JPH05163157A - Fixed alginic acid-thrombin preparation, its production and hemostatic agent - Google Patents

Abstract

PURPOSE:To obtain a fixed alginic acid-thrombin preparation containing alginic acid and thrombin bonded with each other through an amide bond and having a thrombin activity comparable to that of thrombin before reaction. CONSTITUTION:Alginic acid is made to react with an active esterifying agent in the presence of a water-soluble carbodiimide to obtain an active ester of alginic acid. The objective fixed alginic acid-thrombin preparation is produced by reacting the active ester with thrombin. The product has a thrombin activity corresponding to about >=80% of the activity before reaction and is useful as an organic component for a hemostatic agent or a hemostatic medical assisting material. The hemostatic agent is applicable to the diseased part or by oral administration. The fixed preparation can be used as a hemostatic medical assisting material by physically adsorbing the compound to a cotton, paper, nonwoven fabric, film, sponge, etc. It is resistant to the decomposition by gastric acid to prolong the retention time in the body and, accordingly the action period is prolonged. The preparation has excellent stability and storability.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an alginic acid / thrombin-immobilized product having a high thrombin activity, a method for producing the same, a hemostatic agent and a medical auxiliary material based thereon.

[0002]

2. Description of the Related Art Thrombin preparations have long been known as hemostatic agents. A material for treating a wound, which is obtained by immobilizing thrombin on a structure such as a monofilament, a fiber assembly, a film, or a sponge, is known as a material for application to a wound on a wound or a surgical wound surface (Japanese Patent Publication No. 61-61). -59737). An immobilized enzyme in which an enzyme other than thrombin is immobilized by an entrapping method using alginic acid as a carrier for immobilization is also known (Japanese Patent Publication No. 59-28).
475 and JP-A-59-109177). Furthermore, immobilized thrombin having Sepharose as a carrier is known [Haemostasis, Vol. 6, 225-2.
35 (1977) and Thrombosis and Hemostasis (Thromb. Haemostas.) Vol. 1, 27-31.
Page (1984)].

Thrombin has recently been used orally for gastrointestinal bleeding. However, thrombin has an optimal pH of 7.
It is an enzyme having 5-7.8 and has the property of being extremely unstable and deactivating in strong acids and strong alkalis. Therefore, in order to expect more appropriate effect expression in the stomach, which is prone to strong acidity, physical removal of gastric juice, suppression of gastric acid secretion by H ₂ receptor antagonist, combination with antacid, etc. Appropriate measures are necessary. In addition, this topical thrombin is often administered in addition to conventional mucosal protective agents, etc.
Since it is inactivated by (for example, pH 5 or less), the pH in the stomach is always
The troublesome operation of checking was required. Furthermore, since the thrombin clotting rate is concentration-dependent, it was necessary to administer a large amount of thrombin, although it was relatively expensive. These have limited topical intragastric application of thrombin.

On the other hand, alginic acid and its salts are the main constituents of the cell membrane of brown algae, have a high molecular weight, and become highly viscous when made into an aqueous solution. This substance is highly safe for the human body, and by itself strongly adhering to the wound surface, it exerts a physical hemostatic action, and is also said to crosslink to erythrocytes to promote the hemostatic action. Therefore, it can be used for the purpose of hemostasis, but its action is mild and is not suitable for emergency hemostasis.

The inventor of the present invention has previously attempted to produce an immobilization product by making the best use of the characteristics of both by directly making an amide bond between thrombin and alginic acid by the carbodiimide method.
(JP-A-3-201982). However, this immobilized product has a low reaction rate when verified thereafter, and has a thrombin activity per protein weight of about 0.35 unit / μg,
I was not completely satisfied with the low point.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have aimed to produce thrombin-immobilized products with higher activity,
As a result of various studies, when an active ester of alginic acid was first produced in the presence of carbodiimide and then an active ester of alginic acid was reacted with thrombin, a two-step reaction was taken It was found that the reaction proceeds with higher efficiency than the one-step direct condensation, and a thrombin-immobilized product with high activity can be obtained.

That is, the present invention provides (1) alginic acid / thrombin, in which an immobilized product in which alginic acid or a salt thereof and thrombin are bound by an amide bond between them has a thrombin activity almost equal to that of thrombin before the reaction. Reacting alginic acid with an active esterifying agent in the presence of an immobilized product, (2) water-soluble carbodiimide or a salt thereof,
A method for producing an alginic acid-thrombin-immobilized product, which comprises reacting the obtained alginic acid active ester with thrombin, and (3) a method for producing an alginic acid-thrombin-immobilized product, which comprises reacting alginic acid active ester with thrombin. Method (4) The alginic acid according to (1) above.
A hemostatic agent containing a thrombin-immobilized substance as an active ingredient, and
(5) A medical auxiliary material for hemostasis, which comprises the alginic acid-thrombin-immobilized product according to (1) above.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The alginic acid-thrombin-immobilized product of the present invention has a thrombin activity almost equal to that of the thrombin before the reaction as shown in the above (1). It means an activity of 80% or more, preferably about 85% or more. Such an alginic acid-thrombin immobilized product is obtained by reacting alginic acid or a salt thereof with an active esterifying agent in the presence of a water-soluble carbodiimide to give an alginic acid active ester, and reacting thrombin with this, as shown in (2) above. It is obtained by

The alginic acid used in the present invention is (C ₅ H ₇
O ₄ COOH) n is a substance containing a polysaccharide represented by the general formula (polymer of D-mannuronic acid and L-guluronic acid) as a main component, and usually has a molecular weight of about 5 to 200,000, for example, obtained from brown algae. And suitable for use are commercially available. Examples of the salt of alginic acid include alkaline metal salts such as sodium salt and potassium salt, and nitrogen compound salts such as ammonium salt and amine salt. Thrombin is a blood coagulation protein having a proteolytic enzyme activity and a molecular weight of about 38,000.
Suitable products made by extraction from plasma (human, bovine, etc.) are commercially available and can also be synthesized by biotechnology.

Water-soluble carbodiimides include water-soluble carbodiimides (compounds having -N = C = N- bond)
Although any of these can be used, those represented by the following general formula or salts thereof are preferable. X-N = C = NY (I) [wherein, X is a hydrogen atom, or a monovalent chain or cyclic hydrocarbon group optionally substituted with lower alkoxyl or phenylazo, and Y is defined for X. Or a group represented by the formula -R ₁ -R ₂ (wherein R ₁ represents a divalent chain or cyclic hydrocarbon group, and R ₂ represents a basic amino group). However, when both X and Y mean a hydrocarbon group having no amino group, each hydrocarbon group contains 8 or less (preferably 6 or less) carbon atoms.] The monovalent chain formula Examples of the hydrocarbon group include a group (lower alkyl group) having 8 or less (preferably 6 or less) carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl and nonyl, decyl, Larger groups such as undecyl, dodecyl, etc. are included and unsaturated bonds may be present other than methyl. The monovalent cyclic hydrocarbon group includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl, ethylcyclohexyl and the like groups having 8 or less (preferably 6 or less) carbon atoms (lower cycloalkyl group), propyl Larger groups such as cyclohexyl, cyclohexylcyclohexyl, etc., their unsaturated forms (including phenyl) and groups formed by combining them with a lower alkylene group are included. The divalent chain hydrocarbon group includes a group obtained by removing one hydrogen from the above monovalent chain hydrocarbon group, and examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene and hexamethylene. , 2-methylethylene, 2-ethyltetramethylene and the like. The divalent cyclic hydrocarbon group includes a group obtained by removing one hydrogen from the monovalent cyclic hydrocarbon group, and examples thereof include cyclopropanediyl, cyclohexanediyl, methylcyclohexanediyl and the like. .. A lower alkoxyl group has the formula RO-
(Wherein R is a lower alkyl group). As the basic amino group, an amino group, a lower alkylamino group (for example, methylamino, ethylamino, propylamino, etc.), a di-lower alkylamino group (for example, dimethylamino, diethylamino, methylethylamino, etc.),
Lower alkyleneamino group (for example, aziridinyl, azetidinyl, pyrrolidinyl, piperidino, morpholino, 4-
Methylpiperazinyl etc.) and their unsaturated forms. Phenyl of phenylazo may be substituted with a group which is common as a benzene ring substituent, such as lower alkyl, lower alkoxyl and halogen. Examples of the salt include inorganic acid salts such as hydrogen halide salts, halogen acid salts and sulfates, and organic acid salts such as methane sulfonate and toluene sulfonate. A typical group of water-soluble carbodiimides is N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide and its hydrochloride, N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide and its toluenesulfonate. , N-benzyl-N'-
(3-Dimethylaminopropyl) carbodiimide toluenesulfonate, N-cyclohexyl-N '-[2-
(4-Methylmorpholino) ethyl] carbodiimide toluenesulfonate, N-methyl-N ′-(3-morpholinopropyl) carbodiimide, 3-[(ethylcarboximidoyl) amino-N, N, N-trimethyl-1- Propanaminium iodide, 3-[(ethylcarbonimidoyl) amino-N, N, N-trimethyl-1-propaneaminium perchlorate, N, N, N-trimethyl-
3-[(phenylcarbonimidoyl) amino] -1-
Propanaminium perchlorate, N, N, N-trimethyl-3-[(phenylcarbonimidoyl) amino]
-1-propaneaminium iodide, N-ethyl-
N '-(3-morpholinopropyl) carbodiimide, N,
N, N-trimethyl-3-[{(4- (phenylazo) phenyl) carboximidoyl} amino] -1-propaneaminium iodide and the like. Another group is cyanamide (carbodiimide), diisopropylcarbodiimide, bis [di (methoxymethyl)] carbodiimide, N
-2- [tri (methoxymethyl)] ethyl-N '-[di (methoxymethyl) methyl] carbodiimide, N-2-
[Tri (methoxymethyl)] ethyl-N '-[tri (methoxymethyl) methyl] carbodiimide and N-di (methoxymethyl) methyl-N'-[tri (methoxymethyl)] carbodiimide.

As active esterifying agents, those used in peptide synthetic chemistry (for example, "Basic Experiments of Peptide Synthesis" (Maruzen) pages 91-100 and "Development of Pharmaceuticals" (Hirokawa Shoten) Vol. 14, No. 162. Any of (-173 pages) can be used, but those represented by the following general formula are preferable. H-O-Z (II) [In the formula, Z means a secondary or tertiary amino group, a methyl group substituted with an electron-withdrawing group, or a phenyl group or a naphthyl group optionally substituted with an electron-withdrawing group. Examples of the secondary or tertiary amino group include a succinimide group and its homologues, substituted products, di-lower alkylamino groups and ring-closed products in the di-lower alkyl portion, methyleneamino groups and their substituted products, acylamino groups, triazoles or Included are triazin-N-yl groups and benzene ring condensates thereof. Although there are various specific examples, they are illustrated on page 168 of the above-mentioned "Development of Pharmaceuticals" Vol. 14, and they can be used in the same manner. Examples of the electron withdrawing group include a halo lower alkyl group (eg trifluoromethyl, trichloromethyl, pentachloroethyl etc.), a nitro group, a cyano group, a lower alkanoyl group (eg formyl, acetyl etc.) and a lower alkoxycarbonyl group (eg methoxycarbonyl). , Ethoxycarbonyl etc.), carboxyl group, N-lower alkylcarbamoyl group, lower alkanoyloxy group, phenyl lower alkanoyloxy group, lower alkylsulfonyl group (eg methylsulfonyl etc.), sulfamoyl group, N-lower alkanoylsulfamoyl group, N
-Phenyl or heteroaromatic sulfamoyl group, sulfo group, halogen, nitrovinyl group, phenylazo group and the like are included. It should be noted that the above compound does not prevent the coexistence of a group having no electron-withdrawing property. A typical active esterifying agent is N
-Hydroxysuccinimide, 1-hydroxybenzotriazole (hydrate), N-hydroxypiperidine, N
-Hydroxyphthalimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxymaleimide and the like. ..

An example of a preferred method of implementing the present invention is as follows. To an aqueous solution of alginic acid or a salt thereof, an aqueous solution of a water-soluble carbodiimide and an active esterifying agent is added respectively under acidic conditions (pH of about 5), and at a moderate temperature (eg 0-50 ° C.) for an appropriate time (eg 2 ° C.).
Stir for 0-120 minutes) to form an aqueous solution of alginic acid active ester. Unreacted water-soluble carbodiimide and active esterifying agent are removed from the aqueous solution by a separation means (for example, gel filtration or dialysis). Next, an aqueous solution of thrombin is added to this aqueous solution while maintaining the temperature at a relatively low temperature (for example, 10 ° C. or lower, preferably about 4 ° C.),
Stir the time required for the reaction to be almost completed (for example, 4 to 24 hours). Then, the alginic acid / thrombin-immobilized product is separated and purified by a commonly used separation and purification method (eg, gel filtration, dialysis, etc.). The alginic acid / thrombin immobilized product thus obtained is made into a concentrated liquid or a dried product by a further commonly used method (for example, lyophilization). The immobilized product in the form of a concentrated solution can be used as a liquid agent, and the lyophilized product can be used as a solid preparation or a dissolved preparation at the time of use. In the above reaction, the ratio of thrombin to alginic acid was such that the weight of alginic acid corresponding to 600 units of constituent monosaccharides (600 carboxyl groups) in alginic acid was about 3800 molecular weight.
0) 0.01-100 moles of molecule are suitable and 0.1-2
0 mol is preferred. Further, the water-soluble carbodiimide and the active esterifying agent are appropriately added in an amount of 1 to 500 mol, preferably 50 to 300 mol, based on the weight of alginic acid.

The above reaction can be represented by the following equation. (AL) -COOH + HO- (AE ) + WSC → (AL) -COO- (AE) + urea derivative _{(AL) -COO- (AE) +} H 2 N- (TH) → (AL) -CONH- (TH) + HO -(AE) [In the formula, (AL) is a group in which COOH involved in the reaction is removed from the alginate sugar chain, (AE) is a residue in which OH is removed from the active esterifying agent, and (TH) is a reaction from thrombin to the reaction. Residues except NH ₂ involved, WSC is a water-soluble carbodiimide]

According to the method of the present invention as described above, the immobilization rate is high because the reaction rate of thrombin with respect to alginic acid is high. Moreover, the specific activity of the obtained alginic acid-thrombin-immobilized product is high, and the specific activity is close to that of thrombin before the reaction.

The hemostatic agent containing the immobilized product of the present invention as an active ingredient can be administered locally or orally.
For topical administration, the dried immobilizate is dissolved in physiological saline (20-100 units / ml as thrombin) and sprayed or irrigated or sprayed as a powder or applied in the form of an ointment. In the case of upper gastrointestinal bleeding, a solution dissolved in an appropriate buffer solution (1
(00-400 units / ml) is orally administered. Dosage forms used for oral and / or topical administration include powders,
Granules, microcapsules, tablets, pills, capsules, solutions, suspensions, emulsions, ointments and the like are included. In preparing the formulation, a buffer, a tonicity agent, an excipient, a stabilizer,
Preservatives, flavoring agents, coloring agents can be added. Further, the immobilized product of the present invention can be physically adsorbed on cotton, paper, non-woven fabric, film, sponge and the like to be used as a medical aid for hemostasis. The immobilization product according to the present invention has many advantages as described below. That is, it has a high thrombin activity, is resistant to decomposition by gastric acid, and has a long residence time in the body, resulting in a prolonged action time, and further improves the stability and storage stability of the preparation.

Next, the present invention will be specifically described based on examples, but these do not limit the invention. (Example 1) Dry sodium alginate (manufactured by Kyosei Pharmaceutical)
1 ml of N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (2.54 × 10 ^-5 ) in 2 ml of a 2 W / W% sodium alginate aqueous solution (36 mg as alginic acid) of pH 5 prepared from
Mol) and an N-hydroxysuccinimide (HOSu) 7.5 mg (6.54 × 10 ⁻⁵ mol) aqueous solution (1 ml) were added to make the whole 4 ml, and the mixture was stirred at room temperature for 40 minutes. 2m of this solution
l was subjected to gel filtration (Biorad desalting column, Econopack 10DG) to remove low molecular weight components, and then 4 ml of a fraction corresponding to alginic acid-activated ester.
About 2 ml was taken and adjusted to pH 7, and thrombin (Mochida Pharmaceutical Co., Ltd.) 3.1 mg (8.17 × 10 ⁻⁸ mol) was added.
0.23 ml of an aqueous solution containing was added and stirred at 4 ° C. for 18 hours. The reaction solution was again subjected to gel filtration under the above conditions to remove the active esterifying agent and other low molecular weight components.
This solution was subjected to SDS polyacrylamide gel electrophoresis. The results are shown in Figure 1. In FIG. 1, the leftmost lane is a marker showing a standard molecular weight, the 2nd to 5th lanes from the left are thrombin before reaction at each concentration, and the rightmost lane is a reaction product. Almost all of the reaction products have a high molecular weight of over 200,000.
It was confirmed that a thrombin-immobilized product was produced. This electrophoretic gel was applied to a densitometer (Shimazu Seisakusho Spot Scanner-CS-9000) to measure the amount of unreacted thrombin, and the reaction rate of thrombin was calculated. The results are shown in Table 1. In addition, the activity of this solution against a synthetic substrate and the amount of protein were measured, and the activity against a synthetic substrate per 1 μg of thrombin was calculated, which was taken as the specific activity. The results are shown in Table 1.

(Example 2) 2 w / w% adjusted to pH 5
Alginic acid [(AL) -COOH] 2 g, EDC
6.3 mg (3.27 × 10 ⁻⁵ mol) / 1 ml, N
-Hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) 5.9 mg (same as above) / 1 ml,
And thrombin [H ₂ N- (TH)] 31.0 mg
The same procedure as in Example 1 was performed using (8.17 × 10 ⁻⁷ mol) /1.2 ml. The results are shown in Table 1. (Example 3) 3 w / w% alginic acid [(AL) -COOH] 2 g adjusted to pH 6 and EDC 4.8 mg
(2.53 × 10 ⁻⁵ mol) / 1 ml, 1-hydroxybenzotriazole (HOBt) 3.9 mg (same as above) /
1 ml, and thrombin [H ₂ N- (TH)] 18
The same procedure as in Example 1 was conducted using 2 ml of 6.3 mg (4.90 × 10 ⁻⁶ mol). The results are shown in Table 1. Example 4 2 w / w% alginic acid [(AL) -COOH] 2 g adjusted to pH 5, N-cyclohexyl-
N '-(2-morpholinoethyl) carbodiimide (CD
I) 57.0 mg (1.35 × 10 ⁻⁴ mol) / 1 m
l, carried out in the same manner as HOSu 15.5 mg (ibid) / 1 ml Example 1 using and _[H 2 N- (TH)] 6.4mg (1.68 × 10 ^-7 mol) /0.25ml, It was The results are shown in Table 1. (Example 5) 2 w / w% alginic acid [(AL) -COOH] 2 g adjusted to pH 5 and CDI 42.8 mg
(1.01 × 10 ⁻⁴ mol) / 1 ml, HONB 1
8.1 mg (same as above) / 1 ml, and thrombin [H ₂
N- (TH)] 3.1 mg (8.17 × 10 ^-8 mol)
The same procedure as in Example 1 was performed using /0.23 ml. The results are shown in Table 1. (Comparative Example 1) The same procedure as in Example 1 was performed except that HOSu was omitted. The results are shown in Table 1.

[Table 1]

Activity measurement method The test solution was diluted 250 times, 100 μl of the solution was collected in a plastic test tube and heated at 37 ° C. for about 5 minutes. Chromogenic substrate solution (mold team test team S-223
(8, 25 mg dissolved in 40 ml of purified water) 250
Add μl and mix and warm at 37 ° C. for exactly 5 minutes. Stop solution (citric acid 2w / v% aqueous solution) 1.0m
Add 1 and mix. Use a spectrophotometer to compare the sample blank with a wavelength of 405.
Measure the absorbance at nm. The absorbance is measured by performing the same operation for the known unit of thrombin to obtain a calibration curve. Protein measurement method The test solution is diluted 250 times, and 200 μl of Biorad's protein assay staining solution is added to 800 μl thereof and mixed. Use a spectrophotometer to compare the sample blank to a wavelength of 595
Measure the absorbance in mm. A thrombin having a known protein amount is similarly operated to measure the absorbance, and the obtained curve is used as a calibration curve.

Example 6 The amounts of the aqueous sodium alginate solution, N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and thrombin in Example 1 were each scaled 10 times. An aqueous solution of immobilized alginic acid / thrombin was obtained in the same manner as in Example 1. This solution was freeze-dried to obtain a freeze-dried product containing 96.8 mg of an alginic acid-thrombin-immobilized product.

(Example 7) The solution obtained in Example 6 was desalted and then freeze-dried to obtain a solid immobilized product. This was formulated as follows to obtain a powder. Parts by weight-immobilized product 70.0-lactose 29.9-medical essence No. 52523 0.1 (manufactured by Sanei Chemical Co., Ltd.) 100.0

(Example 8) 2.0 ml of the desalted and immobilized product solution obtained in Example 7 was sampled and diluted to 50 ml. Add 15 g of surgical gauze (15 cm x 15 cm) to this solution.
The gauze to which the immobilized product was adsorbed was obtained by immersing at 30 ° C. for 30 minutes and then vacuum drying (25 ° C. or less).

[Brief description of drawings]

1 is a diagram showing the results of SDS polyacrylamide gel electrophoresis performed in Example 1. FIG.

Claims (5)

[Claims] 1. An immobilization product of alginic acid or a salt thereof and thrombin, wherein thrombin activity is almost the same as that of thrombin before the reaction, in the immobilization product in which thrombin is bound by an amide bond between them. 2. A process for producing an immobilized alginic acid / thrombin, which comprises reacting alginic acid with an active esterifying agent in the presence of a water-soluble carbodiimide, and reacting the obtained alginic acid active ester with thrombin. 3. A process for producing an alginic acid-thrombin-immobilized product, which comprises reacting an alginic acid active ester with thrombin. 4. A hemostatic agent comprising the alginic acid-thrombin immobilized product according to claim 1 as an active ingredient. 5. A medical auxiliary material for hemostasis, which comprises the alginic acid-thrombin-immobilized product according to claim 1.