Crosslinked gelatin-based absorbable hemostatic composition for use in body

Abstract

The present invention relates to a paste-type absorbable hemostatic composition based on cross-linked gelatin and a preparation method therefor. Being prepared on the basis of a quantified degree of gelatin crosslinking, the paste-type absorbable hemostatic composition according to the present invention exhibits superior hemostatic efficacy compared to existing commercialized hemostatic products, even when used alone, and furthermore, when needed in clinical settings, can further enhance hemostatic efficacy by additionally mixing plasma-derived medicinal products.

Description

Absorbable hemostatic composition for internal use based on cross-linked gelatin

[0001] The present invention relates to a paste-type absorbable hemostatic composition for internal use based on cross-linked gelatin and a method for producing the same. Furthermore, the present invention relates to a method for measuring the degree of cross-linking of the produced cross-linked gelatin hemostatic composition.

[0002] Blood is composed of plasma, a liquid component that accounts for 55% of the total blood volume, and blood cells, which are cellular components that account for the remaining 45%. Blood cells include red blood cells, white blood cells, other blood cells, and platelets dispersed within the liquid phase.

[0003] In general, hemostasis is performed at the site of bleeding in the surgical field. Hemostasis methods include ligation, compression, electrocoagulation, and the use of physiologically active substances such as thrombin or fibrin glue. In the case of arterial bleeding with a clear bleeding point, ligation or electrocoagulation are used, and in the case of venous bleeding, ligation or compression are generally used.

[0004] However, when capillary bleeding from a real organ or bleeding from a vascular anastomosis occurs, the above-described hemostatic method may not be effective, and this may cause difficulties in hemostasis in cases such as when performing surgery using heparin, an anticoagulant, in the field of cardiovascular surgery, in cases where there is a tendency to bleed due to liver failure, or in cases involving extensive dissection during general surgery.

[0005] When hemostasis is difficult, topical absorbable hemostatic agents are used. Simply applying them to the bleeding area promotes blood clotting and rapidly forms a clot, thus stopping the bleeding. These agents are frequently used in surgical procedures, not only shortening the surgical time but also preventing postoperative rebleeding.

[0006] Common local absorbable hemostatic agents include cellulose-based hemostatic agents, gelatin-based hemostatic agents, atelocollagen-based hemostatic agents, and microfibrillar collagen-based hemostatic agents.

[0007] Protein-based hemostatic materials are commercially available in solid sponge or powder form for use in surgical procedures. When the powder is mixed with a fluid, such as saline or thrombin solution, a paste or slurry can be formed, depending on the mixing conditions and the relative proportions of the ingredients, which is useful as a hemostatic composition, particularly for diffuse bleeding from uneven surfaces or difficult-to-reach areas.

[0008] Conventional hemostatic pastes are manufactured by mixing natural or synthetic polymers as hydrophilic, blood-absorbing matrix materials with a liquid (e.g., thrombin solution) prior to use. Mixing to form a paste typically requires extensive mixing, such as kneading or transfer between two syringes.

[0009] Natural or synthetic polymers can be used as hydrophilic, blood-absorbing matrix materials. Known natural polymers include biodegradable materials such as collagen, chitosan, and gelatin. Collagen, a key component of connective tissues such as skin, bone, cartilage, tendons, and ligaments, is widely used as a medical material for hemostatic agents and tissue engineering scaffolds. However, its mechanical properties are limited, requiring chemical crosslinking to enhance its physical properties. Potential immunogenicity and high cost are drawbacks. Chitosan, the second most abundant natural polymer found in nature, is frequently used in medical applications and boasts excellent biocompatibility and low toxicity. However, its general drawbacks include low solubility in organic solvents, relatively slow biodegradation, and a tendency to crystallize. In contrast, gelatin, a natural polymer that transforms the triple helix structure of collagen into a single helix, offers excellent biocompatibility and hygroscopicity, ease of processing, and a relatively low raw material price. This makes it a valuable tool for tissue engineering and hemostatic agents.

[0010] Uncross-linked gelatin is vulnerable to sterilization during the manufacturing process and degrades in the body immediately after administration, impairing its hemostatic function. Therefore, a cross-linking process is necessary for gelatin. The degree of cross-linking within a hemostatic composition significantly impacts the physical properties and efficacy of the developed hemostatic agent. While there are numerous commercially available absorbable gelatin- or collagen-based hemostatic agents for internal use, no product has been developed to date that quantifies the degree of cross-linking within the composition to enhance hemostatic function.

[0011] Meanwhile, an analytical method that can quantitatively analyze the degree of cross-linking of gelatin is very important, but there are limitations to the existing methods utilizing nuclear magnetic resonance (NMR) or Fourier transform infrared spectroscopy (FT-IR).

[0012] Accordingly, the inventors of the present invention have discovered a method for quantitatively quantifying the degree of crosslinking of gelatin using a high performance liquid chromatography (HPLC) device, and based on this, have confirmed the effect of significantly improving the hemostatic ability of a paste-type absorbable hemostatic composition for use in the body containing crosslinked gelatin having an appropriate degree of crosslinking, thereby completing the present invention.

[0013] Object 1 of the present invention is to provide an absorbable hemostatic composition for internal use in a paste type.

[0014] Object 2 of the present invention is to provide a method for producing cross-linked gelatin having a cross-linking degree of 2-15%.

[0015] Object 3 of the present invention is to provide a method for quantitatively analyzing the degree of cross-linking of cross-linked gelatin.

[0016] Object 4 of the present invention is to provide a hemostatic method for administering a paste-type absorbable hemostatic composition for use in the body containing cross-linked gelatin having a cross-linking degree of 2-15%.

[0017] Object 5 of the present invention is to provide a use of cross-linked gelatin having a cross-linking degree of 2-15% in the manufacture of an absorbable paste-type composition for internal use for hemostasis.

[0018] Absorbable hemostatic composition for internal use in paste form

[0019] The present invention provides an absorbable hemostatic composition for use in the body in a paste form containing cross-linked gelatin having a cross-linking degree of 2-15%.

[0020] The present invention provides a hemostatic method for administering a paste-type absorbable hemostatic composition for internal use containing cross-linked gelatin having a cross-linking degree of 2-15%.

[0021] The present invention provides a use of cross-linked gelatin having a degree of cross-linking of 2-15% in the manufacture of an absorbable paste-type composition for internal use for hemostasis.

[0022] The above cross-linking degree may preferably be 3-12%, more preferably 4-10%, and particularly preferably 8-10%. If the cross-linking degree is below the lower limit of the above range, the gelatin may decompose, making storage and distribution difficult. If it exceeds the upper limit, the gelatin may not be completely decomposed within the body for several months and may remain, causing side effects such as allergic reactions to animal proteins.

[0023] The above cross-linked gelatin may be 9-20 wt%, preferably 11-17 wt%, and more preferably 12-15 wt% of the total weight of the composition. If it is less than the lower limit of the above content range, the formulation may be runny when mixed, making it difficult to produce a paste, and if it exceeds the upper limit, the formulation may be crumbly when mixed, making it difficult to produce.

[0024] The above cross-linked gelatin can be prepared in a paste form by mixing it with a biocompatible solvent such as a CaCl2 aqueous solution, PBS (phosphate-buffered saline), physiological saline, or sterile distilled water, and any known biocompatible solvent capable of dispersing cross-linked gelatin can be used without any restrictions.

[0025] In the composition according to the present invention, a plasma fractionation agent may be further included.

[0026] Examples of the above plasma fractions include thrombin; fibrinogen; prothrombin; tissue thromboplastin; Ca 2+ ; Ac globulin; proconvertin; antihemophilic factor; Christmas factor; Stewart-Prawer factor; plasma thromboplastin precursor; Hagemann factor; fibrin stabilizing factor; Fletcher factor; Fizgerald, Williams, Flaujeac factor; protein C; protein S; thrombomodulin; coagulant factor VIII; coagulant factor IX; antithrombin III; albumin, etc. can be used alone or in combination of two or more.

[0027] In the composition according to the present invention, the gelatin may be gelatin derived from pig dermis or bovine dermis, but is not limited thereto.

[0028] The above cross-linking degree is the ratio of cross-linked lysine among the total lysine of the constituent amino acids of gelatin, and can be expressed numerically using the following mathematical formula 1:

[0029] [Mathematical Formula 1]

[0030] .

[0031]

[0032] Method for producing cross-linked gelatin with a cross-linking degree of 2-15%

[0033] The present invention comprises a step (step 1) of dispersing gelatin particles in a water-soluble solvent to obtain a gelatin dispersion;

[0034] A step (step 2) of adding a cross-linking agent to the above gelatin dispersion to obtain cross-linked gelatin with a cross-linking degree of 2-15%; and

[0035] A step (step 3) comprising: after washing the cross-linked gelatin, adding a reducing agent to obtain reduced cross-linked gelatin;

[0036] A method for producing cross-linked gelatin having a cross-linking degree of 2-15% is provided.

[0037] In the manufacturing method according to the present invention, the water-soluble solvent of step 1 may be PBS (phosphate-buffered saline), physiological saline, sterile distilled water, etc., and any known biocompatible solvent capable of dispersing gelatin may be used without any limitation.

[0038] In the manufacturing method according to the present invention, the cross-linking agent of step 2 may be glutaraldehyde (GTA), transglutaminase, formaldehyde, 1,4-butanediol diglycidyl ether (BDDE), genipin, etc., used alone or in combination of two or more thereof, and any known cross-linking agent for gelatin cross-linking that is not harmful to the body may be used without any restrictions.

[0039] In the manufacturing method according to the present invention, the reducing agent of step 3 is sodium borohydride (NaBH4), sodium cyanoborohydride (NaBH3CN), potassium borohydride (KBH4), lithium borohydride (LiBH4), lithium triethylborohydride (C6H 13 BLi), borane pyridine complex (C5H8BN), hydrazine (N2H4), etc. can be used, and among the known reducing agents for cross-linked gelatin reduction, if it is a non-toxic ingredient, it can be used without any restrictions.

[0040]

[0041] Method for quantitatively analyzing the degree of cross-linking of cross-linked gelatin

[0042] The present invention comprises a step (step 1) of hydrolyzing cross-linked gelatin particles to obtain a hydrolyzate;

[0043] A step of drying the above hydrolyzate (step 2);

[0044] Step 3: Dissolving the above dried hydrolyzate and then derivatizing it with OPA (o-phthalaldehyde) to prepare a sample; and

[0045] A step (step 4) of quantitatively analyzing the degree of cross-linking of the above sample using a high-performance liquid chromatography (HPLC) device;

[0046] In the above step 4, the degree of cross-linking is normalized to glycine by normalizing lysine in cross-linked gelatin reacted with OPA (o-phthalaldehyde), and a method for quantitatively analyzing the degree of cross-linking of cross-linked gelatin is provided, which is expressed as a ratio of cross-linked lysine among the total lysine of the constituent amino acids of gelatin, using the following mathematical formula 1:

[0047] [Mathematical Formula 1]

[0048] .

[0049] The absorbable hemostatic composition for internal use in a paste type according to the present invention is manufactured based on a quantified degree of gelatin cross-linking, and not only has a superior hemostatic effect compared to existing commercial hemostatic products even when used alone, but also has the effect of further improving the hemostatic effect by additionally mixing a plasma fraction preparation when necessary in a medical setting.

[0050] Figure 1 is a schematic diagram illustrating the mechanism of gelatin crosslinking and reduction using the crosslinking agent glutaraldehyde (GTA). Lysine in gelatin molecules reacts with glutaraldehyde, forming crosslinks between adjacent gelatin molecules.

[0051] Figure 2a shows the results of measuring absorbance according to wave number in gelatin before and after crosslinking using a Fourier-transform infrared spectroscopy (FT-IR) device.

[0052] Figure 2b is a proton nuclear magnetic resonance (PNMR) 1 This is the result of showing the spectrum according to the resonance frequency in gelatin before and after crosslinking (before crosslinking: Gelatin, after crosslinking: Crosslinked Gelatin) using a H-NMR device.

[0053] Figure 2c shows the results of quantifying the area of ​​the lysine peak of cross-linked gelatin according to the amount of glutaraldehyde cross-linking (0, 8, 15, 27%) using a high-performance liquid chromatography (HPLC) device (top), and measuring the degree of cross-linking from this (bottom).

[0054] Figure 3 shows the results of confirming the stability of gelatin particles with various degrees of cross-linking (0, 1, 2, 5, 7, 8%) after storage at 50°C for 1 hour.

[0055] Figure 4a shows the results of comparing and confirming the in vivo degradation pattern of cross-linked gelatin injected into the dorsal part of an SD rat animal model (n=2) with gelatin particles having cross-linking degrees of 8% and 15% by observing and measuring the size of the remaining gelatin 1 week, 2 weeks, 3 weeks, and 7 weeks after injection.

[0056] Figure 4b shows the in vivo degradation patterns of cross-linked gelatin injected into the dorsal part of an SD rat animal model (n=2) with gelatin particles having cross-linking degrees of 8% and 15%. The results were compared and confirmed by sectioning the area 2, 3, and 7 weeks after injection, and then observing and measuring under a microscope through H&E (Hematoxylin and eosin staining).

[0057] Figure 5 is an image showing the process of evaluating the hemostatic efficacy using an SD rat animal model in the following order: (i) hair removal, (ii) disinfection, (iii) incision and exposure of liver tissue, (iv) induction of bleeding, and (v) application of sample material.

[0058] Figure 6a shows the results of measuring the hemostasis time (sec) of samples that did not contain plasma fraction (thrombin) using an SD rat animal model (n=10) and comparing their effectiveness.

[0059] Sample #1: Sterilized gauze, negative control

[0060] Sample #2: Commercial collagen-based hemostatic agent (Osgen, Korea), positive control

[0061] Sample #3: Cross-linked 4% gelatin-based hemostatic agent

[0062] Sample #4: Cross-linked 9% gelatin-based hemostatic agent

[0063] Figure 6b shows the results of comparing the effectiveness of samples that did not contain plasma fractions (thrombin) and measured the amount of bleeding (total blood loss, mg) using an SD rat animal model (n=10).

[0064] Figure 6c shows the results of comparing the effectiveness of samples that did not contain plasma fractions (thrombin) and the 3-minute hemostasis success rate (hemostasis in 3 minutes, %) using an SD rat animal model (n=10).

[0065] Data in Figure 6 = mean ± standard error; and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0066] Figure 7a shows the results of measuring the hemostasis time (sec) of samples including sample #1 and plasma fraction (thrombin) using an SD rat animal model (n=10) and comparing their effectiveness.

[0067] Sample #1: Sterilized gauze, negative control

[0068] Sample #5: Commercial gelatin-based hemostatic agent (Baxter, USA) with thrombin, positive control

[0069] Sample #6: Cross-linked 4% gelatin-based hemostatic agent w / thrombin

[0070] Sample #7: Cross-linked 9% gelatin-based hemostatic agent w / thrombin

[0071] Figure 7b shows the results of comparing the effectiveness of the blood loss (total blood loss, mg) of samples #1 and plasma fraction (thrombin) using an SD rat animal model (n=10).

[0072] Figure 7c shows the results of comparing the effectiveness of the 3-minute hemostasis success rate (hemostasis in 3 minutes, %) of samples including sample #1 and plasma fraction (thrombin) using an SD rat animal model (n=10).

[0073] Data in Figure 7 = mean ± standard error; and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0074] Hereinafter, the present invention will be described in detail.

[0075]

[0076] Hemostatic agents are available in powder, paste, and sheet types. The "paste type" hemostatic agent of the present invention can be widely used in surgical operations without limitation in the scope of application, including a powder formulation within a sealing unit suitable for adding liquid to the agent and mixing the contents within the container.

[0077]

[0078] Biodegradable materials such as collagen, chitosan, and gelatin are known as natural hemostatic agents. Collagen, a key component of connective tissues such as skin, bone, cartilage, tendons, and ligaments, is widely used as a hemostatic agent and scaffold for tissue engineering. However, its mechanical properties are limited, requiring chemical cross-linking to enhance its physical properties. Potential immunogenicity and high cost are drawbacks. Chitosan, the second most abundant natural polymer found in nature, is frequently used in medical applications and boasts excellent biocompatibility and low toxicity. However, its general drawbacks include low solubility in organic solvents, relatively slow biodegradation, and a tendency to crystallize. In contrast, gelatin, a natural polymer that transforms the triple helix structure of collagen into a single helix, offers excellent biocompatibility, hygroscopicity, and processability, making it a valuable tool for tissue engineering and hemostatic applications.

[0079]

[0080] The paste-type absorbable hemostatic composition for internal use containing the cross-linked gelatin of the present invention must be manufactured through a cross-linking process of gelatin, which is the matrix of the hemostatic agent, to have appropriate physical properties, hygroscopicity, and biodegradability as a hemostatic agent. "Crosslinking" is used for the purpose of improving the physicochemical properties of a polymer by connecting polymer chains through a chemical or physical method, and generally, the higher the degree of crosslinking (degree of crosslinking), the lower the thermoplastic properties of the polymer material, while the higher the mechanical strength and heat resistance. As a cross-linking agent for cross-linking gelatin, glutaraldehyde (GTA), transglutaminase, formaldehyde, 1,4-butanediol diglycidyl ether (BDDE), genipin, etc. can be used, but are not limited thereto.

[0081]

[0082] Since the degree of crosslinking is an important factor that determines the physicochemical and biological properties of polymer materials, it is necessary to analyze the degree of crosslinking of gelatin synthesized during the manufacturing process. The currently used method for analyzing the degree of crosslinking of gelatin is proton nuclear magnetic resonance (PNMR). 1 There is a method using a H-NMR instrument. Since the integrated intensity of a proton nuclear magnetic resonance spectroscopy signal is proportional to the nuclide appearing in the signal, quantification is possible based on a specific signal of the compound to be quantified. In other words, if a standard material with a known molecular structure is added to the sample for accurate quantitative analysis, 1In the H-NMR spectrum, the signal integral intensity of the standard substance and the specific signal integral intensity of the compound to be quantified can be obtained, and since the molecular structure and amount of the standard substance are known, quantitative analysis of the compound in the sample is possible. The standard substance added with the sample in this way is called an 'internal standard'. However, the above quantitative analysis has the problem that it is difficult to apply to insoluble samples such as gelatin. Significant 1 To obtain a H-NMR spectrum, the sample and standard substance must be uniformly mixed, but in the case of an insoluble sample, it is difficult to uniformly mix it in a solvent.

[0083]

[0084] Another analytical method, Fourier-transform infrared spectroscopy (FT-IR), can be used to determine the presence of bonds between gelatin and polymer chains. FT-IR allows for the quantification of gelatin crosslinking in situ and in real time by measuring the vibrational energy changes occurring at the solid-liquid interface. However, this method has limitations, as the measured absorbance bands can easily overlap depending on the vibration mode, system complexity, or the similarity of the gelatin crosslinking degree.

[0085]

[0086] Gelatin is produced by the denaturation of collagen found in materials such as pig skin, cow skin, hides, and animal bones. Like collagen, gelatin is also identified by its unique blend of amino acids. Native collagen is based on polypeptide chains containing approximately 1,050 amino acids. Due to its three-dimensional network structure, collagen is generally insoluble in solvents and forms a mixture. When collagen is degraded, it becomes gelatin, and when gelatin undergoes hydrolysis, it becomes soluble as a hydrolysate. After hydrolysis, approximately one-third of the amino acids are glycine, 22% are proline and 4-hydroxyproline, and the remaining 45% are 17 different amino acids. Thus, gelatin can be analyzed by amino acid composition through hydrolysis. Cross-linked gelatin can be quantitatively analyzed using high-performance liquid chromatography (HPLC) by measuring the differential amino acid distribution depending on the concentration and type of cross-linking agent.

[0087]

[0088] Absorbable hemostatic compositions for internal use are intended for application to the human body during surgical procedures. Therefore, they must be free of side effects such as fever, inflammation, and toxicity, and exhibit excellent biodegradability after administration. Gelatin-based hemostatic compositions naturally degrade in vivo over time in response to various environmental factors, including moisture, microorganisms, and temperature. However, if degradation is not sufficient, they can trigger an excessive immune response, leading to adverse effects in the body. Therefore, it is generally recommended that absorbable hemostatic compositions for internal use completely degrade within several months of administration.

[0089]

[0090] Plasma derivatives are specific proteins contained in blood, such as blood coagulation factors, immunoglobulins (proteins containing antibodies), and plasma proteins (albumin, globulins). These various plasma derivatives can be manufactured from natural or recombinant proteins. However, natural substances are difficult to obtain due to regulatory restrictions, and synthetic substances have limitations such as high production costs. The absorbable, paste-type hemostatic composition for internal use comprising cross-linked gelatin proposed in the present invention is utilized as a hemostatic matrix, and binds to platelets to form platelet clumps and activates blood coagulation factor XII, which in turn induces the initiation of the intrinsic pathway of the secondary hemostatic process. This process can promote thrombin generation, thereby demonstrating excellent hemostatic performance during surgical procedures. Furthermore, the gelatin-based hemostatic composition of the present invention can be used in combination with existing commercially available plasma derivatives (typically blood coagulation factors such as thrombin).

[0091]

[0092] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0093]

[0094] <Manufacturing Example 1> Manufacturing of an absorbable in vivo hemostatic composition based on cross-linked gelatin

[0095] In one manufacturing example of the present invention, to prepare an absorbable hemostatic composition for internal use, first, 0.05 to 0.1 M PBS, 0.15 to 0.5 M NaCl, and 0.5 to 1.0% Triton X-114 were dissolved in distilled water at 4°C to room temperature to prepare a dispersion solvent for gelatin particles. Gelatin particles were added to the dissolved solvent, and glutaraldehyde (GTA) was added according to the crosslinking concentration, and stirred for 10 to 16 hours. Gelatin crosslinking is performed by forming a bond between the amine group of lysine of gelatin and the aldehyde group of glutaraldehyde (GTA), as shown in Fig. 1. After the crosslinking reaction, a first washing was performed using distilled water on a vacuum-state PP (polypropylene) filter. After the first washing, sodium borohydride (NaBH4) reducing agent was used at a ratio of 2 to 4 times that of the cross-linking agent to remove the uncross-linked amine groups in the gelatin particles and the aldehyde groups in glutaraldehyde (GTA) that did not undergo cross-linking reaction at room temperature for 1 to 2 hours. After the reduction reaction, the second washing was performed using distilled water in a vacuum PP filter, and the cross-linked gelatin particles were then freeze-dried for 16 to 48 hours. The freeze-dried gelatin particles were ground with a cutting mill equipped with a 500 to 750 μm sieve to prepare a hemostatic composition. The prepared cross-linked gelatin powder was hydrated with a 0.1 to 1% CaCl2 solution to prepare a finished composition, and when the composition included a plasma fraction, 500 IU of thrombin was added to the composition.

[0096]

[0097] <Analysis Example 1> Analysis of the degree of crosslinking of a crosslinked gelatin composition using FT-IR

[0098] In order to measure the degree of crosslinking of the crosslinked gelatin-based absorbable hemostatic composition for body use prepared in Manufacturing Example 1 using a Fourier transform infrared spectroscopy (FT-IR) device, two samples of gelatin before and after crosslinking were mixed with KBr (Spectrum pure) at a weight ratio of 1:10 to 100 and pressed into transparent sheets. Afterwards, FT-IR (Perkin Elmer, Inc., USA) was used to measure the degree of crosslinking at room temperature in the range of 400 to 4000 cm -1 The transmittance was measured in the wave number range of 1620-1690 cm in gelatin before and after crosslinking, as shown in Fig. 2a. -1 Imine bonds were observed at the wavelength of 1630 cm -1 It was confirmed that quantitative cross-linking analysis was difficult due to overlap with the amide I peak (Fig. 2a).

[0099]

[0100] <Analysis Example 2> 1 Analysis of the degree of cross-linking of cross-linked gelatin compositions using H-NMR

[0101] Proton nuclear magnetic resonance (NPM) of the gelatin-based absorbable hemostatic composition for body use manufactured through the above manufacturing example 1 1 To measure the degree of crosslinking using H-NMR, two samples of gelatin before and after crosslinking were dissolved in D2O, an NMR solvent, and the nuclide 1 It was set to H. Afterwards, the molecular structure was confirmed by measuring the absorbance using a 400MHz FT-NMR spectrometer (Avance III HD 400, Bruker, USA). However, as shown in the results of Fig. 2b (gray shaded area), it was difficult to obtain reliable data due to the overlapping of identical peaks (1.2-1.5 ppm) of gelatin before and after crosslinking (Fig. 2b).

[0102]

[0103] <Analysis Example 3> Analysis of the degree of cross-linking of cross-linked gelatin composition using HPLC

[0104] In order to measure the degree of crosslinking of the composition manufactured using high-performance liquid chromatography (HPLC) proposed in the present invention, hydrolysis of crosslinked dried gelatin particles was first performed. After placing the crosslinked gelatin in a glass tube, vacuum purging was performed with 1 to 6 N HCl for 10 to 20 minutes, and then hydrolysis was performed at 90 to 110°C for 10 to 16 hours using a heat block. The sample after hydrolysis was filtered using a syringe filter, completely dried in vacuum, washed with triple-distilled water, and then vacuum-dried again. The dried sample was prepared by dissolving it in 0.1 to 1 N HCl and filtering it through a 0.45 μm PTFE (poly-tetra-fluoro-ethylene) syringe filter. Afterwards, the product was derivatized by mixing borate buffer, dissolved gelatin, OPA (o-phthalaldehyde), and phosphate buffer, and the peak position and size were measured three times using an Agilent 1260 series LC (USA) instrument at a flow rate of 1.3 ml / min, a sample injection volume of 1 μl, and absorbance conditions of 338 and 262 nm.

[0105] The degree of crosslinking of crosslinked gelatin was normalized by converting lysine reacted with OPA (o-phthalaldehyde) to glycine, and the ratio of crosslinked lysine to the total lysine was expressed as a numerical value using the following mathematical formula 1 (see Figure 1).

[0106] [Mathematical Formula 1]

[0107]

[0108]

[0109] Figure 2c shows the results of quantifying the peak area of ​​lysine according to the amount of glutaraldehyde added (top) using a high-performance liquid chromatography (HPLC) device using the above method, and measuring the degree of crosslinking (bottom, n=3) from this. From this, it was found that the degree of crosslinking of crosslinked gelatin particles can be numerically quantified using the HPLC device proposed in the present invention.

[0110]

[0111] <Example 1> In vitro thermal stability of cross-linked gelatin particles

[0112] FIG. 3 is an image showing the decomposition pattern of gelatin particles after diluting 1 g of gelatin particles with crosslinking degrees of 0, 1, 2, 5, 7, and 8% in 10 ml of distilled water and storing them at 50°C for 1 hour. The degree of crosslinking of each test sample was measured by the method of Analysis Example 3 by varying the amount of glutaraldehyde added. As shown in the image of FIG. 3, if the degree of crosslinking of the absorbable hemostatic gelatin particles for internal use is less than 2%, it may decompose in a short period of time at 50°C, which may limit storage and distribution when commercialized as a medical device, and may also limit its functionality as a hemostatic agent after internal administration. From this, it was found that the degree of crosslinking of the crosslinked gelatin particles in the gelatin-based absorbable hemostatic composition for internal use must be 2% or more.

[0113]

[0114] <Example 2> Confirmation of in vivo decomposition of cross-linked gelatin particles

[0115] To confirm the in vivo degradation of the cross-linked gelatin-based absorbable hemostatic composition manufactured, SD rats (n=2) were anesthetized with 3% isoflurane, the hair on the back was removed with clippers, and pockets for transplanting the cross-linked gelatin composition were created in the subcutaneous tissue on the left and right sides based on the spinal line of the back using Iris scissors. Thereafter, as shown in Fig. 4a (left column image of Fig. 4a), 400 μL of the cross-linked 8% and 15% gelatin compositions were injected into the left and right backs of the SD rats using a syringe, respectively, and the incision sites were sutured.

[0116] After 1, 2, 3, and 7 weeks of injection of the gelatin hemostatic composition, SD rats were sacrificed to obtain samples, and the size of the residual gelatin composition was measured by visual observation. The middle and right columns of Fig. 4a show the injected composition observed in the subcutaneous pocket after 1, 2, 3, and 7 weeks when the cross-linking degree was 8 and 15%, respectively. As shown in Fig. 4a, when the cross-linking degree was 8%, most of the injected composition was degraded after 1 week, and no injected cross-linked gelatin was observed after 2 weeks. On the other hand, when the cross-linking degree was 15%, residual composition was observed even after 7 weeks of injection. Table 1 shows the data obtained by measuring and quantifying the horizontal and vertical sizes of the residual cross-linked gelatin after 1, 2, 3, and 7 weeks of composition injection (n=2, data=mean±standard error).

[0117] Figure 4b shows the results of H&E (Hematoxylin & Eosin) staining, which confirmed the remaining gelatin particles in samples extracted 2, 3, and 7 weeks after injection of the cross-linked gelatin composition. As shown in the purple staining results in Figure 4b, when the cross-linking degree was 8%, no gelatin remained in vivo 2 weeks after injection, and when the cross-linking degree was 15%, residual gelatin particles were found up to 7 weeks after injection. From this, it was found that the gelatin cross-linking degree of the cross-linked gelatin-based absorbable hemostatic composition for in vivo use should be less than 15%.

[0118] From Examples 1 and 2, it was confirmed that the degree of crosslinking of the gelatin-based absorbable hemostatic composition for body use should be 2 to 15% to be suitable for distribution and storage, and also to enable complete decomposition in the body within several months.

[0119]

[0120]

[0121] <Example 3> Evaluation of hemostatic activity in an in vivo animal model (excluding plasma fractions)

[0122] To evaluate the hemostatic activity of the cross-linked gelatin-based hemostatic composition manufactured using an in vivo animal model, SD rats (n=10) were anesthetized with intraperitoneal injection of Zoletil 50 (30 mg / kg) / Rompun (10 mg / kg). The breathing and anesthesia state were observed. The abdominal area was shaved and disinfected three times with alcohol and povidone. An incision of approximately 4-5 cm was made in the center of the abdomen of the SD rat using surgical scissors. The liver inside the incision was exposed to the outside of the abdominal cavity. After wiping the body fluid and blood on the liver surface with sterile gauze, a round filter paper was placed under the median lobe of the liver. Afterwards, a 5.0 mm biopsy punch was used to puncture the central part of the median lobe surface to the marked line located approximately 4 mm from the center, and the median lobe was removed to induce bleeding. After confirming the bleeding, the test substance was immediately applied. Figure 5 shows the sequential processes of (i) hair removal, (ii) disinfection, (iii) incision and exposure of liver tissue, (iv) induction of bleeding, and (v) application of experimental substances for the production of this SD rat animal model.

[0123] The results of evaluating the hemostatic ability of hemostatic compositions not containing plasma fractions (Thrombin) applied to the SD rat bleeding animal model are shown in Fig. 6 and Table 2.

[0124] Sample #1: Sterilized gauze, negative control

[0125] Sample #2: Commercial collagen-based hemostatic agent (Osgen, Korea), positive control

[0126] Sample #3: Cross-linked 4% gelatin-based hemostatic agent

[0127] Sample #4: Cross-linked 9% gelatin-based hemostatic agent

[0128] The results of measuring the hemostasis time after applying each sample are shown in Fig. 6a and Table 2 (n=10, data=mean±standard error). Compared to sterile gauze (negative control), the commercial collagen hemostat (positive control), cross-linked 4% gelatin hemostat, and cross-linked 9% gelatin hemostat all showed statistically significant improvement in hemostasis time. However, the average hemostasis times of the cross-linked 4% gelatin hemostat and cross-linked 9% gelatin hemostat were 183.0 and 161.7 s, respectively, which were 9.8 and 20.3% shorter than the hemostasis time of 202.9 s of the commercial collagen hemostat.

[0129] The results of measuring the amount of bleeding (total blood loss) after applying each sample are shown in Fig. 6b and Table 2 (n=10, data=mean±standard error). Compared to sterile gauze (negative control), the commercial collagen hemostat (positive control), cross-linked 4% gelatin hemostat, and cross-linked 9% gelatin hemostat all showed statistically significant improvement in bleeding amount. However, the average bleeding amounts of the cross-linked 4% gelatin hemostat and the cross-linked 9% gelatin hemostat were 1,467 and 1,160 mg, respectively, which were 8.6 and 27.7% less than the bleeding amount of the commercial collagen hemostat of 1,606 mg.

[0130] The results of measuring the hemostasis success rate (hemostasis in 3 min, %) within 3 minutes after applying each sample are shown in Fig. 6c and Table 2 (n=10, data=mean±standard error). Compared to sterile gauze (negative control), the commercial collagen hemostat (positive control), cross-linked 4% gelatin hemostat, and cross-linked 9% gelatin hemostat all showed a significant improvement in the hemostasis success rate within 3 minutes. However, the hemostasis success rate of the cross-linked 4% gelatin hemostat and the cross-linked 9% gelatin hemostat was 90%, which was 20% higher than the hemostasis success rate of the commercial collagen hemostat within 3 minutes of 70%.

[0131]

[0132]

[0133]

[0134] <Example 4> Evaluation of hemostatic activity in an in vivo animal model (including plasma fractions)

[0135] The cross-linked gelatin-based paste-type absorbable hemostatic composition for use in the body of the present invention can be used in a surgical medical setting by being physically mixed with a plasma fraction preparation.

[0136] The results of evaluating the hemostatic ability of hemostatic compositions physically mixed with 500 IU of plasma fraction (thrombin) in Fig. 7 and Table 3 are shown in the SD rat bleeding animal model.

[0137] Sample #5: Commercial gelatin-based hemostatic agent (Baxter, USA) with thrombin, positive control

[0138] Sample #6: Cross-linked 4% gelatin-based hemostatic agent w / thrombin

[0139] Sample #7: Cross-linked 9% gelatin-based hemostatic agent w / thrombin

[0140] The results of measuring the hemostasis time after applying each sample to Fig. 7a and Table 3 (n=10, data=mean±standard error) are shown. Compared to sterile gauze (negative control), the commercial gelatin hemostat (positive control), the cross-linked 4% gelatin hemostat, and the cross-linked 9% gelatin hemostat all showed statistically significant improvement in hemostasis time. However, the average hemostasis times of the cross-linked 4% gelatin hemostat and the cross-linked 9% gelatin hemostat were 178.8 and 162.3 s, respectively, which were shortened by 6.24 and 14.89%, respectively, compared to the hemostasis time of 190.7 s of the commercial gelatin-based hemostat. It was found that the hemostatic composition of the present invention showed a faster hemostasis time than the existing commercial gelatin hemostat.

[0141] The results of measuring the amount of bleeding (total blood loss) after applying each sample are shown in Fig. 7b and Table 3 (n=10, data=mean±standard error). Compared to sterile gauze (negative control), the commercial gelatin hemostat (positive control), cross-linked 4% gelatin hemostat, and cross-linked 9% gelatin hemostat showed statistically significant improvement in bleeding amount. Compared to the bleeding amount of the commercial gelatin hemostat, the bleeding amount of the cross-linked 4% gelatin hemostat increased slightly, but the average bleeding amount of the cross-linked 9% gelatin hemostat was 785 g, which was 33.86% less than the average bleeding amount of 1,187 g of the commercial gelatin hemostat.

[0142] The results of measuring the hemostasis success rate (hemostasis in 3 min) within 3 minutes after applying each sample are shown in Fig. 7c and Table 3 (n=10, data=mean±standard error). Compared to sterile gauze (negative control), the commercial gelatin hemostat (positive control), the cross-linked 4% gelatin hemostat, and the cross-linked 9% gelatin hemostat all showed a significant improvement in the hemostasis success rate within 3 minutes. The hemostasis success rates of the cross-linked 4% gelatin hemostat and the cross-linked 9% gelatin hemostat were 90 and 100%, respectively, which were superior to the hemostasis success rate of 80% within 3 minutes of the commercial gelatin hemostat.

[0143]

[0144]

[0145] From the results of Examples 3 and 4, it was found that the gelatin-based absorbable hemostatic composition for internal use with a crosslinking degree of 4 to 9% manufactured by the present invention can exhibit hemostatic efficacy superior to that of existing commercial products in general, both when used without mixing and when used with a plasma fraction.

[0146]

[0147] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. An absorbable hemostatic composition for internal use in a paste form containing 2-15% of cross-linked gelatin.

2. In paragraph 1, A composition having a cross-linking degree of 3-12%.

3. In paragraph 2, A composition having a cross-linking degree of 4-10%.

4. In paragraph 3, A composition having a cross-linking degree of 8-10%.

5. In paragraph 1, A composition wherein the cross-linked gelatin is 9-20 wt% of the total weight of the composition.

6. In paragraph 1, A composition further comprising a plasma fractionation agent.

7. In paragraph 6, The above plasma fractions include thrombin; fibrinogen; prothrombin; tissue thromboplastin; Ca 2+ A composition comprising at least one selected from the group consisting of; Ac globulin; proconvertin; antihemophilic factor; Christmas factor; Stewart-Prawer factor; plasma thromboplastin precursor; Hagemann factor; fibrin stabilizing factor; Fletcher factor; Fizgerald, Williams, Flaujeac factor; protein C; protein S; thrombomodulin; blood coagulation factor Ⅷ; blood coagulation factor Ⅸ; antithrombin III; and albumin.

8. In paragraph 1, A composition wherein the above gelatin is gelatin derived from pig dermis or bovine dermis.

9. In paragraph 1, The above cross-linking degree is the ratio of cross-linked lysine among the total lysine of the constituent amino acids of gelatin, and is expressed numerically by the following mathematical formula 1: [Mathematical Formula 1] .

10. A step of dispersing gelatin particles in a water-soluble solvent to obtain a gelatin dispersion (step 1); A step (step 2) of adding a cross-linking agent to the above gelatin dispersion to obtain cross-linked gelatin with a cross-linking degree of 2-15%; and A step (step 3) comprising: after washing the cross-linked gelatin, adding a reducing agent to obtain reduced cross-linked gelatin; A method for producing cross-linked gelatin having a cross-linking degree of 2-15%.

11. In paragraph 10, A manufacturing method, wherein the water-soluble solvent of step 1 is at least one selected from the group consisting of PBS, saline solution, and sterile distilled water.

12. In paragraph 10, A manufacturing method, wherein the crosslinking agent of step 2 is at least one selected from the group consisting of glutaraldehyde, transglutaminase, formaldehyde, 1,4-butanediol diglycidyl ether (BDDE), and genipin.

13. In paragraph 10, The reducing agent of the above step 3 is sodium borohydride (NaBH4), sodium cyanoborohydride (NaBH3CN), potassium borohydride (KBH4), lithium borohydride (LiBH4), lithium triethylborohydride (C6H 13 A manufacturing method, wherein the method comprises at least one selected from the group consisting of BLi), borane pyridine complex (C5H8BN), and hydrazine (N2H4).

14. A step of hydrolyzing cross-linked gelatin particles to obtain a hydrolyzate (step 1); A step of drying the above hydrolyzate (step 2); Step 3: Dissolving the above dried hydrolyzate and then derivatizing it with OPA (o-phthalaldehyde) to prepare a sample; and A step (step 4) of quantitatively analyzing the degree of cross-linking of the above sample using a high-performance liquid chromatography (HPLC) device; In the above step 4, the degree of cross-linking is normalized to glycine by normalizing lysine in cross-linked gelatin that has reacted with OPA (o-phthalaldehyde), and the cross-linking ratio of lysine among the total lysine of the constituent amino acids of gelatin is quantified using the following mathematical formula 1: [Mathematical Formula 1] .

15. A hemostatic method comprising the step of administering to a subject an absorbable hemostatic composition for body use in a paste form containing an effective amount of cross-linked gelatin of 2-15%.

16. Use of cross-linked gelatin having a degree of cross-linking of 2-15% for use in the manufacture of an absorbable paste-type composition for internal use for hemostasis.

Images