Method for excluding factor XIa inhibitors in patient samples - Patent Application 20070122997

Abstract

Method for excluding factor XIa inhibitors in patient samples - Patent Application 20070122997 The present invention is in the field of coagulation diagnostics and relates to a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample.

Description

[Technical Field]

[0001] The present invention is in the field of coagulation diagnostics and relates to a method for excluding anticoagulants from the group of factor XIa inhibitors in patient samples. [Background technology]

[0002] The role of the blood coagulation system (hemostasis) is, first, to ensure continuous blood flow and, second, to induce rapid, localized wound closure to minimize blood loss upon injury. A cascade of enzymatic processes, each activating additional enzymes via proteolytic processes, culminates in fibrin formation, which contributes to the formation of a wound-clotting clot. Examples of such procoagulant enzymes are thrombin (factor IIa), factor Xa, and factor XIa. Thrombin, as well as factors Xa and XIa, are serine proteases normally present in the blood in the form of inactive proenzymes and are activated only upon activation of coagulation.

[0003] Factor XI is a component of the intrinsic blood coagulation system and is activated by activated factor XII or thrombin, which itself activates factor IX, which then finally influences thrombin formation via the activation of factor X.

[0004] In anticoagulation therapy, the use of new direct coagulation factor inhibitors, particularly direct thrombin and factor Xa inhibitors, and more recently factor XI inhibitors, is increasing. These new anticoagulants have the potential to replace the traditionally used indirect anticoagulants, particularly heparin and its derivatives, which exert their anticoagulant effect only in conjunction with cofactors, such as antithrombin or heparin cofactor II. Therefore, it is important to know the amount of therapeutic inhibitor for therapy control and drug administration. Therefore, there is a need to have diagnostic methods that allow for the determination of the presence, activity, or plasma concentration of therapeutic coagulation factor inhibitors.

[0005] Various test methods are known in the prior art for the quantitative detection of direct inhibitors of proteolytically active coagulation factors, such as inhibitors of thrombin, factor Xa or factor XIa, in human plasma samples.

[0006] Inhibitors of proteolytically active coagulation factors are typically determined using chromogenic or coagulometric test methods. In chromogenic tests, for example, a patient sample suspected of containing a thrombin or factor Xa inhibitor is mixed with a defined excess of the corresponding activated coagulation factor and a chromogenic substrate for the activated coagulation factor, and the residual activity of the coagulation factor in the reaction is measured photometrically. Established, commercially available chromogenic tests utilize the chromophores para-nitroaniline (pNA) and 5-amino-2-nitrobenzoic acid (ANBA), which have an absorption maximum at 405 nm. The yellow color formed is generally determined photometrically. The greater the concentration of the therapeutic inhibitor in the patient sample, the greater the inhibition of the activity of the added coagulation factor and the less substrate is cleaved. When determining the inhibitor, the color intensity in the test is inversely proportional to the inhibitor concentration in the sample. For example, U.S. Patent No. 5,929,949 or U.S. Patent No. 5,929,949 describe such thrombin- or factor Xa-based chromogenic test methods. Using these tests, therapeutic inhibitors that inhibit the activity of blood coagulation factors can be quantitatively determined in patient samples. US Pat. No. 5,629,999 describes a method for determining factor Xa inhibitors, and US Pat. No. 5,629,999 describes a method for determining thrombin inhibitors in serum and urine samples.

[0007] The increasing use of a wide variety of coagulation factor inhibitors for the treatment or prevention of thrombotic disorders has led to an increasing need for rapid and simple diagnostic methods that allow determining whether a patient, for example, a patient admitted in an unconscious state with no information available about their medical history or medications, is being treated with anticoagulants, and if so, what their anticoagulant therapy is. Particularly in the case of surgical procedures that must be performed urgently, knowledge of the patient's coagulation status and anticoagulant therapy is important in assessing the risk of bleeding complications.

[0008] In this situation, it would be desirable to have a simple and rapid test by which the presence of anticoagulants from the group of factor XIa inhibitors in patient samples can be excluded. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] EP-B1-0034320 [Patent Document 2] EP-A2-0004271 [Patent Document 3] WO-A1-2012069139 [Patent Document 4] WO-A1-2012175183 Summary of the Invention [Problem to be solved by the invention]

[0010] It is therefore an object of the present invention to provide a simple method for excluding anticoagulants from the group of factor XIa inhibitors in patient samples, which method has high sensitivity and specificity for the absence of factor XIa inhibitors. [Means for solving the problem]

[0011] This object is achieved by providing a method based on the conventional APTT test typically available in clinical laboratories, in which a patient sample and a normal plasma sample are first mixed with factor XI-deficient plasma and at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors, and the APTT (activated partial thromboplastin time) is determined in the two samples. The clotting times thus determined of the two samples are related to each other by forming an index, which is compared with a predetermined reference value; if the index is below the predetermined reference value, it is possible to exclude the presence of a factor XIa inhibitor in the patient sample.

[0012] The present invention therefore provides a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample, comprising: a) A first reaction mixture containing a patient sample and a second reaction mixture containing a normal plasma sample are prepared by adding each of the two samples to a reaction mixture containing the patient sample. i) Factor XI deficient plasma, and ii) at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors, and iii) Coagulation activators of the intrinsic blood coagulation system and mixing the resulting mixture with the b) Clotting time (APTT XI) in the first reaction mixture 試料 ), and the clotting time in the second reaction mixture (APTT XI 正常 ) measuring; c) Two clotting times are used to calculate the index APTT XI 試料 / APTT XI 正常 forming a d) comparing the index with a predetermined reference value; and e) Index APTT XI 試料 / APTT XI 正常 excluding anticoagulants from the group of factor XIa inhibitors in the patient sample if The present invention provides a method comprising:

[0013] The term "anticoagulants from the group of factor XIa inhibitors" refers to therapeutically active substances that do not naturally occur in the human body and that specifically reduce the proteolytic activity of coagulation factor XIa, typically by directly binding to factor XIa (or factor XI). The term "anticoagulants from the group of factor XIa inhibitors" expressly excludes anticoagulant proteins or protein complexes that naturally occur in the human body and are capable of directly or indirectly inhibiting the proteolytic activity of coagulation factor XIa, such as antithrombin or heparin cofactor II.

[0014] Currently known examples of anticoagulants from the group of factor XIa inhibitors include asundexian, milvexian and avelacrimab, as well as osocimab, gruticibart (xisomab), REGN9933, MK-2060, ONO-7684 or ONO-5450598.

[0015] "Patient sample" should be understood to mean a plasma sample from an individual, for example a citrated plasma sample.

[0016] A "normal plasma sample" should be understood to mean a plasma sample from an apparently healthy blood donor or a sample of a mixture of plasma from several, e.g., 20, apparently healthy blood donors, i.e., a normal plasma pool, in which the values ​​of all coagulation factors are within the normal range.

[0017] The term "factor XI-deficient plasma" refers to plasma of human or other animal origin from which factor XI has been completely or at least substantially removed, e.g., by immunoadsorption, but from which all other coagulation factors are present in normal concentrations. Typically, plasma from multiple apparently healthy blood donors (human or animal) is pooled to form a normal plasma pool, and then factor XI is removed or substantially removed from said normal plasma pool by adsorption, e.g., using factor XI-specific monoclonal and / or polyclonal antibodies, thereby resulting in factor XI-deficient plasma.

[0018] According to the present invention, a patient sample and a normal plasma sample are mixed with factor XI-deficient plasma, preferably by adding factor XI-deficient plasma in a volume ratio of 2 to a patient sample or normal plasma sample in a volume ratio of 1. Diluting the patient sample with factor XI-deficient plasma compensates for any clotting factor deficiencies in the patient sample, meaning that they have no effect on the subsequent clotting reaction.

[0019] At least one neutralizing substance that neutralizes anticoagulants other than anticoagulants from the group of factor XIa inhibitors is also added to patient samples and normal plasma samples. The purpose is to eliminate the clotting time-prolonging effect of any other anticoagulants that may be present in the patient sample, meaning that they have no effect on the subsequent clotting reaction. The anticoagulant that should preferably be neutralized may vary depending on time and place, as the prevalence of specific anticoagulants may vary over the years or in the health systems of different countries. Multiple neutralizing substances that neutralize anticoagulants other than anticoagulants from the group of factor XIa inhibitors or various anticoagulants other than anticoagulants from the group of factor XIa inhibitors can also be added to patient samples and normal plasma samples.

[0020] Preferably, at least one anticoagulant neutralizing substance is a substance that neutralizes an anticoagulant from the group of thrombin and FXa inhibitors. A suitable thrombin inhibitor neutralizing substance is, for example, idarucizumab, and a suitable FXa inhibitor neutralizing substance is, for example, andexanet alfa and ciraparantag.

[0021] Particularly preferably, the at least one anticoagulant-neutralizing substance is a substance that neutralizes heparin. A suitable heparin-neutralizing substance is, for example, protamine sulfate.

[0022] To prepare a first reaction mixture, a coagulation activator of the intrinsic blood coagulation system is then added to a patient sample mixed with factor XI-deficient plasma and at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors. Similarly, to prepare a second reaction mixture, the same coagulation activator of the intrinsic blood coagulation system is added to a normal plasma sample mixed with factor XI-deficient plasma and at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors.

[0023] Clot activators of the intrinsic blood clotting system typically consist of a combination of phospholipids (so-called partial thromboplastin), calcium ions and a surfactant, such as kaolin or ellagic acid or silica. When a clot activator of the intrinsic blood clotting system is added to a blood or plasma sample, blood clotting is initiated in the reaction mixture.

[0024] The time (in seconds) until clot formation occurs, i.e., clotting time, or more precisely, activated partial thromboplastin time (APTT), is then measured in the two reaction mixtures. Clotting time can be determined manually or by automated methods. In the case of automated determinations, it is very common to measure a mechanical or optical property of the reaction mixture, such as viscosity or turbidity. In all automated measurements, the property of the reaction mixture is continuously measured, and conventional evaluation methods can be used to determine the clotting time as the endpoint, using the change in property over time.

[0025] According to the present invention, what is measured is the clotting time (APTT XI) in the first reaction mixture containing the patient sample. 試料 ), and the clotting time (APTT XI) in a second reaction mixture containing a normal plasma sample. 正常 The two clotting times thus determined are then calculated as the index APTT XI 試料 / APTT XI 正常 is used to form an index, which is then compared with a predetermined reference value.

[0026] The reference index is typically determined in advance by determining the clotting times of a statistically sufficient number of samples from patients known to have received therapy with anticoagulants from the group of factor XIa inhibitors, and from apparently healthy normal donors who have not received treatment, and optionally from patients known to have received therapy with anticoagulants other than anticoagulants from the group of factor XIa inhibitors (e.g., heparin, direct thrombin, or factor Xa inhibitors) using the modified APTT test according to the present invention (addition of FXI-deficient plasma and an anticoagulant-neutralizing substance), forming indices of clotting times for factor XIa inhibitor-positive samples and factor XIa inhibitor-negative samples, and relating these to the clotting times of normal plasma samples. From the series of indices formed, reference values ​​(reference indexes, limit values, cutoff values) can then be determined that allow highly reliable discrimination between factor XIa inhibitor-negative and factor XIa inhibitor-positive patient samples.

[0027] Index APTT XI 試料 / APTT XI 正常 It has been found that if β is below a given reference value, the presence of an anticoagulant from the group of factor XIa inhibitors in the patient sample can be excluded.

[0028] The present invention relates to a test kit for use in a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample, comprising: a) Factor XI deficient plasma, and b) at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors Further provided is a test kit containing:

[0029] The factor XI-deficient plasma and at least one neutralizing substance (both as described above) can be provided as separate reagents, each in liquid form or as a resuspendable lyophilizate. Alternatively, the test kit can contain a reagent containing a mixture of factor XI-deficient plasma and at least one neutralizing substance.

[0030] The reagent containing at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors, whether it is a separate reagent or a reagent that additionally contains factor XI-deficient plasma or a reagent that additionally contains a coagulation activator of the intrinsic blood coagulation system (see below), preferably contains a neutralizing substance that neutralizes anticoagulants from the group of thrombin and FXa inhibitors, such as at least one of the thrombin inhibitor neutralizing substance idarucizumab and / or the FXa inhibitor neutralizing substances andexanet alfa and ciraparantag. Particularly preferably, the reagent contains the heparin neutralizing substance protamine sulfate.

[0031] Preferably, the test kit according to the present invention further contains a coagulation activator of the intrinsic blood coagulation system. The coagulation activator is preferably provided as a separate reagent, in liquid form or as a resuspendable lyophilizate, and preferably contains a combination of a phospholipid and a contact activator, preferably a combination of a phospholipid and a contact activator from the group consisting of ellagic acid, kaolin, and silica. The coagulation activator reagent may also contain calcium chloride. Alternatively, calcium chloride may be present in the test kit as a separate reagent.

[0032] In yet another embodiment, the test kit may contain a reagent containing a mixture of a coagulation activator and at least one neutralizing substance of the intrinsic blood coagulation system.

[0033] The present invention further provides the use of a test kit according to the invention in a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample, preferably in a method as described above. [Brief explanation of the drawings]

[0034] [Figure 1]1 is a graphical representation of the index APTT XI sample / APTT XI normal formed for various samples and calculated from the APTT clotting times of the samples determined by the present invention and normal plasma samples, compared to the index APTT sample / APTT normal calculated from the APTT clotting times of the samples determined by conventional methods and normal plasma samples. For samples with an index APTT XI sample / APTT XI normal below the cutoff value of 1.21, the presence of 1 μM or more of asundexian (therapeutic factor XIa inhibitors) can be excluded. DETAILED DESCRIPTION OF THE INVENTION

[0035] The following examples serve to illustrate and should not be understood as limiting. [Example]

[0036] Determination of the conventional APTT and the modified APTT according to the invention (APTT XI) in samples differing in terms of blood coagulation status To activate the intrinsic blood coagulation system, an APTT reagent containing ellagic acid and phospholipids (Dade Actin FS Activated PTT Reagent, Siemens Healthineers) was used. To determine conventional APTT, 50 μL of (undiluted) sample was mixed with 50 μL of APTT reagent. After 3 minutes of incubation at 37°C, 50 μL of 25 mM calcium chloride solution was added to the reaction mixture, and the APTT clotting time in seconds was determined using an automated analyzer.

[0037] To perform the method of the present invention, the APTT reagent was modified to contain the heparin-neutralizing substance protamine sulfate (2 μg / mL) in addition to ellagic acid and phospholipids. Furthermore, before adding the modified APTT reagent, the sample was mixed with factor XI-deficient plasma by mixing a volume ratio of 1 sample with a volume ratio of 2 factor XI-deficient plasma. To determine the modified APTT XI of the present invention, 50 μL of the sample mixed with factor XI-deficient plasma was mixed with 50 μL of the modified APTT reagent. After 3 minutes of incubation at 37°C, 50 μL of 25 mM calcium chloride solution was added to the reaction mixture, and the APTT XI clotting time in seconds was determined using an automated analyzer.

[0038] Plasma samples known to have various coagulation disorders and longer than normal APTT clotting times, such as samples with coagulation factor deficiencies, plasma samples containing various anticoagulants or containing other interfering factors (e.g., lupus anticoagulant), were tested (see Table 1).

[0039] In particular, the following sample types were tested:

[0040] [Table 1]

[0041] The conventional APTT (APTT) and the modified APTT according to the invention (APTT XI) in seconds were determined for each sample, and for each sample: 1.) the APTT determined for the sample and the index of the APTT of a normal plasma pool (APTT 試料 / APTT 正常 ) and 2.) the APTT XI determined for the sample and the index of the APTT XI of the normal plasma pool (APTT XI 試料 / APTT XI 正常 ) was calculated (see Table 1). The results are also shown graphically in Figure 1.

[0042] [Table 2]

[0043] The results are shown in Table 1. The APTT XI index determined according to the invention is used in the group of samples tested. 試料 / APTT XI 正常 A value of less than 1.21 excludes the presence of 1 μM or more of asundexian (a therapeutic factor XIa inhibitor) in the sample. Only samples with a severe factor XI deficiency (less than 50%) are reliably excluded; however, due to the rarity of this disease, this does not have a discernible effect on the specificity of the method. On the other hand, the index APTT, calculated from the APTT clotting times of samples determined by conventional methods and normal plasma samples, 試料 / APTT 正常 cannot distinguish between factor XIa inhibitor-positive and factor XIa inhibitor-negative samples.

[0044] The exclusion of factor XIa inhibitors using the cutoff value of 1.21 determined according to the present invention is unlikely to be completely selective in large patient populations. The most likely cutoff value must—as is customary in such cases—be determined using receiver operating characteristic analysis in clinical trials with a statistically sufficient number of patient samples, and a balance must be struck between sensitivity and specificity.

Claims

1. 1. A method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample, comprising: a) A first reaction mixture containing a patient sample and a second reaction mixture containing a normal plasma sample are prepared by adding each of the two samples to a reaction mixture containing the patient sample. i) factor XI-deficient plasma, and ii) at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors, and iii) Coagulation activators of the intrinsic blood coagulation system and mixing the resulting mixture with the b) Clotting time (APTT XI) in the first reaction mixture 試料 ), and the clotting time in the second reaction mixture (APTT XI 正常 ) measuring the c) The index APTT XI from two clotting times 試料 / APTT XI 正常 forming a d) comparing the index with a predetermined reference value; and e) Index APTT XI 試料 / APTT XI 正常 excluding anticoagulants from the group of factor XIa inhibitors in the patient sample if A method comprising:

2. 10. The method of claim 1, wherein the at least one neutralizing substance neutralizes an anticoagulant from the group of thrombin and FXa inhibitors.

3. 3. The method according to claim 2, wherein at least one neutralizing substance for anticoagulants from the group of thrombin inhibitors is idarucizumab and at least one neutralizing substance for anticoagulants from the group of FXa inhibitors is andexanet alfa or ciraparantag.

4. 3. The method of claim 2, wherein the at least one neutralizing agent neutralizes heparin.

5. 5. The method of claim 4, wherein the at least one heparin neutralizing agent is protamine sulfate.

6. The method according to any one of claims 1 to 5 for excluding XIa factor inhibitors from the group consisting of Asundexian, Milvexian, Abelacimab, Osocimab, Glutisivalt (Xomab), REGN9933, MK-2060, ONO-7684 and ONO-5450598.

7. 1. A test kit for use in a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample, comprising: a) factor XI-deficient plasma, and b) at least one neutralizing substance that neutralizes anticoagulants other than those from the group of factor XIa inhibitors; A test kit comprising:

8. 8. The test kit of claim 7, wherein the at least one neutralizing substance neutralizes an anticoagulant from the group of thrombin and FXa inhibitors.

9. 9. The test kit according to claim 8, wherein at least one neutralizing substance for anticoagulants from the group of thrombin inhibitors is idarucizumab and at least one neutralizing substance for anticoagulants from the group of FXa inhibitors is andexanet alfa or ciraparantag.

10. 9. The test kit of claim 8, wherein the at least one neutralizing agent neutralizes heparin.

11. 11. The test kit of claim 10, wherein the at least one neutralizing agent is protamine sulfate.

12. A test kit according to any one of claims 7 to 11, further comprising a coagulation activator of the intrinsic blood coagulation system.

13. 13. The test kit according to claim 12, wherein the coagulation activator of the intrinsic blood coagulation system is a combination of a phospholipid and a contact activator, preferably a combination of a phospholipid and a contact activator from the group consisting of ellagic acid, kaolin and silica.

14. A test kit according to any one of claims 7 to 13, further comprising calcium chloride.

15. A test kit according to any one of claims 7 to 14, which contains a reagent comprising a mixture of factor XI-deficient plasma and at least one neutralising substance.

16. A test kit according to any one of claims 12 to 14, containing a reagent comprising a mixture of a coagulation activator of the intrinsic blood coagulation system and at least one neutralizing substance.

17. Use of a test kit according to any one of claims 7 to 16 in a method for excluding anticoagulants from the group of factor XIa inhibitors in a patient sample.

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