Method for preparing collagen slurry, and hemostatic kit comprising same

Abstract

The present invention relates to a method for preparing a collagen slurry, and a hemostatic kit comprising same, the method comprising implementing a stable formulation in which active ingredients containing collagen are uniformly dispersed while lowering dependence on mixing, thereby enabling consistent and reproducible hemostatic performance to be exhibited even in an emergency bleeding situation.

Description

Method for manufacturing a collagen slurry and a hemostatic kit containing the same

[0001] The present invention relates to a method for manufacturing a collagen slurry and a hemostatic kit containing the same.

[0002] Rapidly and effectively controlling bleeding occurring during trauma, surgery, or medical procedures is a critical factor in improving patient survival rates and preventing infections or complications. Accordingly, various types of hemostatic agents are used in medical settings, and among them, collagen-based hemostatic agents, which possess excellent biocompatibility and are biodegradable within the human body, are widely utilized.

[0003] Collagen is known to exhibit excellent hemostatic efficacy due to its properties of promoting platelet adhesion and aggregation and inducing the activation of blood coagulation factors. Based on these characteristics, collagen is manufactured in various forms, such as sponges, powders, and sheets, and applied to bleeding sites. However, these types of hemostatic agents have limitations; when the shape of the bleeding site is irregular or deep, sufficient adhesion is difficult, leading to reduced applicability and potential displacement or detachment during the procedure. In the case of powder forms, there is a risk of loss due to scattering or contamination of surrounding tissues, and the hemostatic effect rapidly decreases due to blood saturation in cases of heavy bleeding or in humid environments. To address these issues, liquid hemostatic agents have been proposed. Liquid hemostatic agents can be applied or injected into the bleeding site, allowing for uniform application even to tissues with complex shapes or fine crevices. They also have the advantage of rapidly inducing an initial hemostatic response by increasing the contact area with the tissue, and enable minimally invasive application using syringes.

[0004] However, some currently commercialized hemostatic products adopt a dual syringe structure in which collagen, thrombin, and mannitol are filled in powder form in the first syringe, and an aqueous calcium chloride solution is filled in the second syringe. These products are used by connecting the two syringes and mixing them immediately before application, followed by application to the bleeding site. In this case, multiple mixing cycles and a sufficient mixing process lasting for a certain amount of time are required to form a formulation in which the active ingredients, including collagen in dried powder form, are sufficiently hydrated and uniformly dispersed.

[0005] However, if the mixing process is not sufficiently carried out, the hydration and dispersion of the collagen polymer matrix may proceed incompletely, leading to an uneven distribution of the active ingredient within the formulation, which could potentially cause variations in hemostatic performance. Furthermore, this dependence on mixing can result in variations in formulation quality depending on user proficiency or mixing conditions, posing a problem that can undermine ease of use and the reliability of hemostatic performance in actual clinical settings.

[0006] Therefore, there is a need to develop a hemostatic kit that provides a stable formulation with uniformly dispersed active ingredients containing collagen while reducing reliance on the mixing process, and can exhibit consistent and reproducible hemostatic performance even in situations of urgent bleeding.

[0007] The objective of the present invention is to solve the problems described above, and to provide a method for manufacturing a collagen slurry and a hemostatic kit containing the same, which can achieve a stable formulation in which an active ingredient containing collagen is uniformly dispersed while reducing dependence on the mixing process, and exhibit consistent and reproducible hemostatic performance even in urgent bleeding situations.

[0008] To achieve the above objective, one aspect of the present invention provides a method for preparing a collagen slurry.

[0009] Referring to Fig. 1, first, bovine-derived skin is prepared (S10).

[0010] After selecting the skin obtained from cattle, it is desirable to wash it to remove blood, hair, foreign substances, etc. attached to the surface. Meanwhile, cattle skin has uniform collagen fiber length and arrangement, and has a high Type I collagen content, resulting in excellent gel-forming ability and structural stability. It also has excellent processability for alkali and acid treatment processes, leading to high reproducibility in slurrying and homogenization processes, and may be more advantageous than pig skin in terms of safety and biocompatibility as a medical and hemostatic material.

[0011]

[0012] Then, primary virus inactivation is performed by pre-treating bovine-derived skin with a 1-5% by weight calcium hydroxide aqueous solution and then recovering it by pressing and filtration (S20).

[0013] Primary virus inactivation can be induced by pre-treating the bovine-derived skin prepared in step S10 with a calcium hydroxide aqueous solution of 1 to 5 wt%, e.g., 2 wt%, at 20 to 35°C, e.g., 25 to 30°C, for 6 to 48 hours, e.g., 12 to 24 hours, and then recovering it by compression filtration. At this time, the crude collagen slurry can be recovered at approximately 1 / 10 of its weight compared to before compression filtration by compression filtration. The calcium hydroxide aqueous solution is sufficient to fully submerge the bovine-derived skin and can be provided, for example, at 1 to 2 times the total weight percentage of the bovine-derived skin. If the pre-treatment temperature is below 20°C, the virus inactivation reaction rate by calcium hydroxide is reduced, which may lead to an excessive increase in treatment time or insufficient virus inactivation; if it exceeds 35°C, excessive swelling of collagen fibers or protein denaturation may occur, which may result in a deterioration of physical properties in the next step.

[0014] If the pretreatment time is less than 6 hours, there is a concern that virus safety may not be ensured because virus inactivation under alkaline conditions is not sufficiently carried out, and if it exceeds 48 hours, excessive swelling or damage to the molecular structure of the collagen may occur, which may reduce the homogeneity and viscoelastic properties of the collagen slurry.

[0015] When the aforementioned temperature and time are satisfied, sufficient virus inactivation by calcium hydroxide can be ensured, while simultaneously maintaining the structural stability and physical properties of collagen.

[0016]

[0017] After that, the recovered crude collagen slurry is cut into a predetermined size and then degreased (S30).

[0018] In order to improve the efficiency of the degreasing process, the crude collagen slurry recovered in step S20 can be cut into pieces of a predetermined size and then degreasing using an organic solvent, a surfactant, or a combination thereof. By cutting, the surface area of ​​the crude collagen slurry can be increased, allowing the degreasing solvent or degreasing conditions to act uniformly throughout the slurry. Furthermore, by effectively removing non-collagenous impurities from the collagen slurry through degreasing, the efficiency of subsequent acid treatment, purification, and homogenization processes can be improved, and the viscoelasticity and structural stability of the finally produced collagen slurry can be improved.

[0019]

[0020] Next, secondary virus inactivation is performed by treating the degreased crude collagen slurry with a 0.1~1.5M aqueous acetic acid solution and then recovering it by pressing and filtration (S40).

[0021] Secondary virus inactivation can be induced by treating the crude collagen slurry, which has been degreased in step S30, with a 0.1 to 1.5 M aqueous acetic acid solution, for example, 0.5 M, at a temperature of 1 to 30°C, for example, 4 to 20°C, for 24 to 96 hours, for example, 48 to 72 hours, and then recovering it by compression filtration. At this time, the crude collagen slurry can be recovered at approximately 1 / 10 of its weight compared to before compression filtration by compression filtration. The aqueous acetic acid solution can be provided in an amount of 1 to 2 times the total weight percentage of the crude collagen slurry. If the treatment temperature is less than 1°C, the efficiency of collagen swelling and virus inactivation by acetic acid may decrease, and if it exceeds 30°C, the triple helix structure of the collagen may be partially denatured, which may reduce viscoelasticity and structural stability.

[0022] If the treatment time is less than 24 hours, the acetic acid may not penetrate sufficiently into the crude collagen slurry, resulting in incomplete virus inactivation and removal of impurities, and if it exceeds 96 hours, the bonds between collagen fibers may be weakened or unnecessary molecular weight reduction may occur due to excessive acid treatment.

[0023] If the aforementioned temperature and time are satisfied, the virus inactivation effect can be maximized while maintaining the structural stability of collagen.

[0024]

[0025] Finally, the recovered crude collagen slurry is purified and homogenized (S50).

[0026] By purifying and homogenizing the crude collagen slurry recovered in step S40, physical uniformity and reproducibility required for in vivo application of the crude collagen slurry can be secured, and variations in hemostatic performance can be minimized. Purification can be carried out by one or more methods selected from filtration, centrifugation, and washing, for example, by washing with isopropyl alcohol, thereby improving the purity and stability of the crude collagen slurry. Furthermore, homogenization can be carried out by one or more methods selected from stirring, high-speed mixing, and high-pressure homogenization, thereby homogenizing the size distribution of collagen fibers or particles within the crude collagen slurry and achieving a stable slurry state in which precipitation or phase separation is suppressed even during long-term storage or application.

[0027]

[0028] Additionally, cobalt 60 A sterilization step can be performed by irradiating with gamma rays at a dose of 25 to 60 kGy. Through sterilization, the physical structure and hemostatic performance of the collagen slurry can be stably maintained while ensuring sufficient sterilization reliability. If the dose is less than 25 kGy, the sterilization level required for medical hemostatic agents may not be satisfied, and if it exceeds 30 kGy, viscoelasticity and hemostatic performance may be reduced due to damage to the collagen molecular structure.

[0029]

[0030] Another aspect of the present invention provides a hemostatic kit.

[0031] The hemostatic kit of the present invention may be used for hemostasis during surgical procedures or endoscopic procedures.

[0032] Conventional hemostatic kits are applied by mixing a syringe filled with powdered collagen, thrombin, and mannitol with a syringe filled with an aqueous calcium chloride solution immediately before use. This requires a repetitive and sufficient mixing process to ensure that the collagen, a polymer matrix, is adequately hydrated and uniformly dispersed within the formulation. Consequently, if mixing is insufficient, the distribution of the active ingredients becomes uneven, raising concerns about variations in hemostatic performance.

[0033] In order to solve the problem that the reproducibility of hemostatic performance may be reduced due to the requirement of repeated mixing in conventional hemostatic kits, the present invention applies collagen in the form of a sufficiently hydrated slurry, thereby enabling the rapid formation of a uniform formulation regardless of the user's skill level and consistent hemostatic performance even in urgent bleeding situations.

[0034] For example, the present invention provides collagen having a three-dimensional microfiber network structure in the form of a slurry, thereby ensuring improved structural stability compared to conventional particulate collagen. By effectively capturing platelet and blood cell components through this structure and inducing uniform thrombus formation, physical stability can be superior compared to conventional collagen hemostatic agents.

[0035] As the collagen slurry has been explained in detail above, a redundant explanation will be omitted.

[0036] As illustrated in FIG. 2, the hemostatic kit includes a first syringe and a second syringe, and the first syringe and the second syringe can be mixed upon use and applied to the hemostatic site in a mixed state. In addition to the first syringe and the second syringe, the hemostatic kit may further include a connector device and a catheter. The mixture may satisfy at least one of the following: having a three-dimensional microfiber network structure; having a viscosity in the range of 100 to 200 cP; having a storage modulus in the range of 6,000 to 8,000 Pa, for example, 6,500 to 7,500 Pa; having a loss modulus in the range of 1,000 to 2,000 Pa; satisfying the condition that storage modulus > loss modulus; having a blood coagulation time of less than 19 seconds; and having an absorbance in the range of 1.0 to 1.5 after 24 hours at a wavelength of 540 nm.

[0037] A first composition comprising 93 to 99 weight percent of collagen slurry and 1 to 7 weight percent of a first additive may be filled into a first syringe, wherein the first additive may be mannitol. If the collagen slurry is less than 93 weight percent, the formation of a collagen network for hemostasis is insufficient, resulting in reduced hemostatic performance and reduced viscosity and injection stability of the formulation; if it exceeds 99 weight percent, the content of the first additive becomes relatively insufficient, making it difficult to provide sufficient osmotic pressure control and formulation stabilization effects. Consequently, the viscosity of the collagen slurry may increase excessively, or aggregation may occur during storage and discharge failure may occur during injection. Therefore, when the above-described content range is satisfied, the first composition satisfies the injection force calculated from the maximum load value among the load values ​​measured until the sample is completely injected by pressurizing the syringe plunger, which satisfies the range of 5 to 16 N, thereby facilitating mixing with the second composition of the second syringe.

[0038] Meanwhile, the collagen slurry may satisfy at least one of the following: a viscosity in the range of 100 to 200 cP and an injection force in the range of 5 to 25 N.

[0039] For example, a collagen slurry may contain 4 to 6 weight percent of collagen solids based on the total weight percent of the collagen slurry. If the content of collagen solids is less than 4 weight percent, the viscosity of the slurry decreases to less than 100 cP, which reduces retention at the target site after injection and may impair hemostatic performance because the contact area with blood is not sufficiently secured. On the other hand, if the content of collagen solids exceeds 6 weight percent, the viscosity of the slurry exceeds 200 cP, which increases ejection resistance within the syringe and requires excessive pressure during injection, making injection difficult and consequently reducing usability.

[0040] The second syringe may be filled with a second composition comprising 1 to 10 weight percent of thrombin and 90 to 99 weight percent of a second additive, wherein the second additive may include mannitol and calcium chloride.

[0041] Thrombin is an enzyme that converts fibrinogen into fibrin during the blood coagulation process, and when mixed with collagen slurry, it can shorten the time to start hemostasis and accelerate thrombus formation. The thrombin according to the present embodiment may be a highly active bovine-derived thrombin with a residual amount of blood coagulation factor V, an immunogenic protein, of 0.3 ng / mL or less, which is significantly lower than that of existing commercial bovine-derived thrombin. Such thrombin may be included in an amount of 1 to 10% by weight. If the thrombin is less than 1% by weight, the fibrinogen-to-fibrin conversion reaction is not sufficiently induced, which lowers the rate of thrombus formation and may not significantly shorten the time to start hemostasis even when mixed with collagen slurry. If it exceeds 10% by weight, the blood coagulation reaction is excessively promoted, which may lead to the formation of locally non-uniform thrombi, which may result in increased fragility of the thrombus structure and reduced stability of the formulation.

[0042] Mannitol is a substance added to regulate osmotic pressure and maintain water balance, and it can inhibit enzyme denaturation during storage while maintaining the activity stability of thrombin. This mannitol may be included in an amount of 70 to 90 weight percent. If the amount of mannitol is less than 70 weight percent, the osmotic pressure regulating effect is not sufficiently expressed, which lowers the activity stability of thrombin and increases the likelihood of enzyme denaturation or reduced activity during storage. If the amount exceeds 90 weight percent, the calcium chloride content is relatively reduced, resulting in insufficient supply of calcium ions required for the blood coagulation reaction, which may consequently lower the rate of thrombus formation and hemostatic efficiency.

[0043] Calcium chloride (CaCl2) is a substance added to promote thrombin activity and fibrin formation reactions, which can improve the structural stability of blood clots. This calcium chloride may be included in an amount of 9 to 20 weight percent. If the calcium chloride is less than 9 weight percent, there is insufficient supply of calcium ions required for the blood coagulation process, so thrombin activity and fibrin formation reactions are not sufficiently promoted and the onset of hemostasis may be delayed. If it exceeds 20 weight percent, the osmotic balance of the formulation is disrupted due to an increase in local ion concentration, and usability may be reduced due to instability of thrombin activity or irritation of blood components.

[0044] A method for preparing a collagen slurry according to one embodiment of the present invention includes a multi-stage virus inactivation step, which can significantly reduce the risk of virus and microbial contamination resulting from the use of bio-derived raw materials, thereby greatly improving safety as a medical hemostatic agent.

[0045] In addition, a hemostatic kit according to another embodiment of the present invention provides collagen in the form of a sufficiently hydrated slurry, thereby significantly reducing reliance on a repetitive and skill-requiring mixing process immediately before use, and can rapidly form a uniform formulation with only a short mixing time even in urgent bleeding situations, thereby reducing the burden of handling on medical staff and improving ease of use.

[0046] In addition, the collagen slurry forms a dense polymer network based on a microfiber structure, thereby expanding the contact area with blood and effectively inducing the capture and aggregation of blood coagulation proteins, which enables excellent physical hemostatic performance and stable thrombus formation even in the absence of chemical hemostatic factors such as thrombin.

[0047] In addition, through elastic-dominant behavior attributable to a high storage modulus (G′), the thrombus structure is stably maintained without being easily relaxed or dispersed by external pressure or blood flow even after application to the bleeding site, thereby suppressing the increase in hemolysis over time and effectively reducing the possibility of rebleeding.

[0048] In addition, by maintaining a viscosity of 100 to 200 cP and an appropriate syringe injection force, quantitative injection of the hemostatic composition is possible when applied using a syringe, and since it does not require excessive force from the user, it offers excellent applicability in actual clinical settings and simultaneously improves user convenience, thereby enabling stable hemostatic treatment for various bleeding sites.

[0049] FIG. 1 is a process diagram for explaining a method for manufacturing collagen according to one embodiment of the present invention.

[0050] FIG. 2 is a schematic photograph showing a hemostatic kit according to another embodiment of the present invention.

[0051] Figures 3 and 4 are a photograph and a graph showing the results of Experimental Example 1.

[0052] Figure 5 is a figure illustrating the experimental method of Experimental Example 4.

[0053] Figures 6 and 7 are graphs showing the results of Experimental Example 4.

[0054] Figure 8 is a photograph showing the results of Experimental Example 5.

[0055] Figures 9 and 10 are graphs showing the results of Experimental Example 6.

[0056] Figures 11 to 14 are photographs and graphs showing the results of Experimental Example 7.

[0057] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0058]

[0059] Preparation Example 1. Preparation of Thrombin

[0060] Bovine plasma was thawed to prepare it in a liquid state, and impurities were removed and the pH adjusted through a filtration process. Subsequently, protein components were separated and purified by performing anion exchange chromatography. Then, calcium chloride and thromboplastin were added to generate thrombin, and the fraction containing thrombin was selectively recovered through filtration and ion exchange chromatography. Primary virus inactivation was induced by performing solvent / surfactant (S / D) treatment on the recovered fraction. Subsequently, residual solvent and surfactant were removed through a polymer adsorption process, and the purity of the thrombin was improved by performing ion exchange chromatography again. Afterward, the concentration of the thrombin solution was adjusted through concentration and ultrafiltration processes. Next, secondary virus inactivation was induced by performing a virus filtration process, and then thrombin in powder form was prepared through sterile filtration and freeze-drying processes.

[0061]

[0062] Examples 1 to 3. Preparation of collagen slurry

[0063] Bovine skin was immersed in a 1–5 wt% aqueous calcium hydroxide solution and stirred at 20–35°C for 6–48 hours to induce primary virus inactivation. Subsequently, the crude collagen solution was subjected to press filtration to obtain a crude collagen slurry, with the slurry recovered at approximately 1 / 10 of its weight before treatment. Next, the crude collagen slurry was cut into small pieces of a predetermined size and subjected to a degreasing process to remove residual fatty components within the collagen. Afterward, the crude collagen slurry was immersed in a 0.1–1.5 M acetic acid solution and stirred at 1–30°C for 24–96 hours to induce secondary virus inactivation. Subsequently, press filtration was performed again to recover the crude collagen slurry at approximately 1 / 10 of its weight before treatment. Then, the crude collagen slurry was purified and homogenized to prepare a collagen slurry containing the collagen solids listed in Table 1.

[0064] The content listed in Table 1 is based on weight %.

[0065] Classification Example 1 Example 2 Example 3 Collagen solid content 456

[0066]

[0067] Examples 4 to 7. Preparation of the first syringe

[0068] A first composition was prepared by mixing the components and amounts listed in Table 2 below, and then a first syringe was prepared by filling.

[0069] The content listed in Table 2 is based on weight %.

[0070] Classification Example 4 Example 5 Example 6 Example 7 Example 299989795 Mannitol 1235

[0071]

[0072] Example 8. Preparation of the second syringe

[0073] A second composition was prepared by mixing the components and content listed in Table 3 below, and then filled to produce a second syringe.

[0074] The content listed in Table 3 is based on weight %.

[0075] Ingredient Content Preparation Example 1 (Thrombin) 1~10 Calcium Chloride 9~20 Mannitol 70~90

[0076]

[0077] Example 9. Hemostatic kit

[0078] The first syringe according to Example 5 and the second syringe according to Example 8 were manufactured as a hemostatic kit. When using the first syringe and the second syringe, a connector device was connected and then mixed for use.

[0079]

[0080] Example 10. Hemostatic kit

[0081] A hemostatic kit was prepared in the same manner as in Example 9, except that thrombin was not applied.

[0082]

[0083] Comparative Example 1. Preparation of collagen slurry

[0084] A collagen slurry was prepared in the same manner as in Examples 1 to 3, except that it was prepared to contain 2% by weight of collagen solids.

[0085]

[0086] Comparative Example 2. Preparation of Collagen Slurry

[0087] A collagen slurry was prepared in the same manner as in Examples 1 to 3, except that it was prepared to contain 8% by weight of collagen solids.

[0088]

[0089] Comparative Example 3. Manufacture of a hemostatic kit

[0090] A first syringe was prepared by filling it with a 2% mannitol solution. A second syringe was prepared by mixing 59% by weight of collagen powder, 1% by weight of thrombin, 35% by weight of mannitol, and 5% by weight of calcium chloride, and then filling it. When using the first and second syringes, a connector device was connected and the syringes were mixed for use.

[0091]

[0092] Comparative Example 4. Manufacture of a hemostatic kit

[0093] A first syringe was prepared by filling it with a 2% mannitol solution. A second syringe was prepared by mixing 60% by weight of collagen powder, 35% by weight of mannitol, and 5% by weight of calcium chloride, and then filling it. When using the first and second syringes, a connector device was connected and the syringes were mixed for use.

[0094]

[0095] Experimental Example 1. Evaluation of Viscosity Characteristics of Collagen Slurry

[0096] To confirm the viscosity characteristics of the collagen slurry according to the collagen solid content, an experiment was conducted as follows, and the results are shown in Table 4 and Figure 4.

[0097] Viscosity was measured according to Method 2 (Rotational Viscometer Method) of Article 57 (Viscosity Measurement Methods) of the Korean Pharmacopoeia (KP) General Test Methods, b) using a single-cylindrical rotational viscometer.

[0098] Prior to measurement, calibrate the rotational viscometer using a viscosity calibration standard solution, and based on the results, the device constant (K B ...was calculated. Afterwards, the sample to be measured was mounted on a single-cylindrical rotary viscometer, and the torque (T) acting on the cylindrical surface was measured while rotating it at a constant angular velocity (ω). Using the measured value, the viscosity (η) was calculated according to Equation (1). The viscosity value was calculated as the average value of the results of continuous measurements taken at 5-second intervals for 1 minute.

[0099] … … … … … … … … … … Equation (1)

[0100] In Equation (1), η is the viscosity of the sample (mPa·s or cP), K B is the device constant (rad / cm²) 3 ), ω is angular velocity (rad / s), T is torque acting on the cylindrical surface (10 -7 It means N·m).

[0101] All results listed in Table 4 represent viscosity values ​​measured in cP units.

[0102] Classification Viscosity (cP) Test Criteria Compliance Example 1 11.3 100~200 cP Compliance Example 2 124.4 Compliance Example 3 161.5 Compliance Comparative Example 183.9 Non-compliance Comparative Example 2237.3 Non-compliance

[0103] Referring to FIG. 3, Comparative Example 1 and Example 2 show a significant difference in viscosity; while Comparative Example 1 exhibits flowability due to its low viscosity, Example 2 has a relatively high viscosity, which can be confirmed by visual observation alone that flow is inhibited. Referring to Table 4 and FIG. 4, Examples 1 to 3 were found to be suitable as they fall within the test standard viscosity range, whereas Comparative Examples 1 and 2 were found to be unsuitable as they fall outside the test standard viscosity range. Therefore, it was found that ease of injection and convenience of use can be simultaneously ensured by stably satisfying the viscosity range required for injection only when the collagen solid content is applied at 4 to 6 weight%.

[0104]

[0105] Experimental Example 2. Evaluation of Injection Force Characteristics of Collagen Slurry

[0106] To confirm the injection force characteristics of collagen slurry according to the collagen solid content, an experiment was conducted as follows, and the results are shown in Table 6.

[0107] A jig was mounted horizontally on the measuring equipment, and a syringe support was placed at the bottom of the equipment. Then, a catheter was connected to the syringe and fixed to the support. With the jig in close contact with the syringe plunger, the injection force was measured by applying pressure to the syringe plunger at a speed according to the conditions set for each product until the sample was completely injected. The injection force was calculated in Newtons (N) based on the maximum load value among the load values ​​measured during the injection process, and the injectability was evaluated according to Table 5 below.

[0108] Injection Power (N, in vitro) Injectability (in vivo) 0~10 Very easy to inject, smaller needle size can be used 11~25 Easy to inject 26~50 Injectable 51~100 Somewhat difficult to inject 101~130 Difficult to inject, use of larger needle recommended 130 Very difficult to inject, use of larger needle required

[0109] Classification Input (N) Injectability Example 17.6 Very easy to inject, smaller needle size applicable Example 212.6 Easy to inject Example 323.8 Easy to inject Comparative Example 252.7 Somewhat difficult to inject

[0110] Referring to Table 6, Examples 1 to 3 were evaluated as having very easy or easy injection, with the injection force measured in the range of 7 to 24 N, whereas Comparative Example 2 was measured with an injection force of 52.7 N, requiring a relatively large force during injection, and thus it was confirmed that injection was somewhat difficult. From these results, it was found that when the content of collagen solids increases to 8% by weight or more, the viscosity and internal resistance of the composition increase, causing the injection force to increase significantly, and consequently, the injectability decreases.

[0111] Meanwhile, for products applied using a syringe, it is essential to evaluate the syringe injection force to ensure applicability in actual usage environments. If the injection force is excessively low, it is difficult to precisely control the amount of contents discharged, which may result in an excessive amount being ejected at once; conversely, if the injection force is excessively high, the user must apply excessive force to operate the syringe, which may cause inconvenience in use. Therefore, Examples 1 to 3 confirmed that quantitative injection of the contents is possible and user convenience can be simultaneously ensured by maintaining an injection force within an appropriate range.

[0112]

[0113] Experimental Example 3. Evaluation of Injection Force Characteristics of the First Syringe

[0114] To confirm the injection power characteristics according to mannitol content, an experiment was conducted using the same method as in Experimental Example 2, and the results are shown in Table 7.

[0115] Classification Input (N) Injectability Example 2 15.6 Easy injection Example 4 10.6 Very easy injection, smaller needle size applicable Example 5 8.6 Example 6 7.1 Example 75.1

[0116] Referring to Table 7, it was confirmed that the injectability was reduced in Examples 4 to 7, which contain mannitol, compared to Example 2, which does not contain mannitol. Therefore, it was found that when mannitol is added, the injectability is reduced and injectability is improved.

[0117]

[0118] Experimental Example 4. Evaluation of the ease of mixing of the first syringe and the second syringe

[0119] To confirm the ease of mixing according to collagen formulations, an experiment was conducted as follows, and the results are shown in Table 8, Figure 6, and Figure 7.

[0120] After connecting two syringes, consisting of a first syringe and a second syringe, with a connector device, the contents were mixed by moving the plungers of each syringe back and forth. To determine the drug distribution pattern within the formulation according to mixing efficiency, the number of mixing cycles was set to 5, 10, 15, and 20, respectively, and independent test samples were prepared for each mixing condition.

[0121] To evaluate the uniformity of thrombin distribution in the mixed liquid hemostatic composition, the entire amount filled in the syringe was discharged, and fractions were collected by dividing it equally into three sections: the upper section (1 / 3), the middle section (2 / 3), and the lower section (3 / 3), as shown in FIG. 5. Each fractionated sample was immediately used for quantitative analysis, thereby evaluating the variation in thrombin concentration according to the position within the syringe.

[0122] To quantify the thrombin activity for each fractionally collected sample, a calibration curve was constructed by applying the thrombin quantification method according to the Korean Pharmacopoeia (KP). Standard thrombin solutions were prepared by stepwise dilution to concentrations of 2.5, 4, 5, 6.25, and 7.5 Units, and each standard solution was reacted with a fibrinogen solution to measure the coagulation time (seconds) at each concentration. To derive the correlation between the measured coagulation time and thrombin activity (Unit), a logarithm was taken for each measurement value and a linear regression analysis was performed, and Equation (2) was applied to the data analysis.

[0123] log[(Unit)]=log[(sec)-Intercept] / Slope… … … … … … … … Equation (2)

[0124] Based on the slope and intercept derived from this, a regression equation (y = ax + b) was established, and the correlation coefficient (R) and coefficient of determination (R²) 2 The linearity and reliability of the calibration curve were verified by calculating ).

[0125] Subsequently, coagulation times were measured for three fractions (upper, middle, and lower) of samples collected according to the number of mixing cycles (5 to 20 times), and thrombin activity (Unit) for each position was calculated by substituting them into a calibration curve regression equation. The uniformity of the thrombin distribution according to the number of mixing cycles of the liquid hemostatic agent composition was evaluated by calculating the mean value and relative standard deviation (RSD, %) for the calculated thrombin activity values.

[0126] Example 9 Number of Mixings Classification 1 / 3 Fraction (Top) 2 / 3 Fraction (Middle) 3 / 3 Fraction (Bottom) Judgment 5 times Coagulation Time (sec) 98.0 98.6 98.2 Homogeneous Thrombin Unit 1.7 91.7 81.79 10 times Coagulation Time (sec) 98.8 98.6 99.2 Homogeneous Thrombin Unit 1.7 71.7 81.76 15 times Coagulation Time (sec) 99.0 99.0 98.6 Homogeneous Thrombin Unit 1.7 71.7 71.78 20 times Coagulation Time (sec) 98.4 99.4 98.4 Homogeneous Thrombin Unit 1.7 81.76 1.78 Comparative Example 3 Number of Mixings Classification 1 / 3 Fraction (Top) 2 / 3 Fraction (Middle) 3 / 3 Fraction (Bottom) Judgment 5 times Coagulation Time (sec) 75.4 118.8 128.0 Heterogeneous thrombin Unit 2.5 81.3 81.25 10 times coagulation time (sec) 83.6 105.0 121.8 Heterogeneous thrombin Unit 2.2 31.6 31.33 15 times coagulation time (sec) 98.6 100.4 102.4 Homogeneous thrombin Unit 1.7 81.7 31.69 20 times coagulation time (sec) 99.6 100.29 9.0 Homogeneous thrombin Unit 1.7 51.7 41.77

[0127] Referring to Table 8, Figures 6 and 7, in the case of Example 9, which uses a collagen slurry, it was confirmed that a uniform distribution was formed with only 5 mixing cycles, as almost no variation in thrombin activity was observed between the upper, middle, and lower fractions. On the other hand, in the case of Comparative Example 3, which uses collagen powder, more than 15 mixing cycles were required to secure a uniform distribution, and it was confirmed that a distinct variation in distribution was observed when a sufficient number of mixing cycles was not secured. Therefore, it was found that the hemostatic composition using a collagen slurry can form a uniform drug distribution with only a small number of mixing cycles, demonstrating excellent ease of mixing and drug distribution stability. These characteristics can simplify the user's preparation process and serve as an advantage that enables rapid treatment even in urgent bleeding situations.

[0128]

[0129] Experimental Example 5. Evaluation of Microfiber Structure

[0130] To observe the microfiber structure, an experiment was conducted as follows, and the results are shown in Fig. 8.

[0131] A hemostatic composition comprising a mixture of the first syringe and the second syringe was mixed with 1 mL of anticoagulant-treated porcine plasma to form a thrombus. The formed thrombus was lightly washed 1 to 2 times with 1×PBS and then freeze-dried. The freeze-dried sample was fixed to a carbon tape attached to a sample holder, and then coated with a thin film of approximately 5 to 10 nm using platinum (Pt). Subsequently, the sample was observed using an electron microscope under low voltage conditions of 2 kV, and images acquired under low magnification (X500 to X1,000) and high magnification (X5,000 to X30,000) conditions were used for analysis.

[0132] Referring to Fig. 8, in the case of Example 9, to which a collagen slurry was applied, a porous network maintaining a three-dimensional microfiber structure was observed, whereas in the case of Comparative Example 3, to which collagen powder was applied, a relatively dense and flat structure was observed. This difference in microfiber structure implies that the collagen slurry formulation is more advantageous for effectively capturing blood components and stably inducing thrombus formation, and consequently, it was found to contribute to the improvement of hemostatic performance.

[0133]

[0134] Experimental Example 6. Evaluation of Viscosity and Complex Viscosity

[0135] Experiments were conducted as follows to evaluate viscosity and complex viscosity according to the presence or absence of microfiber structure, and the results are shown in Table 9, Figure 9, and Figure 10.

[0136] Specifically, to evaluate elastic properties closely related to the cohesiveness and adhesiveness of the formulation, storage modulus (G′) was set as a key indicator, and through this, it was evaluated whether the formulation could maintain its structure against blood flow or pressure in an actual hemostatic environment and induce stable thrombus formation.

[0137] Viscosity was measured using the same method as in Experimental Example 1.

[0138] For complex viscosity, the zero point was calibrated by performing a Zero Gap setting after aligning the lower and upper plates of the equipment. Subsequently, the test sample was carefully loaded into the center of the lower plate, and the upper plate was lowered to set the measurement gap to 1 mm. At this time, any sample protruding outside the plate was removed using a spatula to prevent deformation or errors during measurement. Complex viscosity measurements were performed in oscillation mode, varying the frequency while maintaining a constant strain. The measurement temperature was set to 37℃, and measurements were taken at a total of five frequency points: 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, and 2.5 Hz. After the measurements were completed, the measurement values ​​for each frequency condition were extracted in a table format using the Analysis tab of the analysis software connected to the equipment. Storage modulus (G′) and loss modulus (G″) were calculated. Here, storage modulus (G′) is an indicator representing the elastic properties of the sample, i.e., solid properties, and is related to the strength of the gel, while loss modulus (G″) is an indicator reflecting the viscous properties of the sample, i.e., liquid properties, and is related to the flowability of the sample.

[0139] Classification Example 9 (Collagen Slurry) Comparative Example 3 (Collagen Powder) Viscosity (cP) 124.9 97.6 Suitable Yes / No Suitable Unsuitable Average Storage Modulus (G', Pa) 6993.3 5970.91 Average Loss Modulus (G'', Pa) 1168.1 1231.47

[0140] Referring to Table 9, Fig. 9, and Fig. 10, viscosity measurements revealed a distinct difference in appearance between Example 9 and Comparative Example 3 immediately after mixing. Specifically, Example 9 exhibited a homogeneous appearance similar to dough by forming a continuous matrix through interactions between polymer chains, whereas Comparative Example 3 showed an appearance similar to slush due to a predominantly particulate structure. This difference in appearance was also quantitatively confirmed by viscosity measurements; Example 9, which used a collagen slurry, fell within the standard viscosity range (100–200 cP) and was evaluated as suitable, whereas Comparative Example 3, which used a collagen powder, fell outside the standard viscosity range and was evaluated as unsuitable. Through this, it was confirmed that the collagen slurry formulation forms a more stable structure in a liquid environment compared to the powder formulation.

[0141] Meanwhile, as a result of viscoelastic analysis, it was confirmed that Example 9 showed a storage modulus (G′) of 6993.35 Pa, which is significantly higher than the 970.91 Pa of Comparative Example 3. This can be interpreted as Example 9 exhibiting elastic-dominant behavior based on high cohesiveness and forming a stable gel structure.

[0142] In addition, regarding the loss modulus of elasticity (G″), Example 9 showed a value of 1168.11 Pa, which is higher than Comparative Example 3's 231.47 Pa; however, since the relationship G′ > G″ was maintained in both formulations, it was confirmed that the elastic component is dominant over the viscous component. In particular, Example 9 showed a large difference in absolute values ​​between G′ and G″, indicating that it simultaneously secures appropriate flow characteristics along with the ability to maintain shape against external forces.

[0143] In summary, it was confirmed that the collagen slurry formulation of Example 9 can perform physical hemostatic action by absorbing blood and swelling or effectively capturing blood proteins in a hemostatic environment, and also maintains sufficient cohesiveness and an elastic structure so that the applied formulation does not easily disperse even under conditions of high bleeding volume or intravascular pressure, thereby stably blocking the site of vascular damage and inducing thrombus formation. On the other hand, it was found that the powder formulation of Comparative Example 3 has relatively low structural stability, making it highly likely that the formulation will easily scatter under the same conditions, and as a result, effective thrombus formation is inhibited, which may increase the possibility of hemostatic failure.

[0144]

[0145] Experimental Example 7. Evaluation of Hemostasis Time and Hemolytic Activity

[0146] To evaluate hemostatic and hemolytic properties depending on the presence or absence of a microfiber structure, experiments were conducted as follows, and the results are shown in Table 10 and Figures 11 to 14.

[0147] When bleeding occurs from a damaged blood vessel, applying a hemostatic agent blocks the leakage site by rapidly forming a blood clot through interaction with blood components. At this time, since it is an important factor in determining hemostatic performance whether the formed blood clot maintains structural stability until the tissue at the site of the blood vessel injury is sufficiently recovered rather than stopping at the initial formation, a hemolytic test was performed as an experiment to evaluate the structural stability of the blood clot.

[0148] Meanwhile, chemical hemostasis using thrombin has the characteristic of proceeding with a very rapid and strong hemostatic reaction compared to physical hemostasis based on collagen. Due to this, the powerful hemostatic effect of thrombin may relatively mask differences in thrombotic stability or structural characteristics resulting from differences in polymer matrix formulations. Accordingly, a formulation not containing thrombin was separately prepared, and a hemolytic test was performed under the same conditions to more clearly compare and analyze the physical hemostatic performance and thrombotic stability of the collagen-based hemostatic composition itself.

[0149] After weighing 10 mg of each sample and preparing at least three, with a bottom area of ​​approximately 1 cm 2 Each sample was placed into a transparent tube. Subsequently, 1 mL of anticoagulated porcine blood was injected into each tube. The tubes containing the sample and blood mixture were immersed in a water bath maintained at 37°C, and the coagulation of the blood was confirmed by inverting the tubes at 5-second intervals to observe the fluidity of the blood. The point at which complete coagulation was confirmed, indicating the loss of blood fluidity, was defined as the completion of hemostasis, and the hemostasis time of each sample was evaluated by calculating the average value of the measured hemostasis times.

[0150] Each thrombus formed after the hemostasis time test was collected and transferred to a 15 mL tube, and the blood remaining on the surface of the thrombus was gently washed with 1×PBS. 10 mL of 1×PBS was injected into each tube containing the washed thrombus, sealed, and stored in a 37°C incubator. Subsequently, 200 μL of the supernatant was collected from each tube at intervals of 0.5, 1, 2, 3, 18, and 24 hours, dispensed into a 96-well plate, and the degree of hemolysis over time was quantitatively evaluated by measuring the absorbance at a wavelength of 540 nm using a microplate reader.

[0151] Red blood cells released into the solvent after dissociating from the thrombus were set as an indicator, and the measured absorbance was interpreted as an indicator reflecting the degree to which the thrombus structure relaxed or collapsed over time.

[0152] Then, the absorbance value was applied to Equation (3) to calculate the hemolysis inhibition rate (%).

[0153] Hemolysis inhibition rate (%) = (1 - absorbance of sample / absorbance of control group) X 100… … … Equation (3)

[0154] Blood coagulation time (s) Example 9 (Collagen Slurry) Comparative Example 3 (Collagen Powder) 120.0 20.0 220.0 20.0 315.0 20.0 Average 18.3 20.0 Blood coagulation time (s) Example 10 (Collagen Slurry) Comparative Example 4 (Collagen Powder) 1190.0 270 2200.0 250 3190.0 280 Average 193.3 266.7

[0155] Referring to Table 10 and Figure 11, for Example 9 (collagen slurry) and Comparative Example 3 (collagen powder) performed under conditions containing thrombin, the average blood coagulation times were within 19 seconds and 20.0 seconds, respectively, confirming that no significant difference was observed between the two samples. This indicates that the influence of differences in the physical form of collagen was relatively reduced as the coagulation chain reaction was directly activated by thrombin, causing chemical hemostasis to act predominantly.

[0156] On the other hand, referring to FIG. 12, for Example 10 (collagen slurry) and Comparative Example 4 (collagen powder) performed under conditions not containing thrombin, the average blood coagulation times were 193.3 seconds and 266.7 seconds, respectively, indicating that the collagen slurry exhibited a significantly shorter coagulation time compared to the collagen powder. This suggests that the dense network of the microfiber structure of the collagen slurry increased the effective contact area with blood, while simultaneously inducing blood coagulation proteins present in the body to be effectively captured and aggregated within the network, thereby promoting thrombus formation and resulting in an enhanced hemostatic effect. Accordingly, it was confirmed that excellent hemostatic performance can be exhibited due to structural characteristics even under conditions where chemical hemostatic factors such as thrombin are not separately added.

[0157] Referring to Table 11 and Figures 13a and 13b, in the case of Example 9, it was observed that the absorbance increased gradually during the initial period (0–3 h), followed by a gradual increasing trend until 24 hours, and finally reached a level of approximately 1.4–1.5. This gradual increase indicates that the collagen slurry induced relatively uniform aggregation and structure formation upon contact with blood, without excessive precipitation or rapid changes in turbidity.

[0158] On the other hand, Comparative Example 3 showed a higher absorbance than Example 9 from the initial time period and continuously increased over time, reaching a value of approximately 2.1 or higher at the 24-hour mark. This indicates that the optical instability increased because the collagen powder formed relatively non-uniform aggregation or particulate precipitation in the blood.

[0159] Therefore, according to the hemolysis inhibition rate calculated from the absorbance measured after 24 hours at a wavelength of 540 nm, it was found that Example 9, to which collagen slurry was applied, showed a result improved by more than 30% compared to Comparative Example 3, to which collagen powder was applied.

[0160] Classification 0 h0.5 h1 h2 h3 h19 h24 h Untreated 3.449 3.517 3.514 3.517 3.553 3.324 3.300 Example 90.205 0.389 0.470 0.658 0.817 1.346 1.472 Comparative Example 30.304 0.572 0.720 1.000 1.201 1.893 2.148

[0161] Referring to Table 12 and Figures 14a and 14b, in the case of Example 10, very low absorbance was maintained during the initial period (0 to 3 h), and a gradual increase was observed until 18 hours, after which it increased to approximately 1.2 at 24 hours. In other words, the range of change in absorbance was generally limited, and relatively stable behavior was maintained even after a long period of time.

[0162] In contrast, Comparative Example 4 exhibited higher absorbance than Example 10 from the initial time point, and it was confirmed that the absorbance increased rapidly, particularly after 18 hours, reaching approximately 2.5 or higher at the 24-hour mark. This rapid increase may be due to the fact that the collagen powder, with its relatively dense and flat structure, failed to form a uniform and stable thrombus network upon contact with blood, causing the thrombus structure to easily relax or collapse over time. If the thrombus structure becomes loose in this manner, it may fail to sufficiently seal the leakage site of the blood vessel, potentially causing the damaged area to reopen and leading to rebleeding. This decrease in thrombus stability manifests as an increase in the amount of red blood cells dissociating from the thrombus, which can be quantitatively evaluated through an increase in the absorbance of the supernatant. Furthermore, the occurrence of rebleeding may require medical personnel to perform additional hemostatic measures, leading to increased procedure time and clinical discomfort.

[0163] Therefore, according to the hemolysis inhibition rate calculated from the absorbance measured after 24 hours at a wavelength of 540 nm, it was found that Example 10, to which collagen slurry was applied, showed a result improved by more than 50% compared to Comparative Example 4, to which collagen powder was applied.

[0164] Classification 0 h0.5 h1 h2 h3 h19 h24 h Untreated 3.20 23.19 63.21 23.23 53.30 13.27 23.169 Example 100.03 50.09 30.13 00.13 50.17 70.48 41.181 Comparative Example 40.04 10.12 90.34 10.46 60.54 51.36 52.571

[0165]

[0166] Although exemplary embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

[0167] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

Claims

1. A first syringe filled with a first composition comprising a collagen slurry and a first additive, and It includes a second syringe filled with a second composition comprising thrombin and a second additive, and A hemostatic kit in which the first syringe and the second syringe are mixed upon use and applied to a hemostatic site in a mixed state.

2. In Paragraph 1, The above collagen slurry is a hemostatic kit having a viscosity of 100 to 200 cP as measured by the following formula (1). … … … … … … … … … … Equation (1) In the above equation (1), η is the viscosity (cP) of the sample, K B is the device constant (rad / cm²) 3 ), ω is angular velocity (rad / s), T is torque acting on the cylindrical surface (10 -7 It means N·m).

3. In Paragraph 1, The above collagen slurry is a hemostatic kit having an injection force of 5 to 25 N, calculated as the maximum load value among the load values ​​measured until the sample is completely injected by pressurizing the syringe plunger.

4. In Paragraph 1, The above-mentioned first composition is a hemostatic kit having an injection force of 5 to 16 N, calculated as the maximum load value among the load values ​​measured until the sample is completely injected by pressurizing the syringe plunger.

5. In Paragraph 1, A hemostatic kit comprising 4 to 6 weight percent of collagen solids based on the total weight percent of the collagen slurry.

6. In Paragraph 1, The first additive above includes mannitol, and The above second additive is a hemostatic kit comprising mannitol and calcium chloride.

7. In Paragraph 1, The above mixture is a hemostatic kit having a porous network structure in which a three-dimensional microfiber structure is maintained.

8. In Paragraph 1, The above mixture is a hemostatic kit having a viscosity of 100 to 200 cP as measured by the following formula (1). … … … … … … … … … … Equation (1) In the above equation (1), η is the viscosity (cP) of the sample, K B is the device constant (rad / cm²) 3 ), ω is angular velocity (rad / s), T is torque acting on the cylindrical surface (10 -7 It means N·m).

9. In Paragraph 1, The above mixture is a hemostatic kit in which the storage modulus measured at 37°C under conditions of 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, and 2.5 Hz is higher than the loss modulus.

10. In Paragraph 1, The above mixture is a hemostatic kit satisfying a storage modulus of 6000 to 8000 Pa measured at 37℃ under conditions of 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, and 2.5 Hz.

11. In Paragraph 1, The above mixture is a hemostatic kit satisfying a loss modulus of 1000 to 2000 Pa measured at 37℃ under conditions of 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, and 2.5 Hz.

12. In Paragraph 1, The above mixture is a hemostatic kit having a blood coagulation time of 19 seconds or less, defined as the point at which the fluidity of the blood is lost while the tube is inverted at 5-second intervals while mixed with anticoagulant-treated blood in a tube immersed in a water bath maintained at 37°C.

13. In Paragraph 1, The above mixture is a hemostatic kit having an absorbance of 1.0 to 1.5 measured after 24 hours at a wavelength of 540 nm.

14. In Paragraph 1, The above mixture is a hemostatic kit in which the hemolysis inhibition rate calculated from the absorbance measured after 24 hours at a wavelength of 540 nm is improved by more than 30% compared to a mixture to which collagen powder is applied. 15.(a) Step of preparing bovine skin; (b) A primary virus inactivation step in which the bovine-derived skin is pretreated with a 1-5% by weight aqueous calcium hydroxide solution and then recovered by pressing and filtration; (c) A step of cutting the recovered crude collagen slurry into a predetermined size and then degreasing it; (d) a secondary virus inactivation step in which the above-degreased crude collagen slurry is treated with a 0.1–1.5 M aqueous acetic acid solution and then recovered by press filtration; and (e) a step of purifying and homogenizing the recovered crude collagen slurry; comprising a method for preparing a collagen slurry.

16. In Paragraph 15, A method for preparing a collagen slurry, wherein in step (b) above, the primary virus inactivation is carried out at 20 to 35°C for 6 to 48 hours.

17. In Paragraph 15, A method for preparing a collagen slurry, wherein in step (d) above, secondary virus inactivation is carried out at 1 to 30°C for 24 to 96 hours.

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