CN114177346A

CN114177346A - Hemostatic composition, hemostatic patch and application thereof

Info

Publication number: CN114177346A
Application number: CN202111603820.6A
Authority: CN
Inventors: 马玉媛; 袁方; 贾俊婷; 张欢; 章金刚; 赵雄; 王强; 王东根; 付秋霞; 阎少多; 孙珍珠; 吕茂民; 皇甫超济; 王蕊
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-15
Anticipated expiration: 2041-12-24
Also published as: CN114177346B

Abstract

The invention discloses a hemostatic composition, a hemostatic patch and application thereof, wherein the hemostatic patch comprises 16-46 parts of fibrinogen, 38-42 parts of thrombin and 100-121 parts of calcium chloride. The hemostatic composition and the hemostatic plaster have good hemostatic effect and good biocompatibility, can be completely degraded in vivo, do not need secondary debridement, avoid secondary injury to wounded persons, can be applied to hemostasis of wounded persons (particularly hemostasis of visceral hemorrhage) in various emergencies, natural disasters (particularly violent earthquakes) and other situations, and have good commercial value and wide application prospect.

Description

Hemostatic composition, hemostatic patch and application thereof

Technical Field

The invention relates to the technical field of medical hemostatic materials, in particular to a hemostatic composition, a hemostatic patch prepared from the hemostatic composition, and applications of the hemostatic composition and the hemostatic patch.

Background

Bleeding is one of the most common and difficult to control problems in surgery and trauma, and especially bleeding from vital organs, such as liver, spleen, kidney rupture is often directly life threatening. The traditional hemostasis methods (such as ligation and compression hemostasis) are only suitable for the hemostasis of large vessel hemorrhage, and have limited hemostasis effect on diffuse hemorrhage and visceral hemorrhage. Traumatic hemorrhage is also a big lethal factor, and the rapid and effective hemostasis after injury is the most important means for saving the lives of the sick and wounded.

However, when the body is subjected to a severe trauma, auto-coagulation is not sufficient to rapidly coagulate blood, and it is therefore necessary to apply a hemostatic material to the wound to control bleeding. The ideal hemostatic material has the following requirements in terms of performance: (1) hemostasis is rapid (i.e., clotting time is short); (2) has good biocompatibility and no short-term or long-term adverse effect; (3) secondary debridement is not needed, and secondary injury to the wounded is avoided; (4) the application is convenient, and even non-professional personnel can use the product easily; (5) is stable under various environmental conditions and has long shelf life.

At present, the hemostatic materials used for surgical operation and wound treatment are various, such as fibrin and chitosan natural biological hemostatic materials, mineral hemostatic materials such as zeolite, kaolin and montmorillonite, and synthetic hemostatic materials such as gelatin-resorcinol-formaldehyde or propylene cyanide, and the hemostatic mechanisms of the hemostatic materials are different. The hemostatic materials have respective defects, for example, the common chitosan hemostatic materials have poor adhesion at the wound and are easy to be washed away by blood, so the hemostatic effect is greatly reduced; mineral hemostatic materials are not absorbable and need secondary debridement, so that the hemostatic material used for visceral hemorrhage can cause secondary injury to the wounded, and the safety is poor; the artificially synthesized hemostatic material has poor biocompatibility and degradability and many potential safety hazards.

In conclusion, compared with the materials, the hemostatic patch product prepared from the fibrin hemostatic material has the advantages of strong adhesion, no need of secondary debridement, no secondary injury, degradability, low immunogenicity, convenient use and the like, can be used for light and medium level hemorrhage without fixed bleeding points, and is particularly suitable for hemostasis of visceral hemorrhage.

Chinese patent application publication No. CN1507358A discloses a carrier having solid fibrinogen and solid thrombin uniformly dispersed and immobilized thereon. The product only contains fibrinogen and thrombin, and has long coagulation time and unsatisfactory hemostatic effect. Moreover, the liquid medicine layer (the liquid medicine layer is a layer containing solid fibrinogen and solid thrombin) of the product is easy to fall off in the use process (see figure 3), and the hemostatic effect of the product cannot be ensured.

Disclosure of Invention

The invention aims to solve the technical defects in the prior art, and provides a hemostatic composition with short coagulation time in a first aspect, which comprises 16-46 parts (preferably 16-20 parts) of fibrinogen, 38-42 parts of thrombin and 100-121 parts of calcium chloride, wherein the fibrinogen is one part in mg, the thrombin is one part in IU, and the calcium chloride is one part in mu g.

Comprises 16-19 parts of fibrinogen, 39-41 parts of thrombin and 103-118 parts of calcium chloride; preferably, the fibrinogen composition comprises 17 parts of fibrinogen, 40 parts of thrombin and 115 parts of calcium chloride (CaCl)₂)。

The viscoelastic property value tan delta of the fibrin polymer formed by the hemostatic composition after meeting water is less than 1, and preferably 0.13-0.31.

In a second aspect, the invention provides a hemostatic patch, which comprises a collagen carrier prepared from collagen and a liquid medicine layer uniformly dispersed and fixed on the collagen carrier, wherein the liquid medicine layer is prepared from the hemostatic composition; the collagen carrier is in a sheet shape.

Each square centimeter of hemostatic plaster contains 16-46mg of fibrinogen, 38-42IU of thrombin, 121 mu g of calcium chloride and more than 0.5mg of collagen.

The liquid medicine layer is formed by dispersing the hemostatic composition in ethanol to prepare a suspension and then spraying the suspension on a collagen carrier; optionally, after dispersing the hemostatic composition in ethanol, placing the mixture in a dispersion machine for homogeneous stirring, wherein the stirring speed is (10-20) × 10³The rpm is 5-10 times, each time is 2min, and the stirring time is 10-20min in total.

When spraying, the amount of the liquid medicine sprayed on the collagen carrier per square centimeter is 0.049-0.563ml, and the thickness of the liquid medicine layer is 0.049-0.247 mm.

The mean diameter of the particles of the hemostatic composition in the suspension is 33-50 μm (preferably 37-42 μm), and/or the 5min sedimentation volume of the suspension is > 90% (preferably ≧ 95%).

Before spraying, the collagen carrier is a vitreous body with smooth surface and compact internal structure, which is obtained by freezing collagen for 19-23h in the process of reducing the temperature of 20 ℃ to-50 ℃; optionally, the thickness is 0.3-0.5 cm.

In a third aspect, the present invention provides the use of the above-described hemostatic composition and hemostatic patch in the manufacture of a hemostatic product, preferably a visceral bleeding hemostatic product.

The hemostatic composition provided by the invention comprises fibrinogen, thrombin and calcium chloride, and a large number of experimental studies find that the fibrinogen, the thrombin and the calcium chloride can synergistically exert the hemostatic effect to reduce the blood coagulation time under a specific ratio, the specific ratios of the fibrinogen, the thrombin and the calcium chloride in the hemostatic composition are respectively 16-46 parts, 38-42 parts and 100-121 parts (one part is calculated by mg of the fibrinogen, one part is calculated by IU of the thrombin and one part is calculated by mu g of the calcium chloride), and experiments prove that the hemostatic composition under the specific ratio can achieve a good hemostatic effect.

The invention also provides a hemostatic plaster, which comprises a collagen carrier and a liquid medicine layer formed by the hemostatic composition, wherein the liquid medicine layer is formed by uniformly dispersing and fixing a suspension containing the hemostatic composition on the collagen carrier. The hemostatic plaster can be used for hemostasis during visceral hemorrhage, can be directly pasted on the viscera without fixed bleeding points during use, has the advantages of simple operation, strong adhesion, good hemostatic effect, good biocompatibility, complete degradation in vivo, no need of secondary debridement, and avoidance of secondary injury to wounded. The hemostatic patch has stable performance, the liquid medicine layer is not easy to fall off, and the hemostatic patch can be applied to hemostasis (especially to hemostasis of visceral hemorrhage) of wounded persons in various emergencies, natural disasters (such as violent earthquakes) and other situations, and has good commercial value and wide application prospect.

Drawings

FIG. 1 is a side view of a hemostatic patch prepared from the hemostatic composition of example 1;

FIG. 2 is a top view of the hemostatic patch of FIG. 1;

FIG. 3 is a top view of a prior art carrier;

FIG. 4 is a schematic perspective view of the automatic coating apparatus;

fig. 5 is a schematic configuration diagram of the liquid ejection mechanism 5.

Detailed Description

The invention provides a fibrin hemostatic composition, which comprises 16-46 parts of fibrinogen (from human, mammal or recombinant fibrinogen), 38-42 parts of thrombin (from human, mammal or recombinant thrombin) and 100-121 parts of calcium chloride, wherein the fibrinogen is one part in mg, the thrombin is one part in IU, and the calcium chloride is one part in mu g; preferably, the fibrinogen composition comprises 17 parts of fibrinogen, 40 parts of thrombin and 115 parts of calcium chloride (CaCl)₂). Wherein the content of the first and second substances,

fibrinogen (Fibrinogen), coagulation factor i, is a macromolecular glycoprotein containing 2964 amino acids, has a relative molecular mass of 340kDa, and is composed of two subunits (fibrinopeptides a and B) arranged bilaterally symmetrically, each subunit containing three peptide chains of α, β, and γ, and the middle of which is connected by 29 disulfide bonds (aalpha B β γ) 2. Fibrinogen is a precursor of fibrin, and sufficient fibrinogen levels are essential requirements for effective coagulation. The hemostatic composition contains enough fibrinogen, can supplement additional fibrinogen to a blood loss part, the fibrinogen is hydrolyzed into fibrin by thrombin, and the fibrin is polymerized to form a fibrin polymer in a solid blood coagulation block shape under the combined action of the thrombin, blood coagulation factors XIII and XIIIa in blood, so as to block the surface of a bleeding blood vessel or a wound, prevent the bleeding blood vessel or the wound from continuing bleeding and achieve the aim of hemostasis. The invention can use human fibrinogen, and can also use mammal fibrinogen and recombinant fibrinogen, and can be purchased from Hualan bioengineering GmbH, Shanghai Yuan leaf science and technology GmbH, Nanjing Sai hong Rui Biotechnology GmbH; the human fibrinogen is white, off-white or light yellow loose body, and can be pulverized into powder.

Thrombin (Thrombin) is a serine protease, and has a main function of hydrolyzing water-soluble fibrinogen into water-insoluble fibrin, thereby playing an important role in hemostasis and coagulation, tissue repair, wound healing, and the like. Thrombin is produced by activation of prothrombin (factor ii) and is converted primarily to alpha-thrombin in activated form (content > 97% in thrombin, with small amounts of beta-and gamma-thrombin remaining). The invention can use human thrombin, or mammal thrombin and recombinant thrombin which can be purchased from Hualan bioengineering GmbH, Shanghai Yuanye science and technology Co, Nanjing hong Rui biotechnology Co, and is white, grey white or light yellow loose body, and can be pulverized into powder.

Calcium ion (Ca)²⁺) I.e. factor iv. Ca is generally supplied by calcium chloride²⁺，Ca²⁺Are indispensable in each coagulation pathway. Such as in the intrinsic coagulation pathway, Ca²⁺Can assist in activating the blood coagulation factor XI (existing in blood), and the blood coagulation factor XI can participate in other blood coagulation pathways to play a blood coagulation role; and in Ca²⁺And platelet phospholipids, factor x (present in the blood) is also activated and thus functions in the common coagulation pathway. In the extrinsic coagulation pathway, Ca²⁺Factor iii (present in blood), and factor vii (present in blood) together activate factor x and thus function in a common coagulation pathway. In the common coagulation pathway, Ca²⁺Binding thrombin, factor V (present in blood) and activated factor X (activated in the intrinsic and extrinsic coagulation pathways) converts fibrinogen into fibrin monomers, exerting hemostatic and blood coagulation effects. In addition, Ca²⁺It may also assist in activating factor XIII (present in the blood), which in turn activates factor XIIIa (present in the blood), which in turn acts on fibrin monomers, turning them into a stable fibrin polymer clot. The calcium chloride used in the invention is purchased from Beijing Hongxing chemical plant and is white granular.

The hemostatic composition provided by the invention takes fibrinogen, thrombin and calcium ions as main hemostatic components, and the hemostatic principle is the final stage of simulated biological coagulation cascade reaction, namely: fibers when the hemostatic composition contacts the bleeding siteProteinogen in thrombin and Ca²⁺Forming fibrin monomers under the action of the catalyst; fibrin monomers are further polymerized under the action of blood coagulation factor XIIIa to form fibrin polymers, and the fibrin polymers exist in the form of blood clots and are used as barriers to block bleeding vessels or wound surfaces, so that the bleeding vessels or the wound surfaces can be effectively prevented from continuously bleeding. Meanwhile, the thrombin can promote the adhesion, activation and aggregation of the platelets, and the aggregated and activated platelets further release blood coagulation active substances, such as Adenosine Diphosphate (ADP), Adenosine Triphosphate (ATP), thromboxane A2, serotonin and the like; while Ca²⁺Can activate some blood coagulation factors, and the blood coagulation active substances and the blood coagulation factors can increase the generation of thrombin, thereby increasing and accelerating the formation of fibrin and polymers, so as to positively accelerate blood coagulation and hemostasis in a virtuous cycle.

On the basis, the invention also provides a hemostatic patch which comprises the hemostatic composition and a solid carrier. The solid carrier needs to be degradable, and commonly used degradable solid carrier materials comprise oxidized regenerated cellulose, polylactic acid protein, collagen and the like. The present invention uses Collagen as a solid carrier of a hemostatic patch because Collagen (Collagen) has a developed quaternary structure, can form a scaffold-like structure, and can be used as a carrier. In addition, the collagen can play a role in hemostasis by promoting platelet aggregation, can promote the formation and growth of blood vessels and has a repair function on local tissues. Common collagen is bovine collagen, porcine collagen, equine collagen, etc., which are derived from mammals and have low immunogenicity. In the specific embodiment of the invention, bovine collagen is used as the solid carrier of the hemostatic patch, so the solid carrier is also called a collagen carrier. The bovine collagen protein liquid is purchased from Tianjin century Kangtai biomedical engineering Co., Ltd, and is in transparent gel form. The hemostatic plaster comprises the following raw materials: 16-46mg/cm²38-42IU/cm of fibrinogen²100-121. mu.g/cm²Calcium chloride of not less than 0.5mg/cm²The collagen of (1).

The process for preparing the hemostatic plaster comprises the following steps:

(1) putting a square mould (made of medical grade PVC) into a 316 stainless steel liquid medicine tray, taking a small amount of bovine collagen (transparent gel, which is not treated after purchase and is directly used) into the mould, and scraping the bovine collagen by using a collagen scraper (made of 316 stainless steel) to ensure that the collagen forms a collagen layer with the thickness of 0.3-0.5cm on the mould; after removing the redundant bovine collagen, sticking a pressure film (made of medical grade PVC) on the surface of the scraped collagen layer, wherein large air bubbles are prevented from appearing between the pressure film and the collagen layer in the sticking process, finally freezing for 19-23h at the temperature of 20-50 ℃ in a gradient manner (namely, the temperature is reduced from 20 ℃ to 50 ℃ below zero in a gradient manner), removing the pressure film, and taking the frozen collagen layer as a solid carrier of the hemostatic patch, namely, a collagen carrier.

The gradient freezing may be specifically: the temperature is reduced from 20 ℃ to 0 ℃ within 1h, then the temperature is reduced to-50 ℃ within 18-22h, and the temperature is maintained for 2 h.

(2) Mixing fibrinogen, thrombin, calcium chloride and low temperature anhydrous alcohol (-20 deg.C for precooling) to obtain medicinal liquid, homogenizing and stirring in a disperser (IKA, T25 easy clean) at stirring speed of (10-20) x 10³The rpm is 5-10 times, and the stirring time is 10-20 min. Homogenizing to obtain suspension containing the hemostatic composition, and placing in a low temperature environment (-50-20 deg.C).

(3) And parameters for setting the automatic coating device (the structure of the automatic coating device is described in patent No. CN 214021579U): the flow rate of the peristaltic pump is 50-80ml/min, the liquid spraying tube swings for 1-6 times, and the sprayed liquid medicine is 0.049-0.563ml/cm²The single movement distance of Y-axis 3000-4500cm (1cm is 1 × 10)^-5m); the total X-axis travel distance is 3000-4500 cm.

(4) And (3) after the parameters of the step (3) are set, putting an inlet of a pump pipe (19#, made of silica gel, with the inner diameter of 2.4mm and the outer diameter of 5.6mm, purchased from Zengron constant flow pump Co., Ltd.) into the suspension obtained in the step (2), uniformly spraying the suspension on the surface of the frozen collagen carrier by using an automatic spraying device, and forming a liquid medicine layer with the thickness of 1-5mm on the surface of the collagen carrier by using the suspension.

(5) After spraying, putting a stainless steel liquid medicine tray into a freeze dryer (CHRIST, Epsilon 2-4 LSC plus) precooled to-50 ℃ in advance, freeze-drying for 49h40min-63h (the freeze-drying time is too short, the moisture content in a collagen carrier and a liquid medicine layer is too high, a sheet patch product cannot be formed), the freeze-drying time is too long, the moisture content in the collagen carrier and the liquid medicine layer is too low, the liquid medicine layer can be dried and cracked due to over drying, the appearance of the product is influenced, and the hemostatic effect is influenced), the liquid medicine layer can be attached to the surface of the collagen carrier in the freeze-drying process and forms an integrated hemostatic patch with the collagen carrier until the moisture content of the hemostatic patch is not higher than 10%, stopping freeze-drying, taking out, packaging and sterilizing to obtain the hemostatic patch product.

In this method, unlike the prior art, the collagen carrier is formed by freezing (rather than freeze-drying) the collagen in step (1), so that the collagen carrier is formed without a sponge-like structure, and is a glassy solid with a smooth surface and a dense interior. And (4) when the suspension is sprayed, the suspension does not penetrate into the collagen carrier, but is positioned on the smooth surface of the collagen carrier. And (3) when the freeze-drying in the step (5) is carried out, the water in the collagen carrier and the solvent in the liquid medicine layer are volatilized together, and substances left in the collagen carrier and the liquid medicine layer are mutually permeated in the process, so that the collagen carrier and the liquid medicine layer are tightly connected together, and the liquid medicine layer is not easy to fall off from the collagen carrier.

The automatic spray device used in the method is used to spray the hemostatic composition to each area of the square mold in the 316 stainless steel liquid drug tray to make the hemostatic patch. Fig. 4 is a structural example of the automatic coating device. The automatic spraying device comprises a reagent tray translation mechanism 3, a liquid spraying mechanism 5 and a nozzle translation mechanism 6, wherein the reagent tray translation mechanism 3 is provided with a translation driving motor for driving a reagent tray arranged on the reagent tray translation mechanism 3 to do translation reciprocating motion (namely, horizontal movement left and right and X axis), the nozzle translation mechanism 6 is provided with a translation driving motor for driving a nozzle 65 arranged on the nozzle translation mechanism 6 to do translation reciprocating motion (namely, horizontal movement front and back and Y axis), the nozzle is positioned above a reagent tray 4, the liquid spraying mechanism 5 is provided with a peristaltic pump 51 (see figure 5) for pushing liquid medicine into the nozzle 65 through a spray pipe 52, and the liquid medicine is sprayed on each area of the reagent tray provided with a bottom plate in a quantitative mode. The translation driving motor can rotate forwards and backwards to enable the reagent tray 4 arranged on the reagent tray translation mechanism 3 to do translation reciprocating motion, and the traverse driving motor can rotate forwards and backwards to enable the nozzle 65 arranged on the nozzle traverse mechanism 6 to do linear reciprocating motion along the direction vertical to the translation direction of the reagent tray 4; the peristaltic pump of the liquid spraying mechanism 5 extracts a preset amount of liquid medicine for each area of the reagent tray 4, so that the liquid medicine is quickly and uniformly sprayed into each area of the reagent tray 4 according to a preset route, the activity of the medicine is kept, the loss of the liquid medicine is reduced, and the spraying uniformity is ensured.

The present invention will be described more specifically and further illustrated with reference to specific examples, which are by no means intended to limit the scope of the present invention.

Example 1

The hemostatic composition of this example consisted of 17mg fibrinogen, 40IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Example 2

The hemostatic composition of this example consisted of 16mg fibrinogen, 38IU thrombin, 100 μ g calcium chloride (CaCl)₂) And (4) forming.

Example 3

The hemostatic composition of this example consisted of 20mg fibrinogen, 42IU thrombin, 121 μ g calcium chloride (CaCl)₂) And (4) forming.

Example 4

The hemostatic composition of this example consisted of 18mg fibrinogen, 39IU thrombin, 110 μ g calcium chloride (CaCl)₂) And (4) forming.

Example 5

The hemostatic composition of this example consisted of 17mg fibrinogen, 40IU thrombin, 103 μ g calcium chloride (CaCl)₂) And (4) forming.

Example 6

The hemostatic composition of this example consisted of 46mg fibrinogen, 40IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 1

Ben Bian (Ben)A comparative hemostatic composition consists of 10.3mg fibrinogen, 28.6IU thrombin, 2500 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 2

The hemostatic composition of this comparative example consisted of 15mg fibrinogen, 37.5IU thrombin, 117. mu.g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 3

The hemostatic composition of this comparative example consisted of 13mg fibrinogen, 40IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 4

The hemostatic composition of this comparative example consisted of 47mg fibrinogen, 40IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 5

The hemostatic composition of this comparative example consisted of 17mg fibrinogen, 45IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 6

The hemostatic composition of this comparative example consisted of 17mg fibrinogen, 30IU thrombin, 115 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 7

The hemostatic composition of this comparative example consisted of 17mg fibrinogen, 40IU thrombin, 80 μ g calcium chloride (CaCl)₂) And (4) forming.

Comparative example 8

The hemostatic composition of this comparative example consisted of 17mg fibrinogen, 40IU thrombin, 130 μ g calcium chloride (CaCl)₂) And (4) forming.

Experiment I, detection of mechanical properties

The experiment aims to simulate the fibrin polymer blood clot formed after the hemostatic composition contacts blood and then detect the mechanical property of the fibrin polymer blood clot. The mechanical property indexes are as follows: the storage modulus (G '), the loss modulus (G') and the viscoelastic property tan delta (G '/G') are detection indexes.

1. Preparation of samples

Dissolving fibrinogen in the hemostatic compositions of examples 1-6 in 1ml of water to prepare an aqueous solution A, dissolving thrombin and calcium chloride in the hemostatic compositions of examples 1-6 in 1ml of water to prepare an aqueous solution B, and finally mixing the aqueous solution A and the aqueous solution B to form a clot sample, and detecting the mechanical property index of the clot sample. The water added during the process was used to simulate blood and the formed clot was used to simulate the formed fibrin polymer blood clot.

2. Detection of mechanical property index

The clot sample was placed on a test plate of a rheometer (antopa, MCR302), heated to 37 ℃, with a test rotor diameter of 12mm and a test rotor to test plate distance of 1 mm. A dynamic method with small amplitude (1%) is used, namely: shearing a coagulum sample under small-amplitude oscillation with the frequency of 0.1-10 Hz, then measuring the storage modulus (G ') and the loss modulus (G ") of the sample by frequency scanning, and calculating to obtain a viscoelastic characteristic value tan delta (G '/G '); tan delta <1, indicating that the sample tends to gel and is not susceptible to deformation, i.e.: the hemostatic composition is not easy to be broken by blood when in use, and can maintain the original shape; tan δ > 1, indicating that the sample tends to be fluid and is subject to deformation, i.e.: the hemostatic composition is easily broken by blood when used, and cannot maintain its original form. The results are shown in Table 1, using example 1 and example 5 as examples.

Table 1 results of mechanical property testing of hemostatic compositions of examples 1 and 5

Examples	Storage modulus G	Loss modulus G "	Viscoelastic characteristic value tan delta
				Example 1	180.85-285.5	46.781-77.814	0.1396-0.3091
Example 5	353.32-418.99	55.866-90.722	0.1581-0.2265

The results in Table 1 show that the clot samples formed from the hemostatic compositions of examples 1 and 5 have tan delta <1, indicating that the clot samples formed tend to gel and are not susceptible to deformation, i.e.: the hemostatic composition of the present invention is not easily broken by blood when used, and can maintain its original form.

Experiment II, determination of average particle size and precipitation volume ratio

The invention finds that in step (2) of the method for preparing the hemostatic patch, the average diameter of the particles in the obtained suspension and the 5min sediment volume ratio (standing for 5min after the suspension is prepared, and measuring the overall initial height (marked as H) of the particles in the suspension before standing₀) And the height of the particles in the suspension after standing (noted as H), 5min sediment volume ratio ═ H/H₀) The uniform effect of the subsequent spraying can be influenced: when the particle diameter is large, the sedimentation speed of particles in the suspension is high, and the particles are easy to deposit and block a pump pipe, so that the suspension cannot be sprayed or the spraying is uneven. This experiment was therefore carried out by adjusting the parameters of step (2) of the preparation process to obtain suspensions containing hemostatic compositions with different particle sizes and 5min sediment volume ratios. After the liquid medicine in the step (2) is homogenized by a dispersion machine, the temperature can change, and the protein activity of fibrinogen and thrombin can be influenced by overhigh temperature, so the temperature of the suspension before and after homogenization is also detected in the experiment.

The specific process of the experiment is as follows: the hemostatic compositions of examples 1-6 were each added to absolute ethanol pre-cooled to-20 deg.C and stirred to form a liquid medicine, the final concentration of each substance in the liquid medicine was fibrinogen 140.5mg/ml, thrombin 330.6IU/ml, calcium chloride 950.5 μ g/ml. Then theHomogenizing and stirring the medicinal liquid with a disperser (IKA, T25 easy clean) at a stirring speed of 15 × 10³Stirring for 5-10 times at rpm for 2min (30 s for subsequent spraying to block the pump tube), and homogenizing for 10-20 min. Three samples were taken after each homogenization and the temperature before and when homogenization stopped and the 5min sediment volume fraction were recorded. Two experiments were performed, using the hemostatic composition of example 1 as an example, the two experiments (experiment a and experiment B) differing only in the number of stirring. The stirring frequency in experiment A is 5 times, and the results are shown in Table 2; the number of stirring times in experiment B was 10, and the results are shown in Table 3.

TABLE 2 temperature, mean diameter and 5min sediment volume ratio before and after homogenization for experiment A

The results in table 2 show that the temperature difference of the suspension before and after homogenization in step (2) does not exceed 1.5 ℃, and the activity of the protein is not affected; the average diameter of the particles of the hemostatic composition in the suspension after homogenization is 37.78 mu m, the 5min precipitation volume ratio after each homogenization is higher than 90 percent, and the 5min precipitation volume ratio of the suspension obtained after the homogenization can reach 95 percent, which shows that the suspension obtained in the homogenization process can reach higher 5min precipitation volume ratio.

Table 3 temperature, average diameter and setting time before and after homogenization for experiment B

The results in Table 3 show that the mean diameter of the particles of the hemostatic composition in the suspension after homogenisation was 41.63 μm and that the 5min sedimentation volume after each homogenisation averaged over 90% (not shown), indicating that a higher 5min sedimentation volume ratio was achieved for the suspension obtained during homogenisation. The temperature difference of the suspension before and after homogenization in the step (2) is not more than 2.5 ℃, and the influence on the activity of the protein is low.

In addition, the effect of the homogenization process on the protein activity was also characterized by the clotting time, which is given as an example for fibrinogen, in table 3. The results in Table 3 show that the clotting time of fibrinogen after each homogenisation did not exceed 22s, and met the criteria that the clotting time is less than 60s in the Standard of human fibrinogen Activity in the Chinese pharmacopoeia (2020 edition).

The results in tables 2 and 3 show that the mean diameter of the particles of the hemostatic composition in the suspension obtained in step (2) of the method for preparing the hemostatic patch is 37.78-41.63 μm, and the precipitation volume ratio in 5min is higher than 90%, which indicates that the obtained suspension has concentrated particle size, small span, slow sedimentation rate of the particles, and is not easy to deposit and block the pump pipe, and uniform spraying effect can be achieved in subsequent spraying. The temperature difference before and after homogenizing treatment of the suspension is less than 10 ℃, the fibrinogen coagulation time after homogenizing is less than 60s, the requirements of Chinese pharmacopoeia (2020 edition) are met, and the homogenization process does not influence the protein activity of the fibrinogen and the thrombin.

Since the content of each substance in the raw materials is not related to the homogenization effect, the results of example 1 in this experiment can be used to show that other examples have the same results as example 1, and are not repeated herein.

Experiment III, determination of coagulation time

Sample preparation:

control sample 1: prepared according to the content disclosed in the Chinese patent application with the publication number CN1507358A, and the hemostatic component of the hemostatic component is 5.5mg/cm fibrinogen²And thrombin 2.0IU/cm²And (4) forming.

Control sample 2: the hemostatic component is composed of fibrinogen 15mg/cm²Thrombin 37.5IU/cm²And 117. mu.g/cm calcium chloride²And (4) forming.

Experimental samples: the hemostatic compositions of examples 1-6.

Comparison of samples: the hemostatic compositions of comparative examples 1, 3-8.

This experiment was conducted by adding the hemostatic composition/hemostatic composition contained in square centimeters per unit to water to simulate clotting in blood. According to literature reports, the blood-sucking amount per unit area of the control samples 1 and 2 does not exceed 4 ml. Therefore, in this experiment, a magnetic bead method is used to add fibrinogen in the hemostatic composition/hemostatic component contained in a unit square centimeter into 5ml of water to form a solution A, then add the remaining hemostatic components (thrombin or thrombin and calcium chloride) into 5ml of water to form a solution B, and add 100. mu.l of the solution A and 100. mu.l of the solution B into a reaction cup of a hemagglutination instrument in equal volume, and the hemagglutination instrument automatically detects the time when the solution A and the solution B react to form fibrin polymer clot and records the clotting time. The results are shown in Table 4, using examples 1, 2, 5 and 6 as examples.

TABLE 4 coagulation time of the samples

The results in table 5 show that the hemostatic compositions of examples 1, 2, 5, and 6 are superior to control samples 1 and 2 in clotting time at equal dilution ratios, with significant differences; it is shown that the hemostatic composition of the present invention is significantly superior in hemostatic effect to the control samples 1 and 2. In addition, the hemostatic compositions of examples 1, 2, 5 and 6 were also superior to comparative examples 1-8 in clotting time, with significant differences; the hemostatic composition of the invention is obviously superior to the comparative examples 1-8 in hemostatic effect; in particular, in comparative example 4, the content of fibrinogen is only slightly higher than that in example 6, and the clotting time is significantly improved, which indicates that the hemostatic composition provided by the present invention can synergistically exert more excellent hemostatic effects by adjusting the contents of fibrinogen, thrombin and calcium chloride.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the content of the present invention.

Claims

1. A haemostatic composition comprising 16-46 parts (preferably 16-20 parts) fibrinogen, 38-42 parts thrombin and 100 parts calcium chloride, wherein fibrinogen is present in mg, thrombin is present in IU and calcium chloride is present in μ g.

2. The hemostatic composition of claim 1, comprising 16-19 parts fibrinogen, 39-41 parts thrombin and 103-118 parts calcium chloride; preferably, the fibrinogen composition comprises 17 parts of fibrinogen, 40 parts of thrombin and 115 parts of calcium chloride (CaCl)₂)。

3. Hemostatic composition according to claim 1 or 2, wherein the fibrin polymers formed after the composition has been exposed to water have a viscoelastic property value tan δ <1, preferably between 0.13 and 0.31.

4. A hemostatic patch comprising a collagen carrier prepared from collagen and a liquid drug layer uniformly dispersed and fixed on the collagen carrier, wherein the liquid drug layer is prepared from the hemostatic composition according to any one of claims 1 to 3.

5. The hemostatic patch according to claim 4, wherein each square centimeter of the hemostatic patch contains 16-46mg of fibrinogen, 38-42IU of thrombin, 100 μ g of calcium chloride and > 0.5mg of collagen.

6. The hemostatic patch according to claim 3 or 4, wherein the liquid drug layer is formed by dispersing the hemostatic composition in ethanol to form a suspension and then spraying the suspension onto a collagen carrier; optionally, after dispersing the hemostatic composition in ethanol, placing the mixture in a dispersion machine for homogeneous stirring, wherein the stirring speed is (10-20) × 10³The rpm is 5-10 times, each time is 2min, and the stirring time is 10-20min in total.

7. The hemostatic plaster of claim 6, wherein the amount of the liquid medicine sprayed per square centimeter of the collagen carrier is 0.049-0.563ml, and the thickness of the liquid medicine layer is 0.049-0.247 mm.

8. Hemostatic patch according to any one of claims 5-7, wherein the mean diameter of the particles of the hemostatic composition in the suspension is 33-50 μm (preferably 37-42 μm) and/or the 5min sediment volume of the suspension is > 90% (preferably ≥ 95%) by weight of the mean.

9. The hemostatic patch according to any one of claims 5-8, wherein the collagen carrier before spraying is a vitreous body with smooth surface and dense internal structure obtained by freezing collagen for 19-23h in the process of reducing the temperature of 20 ℃ to-50 ℃; optionally, the thickness is 0.3-0.5 cm.

10. Use of a hemostatic composition according to claim 1 or 2 and a hemostatic patch according to any one of claims 3 to 9 for the preparation of a hemostatic product.