JP2005289883A - Bioabsorbable hemostatic agent and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a hemostatic agent made of an oxidized cellulose composed of β-1,4-polyglucuronic acid units, without containing β-1,4-D-glucose units, and to provide a method for producing the same. <P>SOLUTION: The bioabsorbable hemostatic agent which comprises β-1,4-polyglucuronic acid expressed by formula (I) (X is H or an alkali metal; and (n) is an integer not less than 2) is obtained by a method having a process for dispersing regenerated cellulose in water, a process for selectively oxidizing primary hydroxy groups of the regenerated cellulose in the water, so as to obtain the β-1,4-polyglucuronic acid units, and a process for purifying the β-1,4-polyglucuronic acid units. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bioabsorbable hemostatic agent comprising β-1,4-polyglucuronic acid and a method for producing the same. Specifically, the primary hydroxyl group of regenerated cellulose is selectively oxidized by an oxidation reaction using an oxidizing agent in the presence of 2,2,6,6-tetramethyl-1-piperidine-N-oxyl and an alkali metal bromide. This invention relates to the use of β-1,4-polyglucuronic acid obtained as a bioabsorbable hemostatic agent.

  In surgery, a hemostatic agent contributes to shortening the operation time and reducing the amount of bleeding by quickly stopping bleeding due to a surgical operation, which contributes to reducing the amount of blood transfused. In particular, as a hemostatic agent used in a living body, for example, a hemostatic agent used for bleeding from an anastomosis portion or suture portion of a blood vessel or a suture line of an organ, a bioabsorbable agent is useful. This is because it is not necessary to remove the hemostatic agent after the hemostasis is achieved, and the risk of rebleeding can be reduced because the thrombus formed as a result of hemostasis can be retained.

  Currently, bioabsorbable hemostatic agents that have a clinical track record use collagen and gelatin, which are bio-derived materials (among biomaterials, materials obtained from human or animal bodies). Blood coagulation is promoted by the positive adhesion of platelets to these materials. It is well known that these biological materials are absorbed in the biological metabolic system. In the manufacturing process of such hemostatic agents, safety measures such as raw material management and virus inactivation and removal treatment are taken, but the risk of virus infection due to the use of biological materials is completely denied. Because it is not possible, it must be used carefully.

  On the other hand, oxidized cellulose, which is a synthetic material with a low risk of virus infection, is often used. In this case, blood coagulation is enhanced by the red blood cell agglutination action of oxidized cellulose. Moreover, since it gradually becomes water-soluble under physiological conditions, it disappears from the application site in about 30 days, and since oxidized cellulose itself originally has a low molecular weight, it is finally excreted through the kidney. The conventional oxidized cellulose used in this hemostatic agent is obtained by oxidizing regenerated cellulose (β-1,4-D-polyglucose) using an oxidation reaction with nitrogen dioxide. Since this oxidation reaction is a heterogeneous reaction, it is impossible to oxidize completely regenerated cellulose, and as a result, the obtained oxidized cellulose contains at least β-1,4-D-glucose units, β- It becomes a polysaccharide consisting of 1,4-glucuronic acid and β-1,4-D-glucose. From the point of view of biological metabolism, particularly liver metabolism, oxidized cellulose composed of β-1,4-polyglucuronic acid units that do not contain β-1,4-D-glucose units is preferable.

  In view of the above circumstances, an object of the present invention is to provide a hemostatic agent made of oxidized cellulose comprising a β-1,4-polyglucuronic acid unit and not containing a β-1,4-D-glucose unit, and a method for producing the hemostatic agent. Is to provide.

Said subject is achieved by the bioabsorbable hemostatic agent which consists of (beta) -1,4-polyglucuronic acid represented by Structural formula (I).

(X in the formula represents hydrogen or an alkali metal, and n is an integer of 2 or more)
The hemostatic agent includes a step of dispersing regenerated cellulose in water, a step of selectively oxidizing primary hydroxyl groups of regenerated cellulose in water to obtain β-1,4-polyglucuronic acid, and β-1. , 4-polyglucuronic acid is obtained by a method for producing a bioabsorbable hemostatic agent comprising a step of purifying.

  In the bioabsorbable hemostatic agent of the present invention, the oxidized cellulose constituting the bioabsorbable hemostatic agent does not contain a β-1,4-D-glucose unit, so that its biometabolism, particularly liver clearance can be improved.

  The bioabsorbable hemostatic agent of the present invention is characterized in that the chemical structure is clear and uniform.

The bioabsorbable hemostatic agent of the present invention comprises β-1,4-polyglucuronic acid represented by the following chemical formula (I). Here, X in the formula is almost hydrogen, but contains an alkali metal such as sodium derived from the production method, an alkaline earth metal, or the like. That is, it contains a salt of glucuronic acid.

(X in the formula represents hydrogen or an alkali metal, and n is an integer of 2 or more)
Although β-1,4-polyglucuronic acid itself is not highly soluble in water, its alkali metal salt exhibits a solubility of 10% or more in distilled water at 25 ° C. Therefore, analysis by nuclear magnetic resonance spectroscopy (NMR) is possible by dissolving the sodium salt in heavy water. The ¹³ C-NMR peaks of β-1,4-polyglucuronic acid sodium salt dissolved in heavy water are as follows: C1: δ = 105.2 ppm, C2: δ = 75.6 ppm, C3: δ = 77.0 ppm, C4 : Δ = 83.7 ppm, C5: δ = 78.1 ppm, C6: δ = 177.8 ppm. The bioabsorbable hemostatic agent of the present invention shows six peaks having the above-mentioned chemical shift values clearly determined by NMR analysis, thereby supporting a uniform structure of the chemical formula (I).

Conventionally, nitrogen dioxide-oxidized cellulose has been used as a hemostatic agent. In the case of nitrogen dioxide oxidation, from the analysis result of ¹³ C-NMR, it is derived from unoxidized C6, that is, derived from the 6-position carbon of β-1,4-glucopyranose. Peaks are detected in the vicinity of δ = 63 ppm, and many peaks of decomposition products due to side reactions are observed. On the other hand, those peaks are not substantially detected in the bioabsorbable hemostatic agent of the present invention.

  Furthermore, the bioabsorbable hemostatic agent of the present invention is characterized by a narrow molecular weight distribution while having a high molecular weight, and can be said to be highly uniform in terms of molecular weight distribution. In the case of the above-mentioned nitrogen dioxide-oxidized cellulose, when the 6-position carbon of β-1,4-glucopyranose is completely oxidized, decomposition due to side reactions increases and the molecular weight is drastically reduced. In addition, the molecular weight distribution tends to be wide due to non-uniform oxidation reaction, and it is difficult to obtain oxidized cellulose having a narrow molecular weight distribution. In contrast, the bioabsorbable hemostatic agent of the present invention is characterized in that β-1,4-polyglucuronic acid has a number average molecular weight (Mn) of 10,000 to 50,000, and has a molecular weight distribution. The ratio (Mw / Mn) of the weight average molecular weight (Mw) and the number average molecular weight (Mn), which serves as an index of the width, is 1.2 to 2.4.

  The number average molecular weight (Mn) and the weight average molecular weight (Mw) described here are an average molecular weight in terms of pullulan measured by size exclusion chromatography using pullulan as a standard substance.

  Furthermore, the bioabsorbable hemostatic agent of the present invention is characterized in that β-1,4-polyglucuronic acid is amorphous. That is, as a result of measuring the X-ray diffraction of the powder sample, this powder was shown to be amorphous. When cellulose with high crystallinity is oxidized, crystallinity may be observed in the product, but the β-1,4-polyglucuronic acid produced by the method of the present invention is uniformly amorphous. This shows that the oxidation reaction progressed well and polyglucuronic acid having a uniform structure was obtained. Moreover, it can be said that the polyglucuronic acid in the present invention is completely absorbed even in a living body and easily disappears due to being amorphous.

  Furthermore, the bioabsorbable hemostatic agent of the present invention is characterized in that the dosage form is powder. Since it is a powder, it can be easily stored, and a method of use such as spraying or applying the powder to the affected area can be employed. Furthermore, in the present invention, by using the method for producing the bioabsorbable hemostatic agent of the present invention, which will be described later, the product can be recovered directly as a powder rather than being powdered by physical treatment such as pulverization. Is good.

  Subsequently, the production of the bioabsorbable hemostatic agent of the present invention will be described.

  The method for producing a bioabsorbable hemostatic agent of the present invention comprises a step of dispersing regenerated cellulose in water (dispersing step), selectively oxidizing primary hydroxyl groups of regenerated cellulose in water, and β-1,4-polyglucuron. A step of obtaining an acid (oxidation step) and a step of purifying β-1,4-polyglucuronic acid (purification step).

  Nitrogen dioxide oxidized cellulose conventionally used as a hemostatic agent is obtained by oxidizing a cellulose raw material with a gas, whereas the method for producing a bioabsorbable hemostatic agent of the present invention is characterized by being oxidized in water. As described above, the alkali metal salt of β-1,4-polyglucuronic acid has high water solubility, and when the regenerated cellulose is completely oxidized to β-1,4-polyglucuronic acid, the reaction solution is a uniform aqueous solution. Become.

  As a raw material for the production method of this bioabsorbable hemostatic agent, cellulose composed of β-1,4-D-glucose is preferably used. Cellulose used as a raw material can be used without limitation from general cellulose derived from wood and cotton to bacterial cellulose, etc., but once dissolved and regenerated cellulose is used, side reactions are suppressed, and a high-molecular-weight completely water-soluble polymer. Since glucuronic acid is obtained, it is desirable to use regenerated cellulose as a raw material. Here, as a regeneration treatment method of cellulose, a known regeneration treatment method such as a cupra ammonium method, a viscose method, or a lithium chloride / dimethylacetamide method can be used.

  In the method for producing a bioabsorbable hemostatic agent of the present invention, first, in the step of dispersing regenerated cellulose in water, the regenerated cellulose is dispersed in water as a reaction solvent. Here, it is preferable to adjust the fiber length of the regenerated cellulose to 2 cm or less and to cool the dispersion to 5 ° C. or less so that the oxidation reaction in the next step can easily proceed uniformly.

  In the present invention, in the step of selectively oxidizing the primary hydroxyl group of the regenerated cellulose in water, all the pyranose is selectively oxidized under the mild reaction conditions without oxidizing the 2nd and 3rd positions of the pyranose ring. In order to oxidize the 6-position of the ring to a carboxyl group and obtain a high molecular weight and uniform β-1,4-polyglucuronic acid, an oxidation method using an N-oxyl compound catalyst is used.

  The oxidation method using the N-oxyl compound catalyst is a method of oxidizing in an aqueous system using an oxidizing agent in the presence of the N-oxyl compound catalyst. As the N-oxyl compound, 2, 2, 6, 6- Tetramethyl-1-piperidine-N-oxyl (hereinafter referred to as TEMPO) is preferably used, and as the oxidizing agent, halogen, hypohalous acid, halous acid, perhalogen acid or salts thereof, halogen oxide Any oxidizing agent can be used as long as it can promote the target oxidation reaction, such as nitrogen oxide and peroxide. Furthermore, it is more preferable to oxidize in the presence of bromide or iodide because the oxidation reaction can proceed smoothly even under mild conditions and the efficiency of introducing carboxyl groups can be greatly improved.

  In the present invention, in the step of selectively oxidizing the primary hydroxyl group of regenerated cellulose in the above water, TEMPO is used as the N-oxyl compound, and sodium hypochlorite is used as the oxidizing agent in the presence of sodium bromide. It is particularly preferable to carry out.

  Here, the amount of the N-oxyl compound is sufficient as a catalyst. For example, 10 ppm to 5% is sufficient with respect to the number of moles of β-1,4-D-glucose, but 0.05% to 3%. The amount of bromide or iodide used can be selected within a range that can promote the oxidation reaction. For example, 0 to 100%, more preferably 1 to 50, relative to the number of moles of β-1,4-D-glucose. %.

  In order to increase the selectivity of oxidation to primary hydroxyl groups and suppress side reactions, the reaction temperature is preferably room temperature or lower, more preferably 5 ° C. or lower. Furthermore, it is preferable to keep the inside of the system alkaline during the reaction. The pH at this time is preferably 10.0 to 11.5, more preferably pH 10.5 to 11.0.

  Further, in this oxidation method, the degree of oxidation can be controlled by the amount of the oxidant and the amount of alkali added when the pH is kept constant. For example, when an alkali is added in an equimolar amount with a glucose residue, almost all primary hydroxyl groups at the 6-position of the pyranose ring are oxidized to carboxyl groups.

  Here, in the step of selectively oxidizing the primary hydroxyl group of the regenerated cellulose in the water of the present invention, for example, an aqueous solution in which TEMPO and sodium bromide are dissolved is added to the regenerated cellulose dispersion, and the inside of the system is 5 Cool to below ℃ and adjust pH to 10. When a small amount of sodium hypochlorite solution is added here, the pH temporarily rises, but if the stirring is continued, the pH in the system gradually decreases. The pH is kept constant in the range of 10.0 to 11.5. Further, by dropping the sodium hypochlorite solution, which is an oxidizing agent, while adjusting according to the progress of the reaction, it is possible to suppress the occurrence of a side reaction due to the action of excess oxidizing agent present in the system. During the reaction, the temperature in the system is maintained at 5 ° C. or lower. As the reaction proceeds, the system becomes a homogeneous solution. Under these reaction conditions, the amount of sodium hydroxide added corresponds approximately to the amount of carboxyl groups introduced by oxidation, and when the amount of glucose residues in the raw material and the amount added in an equimolar amount are reached, Ethanol is added to quench excess oxidant. In this way, the primary hydroxyl group of the regenerated cellulose can be selectively oxidized to form water-soluble sodium β-1,4-polyglucuronate.

  Subsequently, in the purification step of β-1,4-polyglucuronic acid, undissolved components derived from the raw material may float in the reaction solution dissolved by the oxidation reaction, and this is removed by filtration. Then, when the filtrate is poured into an excessive amount of ethanol, the product is precipitated as a white precipitate. The precipitate is sufficiently washed with a mixed solution of acetone and water, dehydrated with acetone, and then dried under reduced pressure to obtain β-1,4-polyglucuronic acid sodium salt in the form of white powder. This powder exhibits a solubility of 10 g or more with respect to 100 g of distilled water at 25 ° C.

  Furthermore, the polyglucuronic acid sodium salt (COONa type) obtained as described above was dissolved in water, acid-treated, and reprecipitated with the above ethanol, washed, and dried to repeat the desalination to the COOH type. Thus, β-1,4-polyglucuronic acid in the form of white powder, which is the bioabsorbable hemostatic agent of the present invention, is obtained.

  Here, the desalting operation by acid treatment may be performed on the reaction solution before precipitation in ethanol, and after removing the insoluble matter by filtration, the pH of the reaction solution is lowered by adding an aqueous hydrochloric acid solution or the like. By precipitating in an excessive amount of ethanol, the washing operation can be reduced and β-1,4-polyglucuronic acid can be obtained. The desalting treatment is not limited to acid treatment, and any method such as treatment with an ion exchange resin may be used.

  In the purification step of β-1,4-polyglucuronic acid, the purpose is to remove TEMPO, sodium bromide, and sodium chloride generated by the selective oxidation reaction, so long as the purpose is achieved. In addition to the above-described purification method using an organic solvent, various purification methods can be used.

  Further, when the organic solvent used for purification cannot be completely removed by drying under reduced pressure, the solvent may be completely removed by dissolving or dispersing in distilled water and freeze-drying.

  The use of the bioabsorbable hemostatic agent for spraying or applying the bioabsorbable hemostatic powder of the present invention to the affected area is performed by spraying to coat the bleeding site, if necessary, at or near the bleeding site of the living body. Or achieved by painting. In the case of spraying, the bioabsorbable hemostatic agent powder of the present invention may be sprayed using a volatile solvent such as compressed air, nitrogen, aqueous alcohol, liquid propane or chlorofluorocarbon. Moreover, in order to improve the attachment of the powder to the surface of the living body, a lipophilic substance such as isopropyl myristate can be used in combination as an additive.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail about an Example, this invention is not limited to these.

  5.0 g of Benlyse (manufactured by Asahi Kasei Kogyo Co., Ltd.), a commercially available regenerated cellulose, was uniformly dispersed in distilled water at a concentration of 5%. An aqueous solution in which 96 mg of TEMPO and 1.27 g of sodium bromide were dissolved was added thereto to prepare a cellulose solid content concentration of about 2 wt%. The reaction system is cooled, and 15 g of 11% aqueous sodium hypochlorite solution is added to start the oxidation reaction. The reaction temperature was always kept below 5 ° C. During the reaction, the pH in the system decreases, but 0.5N-NaOH aqueous solution is sequentially added to adjust the pH to around 10.8, and 35 g of 11% sodium hypochlorite aqueous solution is adjusted according to the progress of the reaction. While dripping. When approaching the amount of alkali added corresponding to 100% of the total number of moles of glucose residues, the rate of alkali addition slows down and the system is completely dissolved, resulting in a uniform yellow solution. When the amount of alkali added reached 100% (61.73 ml), ethanol was added to stop the reaction. The reaction time was 2 hours. This reaction solution was filtered to remove insoluble impurities, and then poured into an excess amount of ethanol to reprecipitate the product. Further, after thoroughly washing with a solution of water: acetone = 1: 7, dehydrated with acetone and dried under reduced pressure at 40 ° C. to obtain 5.9 g of white powdery β-1,4-polyglucuronic acid sodium salt. It was.

  2.0 g of sodium salt of β-1,4-polyglucuronic acid of Example 1 was dissolved in 80 ml of distilled water, and 2N-hydrochloric acid was added to pH 1 while stirring. The solution became a white uniform dispersion. This dispersion was put into an excess amount of ethanol to reprecipitate the product. Further, after thoroughly washing with a solution of water: acetone = 1: 7, dehydrated with acetone and dried under reduced pressure at 40 ° C. to obtain 1.6 g of desalted β-1,4-polyglucuronic acid as a white powder. It was.

  The filter paper pulp was completely dissolved in a solution of copper ethylenediamine, regenerated in water and thoroughly washed while neutralizing. 2.0 g of this wet regenerated cellulose was suspended in water at a concentration of 5%. An aqueous solution in which 39 mg of TEMPO and 0.50 g of sodium bromide were dissolved was added thereto to prepare a cellulose solid content concentration of about 2 wt%. The reaction system is cooled and 6 g of 11% aqueous sodium hypochlorite solution is added to start the oxidation reaction. The reaction temperature was always kept below 5 ° C. During the reaction, the pH in the system decreases, but 0.5N-NaOH aqueous solution is sequentially added to adjust the pH to around 10.5, and further 15 g of 11% sodium hypochlorite aqueous solution is adjusted according to the progress of the reaction. While dripping. When the alkali addition amount corresponding to 100% of the number of moles of glucose residues was approached, the alkali addition rate was slow. When the amount of alkali added reached 100% (24.67 ml), ethanol was added to stop the reaction. The reaction time was 1 hour 40 minutes. The reaction solution was filtered to remove insoluble impurities, and then 2N hydrochloric acid was added until the pH reached 1. The solution became a white uniform dispersion. This dispersion was poured into an excess amount of ethanol to reprecipitate the product. Further, it was sufficiently washed with a solution of water: acetone = 1: 7, dehydrated with acetone, and dried under reduced pressure at 40 ° C. to obtain 2.0 g of β-1,4-polyglucuronic acid in the form of white powder.

<Comparative example>
As comparative examples, three commercially available hemostatic agents of nitrogen dioxide-oxidized cellulose (SURGICELL (Fibrillar cotton), SURGICELL (NU-KNIT woven fabric) (both manufactured by Johnson & Johnson), OXYCEL (COTTON-TYPE cotton) Sankyo Co., Ltd.)) and nitrogen dioxide-oxidized cellulose commercially available adhesion prevention material 1 (INTERCEEDED woven cloth) (manufactured by Johnson & Johnson) were used. The following evaluations were performed on these samples.

<Evaluation>
(Structural analysis by IR)
The IR spectra of the β-1,4-polyglucuronic acid of Example 2 and the commercially available oxidized cellulose of the comparative example were measured by the KBr tablet method and compared. As a result, as shown in FIG. 1, in both the example and the comparative example, large absorption was observed in the vicinity of 1750 cm ⁻¹ derived from the carboxyl group introduced by oxidation. As long as comparison was made by IR analysis, no significant difference was found between the examples and the comparative examples.

(Structural analysis by NMR)
^13C -NMR was measured by dissolving β-1,4-polyglucuronic acid sodium salt of Example 1 in deuterated water and commercially available oxidized cellulose of Comparative Example in deuterated sodium hydroxide heavy aqueous solution. . The NMR spectrum is shown in FIG. In the β-1,4-polyglucuronic acid of the example, the peak derived from carbon having a hydroxyl group at the 6-position of the pyranose ring (around δ = 60-65 ppm) disappeared completely, and the carboxyl group (around δ = 170-180 ppm) ), The peaks derived from the carbons at the 2nd and 3rd positions were not changed, and the peaks of ketones and the like (near δ = 200 to 210 ppm) were not confirmed. Therefore, it can be said that the bioabsorbable hemostatic agent of the present invention is β-1,4-polyglucuronic acid having a uniform structure. On the other hand, the INTEREDED and SERGICELL of the comparative example showed a residual peak (around δ = 60-65 ppm) derived from carbon having a hydroxyl group at the C6-position of unoxidized pyranose ring, and the complete β-1,4-poly Not glucuronic acid. In addition, in OXYCEL, although the peak near δ = 60-65 ppm has almost disappeared, it is recognized that the peak that seems to be derived from the decomposition product due to the side reaction is increased.

(Measurement of average molecular weight)
The number average molecular weight (Mn) and weight average molecular weight (Mw) of β-1,4-polyglucuronic acid of Examples 2 and 3 and commercially available oxidized cellulose of Comparative Example were measured by GPC method. Each sample was dissolved in 0.05N sodium hydroxide aqueous solution, precipitated and isolated with ethanol, washed with a solution of water: acetone = 1: 7, dehydrated with acetone, dried under reduced pressure, and used for GPC measurement. A sample was used. TSK gel G6000PWXL and TSKgel G3000PWXL were used as columns, and 0.1 M NaCl was used as an eluent and measurement was performed using an RI detector. A calibration curve was prepared from a standard pullulan having a known molecular weight, and a number average molecular weight and a weight average molecular weight in terms of pullulan were calculated. The results are shown in Table 1. The GPC chart of Example 2 is shown in FIG. The oxidized cellulose of the comparative example has a distribution of two peaks on the low molecular weight side and the high molecular weight side, and the weight average molecular weight is high, but the molecular weight distribution is wide and other than the oxidation of the primary hydroxyl group at the 6-position of the pyranose ring. In addition, it was thought that side reactions such as depolymerization were taking place. This tendency was most remarkable in OXYCEL, in which it was confirmed by NMR that almost all of the 6-position primary hydroxyl groups were oxidized. On the other hand, the β-1,4-polyglucuronic acid of the example has a narrow molecular weight distribution and is suggested to have a uniform structure. Further, it can be seen that the molecular weight distribution is also on the high molecular weight side rather than the low molecular weight side of the comparative example.

(crystalline)
The samples of Example 2 and Comparative Example were evaluated for crystallinity using Rigaku RAD-rX (X-ray source = CuKα, voltage 40 kV, current 100 mA) by the powder method. The sample of the comparative example was powdered by freeze pulverization. As a result, no peaks were detected in SERGICELL of Example 2 and Comparative Example, and it was found that the SERGICELL was amorphous. On the other hand, in OXYCEL of the comparative example, a peak derived from cellulose appeared. FIG. 4 shows spectra of X-ray diffraction gratings obtained by SERGICELL and OXYCEL of Example 2 and Comparative Example.

(Whole blood clotting time measurement (Lee-White method))
Dispense 0.75 mL of whole blood into polystyrene tubes (for RIA, manufactured by Eiken Co., Ltd.) φ10 mm and 2 tubes (test tubes 1 and 2) in which 1 mg of a sample has been placed in advance. Add 0.25 mL of a 0.025 M calcium chloride solution (manufactured by Sigma), invert the tube quickly, and then incubate in a 37 ° C. thermostat. After 3 minutes, tilt one test tube and observe whether the blood flows to form a horizontal plane. If solidified, a horizontal plane will not be formed. Return to the thermostat again and observe whether it solidifies in the same manner every 30 seconds. When this solidifies (t1 minutes), the second test tube is immediately inspected every 30 seconds in the same manner, and when solidified (t2 minutes), the measurement is terminated. The average of t1 and t2 is set as the solidification time. A blank measurement was also performed in the same manner except that no sample was added. The coagulation time is shown below.

Example 2: 11.5 minutes Example 3: 12.0 minutes Comparative Example
SURGICELL (F): 15.5 minutes
OXYCEL: 14.5 minutes
INTERCEED: 17.0 minutes (blank measurement: 20.5 minutes).

(Observation of erythrocyte aggregation)
After whole blood is centrifuged (2,000 G, 10 minutes), the plasma and the buffy coat layer are removed with a pipette. Saline (manufactured by Terumo) is added and gently mixed by inversion. Centrifuge and remove plasma and buffy coat layer again. This was performed three times, and finally washed red blood cells obtained by adding physiological saline were used for the test.

  9 mL of washed erythrocytes (hematocrit value 5%) is added to a 15 mL centrifuge tube (manufactured by Iwaki Co.) containing 45 mg of sample in advance. After gently mixing by inversion, it was left at room temperature for 2 h. After slowly mixing by inversion, blood was collected and immediately observed with a microscope (100 times). In addition, the blank was also observed in the same manner except that no sample was added.

  Hemagglutination was observed in the β-1,4-polyglucuronic acid of Example 2 and Example 3 and the three kinds of oxidized celluloses of Comparative Examples, and it was confirmed that these had a hemagglutination effect. On the other hand, no hemagglutination was observed in the blank.

It is IR spectrum which measured the sample of Example 2 and the comparative example by KBr method. It is the ¹³ C-NMR spectrum which measured the sample of Example 1 and the comparative example by melt | dissolving in heavy water. It is a GPC chart of the sample of Example 2 and a comparative example. It is a spectrum of the X-ray diffraction grating of the sample of Example 2 and a comparative example.

Claims (10)

A bioabsorbable hemostatic agent comprising β-1,4-polyglucuronic acid represented by the structural formula (I).

(X in the formula represents hydrogen or an alkali metal, and n is an integer of 2 or more)   The bioabsorbable hemostatic agent according to claim 1, wherein the number average molecular weight (Mn) of β-1,4-polyglucuronic acid is 10,000 to 50,000.   The ratio (Mw / Mn) of the weight average molecular weight (Mw) and the number average molecular weight (Mn) of β-1,4-polyglucuronic acid is 1.2 to 2.4. The bioabsorbable hemostatic agent described.   The bioabsorbable hemostatic agent according to claim 1, wherein β-1,4-polyglucuronic acid is amorphous.   The bioabsorbable hemostatic agent according to claim 1, wherein the dosage form is powder. A step of dispersing regenerated cellulose in water;
Bioabsorption comprising the steps of selectively oxidizing primary hydroxyl groups of regenerated cellulose in water to obtain β-1,4-polyglucuronic acid, and purifying β-1,4-polyglucuronic acid A method for producing a hemostatic agent.   The step of selectively oxidizing the primary hydroxyl group of regenerated cellulose in water is an oxidation using an oxidizing agent in the presence of 2,2,6,6-tetramethyl-1-piperidine-N-oxyl and an alkali metal bromide. 7. The method for producing a bioabsorbable hemostatic agent according to claim 6, wherein the method is a reaction.   The bioabsorption according to claim 7, wherein the pH of the reaction solution in the step of selectively oxidizing the primary hydroxyl group of regenerated cellulose in water is maintained at a pH of 10.0 to 11.5 while adding an alkali. A method for producing a hemostatic agent.   The method for producing a bioabsorbable hemostatic agent according to claim 7, wherein the reaction temperature in the step of selectively oxidizing the primary hydroxyl group of regenerated cellulose in water is maintained at 5 ° C. or lower.   A method for using a bioabsorbable hemostatic agent, wherein the bioabsorbable hemostatic agent powder according to claim 5 is sprayed or applied to an affected area.

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