Peptides with coagulation activity and uses thereof

Abstract

The present invention relates to a novel sequence of a peptide, characterized by gelling depending on the pH conditions. Since the peptide can induce coagulation, a composition comprising the peptide can be used for inducing hemostasis.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel peptide capable of inducing coagulation and use thereof. BACKGROUND

[0002] The vascular system plays an important role in transporting various nutrients and transporting oxygen and carbon dioxide in the animal body, thereby exchanging blood and body fluids between blood vessels and tissues. However, when skin tissue or blood vessels are damaged, blood and body fluids can flow out. When bleeding is excessive, the transport of oxygen cannot occur normally, which can cause death. Therefore, hemostatic methods for reducing bleeding have been developed.

[0003] In living organisms, there is a system that induces blood coagulation by itself. By aggregating blood cells to form a blood clot or a blood plug through thrombin formed by an enzymatic reaction and fibrin fibers generated therefrom, the outflow of blood or body fluid components is inhibited, thereby performing hemostasis. However, when excessive bleeding occurs due to the presence of a deep large wound, it can not be sufficient to use only the coagulation system of the organism, and there is also an urgent need to control excessive bleeding that inevitably occurs at the time of surgery. Therefore, it can be considered that the demand for a method for effective hemostasis and a substance having hemostatic activity has been high.

[0004] Chitosan, collagen, starch, beeswax, etc. have been used as ingredients of conventional preparations for hemostatic use, and these ingredients are manufactured and applied in the form of powder, sponge, sheet, gel, etc. In Korean Laid-Open Patent Publication No. 2018-0027126, a composition comprising a cross-linked hyaluronic acid derivative matrix is used to induce a hemostatic effect.

[0005] However, hemostatic preparations should be harmless to the organism because they are basically applied to damaged or injured areas, and should not cause side effects such as hemolysis. Hemostatic preparations using chitosan, polymers, etc. developed as conventional medical devices have a minimal hemostatic efficacy, and there is a problem in that, after being applied to the organism, the residue must be decomposed or excreted from the body, and the residue can induce an inflammatory response. In addition, in the case of conventional biological drug preparations having a coagulation mechanism, there is a disadvantage in that, since these biological drug preparations are biological derivative ingredients, they are not easy to manufacture and transport / store. These conventional biological drug preparations also have the disadvantage of poor availability because a long time is required to be applicable, for example, a thawing process is required before use, or the biological drug preparations can be used only after mixing the first and second doses. Therefore, it is necessary to develop a peptide preparation for promoting coagulation, which is synthesized using amino acid ingredients as biological ingredients, while having excellent manufacturing and storage stability and hemostatic efficacy. SUMMARY

[0006] [PROBLEMS]

[0007] An object of the present application is to provide a peptide that can be gelled under specific conditions and used to induce coagulation.

[0008] Further, an object of the present application is to provide a composition capable of effectively inducing hemostasis using the peptide.

[0009] Further, an object of the present application is to provide a method of hemostasis using the composition.

[0010] [Technical Solution]

[0011] To achieve the above object, one aspect of the present application provides a peptide having a pH-dependent gelling property, which includes the amino acid sequence of SEQ ID NO: 1.

[0012] Another aspect of the present application provides a composition for inducing hemostasis comprising the peptide.

[0013] Still another aspect of the present application provides a method for hemostasis, comprising the step of treating a bleeding site of an animal that has bled with the composition.

[0014] [Advantageous Effects]

[0015] The peptide provided by the present application has a self-gelling property under neutral pH conditions. Specifically, the peptide can be gelled to form aggregates under specific pH conditions, and thus, when blood or a body fluid is treated with the peptide or a composition comprising the peptide, the peptide can be gelled according to the pH conditions in the blood. The peptide aggregates with blood cells, or a nanofiber structure formed by gelling of the peptide surrounds and captures blood cells and blood components other than blood cells to form a mass. Thus, it has an effect of inducing hemostasis by precipitating or hindering the flow or outflow of blood.

[0016] The peptide of the present application and the composition comprising the peptide have an activity of inducing coagulation at a similar level to conventional hemostatic agents, have an excellent effect of shortening the hemostatic time, and do not cause hemolysis, and thus the peptide of the present application and the composition comprising the peptide can be effectively used to induce hemostasis.

[0017] In particular, under acidic conditions, gelling does not occur, and it can be in the form of a liquid or sol. Thus, since the peptide and the composition of the present application maintain an acidic pH condition until application to actual blood or a body fluid, the peptide and the composition of the present application can exhibit a more suitable property for storage or application due to fluidity. Further, when applied to blood or a body fluid, gelling occurs according to the neutral pH of the blood or the body fluid, which has an advantage of effectively exhibiting hemostasis at an actual bleeding site.

[0018] Further, the peptide of the present application and the composition comprising the same are prepared as an amino acid component constituting an organism. Accordingly, when applied to an organism, there is an advantage in that it is less likely to cause side effects or problems of decomposition / elimination, and thus the possibility of an inflammatory reaction is very low. Further, the peptide of the present application and the composition comprising the same are easy to manufacture and have excellent storage stability compared to conventional hemostatic preparations.

[0019] However, the effects of the present application are not limited to the above-mentioned effects, and other unmentioned effects will be clearly understood by those skilled in the art through the following description. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A graph showing the light scattering intensity kcps value and the particle size of the peptide of the present application measured according to the change in pH.

[0021] Figure 2 A graph showing the optical density value (OD 600) of a solution comprising the peptide of the present application measured according to the change in the concentration of a salt (NaCl), and a photograph showing the solution form according to the concentration of the salt.

[0022] Figure 3 A photograph showing the difference in the degree of gelation according to the difference in the concentration of the peptide of the present application.

[0023] Figure 4 A result of a red blood cell agglutination test observed by mixing a composition comprising the peptide of the present application with a red blood cell suspension collected from a mouse at different concentrations. The area marked with a circle represents the experimental result of the concentration at which blood cells do not precipitate and start to form a blood clot.

[0024] Figure 5 A photograph showing that coagulation occurs after 13 seconds due to a coagulation test using a composition comprising the peptide of the present application.

[0025] Figure 6 A result of a coagulation test using a composition comprising the peptide of the present application, and a result of measuring and comparing the optical density of the supernatant of a centrifuged sample. The relative value is shown based on the value of a negative control group (DW) treated with distilled water, and groups treated with Greenplast (Greenplast Q) and RADA16 peptide preparations are used as control groups.

[0026] Figure 7The results of animal experiments for the hemostatic effect of the composition comprising the peptide of the present application, and the pictures showing the bleeding site, blood loss amount, and bleeding time values of the control group treated with no reagent (untreated), the control group treated with Greenplast (Greenplast Q), and the groups treated with the peptide of the present application at concentrations of 1% and 2.5%, respectively. For the control group treated with no reagent, the blood loss amount was measured after 90 seconds from the start of bleeding.

[0027] Figure 8 The results of animal experiments for the hemostatic effect of the composition comprising the peptide of the present application, and the pictures showing the bleeding site, blood loss amount, and bleeding time values of the control group treated with no reagent (untreated), the control group treated with Greenplast (Greenplast Q), and the groups treated with the peptide of the present application at concentrations of 1% and 2.5%, respectively. For the control group treated with no reagent, the blood loss amount was measured after 90 seconds from the start of bleeding.

[0028] Figure 9 The results of animal experiments for the hemostatic effect of the composition comprising the peptide of the present application, and the pictures showing the bleeding site, blood loss amount, and bleeding time values of the control group treated with no reagent (untreated), the control group treated with Greenplast (Greenplast Q), and the groups treated with the peptide of the present application at concentrations of 1% and 2.5%, respectively. For the control group treated with no reagent, the blood loss amount was measured after 90 seconds from the start of bleeding.

[0029] Figure 10 The pictures and graphs for the comparison of the results of hemolysis tests using the composition comprising the peptide of the present application. As a control, treatment was performed using 1% SDS and PBS instead of the composition of the present application, and the results were confirmed. DETAILED DESCRIPTION

[0030] Hereinafter, the present application will be described in detail.

[0031] 1. Peptide having pH-dependent gelation property

[0032] One aspect of the present application provides a novel peptide having a pH-dependent gelation property.

[0033] In the present application, the term "peptide" refers to a polymer composed of two or more amino acids connected by a peptide bond.

[0034] In the present application, the term "gelation" refers to a phenomenon of becoming a gel form (morphology), and means that colloidal particles in a solution lose fluidity and have a certain form of solid or semi-solid. In general, a gel can be gelated when a chemical bond is formed in a sol in which solid particles are dispersed in a liquid.

[0035] The peptide includes the amino acid sequence of SEQ ID NO: 1. Within a range that does not affect the properties or activities of the peptide, the peptide can include a mutant peptide having a different sequence through deletion, insertion, and substitution of amino acid residues, or a combination thereof, or can be in the form of a protein fragment having the same function. Amino acid modifications at the protein and peptide level that do not substantially alter the properties or activities of the included amino acid sequence of SEQ ID NO: 1 are known in the art. In some cases, the modifications can be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Accordingly, the peptide includes not only the amino acid sequence of SEQ ID NO: 1, but also peptides having substantially the same amino acid sequence as the peptide and variants thereof. The peptide having substantially the same amino acid sequence can be a peptide including an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% amino acid sequence homology to the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Any peptide including a sequence having at least 90% amino acid sequence homology to the amino acid sequence of SEQ ID NO: 1 and having the same activity is included in the scope of the present application.

[0036] The peptide of the present application includes the amino acid sequence of SEQ ID NO: 1, and can consist of 20 or less amino acids exhibiting a gelation characteristic in a pH-dependent manner. Specifically, the peptide can consist of 20 or less amino acids, 18 or less amino acids, 15 or less amino acids, or 12 amino acids.

[0037] Further, the peptide of the present application can be obtained by various methods well known in the art. For example, the peptide of the present application can be prepared using polynucleotide recombination and protein expression systems, or synthesized in vitro by chemical synthesis such as peptide synthesis and cell-free protein synthesis, but is not limited to these preparation methods.

[0038] Further, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activity), and reduced antigenicity, a protecting group can be bound to the N- or C-terminus of the peptide. For example, the protecting group can be acetyl, fluorenylmethyloxycarbonyl, formyl, palmitoyl, myristyl, stearyl, or polyethylene glycol (PEG), however, the protecting group can include any ingredient capable of modifying the peptide, particularly enhancing the stability of the peptide, without limitation. "Stability" refers to storage stability (e.g., room temperature storage stability) as well as in vivo stability that protects the peptide of the present application from attack by protein cleaving enzymes in vivo.

[0039] The peptide of the present application can be a peptide that gels in a neutral pH. Specifically, the peptide can gel under pH conditions ranging from pH 4 to pH 8, pH 4 to pH 7, pH 4.5 to pH 7, or pH 5 to pH 7, but is not limited thereto. The peptide can gel within a pH range that can be exhibited by the blood or body fluid of a human or an animal other than a human.

[0040] Further, the peptide of the present application can exhibit a sol or liquid form in an acidic pH. Specifically, a solution comprising the peptide can be in a sol or liquid form when, for example, an acidic pH ranging from pH 0 to pH 3.9. The peptide of the present application can have a gelling property that loses fluidity and forms a solid or semi-solid gel when the pH is changed and given a neutral pH condition.

[0041] In a specific embodiment of the present application, the light scattering intensity kcps value and the size of the peptide are measured while changing the pH, and it is confirmed that gelling has occurred by confirming that the size of the particles in the solution increases and the kcps value gradually increases under conditions above pH 4. In particular, the peptide is observed to aggregate with the naked eye under conditions above pH 6. Thus, it is confirmed that the peptide of the present application has a gelling property in a pH-dependent manner, and more specifically, has a gelling property in a pH-dependent manner under a neutral pH condition.

[0042] The peptide can have a gelling property in the presence of a salt, for example, the salt can be sodium chloride (NaCl), but is not limited thereto. The peptide can gel under conditions in which the salt concentration is 50 mM or more, 60 mM or more, 70 mM or more, or 100 mM or more, and the degree of gelling can increase as the salt concentration increases. The peptide can gel within a salt concentration range that can be exhibited by the blood or body fluid of a human or an animal other than a human.

[0043] In a specific embodiment of the present application, the degree of aggregation of the peptide is visually confirmed by measuring the optical density while changing the salt concentration. As a result, it is confirmed that when the concentration of sodium chloride (NaCl) is 100 mM or more, the turbidity gradually increases, and gelling occurs due to an increase in viscosity.

[0044] According to the properties of the above-described peptide, the peptide can induce coagulation. Coagulation means blood solidification, and thus the peptide can induce a reaction in which liquid blood is solidified into a solid thrombus and blood clot. More specifically, since the peptide has a gelling property, a nanofiber structure formed due to gelling of the peptide surrounds blood cells and other blood components in the blood and induces aggregation. The blood cells aggregated with the peptide of the present application can form a mass, which can increase the volume and precipitate or hinder blood flow and outflow. Thus, since the peptide of the present application has an activity capable of inducing coagulation when bleeding occurs, the peptide of the present application or a composition comprising the same can be effectively used for hemostasis.

[0045] 2. Composition for inducing hemostasis and method for hemostasis using the same

[0046] Another aspect of the present application provides a composition for inducing hemostasis comprising the peptide.

[0047] The peptide comprises the amino acid sequence of SEQ ID NO: 1, and its description is the same as that of the peptide in the section of "1. Peptide having pH-dependent gelation property", so this specific description is incorporated into this section.

[0048] In the present application, the term "hemostasis" means reduction of bleeding, which can mean both reduction of blood outflow and reduction of outflow of body fluids other than blood. In addition, the concept of hemostasis includes all of the following: induction or promotion of coagulation of blood or body fluids, reduction of amount of blood loss, shortening of bleeding time, stopping of bleeding, promotion of formation of thrombus or blood clot, etc.

[0049] The peptide of the present application is characterized by being gelated in a pH-dependent manner. Accordingly, the composition comprising the peptide can also be aggregated and gelated according to pH conditions, and can be used for inducing hemostasis because the composition comprising the peptide aggregates with blood cells. Specifically, gelation of the peptide contained in the composition can occur at a bleeding site, and the gel or nanofiber formed thereby can aggregate while surrounding blood cells and other blood components. The aggregate formed in this way can precipitate into a large mass or obstruct the flow or outflow of blood. Accordingly, the composition of the present application can be effectively used for inducing hemostasis at a bleeding site.

[0050] The composition can exhibit an acidic pH. For example, the pH of the composition can range from 0 to 3.9, so the composition can be in the form of a liquid or sol that has not been gelated.

[0051] The concentration of the peptide contained in the composition can be 0.5 mg / ml to 50 mg / ml. Specifically, the concentration of the peptide can be 0.5 mg / ml to 50 mg / ml, 1 mg / ml to 40 mg / ml, 10 mg / ml to 35 mg / ml, or 20 mg / ml to 30 mg / ml, but is not limited thereto. When the concentration of the peptide contained in the composition of the present application is within the above range, the peptide can be sufficiently gelated, thereby inducing coagulation and sufficiently exhibiting a hemostatic effect.

[0052] The composition can further comprise calcium ions. The calcium ions can be added to the composition of the present application, for example, in the form of calcium chloride (CaCl2). It is known that calcium ions induce and accelerate the coagulation reaction in blood, and more specifically, calcium ions can help the reaction of enzymes such as thromboplastin involved in the coagulation reaction to form thrombin. Accordingly, calcium ions can contribute to the coagulation or hemostatic effect of the composition of the present application, and the contained calcium ions can also form an appropriate salt concentration.

[0053] The composition can coagulate blood at neutral pH. More specifically, the composition can coagulate blood at a pH condition of pH 4 to pH 8, pH 4 to pH 7, pH 4.5 to pH 7, or pH 5 to pH 7, but is not limited thereto. The composition can gelate within a pH range that can be exhibited by blood or body fluid of a human or an animal other than a human.

[0054] The composition can coagulate blood at a salt concentration of 50 mM or more. More specifically, the composition can coagulate blood at a salt concentration of 50 mM or more, 60 mM or more, 70 mM or more, or 100 mM or more, but is not limited thereto. The composition can coagulate blood within a salt concentration range that can be exhibited by blood or body fluid of a human or an animal other than a human.

[0055] The composition can not cause hemolysis. Hemolysis refers to a phenomenon in which blood cells are destroyed, and more specifically, a phenomenon in which red blood cells are destroyed. Since the composition of the present application does not cause hemolysis, the composition is suitable for application to blood or a bleeding site.

[0056] In a specific embodiment of the present application, red blood cells isolated from blood collected from a mouse are treated with the composition of the present application, and then centrifuged to measure the optical density of the supernatant. As a result, the measured value is significantly smaller than that when treated with 1% SDS. This is a result that occurs because hemolysis of red blood cells does not occur, and thus it has been confirmed that the composition of the present application does not induce hemolysis of blood.

[0057] The composition can be manufactured in various types of formulations to induce hemostasis. More specifically, the composition can be at least one form selected from the group consisting of a powder, a patch, a gauze, a spray, and an injection, but is not limited thereto. When the peptide contained in the composition can gelate and aggregate with blood cells and other blood components, the composition can be provided in any form.

[0058] Another aspect of the present application provides a method of using the composition to induce hemostasis.

[0059] The hemostasis method of the present application includes a step of treating a bleeding site of an animal in which bleeding occurs with the composition of claim 4. The animal includes both a human and a non-human animal, and the non-human animal includes, for example, a cow, a pig, a sheep, a goat, a deer, a horse, a rat, or poultry (chickens, ducks, geese, turkeys, ostriches, turkeys, pheasants), but is not limited thereto, and can be applied to any animal, not limited to an animal having a vascular system.

[0060] The bleeding site includes all sites from which blood or a body fluid other than blood flows out. For example, the bleeding site can include all areas from which blood or a body fluid flows out due to damage of a skin surface, areas from which blood flows out due to damage or cutting of a blood vessel, and areas of intestinal bleeding.

[0061] The method of treating with the composition can vary depending on the form of the composition. For example, when the composition is a powder, a spray, or the like, the treatment can be performed by spraying or coating to the bleeding area, and when the composition is a patch, a gauze agent, or the like, the treatment can be performed by adhering to the surface of the bleeding area. In addition, when the composition is an injection, the treatment can be performed by injecting the bleeding site using a syringe, but is not limited thereto.

[0062] The hemostatic method can further include a step of applying physical pressure to the bleeding site after the step of treating with the composition. However, the hemostatic method of the present application is not limited thereto, and any hemostatic method generally used to inhibit bleeding can be applied together with the step of treating with the composition of the present application, but is not limited thereto.

[0063] Hereinafter, the present application will be described in detail through examples.

[0064] However, the following examples specifically illustrate the present application, and the scope of the present application is not limited by the following examples.

[0065] [Preparation Example] Preparation of peptide

[0066] A peptide having an amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse phase high performance liquid chromatography (HPLC, Waters Associates, USA). ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co, USA) was used as a chromatographic column.

[0067] [Table 1]

[0068] SEQ ID NO: Peptide sequence 1 SASQAYLAGNIT

[0069] [Experimental Example 1] Confirmation of gelation of peptide according to pH change

[0070] To confirm the gelation phenomenon of the peptide of the present application having the amino acid sequence of SEQ ID NO: 1, the change in the degree of gelation was confirmed by measuring the change in the physical properties of the peptide of the present application while adjusting the pH. Specifically, the peptide of the present application was dissolved at a concentration of 1 mg / ml using 10 mM acetate buffer (pH 3.4 to pH 5.5) and 10 mM phosphate buffer (pH 6.0 to pH 7.4). And when the pH was 3.4, 4, 4.5, 5, 5.5, 6, 6.5, 7, and 7.4, respectively, the size and light scattering intensity (unit: kcps) of the peptide were measured using a light scattering analyzer (ZetaSizer, Malvern panalytical, UK).

[0071] The results can be seen in Table 1. Figure 1 At pH 3.4 and 4, the kcps value of the light scattering intensity did not change much, but when the pH increased to 4.5, a significant increase in the kcps value was measured. Therefore, it was confirmed that the gelation point, i.e., the pH at which the peptide of the present application began to gel, was between pH 4 and pH 4.5. And as the pH gradually increased, the kcps value also gradually increased, and the highest value was measured at pH 5.5. It is expected that this is because the peptide of the present application induces a structural change as the pH increases, and the arrangement of particles resulting therefrom causes the gelation property. Even at pH 6, the kcps value of the peptide remained high, and it was visually confirmed that peptide aggregation occurred. As the pH increased, the size of the particles gradually increased, confirming that peptide aggregation and gelation occurred at neutral pH.

[0072] From the above results, it can be confirmed that the peptide has a characteristic of not undergoing gelation under acidic pH conditions, and therefore no aggregation phenomenon was observed, but began to gel when the pH gradually increased to pH 4 to 4.5, and when it reached neutral pH, the peptide aggregated to form a gel. Therefore, the peptide can form aggregates depending on its pH conditions, and it is possible that it can be used to induce coagulation of blood. In particular, considering the characteristic of the peptide of the present application that gelation occurs in a pH range similar to that of the blood and body fluids of animals, it was confirmed that the peptide of the present application is suitable for application to living organisms.

[0073] [Experimental Example 2] Confirmation of gelation of peptide according to salt concentration

[0074] In addition to confirming that the peptide of the present application has a gelation characteristic as the pH increases according to Experimental Example 1, it was also confirmed how the gelation of the peptide changes according to the concentration of a salt such as NaCl. Specifically, 0 mM, 10 mM, 50 mM, 100 mM, 150 mM, and 200 mM NaCl aqueous solutions were prepared, and 25 mg / ml of the peptide of the present application having the amino acid sequence of SEQ ID NO: 1 was added to 200 μl of the NaCl aqueous solution, respectively, followed by mixing by vortexing at 3,500 rpm. The optical density of the mixture of the peptide and the salt solution was measured at a wavelength of 600 nm.

[0075] The results can be seen in Table 1 Figure 2 When the concentration of NaCl was 100 mM, the turbidity of the mixture began to increase, and the turbidity of the mixture continued to increase until the concentration of NaCl reached 200 mM. It is expected that this is due to the fact that aggregation is facilitated as the interaction between the molecules of the peptide of the present application increases. In addition, it was confirmed that the viscosity tended to increase up to a concentration of 150 mM of NaCl.

[0076] Since a certain amount of salt is dissolved in the blood or body fluid of an animal, it is necessary to examine whether the peptide of the present application sufficiently undergoes gelation even in an environment similar to blood or body fluid. Since the peptide is gelated according to the concentration of NaCl, it was confirmed that the peptide of the present application can be effectively used to induce coagulation of blood by forming a gel phase in an in vivo environment.

[0077] [Experimental Example 3] Confirmation of gelation of peptide according to peptide concentration

[0078] The degree of gelation of the peptide was confirmed according to the concentration of the peptide of the present application having the amino acid sequence of SEQ ID NO: 1. Specifically, the peptide was mixed with distilled water at a concentration of 5 mg / ml, 10 mg / ml, and 25 mg / ml, respectively, and dissolved in a 1.5 ml microtube. By gradually increasing the pH by adding 5N NaOH to the mixture containing the peptide, while visually observing the phase transition of the mixture containing the peptide.

[0079] The results can be seen in Table 2 Figure 3 The peptide of the present application mainly showed a sol phase under the initial acidic pH conditions, but gelation proceeded as the pH increased. When the concentration of the peptide was 0.5 mg / ml, gelation did not occur even when the pH reached 7.5, but as the concentration of the peptide increased, the gelation of the peptide was further facilitated. It was confirmed that gelation began under the conditions of pH 4.2 in a test tube containing the peptide at a concentration of 25 mg / ml.

[0080] [Experimental Example 4] Confirmation of erythrocyte agglutination effect of peptide of the present application (erythrocyte agglutination test)

[0081] Using the peptide of Preparation Example confirmed to have a gelation and aggregation property under neutral pH conditions, it was confirmed whether the peptide has an aggregation effect on red blood cells in blood. Specifically, blood collected from a rat was distributed into a 1.5 ml EP tube, centrifuged at 1,500 g for 10 minutes, and then the upper plasma was removed and washed three times with 0.9% sodium chloride aqueous solution. The red blood cells and the sodium chloride aqueous solution were mixed at a ratio of 3:11 (v / v) to prepare a red blood cell suspension. As a control of the peptide of the present application, a research-use commercially available RADA16 peptide sample (3D Matrix) known to have a hemostatic activity was used. The peptide of Preparation Example of the present application and the control peptide were dissolved in pure water, and treated at a respective concentration of 0 μg / ml, 62.5 μg / ml, 125 μg / ml, 250 μg / ml, 500 μg / ml, and 1000 μg / ml to 10 μl in a v-type 96-well plate, and then 90 μl of the sodium chloride aqueous solution was added thereto. After 10 μl of the red blood cell suspension was further added thereto, the mixture was mixed at 300 rpm for 10 minutes, and reacted at 4°C for 2 hours or more. In addition, the degree of aggregation of the red blood cells due to the formation of nanofibers of each sample was evaluated by visually checking the degree of precipitation of the red blood cell suspension.

[0082] The results are shown in Table 1. Figure 4 As shown in Table 1, it was found that when the concentration of the peptide of the present application was 500 μg / ml or more, the red blood cell suspension did not precipitate and formed a blood clot. This is an effect induced by the aggregation of red blood cells due to the nanofibers formed by the peptide of the present application, and it was confirmed that the blood coagulation promoting effect was similar to that of the control peptide sample known to have an aggregation effect on blood cells. Therefore, it was confirmed that the peptide of the present application or a composition comprising the same can be effectively used for hemostasis by inducing the aggregation of blood cells.

[0083] [Experimental Example 5] Confirmation of coagulation effect of peptide of the present application (coagulation test)

[0084] To confirm whether the peptide of the present application has an effect of inducing blood coagulation, blood collected from a rat was treated with the peptide of the present application, and then it was confirmed whether the blood was coagulated.

[0085] First, the blood clotting time of the blood treated with the peptide according to the present application was evaluated. In a state in which the blood clotting reaction of the collected rat blood was inhibited in a heparinized vacuum collection tube, 300 μl of the blood was transferred to an EP tube, and 150 μl of a solution of the peptide of the present application dissolved in pure water was treated in the EP tube. The blood clotting time was measured by inverting the tube at 10 second intervals and visually evaluating the flowability of the blood. As a control group, in the same manner as the peptide of the present application, a RADA16 peptide sample and Greenplast (Greenplast Q, GC Green Cross) were used to perform the experiment.

[0086] The results can be seen in Table 2 below and Figure 5 When treated with the peptide of Preparation Example at a concentration of 2.5%, it was confirmed that the blood coagulated into a clot after 13 seconds. When treated with Greenplast, the blood coagulated in less than 20 seconds, and when treated with the RADA 16 peptide, in less than 30 seconds. Thus, it was confirmed that the peptide of the present application has a coagulation efficacy similar to or superior to that of the commercial agent previously used as a hemostatic agent.

[0087] [Table 2]

[0088] Coagulation time Distilled water - Greenplast Q < 20 seconds RADA16 2.5% < 30 seconds Peptide of the present application 2.5% 13 seconds

[0089] In addition, in order to evaluate the coagulation efficacy of the peptide of the present application, rat blood was treated with the peptide of Preparation Example, and the optical density thereof was measured. Specifically, 300 μl of blood was transferred to a 15 ml conical tube in a state in which the coagulation reaction of the collected rat blood was inhibited in a heparinized vacuum collection tube, and 300 μl of a peptide solution of the present application was added thereto and mixed gently. Then, 30 μl of a 0.2 M CaCl2solution was added thereto, and incubated at 37°C for 10 minutes to accelerate the coagulation reaction. Then, 10 ml of distilled water was added thereto carefully, and 200 μl of the uniformly dispersed supernatant was added to a 96-well plate, and the optical density at a wavelength of 540 nm was measured using a spectrophotometer. Likewise, Greenplast and a RADA 16 peptide preparation were used as controls.

[0090] The results can be seen in Table 2 below and Figure 6 Based on the degree of coagulation of the negative control group treated with distilled water, the optical density measured when treated with the peptide of the present application was 0.41, which indicates that the amount of blood cells in the supernatant was significantly reduced. This shows a better effect than when treated with the RADA 16 peptide preparation, which is a commercial hemostatic agent (0.7). Thus, it was confirmed that the peptide of the present application and the composition comprising the same can be effectively used to promote coagulation.

[0091] [Experimental Example 6] Animal experiment for confirming hemostatic effect of peptide of the present application (in vivo test)

[0092] An animal experiment was performed to confirm whether the blood coagulation effect of the peptide of the present application was also exhibited in actual animals. Specifically, a bleeding model was constructed by incising the leg of an 8-week-old female rat (SD rat) to expose an artery, and then wounding the exposed artery with a 21G needle. Then, the bleeding site was treated with 200 μl of a sample prepared by dissolving the peptide of the present application in pure water at a concentration of 1% and 2.5%, respectively, immediately after bleeding, and then the bleeding was absorbed with a pre-weighed gauze. After measuring the time required for bleeding to stop, the amount of blood loss was confirmed by measuring the weight of the gauze that absorbed the blood. A Greenplast preparation was used as a positive control group, and in the case of a negative control group that was not treated with any agent, the experiment was stopped 90 seconds after bleeding began, and the amount of blood loss at the stopping point was checked.

[0093] The results can be seen in Table 1 Figures 7 to 9 When treated with the peptide of the present application, it was found that the hemostatic effect was considered to be the bleeding of the rat stopping. In the case of treatment with 1% peptide, bleeding stopped after 96 seconds, and in the case of treatment with 2.5% peptide, hemostasis occurred after 59 seconds. Thus, it was confirmed that the hemostatic effect when treated with the peptide of the present application was similar to or better than the hemostatic time (84 seconds) of the positive control group treated with Greenplast. Even when comparing the amount of blood loss, when treated with 2.5% of the peptide of the present application, the amount of blood loss was significantly reduced compared to the negative control group in which bleeding occurred for 90 seconds without any treatment. Thus, it was confirmed that the peptide of the present application showed a hemostatic effect even when treating an actual animal in which bleeding occurred, and can show an effect similar to that of a commercially available hemostatic agent.

[0094] [Experimental Example 7] Confirmation of whether peptide of the present application is hemolytic (hemolysis test)

[0095] In addition, it was confirmed whether hemolysis in which blood cells were destroyed occurred by treatment with the peptide of the present application. The blood of a rat was transferred to an EP tube in a state in which the coagulation reaction of the collected blood of the rat was inhibited in a heparinized vacuum collection tube, and centrifuged at 1,500 g for 10 minutes. After removing the supernatant plasma, the precipitated red blood cells were treated with 200 μl of 2.5% and 5% of the peptide of the present application, respectively. A sample treated with 1% SDS was used as a positive control for hemolysis. Each sample was stored in a 37°C incubator for 1 hour, and then centrifuged again at 1,500 g for 10 minutes. 200 μl of the supernatant was transferred to a 96-well plate, and the optical density at a wavelength of 540 nm was measured.

[0096] The results can be seen in Table 2 Figure 10 Hemolysis occurred in the SDS-treated group, but both the peptide of the present application at a concentration of 2.5% and 5% did not induce hemolysis like the negative control group. Thus, since the peptide of the present application does not destroy blood cells, it can be confirmed that there is no problem even in the case of use for the purpose of inducing blood coagulation, and a composition containing the peptide can be effectively used for hemostasis.

[0097] In the foregoing, the present application has been described in detail only with respect to the embodiments described, but it will be obvious to those skilled in the art that various changes and modifications can be made within the technical idea of the present application, and such changes and modifications naturally fall within the scope of the appended claims. <110> KIRGEN LTD. <120> Peptides having coagulation activity and uses thereof <130> 2021OPA6441 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptides having coagulation activity <400> 1 Ser Ala Ser Gin Ala Tyr Leu Ala Gly Asn lie Thr 1 5 10

Claims

1. A peptide having a pH-dependent gelation property, consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the peptide is gelled at pH 4 to pH 8.

3. The peptide according to claim 1, wherein the peptide can induce blood coagulation.

4. A composition for inducing hemostasis, comprising the peptide of claim 1.

5. The composition for inducing hemostasis according to claim 4, wherein the concentration of the peptide contained in the composition is 0.5 mg / ml to 50 mg / ml.

6. The composition for inducing hemostasis according to claim 4, wherein the composition further comprises calcium ions.

7. The composition for inducing hemostasis according to claim 4, wherein the composition coagulates blood at pH 4 to pH 8.

8. The composition for inducing hemostasis according to claim 4, wherein the composition coagulates blood under conditions where the salt concentration is 50 mM or more.

9. The composition for inducing hemostasis according to claim 4, wherein the composition does not cause hemolysis.

10. The composition for inducing hemostasis according to claim 4, wherein the form of the composition is at least one selected from the group consisting of a powder, a patch, a gauze, a spray, and an injection.

11. Use of the peptide according to claim 1 in the manufacture of a medicament for hemostasis.

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