Factor xa reagent

Abstract

To provide a factor Xa reagent.SOLUTION: Exemplary compositions include Factor Xa, a substrate, one or more guanidine derivatives, and in some cases heparin. An exemplary method of detecting an anticoagulant in a sample includes combining or contacting a composition with a sample and measuring factor Xa activity based on substrate status. The composition modulates the activity of factor Xa in the sample, and the modulation includes a reduction in the rate of clotting of the sample caused by factor Xa. The presence of an anticoagulant is based on the activity of factor Xa.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to compositions of factor Xa reagents and their uses. [Background technology]

[0002] Factor Xa (FXa) is the activated form of factor X, or thrombokinase, involved in coagulation. Factor X is an enzyme that promotes clotting of a test sample, such as whole blood or plasma. An exemplary factor Xa inhibitor is an anticoagulant that functions by selectively blocking the activity of factor Xa, thereby preventing or inhibiting clot formation in the test sample. Summary of the Invention [Means for solving the problem]

[0003] Exemplary compositions include Factor Xa, a substrate, and one or more guanidine derivatives. The composition may include one or more of the following features, alone or in combination:

[0004] The composition may include heparin. The one or more guanidine derivatives may be or include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, or N-benzyl-N-methylguanidine (N indicates a substituent on the nitrogen atom). The one or more guanidine derivatives may be or include monoalkylated guanidine derivatives. The one or more guanidine derivatives may be or include dialkylated guanidine derivatives. The one or more guanidine derivatives may be represented by the following formula:

[0005] [ka]

[0006] It may be or include an alkylated guanidine having the chemical structure represented by the formula: wherein R1 is H, methyl, ethyl, or benzyl; R2 is H, methyl, ethyl, or benzyl; N is nitrogen; and H is hydrogen.

[0007] The one or more guanidine derivatives may include monoalkylated guanidines having an alkyl group length different from that of 1-methylguanidine, or dialkylated guanidines having an alkyl group length different from that of 1,1-dimethylguanidine or 1,1-diethylguanidine. The one or more guanidine derivatives may be or may include a sulfate salt. The substrate may be or may include a chromogenic substrate. The composition may be a kit or may be included in a device containing a kit, or in a device that is a cartridge or includes a cartridge.

[0008] An exemplary method for detecting an anticoagulant in a sample includes combining or contacting the sample with a composition comprising factor Xa, a guanidine derivative, and a substrate, and measuring the activity of factor Xa based on the status of the substrate, wherein the presence of the anticoagulant is based on the activity of factor Xa. The method may include one or more of the following features, alone or in combination:

[0009] The anticoagulant may include a factor Xa inhibitor, which may be a direct factor Xa inhibitor. The anticoagulant may be or include a factor Xa inhibitor, including one or more of rivaroxaban, apixaban, edoxaban, or betrixaban. The anticoagulant may be or include an indirect factor Xa inhibitor based on antithrombin. The indirect factor Xa inhibitor may include one or more of low molecular weight heparin, unfractionated heparin, fondaparinux, or danaparoid. The substrate may be or include a color change within the substrate. The composition may modulate the activity of factor Xa in a sample, where the modulation includes a reduction in the rate of coagulation or substrate cleavage caused by factor Xa.

[0010] Any two or more features described in this specification (including the Abstract) may be combined to form an embodiment not specifically described herein.

[0011] The systems and processes described herein, or portions thereof, may be implemented as one or more devices or methods and may include one or more processing devices and computer memory for storing executable instructions for implementing the control of various functions. The systems, processes, and compositions described herein, including but not limited to devices, methods, and / or reagents, can be configured through, for example, design, manufacture, construction, combination of two or more substances, arrangement, placement, programming, operation, activation, deactivation, and / or control.

[0012] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will become apparent from the description, drawings, and claims. [Brief explanation of the drawings]

[0013] [Figure 1A]1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1B] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1C] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1D] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1E] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1F] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 1G] 1 shows the structural formulas of example guanidine derivatives that may be used in the exemplary Factor Xa reagents described herein. [Figure 2A] 1 is a flow chart illustrating an exemplary method that may be performed to detect a Factor Xa inhibitor in a test sample using a chromogenic assay. [Figure 2B] 1 is a flow chart illustrating an exemplary method that may be performed to detect a Factor Xa inhibitor in a test sample using fluorogenic analysis. [Figure 3] 1 is a graph containing exemplary plots showing Factor Xa inhibition, shown on the Y-axis, against various concentrations of salt or guanidine derivative, shown on the X-axis. [Figure 4] 1 is a graph containing exemplary plots showing Factor Xa inhibition on the Y-axis against various concentrations of guanidine derivatives on the X-axis. [Figure 5] 1 is a graph containing exemplary plots showing factor Xa activity on the Y-axis against various concentrations of direct factor Xa inhibitors on the X-axis. [Figure 6]1 is a graph containing exemplary plots showing substrate exchange times on the Y-axis against various levels of direct Factor Xa inhibitors on the X-axis for both dimethylguanidine and salts. [Figure 7] 1 is a graph containing exemplary plots showing chromogenic activity on the Y-axis against various reaction rates on the X-axis for reactions containing various amounts of dimethylguanidine. 2 is a graph containing exemplary plots showing chromogenic activity on the Y-axis with different reaction rates. [Figure 8] 1 is a graph containing exemplary plots showing the linear correlation between the concentration of unfractionated heparin, shown on the X-axis, and Factor Xa activity, shown on the Y-axis, in reaction mixtures containing dimethylguanidine. [Figure 9] 1 is a graph containing exemplary plots showing factor Xa activity, shown on the Y-axis, compared to antithrombin activity, shown on the X-axis, in the absence of dimethylguanidine. [Figure 10] 1 is a graph containing exemplary plots showing factor Xa activity compared to antithrombin activity in the presence of 20 millimolar (mM) dimethylguanidine for various factor Xa concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0014] Like reference symbols in the various drawings indicate like elements.

[0015] Described herein are examples of reagents (herein referred to as "factor Xa reagents") that modulate the activity of factor Xa in a reaction under analysis. Modulation in this context may include affecting the rate of clotting of a test sample caused by factor Xa. For example, modulation may include reducing the rate of clotting of a test sample caused by factor Xa relative to the rate of clotting of the test sample that would have occurred in the absence of the factor Xa reagent. Modulation of factor Xa activity may have advantages in assays detecting clotting of a test sample caused by factor Xa, such as allowing for optimization of reaction kinetics and sample-to-reagent volume ratios, and may allow for the use of larger amounts of test sample and / or higher amounts and / or concentrations of factor Xa in an assay compared to the amount of test sample, amount, and concentration of factor Xa that would have been used in an identical assay in the absence of the factor Xa reagent. Larger amounts of test sample and / or higher amounts and / or concentrations of factor Xa may facilitate the detection of factor Xa inhibitors or reversal of such inhibitors in an assay. Slower reaction rates may allow for more accurate detection of factor Xa inhibitors or neutralization of the same in the assay.

[0016] The Factor Xa reagent may be in a solid form, such as a powder or a film. In some embodiments, the Factor Xa reagent may be lyophilized or desiccated. The Factor Xa reagent may be in liquid or solid form at least in the temperature range of 0°C to 100°C. Temperatures described herein are at standard pressure of 1 atmosphere (101.325 kPa).

[0017] The test sample may be whole blood, a portion of whole blood, a sample derived from whole blood, a portion of whole blood depleted of one or more blood components (e.g., depleted of white blood cells, red blood cells, platelets, or proteins (e.g., albumin)), or an isolated component of whole blood. In one example, the test sample may be platelet-free plasma (which is whole blood depleted of blood components (platelets)). The test sample may be collected from a subject, such as a human, using standard techniques.

[0018] An exemplary factor Xa reagent may include factor Xa, a substrate, and one or more components that modulate the activity of factor Xa in a test sample. In some embodiments, an aqueous buffer may be included. Examples of one or more components that may be included in a factor Xa reagent to modulate the activity of factor Xa contained in a test sample include guanidine or one or more guanidine derivatives. Guanidine is a nitrogen-rich organic compound having the formula HNC(NH2)2. The structural formula of guanidine 100 is shown in Figure 1A.

[0019] An example of a guanidine derivative that may be included in a factor Xa reagent to regulate factor Xa activity may be alkylated guanidine. Guanidine derivatives, such as alkylated guanidine and other compounds described herein, regulate the amidolytic activity of factor Xa. Amidolytic activity refers to the cleavage of peptide bonds in polypeptides or proteins by protease enzymes (including factor Xa). Unless otherwise specified, factor Xa activity as described herein includes amidolytic activity.

[0020] In alkylated guanidines, one or both of the hydrogen (H) atoms (101 and 102) may be replaced with larger chemical groups (shown as R1 (111) and R2 (112) in Figure 1B). Here, R1 (111) may be a methyl group (131) (Figure 1D), an ethyl group (141) (Figure 1E), or a benzyl group (151) (Figure 1F), and R2 (112) may be a methyl group, an ethyl group, or a benzyl group. The methyl group (131) contains an alkyl derived from methane, contains one carbon atom with three hydrogen atoms attached, and has the chemical formula -CH3. The ethyl group (141) contains an alkyl substituent of the formula -CH2CH3 derived from ethane (C2H6). The benzyl group (151) contains a substituent or molecular fragment with the structure R-CH2-C6H5. Benzyl is characterized by a phenyl group (C6H5) attached to a methylene group (-CH2-).

[0021] Other examples of guanidine derivatives that may be included in a Factor Xa reagent to modulate Factor Xa activity may include mono-alkylated guanidine, di-alkylated guanidine, or a combination of mono- and di-alkylated guanidine. Non-limiting examples of guanidine derivatives that may be included in a Factor Xa reagent include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine (CH 13 N3), or N-benzyl-N-methylguanidine (N indicates a substituent on the nitrogen atom). Figure 1C shows the structural formula of 1-methylguanidine (120), Figure 1D shows the structural formula of 1,1-dimethylguanidine (130), Figure 1E shows the structural formula of 1,1-diethylguanidine (140), and Figure 1F shows the structural formula of N-benzyl-N-methylguanidine (150).

[0022] In one example, methylated guanidinium (C2H8N3 + Methylguanidine, CH, regulates factor Xa activity. The rate of regulation is determined by the second 1N-methylation (1,1-dimethylguanidine, CH). 10 N3 +) The rate can be adjusted by the length of the alkyl group or by adding bulky groups (such as aromatic rings) to the second 1N position.

[0023] Other examples of guanidine derivatives that may be included in a Factor Xa reagent to modulate Factor Xa activity may include mono-alkylated guanidine derivatives containing an alkyl group of a different length than the alkyl group in 1-methylguanidine (120) of FIG. 1C. For example, the alkyl group of a different length than the alkyl group in 1-methylguanidine (120) may contain two or more carbon atoms. An alkyl group having two carbon atoms has the formula -CH2CH3. A mono-alkylated guanidine derivative containing an alkyl group having two carbon atoms has the formula CH9N3. An alkyl group having three carbon atoms has the formula -CH2CH2CH3. A mono-alkylated guanidine derivative containing an alkyl group having three carbon atoms has the formula CH 11 N3. An alkyl group having four carbon atoms has the formula -CH2CH2CH2CH3. A monoalkylated guanidine derivative containing an alkyl group having four carbon atoms has the formula CH 13 Has N3.

[0024] Other examples of guanidine derivatives that may be included in Factor Xa reagents to modulate Factor Xa activity include dialkylated guanidine derivatives containing one or both alkyl groups that are different in length from the alkyl groups in 1,1-dimethylguanidine (130) of FIG. 1D. The alkyl groups that are different in length from the alkyl groups in 1,1-dimethylguanidine (130) may contain one or more carbon atoms. An alkyl group with one carbon has the formula -CH3. A dialkylated guanidine derivative containing one alkyl group with one carbon and one alkyl group with two carbon atoms has the formula CH4. 11 Has N3.

[0025] Other examples of guanidine derivatives that may be included in Factor Xa reagents to modulate Factor Xa activity include dialkylated guanidine derivatives containing one or both alkyl groups that differ in length from the alkyl groups in 1,1-diethylguanidine (140) of FIG. 1E. The alkyl groups that differ in length from the alkyl groups in 1,1-diethylguanidine 140 may contain three or more carbon atoms. An alkyl group having three carbon atoms has the formula -CH2CH2CH3. A dialkylated guanidine derivative containing one alkyl group having two carbon atoms and one alkyl group having three carbon atoms has the formula CH 15 N3. An alkyl group having four carbon atoms has the formula -CH2CH2CH2CH3. A dialkylated guanidine derivative containing one alkyl group having two carbons and one alkyl group having four carbons has the formula CH 17 Has N3.

[0026] Other examples of guanidine derivatives that may be included in Factor Xa reagents to modulate Factor Xa activity include dialkylated guanidine derivatives containing one alkyl group longer than that of 1,1-dimethylguanidine (130) or 1,1-diethylguanidine (140) and another alkyl group of the same or shorter length than that of 1,1-dimethylguanidine (130) or 1,1-diethylguanidine (140). The alkyl group of the same length as that of 1,1-dimethylguanidine (130) has the formula -CH3. The alkyl group of the same length as that of 1,1-diethylguanidine (140) has the formula -CH2CH3.

[0027] Another example of a guanidine derivative that may be included in a Factor Xa reagent to modulate Factor Xa activity is the conjugate acid of guanidine (chemical formula C(NH2)3 +(having the formula: ##STR00001##). Examples of guanidine derivative salts include a conjugate acid of guanidine, a carbonate salt containing the conjugate acid of guanidine, a chloride salt containing the conjugate acid of guanidine, a nitrate salt containing the conjugate acid of guanidine, a perchlorate salt containing the conjugate acid of guanidine, and / or a picrate salt containing the conjugate acid of guanidine. An example of a guanidine derivative salt that may be included in the Factor Xa reagent may include the sulfate form of guanidine (160), which has the structural formula shown in FIG. 1G.

[0028] Guanidine derivatives that may be included in factor Xa reagents to modulate factor Xa activity may include combinations of two or more of the examples of guanidine derivatives described herein. Exemplary combinations of guanidine derivatives that may be included in a Factor Xa reagent to modulate Factor Xa activity may be a combination of 1-methylguanidine (120) and 1,1-dimethylguanidine (130), a combination of 1-methylguanidine (120) and 1,1-diethylguanidine (140), a combination of 1-methylguanidine (120) and N-benzyl-N-methylguanidine (150), a combination of 1,1-dimethylguanidine (130) and 1,1-diethylguanidine (140), a combination of 1,1-diethylguanidine (140) and N-benzyl-N-methylguanidine (150), and / or a combination of 1,1-dimethylguanidine (130) and N-benzyl-N-methylguanidine (150).

[0029] Guanidine derivatives that may be included in a Factor Xa reagent to modulate Factor Xa activity may include one or more exemplary guanidine derivatives described herein in combination with guanidine itself (FIG. 1A). Exemplary combinations of guanidine derivatives that may be included in a Factor Xa reagent to modulate Factor Xa activity may be guanidine and 1-methylguanidine, guanidine and 1,1-dimethylguanidine, guanidine and 1,1-diethylguanidine, and / or guanidine and N-benzyl-N-methylguanidine.

[0030] In some embodiments, the guanidine and / or individual guanidine derivatives described herein may each be present in a Factor Xa reagent at a concentration of about 0.1 to 100 millimoles (mM) of guanidine derivative per 1 nkat / mL of Factor Xa. One kat (kat) is the amount of enzyme that converts one mole of substrate per second. As used herein, "kat" refers to nanokat. As used herein, "about" allows for deviations from a given number, such as, but not limited to, deviations of 1%, 2%, 3%, 4%, or 5%.

[0031] In some embodiments, the aqueous buffer that may be included in the liquid form of the Factor Xa reagent may be or may include 2-morpholin-4-ylethanesulfonic acid (MES) or the structural analog of MES, 3-(N-morpholino)propanesulfonic acid (MOPS). In some implementations, the aqueous buffer in the Factor Xa reagent may be HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-Tris buffer, citrate, ADA (N-(2-acetamido)iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid) buffer, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffer, imidazole / iminodiacetate, or the like. The buffer may be or include dazolium buffer, bis-trispropane buffer, maleic acid buffer, phosphate buffer, MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) buffer, BES (bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid) buffer, MOPS buffer, TES (tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid) buffer, and / or MOPS (3-(N-morpholino)propanesulfonic acid) buffer.

[0032] In some embodiments, the aqueous buffer in the Factor Xa reagent may be or may contain one or more carboxylic acid derivatives. Examples of carboxylic acid derivatives that may be included in the aqueous buffer may include carboxylate acetate, ethylenediaminetetraacetate, butanetetracarboxylate, propanetricarboxylate, citrate, succinate, tartrate, malonate, and / or gluconate.

[0033] In some embodiments, the aqueous buffer in the Factor Xa reagent may contain a preservative. Examples of preservatives that may be contained in the aqueous buffer include Proclin™ 300 (3% 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT)), sodium azide, gentamicin, thimerosal, butylated hydroxytoluene (BHT), sucrose, trehalose, glycerin, sodium citrate, poloxamer, cetyltrimethylammonium bromide (CTAB), or a combination of two or more thereof.

[0034] In some embodiments, the factor Xa reagent may contain one or more substrates to measure the amount of factor Xa activity in a test sample in the presence of a factor Xa inhibitor. An example of a substrate that may be included in the factor Xa reagent includes a chromogenic substrate. In some examples, the chromogenic substrate is a colorless substance that changes into a colored substance in response to an enzyme reaction. In one example, the chromogenic substrate changes its state or color based on the amount of factor Xa activity in the test sample. Therefore, as described below, the chromogenic substrate allows the activity of factor Xa to be measured based on the color of the substrate. An example of a chromogenic substrate that may be used in a Factor Xa reagent is (S)-2-((2S,3S)-2-(2,5-dioxopyrrolidin-1-yl)-3-methylpentanamido)-N-(2-(((S)-5-guanidino-1-((4-nitrophenyl)amino)-1-oxopentan-2-yl)amino)-2-oxoethyl)-5-oxo-5-(piperidin-1-yl)pentanamide hydrochloride (Pefachrome® Factor Xa Examples of suitable nucleotides include, but are not limited to, S-2732™), S-2222™ (Bz-Ile-Glu(-OR)-Gly-Arg-pNA·HCl), S-2765™ (ZD-Arg-Gly-Arg-pNA·2HCl), and Spectrozyme FXa (CHO-CO-D-CHG-Gly-ArgpNA.AcOH).

[0035] Other examples of substrates that may comprise a factor Xa reagent include fluorogenic substrates. Exemplary fluorogenic substrates include non-fluorescent materials that generate a fluorescent compound upon enzymatic action. In one example, the fluorogenic substrate shines or glows due to fluorescence based on the amount of factor Xa activity in the test sample. The greater the amount of fluorescence, the greater the amount of factor Xa activity in the test sample. Thus, the fluorogenic substrate can enable measurement of factor Xa activity based on the amount of fluorescence, as described below. Examples of fluorogenic substrates include, but are not limited to, Z-Gly-Gly-Arg-AMC, Technothrombin®, a 6-amino-1-naphthalenesulfonamide (ANSN)-based fluorogenic substrate (Mes-D-LGR-ANSN(C2H5)2), and / or a calibrated automated thrombogram.

[0036] Examples of factor Xa inhibitors that may be detected using the factor Xa reagent described herein include direct factor Xa inhibitors. Direct factor Xa inhibitors block factor Xa and prevent the conversion of prothrombin to thrombin in the final common pathway of thrombus formation. Examples of such direct factor Xa inhibitors include, but are not limited to, rivaroxaban, apixaban, edoxaban, and betrixaban.

[0037] Examples of factor Xa inhibitors that may be detected using the factor Xa reagent described herein include indirect factor Xa inhibitors. Examples of indirect factor Xa inhibitors are based on antithrombin. Antithrombin is a plasma glycoprotein that inhibits thrombin and other activated serine proteases, including factor Xa. Examples of indirect factor Xa inhibitors include, but are not limited to, one or more of low molecular weight heparin, unfractionated heparin, fondaparinux, and danaparoid.

[0038] (Chromogenic and fluorogenic assays) In some embodiments, detection of a factor Xa inhibitor or neutralization of the inhibitor in a test sample may be performed using a chromogenic or fluorogenic assay. Chromogenic assays involve detection using a colored substrate. Fluorogenic assays involve detection using a fluorescent substrate. For each assay, one or more curves may be pre-generated based on the known amount of factor Xa activity, as defined by fluorescence or absorbance values, and the corresponding known concentration of the factor Xa inhibitor in the reaction mixture. In some embodiments, one or more curves may be generated for different factor Xa inhibitors. When the assay is performed on a diagnostic test device or clinical analyzer, the curves may be stored in memory for retrieval when the test sample is analyzed. The curves may be used to detect the factor Xa inhibitor and its concentration in the reaction mixture.

[0039] FIG. 2A illustrates an exemplary method 220 that may be performed to detect a factor Xa inhibitor in a test sample using a chromogenic assay. According to method 220, a test sample is received (221) and mixed, combined, or contacted with a factor Xa reagent containing a chromogenic substrate (222). The absorbance of the cleaved chromogenic substrate, representing the remaining factor Xa activity in the resulting mixture, is measured spectrophotometrically (223). In this example, the absorbance of a series of samples with known concentrations of factor Xa inhibitor was previously measured and used to generate a curve relating absorbance values ​​to known factor Xa inhibitor concentrations. In operation 224, this curve is retrieved. The factor Xa inhibitor concentration in the test sample is determined by comparing the absorbance measurements of the mixture to the curve (225). That is, the absorbance of the mixture is identified on the curve, and the corresponding factor Xa inhibitor concentration is obtained from the curve. If the test sample is previously known to contain a known concentration of a factor Xa inhibitor, and the determined factor Xa activity does not correspond to the expected effect of the known factor Xa inhibitor on the test sample, the results of this analysis may indicate that the effect of the factor Xa inhibitor has been reversed.

[0040] Determining the factor Xa inhibitor concentration is not limited to methods using the curve prepared in this example. In some embodiments, additional methods, such as machine learning algorithms, may use the results of the colorimetric assay (e.g., absorbance and / or other parameters derived from the results) to determine the factor Xa inhibitor concentration in the test sample.

[0041] In some embodiments, the chromogenic assay may be performed on the ACL TOP® Family Coagulation Test System offered by Werfen® SA. This system includes a fully automated system for loading and unloading cuvettes that hold a mixture of a test sample, such as blood, and a Factor Xa reagent described herein. In another non-limiting example, the chromogenic assay may be performed on a SPECTROstar Nano® absorbance microplate reader offered by BMG LABTECH® Inc. The system includes a microplate- or cuvette-based system for reading the full spectrum absorbance of a mixture of a test sample and a Factor Xa reagent described herein.

[0042] FIG. 2B illustrates an example of a method 230 that may be performed to detect a factor Xa inhibitor in a test sample using a fluorogenic assay. The test sample is received (231) and mixed, combined, or contacted (232) with a factor Xa reagent containing a fluorogenic substrate. The fluorescence of the cleaved fluorogenic substrate in the resulting mixture, representing the remaining factor Xa activity, is measured (233). In this example, the fluorescence from a series of samples with known factor Xa inhibitor concentrations is previously measured and used to generate the aforementioned curve relating fluorescence values ​​to known factor Xa inhibitor concentrations. In operation 234, the curve is searched. The factor Xa inhibitor concentration in the test sample is determined (235) by comparing the fluorescence of the mixture with the curve of known factor Xa inhibitor concentrations. That is, the fluorescence of the mixture is identified on the curve, and the corresponding factor Xa inhibitor concentration is obtained from the curve. As before, if the test sample is known in advance to contain a known concentration of a factor Xa inhibitor, and the determined factor Xa activity does not correspond to the expected effect of the known factor Xa inhibitor on the test sample, the results of this analysis may indicate that the effect of the factor Xa inhibitor has been neutralized.

[0043] Determining the concentration of a factor Xa inhibitor is not limited to methods using the curve prepared in this example. In some embodiments, additional methods, such as machine learning algorithms, use the results of the fluorogenic analysis (e.g., fluorescence and / or other parameters derived from the results) to determine the concentration of a factor Xa inhibitor in a test sample.

[0044] In some embodiments, fluorogenic analysis may be performed on a PHERAstar® FSX fluorescence microplate reader provided by BMG LABTECH® Inc. This system includes a microplate-based system for high-throughput and multimode reading of test samples and Factor Xa reagents described herein. In another non-limiting example, fluorogenic analysis may be performed on a Xenius XOF spectrofluorometer provided by SAFAS® Ltd. This system includes a microplate-based or cuvette-based system for in situ and high-throughput reading of test samples and Factor Xa reagents described herein.

[0045] (antithrombin detection) In some embodiments, the Factor Xa reagent composition may include heparin, such that the reagent composition includes Factor Xa, a substrate, such as a chromogenic or fluorogenic substrate, one or more guanidine derivatives, and heparin. In some embodiments, the heparin may be included in the reagent at a concentration ranging from about 100 to 4000 U / L (units per liter). An aqueous buffer may be included in the liquid form of the reagent.

[0046] Antithrombin in a test sample may be detected by measuring factor Xa activity using a factor Xa reagent containing heparin. Antithrombin is a natural anticoagulant consisting of a glycoprotein that inactivates enzymes in the coagulation system. The heparin and factor Xa in the reagent form a complex with antithrombin and inhibit the activity of factor Xa in the test sample. The resulting residual factor Xa activity in the test sample is inversely proportional to the amount of antithrombin in the test sample. For example, a larger amount of antithrombin in the test sample will result in a lower factor Xa activity value, and a smaller amount of antithrombin in the test sample will result in a higher factor Xa activity value.

[0047] The above-described systems may be used to perform chromogenic or fluorogenic analyses of test samples. For example, factor Xa activity in a test sample may be measured after contact with a factor Xa reagent containing heparin, which may be performed by measuring the absorbance or fluorescence of a substrate in the test sample as described above. The resulting measurements may be compared to a predefined curve relating absorbance or fluorescence to known values ​​of antithrombin in a manner similar to that described with respect to Figures 2A and 2B. Thus, the concentration of antithrombin in the test sample can be determined to be the value of antithrombin on the curve corresponding to the absorbance or fluorescence measurement of the substrate in the test sample.

[0048] Modulation of Factor Xa Activity by Alkylating Guanidines This experiment is presented to demonstrate that alkylated guanidine derivatives modulate factor Xa activity.

[0049] In this experiment, the activity of factor Xa in the presence of guanidine sulfate and its derivatives (e.g., 1-methylguanidine sulfate, 1,1-dimethylguanidine sulfate, 1,1-diethylguanidine, N-benzyl-N-methylguanidine sulfate, 1,1,3,3-tetramethylguanidine, and 1,1-dimethylbiguanide hydrochloride) was assessed using a chromogenic assay. In the experiment, each guanidine derivative was dissolved in deionized (DI) water to a final concentration of 100 mM. Multiple such guanidine dilutions were prepared. The guanidine derivative levels in these solutions were tested in triplicate at 37°C as follows: 40 μL of each guanidine derivative level solution was incubated for 20 to 60 seconds (seconds) before adding 40 μL of a 6 nkat / mL factor Xa solution in 20 mM Hepes buffer (pH 7.5) to the reaction. After an incubation period of 150 to 180 seconds, 100 μL of a 1.5 mM solution of the Factor Xa chromogenic substrate S-2732™ was added to the reaction, and milliabsorbance per minute (mAbs / min) was measured at 405 nm (nanometers) after a 20-second delay.

[0050] Figure 3 shows the results of this experiment, including factor Xa activity plotted against the concentration of guanidine derivative (301) or NaCl (salt) (302) (in millimolar units). As shown, NaCl (302) activates factor Xa activity. In contrast, 1-methylguanidine sulfate (303) dose-dependently modulates factor Xa. 1,1-dimethyl-biguanide (304), the active ingredient in metformin, and guanidine sulfate (305) also dose-dependently modulate factor Xa, albeit to a lesser extent than 1-methylguanidine sulfate (303).

[0051] (Effect of alkylated guanidines on factor Xa activity) This experiment was performed to demonstrate that guanidine and its derivatives modulate factor Xa activity.

[0052] In this experiment, a chromogenic assay was used to analyze the effects of guanidine sulfate and guanidine derivatives (1-methylguanidine sulfate, 1,1-dimethylguanidine sulfate, 1,1-diethylguanidine, and N-benzyl-N-methylguanidine sulfate). Using the types of guanidine or guanidine derivatives described herein, multiple dilutions were prepared in water. 40 μL of a 6 nkat / mL solution of factor Xa diluted in 20 mM HEPES buffer (pH 7.5) was mixed with 40 μL of each guanidine or guanidine derivative solution for 20–60 seconds. After an incubation period of 150–180 seconds, 100 μL of a solution of factor Xa chromogenic substrate S-2732™ (1.5 mM) was added to each guanidine and factor Xa mixture. After a 20-second delay, each mixture was measured for color development at 405 nm at mAbs / min.

[0053] Figure 4 shows the results of this experiment. As shown in Figure 4, the greatest degree of factor Xa inhibition was observed in mixture 401 containing dialkylated guanidines (1,1-dimethylguanidine sulfate and 1,1-diethylguanidine sulfate). Compared to the factor Xa inhibition of mixture 401 containing dialkylated guanidine derivatives, mixture 403 containing 1-methylguanidine sulfate and a guanidine derivative containing a benzyl group (N-benzyl-N-methylguanidine sulfate) resulted in reduced factor Xa inhibition. The reduction in factor Xa inhibition was minimal in mixtures containing guanidine sulfate 404 alone.

[0054] We also show that methylation at the second 1N position enhances the inhibition of factor Xa activity by methylated guanidines. Introduction of a bulky benzyl group at the 1N position reduces factor Xa inhibition. The inhibition can be tuned by varying the length of the alkyl group.

[0055] In some embodiments, different guanidine derivatives (e.g., two or more of the guanidine derivatives described herein) can be combined in a composition to modulate Factor Xa activity. In other embodiments, one or more guanidine derivatives and a Factor Xa activator, such as NaCl, can be combined in a composition to modulate Factor Xa activity.

[0056] (Alkyl guanidine enables the detection of factor Xa inhibitors) This experiment was performed to demonstrate that guanidine and its derivatives may be included in reagents for detecting multiple factor Xa inhibitors.

[0057] In this experiment, 100 mM dimethylguanidine was used to replace NaCl in a liquid anti-factor Xa assay for the measurement of factor Xa inhibitors (500). While other factor Xa inhibitor calibrators may be used, in this example, a set of apixaban calibrators was used to specify concentrations (in nanomolar units) for plasma samples spiked with either apixaban, rivaroxaban, edoxaban, or betrixaban. A series of varying amounts of each factor Xa inhibitor was added to spiked samples and neat plasma samples containing no factor Xa inhibitor. Each sample was tested in triplicate, and linear regressions were generated for the four factor Xa inhibitors combined based on the logarithm of the response versus direct factor Xa inhibitor concentration (log(DmAbs)). As shown in the exemplary plot in Figure 5, a combination of factor Xa reagents and calibrators may be used to detect various factor Xa inhibitors, such as apixaban, rivaroxaban, edoxaban, and betrixaban. Therefore, a calibration curve generated using a first factor Xa inhibitor can serve as a universal calibration curve for quantifying calibration curves of different factor Xa inhibitors (e.g., second, third, fourth, etc. factor Xa inhibitors) and provide concentrations equivalent to the first factor Xa inhibitor.

[0058] (Alkylated guanidine allows for the use of different factor Xa concentrations in the assay)

[0059] This experiment demonstrates that guanidine and its derivatives allow the use of different concentrations of factor Xa in the assay.

[0060] As previously described, guanidine derivatives modulate the activity of factor Xa enzyme during the reaction, thereby slowing the reaction. Consequently, when guanidine derivatives are incorporated into the reaction, a larger amount and / or higher concentration of factor Xa can be used in the reaction. In this experiment, factor Xa levels were tested in triplicate reactions at 37°C as follows: 40 μL of a factor Xa solution was incubated for 20–60 seconds before adding 40 μL of a 100 mM aqueous 1,1-dimethylguanidine solution or 40 μL of a 150 mM aqueous NaCl solution to the reaction. After an incubation period of 150–180 seconds, 100 μL of a 1.5 mM solution of factor Xa chromogenic substrate S-2732™ was added to the reaction, and mAb / min was measured at 405 nm after a 20-second delay. In Figure 6, the substrate turnover (e.g., substrate coloration) in the presence of 22 mM 1,1-dimethylguanidine (601) is compared to the substrate turnover in the presence of 33 mM NaCl (602). As can be seen from Figure 6, a larger amount and / or higher concentration of Factor Xa can be added to the reaction to produce the same substrate turnover in the presence of 1,1-dimethylguanidine compared to NaCl. This is because the reaction is slowed by 1,1-dimethylguanidine. As previously mentioned, a larger amount and / or higher concentration of Factor Xa facilitates the detection of Factor Xa inhibitors or neutralization of such inhibitors in the assay.

[0061] (Effect of alkylated guanidine on reaction activity) This experiment was carried out to demonstrate the effect of guanidine derivatives on reaction activity.

[0062] In an exemplary factor Xa inhibitor measurement setup, about 5% of the sample is mixed with a reagent and factor Xa at a concentration of about 2 nkat / mL. A relatively low sample-to-reagent volume ratio, such as 1:20 (where "1" is the sample ratio and 20 is the reagent ratio), may result in clinically irrelevant test results due to dissociation of the complex in the sample or insufficient inhibition of factor Xa by the reagent. These issues may be addressed by using factor Xa reagents of the type described herein. In some embodiments, the factor Xa reagents enable clinically relevant test results using sample-to-reagent volume ratios ranging, for example, from about 1:10 to about 50:1 (i.e., from 1 part sample to 10 parts reagent to 1 part reagent to 50 parts sample).

[0063] In this experiment, 180 μL of plasma sample containing dimethylguanidine (DMG) and 1.5 mM substrate S-2732™ was mixed with 20 μL of factor Xa solution (9:1 sample to factor Xa ratio). Factor Xa activity was determined at 37°C based on the chromogenic activity of the substrate. Factor Xa activity is linearly correlated with factor Xa concentration in the presence of dimethylguanidine. As shown in FIG. 7, 50 mM dimethylguanidine (702) retards the factor Xa reaction in samples with greater than 25 mM dimethylguanidine (701). Thus, increasing the amount of dimethylguanidine in the sample decreases the reaction rate. In some embodiments, dimethylguanidine can be added to the reaction at a concentration of up to about 500 mM.

[0064] Effect of alkylated guanidines on the activity of reactions containing unfractionated heparin (UFH). This experiment was performed to demonstrate the effect of adding a guanidine derivative to a reaction containing unfractionated heparin (UFH). In this experiment, 180 μL of a plasma sample containing 0-4.5 IU / mL (international units per milliliter) UFH, 56 mM dimethylguanidine, and 1.5 mM substrate S-2732™ was mixed with 20 μL of a 475 kJ / mL solution of factor Xa. In the resulting reaction, UFH and antithrombin in the plasma sample form a ternary complex with factor Xa, inhibiting its activity. The residual activity of unbound factor Xa was measured at 37°C. As shown in Figure 8, the measured value of factor Xa activity, shown on the Y-axis, is linearly correlated with the UFH concentration in the plasma sample in the presence of dimethylguanidine, shown on the X-axis (801). Thus, by determining the factor Xa activity in the reaction based on, for example, chromogenic activity, this linear correlation can be used to determine the concentration of an indirect factor Xa inhibitor, such as UFH, in a test sample.

[0065] (antithrombin activity) These experiments were performed to demonstrate the amidolytic activity of factor Xa compared to the antithrombin activity in the absence and presence of a guanidine derivative (dimethylguanidine in this example).

[0066] In these experiments, plasma samples containing 99% antithrombin activity were diluted 15-fold with antithrombin-deficient plasma and tested for inhibition of factor Xa activity in the presence of heparin.

[0067] In the experiment producing the plot in Figure 9, residual factor Xa activity was measured by mixing 50 µL of the aforementioned plasma sample with 50 µL of a reagent containing 10 nkat / mL factor Xa and heparin, followed by the addition of 50 µL of a solution of the chromogenic substrate S-2765™. A panel of plasma samples containing various levels of antithrombin was prepared and tested by diluting the plasma sample with saline. As shown in Figure 9, low levels of antithrombin activity result in high levels of factor Xa activity, and high levels of antithrombin activity result in low levels of factor Xa activity.

[0068] In the experiment producing the plot of Figure 10, 50 μL of the aforementioned plasma sample was mixed with 50 μL of Reagent (1001) containing 20 nkat / mL of factor Xa and heparin, 50 μL of Reagent (1002) containing 30 nkat / mL of factor Xa and heparin, and 50 μL of Reagent (1002) containing 40 nkat / mL of factor Xa and heparin. In each case, residual factor Xa activity was measured by adding 50 μL of a solution of factor Xa chromogenic substrate S-2765™ containing 60 mM dimethylguanidine. As shown in Figure 10, factor Xa activity, when modulated by dimethylguanidine, responded linearly across various antithrombin activities. Using a plot such as Figure 10, the antithrombin activity and, therefore, the amount of antithrombin in a test sample can be determined by knowing the concentration of factor Xa in the reagent and the resulting measured factor Xa activity in the test sample.

[0069] The analysis using the factor Xa reagent described herein can be performed manually or automatically. For example, the reagent can be activated manually or automatically by using a robotic manipulator or a flow-through fluidic device to mix the reagent with the test sample. A software application can be used to analyze the test results and provide or calculate parameters. The software application can also be used to generate additional information that can be used, for example, to inform clinical treatment.

[0070] The use of the Factor Xa reagent is not limited by the examples described herein, and applicable assays are not limited by any particular system for implementation. Systems can be used in laboratories, such as central laboratories for clinical testing, to point-of-care applications, such as first responders or operating rooms.

[0071] The factor Xa reagent may be packaged and stored in bulk, single-use, multi-use, or test quantities. The factor Xa reagent may be packaged and stored in any format, including kits or cartridges. For example, a cartridge may store the factor Xa reagent in liquid or dry form, or its components (such as factor Xa, substrate, and one or more guanidine derivatives) in liquid and / or dry form. The cartridge may be designed for connection to or insertion into a device and for measuring factor Xa inhibitors. The cartridge may also be configured for connection to or insertion into a device to receive a test sample, such as whole blood; mix the test sample with the factor Xa reagent; and move the resulting mixture to a location for measuring the mixture's absorbance or fluorescence. The cartridge may be for single or multiple use and may be disposable after the reagent is consumed. The cartridge may contain reference or calibration reagents to verify that the cartridge and / or instrument are operating within acceptable parameters or to generate a calibration curve. In some embodiments, the reference or calibration reagent is a direct or indirect factor Xa inhibitor described herein.

[0072] For example, the kit may contain components in liquid and / or dry form, such as a factor Xa reagent, or factor Xa, a substrate, and one or more guanidine derivatives. These may be packaged in a container for multiple use or a container for single use. The kit may also include an aqueous buffer, examples of which are described herein. The kit may also include a calibration or reference reagent to verify that the kit and / or instrument are operating within acceptable parameters or to generate a calibration curve. In some embodiments, the calibration or reference reagent is a direct or indirect factor Xa inhibitor described herein.

[0073] The diagnostic testing instruments, clinical analyzers, and / or analyses described herein may be controlled using a computing system or any other suitable computing device. The computing system or computing device may have one or more processing devices, such as one or more microprocessors, one or more microcontrollers, or programmable logic, such as one or more field programmable gate arrays (FPGAs) or one or more application specific integrated circuits (ASICs). The diagnostic testing instruments and clinical analyzers described herein may execute one or more computer program products (e.g., one or more computer programs tangibly embodied in one or more information carriers (one or more non-transitory machine-readable media)) to control the analyses and perform all or part of the methods described herein.

[0074] Elements of different embodiments described herein may be combined to form other embodiments not specifically set forth above. Elements may be omitted from structures described herein without adversely affecting their operation. Furthermore, various separate elements may be combined into one or more independent elements to perform the functions described herein.

Claims

1. A composition comprising Factor Xa, a substrate, and one or more guanidine derivatives.

2. The composition of claim 1 further comprising heparin.

3. 2. The composition of claim 1, wherein the one or more guanidine derivatives comprise 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, or N-benzyl-N-methylguanidine, where N represents a substituent on a nitrogen atom.

4. 10. The composition of claim 1, wherein the one or more guanidine derivatives comprise a monoalkylated guanidine derivative.

5. 10. The composition of claim 1, wherein the one or more guanidine derivatives comprise a dialkylated guanidine derivative.

6. The one or more guanidine derivatives may be of the formula: 【Chemistry 1】 wherein R1 is H, methyl, ethyl, or benzyl; R2 is H, methyl, ethyl, or benzyl; and N is nitrogen; and H is hydrogen. The composition of claim 1, comprising an alkylated guanidine having the chemical structure:

7. The one or more guanidine derivatives are a monoalkylated guanidine containing an alkyl group having a length different from the length of the alkyl group of 1-methylguanidine, or Dialkylated guanidines containing alkyl groups with lengths different from those of 1,1-dimethylguanidine or 1,1-diethylguanidine The composition of claim 1 comprising:

8. 10. The composition of claim 1, wherein the one or more guanidine derivatives comprise a sulfate salt.

9. The composition of claim 1 , wherein the substrate comprises a chromogenic substrate.

10. A device comprising the composition of claim 1.

11. 11. The device of claim 10, wherein the device is a cartridge.

12. 11. The device of claim 10, wherein the device is a kit.

13. 1. A method for detecting an anticoagulant in a sample, the method comprising: combining or contacting the sample with a composition comprising Factor Xa, a guanidine derivative, and a substrate; measuring factor Xa activity based on the substrate status, wherein the presence of an anticoagulant is based on factor Xa activity; A method comprising:

14. 14. The method of claim 13, wherein the anticoagulant comprises a direct factor Xa inhibitor.

15. 14. The method of claim 13, wherein the anticoagulant comprises a factor Xa inhibitor, including one or more of rivaroxaban, apixaban, edoxaban, or betrixaban.

16. 14. The method of claim 13, wherein the anticoagulant comprises an antithrombin-based indirect factor Xa inhibitor.

17. 17. The method of claim 16, wherein the indirect factor Xa inhibitor comprises one or more of low molecular weight heparin, unfractionated heparin, fondaparinux, or danaparoid.

18. 14. The method of claim 13, wherein the state of the substrate comprises a color change of the substrate.

19. 14. The method of claim 13, wherein the composition modulates the activity of factor Xa in a sample, the modulation comprising a decrease in the clotting rate of the sample caused by factor Xa.

20. 14. The method of claim 13, wherein the composition further comprises heparin and the anticoagulant is antithrombin.

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