Blood coagulation composition comprising calcium lactate as active ingredient and use thereof

Abstract

The present invention relates to a blood coagulation composition comprising calcium lactate as an active ingredient, a method for treating a wound or reducing bleeding at a bleeding site, the method comprising a step of administering the composition of the present invention to the wound or the bleeding site, an anti-adhesion composition comprising calcium lactate as an active ingredient, and a method for preventing adhesion during surgery and after surgery, the method comprising a step of administering the composition of the present invention during surgery and after surgery.

Description

Blood coagulation composition containing calcium lactate as an active ingredient and uses thereof

[0001] The present invention relates to a blood coagulation composition comprising calcium lactate as an active ingredient and its use, and more specifically, the present invention relates to a composition comprising calcium lactate as an active ingredient that promotes rapid hemostasis and prevents adhesion.

[0002] New technologies, devices, and drugs for bleeding and / or hemorrhage control have been developed, particularly for severe hemorrhage. Despite all currently available technologies, bleeding and hemorrhage control remain major unresolved issues in emergency medical management. Nearly 50% of all deaths within the first 48 hours of hospitalization are associated with the inability to adequately control bleeding. Failure to stop bleeding within the first 24 hours is almost always fatal, especially when multiple trauma sites are involved. This generally means that hemostatic products for post-treatment management on the battlefield must rapidly control bleeding, be ready to use, simple to apply, have a shelf life approaching two years at ambient temperature, and ideally prevent bacterial growth or viral transmission / reactivation. To meet the needs of both military and civilian sectors, the timing of the product's hemostatic action is critical.

[0003] Devices investigated or used as external wound treatment methods are in the range of adhesive pads containing coagulants, compression bandages, gauze, tourniquets for limbs, and trauma kits for wounds on the body.

[0004] Agents designed to stop external bleeding vary in composition and ingredients, but are often designed to facilitate the rapid formation of a coagulation at the site of application. Coagulation products are generally varied but often contain high concentrations of substances, such as human fibrinogen, thrombin, calcium, factor XIII, and / or antifibrinolytic agents. In addition to fibrin, microporous polysaccharide macrobeads, inorganic salts, synthetic zeolites, and chitosan (poly-N-acetylglucosamine) are also available for use as hemostatic agents. A number of novel hemostatic products, such as a chitosan-based bandage product (deacetylated poly-N-acetylglucosamine base, HemCon Inc., T-Guard, USA), are available for treating wound trauma, which is a freeze-dried chitosan dressing designed to optimize the mucosal adhesive surface density of chitosan and the structural integrity of chitosan at the wound site. Although there is evidence suggesting that HemCon (trademark) chitosan bandages may also enhance platelet function and incorporate red blood cells into clots to form clots on wounds, they clearly exert their hemostatic effect primarily through adhesion to the wound. While these bandages demonstrated improved hemostasis and reduced blood loss in several animal models of arterial hemorrhage, significant variability was observed among the bands, including some failures attributed to inadequate adhesion to the wound. (See reference [McManus et al, Business Briefing: Emergency Medical Review 2005, 79]).

[0005] Another commonly used hemostatic product is Combat Gauze, a kaolin-coated surgical gauze currently used as a standard dressing in the U.S. military.

[0006] Austrian company Nycomed Pharma markets horse collagen matrices coated with human fibrinogen and thrombin under the trade names Tachocomb (registered trademark) and Tachosil (registered trademark), which are available for operating room use in many European countries (see reference [U. Schiele et al, Clin. Materials 9: 169-177 (1992)]). Although these fibrinogen-thrombin dressings do not require the pre-mixing necessary for liquid fibrin sutures, their utility for site application is limited by the usual need to pre-moisten the product with saline to provide suitable solubility for application to bleeding sites on the toes. In fact, their site usability has not been observed to date, and it is also known that these dressings are not effective for high pressure, high-pressure bleeding (see reference [Sondeen et al. Trauma 54:280-285 (2003)]). Another dry fibrinogen / thrombin dressing for treating wounded tissue is disclosed in U.S. Patent No. 6,762,336 of the American Red Cross (ARC). This particular dressing consists of a backing material and multiple layers, namely, two outer layers containing fibrinogen (but not thrombin) and an inner layer containing thrombin and calcium chloride (but not fibrinogen). Although this dressing has been shown to be successful in several animal models of hemorrhage, the bandage is loose, insoluble, and tends to break when handled (see references [McManus et al, Business Briefing: Emergency Medical Review 2005, page 78; Kheirabadi et al. Trauma 59:25-35 (2005)]).It has been found that the mixing of fibrinogen and thrombin is very important during the freeze-drying / manufacturing process, which indicates that the complete mixing of the fibrinogen and thrombin active ingredients is essential for the full efficacy of the product.

[0007] Other fibrinogen / thrombin-based dressings have also been proposed. For example, U.S. Patent No. 4,683,142 discloses a reabsorbable sheet material for wound closure and healing comprising a glycoprotein matrix such as collagen and containing coagulating proteins such as fibrinogen and thrombin. U.S. Patent No. 7,189,410 discloses a band comprising (i) particles of fibrinogen; (ii) particles of thrombin; and (iii) a backing material having calcium chloride. U.S. Patent No. 2008 / 003272 and WO 00 / 38752 disclose fibrin glue in the form of mixed granules or a mixture of granules coated on a support material. European Patent No. 0059265 discloses a collagen carrier coated with fibrinogen and thrombin particles. WO 97 / 44015 discloses a mixture of thrombin / fibrinogen albumin microparticles. WO 2010 / 002435 discloses a bioresorbable hemostatic pouch containing free microparticles as well as fibrinogen or thrombin microparticles in a core.

[0008] There is currently no complete solution for the treatment of excessive or severe bleeding, particularly for the control of compressive bleeding from arterial or venous hemorrhage. Heat generation from one type of agent is a major problem. The ability of specific dressings to adhere effectively when applied to deep or irregularly shaped wounds creates another major limitation.

[0009] Surgical and traumatic wounds are the most common types of wounds treated in the field of wound management. An additional obstacle to overcome when developing products for this sector is the harmful effects of anticoagulants present in the blood of these surgical patients.

[0010] Excessive bleeding caused by war or civilian accidents triggers hypothermia, coagulation disorders, acidosis, sepsis, and organ failure, becoming a major cause of death and disability due to trauma. Preventing excessive bleeding can prevent 50% of trauma-related deaths. Excessive bleeding can be prevented, particularly in the operating room, by stopping bleeding and stabilizing blood clots.

[0011] In the coagulation process for bleeding, calcium helps activate various coagulation factors. Fibrin clots, which enhance the stability of blood clots including the blood coagulation process caused by platelets, have their stability increased by calcium and Factor XIII, one of the transglutaminases (Tgase).

[0012] Ultimately, calcium is an indispensable element for the activation of the intrinsic pathway during the blood coagulation process; by promoting the coagulation process, it enables rapid and stable hemostasis through the activation of blood coagulation factor 13.

[0013] In particular, fibrin is a cause of postoperative adhesions. Adhesions are currently the leading surgical complication, occurring in over 90% of cases during abdominal surgery. If fibrin remains in the abdominal cavity in a residual or unstable state due to blood leakage caused by damage to the intestinal wall and organ surfaces during surgery, it can lead to adhesions. Therefore, through irrigation, an essential surgical procedure, calcium lactate is supplied to the leaked blood to promote the formation of stable fibrin clots at the surgical site. Furthermore, since free fibrin clots resulting from bleeding can be removed through irrigation, the occurrence of postoperative adhesions can be significantly prevented.

[0014]

[0015] The present invention solves the above problems and was devised in response to the above necessity. The objective of the present invention is to provide a means to prevent excessive bleeding by rapidly stopping the bleeding.

[0016] Another objective of the present invention is to provide a composition that prevents adhesions that may occur after surgery.

[0017] To achieve the above objective, the present invention provides a blood coagulation composition comprising calcium lactate as an active ingredient.

[0018] In one embodiment of the present invention, the calcium lactate is preferably in the range of 1.5 mM or more to less than 162 mM, but is not limited thereto.

[0019] In another embodiment of the present invention, the calcium lactate is preferably dispersed on an absorbent carrier, and

[0020] The above absorbent carrier is cotton, polysaccharide, albumin, cellulose, methylcellulose, alkyl hydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose sulfate, salt of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxidized cellulose; Collagen such as gelatin or collagen-sponge, chitin, carboxymethyl chitin, hyaluronic acid, salt of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfate, carboxymethyl dextran, heparin, heparin sulfate, heparan, heparan sulfate, dermatan sulfate, keratan sulfate, carrageenan, starch, amylose, amylopectin, poly-N-glucosamine, poly-N-acetylglucosamine, polymannuronic acid, polyglucuronic acid, polygluronic acid; It is more preferable, but not limited to, a biocompatible polymer selected from the group consisting of chitosan, chitin, chitin-glucan, chitosan-glucan, carboxymethyl chitosan, chitosan salts, chitosan derivatives thereof, and any combination thereof; polyurethane; oxidized polysaccharides, derivatives or combinations of any of the above.

[0021] In another embodiment of the present invention, the composition is preferably provided as, but not limited to, a dry adhesive coating, aerosol, dry aerosol, pump spray, medical compression bandage; film; coated plaster; sponge or surgical patch containing a drug; hemostatic pad; gauze; ointment, semi-gel, gel, foam, paste, suspension, ointment, emulsion, moldable form, nasal plug, surgical dressing, wound packing, bandage, cotton swab, catheter, optical fiber, syringe, pessary, suppository, or suspension of a liquid or non-aqueous liquid.

[0022] The present invention also provides a method for treating a wound or reducing bleeding at a bleeding site, comprising the step of administering a composition of the present invention to the wound or bleeding site.

[0023] The present invention also provides a use of calcium lactate for the manufacture of a product for hemostasis.

[0024] In one embodiment of the present invention, the calcium lactate is preferably in the range of 1.5 mM or more to less than 162 mM, but is not limited thereto.

[0025] In another embodiment of the present invention, the calcium lactate is preferably dispersed on an absorbent carrier, but is not limited thereto.

[0026] The present invention also provides an anti-adhesion composition comprising calcium lactate as an active ingredient.

[0027] In one embodiment of the present invention, the calcium lactate is preferably in the range of 1.5 mM or more to less than 162 mM, but is not limited thereto.

[0028] The above anti-adhesion composition may be applied to a surface in the form of a liquid, gel, or foam; or may be incorporated into washing water. Additionally, the composition may be applied to a surface with a vehicle such as a wipe.

[0029] Some embodiments of the anti-adhesion composition of the present invention may be suitably prepared with an anti-adhesion agent in an amount of about 0.01% (based on the total weight of the composition) to about 20% (based on the total weight of the composition), preferably about 0.05% (based on the total weight of the composition) to about 15% (based on the total weight of the composition), or more preferably about 0.1% (based on the total weight of the composition) to about 10% (based on the total weight of the composition). However, it is considered that the anti-adhesion composition of the present invention may be outside the above ranges. For example, in some embodiments, the anti-adhesion composition may include an anti-adhesion agent providing 100% of the total weight of the anti-adhesion composition.

[0030] The anti-adhesion composition of the present invention may be formulated with one or more conventional commercially available carrier materials. The anti-adhesion composition may take various forms without limitation, including aqueous solutions, gels, balms, lotions, suspensions, creams, milks, plasters, ointments, sprays, emulsifiers, oils, resins, foams, solid sticks, aerosols, etc. Liquid carrier materials suitable for use in the present invention include those well known in the fields of cosmetics and medical technology for use as bases for ointments, lotions, creams, plasters, aerosols, gels, suspensions, sprays, foams, cleaning solutions, etc., and may be used at established levels thereof.

[0031] Non-limiting examples of suitable carrier materials include water, softeners, wetting agents, polyols, surfactants, esters, perfluorocarbons, silicones, and other pharmaceutically acceptable carrier materials.

[0032] In one embodiment, the anti-adhesion composition may comprise one or more emollients that act to optionally soften, soothe, otherwise smooth, and / or moisturize the skin. Suitable emollients that may be incorporated into the composition include oils, such as alkyl dimethicone, alkyl methicone, alkyl dimethicone copolyol, phenyl silicone, alkyl trimethylsilane, dimethicone, dimethicone crosslinked polymer, cyclomethicone, lanolin and its derivatives, fatty esters, glycerol esters and derivatives, propylene glycol esters and derivatives, alkoxylated carboxylic acids, alkoxylated alcohols, fatty alcohols, and combinations thereof.

[0033] Wetting agents suitable as carriers in the anti-adhesion composition of the present invention include, for example, glycerin, glycerin derivatives, hyaluronic acid, hyaluronic acid derivatives, betaine, betaine derivatives, amino acids, amino acid derivatives, glycosaminoglycans, glycols, polyols, sugars, sugar alcohols, hydrogenated starch hydrolysates, hydroxy acids, hydroxy acid derivatives, PCA salts, etc., and combinations thereof. Specific examples of suitable humectants include honey, sorbitol, hyaluronic acid, sodium hyaluronate, betaine, lactic acid, citric acid, sodium citrate, glycolic acid, sodium glycolate, sodium lactate, urea, propylene glycol, butylene glycol, pentylene glycol, ethoxydiglycol, methylgluceth-10, methylgluceth-20, polyethylene glycol (listed in the International Cosmetic Ingredient Dictionary as PEG-2 to PEG 10), propanediol, xylitol, maltitol, or combinations thereof. Humectants are beneficial in that they prevent or reduce the chance of the anti-adhesion film formed after the anti-adhesion agent is applied to the surface.

[0034] The anti-adhesion composition may contain water. For example, if the anti-adhesion composition is a wetting composition as described below for use with a wetting wipe, the composition will typically contain water. The anti-adhesion composition may suitably contain water in an amount of about 0.01% (based on the weight of the composition) to about 99.98% (based on the total weight of the composition), about 1.00% (based on the total weight of the composition) to about 99.98% (based on the total weight of the composition), or about 50.00% (based on the total weight of the composition) to about 99.98% (based on the total weight of the composition), or about 75.00% (based on the total weight of the composition) to about 99.98% (based on the total weight of the composition).

[0035] In an embodiment where the anti-adhesion composition acts as a detergent (e.g., shampoo; surface cleaner; or hand, face, or body cleaner), the anti-adhesion composition may include one or more surfactants. These may be selected from anionic, cationic, nonionic, zodiacionic, and diionic surfactants.

[0036] Optionally, one or more rheology modifiers, such as thickeners, may be added to the anti-adhesion composition. Suitable rheology modifiers are compatible with the anti-adhesion agent. As used herein, "compatible" refers to a compound that does not adversely affect the anti-adhesion properties of the anti-adhesion agent when mixed with it.

[0037] A thickening system is used in the anti-adhesion composition to adjust the viscosity and stability of the composition. Specifically, the thickening system prevents the composition from leaking out of the hands or body during dispensing and use of the composition. When the anti-adhesion composition is used with a wipe product, a thicker formulation may be used to prevent the composition from migrating from the wipe substrate.

[0038] The thickening system must be compatible with the compound used in the present invention; that is, when the thickening system is used in combination with the anti-adhesion compound, it must not precipitate or form a coacervate.

[0039] Thickeners may include cellulose, gum, acrylate, starch, and various polymers.

[0040] In addition, the present invention provides a method for preventing adhesions during and after surgery, comprising the step of administering the composition of the present invention during and after surgery.

[0041] In one embodiment of the present invention, the surgery is preferably, but is not limited to, at least one of orthopedic surgery, ophthalmic surgery, gastrointestinal surgery, abdominal surgery, thoracic surgery, cranial surgery, cardiovascular surgery, gynecological surgery, arthroscopic surgery, urological surgery, plastic surgery, and musculoskeletal surgery.

[0042]

[0043] In addition, the composition according to the present invention is characterized by applying or impregnating the calcium lactate composition to a fiber substrate. In addition, the fiber substrate may be one selected from the group consisting of nonwoven fabric, woven fabric, knitted fabric, cotton, sheet, hemostatic gauze, bandage, dressing, suture, and tape, and is preferably a nonwoven fabric made of one or more materials selected from the group consisting of rayon, polyethylene terephthalate (Poly(ethyl benzene-1,4-dicarboxylate); PET), cotton, bamboo fiber (SMP bamboo), pulp, polyester, and polypropylene, but is not limited thereto.

[0044] The present invention will be described below.

[0045] The term 'METI' as used in the specification and / or drawings of the present invention means calcium lactate.

[0046] In order to supply calcium, the development of safe calcium must be prioritized. However, inorganic calcium preparations are unsuitable for application to the human body due to their low safety. This patent discovered that blood coagulation is promoted by using calcium lactate, an organic calcium preparation with guaranteed safety, instead of inorganic calcium preparations, and thereby completed an invention capable of preventing traumatic excessive bleeding and postoperative adhesions.

[0047] As can be seen from the present invention, the calcium lactate of the present invention has thrombosis-forming and blood coagulation activity, and in particular, blood treated with calcium lactate shows higher transglutaminase activity than calcium chloride at the same concentration, so it can be utilized in cotton including gauze, nonwoven fabrics, gels, and hemostatic guns, and is considered applicable to hemostatic agents and all hemostatic medical devices. In addition, the efficacy of preventing postoperative intestinal adhesions in a surgical cleansing agent using calcium lactate has been confirmed, so it can be used as an adhesion prevention agent.

[0048] Figure 1 is a graph quantifying the degree of thrombus formation according to the concentration of calcium lactate, and Figure 2 is a photograph of a thrombus formed by calcium lactate.

[0049] Figure 3 is a graph quantifying blood coagulation factor 13, one of the proteins involved in blood coagulation, and Figure 4 is a graph measuring the activity of transglutaminase, which plays an important role in blood coagulation, according to the concentration of calcium lactate.

[0050] Figure 5 shows photographs of gauze coated with different concentrations of calcium lactate and photographs of fibers observed under a microscope at magnification, and Figure 6 is a microscopic photograph confirming whether the gauze is coated with calcium lactate using a calcium staining method.

[0051] Figure 7 is a graph confirming the water absorption capacity of gauze containing calcium lactate, and

[0052] Fig. 8 is a schematic photograph illustrating the experimental process for observing thrombus formation on gauze containing calcium lactate; Fig. 9 is a 2D image of the thrombus and serum formed after dropping blood onto gauze containing calcium lactate; Fig. 10 is a photograph showing the thrombus-forming ability of calcium lactate-coated gauze according to concentration; Fig. 11 is a photograph confirming the thrombus formed on gauze containing calcium lactate through an optical microscope; Fig. 12 is a graph showing the results of measuring the volume of the thrombus formed on gauze containing calcium lactate; and Fig. 13 is a 3D image of the size of the thrombus and serum formed on gauze containing calcium lactate.

[0053] Fig. 14 is a photograph confirming the hemostatic effect of cotton gauze soaked in water and calcium lactate solution, and Fig. 15 is a photograph taken after separating the gauze one by one after drying the blood clot and serum formed on the cotton gauze soaked in water and calcium lactate solution.

[0054] Figure 16 is a graph showing the change in body weight of rats in an anti-intestinal adhesion activity test, Figure 17 is a graph showing an image of intestinal adhesion and the degree of adhesion scored, Figure 18 is a photograph of intestinal tissue stained with MT (Masson trichrome) and a graph scored thereon, and Figure 19 is a photograph of intestinal tissue stained with H&E (Hematoxylin & Eosin) and a graph scored thereon.

[0055] In the drawings of the present invention, the term 'METI' means 'calcium lactate'.

[0056]

[0057] The present invention will be described in more detail below through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention shall not be interpreted as being limited by the following examples.

[0058] Example 1: Blood coagulation reaction caused by calcium lactate

[0059] 1-1. Measurement of Thrombus Weight via Coagulation Activity

[0060] To conduct coagulation reaction experiments using human blood, Single Donor Human Whole Blood sold by Innovative Research was purchased, and the anticoagulant used was Sodium Citrate. This acts to prevent blood coagulation by removing calcium from the serum. Since coagulation factors in the blood are maintained, it was used to conduct experiments on coagulation reactions caused by calcium. To simulate the coagulation reaction of actual human blood, the experiment was conducted by calculating the concentration of calcium capable of neutralizing the anticoagulant activity caused by Sodium Citrate.

[0061] 100 μl of Human Whole Blood was dispensed into each well of a 96-well plate using a micropipette, with the tip trimmed to prevent the blood cells from breaking. Calcium chloride was used as a positive control. The concentrations used for the experiment were 1.5 mM, 2.5 mM, and 5 mM. In the negative control group, calcium chloride was added to a concentration of 15 mM to neutralize the anticoagulant effect caused by sodium citrate; similarly, calcium chloride and calcium lactate were added to both the positive control and the experimental group at concentrations exceeding those used in the experiment. The substances were applied at 30-second intervals, starting with the control group, at their respective concentrations. Afterward, the samples were left at room temperature for 15 minutes, and the weight of the thrombi was measured using an ultra-precision balance.

[0062] As shown in the experimental result photograph in Fig. 1, no thrombi were formed in the control group, while it was confirmed that thrombi formed in the experimental group treated with calcium chloride and calcium lactate (Fig. 2). In the groups treated with calcium chloride and calcium lactate, it was observed that the weight of the thrombi increased as the calcium concentration increased (Fig. 1a). Furthermore, when comparing calcium chloride and calcium lactate, the thrombus weight of calcium lactate increased more at the same concentration, suggesting that it is expected to demonstrate performance superior to the conventionally used calcium chloride (Fig. 1).

[0063] 1-2. Mechanism of Blood Coagulation Factor XIII and Changes in Serum Concentration

[0064] The blood coagulation process occurs through the complex interaction of various blood coagulation factors, ultimately enabling the stable formation of blood clots via fibrin. Factor XIII is a type of transglutaminase that acts in the final stage of blood clot stabilization. Factor XIII is activated by calcium and thrombin to form stable blood clots, thereby creating more organized and solid clots. For this reason, activated Factor XIII is a major factor in determining the size of the blood clot and the red blood cell content of the clot. The activated form of Factor XIII is referred to as Factor XIIIa below.

[0065] Enzyme immunoassay (ELSIA) was performed to quantify the activated form of Factor XIII (Factor XIIIa), which enables the formation of stable blood clots. The enzyme immunoassay kit used in the experiment was the Human Factor XIIIa ELISA kit (Abcam, ab287172). Calcium chloride and calcium lactate were used at concentrations of 1.5 mM, 2.5 mM, and 5 mM. For sample preparation, 1 ml of Human Whole Blood was dispensed, followed by the addition of calcium chloride and calcium lactate at the specified concentrations, and the mixture was slowly inverted for 15 minutes. Subsequently, the serum was separated using a centrifuge. Using the obtained serum, enzyme immunoassay for Factor XIIIa was performed according to the experimental method provided by Abcam.

[0066] As can be seen in Figure 3, Factor XIIIa showed inversely proportional results to the concentrations of calcium chloride and calcium lactate. These results indicate a tendency to decrease in serum because the thrombus formed due to Factor XIII activity by calcium is precipitated and removed during the centrifugation process. The group treated with 1.5 mM calcium lactate showed a reduced form compared to calcium chloride, suggesting that sufficient Factor XIII activity is exhibited at low concentrations.

[0067] Based on the above results, it was confirmed that calcium can convert Factor XIII into its active form, Factor XIIIa, and that calcium lactate, as well as calcium chloride, induces the activity of Factor XIII to an equivalent level (Fig. 3).

[0068] 1-3. Changes in serum protein activity of transglutaminase, a thrombus-stabilizing enzyme

[0069] Transglutaminase is an enzyme that forms protein polymers by inducing isopeptide bonds in proteins. Humans possess nine types of transglutaminase, and Factor XIII, a representative type, plays a key role in the stabilization of blood clots. Other types of transglutaminase assist in the formation of the skin barrier and the extracellular matrix structure. To evaluate the transglutaminase activity induced by calcium lactate, an enzyme-linked immunoassay (ELISA) was performed. The ELISA kit used in the experiment was the Transglutaminase Assay Kit (CS1070) provided by Sigma. Transglutaminase activity tests were conducted using calcium chloride and calcium lactate at concentrations of 1.5 mM, 2.5 mM, and 5 mM. 500 µl of human whole blood was dispensed into EP-tubes, followed by the addition of calcium chloride and calcium lactate according to the above concentrations, and the tubes were slowly inverted for 15 minutes. Afterward, serum and plasma were separated using a centrifuge. Using the obtained serum, the experiment was conducted following the experimental method provided in the transglutaminase activity kit.

[0070] As shown in Figure 4, it was confirmed that the activity of transglutaminase gradually decreased with concentration in the groups treated with calcium chloride and calcium lactate. Since the experiment involved calcium chloride and calcium lactate activating transglutaminase in the blood and quantifying the transglutaminase remaining in the serum, a decrease in experimental activity can be interpreted as an increase in activity in the blood. Therefore, it can be concluded that the activity of transglutaminase in human blood is promoted by calcium, leading to the formation of stable thrombi during the blood coagulation reaction. In particular, at the same concentration, blood treated with calcium lactate showed slightly higher transglutaminase activity than that treated with calcium chloride. (Figure 4)

[0071] Example 2: Confirmation of blood absorption and coagulation in gauze

[0072] 2-1. Gauze coated in molecular form

[0073] Cotton gauze was prepared with aqueous calcium lactate solutions of 0 mM, 10 mM, 25 mM, 54 mM, and 162 mM to confirm blood coagulation by calcium lactate on cotton gauze.

[0074] 10 ml of calcium lactate aqueous solution was used on one piece of cotton gauze, and it was dried in a dry oven until the next day. If the quantity manufactured was large and it did not dry in the dry oven, it was left for a sufficient amount of time until the moisture was gone.

[0075] Visual inspection of the cotton gauze revealed that white powder was observed on the surface starting at 25 mM. The amount of precipitated, crystalline powder increased with increasing concentration. No crystals were visible to the naked eye inside the cotton gauze. (Top photo in Fig. 5)

[0076] Observation using an optical microscope revealed that crystals had formed on the surface of the fibers constituting the 162 mM calcium lactate cotton gauze in a 1,000x magnified image. At concentrations below 162 mM, the appearance of the fibers was observed to be indistinguishable from the control group. (Bottom photo of Fig. 5)

[0077] Von Kossa staining was performed to confirm whether calcium lactate was coated on the surface of the cotton gauze. Von Kossa staining is a metal substitution method in which deposited calcium is treated with a silver nitrate solution to replace it with silver ions, followed by reduction using ultraviolet (UV) light. To verify whether calcium lactate was coated on the cotton gauze through Von Kossa staining, cotton gauze containing calcium lactate was prepared in the same manner as in the previous experiment. The calcium lactate-coated cotton gauze was thoroughly soaked in a 5% silver nitrate solution and then irradiated with UV light for one hour. Afterward, it was washed twice with triple-distilled water, and impurities were removed using a 5% sodium thiosulfate solution.

[0078] For reference, the gauze used in the present invention is [Sterile Gauze-N, Water-based] and the specifications are [5cm X 5cm X 8 layers].

[0079] As shown in Fig. 3b, cotton gauze without calcium lactate was not dyed with Von kossa. On the other hand, cotton gauze coated with calcium lactate was dyed a dark red color. The dyeing became darker as the concentration increased and the surface area of ​​the thread increased. This confirmed that calcium lactate was coated on the surface of the cotton gauze. (Fig. 6)

[0080] 2-2. Changes in the Moisture Absorption Capacity of Cotton Gauze

[0081] Distilled water was used to evaluate the liquid absorption capacity of cotton gauze fused with calcium lactate. The experimental method and result calculation were performed as follows.

[0082] (a) Measure the weight of the dish.

[0083] (b) Place each cotton gauze on a weight dish and measure its weight.

[0084] (c) Transfer the cotton gauze to another weight dish, add 10 ml of distilled water, and wait 30 minutes to allow it to absorb sufficiently.

[0085] (d) Lift one end of the cotton gauze soaked in distilled water and wait for 30 seconds without any water dripping.

[0086] (e) Place a cotton gauze containing distilled water on a weight dish and measure its weight.

[0087] The formula for plotting the graph is as follows: {(Wet cotton gauze) - (Weight of cotton gauze)} / {(Weight of cotton gauze) - (Weight of weight dish)}

[0088] As shown in Fig. 7, the experimental results showed that the calcium lactate-coated cotton gauze exhibited similar absorbency to the control group. This confirmed that it possesses the same liquid absorbency as conventional cotton gauze. (Fig. 7)

[0089] 2-3 Evaluation of blood absorption capacity

[0090] To confirm blood coagulation on calcium lactate-coated cotton gauze, cotton gauze coated at different concentrations (10 mM, 25 mM, 54 mM, 162 mM) was prepared. Each cotton gauze consisted of 12 layers, and 200 μl of blood was dropped vertically from a certain height without interruption (Fig. 8). After waiting for the blood to sufficiently soak into the gauze, the cotton gauze was inspected visually, and observed using an optical microscope to confirm thrombus formation. Additionally, 2D and 3D drawings were used to illustrate this process.

[0091] In the control group, which consisted of ordinary cotton gauze, blood permeated up to 9 layers. In the gauze coated with 10 mM calcium lactate, blood permeated 5 layers; in the coated gauze coated with 25 mM and 54 mM, 7 layers; and in the cotton gauze coated with 162 mM, blood permeated up to 9 layers, the same as the control group. (Fig. 9) Thrombi formed on the calcium lactate-coated gauze. A thrombus with a dark color in the center and a light red serum region at the edge can be observed. (Fig. 10) To confirm this in detail, observation was performed using an optical microscope. In the third gauze, compared to the control group, the surfaces of the 10 mM and 25 mM threads appear black because light is blocked due to the thrombus. In the case of the 10 mM gauze, blood dripped and a thrombus formed rapidly, allowing a clean surface to be observed in the seventh gauze. (Fig. 11)

[0092] It was confirmed that blood clots formed on calcium lactate-coated gauze, and the lengths of the blood clot and serum portions of each gauze were measured. The lengths of the major and minor axes were measured under the assumption that the gauze is elliptical. The measured values ​​were reconstructed into a cross-section to obtain an image as shown in Fig. 9, and then reconstructed in 3D as shown in Fig. 13 using the Solid Edge program. In the 3D imaging process, the spacing between the cotton gauze was set to 1 mm.

[0093] Volume was measured using a 3D program to compare the precise size of thrombus formation. The control group showed zero thrombus, while the 10 mM calcium lactate-coated cotton gauze measured 349.7 mm. 3 , 483.7 mm on 25 mM calcium lactate-coated cotton gauze 3 , 386.6 mm on 54 mM calcium lactate-coated cotton gauze 3 And 473.4 mm on cotton gauze coated with 162 mM calcium lactate 3 The thrombus volume value was obtained. (Fig. 12)

[0094] 2-4 Evaluation of Thrombotic Stability of Cotton Gauze Soaked in Calcium Lactate Solution

[0095] Changes in blood clots were checked on cotton gauze soaked in calcium lactate solution. A sufficient amount of distilled water and 10 mM calcium lactate solution was sprayed onto the cotton gauze. Wait 3 to 5 minutes until the liquid was evenly absorbed. Once the cotton gauze had sufficiently absorbed the liquid, it was lifted and shaken until no liquid dripped, and then used for the experiment. 100 µl of blood was injected while the tip remained slightly attached to the surface of the gauze.

[0096] Standard cotton gauze holds blood to some extent at the surface. However, on cotton gauze soaked in distilled water, blood spread more easily along the distilled water. On gauze soaked in a 10 mM calcium lactate solution, blood also spread widely, but compared to distilled water, the area of ​​blood at the bottom was smaller and the color was lighter. This result is because as the blood spreads, coagulation occurs due to calcium, trapping red blood cells; consequently, the concentration of serum increases and the concentration of blood cells decreases as one moves toward the bottom. Furthermore, the front surface, into which blood was injected, also shows differences in blood cell aggregation due to calcium. (Fig. 14)

[0097] To observe precise differences in serum, all moisture soaked in the cotton gauze was removed in a dry oven. The color of the dried blood on standard cotton gauze is uniform. On cotton gauze soaked in distilled water, some blood cells are captured at the injection site, but most dissolve in the distilled water and spread evenly across the gauze. In contrast, cotton gauze soaked in an aqueous calcium lactate solution forms blood clots very rapidly. In particular, the blood coagulates rapidly from the injection site toward the center, and because only the serum dissolves in the solution and spreads throughout, the color of the serum appears lighter than on the cotton gauze soaked in the aqueous solution. (Fig. 15)

[0098] Example 3: Anti-intestinal adhesion activity test in house rats

[0099] 3-1. Visual Evaluation of the Efficacy of a Calcium Lactate-Based Surgical Cleanser in Preventing Postoperative Intestinal Adhesion

[0100] To investigate the pharmacological effects of calcium lactate, an organ adhesion model was constructed using SD (Sprague-Dawley) rats. Calcium lactate was prepared at 2.5 mM in 0.9% sodium chloride. Four groups were prepared: a control group (G1, 3 rats) treated with physiological saline, a negative control group (G2, 10 rats) treated with physiological saline after inducing intestinal adhesion, an experimental group (G3, 10 rats) treated with 2.5 mM calcium lactate in physiological saline after inducing intestinal adhesion, and a positive control group (G4, 10 rats) treated with Guardix sol® after inducing intestinal adhesion.

[0101] Anesthesia was administered by intramuscular injection of Zoletil (Vibac Korea Co. Ltd, South Korea) 50 mg / kg and Rompun (Bayer Korea Ltd, South Korea) 10 mg / kg in the laboratory of the Rodent SPF area within the Non-Clinical Support Center. The periareolar area of ​​the house rat's abdomen was shaved, and after disinfection with alcohol and povidone, the abdomen was opened along the midline to expose the cecum. For G1, the exposed organ was repositioned and the skin was sutured with non-absorbable sutures, while for G2–G4, the following procedure was followed. Bleeding was induced in the cecum by creating friction with gauze, and bleeding was induced in the abdominal wall by creating a wound using an 8 mm biopsy puncher. Subsequently, an anti-adhesion agent was applied, and the bleeding site was brought into contact with the abdominal wall by suturing the periareolar area. After confirming that the bleeding sites were in contact, the skin was sutured with non-absorbable sutures and disinfected with povidone. Euthanasia was performed in the second week of applying the anti-adhesion agent, and photographs, an assessment of the degree of adhesion, and an autopsy were conducted.

[0102] General symptoms were observed at least once a day for up to one week after transplantation, and thereafter at intervals of at least twice a week until the end of the study (2 weeks). No animals died during the study period, all individuals gained body weight, and no abnormal symptoms were observed. (Fig. 16)

[0103] The degree of adhesion at the sites inducing organ adhesion was assessed visually during the autopsy, and scores were applied based on the Score table. (Top right of Fig. 17) To minimize subjective results, two participants in the study cross-evaluated using a blinded method, and the degree of adhesion was assessed as the average of the final scores. This is a visual confirmation of the results. (Left of Fig. 17) The adhesion scores for each group were evaluated as 0 points for G1, 4.0±0.0 points for G2, 2.5±0.6 points for G3, and 2.7±1.9 points for G4. (Bottom right of Fig. 17)

[0104] 3-2. Histological Analysis for Evaluating the Anti-adhesion Efficacy of Calcium Lactate Detergents

[0105] CO2 euthanasia was performed two weeks after the application of the test substance. CO2 was injected into the chamber at a rate of 30–70% / min, and euthanasia was induced over a period of 5 minutes. Excised specimens and tissues were fixed in 10% formalin for histopathological evaluation. After preparing paraffin blocks and tissue slides, Hematoxylin & Eosin (H&E) and Masson's Trichrome (MT) staining were performed. The degree of inflammatory cell infiltration at the adhesion-inducing site was confirmed via H&E, while the evaluation of the adhesion-induced tissue was performed using MT staining. To quantify the analysis, scores were calculated based on established criteria. (Figure 18, top right; Figure 19, top right)

[0106] The adhesion scores for each group were evaluated as 0 points for G1, 4.0±0.0 points for G2, 2.5±0.6 points for G3, and 2.7±1.9 points for G4. (Bottom right of Fig. 17) In the visual evaluation, G3 treated with calcium lactate showed a good anti-adhesion effect, which was comparable to the results of G4 using Guardix sol.

[0107] As a result of evaluating the degree of adhesion using MT-stained tissue slides, an adhesive response between the abdominal wall and the cecum was observed in some or all individuals of all adhesion-induced groups (left side of Fig. 18). The adhesion scores for each group were evaluated as 0 points for G1, 2.7±0.7 points for G2, 1.9±0.7 points for G3, and 1.1±1.0 points for G4. Compared to G4 treated with Guardix sol, G3 treated with calcium lactate showed a relatively lower effect in preventing adhesion, but was confirmed to exhibit a similar trend. (bottom right side of Fig. 18)

[0108] As a result of evaluating the degree of inflammatory cell infiltration via H&E, infiltration of polymorphonuclear cells, lymphocytes, and macrophages was observed in all groups with induced adhesion. Plasma cells and giant cells were not observed in any group, and necrosis was not confirmed. (Figure 19, left) The inflammatory cell infiltration scores were measured as 0 points in G1, 4.9±1.9 points in G2, 3.3±1.7 points in G3, and 2.6±2.5 points in G4. Although the inflammatory cell infiltration score in G3 was slightly higher compared to G4, there was no statistically significant difference. (Figure 19, bottom right)

Claims

1. A blood coagulation composition comprising calcium lactate as an active ingredient.

2. A blood coagulation composition according to claim 1, characterized in that the calcium lactate is in the range of 1.5 mM or more to less than 162 mM.

3. A blood coagulation composition according to claim 1 or 2, characterized in that the calcium lactate is dispersed on an absorbent carrier.

4. In paragraph 3, the absorbent carrier comprises cotton, polysaccharide, albumin, cellulose, methylcellulose, alkyl hydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose sulfate, salt of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxidized cellulose; Collagen such as gelatin or collagen-sponge, chitin, carboxymethyl chitin, hyaluronic acid, salt of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfate, carboxymethyl dextran, heparin, heparin sulfate, heparan, heparan sulfate, dermatan sulfate, keratan sulfate, carrageenan, starch, amylose, amylopectin, poly-N-glucosamine, poly-N-acetylglucosamine, polymannuronic acid, polyglucuronic acid, polygluronic acid; A pharmaceutical composition comprising a biocompatible polymer selected from the group consisting of chitosan, chitin, chitin-glucan, chitosan-glucan, carboxymethyl chitosan, chitosan salts, chitosan derivatives thereof, and any combination thereof; polyurethane; oxidized polysaccharides, derivatives or combinations of any of the above.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein the composition is provided as a dry adhesive coating, aerosol, dry aerosol, pump spray, medical compression bandage; film; coated plaster; sponge or surgical patch containing a medicinal substance; hemostatic pad; gauze; ointment, semi-gel, gel, foam, paste, suspension, ointment, emulsion, moldable form, nasal plug, surgical dressing, wound packing, bandage, cotton swab, catheter, optical fiber, syringe, pessary, suppository, or suspension of a liquid or non-aqueous liquid.

6. A method for treating a wound or reducing bleeding at a bleeding site, comprising the step of administering a composition of any one of claims 1 to 4 to the wound or bleeding site.

7. Use of calcium lactate for the manufacture of products for hemostasis.

8. The use of calcium lactate according to claim 7, characterized in that the calcium lactate is in the range of 1.5 mM or more to less than 162 mM.

9. The use of calcium lactate according to claim 7 or 8, characterized in that the calcium lactate is dispersed on an absorbent carrier.

10. An anti-adhesion composition comprising calcium lactate as an active ingredient.

11. An anti-adhesion composition according to claim 10, characterized in that the calcium lactate is in the range of 1.5 mM or more to less than 162 mM.

12. A method for preventing adhesions during and after surgery, comprising the step of administering a composition of any one of claims 10 to 11 during and after surgery.

13. A method according to claim 12, wherein the surgery is at least one of orthopedic surgery, ophthalmic surgery, gastrointestinal surgery, abdominal surgery, thoracic surgery, cranial surgery, cardiovascular surgery, gynecological surgery, arthroscopic surgery, urological surgery, plastic surgery, and musculoskeletal surgery.

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