin ⁇ 4-administered group was measured at 5.84 ⁇ 0.82 ug/mg, resulting in improved TAT-thymosin ⁇ 4 confirmed that blood vessel formation in tissues occurred more actively than VEGF, which was a positive control group.
  • HUVEC cells were treated with the improved TAT-thymosin ⁇ 4 at different concentrations (0, 5, 10, 15, 20 ug/ml) for 24 hours, and then dissolved in a radioimmunoprecipitation assay buffer (RIPA).
  • the dissolved protein was quantified by BCA assay, and 20 ⁇ g of protein was separated using a 10% SDS-PAGE gel and transferred to a 0.22- ⁇ m nitrocellulose (NC) membrane.
  • NC 0.22- ⁇ m nitrocellulose
  • the membrane was incubated in 5% skim milk blocking buffer at room temperature for 1 hour and then incubated overnight at 4°C with primary antibody (1:1000 dilution, 3% bovine serum albumin solution).
  • the antibodies used at this time are as follows: Anti-F-actin (ab205, Abcam), Anti-VEGFA (sc-7269, Santacruz), Anti-VEGFR2 (SAB4501645, Sigma), Anti-phospho VEGFR2 (PS1013, Merk) , Anti-Angiopoietin1 (AB3120, Millipore), Anti-Angiopoietin2 (AB3121, Millipore), Anti-phospho AKT (9271, CST), Anti-AKT (9272, CST), Anti-phospho-ERK (9102, CST), Anti- ERK (9101, CST), Anti-CD31 (ab28364, Abcam), Anti- ⁇ -actin (A5441, Sigma).
  • Figure 6 is a result of measuring the relative amounts of VEGFR2, p-VEGFR2, ang1, ang2, which are proteins involved in the VEGFR2 signaling pathway, by treating HUVEC cell lines with improved TAT-thymosin ⁇ 4 at various concentrations and then performing Western blotting,
  • Figure 6a is a band image;
  • 6B is a graph in which band density is measured and quantified.
  • the phosphorylation of VEGFR2 was most increased at the concentration of 10ug/ml, which had the most excellent angiogenic effect in the HUVEC tube formation experiment, and the expression of Ang1 and Ang2, which are markers of angiogenesis, was also It was most potent at a concentration of 10 ug/ml. This means that the angiogenic action of the improved TAT-thymosin ⁇ 4 is achieved through the classical VEGFR2 signaling pathway through phosphorylation of VEGFR2.
  • FIG. 7 shows that HUVEC cell lines were treated with the improved TAT-thymosin ⁇ 4 together with inhibitors of the VEGFR2 signaling pathway (bevacizubam; B, sorafenib; S), and then Western blotting was performed to detect VEGFA, p-, which are proteins involved in the VEGFR2 signaling pathway.
  • VEGFR2 signaling pathway bevacizubam; B, sorafenib; S
  • Figure 7a is a band image confirming the amount of protein secreted out of the cell
  • Figure 7b is a band image confirming the amount of non-secreted intracellular protein
  • 7c is a graph in which band density is measured and quantified.
  • FIGS. 7a to 7c it was confirmed that the amount of VEGFA secreted from cells to the outside of cells increased when the improved TAT-thymosin ⁇ 4 was treated. At this time, it was confirmed that the increased secretion of VEGFA was reduced to the control level by the VEGF inhibitors bevacizubam and sorafenib. This was the same in the concentration of non-secreted intracellular VEGFA, and as in the results of FIGS. 6a and 6b, the phosphorylated p-VEGFR2 was also highly phosphorylated in the improved TAT-thymosin ⁇ 4 treatment group, and it was confirmed that phosphorylation was reduced in the VEGF inhibitor treatment group. .
  • FIG. 8 is a schematic diagram showing intracellular molecular biological signaling pathways of angiogenesis by the improved TAT-thymosin ⁇ 4 derived from the protein analysis results.
  • the improved TAT-thymosin ⁇ 4 protein is introduced into the cells to secrete VEGFA to the outside of the cells, and the VEGFA outside the cells promotes the VEGFR2 signaling pathway of the cells to promote phosphorylation of ERK and AKT to form angiogenesis It can be seen that it promotes the effect.
  • Figure 9 is a result of measuring the amount of VEGFR2 apoptosis-related proteins cleaved PARP, claeaved caspase3, and Procaspase 3 by performing Western blot after treatment with the improved TAT-thymosin ⁇ 4 in HUVEC cell lines by concentration
  • Figure 9a is a band image
  • 9B is a graph in which band density is measured and quantified.
  • TMSB4X Thymosin ⁇ 4
  • a string database https://string-db.org/
  • VEGFA VEGF
  • FIG. 10 analyzes the angiogenesis-related mechanism between thymosin ⁇ 4 (TMSB4X) and VEGF (VEGFA) using a string database capable of analyzing protein-protein interactions, and shows the association as a medium confidence score.
  • FIG. 10A is a result of pictorial representation of the correlation between a median reliability index of 0.400 or more and 20 or less;
  • Figure 10b is a result drawn by culling a protein with a high correlation by culling a high index among them.
  • thymosin ⁇ 4 acts to promote angiogenesis by increasing the angiogenic signal of VEGF through the VEGFR2 signaling pathway.
  • thymosin ⁇ 4 is a substance that can promote the VEGFR2 signal transduction pathway and at the same time, it is a substance that can generate more efficient angiogenesis through higher signaling pathways than EGF and VEGF.

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Abstract

La présente invention se rapporte à une composition qui est destinée à favoriser l'angiogenèse et qui comprend des protéines de fusion de thymosine β-4. Les protéines de fusion de thymosine β-4 selon un aspect de la présente invention peuvent passer très efficacement à travers les membranes cellulaires pour induire une angiogenèse ainsi que pour inhiber l'apoptose, et peuvent induire la cicatrisation d'une plaie, et peuvent ainsi être efficacement utilisées en tant qu'agent thérapeutique pour la régénération de vaisseaux sanguins ou d'autres tissus lésés ou le traitement de maladies ischémiques ou d'ulcères du pied diabétique.
PCT/KR2022/018574 2021-11-23 2022-11-23 Composition permettant de favoriser l'angiogenèse comprenant des protéines de fusion de thymosine bêta-4 Ceased WO2023096330A1 (fr)

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Citations (3)

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US20090169538A1 (en) * 2006-01-17 2009-07-02 Regenerx Biopharmaceuticals, Inc. Methods of Treating or Preventing Tissue Damage Caused by Increased Blood Flow
KR20140120018A (ko) * 2013-04-02 2014-10-13 일양약품주식회사 상처치료, 신생혈관유도 및 발모효능을 증식시키는 펩타이드 및 이의 용도
KR20160107818A (ko) * 2015-03-05 2016-09-19 한국유니온제약 주식회사 개량형 TAT 펩타이드가 융합된 EGF, 티모신β4, hGH 단백질의 생산방법 및 이들 단백질을 포함하는 화장료 조성물

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US20090169538A1 (en) * 2006-01-17 2009-07-02 Regenerx Biopharmaceuticals, Inc. Methods of Treating or Preventing Tissue Damage Caused by Increased Blood Flow
KR20140120018A (ko) * 2013-04-02 2014-10-13 일양약품주식회사 상처치료, 신생혈관유도 및 발모효능을 증식시키는 펩타이드 및 이의 용도
KR20160107818A (ko) * 2015-03-05 2016-09-19 한국유니온제약 주식회사 개량형 TAT 펩타이드가 융합된 EGF, 티모신β4, hGH 단백질의 생산방법 및 이들 단백질을 포함하는 화장료 조성물

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LV SHUMIN, CAI HONGWEN, XU YIFEI, DAI JIN, RONG XIQING, ZHENG LANZHI: "Thymosin‑β4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF‑κB pathway", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 46, 1 January 2020 (2020-01-01), GR , pages 1347 - 1358, XP093069204, ISSN: 1107-3756, DOI: 10.3892/ijmm.2020.4701 *

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