Patent Application
20230190992
\This application claims priority under 35 USC 119 from Japanese Patent Application No. 2021-207606 filed on Dec. 21, 2021, the disclosure of which is incorporated by reference herein in its entirety.
The present disclosure relates to a hemostatic material composition.
Uncontrollable bleeding is still the main cause of death of traumatic or surgical injuries. Accordingly, the development of an effective therapeutic approach for controlling bleeding is of vital clinical and social importance.
As hemostatic materials that are used in a surgical operation, fibrin glue, a non-woven fabric of oxidized cellulose, a collagen sponge, fine starch particles, a thrombin-containing gelatin paste, and the like have been known so far, and these are used in various forms.
Among them, FLOSEAL (registered trade name, manufactured by Baxter International Inc.) and SURGIFLO (manufactured by Ethicon, Inc.) are widely known as thrombin-containing gelatin pastes.
Thrombin activates fibrinogen contained in the blood to form a fibrin network, and the fibrin network has an action of sealing a bleeding part by forming a blood clot together with blood cells. Although thrombin is purified from the human blood that has been properly sorted out through a step of inactivating viruses and a step of removing them, there is a problem that the risk of transmission of an infectious disease cannot be completely eliminated. As a result, there is a demand for the development of a hemostatic material having excellent safety.
JP6195568B discloses a hemostatic material composition containing a polyethylene glycol having an N-hydroxysuccinimide ester group, a polyethylene glycol which is a hydrophilic solvent and containing crosslinked gelatin particles.
Recently, the inventors of the present invention newly found that in the hemostatic material composition disclosed in JP6195568B, there is room for improvement in storage stability since an N-hydroxysuccinimide ester group contained in polyethylene glycol reacts with a trace amount of water contained in a hydrophilic solvent, a hydroxyl group contained in the hydrophilic solvent, and the like and is decomposed, resulting in temporal deterioration of hemostatic properties.
An object to be achieved by one aspect according to the present disclosure is to provide a hemostatic material composition which is excellent in storage stability.
Specific means for solving the object are as follows.
<1> A hemostatic material composition comprising:
<2> The hemostatic material composition according to <1>, in which the crosslinked particle contains the crosslinked protein, and the crosslinked protein is a crosslinked gelatin.
\<3> The hemostatic material composition according to <1> or <2>, in which an average primary particle diameter of the crosslinked particles is 20 µm to 700 µm.
\<4> The hemostatic material composition according to any one of <1> to <3>, in which the reactive group is one or more groups selected from the group consisting of -CON(COCH2)2\, an N-hydroxysuccinimide ester group, an imide ester group, an aldehyde group, a carboxy group, an isocyanate group, a maleimide group, and a haloacetyl group.
<5> The hemostatic material composition according to any one of <1> to <4>, in which the reactive group is an N-hydroxysuccinimide ester group.
<6> The hemostatic material composition according to any one of <1> to <5>, in which the polysaccharide includes a structure derived from one or more polysaccharides selected from the group consisting of cellulose, dextran, dextrin, pullulan, hyaluronic acid, alginic acid, xanthan gum, chitin, chitosan, and a modified product thereof.
\<7> The hemostatic material composition according to any one of <1> to <6>, in which the polysaccharide contains a constitutional unit derived from a monosaccharide having the reactive group, and a content of a constitutional unit derived from the monosaccharide having the reactive group is 1% by mole to 40% by mole with respect to 100% by mole of the polysaccharide.
<8> The hemostatic material composition according to <7>, in which the constitutional unit derived from the monosaccharide having the reactive group is represented by General Formula (1).
In General Formula (1), L is a group represented by the following formula.
In the formula, n and m represent an integer of 1 to 3, * represents a site linked to an oxygen atom on a tetrahydrofuran side, and ** represents a site linked to an oxygen atom on an N-hydroxysuccinimide ester group side.
<9> The hemostatic material composition according to any one of <1> to <8>, in which a weight-average molecular weight of the polysaccharide is \2,000 to 2,500,000.
<10> The hemostatic material composition according to any one of <1> to <9>, in which the hydrophobic solvent is one or more selected from the group consisting of a vegetable oil, a paraffin-based solvent, an olefin-based solvent, a silicone oil-based solvent, a fatty acid ester-based solvent, an alkyl halide-based solvent, and a terpene-based solvent.
<11> The hemostatic material composition according to any one of <1> to <10>, in which a kinematic viscosity of the hydrophobic solvent at 25° C. is 1 mm2/sec to \1,000 mm2/sec.
<12> The hemostatic material composition according to any one of <1> to <11>, in which a content of the hydrophobic solvent is 30% by mass to 80% by mass with respect to a total mass of the hemostatic material composition.
<13> The hemostatic material composition according to any one of <1> to <12>, in which the hemostatic material composition does not contain water or contains water, and a content of the water is 0.5% by mass or less with respect to a total mass of the hemostatic material composition.
According to the present disclosure, it is possible to provide a hemostatic material composition which is excellent in storage stability.
In the present disclosure, a numerical range described using “to” includes numerical values before and after “to” as a minimum value and a maximum value, respectively.
In the numerical ranges described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical range described stepwise in other stages. Further, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in Examples.
In the present disclosure, the “hydrophobic solvent” means a solvent which has low miscibility with water, where 20 g or more of the solvent is not dissolved in 1 L of ion exchange water (pH 6 to 8) at 20° C., and does not form a uniform aqueous solution, that is, it refers to a solvent having a solubility in water of less than 20 g/L. In addition, in the present disclosure, the solvent having a solubility of 20 g/L or more is referred to as a hydrophilic solvent.
Here, the pH of the ion exchange water indicates a value measured at 20° C. within 3 minutes after the ion exchange water comes into contact with air.
A hemostatic material composition according to the present disclosure contains a polysaccharide (hereinafter, also referred to as a “specific polysaccharide”) having a reactive group (hereinafter, also referred to as a “specific reactive group”) that reacts with a protein to form a crosslinking structure, and a hydrophobic solvent, and a crosslinked particle containing one or more compounds selected from the group consisting of a crosslinked protein and a crosslinked polysaccharide.
The hemostatic material composition according to the present disclosure is excellent in storage stability. The reason why the above effect is obtained is presumed as follows, which is not limited thereto.
The hemostatic material composition according to the present disclosure contains a hydrophobic solvent, and the specific polysaccharide is present in a state of being dispersed in the hydrophobic solvent. It is presumed that the specific reactive group contained in the specific polysaccharide is difficult to be decomposed in the hydrophobic solvent and exhibits excellent storage stability.
In addition, since the hemostatic material composition according to the present disclosure contains crosslinked particles, it is presumed that the crosslinked particles absorb a trace amount of water in the composition or the like, and the decomposition of the specific polysaccharide is suppressed, thereby exhibiting excellent storage stability. Further, since the crosslinked gelatin has a protein structure even in the crosslinked particles, it is presumed that the crosslinked gelatin has a high affinity to water, and the effect of the above-described storage stability is more remarkably exhibited.
The state of the hemostatic material composition according to the present disclosure is not particularly limited; however, it is preferably a paste state from the viewpoint of ease of application to a living body.
The specific polysaccharide has a specific reactive group that reacts with a protein to form a crosslinking structure.
\The specific reactive group is not particularly limited as long as it is a group capable of reacting with an amino group or the like contained in a protein to form a crosslinking structure. However, from the viewpoint of improving hemostatic properties, it is preferably one or more groups selected from the group consisting of —CON(COCH2)2\, an N-hydroxysuccinimide ester group (hereinafter, also referred to as an “NHS-ester group”), an imide ester group, an aldehyde group, a carboxy group, an isocyanate group, a maleimide group, and a haloacetyl group, and it is more preferably an NHS-ester group.
The specific polysaccharide may have two or more kinds of specific reactive groups. In addition, the hemostatic material composition according to the present disclosure may contain two or more kinds of specific polysaccharides.
It is noted that in the present disclosure, a polysaccharide that does not have a specific reactive group is also referred to as a non-specific polysaccharide.
The specific polysaccharide contains a constitutional unit derived from two or more monosaccharides.
From the viewpoint of improving storage stability and hemostatic properties and the viewpoint of biocompatibility, the specific polysaccharide preferably includes a structure derived from one or more polysaccharides selected from the group consisting of cellulose, dextran, dextrin, pullulan, hyaluronic acid, alginic acid, xanthan gum, chitin, chitosan, and a modified product thereof, and it is more preferably include a structure derived from dextran.
The above-described structure contained in the polysaccharide is preferably a structure modified with a specific reactive group. In addition, the above-described structure contained in the polysaccharide may be carboxy-modified.
In the present disclosure, the modified product refers to a compound into which a functional group has been introduced by reacting cellulose or the like with a functional group such as a carboxy group.
From the viewpoint of improving storage stability and hemostatic properties, the specific polysaccharide contains a constitutional unit (hereinafter, also referred to as a “specific constitutional unit”) derived from a monosaccharide having a specific reactive group, and the content of the specific constitutional unit is preferably 1% by mole to 40% by mole, more preferably 3% by mole to 30% by mole, and still more preferably 5% by mole to 25% by mole, with respect to 100% by mole of the specific polysaccharide.
From the viewpoint of improving storage stability and hemostatic properties, the viewpoint of biocompatibility, and the like, the specific constitutional unit is preferably a constitutional unit represented by General Formula (1).
In General Formula (1), L is a group represented by the following formula.
In the following formula, n and m represent an integer of 1 to 3, * represents a site linked to an oxygen atom on a tetrahydrofuran side, and ** represents a site linked to an oxygen atom on an N-hydroxysuccinimide ester group side.
Among the above-described groups, from the viewpoint that the reaction rate of the reactive group is highest and the hemostatic properties are improved, L is preferably a group represented by the following formula.
From the viewpoint of biocompatibility and the like, the specific polysaccharide preferably contains a constitutional unit represented by Formula (2) in addition to the constitutional unit represented by Formula (1).
In a case where the specific polysaccharide contains the constitutional unit represented by Formula (2), the content of the constitutional unit represented by Formula (2) is preferably 40% by mole to 90% by mole and more preferably 45% by mole to 80% by mole with respect to 100% by mole of the specific polysaccharide.
The specific polysaccharide can contain a constitutional unit represented by Formula (3), whereby the pH at the time of dissolution in water can be decreased. Since the decomposition of the specific reactive group such as the N-hydroxysuccinimide ester group is suppressed in a solution having a low pH, it is possible to suppress a decomposition reaction of the composition or the like due to a small amount of water in the atmospheric air.
In a case where the specific polysaccharide contains the constitutional unit represented by Formula (3), the content of the constitutional unit represented by Formula (3) is preferably 5% by mole to 40% by mole and more preferably 8% by mole to 30% by mole with respect to 100% by mole of the specific polysaccharide.
The weight-average molecular weight (Mw) of the specific polysaccharide is preferably 2,000 to 2,500,000, more preferably 5,000 to 2,000,000, still more preferably 10,000 to 750,000, and particularly preferably 20,000 to 200,000.
In a case where the Mw of the specific polysaccharide is 2,000 or more, the specific reactive group contained in the specific polysaccharide reacts with a protein to form a good coating film in a case where a crosslinking structure is formed, and the hemostatic properties are further improved. In a case where the Mw of the specific polysaccharide is 2,500,000 or less, the specific polysaccharide can be easily synthesized, and the hemostatic material composition containing the specific polysaccharide is excellent in handleability.
In addition, in a case where the Mw of the specific polysaccharide is 2,000 or more, it is possible to easily adjust the number of specific reactive groups contained in the specific polysaccharide, and it is possible to improve hemostatic properties and storage stability. It is noted that the number of specific polysaccharides can be adjusted by adjusting the proportion of the monosaccharide having a reactive group that is used in the synthesis of the specific polysaccharide.
The excess portion of the hemostatic material composition applied to the hemostasis site is preferably removed after the hemostasis is completed, and it is generally removed by the saline or the like. In a case where the Mw of the specific polysaccharide is 2,500,000 or less, the removability can be improved.
In the present disclosure, Mw is determined by dissolving a specimen in ultrapure water to a concentration of 0.1% by mass, carrying out filtration using a filter having a pore diameter of 0.45 µm, and subjecting the obtained filtrate to the gel permeation chromatography (GPC) measurement. The measurement conditions are as follows.
Measurement condition
From the viewpoint of improving storage stability and hemostatic properties, the content of the specific polysaccharide is preferably 1% by mass to 60% by mass, more preferably 5% by mass to 50% by mass, and still more preferably 15% by mass to 30% by mass, with respect to the total mass of the hemostatic material composition.
From the viewpoint of improving storage stability, hemostatic properties, and removability, the solubility of the specific polysaccharide is preferably 1% by mass or less, more preferably 0.1% by mass or less, and still more preferably 0.5% by mass or less, in 100 g of the hydrophobic solvent at 20° C. The lower limit of the solubility is not particularly limited and may be 0% by mass.
For example, the fact that the solubility is 1% by mass in 100 g of a hydrophobic solvent at 20° C. means that the amount of the specific polysaccharide dissolved in 100 g of the hydrophobic solvent is 1 g.
From the viewpoint of improving storage stability, hemostatic properties, and removability, the hydrophobic solvent preferably has a solubility of less than 20 g/L, more preferably less than 5 g/L, and still more preferably less than 1 g/L in 1 L of ion exchange water at 20° C. The lower limit of the solubility is not particularly limited and may be 0 g/L.
The kind of the hydrophobic solvent is not particularly limited. From the viewpoint of improving storage stability and hemostatic properties and the viewpoint of biocompatibility, the hydrophobic solvent is preferably one or more selected from the group consisting of a vegetable oil, a paraffin-based solvent, an olefin-based solvent, a silicone oil-based solvent, a fatty acid ester-based solvent, an alkyl halide-based solvent, and a terpene-based solvent, more preferably one or more selected from the group consisting of a vegetable oil and a fatty acid ester-based solvent, and still more preferably a vegetable oil.
In the present disclosure, the “vegetable oil” is not particularly limited as long as it is an oil obtained by using a plant as a raw material, where it may be a derivative thereof, and a vegetable oil containing a fatty acid glyceride is preferable. Examples of the vegetable oil include a linseed oil, a wild sesame oil, a olive oil, a grapeseed oil, a corn oil, a coconut oil, a sesame oil, a rice bran oil, a soybean oil, a rapeseed oil, a palm oil, a sunflower oil, safflower oil, a cottonseed oil, a groundnut oil, an avocado oil, an almond oil, an argan oil, a cacao oil, a shea butter, a tea seed oil, a peanut oil, and a castor oil.
Examples of the paraffin-based solvent include octane, decane, dodecane, and tridecane.
Examples of the olefin-based solvent include decene and dodecene.
Examples of the silicone oil-based solvent include a dimethyl silicone oil, a methylphenyl silicone oil, and a cyclic dimethyl silicone oil.
Examples of the fatty acid ester-based solvent include methyl laurate, methyl palmitate, methyl stearate, methyl oleate, palm methyl oleate, caprylic acid 2-ethylhexyl ester, glycerin monostearate, glycerin monobehenate, glycerin mono-12-hydroxystearate, glycerin monooleate, glycerin monocaprylate, glycerin monocaprate, glycerin monolaurate, an alkyl oleate (ethyl oleate, methyl oleate, or the like), triglyceride oleate, an alkyl linoleate (methyl linoleate, ethyl linoleate, or the like), linoleic acid triglyceride, and dioleic acid glyceride.
Examples of the alkyl halide-based solvent include methylene chloride and 1,1,1-trichloroethane.
Examples of the terpene-based solvent include D-limonene.
The hemostatic material composition according to the present disclosure may contain two or more kinds of hydrophobic solvents.
The molecular weight of the hydrophobic solvent is preferably 300 to 1,500.
Due to being used for medical use application, the hydrophobic solvent may become hot instantaneously by the high-frequency treatment or the like. As a result, from the viewpoint of safety assuming an actual operation, the hydrophobic solvent is preferably non-volatile. The non-volatile solvent refers to a solvent having a boiling point of 50° C. or higher, where the boiling point is preferably 100° C. or higher and more preferably 200° C. or higher.
In the present disclosure, the measurement of the boiling point means the boiling point at 1 atmospheric pressure (101,325 Pa). The boiling point is measured by a boiling point meter, and it is measured using, for example, a boiling point measuring device (product name: “DosaTherm 300”) manufactured by Titan Technologies, K.K.
From the viewpoint of improving the ease of application to a living body and the removability by washing with water from the living body, and suppressing the temporal liquid separation, the kinematic viscosity of the hydrophobic solvent at 25° C. is preferably 1 mm2/sec to 1,000 mm2/sec, more preferably 5 mm2/sec to 200 mm2/sec, and still more preferably 20 mm2/sec to 100 mm2/sec.
In the present disclosure, the kinematic viscosity is measured in accordance with JIS K 2283 (2000).
From the viewpoint of improving storage stability and hemostatic properties, the content of the hydrophobic solvent is preferably 25% by mass to 75% by mass, more preferably 30% by mass to 70% by mass, and still more preferably 40% by mass to 60% by mass, with respect to the total mass of the hemostatic material composition.
The hemostatic material composition according to the present disclosure contains a crosslinked particle containing one or more compounds selected from the group consisting of a crosslinked protein and a crosslinked polysaccharide. In a case where the hemostatic material composition containing crosslinked particles is mixed with blood, water in the blood is taken up by the crosslinked particles, the concentration of proteins in the blood is increased, the reactivity with the specific polysaccharide is improved, and the hemostatic properties are further improved.
In addition, for the intended purpose of improving the hemostatic effect, the hemostatic material composition applied to the bleeding site may be pressed with gauze or the like, which has been impregnated with a saline solution or the like. In a case where the hemostatic material composition contains the crosslinked particles, it is possible to suppress the transfer of the hemostatic material composition to a gauze or the like, which is peeled off after pressing. This is presumed to be because the crosslinked particle takes up the saline or the like, with which gauze or the like has been impregnated, and swells depending on the degree of crosslinking, whereby the contact area between the gauze and the hemostatic material composition decreases.
In the present disclosure, the crosslinked particle means a particle in which two or more proteins or the like are crosslinked. In addition, in the present disclosure, the crosslinked protein refers to a crosslinked protein in which two or more proteins are crosslinked, and the crosslinked polysaccharide refers to a product in which two or more polysaccharides are crosslinked.
Examples of the crosslinked protein include crosslinked gelatin, crosslinked collagen, crosslinked albumin, crosslinked hemoglobin, crosslinked fibrinogen, crosslinked fibrin, crosslinked casein, crosslinked elastin, and crosslinked keratin.
Examples of the polysaccharide constituting the crosslinked polysaccharide include crosslinked cellulose, crosslinked dextran, crosslinked dextrin, crosslinked pullulan, crosslinked hyaluronic acid, crosslinked alginic acid, crosslinked xanthan gum, crosslinked chitin, crosslinked chitosan, and a modified product thereof.
The hemostatic material composition according to the present disclosure may contain two or more kinds of crosslinked particles.
From the viewpoint of improving storage stability, hemostatic properties, and transfer suppressibility, it is preferable that the crosslinked particle contains a crosslinked protein, where the crosslinked protein is crosslinked gelatin.
From the viewpoint of improving the properties of taking up water and improving hemostatic properties, the crosslinked protein and the crosslinked polysaccharide are preferably a hydrophilic polymer.
From the viewpoint of improving storage stability and hemostatic properties and the viewpoint of suppressing the transfer of the hemostatic material composition to the gauze, the sum of the contents of the crosslinked protein and the crosslinked polysaccharide with respect to the total mass of the crosslinked particles is preferably 50% by mass or more, more preferably 65% by mass or more, still more preferably 75% by mass or more, and particularly preferably 90% by mass or more, and it may be 100% by mass.
From the viewpoint of improving the properties of taking up water and improving storage stability and hemostatic properties, the viewpoint of suppressing the transfer of the hemostatic material composition to gauze, and the viewpoint of suppressing the invasion into blood vessels, and the like, the average primary particle diameter of the crosslinked particles is preferably 20 µm to 700 µm, more preferably 100 µm to 700 µm, and still more preferably 200 µm to 700 µm.
In the present disclosure, the measurement of the average primary particle diameter is determined by arithmetically averaging the diameters of 50 particles randomly selected from an image of a scanning electron microscope (SEM). It is noted that the primary particle diameter is measured as the particle diameter of the particle.
From the viewpoint of improving storage stability, the viewpoint of improving the properties of taking up water and hemostatic properties, the viewpoint of suppressing the transfer of the hemostatic material composition to gauze, and the viewpoint of suppressing the invasion into blood vessels, and the like, the content of the crosslinked particle is preferably 1% by mass to 50% by mass, more preferably 8% by mass to 40% by mass, and still more preferably 20% by mass to 40% by mass, with respect to the total mass of the hemostatic material composition.
Component other than specific polysaccharide, hydrophobic solvent, and crosslinked particle
The hemostatic material composition according to the present disclosure may contain a component other than the specific polysaccharide, and the hydrophobic solvent, and the crosslinked particle, and examples thereof include a stabilizer such as ascorbic acid, an antibiotic, a vasoconstrictor, a pigment, a growth factor, a proliferation factor, a bone morphogenetic protein, an analgesic agent, an anti-inflammatory agent, and an antifibrinolytic agent such as tranexamic acid.
The hemostatic material composition according to the present disclosure may contain water; however, from the viewpoint of storage stability, the content of water with respect to the total mass of the hemostatic material composition is preferably 5% by mass or less, more preferably 3% by mass or less, still more preferably 1% by mass or less, particularly preferably 0.5% by mass or less, and most preferably 0.1% by mass or less, where it is most preferable that the water is not contained.
Hereinafter, the above-described embodiment will be specifically described with reference to Examples; however, the above-described embodiment is not limited to these Examples. It is noted that the unit of the numerical values in Tables 1 to 3 is % by mass unless otherwise specified.
10 g of a sodium carboxymethyl dextran A (manufactured by Meito Sangyo Co., Ltd., CMD-D40, Mw: 40,000) was dissolved in 100 g of distilled water, and 13 g of a 10% by mass hydrochloric acid aqueous solution was added thereto. The obtained solution was reprecipitated in 1,600 mL of methanol, and the filtrate was washed with methanol and then subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 10.44 g of a carboxymethyl dextran X.
Next, 10 g of the carboxymethyl dextran X was dissolved in 100 g of dimethylacetamide (DMAc), and 3.78 g of N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Corporation) and 6.30 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride, manufactured by FUJIFILM Wako Pure Chemical Corporation) were added thereto, and stirring was carried out at 40° C. for 4 hours.
The obtained solution was reprecipitated in 350 mL of methanol, the filtrate was washed with methanol and acetone and subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 10.6 g of carboxymethyl dextran A having N-hydroxysuccinimide ester group (hereinafter, also referred to as an “NHS-carboxymethyl dextran”). It is noted that the NHS-carboxymethyl dextran A is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
10 mg of the NHS-carboxymethyl dextran A was dissolved in 1 g of heavy water, heated to 40° C., and hydrolyzed for 24 hours.
A hydrolyzate of the NHS-carboxymethyl dextran A was obtained, and in the spectrum obtained using a nuclear magnetic resonance apparatus (manufactured by BRUKER, 400 MHz NMR, number of integrations: 16 times), the ratio of the liberated proton (2.67 ppm to 2.74 ppm) to the proton (4.82 ppm to 5.47 ppm)) at the 1-position of the glucopyranose ring was determined to calculate the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextran A. As a result, it was 14.8% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 15.2% by mole, respectively.
In addition, the Mw of the NHS-carboxymethyl dextran A was 45,000.
19.6 g of sodium hydroxide was dissolved in 106 g of distilled water, and 10 g of pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) was gradually added thereto a little bit at a time to be dissolved, and stirring was carried out for 90 minutes.
After the stirring, an aqueous solution of chloroacetic acid (an aqueous solution obtained by dissolving 7 g of chloroacetic acid in 63 g of distilled water) was added thereto, and a reaction was allowed to proceed at 60° C. for 6 hours.
After cooling to room temperature (25° C.), 65 g of a 20% by mass hydrochloric acid aqueous solution was added thereto, and stirring was carried out for 2 hours. The obtained solution was reprecipitated in 2,500 mL of methanol, and the filtrate was washed with methanol and then subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 10.85 g of carboxymethyl pullulan.
An NHS-carboxymethyl pullulan was obtained in the same manner as in Synthesis Example 1 except that the carboxymethyl dextran X was changed to carboxymethyl pullulan and the adding amounts of N-hydroxysuccinimide and EDC hydrochloride were changed to 1.69 g and 2.81 g, respectively. It is noted that the NHS-carboxymethyl pullulan is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl pullulan, it was 6.3% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 23.7% by mole, respectively.
In addition, the Mw of the NHS-carboxymethyl pullulan was 12,000.
19.6 g of sodium hydroxide was dissolved in 106 g of distilled water, and 10 g of dextrin (manufactured by Sanwa Starch Co., Ltd., Sandek (registered trade name) # 100) was gradually added a little bit at a time to be dissolved, and stirring was carried out for 90 minutes.
After the stirring, an aqueous solution of chloroacetic acid (an aqueous solution obtained by dissolving 7 g of chloroacetic acid in 63 g of distilled water) was added thereto, and a reaction was allowed to proceed at 60° C. for 6 hours.
After cooling to room temperature (25° C.), 65 g of a 20% by mass hydrochloric acid aqueous solution was added thereto, and stirring was carried out for 2 hours. The obtained solution was reprecipitated in 2,500 mL of methanol, and the filtrate was washed with methanol and then subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 10.25 g of carboxymethyl dextrin.
An NHS-carboxymethyl dextrin was obtained in the same manner as in Synthesis Example 1 except that the carboxymethyl dextran X was changed to carboxymethyl dextrin and the adding amounts of N-hydroxysuccinimide and EDC hydrochloride were changed to 3.31 g and 5.51 g, respectively. It is noted that the NHS-carboxymethyl dextrin is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextrin, it was 10% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 20% by mole, respectively.
In addition, the Mw of NHS-carboxymethyl dextrin was 18,000.
10 g of the carboxymethyl dextran X synthesized in Synthesis Example 1 was dissolved in 100 g of DMAc, and each of 5.67 g of N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Corporation) and 9.45 g of EDC hydrochloride (manufactured by Fujifilm Wako Pure Chemical Corporation) was added thereto, and stirring was carried out at 40° C. for 4 hours.
The obtained solution was reprecipitated in 350 mL of methanol, and the filtrate was washed with methanol and acetone and then subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 11.2 g of an NHS-carboxymethyl dextran B. It is noted that the NHS-carboxymethyl dextran B is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextran B, it was 21% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 9% by mole, respectively.
In addition, the Mw of NHS-carboxymethyl dextran B was 47,000.
10 g of the carboxymethyl dextran X synthesized in Synthesis Example 1 was dissolved in 100 g of DMAc, and each of 1.32 g of N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Corporation) and 2.20 g of EDC hydrochloride (manufactured by Fujifilm Wako Pure Chemical Corporation) was added thereto, and stirring was carried out at 40° C. for 4 hours.
The obtained solution was reprecipitated in 350 mL of methanol, and the filtrate was washed with methanol and acetone and then subjected to vacuum drying at room temperature (25° C.) for 24 hours to obtain 10.1 g of an NHS-carboxymethyl dextran C. It is noted that the NHS-carboxymethyl dextran C is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextran C, it was 2.2% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 27.8% by mole, respectively.
In addition, the Mw of NHS-carboxymethyl dextran C was 44,000.
An NHS-carboxymethyl dextran D was obtained in the same manner as in Synthesis Example 1 except that the sodium carboxymethyl dextran A was changed to a sodium carboxymethyl dextran B (manufactured by Meito Sangyo Co., Ltd., CMD-500, Mw: 500,000). It is noted that the NHS-carboxymethyl dextran D is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextran D, it was 15% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 15% by mole, respectively.
In addition, the Mw of NHS-carboxymethyl dextran D was 530,000.
An NHS-carboxymethyl dextran E was obtained in the same manner as in Synthesis Example 1 except that the sodium carboxymethyl dextran A was changed to a sodium carboxymethyl dextran C (manufactured by Meito Sangyo Co., Ltd., CMD-L, Mw: 10,000). It is noted that the NHS-carboxymethyl dextran G is composed of a constitutional unit represented by General Formula (1), a constitutional unit represented by Formula (2), and a constitutional unit represented by Formula (3).
In addition, as a result of calculating the content of the constitutional unit (the constitutional unit represented by General Formula (1)) derived from a monosaccharide having an N-hydroxysuccinimide ester group with respect to 100% by mole of the NHS-carboxymethyl dextran E, it was 15% by mole.
Similarly, the contents of the constitutional unit represented by Formula (2) and the constitutional unit represented by Formula (3) were calculated and were found to be 70% by mole and 15% by mole, respectively.
In addition, the Mw of NHS-carboxymethyl dextran E was 11,000.
5 g of diphenylmethane diisocyanate (manufactured by Fujifilm Wako Pure Chemical Corporation) was dissolved in a mixed solution of 90 g of toluene (manufactured by Fujifilm Wako Pure Chemical Corporation) and 5 g of diphenylmethane diisocyanate (manufactured by Fujifilm Wako Pure Chemical Corporation), and 5 g of chitosan (manufactured by GENERAL SCIENCE CORPORATION, Mw: 15,000, powder-shaped, degree of deacetylation: 84%) was dispersed therein.
The obtained dispersion liquid was stirred at 100° C. for 24 hours, cooled to room temperature (25° C.) and filtered, and then the obtained powder washed with toluene and acetone and subjected to air drying to obtain a powder of isocyanated chitosan.
In addition, the Mw of the isocyanated chitosan was 15,000.
High Grade gelatin (Mw: 60,000) manufactured by Nippi. Inc. was irradiated with γ-rays so that the absorbed dose was 50 kGy to obtain crosslinked gelatin.
The crosslinked gelatin was pulverized with a mortar, and each of the crosslinked gelatin particle A having an average primary particle diameter of 20 µm or more and less than 100 µm, the crosslinked gelatin particle B having an average primary particle diameter of 100 µm or more and less than 200 µm, and the crosslinked gelatin particle C having an average primary particle diameter of 200 µm or more and 700 µm or less was obtained by using a mesh sieve.
Examples 1 to 20 and Comparative Examples 1 to 6
Components shown in Tables 1 to 3 were mixed to produce each paste-shaped hemostatic material composition.
The details of the components shown in Tables 1 to 3 are as follows.
Hydrophobic solvent
When 20 g of the above-described hydrophobic solvent was put into 1 L of ion exchange water (pH 7) at 20° C., it was confirmed that the solution is separated into two layers and is not an uniform aqueous solution, and the solubility in water is less than 20 g/L.
When 20 g of the above-described hydrophilic solvent was put into 1 L of ion exchange water (pH 7) at 20° C., it was confirmed that the solution is an uniform aqueous solution, and the solubility in water is 20 g/L or more.
Each of the hemostatic material compositions produced in Examples 1 to 20 and Comparative Examples 1 to 6 was stored for 10 days in an environment of 50° C.
The abdomen of the pig was opened, and a bleeding wound part having a diameter of 8 mm and a depth of 2 mm was formed on the surface part of the liver with a biopsy trephine.
0.5 g of each hemostatic material composition after storage was applied onto the bleeding wound part so that the bleeding wound part was covered, a coating film was allowed to be formed, and the bleeding wound part was pressed with gauze wetted with the saline.
The state of the bleeding wound part after 1 minute, 2 minutes, 5 minutes, and 10 minutes from the application of the hemostatic material composition was visually observed and evaluated based on the following evaluation standards, and the results were summarized in Table 1 to Table 3. It is noted that it was determined that the storage stability is good in a case where the evaluation is 3 to 5.
5: It could be confirmed that the hemostasis is achieved when observing the state of the bleeding wound part after 1 minute.
4: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 1 minute, the hemostasis is achieved when observing the state of the bleeding wound part after 2 minutes.
3: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 2 minutes, the hemostasis is achieved when observing the state of the bleeding wound part after 5 minutes.
2: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 2 minutes, the hemostasis is achieved when observing the state of the bleeding wound part after 5 minutes.
1: It could be confirmed that the hemostasis is not achieved even when observing the state of the bleeding wound part after 10 minutes.
The abdomen of the pig was opened, and a bleeding wound part having a diameter of 8 mm and a depth of 2 mm was formed in the liver part with a biopsy trephine.
0.5 g of the hemostatic material composition produced in each of Examples 1 to 20 and Comparative Examples 1 to 6 was applied onto the bleeding wound part so that the bleeding wound part was covered, a coating film was allowed to be formed, and the bleeding wound part was pressed with gauze wetted with the saline.
The state of the bleeding wound part after 1 minute, 2 minutes, 5 minutes, and 10 minutes from the application of the hemostatic material composition was visually observed and evaluated based on the following evaluation standards, and the results were summarized in Table 1 to Table 3. It is noted that it was determined that the hemostatic properties are good in a case where the evaluation is 3 to 5.
5: It could be confirmed that the hemostasis is achieved when observing the state of the bleeding wound part after 1 minute.
4: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 1 minute, the hemostasis is achieved when observing the state of the bleeding wound part after 2 minutes.
3: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 2 minutes, the hemostasis is achieved when observing the state of the bleeding wound part after 5 minutes.
2: It could be confirmed that although the hemostasis is not achieved when observing the state of the bleeding wound part after 2 minutes, the hemostasis is achieved when observing the state of the bleeding wound part after 5 minutes.
1: It could be confirmed that the hemostasis is not achieved even when observing the state of the bleeding wound part after 10 minutes.
The hemostatic material composition produced in each of Examples 1 to 20 and Comparative Examples 1 to 6 was applied onto a slide glass plate (2.5 cm x 7.5 cm) to a thickness of 2 mm.
The slide glass plate after the coating was gently immersed in 50 cc of the saline, taken out after 10 seconds and 1 minute, visually observed, evaluated based on the following evaluation standards, and summarized in Tables 1 and 2. It is noted that it was determined that the removability is good in a case where the evaluation is 3 to 5.
5: The residual of the hemostatic material composition was not observed in the slide glass plate taken out 10 seconds after the start of the immersion.
4: Although the slight residual of the hemostatic material composition was observed on the slide glass plate taken out 10 seconds after the start of the immersion, the residual of the hemostatic material composition was not observed in the slide glass plate taken out 1 minute after the start of the immersion.
3: The slight residual of the hemostatic material composition was observed in any of the slide glass plates taken out 10 seconds and 1 minute after the start of the immersion.
2: The large residual of the hemostatic material composition was observed in any of the slide glass plates taken out 10 seconds and 1 minute after the start of the immersion.
1: It was confirmed that in any of the slide glass plates taken out 10 seconds and 1 minute after the start of the immersion, there are no signs that the hemostatic material composition has been removed, and almost the entire amount of the applied hemostatic material composition is remained.
0.5 g of the hemostatic material composition produced in each of Examples 1 to 20 and Comparative Examples 1 to 6 was applied onto the surface of the excised porcine liver, a coating film was allowed to be formed, and the bleeding wound part was pressed for 2 minutes with gauze wetted with the saline.
Then, the gauze was slowly peeled off, and the surface of the gauze and the surface of the excised liver were visually observed to carry out the evaluation based on the following evaluation standards, which are summarized in Table 1 to Table 3. It is noted that it was determined that the transfer suppressibility is good in a case where the evaluation is 3 to 5.
5: No transfer of the hemostatic material composition to the gauze was observed, and no peeling of the coating film on the surface of the excised liver was observed.
4: Although the transfer of the hemostatic material composition to the gauze was slightly observed, no peeling of the coating film on the surface of the excised liver was observed.
3: The transfer of the hemostatic material composition to the gauze was slightly observed, and the peeling of the coating film on the surface of the excised liver was slightly observed.
2: The large transfer of the hemostatic material composition to the gauze was observed, and the large peeling of the coating film on the surface of the excised liver was observed.
1: The large transfer of the hemostatic material composition to the gauze was observed, and most of the coating film on the surface of the excised liver was peeled off.
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From Tables 1 and 2, it can be seen that the hemostatic material composition according to the present disclosure containing the specific polysaccharide, the hydrophobic solvent, and the crosslinked particles is excellent in storage stability.
From Table 3, it can be seen that there is room for improvement in the storage stability of the hemostatic material composition containing the hydrophilic solvent.
In addition, from Table 3, it can be seen that there is room for improvement in the hemostatic property and the storage stability of the hemostatic material composition that does not contain the specific polysaccharide.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-207606 | Dec 2021 | JP | national |
