JP5160102B2 - Amorphous partially deacetylated chitin salt sponge hemostatic material and method for producing the same - Google Patents

Description

  The present invention relates to a sponge hemostatic material mainly composed of an amorphous partially deacetylated chitin salt and a method for producing the same.

Chitin and chitosan are widely used in the cosmetics field, the medical field, the food field, and the like, and are preferably used as natural materials in the same manner as collagen materials. However, the past application examples are mostly used as powders, and no applications utilizing shapes such as molded articles have been found. Since this natural polymer, chitosan, is easily dissolved in an aqueous acid solution such as acetic acid, various molded articles such as films, fibers, and sponges can be prepared by wet molding. However, there have been almost no successful examples of its practical application.
Chitosan is defined as a deacetylated product of chitin. Generally, chitosan has a degree of deacetylation of 70 to 80% or more and is soluble in a dilute acid solution that is insoluble in water but not in chitin. have.

  Attempts to use chitin and chitosan as a medical band have been made for more than 20 years, and there are also disclosures of organic acid salts thereof (Patent Document 1). Attempts have also been made to make chitosan into a sponge form (Patent Document 2). An attempt was made to commercialize an organic acid salt of chitin or a chitin derivative (Patent Document 3) (Patent Document 4) (Patent Document 5). However, none of these products are perfect in terms of usefulness and safety as hemostatic materials. The present inventors have also proposed a novel hemostatic material that improves chitosan itself, adjusts the degree of deacetylation, and makes it amorphous (Patent Document 6) (Patent Document 7). Furthermore, the present applicant has also tried to provide an improved preparation of chitosan sponge (Patent Document 8) (Patent Document 9).

  In spite of many attempts as described above, a hemostatic material using a chitin derivative has not been put on the market in practical use due to the product recovery of the surgical hemostatic material in 1997.

Japanese Patent Laid-Open No. 60-142927 JP 63-90507 A JP-A-9-169654 JP 2003-26578 A Special Table 2005-503197 Japanese Patent Laid-Open No. 11-5803 Japanese Patent Laid-Open No. 11-276189 JP 2003-292501 A JP 2004-256784 A

  An object of the present invention is to develop a manufacturing method having performance that is expected to be put to practical use with respect to a sponge-like hemostatic material, and to develop a high-quality performance equivalent to or higher than that of a sponge-like hemostatic material prepared by collagen. It is.

The present inventors conducted an optimization analysis of the degree of deacetylation of chitin, selection of an amorphous one as partially acetylated chitin, further selection of an organic acid to be salted, and further optimization analysis of the drying method, As a result, the present inventors have succeeded in developing a preparation superior in safety, blood absorbability, biodegradability, and convenience in use as compared with conventional chitin derivative sponge-like hemostatic materials, and completed the present invention.
In other words, the present invention
“1. Amorphous partially deacetylated chitin gluconate or aspartic acid having a degree of deacetylation of 20 to 65% and a viscosity in a 0.5% W / W solution at 20 ° C. of 20 mPa · s to 300 mPa · s. A sponge-like hemostatic material mainly composed of salt.
2. Item 1 above, wherein the acid addition amount of gluconic acid or aspartic acid at the time of production is 0.2 to 1.00 mol per 1 mol of the amino group of amorphous partially deacetylated chitin, and is prepared by a sherbet freezing method The hemostatic material described in 1.
3. Residual acid concentration of the final preparation is 0-7.0% W / W acetic acid, 2-40% W / W gluconic acid or aspartic acid, and the total amount is 1 mol of amino group of amorphous partially deacetylated chitin. 3. The hemostatic material according to item 1 or 2, wherein the acid ion is 0.2 to 1.00 mol.
4). 4. The hemostatic material according to any one of items 1 to ³ , wherein the final preparation has a weight per volume of 0.005 to 0.035 g / cm ³ and a water absorption of 40 to 120 g / g.
5. 5. The hemostatic material according to any one of items 1 to 4, wherein the final preparation has a pore size of 50 to 400 μm and a substantially homogeneous structure.
6). 6. The hemostatic material according to any one of 1 to 5 above, wherein when applied to a wound site, the change in pH of the wound local blood is 0.05 or less.
7). 7. The hemostatic material according to any one of items 1 to 6, which can be easily removed from a wound site without substantially leaving a fragment after blood absorption.
8). 8. The hemostatic material according to any one of 1 to 7 above, wherein the hemostatic material is not adsorbed to the surgical instrument.
9. The preparation of amorphous partially deacetylated chitin is carried out at a treatment temperature of 35 to 60 ° C. and a treatment time of 2 to 5 hours when caustic soda of about 40% W / W is added. The hemostatic material according to any one of the above.
10. After suspending amorphous partially deacetylated chitin having a degree of deacetylation of 20 to 65%, gluconic acid or aspartic acid is added to 0.2 mol per 1 mol of amino group of amorphous partially deacetylated chitin. Add to ~ 1.0 mol, stir, and then freeze the filtrate of the solution after stirring, crush and form into a sherbet, fill it into a product tray, and prepare it by freeze vacuum drying and further secondary drying A method for producing a sponge-like hemostatic material mainly comprising a crystalline partially deacetylated chitin salt.
11. 11. The method according to item 10 above, wherein amorphous partially deacetylated chitin is dissolved by addition of acetic acid before addition of gluconic acid or aspartic acid.
12. The method according to 11 above, wherein the secondary drying and / or heat treatment is performed at 65 to 85 ° C. for 1 to 7 days. Is.

  The sponge-like hemostatic material mainly composed of the amorphous partially deacetylated chitin salt of the present invention has excellent blood absorbability, is substantially homogeneous with a pore size set to 50 to 400 μm, and is a wound local area. This is a hemostatic material that is safe for a living body at a level that has not existed in the past, because it has an excellent hemostatic effect and can be easily and completely removed after hemostasis.

  The partially deacetylated chitin in the present invention is derived from chitin which is a kind of amino polysaccharide contained in the exoskeleton of crustaceans such as crabs and shrimps, and the chemical structure is a repetition of glucosamine and a small amount of N-acetylglucosamine. It is a polymer derived from a natural product that is a structure. In general, chitin obtained by deproteinizing crustacean exoskeletons with alkali such as caustic soda and decalcifying with acid solution such as hydrochloric acid is further obtained by partial deacetylation with high concentration alkaline aqueous solution such as caustic soda. It is done. It is sparingly soluble in water and dissolves in acid aqueous solutions such as acetic acid.

  A method for producing amorphous partially deacetylated chitin (hereinafter sometimes referred to as amorphous chitin) is about 40% W of high-purity chitin having a protein content of 0.1% by weight or less and an inorganic content of 0.01% by weight or less. / W in alkali at 35 to 60 ° C. for 2 to 7 hours, and then placed under cooling conditions (−10 ° C. to −30 ° C.) for several hours (1 to 3 hours). The alkali concentration is about 10% W / Water is added so that it becomes W, and an alkali chitin dope is prepared. During alkaline hydrolysis in a homogeneous system, the chitin dope is aged at 30 ° C or less to the desired viscosity, further neutralized to form a precipitate, dehydrated, washed, freeze-dried, etc., and deacetylated chitin Partially deacetylated so that the rate (also referred to as DAC degree) is generally about 20 to 65%, preferably about 25 to 45%, more preferably about 30 to 40%, and 0.5% W at 20 ° C. An amorphous partially deacetylated chitin having a / W solution viscosity of 20 to 300 mPa · s, more preferably 30 to 250 mPa · s, still more preferably 35 to 200 mPa · s is prepared. Neutralization is dealkalized by addition of acid or alcohol, ion exchange resin or the like.

  The molecular weight of the amorphous partially deacetylated chitin used in the present invention generally has a weight average molecular weight (calculated by GPC molecular weight measurement using pullulan as a standard product) of about 50,000 to 4 million. Preferably, it is 100,000 to 3 million, more preferably 200,000 to 2 million. The deacetylation rate is generally about 20 to 65%, preferably about 25 to 45%, more preferably about 30 to 40%. The viscosity of the 0.5% W / W solution viscosity at 20 ° C. is 20 to 300 mPa · s, more preferably 30 to 250 mPa · s, and still more preferably 35 to 200 mPa · s.

Amorphous partially deacetylated chitin can be prepared as a salt by dissolving it in a certain aqueous acid solution. The acid used for the acid solution of partially deacetylated chitin can be any weak acid such as acetic acid. The acid concentration is generally 0.01 to 10.0 wt%, and 0.05 to 5.0 wt% is particularly preferably used. The concentration of the partially deacetylated chitin is preferably one having a viscosity that is easy to use, generally 0.1 to 5.0 wt%, and particularly preferably 0.5 to 3.0 wt%. The amount of acid added may be calculated by using about 1.0 to 105 moles of acid per mole of amino group of partially deacetylated chitin. In addition to the partially deacetylated chitin, an auxiliary agent such as a surfactant may be added to the aqueous solution as necessary, but the presence or absence of the auxiliary agent does not affect the effect of the present invention. .
Preferably, the amorphous partially deacetylated chitin salt solution dissolved with a weak acid such as acetic acid is stirred until the partially deacetylated chitin is almost completely dissolved.

In the present invention, as described above, it is sufficient to suspend in amorphous water without dissolving amorphous partially deacetylated chitin using weak acid such as acetic acid.
In addition, if it does not melt | dissolve using weak acids, such as an acetic acid, the process of reducing weak acids, such as an acetic acid, in a later process is unnecessary.
Thereafter, gluconic acid or aspartic acid is added as an acid to form amorphous partially deacetylated chitin gluconate or aspartate. The acid is added in an amount of 0.2 to 1.0 mol, preferably 0.3 to 0.8 mol, more preferably 0.4 to 0.6 mol, per mol of amino group of amorphous partially deacetylated chitin.

Amorphous partially deacetylated chitin gluconate or aspartate (sometimes referred to as amorphous partially deacetylated chitin salt) is sufficiently stirred, and if dissolved, the solution after stirring If it is not dissolved, it is not filtered.
Next, the obtained liquid is subjected to sherbet ice production by the “sorbet freezing method”. Here, the “sherbet freezing method” of the present invention does not produce frozen ice by quick freezing or gradually freezing as in the prior art, but once frozen frozen ice is crushed, Sorbet frozen ice can be obtained by refilling and freezing again. Further, when the amorphous chitin solution is pulverized with dry ice, it becomes a sherbet shape, and is similarly filled into a mold and frozen again to obtain frozen sherbet ice. The detailed manufacturing method is as follows. About 500 g to 700 g of dry ice is added to about 1000 g of the amorphous partially deacetylated chitin salt solution, and pulverized with a blender or the like to form a sherbet. The sherbet temperature is adjusted to -1 ° C to + 2 ° C. In the production of the sponge-like hemostatic material of the present invention, the freezing method is not particularly limited as long as the sherbet shape as described above can be obtained.

When prepared by adding gluconic acid or aspartic acid without using acetic acid, acetic acid is not contained, so secondary drying to reduce residual acetic acid is not necessary, but to increase strength. You may perform the process by the heat similar to secondary drying.
The present inventors have newly confirmed that the secondary drying step can increase the breaking strength regardless of the concentration of gluconic acid or aspartic acid in the broken sponge.
Hereinafter, the heat treatment for the purpose of increasing the strength is referred to as heat treatment, and is distinguished from the secondary drying treatment. This completes the preparation of a sponge-like hemostatic material mainly composed of an amorphous partially deacetylated chitin salt.

  Freeze-vacuum drying of the method for producing a sponge-like hemostatic material mainly composed of an amorphous partially deacetylated chitin salt according to the present invention has a certain depth in a container such as a tray, assuming the thickness of the finished sheet. In general, an amorphous partially deacetylated chitin salt sherbet preparation is poured, frozen as it is, and then dried.

  The secondary drying and / or heat treatment is performed at 65 to 85 ° C. for 1 to 7 days.

  Since the thickness of the sheet that can be prepared by the method of the present invention is about 0.5 mm to 20 mm, the thickness of the frozen sherbet-like sample may be set to be substantially the same. The sherbet-like specimen prepared in this way is frozen in contact with the cooling atmosphere. Freezing can be performed at a temperature below the freezing temperature of water, that is, 0 ° C. or less, preferably −20 ° C. or less.

  The freezing method includes placing the sample in a normal air-cooled freezer, immersing it in a refrigerant such as an aqueous solution of ethanol or calcium chloride, which is brine, at −20 ° C. or lower, liquid nitrogen, liquid carbon dioxide, etc. A spraying method can also be used.

  The freezing time varies depending on the freezing method and freezing temperature, but generally it is preferably about 2 to 24 hours or longer and slowly frozen. More preferably, it is about 3 hours or more. Once frozen, frozen ice can be stored in a freezer for any number of days, and when necessary, the frozen ice can be used as the sponge hemostatic material of the present invention by freeze-drying in the next step. .

  The sheet after freeze-drying was prepared by removing the remaining acids by secondary drying such as hot air drying when dissolved with a weak acid such as acetic acid, and adding only gluconic acid or aspartic acid without using acetic acid. In some cases, heat treatment is preferably performed to increase the strength of the sponge, and finally a sponge-like hemostatic material mainly composed of the amorphous partially deacetylated chitin salt of the present invention can be obtained. The present invention is characterized in that the performance of the finished sponge-like hemostatic material is much better than that obtained by the conventional method. That is, there is a feature that, while the thickness is uniform, the water absorption speed is high, the water absorption magnification is high, the wet strength is high, and the biodegradability is high.

  The residual acid concentration of the final preparation obtained by the present invention is 0 to 7.0% W / W or less of acetic acid, 2 to 40% W / W of gluconic acid or aspartic acid, and the amino group of amorphous partially deacetylated chitin The total acid ion is 0.2 to 1.00 mol with respect to 1 mol. More preferably, the residual acid concentration of the final formulation is about 0-6.0% W / W acetic acid, about 2.0-30% W / W gluconic acid or aspartic acid, and the amino group of amorphous partially deacetylated chitin The total acid ion is 0.4 to 0.8 mol with respect to 1 mol.

The weight per volume of the final formulation obtained by the present invention is 0.005 to 0.035 g / cm ³ and the water absorption is 40 to 120 g / g. More preferably, the weight per volume is about 0.011 to 0.0017 g / cm ³ and the water absorption is about 60 to 100 g / g.

  The pore size of the final formulation obtained according to the present invention is 50-400 μm and has a substantially homogeneous structure. Further, when applied to the wound site, the change in pH of the wound local blood is 0.05 or less. Furthermore, it can be easily removed from the wound site without substantially leaving fragments after blood absorption. In addition, the surgical instrument has a property of not adsorbing sticky.

  The measuring method of each concentration used in the present invention is as follows. The following examples and test examples were also used.

  The DAC degree (* 1 in the table) is determined by dissolving a chitosan sample (amorphous chitin or chitosan) in 0.5% (w / w) acetic acid solution to 0.5% (w / w), and toluidine blue solution as an indicator Was used to determine the DAC degree (deacetylation degree) per dry matter by colloidal titration with an aqueous polyvinyl potassium sulfate solution.

  Viscosity (* 2 in the table) is determined by dissolving a chitosan sample (amorphous chitin or chitosan) in 0.5% (w / w) acetic acid solution to 0.5% (w / w) and stirring at room temperature for 3 hours. Stir for 2 minutes with a homogenizer. The rotational viscosity (mPa · s) was measured with a B-type viscometer while keeping this solution at 20 ° C. in a thermostatic bath.

  For the amount of water absorption (* 3 in the table), after measuring the weight (A) of the sample, the sample is immersed in a container containing sufficient pure water for 5 minutes. The sample is then drawn into a colander, drained for 5 minutes, and the weight (B) of the sample is measured. The amount of water absorption is represented by (BA) / A. More specifically, about 0.4 g was weighed (A), soaked in pure water for 5 minutes, lifted into a colander, drained for 5 minutes, weighed (B), and obtained by the following formula. Water absorption = (B−A) / A. In the case of the present invention, the amount of water absorption is 15 g / g or more, and a good one is 40 times or more.

  The water absorption rate (* 4 in the table) is a time measured by adding a drop of pure water to a sample using the JIS L1907 dropping method and absorbing the water so that it does not reflect. More specifically, the sponge is placed horizontally, 50 μl of water is dropped from above with a micropipette or the like, and the time during which water penetrates from the sponge surface into the inside, that is, the swelling due to the surface tension of the dropped water is observed. It can be measured by visually observing the time when it disappears. In the case of the present invention, it is 5 seconds or less, and a good one is 3 seconds or less.

  The breaking strength after water absorption (* 6 in the table) is to cut the sample into 50mm squares, fully immerse in pure water, lift to a colander and drain for 5 minutes. Cut the water-absorbed sample to a diameter of 40 mm and a thickness of 15 mm using a cork borer and cutter, and set it in a special container (diameter of 40 mm and height of h15 mm). When measuring the breaking strength by crushing using the creep meter (Yamaden RHEONERII), measuring speed 1mm / sec, contact diameter 20mm, measuring distortion 99.99%, storage pitch 0.04sec, in the case of the present invention, It is 0.2N or more, and a favorable thing is 0.25N-200N.

In the acid concentration in the sponge (* 7 in the table), for analysis of gluconic acid and acetic acid, weigh about 0.1 g of sample finely, weigh appropriately, then add pure water, add acid not present in sponge, After stirring, dissolve completely. For the added acid not present in the sponge, for example, when gluconic acid is contained in the sponge, 50 to 200 μl of acetic acid, glucurono-δ-lactone 0.2 to 0.3 g when acetic acid is contained in the sponge, and gluconic acid in the sponge If both acetic acids are included, add 50-200 μl of lactic acid. Then, make up to 100 ml of solution. The solution is filtered through a 0.45 μm filter to obtain a sample solution. Using a microsyringe, collect 10 μl of the sample solution, inject it into HPLC (Carboxylic Acid Analyzer, manufactured by Shimadzu Corporation), and measure under the following conditions. The retention time of gluconic acid is around 17.1 minutes and that of acetic acid is around 26.1 minutes. The gluconic acid standard solution, the acetic acid standard solution and the sample solution are measured, and the glucone content of the sample is determined from the ratio of the peak area values of the standard solution and sample solution Acid concentration and acetic acid concentration are calculated.
[Analysis conditions]
Equipment: Carboxylic acid analyzer manufactured by Shimadzu Corporation
<Separation conditions>
Column: Two Shim-Pak SCR-102H units Mobile phase: 5 mM p-toluenesulfonic acid aqueous solution Mobile phase flow rate: 0.8 ml / min
Temperature: 40 ° C
<Detection conditions>
Buffer: 5 mM p-toluenesulfonic acid aqueous solution and
20 mM Bis-tris aqueous solution containing 100 μM EDTA Buffer flow rate: 0.8 ml / min
Detector: CDD-6A Conductivity detector Injection volume: 10μl

In the acid concentration in the sponge (* 7 in the table), aspartic acid is analyzed by finely weighing about 0.15 g of sample, adding about 20 ml of pure water, and preparing a suspension using a homogenizer. Next, add 10 ml of 50 mM phosphate buffer (pH 6.9) and make up to 50 ml. The solution is filtered with a 0.45 μm filter to obtain a sample solution, which is measured using HPLC (manufactured by Shimadzu Corporation) under the following analysis conditions. The retention time of aspartic acid is around 10.6 minutes, but the retention time of acetic acid is close to around 10.8 minutes, so if both the acetic acid and aspartic acid are contained in the sponge, the peaks will overlap and correction is necessary. is there. The correction method is to analyze the acetic acid standard solution by this method, determine the peak area value relative to the acetic acid concentration, calculate the acetic acid area value in this method from the acetic acid concentration determined by the method of paragraph “0030”, and then The sample is analyzed and the area value of acetic acid is subtracted from the area value of the overlapping peak of aspartic acid and acetic acid. The aspartic acid standard solution and the sample solution are measured, and the aspartic acid concentration of the sample is calculated from the ratio of the peak area value of the standard solution to the corrected peak area value of the sample solution.
[Analysis conditions]
<Separation conditions>
Column: Shodex KW-802.5
Mobile phase: 10 mM phosphate buffer (pH 6.9)
Flow rate: 1.0ml / min
Temperature: 40 ° C
Detector: UV detector Measurement wavelength 210nm
Injection volume: 10μl

The determination of the rate of degradation of each sponge-like hemostatic material with an enzyme (FIG. 6) uses a GlcN quantification method by the Rondle-Morgan method. The details of the quantification method are as follows.
Transfer 1.5 mL of the sample (each sponge hemostatic material) solution hydrolyzed with lysozyme to a sample tube, add 1.5 mL of acetic acid buffer (see below), and heat at 40 ° C. for 10 minutes. Next, 1.0 mL of an acetylacetone reagent (refer to the following) is added, dropped to pH 9.80 with HCl, heated in a boiling water bath for 20 minutes, and then cooled to room temperature with running water. Furthermore, EtOH3.0mL and Ehrlich reagent 1.0mL (referred hereafter) are added, and after heating for 10 minutes at 65-70 degreeC, it cools to room temperature with running water. Enzymatic hydrolysis is evaluated by measuring VIS at 530 nm using a spectrophotometer (manufacturer: Shimadzu Rika Kikai Co., Ltd.).
Acetic acid buffer: Add pure water to 0.60 g of acetic acid, make up to 100 mL, make 0.1 M acetic acid solution, add pure water to 4.1015 g of sodium acetate and make up to 500 mL to obtain 0.1 M sodium acetate solution. Next, 10 mL of 0.1 M acetic acid solution and 160 mL of 0.1 M sodium acetate solution are mixed to prepare an acetic acid buffer having pH = 5.60. The pH is confirmed with a HORIBA Castani ACT pH meter.
Acetylacetone reagent: Prepare by dissolving 1 mL of acetylacetone in 50 mL of 0.5 mol / L Na ₂ CO ₃ .
Ehrlich reagent: Prepare by dissolving 0.4 g of DMAB in 15 mL of EtOH and adding 15 mL of conc-HCl.

  Further, the hemostatic material obtained in the present invention may be fragile due to lack of flexibility depending on the specific density and strength of the sponge, or may be fragile. In that case, if it is manufactured in the same manner by adding about 0.01 to 10% W / W of a softening agent such as polyethylene glycol, ethylene glycol, glycerin or oil in the state of an amorphous partially deacetylated chitin salt, The created hemostatic material can have flexibility.

  In the present invention, it is possible to easily prepare a third hemostatic material containing the third component at the time of the solution of the amorphous partially deacetylated chitin salt and the final hemostatic material. For example, it is possible to manufacture hemostatic materials including pharmaceuticals, cosmetics, and quasi drugs. In particular, a substance whose physiological activity is easily denatured by other chemical components or temperature can be retained in the hemostatic material.

  In addition, it is considered that these drugs can be provided with a sustained release function by mixing and molding in an amorphous partially deacetylated chitin salt sponge.

Example 1
Production of sponge hemostatic material mainly composed of amorphous partially deacetylated chitin salt The sponge hemostatic material used in the following Example 2 and each experimental example was produced. The manufacturing method is as follows.

48L NaOH 3kg, water 0.6L, 43 mesh high purity chitin powder (trade name: Chitin L-PC) [viscosity 65mPa · s (NN-dimethylacetamide / 8% (W / W) 0.2% in lithium chloride solution ( W / W) was dissolved and measured at 30 ° C.), 1.8% water, 0.03% ash, 0.96% DAC degree, 0.3 kg], and prepared and dispersed at 35-60 ° C. for 2-5 hours. Then, add 6kg of ice and stir for 3 hours under cooling conditions (-10 to -30 ° C), add 6L of water, chitin concentration about 2% (W / W), alkali concentration about 10% (W / W). Of alkali chitin dope and adjust the DAC degree (deacetylation degree) and viscosity to the target values at 30 ° C. [For example, DAC degree 30%, viscosity 50 mPa · s (0.5% at 20 ° C. (W / W) Solution)] Aged. Thereafter, the solution was filtered under reduced pressure with a 150 mesh nylon mesh and neutralized with 95% aqueous sulfuric acid. The partially deacetylated chitin gel precipitated at this time was transferred to a 50 mesh nylon mesh and manually dewatered. Then, it was desalted by washing with hot water four times and then squeezed. Next, the partially deacetylated chitin gel after dehydration was freeze-dried and then pulverized to give a degree of deacetylation of about 30% and no decrease in viscosity [from 31 mPa · s to 780 mPa · s (0.5% (W Amorphous partially deacetylated chitin (also referred to as amorphous chitin DAC30) was obtained.
Amorphous chitin DAC30 (Deacetylation degree = 30-40%) [Viscosity 20-100 Pa · s (0.5% (W / W) solution) at 20 ° C.]] or chitosan as control (Deacetylation degree = 100% ) [Viscosity 20-100 Pa · s at 20 ° C. (0.5% (W / W) solution)] was added to 1% by weight of pure water. At that time, amorphous chitin DAC30 was stirred and dispersed, and then acetic acid was added. Acetic acid was added in an amount of 1.1 mol of acetic acid to 1 mol of amino group of amorphous chitin.

The amount of acetic acid added (g) was calculated as follows. In the following, amorphous chitin means amorphous partially deacetylated chitin.
Acetic acid addition amount (g) = amorphous chitin amount (g) / amorphous chitin monomer molecular weight x degree of deacetylation (%) / 100 x 1.1 x acetic acid molecular weight (60)

The molecular weight of the amorphous chitin monomer was calculated as follows.
Amorphous chitin monomer molecular weight = chitosan monomer molecular weight (161) × deacetylation degree (%) / 100 + chitin monomer molecular weight (203) × (100−deacetylation degree) / 100

Therefore, the acetic acid addition amount (%) is according to the following formula.
Acetic acid addition amount (%) = acetic acid addition amount (g) / solution weight (g) x 100

After the addition, the solution was stirred until completely dissolved, and then acid (phosphoric acid or aspartic acid or gluconic acid) was added. The amount of acid added is 0.4 to 0.8 mol of acid per 1 mol of amino group of amorphous chitin. The calculation formula is as follows.
Acid addition amount (g) = amorphous chitin amount (g) / amorphous chitin monomer molecular weight x degree of deacetylation (%) / 100 x 0.4 to 0.8 x acid molecular weight where the acid molecular weight is 178.14 for gluconic acid, asparagine The acid is 133.1 and phosphoric acid is 98.

  After the addition of the acid, stirring was performed overnight, and the solution after stirring was filtered (stainless steel mesh 1480 mesh air pressure filtration). The filtrate was then subjected to sherbet ice production. About 500 g to 700 g of dry ice was added to about 1000 g of the filtered amorphous chitin solution, and crushed with a blender to form a sherbet. The sherbet temperature was adjusted to -1 ° C to + 2 ° C.

The obtained sherbet ice was filled in a tray and frozen. Freezing was carried out in a freezer at −40 ° C., and the sherbet was frozen slowly and overnight. If you put it in the freezer, it can be stored for many days. Subsequently, the frozen sample was subjected to freeze vacuum drying. After 4 days of freeze-drying, further secondary drying was treated at 75 ° C. for 3 days to reduce residual acetic acid. Alternatively, the secondary drying was performed under low vacuum.
This completed the preparation of a sponge-like hemostatic material composed mainly of an amorphous partially deacetylated chitin salt having the sample numbers shown in Table 1.
Moreover, the detailed manufacturing method of each sample is as follows.

Sample No. S510204 (See Table 1)
Amorphous chitin prepared as above (deacetylation degree = 37.6%) [viscosity 44mPa · s (0.5% (W / W) solution) at 20 ° C] 10g (1% (W / W)) Dispersed in 987.04 g of pure water to be used, and then 1.2 g (0.12% W / W) of acetic acid (special grade manufactured by Kanto Chemical) was added.
The amount of acetic acid added was according to the following formula.
Acetic acid addition amount = amorphous chitin amount (10 g) / amorphous chitin monomer molecular weight (187.2) x degree of deacetylation (37.6%) / 100 x acetic acid molecular weight (60) = 1.2
The amorphous chitin molecular weight was according to the following formula.
Amorphous chitin molecular weight = chitosan molecular weight (161) × degree of deacetylation (37.6%) / 100 + chitin molecular weight (203) × (100−degree of deacetylation 37.6%) / 100 = 187.2
The solution to which acetic acid had been added was dissolved by stirring, and 1.7 g (0.17% W / W) of glucono-δ-lactone (special grade manufactured by Kanto Chemical) was further added. Glucono-δ-lactone is a powder and becomes gluconic acid when dissolved in water.
The amount of gluconic acid added was according to the following formula.
Gluconic acid addition amount = Amorphous chitin amount (10 g) / Amorphous chitin monomer molecular weight (187.2) x Deacetylation degree (37.6%) / 100 x 0.48 x Glucono-δ-lactone molecular weight (178.14) = 1.7 g
After the addition of gluconic acid, the mixture was stirred overnight, filtered (stainless mesh 1480 mesh air pressure filtration), and the filtrate was subjected to sherbet ice production. 500 g of dry ice was added to about 1000 g of the amorphous chitin solution, and pulverized with a blender (Oster OB-1) to form a sherbet. The sherbet temperature was adjusted to 0 ° C. Thereafter, the tray was filled, frozen at −40 ° C., and stored in a freezer for 3 days.

Sample No. S604182 (See Table 1)
10 g (1% (W / W)) of amorphous chitin prepared in the same manner as above (deacetylation degree = 37.7%) [viscosity 77mPa · s (0.5% (W / W) solution) at 20 ° C] It was dispersed in 988.3 g of pure water for use and suspended.
The amorphous chitin molecular weight was according to the following formula.
Amorphous chitin molecular weight = chitosan molecular weight (161) × degree of deacetylation (37.7%) / 100 + chitin molecular weight (203) × (100−degree of deacetylation 37.7%) / 100 = 187.17
While stirring the suspension, 1.7 g (0.17% W / W) of glucono-δ-lactone (special grade manufactured by Kanto Chemical) was added. Glucono-δ-lactone is a powder and becomes gluconic acid when dissolved in water.
The amount of gluconic acid added was according to the following formula.
Gluconic acid addition amount = Amorphous chitin amount (10 g) / Amorphous chitin monomer molecular weight (187.17) x Deacetylation degree (37.7%) / 100 x 0.48 x Glucono-δ-lactone molecular weight (178.14) = 1.7 g
After addition of gluconic acid, even if stirring was performed overnight, the solution was not completely dissolved and became cloudy. This white turbid solution was subjected to sherbet ice production. 500 g of dry ice was added to about 1000 g of the amorphous chitin solution, and pulverized with a blender (Oster OB-1) to form a sherbet. The sherbet temperature was adjusted to 0 ° C. Thereafter, the tray was filled, frozen at −40 ° C., and stored in a freezer for 4 hours. Next, freeze-drying was carried out, followed by further heat treatment at 75 ° C. for 3 days (NY4 type, dryer Dalton) to obtain a sponge-like hemostatic material mainly composed of amorphous partially deacetylated chitin salt.

Sample NO.S606135
10 g (1% (W / W)) of amorphous chitin prepared in the same manner as above (deacetylation degree = 37.7%) [viscosity 77mPa · s (0.5% (W / W) solution) at 20 ° C] It was dispersed in 987.1 g of pure water for use and suspended.
The amorphous chitin molecular weight was according to the following formula.
Amorphous chitin molecular weight = chitosan molecular weight (161) × degree of deacetylation (37.7%) / 100 + chitin molecular weight (203) × (100−degree of deacetylation 37.7%) / 100 = 187.17
While stirring the suspension, 2.87 g (0.287% W / W) of glucono-δ-lactone (special grade manufactured by Kanto Chemical) was added. Glucono-δ-lactone is a powder and becomes gluconic acid when dissolved in water.
The amount of gluconic acid added was according to the following formula.
Gluconic acid addition amount = Amorphous chitin amount (10 g) / Amorphous chitin monomer molecular weight (187.17) x Deacetylation degree (37.7%) / 100 x 0.8 x Glucono-δ-lactone molecular weight (178.14) = 2.87 g
After adding gluconic acid, the mixture was stirred overnight to dissolve. The solution was filtered (stainless mesh 1480 mesh air pressure filtration), and the filtrate was subjected to sherbet ice production. 500 g of dry ice was added to about 1000 g of the amorphous chitin solution, and pulverized with a blender (Oster OB-1) to form a sherbet. The sherbet temperature was adjusted to 0 ° C. Thereafter, the tray was filled, frozen at −40 ° C., and stored in a freezer for 3 hours. Next, freeze-drying was carried out, followed by further heat treatment at 75 ° C. for 3 days (NY4 type, dryer Dalton) to obtain a sponge-like hemostatic material mainly composed of amorphous partially deacetylated chitin salt.

In the table, the column labeled “chitosan” includes various chitin derivatives exemplified below.
FM is medium viscosity chitosan (DAC degree 100%, viscosity 44mPa · s: sample No.S510194, No.S510195, S510203), DAC30M is medium viscosity amorphous chitin (DAC degree about 25% ~ 40% viscosity 20 ~ 100mPa・ S: Samples No. S509081, No. S510191, No. S510204, No. S604182, No. S606133, No. S606134, No. S606135), DAC30L is low viscosity amorphous chitin (DAC degree of about 25% to 40%) Viscosity 20 mPa · s or less: Samples No. S510192, No. S510193, No. S510201), DAC30H is a high viscosity amorphous chitin (DAC degree of about 25% to 40% Viscosity 100 to 300 mPa · s: Sample No. S601161, No DAC50L is low viscosity amorphous chitin (DAC degree around 50%, viscosity 20mPa · s or less: sample No.S509082, No.S510062, No.S510202), DAC30L + FH (DAC degree 59%, viscosity 86mPa · s: Sample No. S510281) is a mixture of low-viscosity amorphous chitin DAC30L and high-viscosity chitosan FH (DAC degree 100%, viscosity 215 mPa · s) mixed at 13: 7.

Example 2
The structure of the sponge-like hemostatic material (sample No. S510204) obtained in Example 1 was confirmed with a scanning electron microscope.

  The appearance of the sponge-like hemostatic material (sample No. S510204) obtained in Example 1 is shown in FIG. Further, a scanning electron micrograph is shown in FIG. In all cases, the structure and the pore size were substantially uniform. In addition, without using sherbet ice, the medium viscosity chitosan FM (see Experimental Example 1) was filled in a tray and normally frozen in a -40 ° C freezer, and the final freezing temperature was -39 ° C. Scanning electron micrographs are shown in FIGS. 1-3. The figure is obtained by slicing and observing the cross-sectional direction with a cutter, and the structure varies considerably depending on the observation location (cross-section 1: A) (cross-section 2: B). In addition, when the outermost surface of the material was observed (C), the material was further sliced with a cutter in the plane direction, and the upper layer portion (D) and the middle layer portion (E) were observed, the pore size was greatly different between the upper layer portion and the middle layer portion. It was.

Experimental example 1
Results of analysis of sponge-like hemostatic material mainly composed of amorphous partially deacetylated chitin salt The physical characteristics of each sponge-like hemostatic material obtained in Example 1 were analyzed and evaluated. The analysis results are shown in Table 1.

It was confirmed that medium-viscosity amorphous chitin DAC30M, DAC60M and high-viscosity amorphous chitin DAC30H having a DAC degree of about 25% to 60% and a viscosity of 20 to 200 mPa · s are suitable preparation raw materials. Furthermore, gluconic acid is most suitable as the acid, and aspartic acid (Asp) can also be used.
In the table, the thick frame is a sample with good results in animal experiments.

Experimental example 2
Animal Experiment Results Each sponge-like hemostatic material obtained in Example 1 was used for animal experiments. The analysis results are shown in Table 2 below. In addition, it is a summary of 6 trials of animal experiment results.

In the table, the column labeled chitosan includes the various chitin derivatives exemplified below. FM is medium viscosity chitosan (DAC degree 100%, viscosity 44mPa · s), DAC30M is medium viscosity amorphous chitin (DAC degree 38.0%, viscosity 46mPa · s), DAC50L is low viscosity amorphous chitin (DAC degree 53.0) %, Viscosity 8 mPa · s). In animal experiments, x means “bad”, Δ means “not good or bad”, ○ means “good”, and ◎ means “very good”.
As a result, it was confirmed that DAC30M, that is, medium-viscosity amorphous chitin (DAC degree of 30% to 40%, viscosity of 20 to 100 mPa · s) is a suitable drug substance. Furthermore, gluconic acid is most suitable as the acid, and aspartic acid (Asp) can also be used.

Experimental example 3
The hemostatic material prepared with each sponge-like hemostatic material obtained in Example 1 was immersed in bone marrow blood that bleeds from the rabbit femoral neck bone marrow, and blood absorbability was evaluated.
The blood absorbability of the hemostatic material prepared by the sherbet freezing method improved rapidly. When the performances of DAC30M Asp salt and DAC50L Asp salt were compared among the samples prepared by the sherbet freezing method, DAC30M Asp salt showed good blood absorbability (FIG. 2). On the other hand, the DAC50L Asp salt was worse in blood absorption than the DAC30M Asp salt, and after a certain period of time, the specimen contracted and was broken into a tattered state. As a result, it was confirmed that DAC30M and DAC60M, that is, medium-viscosity amorphous chitin (DAC degree of about 30% to 60%, viscosity of about 20 to 100 mPa · s) is a suitable drug substance.

Experimental Example 4
The effect of the hemostatic material of the present invention on microhemorrhage from the rabbit vertebral venous plexus was examined. An in vitro blood absorbability test was used as a specimen for hemostasis experiments. That is, DAC30M Asp salt, DAC30M phosphate, FM phosphate, DAC30M gluconate (acetic acid residual and acetic acid free) and DA60M gluconate. The amount of acid was 0.4 to 0.8 mole per mole of amino group. In DAC30M, the amount of acid added is small, but since FM has many amino groups, naturally the amount of acid added was increased. The pore size of the hemostatic material of the present invention is 50 to 400 μm, and since the pore size is larger and more homogeneous than the conventional method of rapid freezing with an aluminum plate in between, blood absorption and handling are significantly improved, and hemostatic effect is achieved in all specimens Was obtained and reached a level that can be used as a hemostatic material. Handling was preferable in the order of DAC30M gluconate (acetic acid free)> DAC30M gluconate (acetic acid residue)> DAC30M phosphate> FM phosphate> DAC30M Asp salt (Table 2). FM phosphate has a very low pH and is somewhat difficult to handle. Gluconate has almost no decrease in pH when impregnated in blood, has some gelation, and has good physical properties. In addition, even if gluconic acid is absorbed into the living body, it is predicted that there is little adverse effect on the living body, and since the hemostatic effect is clear, it is considered to be a very useful material as a hemostatic material. In addition, handling means the ease of handling as a hemostatic material, and the sponge hemostatic material is dissolved by the liberated acid, and it may be difficult to handle by adsorbing sticky to the surgical instrument. Is.
In addition, FIGS. 3-5 shows the state in the specific hemostatic process by the hemostatic material of this invention. FIG. 3 shows a state where the hemostatic material is applied to the wound site, FIG. 4 shows a completed state of hemostasis at the wound site, and FIG. 5 shows a recovery of the hemostatic material.

Experimental Example 5
The biodegradation rate of the hemostatic material of the present invention was examined. Since most hemostatic materials are buried in the living body after use, it is preferably absorbed and disappeared as soon as possible after the role as the hemostatic material is finished.
Therefore, it was confirmed whether the hemostatic material of the present invention was rapidly decomposed with lysozyme found on the wound surface of the human body. The experimental method and experimental results are as follows.

1. Reagents DAC30 (amorphous chitin: deacetylation degree of about 30%), FM-80, and FH-80 (distributor: Koyo Chemical Co., Ltd.) obtained in Example 1 are used as samples of each hemostatic material. did. In addition, lysozyme (derived from egg white, optimum pH 5.0 to 6.0, sold by Wako Pure Chemical Industries) was used as an in vivo enzyme.
2. Preparation method of sponge of each hemostatic material The above DAC, FH and FM were prepared in a sponge form by the following method.
Conventional quick freezing
FM chitosan was dissolved in 0.75% acetic acid, sandwiched between conductive plates, quenched and freeze-dried in an ethanol refrigerant, and heat-dried to remove acetic acid (FIG. 6: FM-AQ).
FH chitosan was dissolved in 0.75% acetic acid, sandwiched between conductive plates, quenched and freeze-dried in ethanol refrigerant, and heat-dried to remove acetic acid (FIG. 6: FH-AQ).
"Sherbet freezing method" of the present invention
FM chitosan was dissolved in 0.4% acetic acid and then 0.365% phosphoric acid was added. Then, this chitosan compounded liquid was pulverized with a blender together with dry ice, filled into a tray, gradually frozen in a freezer, freeze-dried under vacuum, and secondary dried to remove acetic acid (FIG. 6: FM-PG). ).
DAC30 was dissolved in 0.125% acetic acid and then 0.094% phosphoric acid was added. Then, this chitosan compounded liquid was pulverized with a blender together with dry ice, filled into a tray, gradually frozen in a freezer, freeze-vacuum dried, and secondarily dried to remove acetic acid (FIG. 6: DAC30-PG). ).
DAC30 was dissolved in 0.125% acetic acid, then 0.171% gluconic acid was added. This chitosan compounded solution was pulverized with a blender together with dry ice, filled into a tray, gradually frozen in a freezer, freeze-dried, and dried to remove acetic acid (FIG. 6: DAC30-GG). ).
3. Decomposition measurement with each hemostatic material Add 100 ml of 0.1M acetic acid buffer (pH5.60) containing 2 mg of lysozyme to 10 mg of each hemostatic material (FM-AQ, FH-AQ, FM-PG, DAC30-PG, DAC30-GG) It was. This solution was hydrolyzed with a constant temperature shaker while maintaining at 37 ° C. Enzymatic degradation at each time was evaluated by the Rondle-Morgan method shown below using a spectrophotometer.
Determination of GlcN by Rondle-Morgan method 1.5 mL of a sample solution hydrolyzed with lysozyme was placed in a sample tube, 1.5 mL of acetic acid buffer was added, and the mixture was heated at 40 ° C. for 10 minutes. Subsequently, 1.0 mL of an acetylacetone reagent was added, the pH was lowered to 9.80 with HCl, the mixture was heated in a boiling water bath for 20 minutes, and then cooled to room temperature with running water. After adding EtOH 3.0mL and Ehrlich's reagent 1.0mL and heating at 65-70 degreeC for 10 minute (s), it cooled to room temperature with running water. Enzymatic hydrolysis was evaluated by measuring VIS at 530 nm using a spectrophotometer.

Results of degradation rate of each hemostatic material by lysozyme DAC30, which is the hemostatic material of the present invention, was more degradable than FM and FH (FIG. 6). Thereby, it was confirmed that the hemostatic material of the present invention is more biodegradable than the conventional hemostatic material.

The hemostatic material of the present invention Scanning electron microscope Scanning electron micrograph of medium viscosity chitosan FM Blood absorption test results of hemostatic material Application of hemostatic material to wound site Completion of hemostasis at the wound site Collected hemostatic material Results of biodegradation of the hemostatic material of the present invention

Claims (9)

Mainly gluconate or aspartate of amorphous partially deacetylated chitin with a degree of deacetylation of 20-65% and viscosity at 20 ° C in 0.5% W / W solution of 20mPa · s to 200mPa · s Sponge hemostatic material as an ingredient. The amount of gluconic acid or aspartic acid added during production is 0.2 to 1.00 mol per mol of the amino group of amorphous partially deacetylated chitin, and the frozen ice is crushed and filled into a mold. The hemostatic material according to claim 1, which is prepared by freezing again . Residual acid concentration of the final preparation is 0-7.0% W / W acetic acid, 2-40% W / W gluconic acid or aspartic acid, and the total amount is 1 mol of amino group of amorphous partially deacetylated chitin. The hemostatic material according to claim 1 or 2, wherein the acid ion is 0.2 to 1.00 mol. The hemostatic material according to any one of claims 1 to ³ , wherein the final preparation has a weight per volume of 0.005 to 0.035 g / cm ³ . The hemostatic material according to any one of claims 1 to 4, wherein the final preparation has a pore size of 50 to 400 µm and a substantially homogeneous structure. The preparation of amorphous partially deacetylated chitin is carried out at a treatment temperature of 35 to 60 degrees and a treatment time of 2 to 5 hours when caustic soda of about 40% W / W is added. The hemostatic material according to any one of 5. After suspending amorphous partially deacetylated chitin having a degree of deacetylation of 20 to 65%, gluconic acid or aspartic acid is added to 0.2 mol per 1 mol of amino group of amorphous partially deacetylated chitin. Add to ~ 1.0 mol, stir, and then freeze the filtrate of the solution after stirring, crush and form into a sherbet, fill it into a product tray, and prepare it by freeze vacuum drying and further secondary drying A method for producing a sponge-like hemostatic material mainly comprising a crystalline partially deacetylated chitin salt. The method according to claim 7, wherein amorphous partially deacetylated chitin is dissolved by addition of acetic acid before addition of gluconic acid or aspartic acid. The method according to claim 8, wherein the secondary drying and / or heat treatment is performed at 65 to 85 degrees for 1 to 7 days.