JPH0820440B2 - Blood coagulation promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to a blood coagulation promoter, and more particularly to a blood coagulation promoter that promotes the coagulation of a blood sample obtained from a patient receiving heparin.

(Conventional technology) Blood tests such as serum biochemical tests, serum immunological tests, and blood cell tests have become widespread in combination with the remarkable progress in testing techniques, and are useful for disease prevention and early diagnosis. Most blood tests are serum tests, and the serum required for the test is usually blood clots (fibrin and blood cell mixed gel) with different specific gravities, which are obtained by centrifuging after coagulating the blood collected in blood test containers. Lumps) and collected with a pipette or by decantation.

It takes a relatively long time for the blood collected from the subject to become firm. For example, it takes 40 to 60 minutes for blood to coagulate even if a glass test container, which is said to have a relatively short blood coagulation time, is used. Is required. Therefore, it has a drawback that the serum required for the test cannot be quickly obtained. This is a problem, especially when urgent testing is required.

As described above, according to the conventional serum fractionation method, in addition to the problem that it takes a long time for blood coagulation, when the coagulated whole blood is separated by centrifugation, it is difficult to separate the serum and the blood clot well. There is also. If the separation is poor, erythrocyte contamination is unavoidable, even with the utmost care when pipetting the serum portion and / or decanting. As a result, the clinical laboratory test results are adversely affected and it is necessary to perform centrifugation again.

Another problem arises when dealing with blood samples from patients undergoing hemodialysis and thrombosis. Such patients are given heparin to prevent thrombosis, so 1 to 20 units of heparin are present per 10 ml of blood.
This heparin binds to antithrombin III in blood and significantly inhibits the action of thrombin. In addition,
It is also said to inhibit the action of blood coagulation factors such as factor XII. Therefore, the conversion of fibrinogen to fibrin does not occur and, as a result, blood does not clot. Therefore, it becomes difficult to collect serum.

In order to solve these problems, the inventor has proposed that (a) a cyclic organic compound represented by the following general formula (I), in which adjacent carbonyl groups are present in substantially the same plane. A blood coagulation promoter containing (b) an amine salt and / or an organic compound having a quaternary nitrogen has been proposed (JP-A-60-27858).

(Where A is the residue of the cyclic compound).

Examples of the compound represented by the formula (I) include alkyl gallate oxide and ellagic acid oxide. As the amine salt and / or the organic compound having quaternary nitrogen, alkylamine hydrochloride or the like is used. When a blood coagulation promoter containing these compounds is used, the above-mentioned amine salts contained therein adsorb heparin.
The compound of formula (I) is neutralized and inactivated, and the compound of the formula (I) activates blood coagulation factor XII in the blood to coagulate the blood in a short time. However, after the time of blood coagulation, due to the action of degrading enzymes existing in plasma,
The clot of fibrin begins to dissolve. Therefore, there is a problem regarding stability over time after solidification.

(Problems to be Solved by the Invention) The inventor has further studied the above-mentioned blood coagulation promoter, and can rapidly coagulate not only heparin-free blood but also heparin-containing blood, and stable coagulation after coagulation. We tried to develop a highly effective blood coagulation promoter. An object of the present invention is to provide a blood coagulation promoter that can rapidly coagulate blood regardless of whether heparin is contained, has good serum separability, and has good stability after coagulation.

(Means and Actions for Solving Problems) The blood coagulation promoter of the present invention comprises (a) a compound represented by the following general formula, in which adjacent carbonyl groups are present on substantially the same plane. Cyclic organic compound (I), (b) amine salt and / or organic compound having quaternary nitrogen,
It contains (c) an antifibrinolytic agent and / or an antiplasmin agent, and (d) a hydrolase, whereby the above objects are achieved: (Where A is the residue of the cyclic compound).

A cyclic organic compound represented by the above formula (I), which is the main component of the blood coagulation promoter of the present invention (hereinafter referred to as a cyclic compound)
May be a homocyclic compound or a heterocyclic compound, and may be a monocyclic compound or a polycyclic compound. As described above, each of these cyclic compounds has two arbonyl groups located in substantially the same plane in the molecule. Although the detailed mechanism is unknown, these compounds specifically act on and activate blood coagulation factors. As such a cyclic compound, the ring containing two carbonyl carbons is preferably a 6-membered ring or a 5-membered ring.

Among the homocyclic compounds, preferred 6-membered cyclic compounds include compounds having an o-quinone ring represented by the following general formula (II): (Here, R ₁ , R ₂ , R ₃ and R ₄ represent hydrogen, a hydrocarbon group, a polar substituent or a residue in a polycyclic compound).

In the above formula, the hydrocarbon group is not particularly limited, but an alkyl group, particularly an alkyl group having 1 to 18 carbon atoms is preferable.
The polar substituent is also not particularly limited. Examples include a carboxyl group, a carboxylic ester group, a hydroxyl group, an amino group, and a mercapto group. Examples of the compound having an o-quinone ring include o-quinone and compounds represented by the following formulas (III) to (VII): Alkyl gallate ester oxide (Here, R ₅ represents an alkyl group.) Ellagic acid partial oxide Ellagic acid complete oxide 1.4-di (3.4-dihydroxyphenyl) 2.3
-Dimethylbutane partial oxide 1.4-di (3.4-dihydroxyphenyl) 2.3
-Complete dimethylbutane oxide Among the homocyclic compounds, preferable specific examples of the 5-membered cyclic compound include 1,2,3-triketohydroindene represented by the following formula (VIII).

Examples of the heterocyclic compound include the following general formula (IX)
The compound shown by is mentioned.

(Here, R ₆ represents hydrogen, carbon-hydrogen, or a residue in a polycyclic compound, and R ₇ and R ₈ represent hydrogen, a hydrocarbon group, a polar substituent, or a residue in a polycyclic compound. The group and the polar substituent are the same as those in formula (II).) Preferred specific examples of the compound represented by formula (IX) are:
For example, there is isatin represented by the following formula.

The amine salt and / or the organic compound having a quaternary nitrogen contained in the blood coagulation promoter of the present invention acts as a heparin neutralizing agent that adsorbs and neutralizes heparin to inactivate it. The amine that constitutes the amine salt may be any of primary, secondary and tertiary amines, and the acid that constitutes the amine salt may be either an inorganic acid or an organic acid. The inorganic acid includes hydrohalic acid such as hydrochloric acid, sulfuric acid, and sulfurous acid, and the organic acid includes formic acid and acetic acid. The organic residue of the amine salt is usually an alkyl group, but may be a hydrocarbon group containing a different element such as an imino group or an ether group. The amine salt may be an inner salt.

Specific examples of preferable amine salts include (XI)
Hexadecyldimethylamine hydrochloride represented by the formula,
Tetradecyldi (aminoethyl) represented by formula (XII)
There is glycine.

An organic compound having a quaternary nitrogen is, for example, tetraalkylammonium. It may be a compound having an aryl group instead of an alkyl group or a compound having a hydrocarbon group containing a different element such as an imino group or an ether group. A preferred specific example is dodecyltrimethylammonium chloride represented by the formula (XIII).

Besides such a relatively low molecular weight compound, an organic polymer having a quaternary nitrogen can also be used. Examples of such a polymer include a polycation having a repeating unit represented by the general formula (XIV).

(Wherein R _{9 to} R ₁₂ are hydrogen or an alkyl group, X is a halogen group or an acid group, Y is an alkylene group or an -alkylene group-
SO ₂ − is shown, and the repeating number of the above units is 5 to 2000. Among the compounds represented by (XIV), a polycation having a repeating unit represented by (XV) or (XVI) is particularly preferable.

The amine salt and / or the compound having a quaternary nitrogen (neutralizing agent) is 5-10, relative to 100 parts by weight of the cyclic compound.
It is contained in a proportion of 000 parts by weight. If it is too small, heparin will not be neutralized and blood will not clot. Even if it is excessive, the effect depending on the content cannot be obtained.

As antifibrinolytic agents and / or antiplasmin agents, aprotinin, soybean trypsin inhibitor, ε-aminocaproic acid, p-aminomethylbenzoic acid, aminomethylcyclohexanecarboxylic acid, etc., which have been conventionally used clinically, are used alone or in combination. Used. These are contained in the blood coagulation promoter in an amount that does not affect the clinical test using the obtained serum. For example,
Aprotinin is about 100 to 600 KIU (unit) per ml of blood, soybean trypsin inhibitor is about 500 to 4000 FU (unit) per ml of blood, and ε-aminocaproic acid, p-aminomethylbenzoic acid and aminomethyl are used. Cyclohexanecarboxylic acid is contained in the blood coagulation promoter at a rate of about 10 ^-2 to 10 ^-8 g / ml of blood.

The hydrolase contained in the blood coagulation promoter of the present invention is a protease and is an enzyme capable of hydrolyzing the bond between Arg and any amino acid residue and the bond between Lys and any amino acid residue in the peptide chain. is there. Examples of such protease include trypsin, thrombin,
There are serine proteases such as snake venom thrombin-like enzyme; thiol proteases such as cathepsin B and ficin; and metalloproteases such as kininase I. Particularly, serine protease is preferably used. These proteases alone have a blood coagulation promoting action, but when used in combination with the above cyclic compound, the blood coagulation activating ability is dramatically improved.

The hydrolase is contained in the blood coagulation promoter at 10 ^-2 to 10 ² IU / ml of blood. Even if the amount of the enzyme is too small, the effect of promoting blood coagulation can be obtained if the cyclic compound is contained, but the effect is far smaller than that when the enzyme is blended in the above proportion. Even if it is excessive, the effect proportional to the content cannot be obtained.

The blood coagulation promoter of the present invention is 1 × 10 ⁻¹⁰ to 1 ml of blood.
Used at a rate of 1 x 10 ^-1 g. If it is too small, the blood coagulation promoting effect cannot be obtained. Even if it is excessive, the effect depending on the amount used may not be obtained, and it may affect blood tests in various ways.

The blood test container used when using the blood coagulation promoter of the present invention may be made of glass or resin. To coagulate blood, for example, a blood coagulation promoting agent may be added to the blood collected in the container, or the blood coagulation promoting agent may be previously provided inside the container. The blood coagulation promoter may be used, for example, in the form of powder, or may be dissolved or dispersed in an appropriate solvent in advance. The blood coagulation promoter can be supported on a carrier having a large specific surface area in order to prevent the high concentration of the blood coagulation promoter from contacting blood and denaturing the protein component.

The carrier is not particularly limited as long as it does not adversely affect the blood test and has a large specific surface area. For example, non-woven fabric, woven fabric, resin beads and the like are preferably used. In order to make the carrier support such a blood coagulation promoter, for example, a solution or dispersion thereof is applied to the carrier, or the carrier is dipped in the solution or dispersion to impregnate the carrier and then dried. It is also possible to prepare an aqueous dispersion of a blood coagulation promoter containing an appropriate auxiliary agent such as gum arabic and rapidly freeze-dry this to obtain a blood coagulation promoter-supporting particulate material.

When the blood coagulation promoter of the present invention is added to blood containing heparin, heparin in the blood is adsorbed and neutralized by a neutralizing agent such as an amine salt and precipitates, so that heparin inhibits thrombin and factor XII. Disappears. Therefore, blood restores normal coagulation function. The cyclic compound contained in the blood coagulation promoter has the ability to act on and activate Factor VII in blood. Blood coagulation proceeds in a chain reaction in a short time by activation of factor XII, and finally thrombin produced by activation of prothrombin acts on fibrinogen to form an insoluble fibrin network and blood. Coagulation is complete. Since the blood coagulation promoter further contains a protein hydrolase such as serine protease, the activation of the blood coagulation factor is promoted. It is also considered that the cyclic compound contained in the blood coagulation promoter promotes the enzymatic reaction of this protein hydrolase. As a result, blood coagulates in a short time. The time required for blood coagulation depends on the types of cyclic compounds and neutralizing agents in the blood coagulation promoter, the amount of blood coagulation promoter, the material of the container used,
Although it depends on the amount of heparin in the blood, it is usually 10 to 20 minutes using a synthetic resin container. Since the anticoagulant and / or the antiplasmin agent are further contained in the blood coagulation promoter, the fibrinolytic action of plasmin, which is antagonistically produced in the blood coagulation reaction process, is inhibited. Therefore, blood coagulation is promoted, and the coagulation state can be kept stable even after coagulation. Thus, not only normal blood but also blood containing heparin can be coagulated within a short time, and the coagulation state can be kept stable. Furthermore, since the serum and the blood clot are easily separated, the problem that the blood clot component is mixed in the separated and collected serum is solved.
Since the degree of contraction of the blood clot component is sufficient, the serum yield is high.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 A polyester-based nonwoven fabric is impregnated with an aqueous dispersion containing elasic acid oxide (compound represented by formula (V)), polycation (compound represented by formula (XVI)), aprotinin and trypsin, and dried sufficiently. Let The amount of each of the above components per 1 cm ^{2 of} non-woven fabric is 4 × 10 ^-4 g, 4 × 10 ^-4 g, 500, respectively.
It was KIU and 3IU.

1.0 IU of heparin in a commercially available 5 ml polyethylene spitz
2 ml of human fresh blood containing at a concentration of / ml was infused, then 1 cm ² of the non-woven fabric carrying the above components was added, and after gently stirring, 2
It was left at 0 ° C. The sera after 1 hour and 30 hours after leaving were respectively collected, and the fibrinogen and fibrin degradation products (hereinafter abbreviated as FDP) were measured. The results are shown in the table below. There is no difference in the FDP measurement values after 1 hour and 30 hours, indicating that the clot decomposition reaction is well suppressed. The results of Examples 2 to 7 and Comparative Examples 1 and 2 are also shown in the table below.

Example 2 N-Propyl gallate oxide, tetradecyldi (aminoethyl) glycine, aprotinin and trypsin were added at 0.5% by weight, 0.5% by weight, 10000 KIU / ml and 40 IU / ml, respectively.
A physiological saline solution dispersion containing a concentration of ml was prepared.

1.0 IU of heparin in a commercially available 5 ml polyethylene spitz
2 ml of human fresh blood containing at a concentration of / ml was injected, 50 μl of the above dispersion was added, and then the same treatment as in Example 1 was carried out for evaluation.

Example 3 Isatin 1 g, hexadecyldimethylamine hydrochloride 0.4 g,
A small amount of ethanol was added as a dispersion aid to 50 mg of aminomethylcyclohexanecarboxylic acid and 1 kg of polystyrene bead carrier having an average particle size of 1.5 mm, mixed well, and then dried. Separately, 7,000 IU of trypsin was dissolved in a small amount of physiological saline, which was further coated on the surface of the beads after the above treatment and dried.

1.0 IU of heparin in a commercially available 5 ml polyethylene spitz
2 ml of human fresh blood contained at a concentration of / ml was injected, and then 1 g of the above blood coagulation promoter (beads) was added, and then treated and evaluated in the same manner as in Example 1.

Example 4 Instead of n-propyl gallate oxide, tetradecyldi (aminoethyl) glycine and aprotinin, o-
Same as Example 2 except that quinone, polycation (compound represented by formula (XV)) and ε-aminocaproic acid were used in proportions of 0.5% by weight, 0.5% by weight and 0.1% by weight, respectively.

Example 5 In place of n-propyl gallate oxide, tetradecyldi (aminoethyl) glycine, aprotinin and trypsin, 1,2,3-triketohydroindene, polycation (compound represented by formula (XVI)), ε -The same as Example 2 except that aminocaproic acid and thrombin were used respectively.

Example 6 The same as Example 2 except that dodecyltrimethylammonium chloride and thrombin were used instead of tetradecyldi (aminoethyl) glycine and trypsin, respectively.

Example 7 Instead of n-propyl gallate oxide, tetradecyldi (aminoethyl) glycine and trypsin, 1.4
-Di (3,4-dihydroxyphenyl) 2.3-dimethylbutane oxide, polycation (compound represented by the formula (XV)) and snake venom thrombin-like enzyme were used respectively, but the same as Example 2. .

Comparative Example 1 The same as Example 2 except that aprotinin and trypsin were not used.

Comparative Example 2 A physiological saline solution containing n-propyl gallate, tetradecyldi (aminoethyl) glycine and ε-aminocaproic acid at concentrations of 0.5% by weight, 0.5% by weight and 0.1% by weight, respectively, was prepared. This dispersion (50 μ) was placed in a commercially available 5 ml polyethylene spitz and dried. 2 ml of human fresh blood containing heparin at a concentration of 1.0 IU / ml was injected into this spitz, and the same operation as in Example 1 was carried out for evaluation.

Example 8 A blood coagulation promoter having the formulations of Examples 1 to 7 was prepared. Each blood coagulation promoter was placed in a blood collection spitz, and heparin-containing (1.01 U / ml) human fresh blood was injected into this. After gently stirring, the mixture was allowed to stand at 20 ° C and the time required until the whole blood completely stopped flowing, that is, the blood coagulation time was measured. Blood coagulated in 10 to 20 minutes in each of the examples. Immediately after blood coagulation, at a rotation speed of 3000 rpm, 5
Centrifugation was performed for minutes, the serum separation state was observed, and the state of serum collection with a pipette was examined. In all of the examples, good results were obtained for serum separability and serum yield.

Comparative Example 3 The blood coagulation promoters of the formulations of Comparative Examples 1 and 2 were prepared, and the blood coagulation time was measured in the same manner as in Example 8. The time required was 30 to 35 minutes.

(Effects of the Invention) According to the present invention, it is possible to rapidly coagulate not only a normal blood sample but also blood containing heparin,
In addition, a blood coagulation promoter that can keep the coagulation state stable can be obtained. The serum and blood clot are well separated, and the serum yield is high. Such a blood coagulation accelerating agent is suitably used for fractionation of serum at the time of a blood test of an artificial dialysis patient or a thrombotic patient receiving heparin.

Claims (2)

[Claims] 1. A cyclic organic compound (I) represented by the following general formula, in which adjacent carbonyl groups in the formula are present on substantially the same plane, (b) an amine salt and / or Or a blood coagulation promoter containing an organic compound having a quaternary nitrogen, (c) an antifibrinolytic agent and / or an antiplasmin agent, and (d) a hydrolase, wherein the hydrolase is in a peptide chain. An enzyme capable of hydrolyzing a bond between Arg and an arbitrary amino acid residue and / or a bond between Lys and an arbitrary amino acid residue, a blood coagulation promoter: (Where A is the residue of the cyclic compound). 2. The hydrolase is a serine protease,
The blood coagulation promoter according to claim 1, which is at least one of thiol protease and metalloprotease.