JP4921359B2 - Sensitive hematology assays and reagents - Google Patents

Description

  The present invention relates to reagents, kits and assay methods for use in blood coagulation system assays. More specifically, the present invention is for use in a blood coagulation assay of a direct factor Xa inhibitor containing activated blood coagulation factor X (hereinafter referred to as “factor Xa”), phospholipids and calcium ions. The present invention relates to reagents, kits, and assay methods using them.

  Various methods for quantifying the effect of a blood coagulation factor inhibitor on the blood coagulation system and measuring the blood coagulation factor inhibitor concentration in blood or plasma (hereinafter referred to as “blood coagulation system assay”) are known. This blood coagulation system assay can be classified into two methods: a method of measuring the inhibitory activity of coagulation factors (for example, factor Xa, thrombin, etc.) using a chromogenic synthetic substrate, and a method of measuring the coagulation time.

  A method for measuring the inhibitory activity of factor Xa or thrombin, which is a coagulation factor, using a chromogenic synthetic substrate is usually performed by adding Xa in a solution containing factor Xa or thrombin (antithrombin is also added if necessary). Calculate the concentration of factor Xa or thrombin activity or blood coagulation factor inhibitor by adding chromogenic synthetic substrate and plasma decomposed by factor or thrombin and measuring the absorbance etc. of the resulting chromogenic synthetic substrate degradation product (For example, refer nonpatent literature 1). However, this method requires a dedicated device and is not widely used in the clinical field because the work is complicated.

  The coagulation time measurement method is a method of measuring the time until fibrin formation by activating the coagulation system by adding various activators to blood or plasma, and is also called “coagulation method”. For example, PT (prothrombin time) method (hereinafter referred to as “PT method”), APTT (activated partial thromboplastin time) method (hereinafter referred to as “APTT method”), TT (thrombin time) method, ACT (activated coagulation time) ) Method, ECT (ecarin clotting time) method, PiCT (prothrombinase-induced clotting time) method (hereinafter referred to as “PiCT method”), measuring method using ACCUCLOOT HEPTEST ™ (hereinafter referred to as “ACCUCLOT method”) Etc. are included. These methods are used for blood drug monitoring of anticoagulation therapy with indirect coagulation inhibitors such as heparin and low molecular weight heparin and warfarin which is a coagulation factor synthesis inhibitor (see Non-Patent Document 2).

Below, the example of the typical coagulation time measuring method is demonstrated.
The PiCT method is a method of inducing coagulation with a prothrombinase complex. Specifically, an antithrombin-dependent blood coagulation factor inhibitor in plasma and the added factor Xa are bound in the absence of calcium ions, and then calcium chloride is added to leave the remaining factor Xa and RVV-V. (Factor V activator derived from Russell's viper venom) and factor Va fully activated form a complex with calcium ions on phospholipids, and the clotting time (prothrombinase depending on the enzyme activity) Induced coagulation time, PiCT) is measured (for example, Patent Document 1 and Non-Patent Document 3). Therefore, it is necessary to add a two-step reagent (Pefakit PiCT (trademark), Pentapharma Co., Ltd., activation reagent and starting reagent) for measurement, and the value varies depending on the processing time after addition of the activation reagent. There is a problem.

  The ACCUCLOT method is a bovine plasma fraction rich in RECALMIX ™ (Haemachem) (phospholipid, fibrinogen, blood coagulation factor V (hereinafter referred to as “factor V”) after incubation of citrated plasma with factor Xa. (Including non-patent document 4). Each reagent uses a commercially available kit (ACCUCLOT HEPTEST (trademark), Trinity Biotech).

  In the PT method, thromboplastin and calcium ions (neoplastin reagent, Roche Diagnostics) are added to citrated plasma to enhance the coagulation system and to coagulate with thrombin (prothrombin time, hereinafter, (Referred to as non-patent document 5).

  Among the various methods for measuring the clotting time described above, there are those using factor Xa, phospholipid and calcium ions as activators, and these are simultaneously used as factor V and activated blood coagulation factor V (hereinafter, “ Or a blood coagulation factor V activating enzyme (hereinafter referred to as “factor V activating enzyme”).

  By the way, in recent years, for example, the following general formula (I)

(Wherein R is a hydrogen atom or a lower alkyl group, and Z is a hydrogen atom or a hydroxyl group) a benzenesulfonamide derivative (see Patent Document 2), Razaxaban (Razababan, Bristol-Myers Squibb), DX Direct factor Xa inhibitors such as -9065a (Daiichi Pharmaceutical Co., Ltd.) have been developed (see, for example, Non-Patent Document 6). These direct factor Xa inhibitors are different from heparin, low molecular weight heparin, Fondaparinux (ARIXTRA (trademark), GlaxoSmithKline), etc., and inhibit blood coagulation directly without using antithrombin, and blood coagulation system via thrombin Therefore, it has attracted attention as a therapeutic agent for blood coagulation diseases with few side effects such as bleeding tendency. However, the direct factor Xa inhibitor shows little prolongation of coagulation time such as PT and APTT in the concentration range (therapeutic range) in which antithrombotic action is manifested in the clinic, and thus the effect on the blood coagulation system cannot be evaluated. .

Accordingly, there is an urgent need for early development of a simple and clinically usable method for the appropriate formulation of direct factor Xa inhibitors.
International Publication WO01 / 44819 Pamphlet International Publication WO02 / 28827 Pamphlet Teien, AN and Lie, M .: Thromb. Res. 1977; Volume 10: pp.399-410 Michio Fujimaki, Katsuyuki Fukutake, Katseido Publishing, "Coagulation Handbook", 1992, 2nd revised edition, pp.163-182 Calatzis A, 5 others, Haemostasis, 2000, Volume 30 (Suppl. 2), pp.172-174 Harenberg J, 3 others, Haemostasis, 1989, Vol. 19, pp. 13-20 Proctor RR, 1 other person, Am J Clin Path., 1961, Vol. 41, pp. 212-219 Johannes Ruef, 1 other, Expert Opin. Investig. Drugs., 2003, Vol. 12, pp.781-797

  An object of the present invention is to provide a reagent, a kit, an assay method thereof, and the like that can be directly used for a blood coagulation system assay of a factor Xa inhibitor.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have prepared a mixed reagent containing factor Xa, phospholipid, and calcium ion, and highly activated factor Xa, and then mixed with a blood sample. The method has surprisingly been found to show high sensitivity and prolonged clotting time in a direct factor Xa inhibitor blood coagulation assay, and has led to the present invention.

That is, the present invention
[1] A reagent for use in a blood coagulation system assay of a direct factor Xa inhibitor containing factor Xa, phospholipid and calcium ion (provided that factor V, activated factor V or factor V activating enzyme is used) Except those containing);
[2] The reagent according to [1] above, containing 0.1 to 10 mU of factor Xa and 1 to 100 μg of phospholipid per 1 mL of an aqueous solution when dissolved;
[3] The reagent according to [2] above, containing 0.1 to 10 mU / mL of factor Xa and 1 to 100 μg / mL of phospholipid;
[4] A reagent produced by drying the reagent according to [3];
[5] Direct factor Xa inhibitors are Razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otamixban, Fidedexban, BIBT-986, Apixavan The reagent according to [1] or [2] above, which is a benzenesulfonamide derivative represented by the general formula (I) or a pharmacologically acceptable salt thereof;
[6] A kit for directly using a factor Xa inhibitor, a phospholipid-containing reagent, and a calcium ion-containing reagent in a blood coagulation assay of a factor Xa inhibitor (provided that factor V, Excluding those containing activated factor V or factor V activating enzyme);
[7] The kit according to [6] above, wherein a reagent containing factor Xa and phospholipid and a reagent containing calcium ion are combined;
[8] The kit according to [6] or [7] above, containing 0.1 to 10 mU / mL of factor Xa and 1 to 100 μg / mL of phospholipid;
[9] Direct factor Xa inhibitors are razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otamixavan, Fidedexban, BIBT-986, Apixavan The kit according to any one of [6] to [8], which is a benzenesulfonamide derivative represented by the general formula (I) or a pharmacologically acceptable salt thereof;
[10] (1) A reagent for use in a blood coagulation system assay of a blood sample and a direct factor Xa inhibitor containing factor Xa, phospholipid and calcium ion (provided that factor V, activated factor V or And (2) a method for directly measuring a blood coagulation system of a factor Xa inhibitor, comprising the step of measuring a blood coagulation time;
[11] (1) Mixed reagent for use in blood coagulation system assay of direct factor Xa inhibitor containing factor Xa, phospholipid and calcium ion (provided that factor V, activated factor V or factor V Direct factor Xa inhibition comprising the steps of: (2) preparing a non-containing enzyme), (2) mixing the reagent of (1) with a blood sample, and (3) measuring the blood clotting time. Blood coagulation assay method of drugs;
[12] Reagents containing 0.1 to 10 mU / mL of factor Xa and 1 to 100 μg / mL of phospholipid (except for those containing factor V, activated factor V or factor V activating enzyme) The assay method according to [10] or [11] above, wherein
[13] The assay method according to any one of [10] to [12], wherein the blood sample is human plasma;
[14] The assay method according to [13] above, wherein the blood sample is collected from a human who has been directly administered with the factor Xa inhibitor;
[15] Direct factor Xa inhibitors are razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otamixavan, Fidedexban, BIBT-986, Apixavan , The assay method according to any one of [10] to [14] above, which is a benzenesulfonamide derivative represented by the general formula (I) or a pharmacologically acceptable salt thereof; .

  In the present invention, the direct factor Xa inhibitor refers to an inhibitory action on factor Xa among direct coagulation factor inhibitors that directly inhibit coagulation factors such as factor Xa and thrombin without using antithrombin III (ATIII). For example, Razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otaxixban, Fidexban, BIBT-986, Apixban, M- 55165, M-55551, benzenesulfonamide derivatives represented by the above general formula (I) and other compounds described in Patent Document 2, 3- [N- (3-amidinophenyl) -N- [N- [4- [ 1- (1-Iminoethyl) piperidin-4-yl] phenyl] carbamoylmethyl ] Aminomethyl] phenoxy acetic acid salt (hereinafter, referred to as "Compound 1") and the like. Further, those that are easy to use as oral administration agents or direct coagulation factor inhibitors with a molecular weight of 1000 or less are preferred. For example, Razaxaban, YM-150, DU-176b, ZK807834 (CI-1031), BAY-59-7939, Otamixban, Fidexban, BIBT-986, Apixavan, benzenesulfonamide derivatives represented by the above general formula (I), etc. Is preferred. The benzenesulfonamide derivative represented by the general formula (I) includes [4- [2- (5-amidino-2-hydroxybenzenesulfonylamino) ethyl] -2′-methanesulfonylbiphenyl-3-yloxy. ] Acetic acid (hereinafter referred to as "compound 2"), [4- [2- (5-amidino-2-hydroxybenzenesulfonylamino) ethyl] -2'-methanesulfonylbiphenyl-3-yloxy] acetic acid n-butyl, [ 4- [2-hydroxy-5- (N-hydroxycarbamimidoyl) benzenesulfonylamino] ethyl] -2'-methanesulfonylbiphenyl-3-yloxy] acetic acid, [4- [2- [2-hydroxy-5- (N-hydroxycarbamimidoyl) benzenesulfonylamino] ethyl] -2'-methanesulfonylbipheni 3-yloxy] acetic acid n- butyl, and the like are preferable.

Examples of factor Xa include human plasma-derived factor Xa and bovine plasma-derived factor Xa. The concentration of factor Xa in the reagent at the time of use is preferably in the range of 0.1 to 10 m international unit / mL (hereinafter referred to as “mU / mL”) that provides a practical coagulation time, and is preferably 0.5 to 5 mU / A range of mL is more preferred.
Examples of the phospholipid include cephalin or a mixture containing phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and the like, and preferably cephalin. The concentration of the phospholipid in the reagent at the time of use is preferably in the range of 1 to 100 μg / mL, and more preferably in the range of 4 to 50 μg / mL, which provides a practical clotting time.
As the calcium ion source, calcium chloride is preferable. The concentration of calcium ions in the reagent at the time of use may be appropriately determined depending on, for example, the amount capable of resuming blood or plasma coagulation by citrate blood collection, and is usually in the range of 10 to 100 mmol / L. it can.

  The kit of the present invention comprises a combination of a reagent containing factor Xa, a reagent containing phospholipid and a reagent containing calcium ion; a combination of a reagent containing factor Xa and phospholipid and a reagent containing calcium ion; Xa A combination of a reagent containing factor and calcium ion and a reagent containing phospholipid; a combination of a reagent containing factor Xa and a reagent containing phospholipid and calcium ion; or containing factor Xa, phospholipid and calcium ion A combination of a reagent containing factor Xa, a reagent containing phospholipid and a reagent containing calcium ion; or a reagent containing factor Xa and phospholipid and a reagent containing calcium ion. A combination containing a reagent containing factor Xa and a phospholipid and a calcium ion Combination of more preferable.

  The reagent used in the reagent or kit of the present invention includes, in addition to each component of factor Xa, phospholipid or calcium ion, a buffering agent, a pH adjusting agent (for example, Hepes, Tris HCl, etc.), an osmotic pressure adjusting agent (sodium chloride) , Mannitol, etc.), albumin, preservatives, preservatives (sodium azide, etc.), solvents and the like.

  Examples of the form of the reagent of the present invention include aqueous solutions, powders, freeze-dried powders, and the like.

  The dried reagent can also be produced by vacuum drying or freeze-drying a solution containing factor Xa, phospholipid and calcium ions by a known method or a method based thereon.

In the blood coagulation system assay method of the present invention (hereinafter also referred to as “KXaT method”), the blood sample may be a human or non-administered or non-administered blood coagulation factor inhibitor regardless of whether the blood coagulation ability is normal or abnormal. Examples include blood collected from human animals by a method that can be used for blood coagulation assay (eg, citrate blood collection) or plasma thereof, and commercially available standard human plasma for blood coagulation tests, human control plasma, or normal animal plasma. It is done. Citrated blood or plasma thereof is preferred from a human or non-human animal that has or has not been administered a blood coagulation factor inhibitor, and a human or non-human animal that has been administered a blood coagulation factor inhibitor, particularly a direct factor Xa inhibitor Therefore, citrate-collected blood plasma is more preferable.
In addition, blood or plasma collected from a human or non-human animal is usually incubated before the activator is added. Incubation may be appropriately performed at 25 to 40 ° C. for 0 to 5 minutes, but is generally performed at 37 ° C. for 60 seconds.
The measurement of the blood coagulation time can be performed by using a fully automatic or semi-automatic blood coagulation measuring apparatus whose measurement principle is light scattering, transmitted light, absorbance, a magnetic sensor, or a viscosity change detection type, or can be performed by a method.

FIG. 1 shows the effect of prolonging the coagulation time of Compound 2 by various coagulation time measurement methods. The horizontal axis represents the plasma concentration of Compound 2 (nmol / L), and the vertical axis represents the ratio of clotting time to control. In the figure, circles indicate the coagulation time by the KXaT method, diamonds by the ACCUCLOT method, squares by the PT method, and triangles by the APTT method.

  The contents of the present invention will be described in more detail with reference to the following reference examples and examples, but the present invention is not limited to the contents.

Reference example 1
Using the PT method, the APTT method, and the ACCUCLOT method, the coagulation time by the compound 2 which is a direct factor Xa inhibitor was measured. For the measurement, an automatic blood coagulation measuring apparatus ST4 (manufactured by Roche Diagnostics) was used.

1) Measurement of coagulation time by PT method Various concentrations of a direct factor Xa inhibitor were added to standard human plasma for blood coagulation test (Dade Behring) to prepare a test drug-added plasma sample. Place 50 μL of plasma sample with various concentrations of test drug in measurement cuvette and preincubate for 60 seconds at 37 ° C., then add 100 μL of Neoplastin reagent (Roche Diagnostics) to start coagulation. Time was measured.
2) Measurement of coagulation time by APTT method 50 μL of various drug-contained plasma samples prepared in the same manner as the above PT method are placed in a measurement cuvette, preincubated at 37 ° C. for 40 seconds, and then PTT reagent “RD” (Roche Diagnostics) 50 μL was added and incubated for 180 seconds. 50 μL of 25 mmol / L calcium chloride aqueous solution was added thereto to start coagulation, and the coagulation time was measured.
3) Measurement of clotting time by ACCUCLOT method 50 μL of various concentrations of test drug-added plasma samples prepared in the same manner as the above PT method are placed in a measurement cuvette, preincubated at 37 ° C. for 60 seconds, and then ACCUCLOT HEPTEST ™ Of factor Xa (containing bovine factor Xa, bovine serum albumin, sodium chloride, PEG and trismaleate) was added. After 120 seconds, 100 μL of ACCULOT HEPTEST RECALMIX ™ (containing rabbit brain sephaline, calcium chloride, fibrinogen, factor V, etc.) was added to initiate clotting, and the clotting time was measured.

  The results are shown in Table 1 and FIG. In each of the PT method, the APTT method, and the ACCUCLOT method, an increase in the coagulation time was observed with an increase in the concentration of Compound 2 with respect to the control. However, due to the change in drug concentration in the low concentration range, the extension time of the coagulation time is short, and sufficient sensitivity cannot be obtained.

Reference example 2
1) Measurement of coagulation time by PiCT method Using the PiCT method, the coagulation time was directly measured by Compound 1, Compound 2, Razaxaban hydrochloride and DX-9065a, which are factor Xa inhibitors. For the measurement, an automatic blood coagulation measuring apparatus ST4 (manufactured by Roche Diagnostics) was used. 50 μL of various concentrations of test drug-added plasma samples prepared in the same manner as the PT method described in Reference Example 1 were placed in a measurement cuvette, pre-incubated at 37 ° C. for 60 seconds, and then Pefakit PiCT (trademark, Penta Pharma) Activating reagent (Reagent 1) (containing factor Xa, RVV-V, phospholipid, hepes, mannitol) was added, and further incubated at 37 ° C for 180 seconds. Next, 100 μL of an initial reagent (reagent 2) (containing 25 mM calcium chloride) was added thereto to start coagulation, and the coagulation time was measured.

  The results are shown in Table 2. There was no correlation between the test drug concentration and the clotting time for any of the direct factor Xa inhibitors, Compound 1, Compound 2, Razaxavan hydrochloride, or DX-9065a, and apparently abnormal behavior of clotting time. Indicated. Therefore, the PiCT method may not be used for blood clotting assays of these direct factor Xa inhibitors.

Reference example 3
1) Measurement of coagulation time by PT method and PiCT method In the same manner as the PT method described in Reference Example 1 and the PiCT method described in Reference Example 2, an automatic blood coagulation measuring apparatus ST4 (manufactured by Roche Diagnostics) was used. Each coagulation time with Compound 2 was measured.
As a result, as shown in Table 3, the PT method did not provide sufficient sensitivity. That is, as the concentration of Compound 2 increased, the coagulation time was extended, but the extension range was small with respect to the change in drug concentration in the low concentration range. In the PiCT method, no correlation was found between the concentration of Compound 2 and the coagulation time in the concentration range used.

Example 1
Preparation of Reagent A coagulation accelerating reagent (hereinafter referred to as “KXaT coagulation reagent”) used in the KXaT method was prepared by the following method.
1) One vial of PTT reagent (containing Sephaline) of PTT reagent [RD] (Roche Diagnostics) was dissolved in 5 mL of STA calcium chloride (25 mmol / L).
2) Coagulation Factor Xa, human plasma (trademark) (Calbiochem) 10 U vial was dissolved in 1 mL of distilled water to prepare a 10 U / mL solution.
3) 100 μL of the Factor Xa and human plasma 10 U / mL solution prepared in 2) was taken and added to 9900 μL of the solution prepared in 1).
4) The solution of 3) was further diluted 100 times with the solution prepared in 1) to prepare a KXaT coagulation reagent of the following composition example 1.

Composition example 1
Factor Xa: 1 mU / mL
Calcium chloride: 25mmol / L
Phospholipid:
Phosphatidylethanolamine (PE): 24 μg / mL
Phosphatidylcholine (PC): 10 μg / mL
Phosphatidylserine (PS): 12 μg / mL
pH: 7.0

Example 2
Factor Xa concentration In the same manner as described in Example 1, KXaT coagulation reagents containing Factor Xa at different concentrations of 0 to 10 mU / mL shown in Table 4 were prepared. At this time, the other compositions were set to be the same as in Composition Example 1. 50 μL each of standard human plasma (Dade Bering) supplemented with compound 2 at concentrations of 0 (control), 20, 200, and 2000 nmol / L was placed in a measurement cuvette and preincubated at 37 ° C. for 60 seconds. Coagulation was started by adding 100 μL of each concentration of KXaT coagulation reagent, and the coagulation time (seconds) was measured. For the measurement, an automatic blood coagulation measuring apparatus ST4 (manufactured by Roche Diagnostics) was used.

  The results are shown in Table 4. In the range of 0.1 to 10 mU / mL of the additive concentration of factor Xa, the coagulation time was extended depending on the concentration of compound 2. In general, if the coagulation time of the control is too long, the waiting time becomes long. Conversely, if the control coagulation time is too short, a measurement error tends to occur. Measurement was possible at any concentration, but in particular, in the range of 0.5 mU / mL to 5 mU / mL, the clotting time in the control was about 30 seconds to 1 minute 30 seconds, for example, 200 nmol / L For compound 2 of the above, an increase in clotting time of about 30 seconds to 70 seconds was observed, and a sensitive blood coagulation system assay was possible even in a low concentration range.

Example 3
Phospholipid concentration In the same manner as described in Example 1, KXaT coagulation reagents containing various concentrations of phospholipid were prepared. At this time, using a phospholipid of PE 24 μg / mL, PC 10 μg / mL and PS 12 μg / mL as a reference amount (1 time), a KXaT coagulation reagent containing 0.05 to 2 times the phospholipid concentration was prepared. . The concentrations of Factor Xa and calcium chloride were the same as in Composition Example 1. 50 μL each of standard human plasma (Dade Bering) supplemented with compound 2 at concentrations of 0 (control), 20, 200, and 2000 nmol / L was placed in a measurement cuvette and preincubated at 37 ° C. for 60 seconds. Coagulation was started by adding 100 μL of each KXaT coagulation reagent prepared to a concentration of phospholipid, and coagulation time (second) was measured by the KXaT method. For the measurement, an automatic blood coagulation measuring apparatus ST4 (manufactured by Roche Diagnostics) was used.

  The results are shown in Table 5. Depending on the concentration of compound 2, prolongation of clotting time was observed at phospholipid concentrations ranging from 0.05 to 2 times the reference amount. In general, if the coagulation time of the control is too long, the waiting time becomes long. Conversely, if the control coagulation time is too short, a measurement error tends to occur. Measurement was possible at a phospholipid concentration of 0.1 times or more of the reference amount. Particularly, in the range of 0.1 to 1 times the reference amount, the clotting time in the control was about 30 to 60 seconds. For example, with respect to 200 nmol / L of Compound 2, an extension of the clotting time of about 30 to 60 seconds was observed, and a practical and sensitive blood clotting system assay was possible.

Example 4
Standard human plasma supplemented with KXaT coagulation reagent of simultaneous reproducibility composition 1 and compound 2 at concentrations of 0 (control), 0.02, 0.06, 0.2, 0.6, 2 μmol / L (Dade Bering) ), The coagulation time (second) by the KXaT method was measured three times for the same sample in the same manner as in the method described in Example 2. As shown in Table 6, extremely good simultaneous reproducibility was shown.

Example 5
Standard human plasma supplemented with KXaT coagulation reagent with daily reproducibility composition 1 and compound 2 at concentrations of 0 (control), 0.02, 0.06, 0.2, 0.6, 2 μmol / L (Dade Bering) ), The coagulation time (seconds) by the KXaT method was measured for 3 days for each sample prepared once a day in the same manner as described in Example 2. As shown in Table 7, excellent daily reproducibility was shown.

Example 6
Normal human citrate supplemented with KXaT coagulation reagent of composition 1 and 0, (control), 0.02, 0.06, 0.2, 0.6, 2 μmol / L of compound 2, razaxaban hydrochloride or DX-9065a Using acid plasma (Cosmo Bio), coagulation time (seconds) was measured by the KXaT method in the same manner as described in Example 4. As shown in Table 8, prolongation of the coagulation time was observed depending on the concentration of any test drug, and a correlation was observed between the factor Xa inhibitory activity and the coagulation time extension effect of each compound.

Example 7
Various lots of normal human citrate plasma (Cosmo) supplemented with KXaT coagulation reagent of composition 1 and compound 2 at concentrations of 0 (control), 0.02, 0.06, 0.2, 0.6, 2 μmol / L The coagulation time (seconds) by the KXaT method was measured in the same manner as in the method described in Example 4. As shown in Table 9, in any lot, the coagulation time was extended in a concentration-dependent manner, and the reactivity was equivalent.

Reference example 4
Using Fondaparinux at concentrations of 0 (control), 6, 20, 60, 200, and 600 ng / mL, the coagulation times (seconds) by the PiCT method and the KXaT method, respectively, in the same manner as described in Reference Example 2 and Example 4. ) Was measured. As shown in Table 10, Fondaparinux, which is an antithrombin-dependent indirect factor Xa inhibitor, had a weaker coagulation time-extending effect in the KXaT method than in the PiCT method.

Example 8
Standard human plasma (Dade Bering) supplemented with KXaT coagulation reagent of composition 1 and compound 2 at concentrations of 0 (control), 0.02, 0.06, 0.2, 0.6, 2 μmol / L In the same manner as in the method described in Example 2, the coagulation time (seconds) by the KXaT method was measured. As shown in Table 11, the clotting time was extended depending on the drug concentration.
The results are shown in FIG. 1 together with the results of the PT method, PiCT method and ACCUCLOT method described in Reference Example 1. From FIG. 1, it can be seen that the measurement sensitivity in the low concentration range is significantly higher in the KXaT method than in the other methods compared to the compound 2 which is a direct factor Xa inhibitor.

  From the above results, the KXaT method is more effective against various direct factor Xa inhibitors such as Compound 1, Compound 2, Razaxaban, DX-9065a, etc., compared with the existing methods such as PT method, APTT method, PiCT method, ACCUCLOT method, etc. It was confirmed that the reaction was extremely sensitive. Moreover, the KXaT method showed extremely excellent reproducibility in terms of simultaneous reproducibility, daily reproducibility, and reproducibility between lots of human citrate plasma. Thus, the reagents and methods of the present invention are very useful for direct factor Xa inhibitor blood coagulation assays.

    According to the present invention, it is possible to provide a reagent, a kit, an assay method thereof, and the like that can be directly used for a blood coagulation system assay of a factor Xa inhibitor.

Claims (15)

Factor Xa, containing phospholipids and calcium ions, factor V, does not contain a factor V or factor V-activating enzyme activation reagent for use in blood coagulation system assay directly factor Xa inhibitors. The reagent according to claim 1, comprising 0.1 to 10 mU of factor Xa and 1 to 100 µg of phospholipid per mL of aqueous solution when dissolved. The reagent according to claim 2, comprising 0.1 to 10 mU / mL of factor Xa and 1 to 100 µg / mL of phospholipid. A reagent produced by drying the reagent according to claim 3. Direct factor Xa inhibitors are Razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otaxixban, Fidexban, BIBT-986, Apixban, general formula (I)
A benzenesulfonamide derivative represented by the formula (wherein R is a hydrogen atom or a lower alkyl group, and Z is a hydrogen atom or a hydroxyl group) or a pharmacologically acceptable salt thereof. The reagent described. Direct factor Xa inhibition by combining a reagent containing factor Xa, a reagent containing phospholipid, and a reagent containing calcium ion, each containing no factor V, activated factor V or factor V activating enzyme kit for use in coagulation-based assays medicine. The kit according to claim 6, wherein a reagent containing factor Xa and phospholipid and a reagent containing calcium ion are combined. The kit according to claim 6 or 7, comprising 0.1 to 10 mU / mL of factor Xa and 1 to 100 µg / mL of phospholipid. Direct factor Xa inhibitors are Razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otaxixban, Fidexban, BIBT-986, Apixban, general formula (I)
A benzenesulfonamide derivative represented by the formula (wherein R is a hydrogen atom or a lower alkyl group, and Z is a hydrogen atom or a hydroxyl group) or a pharmacologically acceptable salt thereof. The kit in any one of. (1) For blood coagulation assay of a blood sample and a direct factor Xa inhibitor containing factor Xa, phospholipid and calcium ion and not containing factor V, activated factor V or factor V activating enzyme step of mixing the reagent for use, (2) a step of measuring blood coagulation time, blood coagulation assay method of the direct factor Xa inhibitor. (1) For use in a blood coagulation system assay of a direct factor Xa inhibitor containing factor Xa, phospholipid and calcium ion and not containing factor V, activated factor V or factor V activating enzyme preparing a mixed reagent, (2) mixing with a reagent and blood sample (1), and (3) measuring the blood coagulation time, blood coagulation assay method of the direct factor Xa inhibitor . Factor Xa 0.1~10mU / mL, and characterized in that the phospholipid containing 1-100 [mu] g / mL, using a reagent containing no factor V, activates factor V or factor V-activating enzyme The assay method according to claim 10 or 11. The assay method according to claim 10, wherein the blood sample is human plasma. The assay method according to claim 13, wherein the blood sample is collected from a human who has been directly administered with a factor Xa inhibitor. Direct factor Xa inhibitors are Razaxaban, DX-9065a, YM-150, DU-176b, ZK807834 (CI-1031), JTV-803, BAY-59-7939, Otaxixban, Fidexban, BIBT-986, Apixban, general formula (I)
(Wherein R is a hydrogen atom or a lower alkyl group, and Z is a hydrogen atom or a hydroxyl group), or a pharmacologically acceptable salt thereof. The assay method according to any one of the above.