Hemostatic compositions and methods of using hemostatic compositions

Abstract

[Problem] The present invention provides a hemostatic composition and a hemostatic method using the hemostatic composition, and specifically provides a composition for hemostasis that can be uniformly applied to a bleeding site and exert a high hemostatic effect. [Solution] A composition to be applied as a spray to a subject is provided, wherein the composition is characterized in that the spray is for hemostasis, and the composition comprises a self-assembling peptide that gels due to self-assembly when the composition is applied to a bleeding site of the subject, and the self-assembling peptide is contained in the composition at a concentration with an improved hemostatic ability compared to direct application.

Description

[0001] This application is a divisional application of Chinese patent application filed on December 14, 2016, with application number 201680072150.0 and entitled "Hemostatic Composition and Hemostasis Method Using Hemostatic Composition". Technical Field

[0002] This invention relates to a hemostatic spray containing self-assembling peptides. Background Technology

[0003] Biogels (such as fibrin glue) are used as perioperative or postoperative hemostatic agents, surgical substrates (such as wound healing sheets), drug delivery systems (DDS), etc.

[0004] However, most of these biogels use biologically derived materials, thus posing risks of microbial (e.g., viral) infection, immunogenicity, and disease transmission during use. For example, while fibrin glue is very useful as a perioperative hemostatic agent, its raw material is derived from human blood, and frequent incidents of patient infection due to hepatitis virus contamination in the fibrin glue during actual surgical use have made it a significant social problem. Therefore, there is a need to develop safe hemostatic agents that are not subject to the risk of microbial contamination from the chemical synthesis of their raw materials.

[0005] For example, the use of compositions containing purified amphiphilic self-assembling peptides in promoting wound healing, etc., has been proposed to date (Patent Document 1).

[0006] Citation List

[0007] Patent documents

[0008] Patent Document 1: National Publication of International Patent Application No. 2008-505919 Summary of the Invention

[0009] Technical issues

[0010] The self-assembling peptide composition described in Patent Document 1 is a very useful composition because it can promote tissue regeneration when applied to a wound site (or surgical site) without the risk of microbial contamination.

[0011] The inventors have conducted application studies on the self-assembled peptides described above, and found that when using the self-assembled peptides described above for hemostasis (e.g., for hemostatic agents used in surgery), there is a technical problem: a higher concentration of peptide in the composition to be applied to the bleeding site improves the hemostatic effect, but increases the viscosity of the composition to be applied, making it difficult for the applied composition to spread at the bleeding site and therefore difficult to exert a uniform hemostatic effect at the application site (in other words, a lower concentration of peptide in the composition to be applied to the bleeding site helps the applied composition to spread at the bleeding site, but does not produce a high hemostatic effect).

[0012] The inventors have conducted persistent research on methods for overcoming the above-mentioned technical difficulties, and the present invention was completed by unexpectedly discovering the following: compared with application by conventional methods, by using a composition containing self-assembled peptides as a "spray", the composition containing self-assembled peptides can be uniformly applied to the bleeding site and produce a high hemostatic effect.

[0013] Solution to the problem

[0014] Specifically, in one embodiment, the present invention relates to a composition to be applied as a spray for hemostasis, the spray being for hemostasis, and the composition comprising a self-assembling peptide, wherein when the composition is applied to the bleeding site of the subject, the self-assembling peptide self-assembles and thus gels, and the self-assembling peptide is contained in the composition at a concentration in which the composition has improved hemostatic ability compared to direct application.

[0015] In one embodiment of the invention, the concentration of the self-assembled peptide in the composition is at least 1.5% or higher by weight.

[0016] In one embodiment of the present invention, the self-assembling peptide is

[0017] (a) A peptide comprising an amino acid sequence of 4 to 34 amino acid residues, wherein polar and nonpolar amino acid residues are alternately arranged, or

[0018] (b) A peptide comprising an amino acid sequence of 13 amino acid residues, wherein nonpolar and polar amino acids are arranged alternately in symmetrical positions centered on the 7th nonpolar amino acid residue toward the N-terminus and C-terminus.

[0019] In one embodiment of the invention, the polar amino acid residue is selected from the following amino acid residues: aspartic acid residue, glutamic acid residue, arginine residue, lysine residue, histidine residue, tyrosine residue, serine residue, threonine residue, asparagine residue, glutamine residue, and cysteine ​​residue.

[0020] In one embodiment of the invention, the nonpolar amino acid residue is selected from the following amino acid residues: alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, phenylalanine residue, tryptophan residue, proline residue, and glycine residue.

[0021] In one embodiment of the invention, the polar amino acid residue is selected from the following amino acid residues: aspartic acid residue, glutamic acid residue, arginine residue, lysine residue, histidine residue, tyrosine residue, serine residue, threonine residue, asparagine residue, glutamine residue, and cysteine ​​residue, and the nonpolar amino acid residue is selected from the following amino acid residues: alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, phenylalanine residue, tryptophan residue, proline residue, and glycine residue.

[0022] In one embodiment of the invention, the self-assembled peptide is a peptide containing RADARADARADA (SEQ ID NO: 1), a peptide containing IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide containing RLDLRLALRLDLR (SEQ ID NO: 3).

[0023] In one embodiment of the present invention, the self-assembled peptide is a peptide composed of RADARADARADARADA (SEQ ID NO: 1), a peptide composed of IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide composed of RLDLRLALRLDLR (SEQ ID NO: 3).

[0024] In one embodiment of the invention, the bleeding site of the object is a non-surgically formed wound site or a surgically formed incision site or excision site.

[0025] In an alternative embodiment, the present invention relates to the use of any of the above-described compositions in the preparation of a spray for hemostasis.

[0026] In an alternative embodiment, the present invention relates to a spray comprising any of the above-described compositions.

[0027] In an alternative embodiment, the present invention relates to a method for hemostasis in a subject, comprising the steps of: applying a composition comprising a self-assembling peptide to a bleeding site of the subject by spraying, wherein when the composition is applied to the bleeding site of the subject, the self-assembling peptide self-assembles and thus gels, and the self-assembling peptide is contained in the composition at a concentration in which the composition has improved hemostatic ability compared to direct application.

[0028] In one embodiment of the present invention, the object is a non-human animal.

[0029] Any combination of one or more of the above features of the present invention is also included within the scope of the present invention. Attached Figure Description

[0030] Figure 1 A syringe containing a peptide solution is shown in an embodiment of the present invention.

[0031] Figure 2 The image shows a photograph taken after peptide No.1 (1.0% or 1.5%) was applied to the bleeding site of pig liver via routine or spray application.

[0032] Figure 3 The image shows a photograph taken after peptide No.1 (2.0% or 2.5%) was applied to the bleeding site of a pig liver via routine or spray application.

[0033] Figure 4 The image shows a photograph taken after peptide No.2 (1.0% or 1.5%) was applied to the bleeding site of a pig liver via routine or spray application.

[0034] Figure 5 The image shows a photograph taken after peptide No. 3 (1.5%) was applied to the bleeding site of a pig liver via routine or spray application.

[0035] Figure 6 The results show the time to hemostasis completion and the amount of composition applied until hemostasis was completed when peptide No.1 (1.0%, 1.5%, 2.0% or 2.5%) was applied to the bleeding site of pig liver by conventional application or spray application.

[0036] Figure 7 The results show the amount of composition applied until hemostasis was achieved when peptide No.2 (1.0% or 1.5%) was applied to the bleeding site of pig liver by conventional or spray application.

[0037] Figure 8 The results show the time to hemostasis when peptide No.2 (1.0% or 1.5%) was applied to the bleeding site of pig liver via routine or spray application.

[0038] Figure 9 The results show the time to hemostasis completion and the amount of composition applied until hemostasis was completed when peptide No. 3 (1.5%) was applied to the bleeding site of pig liver by conventional application or spray application. Detailed Implementation

[0039] This invention relates to compositions intended to be applied as a spray. In this invention, "spray" refers to a pharmaceutical agent applied to a recipient in a spray form. In this invention, the method, instrument, or apparatus for applying the composition in a spray form is not limited and can be appropriately selected by those skilled in the art. For example, the composition can be applied in a spray form by using compressed air or high-pressure gas to spray the liquid composition in, for example, a mist or foam state. More particularly, the composition can be applied in a spray form, for example, by using a canister spray based on internal high-pressure gas, an electro-spray based on an electric pump, or an air spray based on external air pressure (e.g., a spray gun or airbrush).

[0040] The present invention also relates to hemostatic agents. In the present invention, "hemostatic agents" broadly refers to agents for stopping bleeding in living organisms, and includes not only agents that completely stop bleeding, but also agents that reduce bleeding.

[0041] The bleeding site for hemostasis according to the present invention can be any location. The present invention can be used for hemostasis at sites such as non-surgically formed wounds or surgically formed incisions or excision sites. Examples of non-surgically formed wound sites may include wounds to body surface tissues (e.g., skin) or body tissues (e.g., organs, muscles, and bones) caused by accidents, etc. Examples of surgically formed incisions or excision sites may include sites where body surface tissues or body tissues are cut or removed during surgery with a scalpel, etc., and sites where body surface tissues or body tissues are dissected, anastomosed, sutured, etc., using surgical instruments. The present invention is applied to the object by spraying, thus making the application operation easier than conventional products, allowing the composition to be uniformly applied to a wide range with a single operation. Therefore, the present invention is also applicable to bleeding sites requiring careful handling, such as those resulting from endoscopic surgery.

[0042] The object of this invention can be a human or a non-human object. A non-human object can be, for example, a non-human animal, and can be, for example, a non-human mammal, a bird, a reptile, an amphibian, or a fish. Examples of non-human mammals include rodents (e.g., mice and rats), dogs, cats, horses, pigs, cattle, sheep, goats, and primates.

[0043] The compositions of the present invention comprise self-assembling peptides. In this specification, the phrase "peptide 'self-assembling' in solution" means that peptide molecules spontaneously assemble in solution through any interaction (e.g., electrostatic interactions, hydrogen bonds, van der Waals forces, or hydrophobic interactions) and should not be interpreted in a limiting sense. In the present invention, a self-assembling peptide means a peptide having the property of forming a self-assembled form in which many peptide molecules are arranged regularly according to their amino acid sequences. Due to this property, when a composition comprising a self-assembling peptide is applied to a bleeding site of a subject, the self-assembling peptide self-assembles at the application site and thereby forms a gel.

[0044] The self-assembling peptides used in this invention may have a given viscosity in their aqueous solution (i.e., the peptide aqueous solution before self-assembly) before application to the object. However, for ease of explanation in this specification, even when having a given viscosity, such a peptide aqueous solution before application to the object is also referred to as a "peptide solution (or peptide aqueous solution)". Furthermore, even when the peptide aqueous solution has a given viscosity before application to the object, further increasing the viscosity of the composition through the self-assembly of the self-assembling peptide after application of the aqueous solution to the object is also referred to as "gelation (or gel formation)".

[0045] The self-assembled peptide used in this invention may be, for example, a peptide comprising an amino acid sequence of 4 to 34 amino acid residues, wherein polar and nonpolar amino acid residues are arranged alternately, or a peptide comprising an amino acid sequence of 13 amino acid residues, wherein nonpolar and polar amino acids are arranged alternately in symmetrical positions centered on the 7th nonpolar amino acid residue toward the N-terminus and C-terminus.

[0046] By having the peptide structure described above, the self-assembling peptides used in this invention form a "β-sheet structure" in aqueous solution, such that only polar amino acid residues can align on one surface of the "β-sheet structure," while only nonpolar amino acid residues can align on the other surface. Therefore, such β-sheet structures can assemble into bilayer structures to hide their hydrophobic surfaces (the surfaces on which only nonpolar amino acid residues align). Furthermore, as the self-assembly of the molecule proceeds, this layered structure of the β-sheets can extend to form a three-dimensional conformational structure (e.g., a hydrogel). In some cases, the "self-assembling peptide" used in this invention is also referred to as a "self-organizing peptide."

[0047] The self-assembled peptides used in this invention may comprise "an amino acid sequence in which polar and nonpolar amino acid residues are arranged alternately". In this case, the amino acid sequence may be an amino acid sequence consisting of 4 to 34 amino acid residues, more preferably an amino acid sequence consisting of 8 to 30 amino acid residues, more preferably an amino acid sequence consisting of 12 to 26 amino acid residues, and most preferably an amino acid sequence consisting of 13 to 20 amino acid residues.

[0048] In this invention, "amino acid" is used in the broadest sense and includes not only amino acids that constitute proteins but also amino acids that do not constitute proteins, such as amino acid variants and derivatives. Given this broad definition, those skilled in the art will understand that examples of amino acids according to the invention include: L-amino acids that constitute proteins; D-amino acids; chemically modified amino acids, such as amino acid variants and derivatives; amino acids that do not constitute proteins, such as ortholeucine, β-alanine, and ornithine; and chemically synthesized compounds having properties known in the art as characteristic of amino acids. Examples of amino acids that do not constitute proteins include α-methyl amino acids (α-methylalanine, etc.), D-amino acids, histidine-like amino acids (2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine, etc.), amino acids having an additional methylene group in their side chain ("homogeneous" amino acids), and amino acids in which the carboxyl functional group in the side chain is replaced by a sulfonic acid group (sulfopropionic acid, etc.). In a preferred aspect of the invention, the amino acids used in the invention may be amino acids that constitute proteins.

[0049] In this invention, polar amino acid residues are not particularly limited, as long as the polar amino acid residue is an amino acid residue whose side chain can have polarity. Polar amino acid residues include, for example, acidic amino acid residues and basic amino acid residues. In this specification, acidic amino acid residues include, for example, aspartic acid (Asp:D) residues and glutamic acid (Glu:E). Basic amino acids include, for example, arginine (Arg:R), lysine (Lys:K), and histidine (His:H).

[0050] In this specification, for example, the term "aspartic acid (Asp:D)" means that the three-letter code "Asp" and the single-letter code "D" can be used as an abbreviation for aspartic acid.

[0051] In this specification, among neutral amino acid residues, amino acid residues containing hydroxyl groups, amide groups, thiol groups, etc., are included among polar amino acid residues due to their polarity. For example, tyrosine (Tyr: Y), serine (Ser: S), threonine (Thr: T), asparagine (Asn: N), glutamine (Gln: Q), and cysteine ​​(Cys: C) are included among polar amino acid residues.

[0052] In this specification, nonpolar amino acid residues are not particularly limited, as long as the nonpolar amino acid residue is an amino acid whose side chain is nonpolar. Nonpolar amino acid residues include, for example, alanine (Ala:A), valine (Val:V), leucine (Leu:L), isoleucine (Ile:I), methionine (Met:M), phenylalanine (Phe:F), tryptophan (Trp:W), glycine (Gly:G), and proline (Pro:P).

[0053] When the self-assembled peptide used in this invention comprises "an amino acid sequence in which polar and nonpolar amino acid residues are alternately arranged", the peptide may have a "RADA" repeat sequence (2 to 8 repeats, preferably 3 to 6 repeats) or an "IEIK" repeat sequence (1 to 4 repeats, preferably 2 to 3 repeats), and more preferably a peptide comprising RADARADARADA (SEQ ID NO: 1) or a peptide comprising IEIKIEIKIEIKI (SEQ ID NO: 2). Further preferably, the self-assembled peptide used in this invention may be a peptide composed of RADARADARADA (SEQ ID NO: 1) or a peptide composed of IEIKIEIKIEIKI (SEQ ID NO: 2).

[0054] When the self-assembled peptide used in this invention is "a peptide comprising an amino acid sequence of 13 amino acid residues, wherein nonpolar and polar amino acids are arranged alternately in symmetrical positions centered on the 7th nonpolar amino acid residue toward the N-terminus and C-terminus", preferably, the "7th nonpolar amino acid" of the peptide can be alanine (Ala: A). More preferably, the peptide can be a peptide comprising RLDLRLALRLDLR (SEQ ID NO: 3), and even more preferably, a peptide composed of RLDLRLALRLDLR (SEQ ID NO: 3).

[0055] Other examples of self-assembled peptides that can be used in this invention may include peptides disclosed in WO2006 / 014570.

[0056] The self-assembled peptides used in this invention may be modified (or labeled) as long as the peptides do not lose the principal properties intended by this invention. Such modified (or labeled) peptides are also included in the “self-assembled peptides” of this invention. Those skilled in the art can choose any method for modifying (or labeling) the self-assembled peptides used in this invention. For example, linking functional groups, linking chemical substances, or linking additional proteins or peptides may be performed. Examples of linking functional groups may include acylation, acetylation, alkylation, amidation, biotinylation, formylation, carboxylation, glutamylation, glycosylation (linking sugar chains), glycylation, hydroxylation, isopreneation, lipoylation, linking nucleotides or derivatives thereof, PEGylation, and linking lipid chains. Examples of linking chemical substances may include linking suitable labeling agents, such as radioisotopes (e.g., 125 I, 131 I, 3 H and 14 C), enzymes (e.g., β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase), fluorescent materials (e.g., fluorescein and fluorescein isothiocyanate), luminescent materials (e.g., luminol, luminol derivatives, luciferin, and luminol), and affinity tags (e.g., biotin). Examples of linking additional proteins or peptides may include ISG-ization, SUMOylation, and ubiquitination.

[0057] The concentration of the self-assembled peptide in the composition of the present invention may be at least about 0.5% by weight, at least about 1% by weight, at least about 1.5% by weight, at least about 2% by weight, at least about 2.5% by weight, at least about 3% by weight, at least about 3.5% by weight, at least about 4% by weight, at least about 4.5% by weight, at least about 5% by weight, at least about 5.5% by weight, at least about 6% by weight, at least about 6.5% by weight, at least about 7% by weight, at least about 7.5% by weight, or at least about 8% by weight. Preferably, the concentration of the self-assembled peptide in the composition of the present invention may be at least about 1.5% by weight.

[0058] The compositions of the present invention may further contain one or more bioactive substances (e.g., substances that can be used for hemostasis and substances that can be used to promote wound healing) without substantially affecting the self-assembly ability of the self-assembling peptides in the composition. Examples of such substances include synthetic or natural organic molecules, nucleic acid molecules, various cytokines, and various chemokines. Growth factors may also be included, alone or in combination with different bioactive substances, in the compositions of the present invention. Examples of growth factors include, but are not limited to, epidermal growth factor, nerve growth factor, transforming growth factor α and β, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, hematopoietic cell growth factor, heparin-binding growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, hepatocyte growth factor, brain-derived neurotrophic factor, keratinocyte growth factor, bone morphogenetic protein, and cartilage-derived growth factor.

[0059] The terminology used in this specification is given as an example of a particular embodiment and is not intended to limit the invention.

[0060] The term “including / comprises” as used in this specification means that the described items (members, steps, factors, numbers, etc.) are present, and does not exclude the presence of other items (members, steps, factors, numbers, etc.) unless the context clearly requires a different interpretation.

[0061] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as broadly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise defined, the terms used herein shall be interpreted as having the meaning consistent with that in this specification and related technical fields, and shall not be construed as idealized or overly formalized.

[0062] Terms such as “first” or “second” may be used to express a variety of factors. However, it should be understood that these factors should not be limited by these terms. These terms are used only to distinguish one factor from others. For example, a first factor may be described as a second factor, and vice versa, without departing from the scope of the invention.

[0063] The invention will be described in more detail below with reference to embodiments. However, the invention may be embodied in many forms and should not be construed as limited to the embodiments described herein.

[0064] Example

[0065] <Test Object>

[0066] Oozing bleeding was induced in pig liver by rubbing it with a metal file. The self-organizing peptide solution was applied to the bleeding site via routine or spray application, and its hemostatic effect and dosage were evaluated.

[0067] <Testing Practice Guide>

[0068] This test was conducted as a non-GLP test.

[0069] Animal Protection

[0070] The test was conducted with the approval of the Institutional Animal Care and Use Committee, based on the Animal Welfare and Management Act (Law No. 105 of 1973), the Standards for the Care and Management of Laboratory Animals and Pain Relief (Ministry of the Environment Announcement No. 88 of 2006), the Basic Guidelines for Conducting Animal Experiments issued by the Ministry of Agriculture, Forestry and Fisheries and the Ministry of Health, Labour and Welfare, and the Guidelines for the Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006), and in accordance with the provisions of the Rules and Regulations on Animal Experimentation Provided by NAS Laboratories Ltd. regarding the conduct of animal experiments from a scientific point of view and with ethical considerations.

[0071] <Test Animals>

[0072] Animal species: Pig

[0073] Variety: LWD

[0074] Sex: Female

[0075] Number of animals introduced: 2

[0076] Age at introduction: arbitrary

[0077] Weight at introduction: 50 kg

[0078] The research institute that receives the animals: Narita Laboratory, NAS Laboratory Co., Ltd.

[0079] Animal dealers: Shimao Livestock

[0080] Adaptation period: Approximately 7 days after introduction

[0081] Quarantine: No abnormalities were confirmed through clinical observation and quarantine upon introduction and during the adaptation period.

[0082] Individual identification: Ear tag

[0083] <Feeding conditions>

[0084] (1) Rearing environment

[0085] The test animals were housed individually in stainless steel cages (W: 690 × D: 960 × H: 740 mm) in a temperature- and humidity-controlled enclosure. Temperature and humidity were controlled using air conditioning systems set to 25 ± 3℃ and 60 ± 10%, respectively. Lighting was set to a 12-hour light cycle and a 12-hour dark cycle (light cycle: 7:00 to 19:00). The cages and feeders, as well as the enclosure, were cleaned and disinfected daily.

[0086] (2) Feed type, feed amount and feeding method

[0087] The feed used was "Breed 74" (JA East Japan Kumiai Feed Co., Ltd.), given to the animals twice a day (AM and PM) at a rate of 250 g per meal. However, on the day of the experiment, the animals were not fed to avoid aspiration due to vomiting during anesthesia.

[0088] (3) Drinking water

[0089] For drinking water, automated watering systems are used to allow animals free access to well water. The concentration of substances and bacterial counts in the drinking water are analyzed annually to ensure that the analysis values ​​meet the water quality standards of the Water Supply Act.

[0090] <Individual Identification and Test Group Composition>

[0091] Ear tags were used for individual animal identification. The entrance to the enclosure was marked with the test number and test period, and the cages indicated the test number, test group, and animal number.

[0092] <Pretreatment and Anesthetic Administration Methods>

[0093] Atropine sulfate was administered intramuscularly to each animal at a dose of 0.05 mg / kg (dose volume: 0.1 mL / kg). Approximately 30 minutes later, a mixture of 20 mg / kg (dose volume: 0.4 mL / kg) of ketamine hydrochloride and 4 mg / kg (dose volume: 0.2 mL / kg) of toluidine hydrochloride was administered intramuscularly. Each anesthetic was introduced into the ear root via intramuscular administration using a disposable syringe (Telmo Co., Ltd.) with an attached 18 G injection needle (Telmo Co., Ltd.). Then, an endotracheal tube was inserted, and anesthesia was maintained using isoflurane via a SUBARU-KJ02 inhalation anesthesia device and a mechanical ventilator (CONPOSS β-EV, METRAN). The perioperative isoflurane concentration was set at 1% to 3%, and the oxygen flow rate was set at 2 L / min. The intrapulmonary pressure of the mechanical ventilator was set at 15 cmH2O, and the respiratory rate was set at 15 breaths / min. Each animal was placed supine on the operating table and covered with a sterile drape.

[0094] Perioperative monitoring

[0095] Set up an electrocardiogram, pulse oximeter, and thermometer, and perform perioperative monitoring. Measure blood pressure without blood flow using a cuff. Maintain blood pressure at an average of 60 mmHg or higher.

[0096] <Test Substance>

[0097] The peptide solutions (test substances) used in this experiment and their storage elastic moduli are shown in the table below.

[0098]

[0099] (Peptide concentration: % by weight)

[0100] Peptide No. 1: RADARADARADARADA (SEQ ID NO: 1)

[0101] Peptide No. 2: IEIKIEIKIEIKI (SEQ ID NO: 2)

[0102] Peptide No. 3: RLDLRLALRLDLR (SEQ ID NO: 3)

[0103] All the peptides described above were synthesized as raw powders by CPC Scientific, Inc. All peptides were N-terminally acetylated and C-terminally amidated. Purified water according to the Japanese Pharmacopoeia was used as the solvent for the peptides.

[0104] Frequency sweep tests were performed from 1 radians / second to 10 radians / second under a shear stress of 1 Pa, and the storage elastic modulus at 10 radians / second was selected as the data. As for the viscosity before gelation, it was tested by loading each peptide solution into 20 mm plates spaced 500 μm apart and soaking for 2 minutes.

[0105] <Testing Methods>

[0106] 1. Using a scalpel and an electric scalpel, each fully anesthetized pig was disemboweled through a midline abdominal incision to expose the liver.

[0107] 2. Inducing oozing bleeding by scraping the liver with a metal file.

[0108] 3. Apply each test substance to the bleeding slant by conventional application (*1) or spray application (*2).

[0109] 4. Use a stopwatch to measure the time from the end of administration until the end of the assessment when hemostasis is achieved (*3). Record the amount of each test substance used up to the end of the test.

[0110] * 1: Fill the syringe with the peptide solution (test substance) and expel the peptide solution from the nozzle at the tip of the syringe, thus applying it to the subject (see...). Figure 1 (The syringe).

[0111] * 2: Insert a syringe containing the peptide solution (test substance) (equivalent to...) Figure 1 A syringe is inserted into a spray gun, and compressed air is blown through the nozzle at the tip of the syringe using a compressor, causing the peptide solution propelled from the nozzle to be sprayed onto the target. The spray pressure is typically 0.5 to 1.5 [kgf / cm2], but varies depending on the viscosity of each test substance, bleeding conditions, and distance from the application site.

[0112] * 3: If bleeding does not decrease after the initial application (if it is determined that this will not lead to hemostasis), administer additional application as appropriate. The time to complete the initial application is defined as the start of the measurement, and the time to complete the hemostasis assessment is defined as the end of the measurement.

[0113] <Results>

[0114] In the above tests, the method by which each peptide solution was applied to the site of liver bleeding was as follows: Figures 2 to 5 As shown in the figure, the peptide solution applied by conventional means is unevenly distributed at the bleeding site, while the peptide solution applied by spray is uniformly distributed at the bleeding site.

[0115] like Figure 6 As shown, although there were no significant differences, in the spray application group, the peptide solution containing 1.0% peptide No. 1 tended to have shorter hemostasis completion time and require less solution to be applied until hemostasis was achieved, compared to the conventional application group. On the other hand, in the spray application group, concentrations of 1.5% or higher significantly resulted in shorter hemostasis completion time and required less solution to be applied until hemostasis was achieved, compared to the conventional application group.

[0116] like Figure 7 and 8 As shown in the results, similar to those for the peptide solution containing peptide No. 1, the peptide solution containing 1.0% peptide No. 2 also did not show a significant difference between the spray application group and the conventional application group. In contrast, in the spray application group, concentrations of 1.5% or higher significantly resulted in shorter hemostasis completion time and less solution required until hemostasis was achieved compared to the conventional application group.

[0117] like Figure 9 As shown, the peptide solution containing peptide No. 3 also successfully achieved hemostasis in the spray application group, but failed to achieve hemostasis by conventional application.

[0118] These results indicate that solutions containing self-assembled peptides unexpectedly exhibit improved hemostatic ability when used as a spray compared to conventional application.

Claims

1. A composition to be applied as a spray to a target, The spray is used to stop bleeding, and The composition comprises self-assembled peptides, wherein When the composition is applied to the bleeding site of the subject, the self-assembling peptide self-assembles and thus gels, and The self-assembling peptide is contained in the composition at a concentration in which the composition has improved hemostatic ability compared to direct application.

2. The composition according to claim 1, wherein... The concentration of the self-assembled peptide in the composition is at least 1.5% or higher by weight.

3. The composition according to claim 1 or 2, wherein The self-assembled peptide (a) A peptide comprising an amino acid sequence of 4 to 34 amino acid residues, wherein polar and nonpolar amino acid residues are alternately arranged, or (b) A peptide comprising an amino acid sequence of 13 amino acid residues, wherein nonpolar and polar amino acids are arranged alternately in symmetrical positions centered on the 7th nonpolar amino acid residue toward the N-terminus and C-terminus.

4. The composition according to claim 3, wherein The polar amino acid residues are selected from the following amino acid residues: aspartic acid residues, glutamic acid residues, arginine residues, lysine residues, histidine residues, tyrosine residues, serine residues, threonine residues, asparagine residues, glutamine residues, and cysteine ​​residues.

5. The composition according to claim 3, wherein The nonpolar amino acid residue is selected from the following amino acid residues: alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, phenylalanine residue, tryptophan residue, proline residue, and glycine residue.

6. The composition according to claim 3, wherein The polar amino acid residue is selected from the following amino acid residues: aspartic acid residue, glutamic acid residue, arginine residue, lysine residue, histidine residue, tyrosine residue, serine residue, threonine residue, asparagine residue, glutamine residue, and cysteine ​​residue. The nonpolar amino acid residue is selected from the following amino acid residues: alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, phenylalanine residue, tryptophan residue, proline residue, and glycine residue.

7. The composition according to claim 6, wherein The self-assembled peptide is a peptide containing RADARADARADARADA (SEQ ID NO: 1), a peptide containing IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide containing RLDLRLALRLDLR (SEQ ID NO: 3).

8. The composition according to claim 7, wherein The self-assembled peptide is a peptide composed of RADARADARADARADA (SEQ ID NO: 1), a peptide composed of IEIKIEIKIEIKI (SEQ ID NO: 2), or a peptide composed of RLDLRLALRLDLR (SEQ ID NO: 3).

9. The composition according to any one of claims 1 to 8, wherein The bleeding site of the object is a non-surgically formed wound site or a surgically formed incision site or excision site.

10. Use of the composition according to any one of claims 1 to 9 in the preparation of a spray for hemostasis.

11. A spray comprising the composition according to any one of claims 1 to 9.

12. A method for stopping bleeding in an object, comprising the following steps: The composition containing self-assembling peptides is applied to the bleeding site of the subject by spraying, wherein... When the composition is applied to the bleeding site of the subject, the self-assembling peptide self-assembles and thus gels, and The self-assembling peptide is contained in the composition at a concentration in which the composition has improved hemostatic ability compared to direct application.

13. The method of claim 12, wherein The concentration of the self-assembled peptide in the composition is at least 1.5% or higher by weight.

14. The method according to claim 12 or 13, wherein The bleeding site of the object is a non-surgically formed wound site or a surgically formed incision site or excision site.

15. The method according to any one of claims 12 to 14, wherein The subject is a non-human animal.

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