JP3002903B2 - Anticoagulant / antiplatelet agent composition of extravascular blood and method for measuring molecular markers for platelet activation using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for suppressing and delaying platelet activation in extravascular blood, particularly in blood collection and after blood collection, and a method for measuring a molecular marker for platelet activation in blood using the same.

[0002]

BACKGROUND OF THE INVENTION Platelets play a major role in so-called primary hemostasis in the event of any damage to blood vessels, and play an important role in maintaining homeostasis of blood circulation physiology. That is, platelets are activated in response to an intravascular stimulus such as trauma or thrombin generation, and primary hemostasis is performed by a series of reactions of platelet morphology change, adhesion and aggregation.

[0003] By the way, during activation of platelets, β-thromboglobulin (β-TG), platelet factor 4 (PF4), thrombospondin (TSP), plasminogen activator inhibitor (PAI), platelet-derived proliferation Factor (PDGF), thromboxane A
₂ (TXA ₂ ), ATP, ADP, and various other substances. Among these substances, β-TG, PF4, PAI
And the like are substances specific to platelets, so that when they are measured as molecular markers, the activation state of platelets in blood can be known. On the other hand, it is known that platelet activation is deeply involved in the onset of thrombosis and arteriosclerosis, which are increasing in recent years, and the activation state of platelets in blood, that is, a molecular marker for platelet activation, is measured. Then, thrombosis and arteriosclerosis can be diagnosed. In fact, β-TG, PF4 and the like have already been established as diagnostic markers for such diseases.

[0004] However, platelets are highly sensitive to physiological and non-physiological stimuli, and are easily activated by a blood collection operation for obtaining a clinical test sample and a plasma separation operation after the blood collection, and the secondary activity of a molecular marker. Cause an increase. Therefore,
There is a problem that the state in a true blood vessel cannot be reflected, and it is difficult to make a diagnosis corresponding to a disease state.

In order to prevent platelet activation during and after blood collection, blood collection and plasma separation are performed under strict control as shown in Table 1.

[0006]

[Table 1]

However, since these management conditions include many conditions that are not always constant, the reliability of measurement results is low, and it is difficult to collect blood under these conditions. Measurement of molecular markers for platelet activation has not become very popular.

Attempts have been made to use a combination of a blood anticoagulant and an antiplatelet agent to avoid these complications during and after blood collection. For example, Ludlam CA et al. [B
r. J.Haematol., 33, 239 (1976) ], the theophylline or a derivative thereof and PGE ₁ as an anti-platelet agents, anticoagulant as ethylenediaminetetraacetic acid or of pH7.4 that combines its salt A composition was proposed. However, in this composition, PGE ₁ is an unstable substance, the effect of suppressing and delaying platelet activation is not sufficient, operation under cooling is essential, and hemolysis may be caused by such cooling operation. There was a problem. In addition, Contan
t G. et al. [Thrombo. Res., 31 , (1983), 365] describe adenosine or a derivative thereof, theophylline or a derivative thereof and dipyridamole as antiplatelet agents, and citric acid or a salt thereof as a blood anticoagulant at pH 5. A compounded composition was proposed. Certainly, the adoption of the present composition improved the inhibitory / delaying effect of platelet activation as compared to the conventional one, but if the cooling operation after blood collection was insufficient, the inhibitory / delaying effect was still insufficient. In addition, dipyridamole, which is one of the constituent elements, has the inconvenience that it hardly dissolves in water and has absorbance in the visible part, which affects other blood component measurements based on the principle of detection in the visible part. .

On the other hand, as another method for avoiding the above-mentioned problem at the time of blood collection, use of a vacuum blood collection tube can be considered. In other words, it is considered that the use of a vacuum blood collection tube can avoid the cause and complexity of the variation in the measurement result such as the suction speed. In addition, in order to further facilitate plasma separation after blood collection, it is desirable that blood is collected after an anticoagulant has been added to a vacuum blood collection tube in advance. However, when a vacuum blood collection tube is used, a large stress is applied to the blood and platelet activation occurs, which is not preferable.

Therefore, it has an excellent effect of suppressing and delaying the activation of platelets in extravascular blood, and enables blood collection and plasma separation without strict control. There has been a demand for a platelet activation inhibitor / retarder that does not induce the activation of, and a method for measuring a platelet activation molecular marker using the same.

[0011]

Under these circumstances, the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that blood anticoagulants ethylenediaminetetraacetic acid or a salt thereof and citric acid or a salt thereof and By using a combination of the antiplatelet agent adenosine or a derivative thereof and theophylline or a derivative thereof, platelet activation is suppressed during and after blood collection even when using a vacuum blood collection tube. The present inventors have found that it is possible to measure a molecular marker of platelet activation in a state within, and completed the present invention.

That is, the present invention provides (a) ethylenediaminetetraacetic acid or a salt thereof, (b) citric acid or a salt thereof,
(C) adenosine or an adenosine derivative selected from 2-chloroadenosine, dideoxyadenosine and 5'-deoxy-5'-methylthioadenosine; and (d)
Inhibition of platelet activation of extravascular blood characterized by containing theophylline or isobutyl-methylxanthine
In the method for measuring a retarder, and a molecular marker for platelet activation in blood, after adding the platelet activation inhibitor / delayer to the collected whole blood, collecting a test plasma from the blood. The present invention provides a method for measuring a molecular marker of platelet activation.

Ethylenediaminetetraacetic acid or its salt (a) and citric acid or its salt (b) used in the platelet activation inhibitor / retarder of the present invention (hereinafter, referred to as “the composition of the present invention”) It has the action of chelating calcium ions and inhibiting the generation of thrombin, and is known as a blood anticoagulant. As these salts, alkali metal salts such as sodium salts and potassium salts are preferable. Further, adenosine or its derivative (c) and theophylline or its derivative (d) mainly have an action of inhibiting platelet phosphodiesterase to increase the concentration of cyclic AMP in platelets, and are known as antiplatelet agents. Things. Derivatives of adenosine include 2-chloroadenosine, dideoxyadenosine and 5'-deoxy-
5′-methylthioadenosine and the like, and theophylline derivatives include isobutyl-methylxanthine and the like.

These (a) to (d) are incorporated into the composition of the present invention.
The mixing ratio of the components varies depending on the purpose of use and the like, but for the purpose of measuring the molecular marker for platelet activation, (a) :( b) :( c) :( d) = 5 to 154: 110 in molar ratio. ~13
0: 2.5 to 40: 5 to 30 is preferred. Further, the composition of the present invention uses citric acid as a buffer, and has a pH of 4 to 8, especially pH 5
It is desirable to use a buffer solution of a certain degree.

When the composition of the present invention is used for the measurement of a molecular marker for platelet activation, it is preferable to previously fill the container with high airtightness. The material of the container is not particularly limited as long as it can maintain airtightness, and various polymer bases, for example, polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polystyrene (PS), silicone-treated glass, and the like However, polyethylene terephthalate is particularly preferred. Further, it is preferable that this container is decompressed and used as it is as a vacuum blood collection tube. Therefore, the shape of the container is preferably a tube or a test tube.

In order to measure a molecular marker for platelet activation using the composition of the present invention, first, the composition of the present invention is added to collected whole blood. The amount of addition is not particularly limited,
The components (a) to (d) are (a) = 0.5 to 15.4 m at the final concentration.
M, (b) = 11-13 mM, (c) = 0.25-4 mM, (d) =
Preferably, the concentration is 0.5 to 3 mM. The method of addition may be added to whole blood after blood collection, but by collecting blood using a vacuum blood collection tube containing the composition of the present invention in advance, blood and the composition of the present invention are mixed at the same time as blood collection. You may. As described above, in the method of the present invention, the excellent platelet activation inhibitory effect of the composition of the present invention enables the use of a vacuum blood collection tube, thereby simplifying the operation and maintaining constant blood collection conditions. Improved reliability of the results can be achieved.

Next, in order to collect a test plasma from blood to which the composition of the present invention has been added, a conventional method, for example, at 1 to 30 ° C. for about 3000
Spinning is performed by centrifugation for about 15 minutes. Measurement of a molecular marker for platelet activation using the obtained test plasma is carried out according to a conventional method. That is, β-T
G and PF4 can be measured, for example, by RIA and ELISA. The molecular markers to which the method of the present invention can be applied include β-TG, PF4, PAI, PDG
F and the like can be exemplified, but at the present time, β-TG and PF4 are convenient because their measurement significance and method have been established.

[0018]

Next, the present invention will be described with reference to examples.

Embodiment 1 FIG. Preparation of test tubes with drug mixture:
0.25 ml of a drug having the composition shown in Table 2 was sealed in a polyethylene terephthalate (PET) blood collection tube (Sekisui Chemical Co., Ltd.) to produce a 2.5 ml blood collection test tube containing the drug mixture.

[0020]

[Table 2]

Embodiment 2 FIG. The composition group of the present invention [ATE (N
a) Comparison between C-added group or ATE (K) C-added group] and conventional composition group [ATDC-added group] The platelets activation inhibition degree was compared using the test tube containing the drug mixture obtained in Example 1. Two weeks before blood collection, healthy subjects without drug administration [AT
DC] group 5 (1 male, 4 female), [ATE (Na)
C] Group 6 (4 males, 2 females), [ATE (K) C] Group 9 (5 males, 4 females) as blood donors, with 21G needles in disposable poly syringes Then, 10 ml of blood was collected from the elbow vein with a light tourniquet. Immediately, [ATDC], [ATE (Na) C] or [AT
E (K) C] is dispensed with a prescribed amount of whole blood into each test tube.
After gently mixing by inversion, the mixture was left at room temperature for about 30 minutes. Thereafter, the mixture was centrifuged at 3,000 rpm for 15 minutes at room temperature to separate plasma. About 1/3 to 0.2 ml of the upper layer of the plasma was used as a test plasma for β-TG measurement. The operation up to this point was completed in about one hour after blood collection. The measurement of β-TG used a commercially available ELISA kit (Acerachrome β-TG, Boehringer Mannheim Yamanouchi). The results are shown in Tables 3, 4 and 5.

[0022]

[Table 3]

[0023]

[Table 4]

[0024]

[Table 5]

As a result, when neither (D) nor (E) was added, the β-TG value (mean ± standard deviation) was ATDC
Test group 61.0 ± 13.8 IU / ml, ATEC test group E (Na)
76.0 ± 12.8 IU / ml and E (K) 60.4 ± 19.9 IU / ml, and the effect of the combination of [ATC] was considered to be in this range. The concentration of (D) was gradually increased.
No decrease in the -TG value was observed, and a remarkable increase was observed in the vicinity of the (D) concentration of 1.98 × 10 ⁻⁴ M of the commercial product (Diatube H, Boehringer Mannheim Yamanouchi) by [ATDC]. This may be due to the effect of ethanol used as the solvent from the effect of (D), but the coexistence of ethanol cannot be avoided due to the solubility of (D) in water itself. In (E), the addition of (ENa) and (EK) significantly reduced the β-TG value. In addition, the tendency was concentration-dependent, but there were many cases where the amount was small and effective. Based on the above, (E) which does not require formulation technical considerations for solubility is [ATC]
Was effective in suppressing and delaying platelet activation.

Embodiment 3 FIG. Comparison with conventional test tube in β-TG measurement (cooling condition) [ATE (K) C] of Example 1 (E (K) concentration 154 mM)
0.2ml test tube For 2.0ml blood collection, commercially available [ATD
C] -containing blood collection tube for β-TG 5.0 ml for blood collection (Diatube H, Boehringer Mannheim Yamanouchi) and 2.5 ml for [ET] -only blood collection tube for β-TG (Amersham Pharmaceutical) were compared. Healthy subjects without drug administration for the last 2 weeks 10
A total of 5 blood donors (5 males and 5 females) were fitted with 21 G needles in disposable poly syringes, and 10 ml of blood was collected from the elbow vein with a light tourniquet. Immediately under cooling (in crushed ice) [ATE (K) C], [ATDC] or [E
T] was dispensed into each test tube in a prescribed amount, and the mixture was gently mixed by inversion.
Left for a minute. Thereafter, the mixture was centrifuged at 3,000 rpm for 15 minutes under cooling (4 ° C.) to separate plasma. Upper layer of this plasma about 1
From // 3, 0.2 ml was collected as test plasma and used for measurement of β-TG. The operation up to this point was completed in one hour after blood collection.
For the measurement of β-TG, a commercially available ELISA kit (Acerachrome β-TG, Boehringer Mannheim Yamanouchi) was used. The results are shown in Table 6.

[0027]

[Table 6]

The results of the β-TG measurement under the three conditions show that the average value is low and the variation width is [ATEC], [ATDC],
[ET].

Embodiment 4 FIG. Comparison with conventional test tube in β-TG measurement (at room temperature) [ATE (Na) C] ((ENa) concentration of 77 m in Example 1)
M) Commercially available [A]
TDC] blood collection tube for β-TG containing 5.0 ml for blood collection (Diatube H, Boehringer Mannheim Yamanouchi), [E
T] and a 2.5 ml blood collection tube dedicated to β-TG (Amersham Pharmaceutical Co., Ltd.) were compared. In the last 2 weeks, 9 healthy volunteers (five males and four females) without drug administration were used as blood donors. A 21G injection needle was attached to a disposable poly syringe and 10 ml was injected from the elbow vein with a light tourniquet. Blood was collected. Immediately, [ATE (Na) C], [ATDC] or [E
[T] was dispensed into each test tube in a prescribed amount, and the mixture was gently mixed by inversion, and then left at room temperature for about 30 minutes.
Thereafter, the mixture was centrifuged again at room temperature for 3000 rpm for 15 minutes to separate the plasma. Approximately 1/3 to 0.2 ml of the upper layer of this plasma was collected as test plasma and used for β-TG measurement. The operation up to this point was completed in one hour after blood collection. For the measurement of β-TG, a commercially available ELISA kit (Acerachrome β-TG, Boehringer Mannheim Yamanouchi) was used. The results are shown in Table 7.

[0030]

[Table 7]

[ET] clearly showed an increase in β-TG, and it was considered impossible to avoid operation under cooling. [ATD
[C] and [ATEC] both exhibited an inhibitory / delaying effect on platelet activation even at room temperature, and the degree was more pronounced with [ATEC].

Embodiment 5 FIG. Preparation of vacuum blood collection tube containing drug mixture: [ATE (K) C] ((E
K) Concentration: 154 mM; A, T, C concentrations are the same as in Table 2) 0.1
After dispensing ml, the pressure was adjusted to a vacuum sufficient to collect 0.9 ml of whole blood to obtain a sealed test tube.

Embodiment 6 FIG. Measurement of β-TG Using Vacuum Blood Collection Tube Vacuum blood collection tube containing drug mixture of Example 5 for 1.0 ml blood collection [A
TEC] Test tube with drug mixture of Example 1 against vacuum
[ATEC] non-vacuum for 2.0 ml blood collection was compared. In the last 2 weeks, 5 healthy subjects (2 males and 3 females) without drug administration were used as blood donors, and blood was collected from the elbow vein as a vacuum blood collection tube using an [ATEC] vacuum according to a conventional method (with an tourniquet). Was done. On the other hand, from the elbow vein on the opposite side, a light tourniquet was applied gently in the same manner as in Examples 2, 3, and 4, and a disposable poly syringe was used.
10 ml of blood was collected with a 21G injection needle attached, and a prescribed amount of whole blood was dispensed. Both were immediately inverted and mixed with the drug mixture and left at room temperature for about 30 minutes. After that, 3000 rotations at room temperature, 15
After centrifugation for minutes, the plasma was separated. About 1/3 to 0.1 ml of the upper layer of this plasma was collected as test plasma and used for β-TG measurement. The operation up to this point was completed in about one hour after blood collection. β
For the measurement of -TG, a commercially available ELISA kit (Acerachrome β-TG, Boehringer Mannheim Yamanouchi) was used. The results are shown in Table 8.

[0034]

[Table 8]

In the case of a vacuum blood collection tube, a non-vacuum specimen 4
There was no spot error like. It was considered that the blood sampling could be performed promptly, and the uniformity of the conditions could be achieved, thereby reducing the variation. Considering that whole blood drawn out of the blood vessels is mixed with the drug mixture during blood collection, the suppression effect appears quickly and the overall measurement value is even lower, and considering that the results are at room temperature, It was considered that the strict relaxation of the management was achieved.

[0036]

According to the present invention, platelet activation during and after blood collection can be suppressed, and blood collection and plasma separation can be performed without strict control. Therefore, the measurement of the platelet activation molecular marker can be performed with a simpler operation and with good reproducibility, and thrombosis and arteriosclerosis can be more accurately diagnosed.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-2-71154 (JP, A) THROMBOSIS RESEARCH CH, VOL. 31, (1983), p. 365 −374 (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/48 G01N 33/49 G01N 33/50 A61K 31/52

Claims (2)

(57) [Claims] 1. (a) ethylenediaminetetraacetic acid or a salt thereof, (b) citric acid or a salt thereof, (c) adenosine or 2-chloroadenosine, dideoxyadenosine and 5 '
An agent for suppressing or delaying platelet activation of extravascular blood, comprising: an adenosine derivative selected from deoxy-5'-methylthioadenosine; and (d) theophylline or isobutyl-methylxanthine. 2. In a method for measuring a molecular marker for platelet activation in blood, a test plasma is collected from the blood after the platelet activation inhibitor / delay agent according to claim 1 is added to the collected whole blood. A method for measuring a molecular marker for platelet activation, characterized in that: