JPH0725672B2 - 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 of 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 blood collected from a subject to coagulate. For example, even if a glass test container, which is said to have a relatively short blood coagulation time, is used, 40
It takes ~ 60 minutes, and if a synthetic resin inspection container is used, it will take 4 hours or more. 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, even with extreme caution when pipetting the serum portion and / or decanting,
Infiltration of red blood cells is unavoidable. 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 tests on patients undergoing hemodialysis and patients with 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 in the formula are substantially coplanar. 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).

(Here, A represents a residue of a cyclic compound.) Examples of the compound represented by the formula (I) include gallic acid alkyl ester oxides and ellagic acid oxides. 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 amine salt or the like contained therein adsorbs / neutralizes heparin to inactivate it, and the compound represented by the formula (I) causes blood coagulation to occur in blood. It can activate factor XII and coagulate blood in a short time.

The blood coagulation promoter is a blood coagulation promoter capable of rapidly coagulating not only heparin-free blood but also heparin-containing blood. However, it forms a complex with a metal component in blood and changes into a serum component. There was a drawback that there is a risk of giving. In addition, a blood coagulation promoter that is more excellent in thermal stability has been desired.

(Problems to be Solved by the Invention) An object of the present invention is to rapidly coagulate blood regardless of whether heparin is contained, good serum separability, and to form a complex with a metal component in blood to form serum. It is an object of the present invention to provide a blood coagulation promoter that has no fear of changing the components and has excellent thermal stability.

(Means and Actions for Solving Problems) The blood coagulation promoting agent of the present invention has a cyclic structure represented by the following general formula, in which adjacent carbonyl groups are present in substantially the same plane. It contains a metal complex having an organic compound (I) as a ligand, and an amine salt and / or an organic compound containing a quaternary nitrogen, whereby the above object is achieved: (Where A is the residue of the cyclic compound).

The compound represented by the above formula (I), which is the ligand of the metal complex as the main component of the blood coagulation promoter of the present invention, may be a homocyclic compound or a heterocyclic compound. Well,
It may be a monocyclic compound or a polycyclic compound. As such a cyclic compound, it is preferable that the reduction containing the above two carbonyl carbons is 6-membered or 5-membered. Among the homocyclic compounds, preferred 6-membered cyclic compounds include compounds having o-quinone reduction 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,
Alkyl groups, especially those having 1 to 18 carbon atoms, are preferred. The polar substituent is also not particularly limited. For example, there are a carboxyl group, a carboxylic ester group, a hydroxyl group, an amino acid, a mercapto group, and the like. Examples of compounds having o-quinone conversion include o-quinone and the following formulas (III) to (V
II) The compounds shown in the following may be mentioned: gallic acid alkyl 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-dimethylbutane perfect oxide Among the homocyclic compounds, a preferred specific example of the 5-membered cyclic compound is 1.2.3 triketohydroindene represented by the following formula (VIII).

Examples of the heterocyclic compound include compounds represented by the following general formula (IX).

(Here, R ₆ represents a hydrogen, a hydrocarbon or a residue in the polycyclic compound, and R ₇ and R ₈ represent a hydrogen, a hydrocarbon group, a polar substituent or a residue in the 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 Ichisan represented by the following formula.

The metal forming the complex is a metal other than the alkali metal having the o, o-coordination property. Particularly, a complex containing Fa, Co, Ni, Al, etc. is preferable because it is easy to handle. The metal complex used in the blood coagulation promoter of the present invention is obtained by adding a salt solution containing the above metal ion to the compound (I) serving as the above ligand. For example, an iron complex of propyl gallate oxide can be obtained by mixing a ferric chloride solution with a solution containing propyl gallate oxide. Such metal complexes include
In order to maintain the electrical neutrality inside the complex, a ligand containing one kind or two or more kinds of a halogen group, a sulfate group, a nitrate group and an ammonium group may be contained. Water may be contained as a ligand.

Amine salt contained in the blood coagulation promoter of the present invention and /
Alternatively, the organic compound having quaternary nitrogen acts as a heparin neutralizing agent that adsorbs and neutralizes heparin to inactivate it. The amine that constitutes the amino 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 amino 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 amino salt may be an inner salt.

Specific examples of preferable amino salts include, for example, hexadecyldimethylamine hydrochloride represented by the formula (XI) and (XI
There is tetradecyldi (aminoethyl) glycine represented by the formula I).

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. ) Of the compounds represented by (XVI), especially (XV) or (X
A polycation having a repeating unit represented by VI) is suitable.

The blood coagulation promoter of the present invention contains the above metal complex and the above neutralizing agent as main components, and the neutralizing agent is 5 parts by weight per 100 parts by weight of the metal complex.
It is contained at a rate of up to 10,000 parts by weight. If the amount of neutralizing agent 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. The blood coagulation promoter may contain an auxiliary agent such as an antifibrinolytic agent in a proportion of 1% by weight or less. The blood coagulation promoter of the present invention is used at a ratio of 1 × 10 ⁻¹⁰ to 1 × 10 ⁻¹ g per 1 ml of blood. 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 cannot be obtained.

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 may be supported on a carrier having a large critical surface area in order to avoid the high concentration of the blood coagulation promoter from denaturing the protein component upon contact with blood.

Such a carrier is not 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, the solution or dispersion liquid thereof is applied to the carrier, or the carrier is immersed 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 carrier particulate.

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 amino salt and precipitates, so that heparin inhibits thrombin and factor XII. Disappears. for that reason,
Blood restores normal clotting function. Furthermore, the metal complex contained in the blood coagulation promoter has the ability to act on and activate factor XII in blood. Blood coagulation proceeds in a chain reaction due to activation of factor XII within a short period of time, and finally thrombin generated by activation of prothrombin acts on fibrinogen to form an insoluble fibrin network to form blood. Coagulation is complete. The time required for blood coagulation depends on the type of metal complex or neutralizing agent in the blood coagulation promoter, the amount of blood coagulation promoter, the material of the container used, the amount of heparin in the blood, etc. When used, it usually takes 20 to 40 minutes. Thus, not only normal blood but blood containing heparin can coagulate in a short time. 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.

Since the compound that is the main component of the blood coagulation promoter of the present invention is a metal complex, it is more stable to heat than the cyclic organic compound (I) contained in the blood coagulation promoter that the inventor previously developed. Excellent in performance. When the above-mentioned cyclic organic compound is used as a blood coagulation promoter, it may form a complex with a metal component in blood to change the serum component. However, the compound contained in the blood coagulation promoter of the present invention is Since it does not react with the metal components inside, accurate test values can be obtained.

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

Example 1 A polyacetate nonwoven fabric is impregnated with an aqueous dispersion containing an iron complex of ellagic acid oxide (compound represented by formula (V) on page 8) and a polycation (compound represented by formula (XVI) on page 15). And allowed to dry thoroughly. The amount of each of the above components per cm ^{2 of the} non-woven fabric was 10 ^-4 g.

2 units of heparin in a commercially available 10 ml polyethylene spitz
8 ml of human fresh blood containing at a concentration of / ml was infused, then 1 cm ² of non-woven fabric carrying the above components was put therein, gently stirred, and then left at 20 ° C. The time required until the whole blood completely stopped flowing was defined as the blood coagulation time.

Immediately after blood coagulation, centrifugation was performed for 5 minutes at a rotation speed of 3000 rpm to observe the state of serum separation, and serum was collected with a pipette, and the amount was used as the serum yield. The respective results are shown in the table below. The results of Examples 2 to 10 and Comparative Example are also shown in the table below.

In addition, when serum iron was measured by the bathophenanthroline method using the obtained serum, it was 92 μg / dl.

Example 2 A physiological saline dispersion containing the iron complex of gallic acid n-propylate compound and tetradecyldi (aminoethyl) glycine at concentrations of 0.5% by weight and 0.2% by weight, respectively, was prepared.

Inject 8 ml of human fresh blood containing heparin at a concentration of 2 units / ml into a commercially available 10 ml polyethylene spitz, and then
After 80 μm of the above dispersion liquid was added, the same treatment as in Example 1 was carried out and the evaluation was carried out.

Example 3 1 g of iron complex of isatin, 0.4 g of hexadecyldimethylaminohydrochloride and polystyrene bead carrier 1 having an average particle size of 1.5 mm
Kg was thoroughly mixed with a small amount of ethanol as a dispersion aid and then dried. The amounts of the iron complex of isatin and hexadecyldimethylaminohydrochloride in 1 g of the particles supporting the blood coagulation promoter were 10 ⁻³ g and 0.4 × 10 ⁻³ g, respectively. 2 units / ml of heparin in a commercially available 10 ml polyethylene spitz
After injecting 8 ml of human fresh blood containing at the concentration of 1 and then adding 1 g of the above blood coagulation promoter-supporting particles, the same treatment as in Example 1 was carried out, and evaluation was performed.

Example 4 Instead of the iron complex of n-propyl gallate oxide and tetradecyldi (aminoethyl) glycine, an iron complex of o-quinone and a polycation (a compound represented by the formula (page 14 (XV)) at 0.5% by weight respectively and Same as Example 2 except that 0.4 wt% was used.

Example 5 Instead of iron complex of n-propyl gallate oxide and tetradecyldi (aminoethyl) glycine, 1.2.3-
Example 2 is the same as Example 2 except that an iron complex of triketohydroindene and a polycation (a compound represented by the formula on page 15 (XVI) were used.

Example 6 The same as Example 2 except that dodecyltrimethylammonium chloride was used instead of tetradecyldi (aminoethyl) glycine.

Example 7 Instead of iron complex of gallic acid n-propyl oxide and tetradecyldi (aminoethyl) glycine, iron complex of 1,4-di (hydroxyphenyl) 2.3-dimethylbutane oxide and polycation (see page 14 ( The same as Example 2 except that the compound represented by the formula (XV) was used.

Example 8 Instead of an iron complex of gallic acid n-propyl oxide and tetradecyldi (aminoethyl) glycine, a cobalt complex of ellagic acid oxide (V) and a polycation (compound represented by the formula (XV) on page 14) were used. Other than that, it is the same as the second embodiment.

Example 9 Instead of iron complex of gallic acid n-propyl oxide and tetradecyldi (aminoethyl) glycine, nickel complex of ellagic acid oxide (V) and polycation (compound represented by formula (XV) on page 14) were used. Other than that, it is the same as the second embodiment.

Example 10 Instead of iron complex of n-propyl gallate oxide and tetradecyldi (aminoethyl) glycine, aluminum complex of ellagic acid oxide (V) and polycation (14
The same as Example 2 except that the compound represented by the formula (XV) is used.

Comparative Example 1 2 units of heparin in a commercially available 10 ml polyethylene spitz
After injecting 8 ml of human fresh blood contained at a concentration of / ml, the same treatment as in Example 1 was carried out without adding a blood coagulation promoter, and evaluation was performed.

Comparative Example 2 An iron complex of ellagic acid oxide (compound represented by the formula on page 8 (V)) which is not a metal complex, and a polycation (page 15 (XV
A non-woven fabric made of polyacetate was impregnated with an aqueous dispersion containing a compound represented by the formula (I)) and dried sufficiently. Non-woven
The amount of each of the above components per cm ² was 10 ⁻⁴ g.

2 units of heparin in a commercially available 10 ml polyethylene spitz
8 ml of human fresh blood containing at a concentration of / ml was injected, then 1 cm ² of the non-woven fabric carrying the above components was put therein, gently stirred, and then left at 20 ° C. Thereafter, in the same manner as in Example 1, blood coagulation time, serum separability, serum yield, and serum iron were evaluated.

As a result, the blood coagulation time was 35 minutes, the serum separability was extremely good, the serum yield was 4.0 ml, and the serum iron measurement value was 68 μg / dl.

Comparative Example 3 A polyacetate nonwoven fabric was impregnated with an aqueous solution containing a polycation (compound represented by the formula (XVI) on page 15) and dried sufficiently. The amount of polycation per 1 cm ^{2 of} non-woven fabric is 10
^-4 g.

8 ml of human fresh blood containing heparin at a concentration of 2 units / ml was injected into a 10 ml hard glass blood collection tube, and then 1 cm ² of the above-mentioned polycation-supported non-woven fabric was put therein, gently stirred, and then left at 20 ° C. . Then, in the same manner as in Example 1,
Blood coagulation time Serum separation, serum yield, and serum iron were evaluated.

As a result, the blood coagulation time was 40 minutes, the serum separability was extremely good, the serum yield was 4.1 ml, and the serum iron measurement value was 89 μg / dl.

Comparing the measured values of serum iron in Example 1, Comparative Example 2, and Comparative Example 3 above, compared with the one using a hard glass tube (Comparative Example 3), the one using ellagic acid oxide (V) (Comparative Example 2) showed a low value, but ellagic acid oxide (V)
With the iron complex of Example 1 (Example 1), substantially no difference was obtained.

Reference Example A 1% by weight purified water suspension of an iron complex of ellagic acid oxide (compound represented by the formula (V) on page 8) was prepared. In addition, a 1% by weight purified water suspension was prepared for the elasic acid oxide itself.

The obtained purified water suspension was subjected to autoclave heat treatment under the conditions of 121 ° C., 2 atmospheres and 1 hour.

As a result, the iron complex of ellagic acid oxide was a deep black powder before and after the autoclave heat treatment, and did not change at all, but the ellagic acid oxide itself was a blackish brown powder before the autoclave heat treatment. However, after the heat treatment, it became a blackish brown powder partially containing a light yellow color.

After the autoclave heat treatment, the ellagic acid oxide itself partially changed to a bright yellow color, probably because the ellagic acid oxide was partially reduced and returned to the original ellagic acid. On the other hand, the iron complex of ellagic acid oxide is considered to be chemically stable against heat treatment and hard to be reduced by using the iron complex.

(Effect of the Invention) According to the present invention, a blood coagulation promoter capable of rapidly coagulating not only a normal blood sample but also blood containing heparin is thus obtained. The separation state between serum and blood clot is also good, and since the blood coagulation promoter does not change the serum components, the test values of various tests using serum can always be obtained accurately and stably. Since the metal complex contained in the blood coagulation promoter is relatively heat stable, it is easy to store the blood coagulation promoter for a long period of time. 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.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 31/40 9454-4C 31/555 9454-4C 31/785 9454-4C

Claims (1)

[Claims] 1. A metal complex having a cyclic organic compound (I) as a ligand, which is represented by the following general formula and in which adjacent carbonyl groups in the formula are substantially on the same plane, and an amine: A blood coagulation promoter containing a salt and / or an organic compound having quaternary nitrogen: (Where A is the residue of the cyclic compound).