JPH065229B2 - Blood coagulation promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a blood coagulation promoter, and more particularly to a blood coagulation promoter capable of promoting the coagulation of a blood sample of an examinee 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 clots of blood collected in a blood test container and then centrifuged to separate blood clots (gels containing fibrin and blood cells) with different specific gravities. 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, it takes 40 to 60 minutes for blood to coagulate even if a glass-made test container that is said to have a relatively short blood coagulation time is used. Is required. Therefore, there is a drawback that the serum required for the test cannot be secured promptly. This becomes a problem especially when the inspection needs to be performed urgently.

As described above, according to the conventional serum fractionation method, in addition to the problem that it takes a long time for blood coagulation, the problem that serum and blood clot are difficult to be separated well when the coagulated whole blood is separated by centrifugation. 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 test results are adversely affected, and it becomes necessary to centrifuge again.

Another problem arises when dealing with blood samples from patients undergoing artificial dialysis and thrombosis. Since such patients are administered heparin to prevent thrombosis, 1 to 20 units of heparin are present per 10 ml of blood. This heparin binds to antithrombin III in blood and remarkably inhibits the action of thrombin. Furthermore, it is 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 coagulate. Therefore, it becomes difficult to collect serum.

In order to solve these problems, the inventor has (a) a cyclic organic compound represented by the following general formula (I), and the adjacent carbonyl groups in the formula are substantially on the same plane: (b)
A blood coagulation promoter containing an amine salt and / or an organic compound having a quaternary nitrogen has been proposed (JP-A-60-2).
7858 publication).

(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.

However, it is unavoidable that the clot of fibrin begins to be dissolved due to the action of the degrading enzyme existing in plasma after a lapse of time after coagulation, and therefore, there is a problem regarding the stability over time after coagulation. It had been.

(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. Attempts were made to develop a blood coagulation promoter that can improve the stability in blood. An object of the present invention is to provide a blood coagulation promoter that can rapidly coagulate blood regardless of whether heparin is contained, stability after coagulation is good, and serum separability is good.

(Means and Actions for Solving Problems) The blood coagulation promoter of the present invention comprises (a) the general formula: (Wherein A represents a residue of a cyclic compound),
And a cyclic organic compound in which the two adjacent carbonyl groups are sterically substantially on the same plane, (b) an amine salt or an organic compound having a quaternary nitrogen, and (c) a wire-removing material Alternatively, it contains an antiplasmin agent, and thereby the above-mentioned object is achieved.

The cyclic organic compound used as a component of the blood coagulation promoter of the present invention may be either a homocyclic or heterocyclic compound formed by two adjacent carbonyl groups and a residue A. Further, such a cyclic compound may be a monocyclic compound or a polycyclic compound, but in the present invention, the ring containing at least two carbonyl carbons is a 6-membered ring or a 5-membered ring. Certain cyclic compounds are preferably used.

Particularly, the 6-membered cyclic compound preferably used in the present invention is represented by the following formula (Provided that R ^{1 to} R ⁴ represent hydrogen, a hydrocarbon group, a polar substituent or a residue in a polycyclic compound) and have an o-quinone ring. Although not particularly limited, it is preferably an alkyl group, and the polar substituent is not particularly limited, and examples thereof include a carboxyl group, a carboxylic acid ester group, a hydroxyl group, an amino group, a mercapto group and the like. Is.
Accordingly, preferred specific examples of the compound having an o-quinone ring include, for example, o-quinone and the following general formula: (However, R ⁵ represents an alkyl group.) A gallic acid alkyl ester oxide represented by the following formula: Ellagic acid partial oxides and complete oxides represented by 1,4-di (3,4-dihydroxyphenyl) 2,3-dimethylbutane partial oxide and complete oxide represented by

As a preferred specific example of the homocyclic compound in which the ring containing two carbonyl carbons is a 5-membered ring, 1,2,3-triketohydroindene represented by

Further, one of the heterocyclic compounds which can be preferably used in the present invention is the following general formula (However, R ⁶ represents hydrogen, a hydrocarbon group or a residue in the polycyclic compound, and R ⁷ and R ⁸ represent hydrogen, a hydrocarbon group, a polar substituent or a residue in the polycyclic compound.) The hydrocarbon group and the polar substituent are the same as described above.

Preferred specific examples of such a compound include isatin represented by the following formula.

These groups of compounds all have two adjacent carbonyl groups on the same sterically or approximately the same plane in the molecule, and the detailed mechanism of action is unclear. Shows a specific effect against.

The second component constituting the blood coagulation promoter of the present invention is an amine salt or an organic compound having quaternary nitrogen.

As the amine salt, any of inorganic acid salts and organic acid salts of primary, secondary and tertiary amines can be used. Specific examples include hydrohalic acid such as acid salt, and sulfuric acid. Examples thereof include inorganic acid salts such as sulfurous acid and organic acid salts such as formic acid and acetic acid. The organic residue of the amine salt is usually an alkyl group, but this alkyl group may be an alkyl group containing a different element such as an imino group or an ether group, and the amine salt shall include an intramolecular salt. .

Therefore, as a specific example of a preferable amine salt, for example, the following formula Hexadecyldimethylamine hydrochloride represented by And tetradecyldi (aminoethyl) glycine represented by

Further, among the organic compounds having a quaternary nitrogen, the organic monomer is preferably tetraalkylammonium, but may have an aryl group in part, and
As mentioned above, the alkyl group may be an alkyl group containing a different element such as an imino group or an ether group. For example, as a preferred specific example, the following formula Dodecyltrimethylammonium chloride represented by

The polymer of the organic compounds having a quaternary nitrogen preferably has the following general formula: (However, R ⁹ is hydrogen or an alkyl group, X is a halogen atom or an acid radical, Y is an alkylene group or an -alkylene group —SO ₂ —
Indicates. ) Is a polycation having a repeating unit represented by:
In particular, Or A polycation having a repeating unit represented by is preferably used.

On the other hand, as an antifibrinolytic agent or antiplasmin agent, aprotinin, soybean trypsin inhibitor, ε-aminocaproic acid, P-aminomethylbenzoic acid, aminomethylcyclohexanecarboxylic acid, etc., which have been conventionally used clinically, are used alone or in appropriate combination. It may be used together.

In the present invention, in order to allow the cyclic organic compound and the amine salt or the monomer or polymer having a quaternary nitrogen and the antifibrinolytic agent or the antiplasmin agent to exist in blood as a blood coagulation promoter in the present invention. , These may be added to blood as they are or after being dissolved or dispersed in an appropriate solvent, or these blood coagulation promoters may be supported on a carrier having a large specific surface area, which may be used in a blood test container. It may be added to blood.

The carrier is not particularly limited as long as it has a large specific surface area without adversely affecting blood tests, and various carriers can be used.
Nonwoven fabric, woven fabric, resin beads and the like can be preferably used. To support the blood coagulation promoter on such a carrier, for example, a solution or dispersion of the carrier may be applied or dipped in the solution, dried and then attached to the carrier. Also,
Mixing with an appropriate auxiliary agent such as gum arabic to give an aqueous dispersion,
A particulate material carrying a blood coagulation promoter can also be obtained by a method such as rapid freeze drying.

The amount of the above compound present in blood is at least 1 × 10 ⁻¹⁰ g per 1 ml of blood, and if it is less than this, the effect of promoting blood coagulation is poor. However, when it is present in an excessively large amount, it may adversely affect various blood tests. Therefore, the amount is preferably 1 × 10 ⁻¹⁰ g or less.

Further, in the blood coagulation promoter of the present invention, the amine salt or the organic compound having a quaternary nitrogen is contained in an amount of 5 to 10,000 parts by weight based on 100 parts by weight of the cyclic organic compound. It is preferable from the viewpoint of promoting coagulation, and it is preferable to use the antifibrinolytic agent or antiplasmin agent in the clinically recommended amount. For example, aprotinin is about 10 per ml of blood.
Soybean trypsin inhibitor is used in an amount of 0 to 600 KIU (unit), and about 500 to 4,000 FU per 1 ml of blood.
(Unit) The amount of ε-aminocaproic acid, P-aminomethylbenzoic acid and aminomethylcyclohexanecarboxylic acid used is about 10 ^-2 to 10 ^-8 g per 1 ml of blood.
It is preferable to use it in the amount used.

According to the present invention, the amine salt or the quaternary nitrogen-containing monomer or polymer described above adsorbs and neutralizes heparin when contacted with blood containing heparin, and precipitates with it, In the blood test container, since the action of heparin is rapidly eliminated, and thus the normal blood coagulation function is restored, and the cyclic compound described above has a specific effect on blood coagulation factors. The blood can be coagulated within a short time, and thus the serum can be obtained with good separation by centrifugation.

To explain this point in more detail, in normal blood, the factor XII in the coagulation factor is immediately activated by contact between the blood and the inner wall surface of the blood container, and this is the starting point for blood reaction in a chain reaction. Coagulation proceeds, and finally thrombin generated by activation of prothrombin acts on fibrinogen to form an insoluble fibrin network and complete coagulation.

On the other hand, it is said that 10 cc to 10 units of heparin are usually present in the blood of a patient receiving heparin. Thus, in the case of blood containing heparin, heparin is present in the blood. It acts synergistically with existing antithrombin to significantly inhibit the action of thrombin. As described above, heparin is said to inhibit not only the action of thrombin but also the action of other coagulation factors including factor XII, and therefore, fibrinogen is usually contained in blood containing heparin. No conversion of fibrin to fibrin occurs and, as a result, serum cannot be separated because blood does not clot.

However, according to the present invention, as described above, since heparin is adsorbed to an amine salt or a monomer or polymer having a quaternary nitrogen and removed from blood, thrombin and other blood coagulation factors are included. Will restore normal blood coagulation action, and the specific action of the cyclic organic compound will significantly accelerate blood coagulation.

Furthermore, antifibrinolytic agents or antiplasmin agents inhibit the fibrinolytic action of plasmin, which is antagonistically generated in the blood coagulation reaction process, to ensure coagulation and stabilize the coagulation state even after coagulation. Can be kept at

As described above, when the blood coagulation promoter of the present invention is allowed to exist in blood, heparin is removed from the blood, and the blood coagulation factor is rapidly activated, and the coagulation after collecting Etsu Station in a container. In addition to significantly shortening the time required for the separation, the separation of serum and blood clot becomes extremely easy, and therefore the problem that blood clot components are mixed in the separated and collected serum is solved.
Further, according to the present invention, as a result of sufficient contraction of the blood clot component, there is an advantage that the yield of serum is remarkably large. Stability problems can be solved.

The present invention is described below with reference to examples, but the present invention is not limited to these examples.

(Example) Example 1 Ellagic acid oxide represented by the formula (V) and the formula (XVI)
An aqueous dispersion of a polycation represented by and an aprotinin was impregnated into a polyester non-woven fabric and dried sufficiently.
The amount of each of the above components per unit area of nonwoven fabric is 4 x 1
It was 0 ⁻⁴ g, 4 × 10 ⁻⁴ g, and 500 KIU.

Commercially available 5 ml polyethylene spitz with 1.0 IU / heparin
2 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. Serum was collected 1 hour and 30 hours after standing, and fibrinogen and fibrin degradation products (hereinafter abbreviated as FDP) were measured. The results are shown in Table 1, and it can be seen that there is no difference in the FDP measurement values after 1 hour and 30 hours, and that the blood clot decomposition reaction is well suppressed. The results of Examples 2 to 7 and Comparative Example are also shown in Table 1.

Example 2 0.5 g of n-propyl gallate oxide and tetradecyldi (aminoethyl) glycine and aprotinin, respectively.
Saline dispersions were prepared containing wt%, 0.5 wt% and concentrations of 10,000 KIU / ml.

Commercially available 5 ml polyethylene spitz with 1.0 I of heparin
2 ml of human fresh blood containing U / ml was injected, 50 μl of the above dispersion was added, and the same treatment as in Example 1 was carried out for evaluation.

Example 3 Isatin 1 g, hexadecyldimethylamine hydrochloride 0.4
g, 50 mg of aminomethylcyclohexanecarboxylic acid and 1 kg of polystyrene bead carrier having an average particle size of 1.5 mm were thoroughly mixed with a small amount of ethanol as a dispersion aid, and then dried.

Commercially available 5 ml polyethylene spitz with 1.0 of heparin
2 ml of human fresh blood containing at a concentration of IU / ml was infused, then 1 g of the above blood coagulation promoter was added, and the same treatment as in Example 1 was carried out for evaluation.

Example 4 In place of n-propyl gallate, tetradecyldi (aminoethyl) glycine and aprotinin, o-quinone, polycation (compound represented by the formula (XV)) and ε-aminocaproic acid were added at 0.5% by weight and 0.5% by weight, respectively. , And 0.1% by weight, but in the same manner as in Example 2.

Example 5 Instead of n-propyl gallate oxide, tetradecyldi (aminoethyl) glycine and aprotinin, 1.2.
Same as Example 2 except that 3-triketohydroindene, polycation (compound represented by formula (XVI)) and ε-aminocaproic acid were used.

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

Example 7 In place of n-propyl gallate oxide and tetradecyldi (aminoethyl) glycine, 1,4-di (3.4-dihydroxyphenyl) 2.3-dimethylbutane oxide and polycation (in the formula (XV) The same as in Example 2 except that the compound shown) was used.

Comparative Example 1 A physiological saline dispersion solution containing n-propyl gallate and tetradecyldi (aminoethyl) glycine at concentrations of 0.5% by weight and 0.5% by weight, respectively, was prepared. 5 ml commercially available
2 ml of human fresh blood containing heparin at a concentration of 1.0 IU / ml was injected into a polyethylene spitz, and then the above dispersion liquid was added to 5
After adding 0 μm, the same treatment as in Example 1 was carried out and the evaluation was performed.

Example 8 The adjustment of the coagulation promoter in each of Examples 1 to 7 and the preparation of blood collecting spitzs and the injection of heparin-containing human fresh blood into the spitzs were repeatedly performed, and after gently stirring, the mixture was allowed to stand at 20 ° C. The time it takes for whole blood to stop flowing completely,
That is, when the blood coagulation time was measured, coagulation was completed in 30 to 35 minutes in any of the examples.

Immediately after blood coagulation, 5 rpm at a rotation speed of 3,000 rpm.
When the serum separation state was observed by centrifuging for minutes and the serum collection state was examined by a pipette, good results were obtained for the serum separability and the serum yield in all Examples.

(Effect of the Invention) According to the present invention, it is possible to provide a blood coagulation promoter capable of rapidly coagulating not only an ordinary blood sample but also blood containing heparin. Furthermore, when the coagulation accelerator of the present invention is used, the coagulation state after coagulation can be kept stable,
Further, the yield of serum increases when the serum and blood clot are separated. Such a blood coagulation promoter is preferably used for collecting serum for blood tests of artificial dialysis patients and thrombosis patients receiving heparin.

Claims (1)

[Claims] 1. (a) General formula (Wherein A represents a residue of a cyclic compound),
And a cyclic organic compound in which the two adjacent carbonyl groups are sterically substantially on the same plane, (b) an amine salt or an organic compound having a quaternary nitrogen, and (c) an antifibrinolytic solvent Alternatively, a blood coagulation promoter containing an antiplasmin agent.