Liquid thrombin reagent

Abstract

To provide a liquid thrombin reagent to improve the stability of thrombin.SOLUTION: The invention provides a liquid that includes thrombin and a constituent that includes one or more of guanidine derivatives or one or more anticoagulants. The one or more guanidine derivatives may include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, or N-benzyl-N-methylguanidine. The one or more anticoagulants may include rivaroxaban, apixaban, edoxaban, betrixaban, or a factor Xa anticoagulant.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present specification generally relates to exemplary liquid thrombin reagent compositions. [Background technology]

[0002] In a test sample such as whole blood, thrombin causes the formation of a dense fibrin clot composed of thin fibrin fibers.

[0003] The thrombin reagent may be in lyophilized or liquid form. For thrombin reagents in liquid form, the stability of the reagent may be adversely affected by the inherent autolytic and denaturing properties of thrombin in the liquid. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 10,175,225 [Patent Document 2] U.S. Patent Application Publication No. 2018 / 0306774 Summary of the Invention [Means for solving the problem]

[0005] An exemplary composition includes thrombin and a component including one or more guanidine derivatives or one or more anticoagulants. The exemplary composition is liquid. The exemplary composition may have one or more of the following characteristics, alone or in combination:

[0006] The one or more guanidine derivatives may include a mono-alkylated guanidine derivative. The one or more guanidine derivatives may include a di-alkylated guanidine derivative. The one or more guanidine derivatives may include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, or N-benzyl-N-methylguanidine (N represents a substituent on the nitrogen). The one or more guanidine derivatives may include an alkylated guanidine having a chemical structure represented by the following formula:

[0007] [ka]

[0008] wherein R1 is H, methyl, ethyl or benzyl; R2 is H, methyl, ethyl or benzyl, and N is nitrogen and H is hydrogen.)

[0009] The one or more guanidine derivatives may include monoalkylated guanidines containing alkyl groups with lengths different from those of 1-methylguanidine or dialkylated guanidines containing alkyl groups with lengths different from those of 1,1-dimethylguanidine or 1,1-diethylguanidine. The one or more guanidine derivatives may include sulfate salts.

[0010] The one or more guanidine derivatives may have a concentration in the composition of about 0.5 millimolar (mM) or more units per milliliter of thrombin (U / mL). The one or more guanidine derivatives may have a concentration in the composition of about 1 mM or more units per milliliter of thrombin. The one or more guanidine derivatives may have a concentration in the composition of about 20 mM (millimolar). The thrombin may have a concentration in the composition of 2 U / mL (units per milliliter) or more, or 20 U / mL (units per milliliter). The one or more guanidine derivatives may have a concentration in the composition of about 70 mM (millimolar).

[0011] The one or more anticoagulants may include rivaroxaban, apixaban, edoxaban, betoxaban, or a factor Xa anticoagulant.

[0012] The composition may include a buffer solution that is an aqueous solution. The buffer solution may include 2-morpholin-4-ylethanesulfonic acid (MES) and a preservative. The buffer solution may include one or more carboxylic acid derivatives.

[0013] The composition can be a liquid reagent used as (i) a Claus reagent, (ii) a thrombin time reagent, or (iii) a dilute thrombin reagent for direct thrombin inhibitors.

[0014] The composition may be a water-based solution. The composition may be liquid at least at 20°C. The composition may be liquid at least at 37°C.

[0015] An exemplary method includes forming a mixture including a liquid reagent and a sample, where the sample is whole blood, a portion of whole blood, or a derivative of whole blood, and the liquid reagent includes thrombin and a guanidine derivative. The method may include determining the amount of thrombin or the amount of an anticoagulant relative to thrombin in the mixture. The method may include one or more of the following features, alone or in combination:

[0016] The liquid reagent and liquid mixture may be in a cartridge. Fibrinogen or an anticoagulant for thrombin can be measured by an instrument into which the cartridge is inserted. The liquid reagent may include a chromogenic substrate that is susceptible to cleavage by thrombin.

[0017] An exemplary method for measuring a thrombin inhibitor in a sample may include forming a mixture containing a liquid reagent and the sample, where the sample is whole blood, a portion of whole blood, or a derivative of whole blood, and the liquid reagent includes thrombin and a guanidine derivative. The method may also include determining the amount of thrombin inhibitor in the mixture. The guanidine derivative may include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, or N-benzyl-N-methylguanidine. Specific examples of thrombin inhibitors that may be determined include DOACs (direct oral anticoagulants) against thrombin, such as dabigatran. Other specific examples of thrombin inhibitors include direct thrombin inhibitors (DTIs), such as lepirudin, desirudin, bivalirudin, and argatroban.

[0018] Any two or more features described in this specification, including the summary of the invention, may be practiced in combination, even if not specifically stated otherwise in this specification.

[0019] The systems and processes described herein, or portions thereof, can be implemented as one or more devices or methods, and may include one or more process devices and computer memories for storing executable instructions that effect the control of various functions. The systems, processes, and articles described herein, including but not limited to devices, methods, and / or reagents, can be formed through, for example, design, manufacture, construction, composition, arrangement, installation, programming, operation, activation, deactivation, and / or control.

[0020] The details of one or more implementations are set forth in the accompanying drawings and the following description. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0021] [Figure 1A]FIG. 1A shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 1B] FIG. 1B shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 1C] FIG. 1C shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 1D] FIG. 1D shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 1E] FIG. 1E shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 1F] FIG. 1F shows the structural formulas of specific examples of guanidine derivatives that may be used in the exemplary liquid thrombin reagents described herein. [Figure 2A] FIG. 2A shows plots of α-thrombin activity and α / β-thrombin activity over time obtained using a clotting assay. [Figure 2B] FIG. 2B shows plots of α-thrombin activity and α / β-thrombin activity over time obtained using the chromogenic assay. [Figure 3] FIG. 3 is a plot showing time to clotting (TTC) for test samples mixed with liquid thrombin reagent containing apixaban and test samples mixed with liquid thrombin reagent without apixaban. [Figure 4] FIG. 4 is a plot showing Factor Xa inhibition for different concentrations of guanidine and guanidine derivatives. [Figure 5] FIG. 5 is a plot showing thrombin activity over time for test samples containing and not containing a guanidine derivative. [Figure 6]FIG. 6 is a plot showing the residual thrombin activity for liquid thrombin reagents containing different concentrations of dimethylguanidine after storage at 37° C. [Figure 7] FIG. 7 is a plot showing the residual thrombin activity for liquid thrombin reagents containing different thrombin concentrations in the presence of a guanidine derivative after storage at 37° C. [Figure 8A] FIG. 8A is a flow chart illustrating an exemplary method for determining the concentration of fibrinogen in a test sample using a clotting assay. [Figure 8B] FIG. 8B is a flow chart illustrating an exemplary method for determining the concentration of a thrombin inhibitor in a test sample using a clotting assay. [Figure 8C] FIG. 8C is a flow chart illustrating an exemplary method for determining the concentration of an anticoagulant for thrombin in a test sample using a fluorescent assay. [Figure 8D] FIG. 8D is a flow chart illustrating an exemplary method for determining the concentration of an anticoagulant for thrombin in a test sample using a chromogenic assay.

[0022] Like reference numbers in different drawings indicate like elements. DETAILED DESCRIPTION OF THE INVENTION

[0023] Specific examples of thrombin reagents are described herein that are liquid over a temperature range of at least 0°C to 100°C and are stable over at least typical reagent storage temperatures, over a range of operating temperatures, and / or at room temperature. All temperatures described herein are at standard 1 atmosphere (101.325 kilopaques). As used herein, "about" allows for deviation from the given figure, including, but not limited to, deviations of 1%, 2%, 3%, 4%, or 5%.

[0024] A liquid thrombin reagent can be characterized as stable if it maintains about 70% to about 100% of its original thrombin activity in situ over an extended period of time. In this context, original thrombin activity includes the activity of the liquid thrombin before the liquid thrombin reagent is exposed to one or more conditions that cause, enable, or promote thrombin autolysis. Examples of such activity include, but are not limited to, storage of the liquid thrombin reagent at room temperature of about 20°C for one day or more, storage of the liquid thrombin reagent at a temperature range of about 2°C to about 8°C or about 2°C to about 45°C for one month or more, and / or testing using the liquid thrombin reagent at body temperature of about 37°C.

[0025] An exemplary liquid thrombin reagent comprises thrombin, an aqueous buffer, and one or more components that improve the stability of the thrombin relative to a liquid thrombin reagent that does not contain the one or more components. The stability of the liquid thrombin reagent can be achieved through reduced autolysis of the liquid thrombin reagent within the exemplary temperature range described above, within which there is no significant decrease in the biological activity of the liquid thrombin reagent.

[0026] In some specific examples, the concentration of thrombin in the liquid thrombin reagent can be within a range including about 2 units per milliliter (U / mL) to about 20 U / mL. In some specific examples, the concentration of thrombin in the liquid thrombin reagent can be within a range including about 7 U / mL to about 70 U / mL. In particular, non-limiting specific examples, the concentration of thrombin in the liquid thrombin reagent can be about 7 U / mL, or about 15 U / mL, or about 20 U / mL, or about 70 U / mL. The concentration of thrombin in the liquid thrombin reagent is not limited to the values ​​or ranges shown above. For example, in another non-limiting specific example, the concentration of thrombin in the liquid thrombin reagent is about 100 U / mL.

[0027] Examples of one or more components that may be included in a liquid thrombin reagent that improve the stability of thrombin, and consequently the stability of the liquid thrombin reagent itself, include one or more guanidine derivatives, one or more anticoagulants, or a combination of one or more guanidine derivatives and one or more anticoagulants.

[0028] Guanidine (100) is a nitrogen-rich organic compound with the formula HNC(NH2)2. The structural formula of guanidine (100) is shown in Figure 1A.

[0029] A specific example of a guanidine derivative that can be included in a liquid thrombin reagent to improve the stability of liquid thrombin is alkylated guanidine. Alkylated guanidines can have one or both of the hydrogen atoms (H) (101) or (102) in guanidine (100) replaced with larger chemical groups, shown as R1 (111) and R2 (112) in Figure 1B. R1 (111) can be a methyl group (131) (Figure 1D), an ethyl group (141) (Figure 1E), or a benzyl group (151) (Figure 1F), and R2 (112) can be a methyl group (132), an ethyl group (142), or a benzyl group (151). Methyl group (131) includes an alkyl group derived from methane, containing one carbon atom bonded to three hydrogen atoms, with the chemical formula -CH3. The ethyl group (141) contains an alkyl substituent with the formula -CHCH, derived from ethane (C2H5). The benzyl group (151) contains a building block or molecular fragment with the structure R-CH2-C6H5. Benzyl is characterized by a phenyl group (C6H5) with a methylene group (-CH2-) attached.

[0030] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent to improve the stability of the liquid thrombin reagent is a mono-alkylated guanidine, a di-alkylated guanidine, or a combination of a mono-alkylated guanidine and a di-alkylated guanidine. Non-limiting examples of guanidine derivatives that can be included in the liquid thrombin reagent include 1-methylguanidine, 1,1-dimethylguanidine, 1,1-diethylguanidine, and N-benzyl-N-methylguanidine (N represents 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).

[0031] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent that improves thrombin stability can include a mono-alkylated guanidine derivative that includes an alkyl group that is different in length from the alkyl group in 1-methylguanidine (120) in FIG. 1C. For example, the alkyl group that is different in length from the alkyl group in 1-methylguanidine (120) can include two or more carbon atoms. An alkyl group having two carbon atoms has the formula -CH2CH3. A mono-alkylated guanidine derivative that includes an alkyl group having two carbon atoms has the formula -C3H9N3. An alkyl group having three carbon atoms has the formula -CH2CH2CH3. A mono-alkylated guanidine derivative that includes 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 mono-alkylated guanidine derivative containing an alkyl group having four carbon atoms has the formula -CH 13 Has N3.

[0032] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent that improves thrombin stability can include a dialkylated guanidine derivative that includes one or both alkyl groups that are different in length from the alkyl group of 1,1-dimethylguanidine (130) in FIG. 1D. For example, the alkyl group that is different in length from the alkyl group of 1,1-dimethylguanidine (130) can include one or more carbon atoms. An alkyl group having one carbon atom has the formula -CH. A dialkylated guanidine derivative that includes an alkyl group having one carbon atom has the formula -CH. 11 Has N3.

[0033] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent to improve the stability of the liquid thrombin reagent can include a dialkylated guanidine derivative containing one or both alkyl groups that are different in length from the alkyl groups in 1,1-diethylguanidine (140) of FIG. 1E. The alkyl groups that are different in length from the alkyl groups in 1,1-diethylguanidine (140) can 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 An alkyl group having four carbon atoms has the formula -CH2CH2CH2CH3. A dialkylated guanidine derivative containing one alkyl group having two carbon atoms and one alkyl group having four carbon atoms has the formula -CH 17 Has N3.

[0034] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent to improve thrombin stability is a dialkylated guanidine derivative having one alkyl group longer in length than the alkyl group in 1,1-dimethylguanidine (130) or 1,1-diethylguanidine (140) and another alkyl group of the same or shorter length than the alkyl group in 1,1-dimethylguanidine (130) or 1,1-diethylguanidine (140). The alkyl group of the same length as the alkyl group in 1,1-dimethylguanidine (130) has the chemical formula -CH3. The dialkylated guanidine derivative can include an alkyl group of the same length as the alkyl group in 1,1-diethylguanidine (140) with the chemical formula -CH2CH3.

[0035] Another specific example of a guanidine derivative that can be included in a liquid thrombin reagent to improve thrombin stability is a guanidine derivative having the formula -C(NH2)3 + Specific examples of the guanidine derivative that can be contained in the liquid thrombin reagent include a sulfate salt containing the 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.

[0036] The guanidine derivatives that can be included in the liquid thrombin reagent for improving thrombin stability can include a combination of two or more of the exemplary guanidine derivatives described herein. Exemplary combinations of guanidine derivatives that can be included in the liquid thrombin reagent can include 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).

[0037] Guanidine derivatives that can be included in liquid thrombin reagents to improve thrombin stability can include one or more of the exemplary guanidine derivatives described herein in combination with guanidine itself (FIG. 1A). Exemplary combinations of guanidine and guanidine derivatives that can be included in liquid thrombin reagents can include a combination of guanidine and 1-methylguanidine, a combination of guanidine and 1,1-dimethylguanidine, a combination of guanidine and 1,1-diethylguanidine, and / or a combination of guanidine and N-benzyl-N-methylguanidine.

[0038] In some embodiments, the individual guanidine derivatives described herein may each be present in the liquid thrombin reagent at a concentration of about 0.5 millimoles per milliliter of thrombin (U / mL) or greater. In some embodiments, the individual guanidine derivatives described herein may each be present in the liquid thrombin reagent at a concentration of about 1.0 mM or greater per milliliter of thrombin. In some embodiments, the individual guanidine derivatives described herein may each be present in the liquid thrombin reagent at a concentration of about 2 mM to about 20 mM or greater. In some embodiments, a combination of two or more guanidine derivatives described herein may each be present in the liquid thrombin reagent at a concentration of about 2 mM to about 20 mM or greater.

[0039] One or more components that can be added to a liquid thrombin reagent to improve thrombin stability can include one or more anticoagulants. Specific examples of anticoagulants that can be added to a liquid thrombin reagent include known Factor Xa anticoagulants, such as rivaroxaban, apixaban, edoxaban, and betoxaban. These anticoagulants can be added to the liquid thrombin reagent individually or in combinations of two or more. Specific examples of combinations of anticoagulants that can be added to a liquid thrombin reagent include, but are not limited to, a combination of apixaban and rivaroxaban, a combination of apixaban and edoxaban, a combination of apixaban and betoxaban, a combination of rivaroxaban and betoxaban, a combination of edoxaban and betoxaban, and / or a combination of edoxaban and rivaroxaban.

[0040] In some implementations, each of the individual anticoagulants described herein may be included in the liquid thrombin reagent at a concentration of about 20 nanograms per milliliter (ng / mL) to 1000 ng / mL.

[0041] In some implementations, the liquid thrombin reagent can include one or more guanidine derivatives described herein and one or more anticoagulants described herein. Specific examples of guanidine derivative and Factor Xa anticoagulant combinations that can be included in the liquid thrombin reagent include, but are not limited to, 1-methylguanidine (120) and apixaban, 1,1-dimethylguanidine (130) and apixaban, 1,1-diethylguanidine (140) and apixaban, 1,1-dimethylguanidine (130) and rivaroxaban, 1,1-dimethylguanidine (130) and edoxaban, and / or 1,1-dimethylguanidine (130) and vetoxaban.

[0042] In some implementations, the aqueous buffer in the liquid thrombin reagent can be or include 2-morpholin-4-yl-ethanesulfonic acid (MES) or 3-(N-morpholino)propanesulfonic acid (MOPS), a structural analog of MES. In some implementations, the aqueous buffer in the liquid thrombin reagent can 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 / isothiazolinone (ISO) buffer, or thrombin buffer. The buffer may be or comprise midazolium buffer, bis-tris propane 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 MOBS (3-(N-morpholino)propanesulfonic acid) buffer.

[0043] In some implementations, the aqueous buffer in the liquid thrombin reagent can be or include one or more carboxylic acid derivatives. Specific examples of carboxylic acid derivatives that can be included in the aqueous buffer can include carboxylate acetate, ethylenediaminetetraacetate, butanetetracarboxylate, propanetricarboxylate, citrate, succinate, tartrate, malonate, and / or gluconate.

[0044] In some implementations, the aqueous buffer in the liquid thrombin reagent may contain a preservative. Specific 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. may include:

[0045] In some implementations, one or more substrates may be added to a mixture of the liquid thrombin reagent and the test sample to measure the amount of thrombin inhibitor in the test sample. Specific examples of substrates that may be added to the liquid thrombin reagent include the chromogenic substrates HD-phenylalanyl-L-pipeconyl-L-arginine-p-nitroaniline dihydrochloride (HD-Phe-Pip-Arg-pNA) (Chromogenix S-2238™) and L-pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline dihydrochloride (Chromogenix S-2366™), or the fluorogenic substrate Z-Gly-Gly-Arg-AMC, Technothrombin®, and / or a calibrated automated thrombogram.

[0046] (clotting assay) Clotting-based assays can be used to determine the concentration of fibrinogen (with excess thrombin) in a test sample and / or to determine the concentration of a thrombin inhibitor (with thrombin serving as the rate-limiting material) in a test sample. In this regard, fibrinogen is a soluble protein that circulates in the blood and, upon vascular injury, is enzymatically converted to fibrin by thrombin, initiating the clotting process. Specific examples of thrombin inhibitors that can be determined include thrombin-directed anticoagulants (DOACs) such as dabigatran. Other specific examples of thrombin inhibitors include direct thrombin inhibitors (DTIs) such as lepirudin, desirudin, bivaldin, and argatroban.

[0047] The test sample may include whole blood, a portion of whole blood, a sample derived from whole blood, a portion of whole blood from which one or more blood components (e.g., white blood cells, red blood cells, platelets, or proteins (e.g., albumin) have been used), or separated whole blood components. In a specific example, the test sample may be platelet-free plasma, which is whole blood from which a blood component (e.g., platelets) has been used. The test sample may be obtained from a subject, such as a human, using standard techniques.

[0048] FIG. 8A shows an exemplary process (800) that can be performed to determine the fibrinogen concentration associated with a test sample using a clotting assay. The clotting assay determines the time it takes for a test sample mixed with a liquid thrombin reagent to clot. According to process (800), a test sample is received (801) and mixed (802) with a liquid thrombin reagent of the type described herein. The TTC (time to clot) of the resulting mixture is measured (803). In this example, the TTC of a series of samples with known fibrinogen concentrations is previously measured and used to generate (e.g., by accessing memory) a standard curve relating TTC values ​​to the obtained fibrinogen concentrations (804). The fibrinogen concentration in the blood sample is determined (805) by comparing the TTC concentration of the mixture to the standard curve of samples with known fibrinogen concentrations. That is, the TTC of the mixture is identified on the standard curve, and the corresponding fibrinogen concentration is obtained from the standard curve. However, determining fibrinogen concentration is not limited to processes using a standard curve. In some implementations, additional methods, such as machine learning algorithms, use the results of the clotting assay (e.g., TTC and / or another parameter derived from the results) to derive the fibrinogen concentration in the test sample.

[0049] FIG. 8B shows an exemplary process (810) that can be performed to determine the concentration of a thrombin inhibitor associated with a test sample using a clotting assay. A clotting assay determines the time it takes for a test sample mixed with a liquid thrombin reagent to clot. According to process (800), a test sample is received (811) and mixed (812) with a liquid thrombin reagent of the type described herein. The TTC of the resulting mixture is measured (813). In this example, the TTC of a series of samples with known thrombin inhibitor concentrations is pre-measured and used to generate a standard curve relating the TTC values ​​to the thrombin inhibitor concentrations obtained in operation (814). The thrombin inhibitor concentration in the blood sample is determined (815) by comparing the TTC concentration of the mixture to the standard curve of samples with known thrombin inhibitor concentrations. That is, the TTC of the mixture is identified on the standard curve, and the corresponding thrombin inhibitor concentration is obtained from the standard curve. However, determining the thrombin inhibitor concentration is not limited to processes using a standard curve. In some implementations, an additional method, such as, for example, a machine learning algorithm, uses the results of the coagulation assay (e.g., TTC and / or another parameter derived from the results) to derive the concentration of a thrombin inhibitor in the test sample.

[0050] An exemplary cartridge for use in a blood testing system can be used to perform a clotting assay and can include one or more chambers for receiving / containing a test sample, a reagent, and / or a mixture of the test sample and reagent. The clotting assay is performed on the mixture of the test sample and reagent to determine clotting characteristics based on the mixture, such as TTC. The cartridge can include multiple fluid paths that allow or facilitate fluid movement of the sample or materials within the cartridge, e.g., controlled by pressure or other mechanisms.

[0051] In a non-limiting example, a coagulation assay can be performed using the system described in U.S. Patent Application Publication No. 2019 / 0129999, entitled "Blood Testing System and Method," which was issued on January 8, 2019, and is incorporated herein by reference. U.S. Patent Application Publication No. 2019 / 01299999 describes a blood testing system that includes an analyzer console and one or more cartridges for performing tests, including a test for determining the amount of fibrinogen in a test sample using a liquid thrombin reagent as described herein. This test can be performed using the cap and pin configuration used in the Rotem® sigma and Rotem® delta systems offered by Werfen® SA. The coagulation profile can be used with an associated analyzer console and implemented in the manner described herein to determine the amount of fibrinogen in a mixture and / or the amount of thrombin inhibitor in a mixture.

[0052] In another non-limiting example, coagulation assays can be performed using the system described in U.S. Patent Application Publication No. 2018 / 0129999, entitled "Disposable System for Analysis of Hemostatic Function," published on October 25, 2018, and incorporated herein by reference. This patent publication describes the Hemosonics® Quantra® Hemostasis Analyzer.

[0053] In yet another non-limiting embodiment, coagulation assays can be performed using the TEG® 6s cartridge-based system provided by Haemonetics®. The system includes a cartridge with one or more chambers capable of holding a test sample mixture, such as blood and a liquid thrombin reagent, as described herein. Testing in the TEG® 6s system is performed using optical analysis combined with resonant frequency technology and is used to determine coagulation characteristics based on a mixture, such as TTC. More specifically, in an embodiment using the TEG® 6s cartridge-based system, Haemonetics® USFDA The 501K abstract states, "The TEG® 6s technology is based on a disposable cartridge containing up to four independent measurement cells. Each cell consists of a short, vertically oriented, infused, molded tube (ring). Detection of coagulation in the TEG® 6s hemostatic system is performed optically. A piezoelectric actuator vibrates the measurement cell through a movement profile consisting of summed sine waves at different frequencies. The movement of the measurement cell induces motion in the sample meniscus, which is detected by a photodiode. The resulting meniscus movement The vibrations are optically detected and analyzed by the instrument to calculate the resonant frequency and elastic modulus (stiffness) of the sample. The resonant frequency can be determined by performing a Fast Fourier Transform (FFT) on the meniscus motion data. The analyzer detects the harmonic motion of the hanging drop of blood in response to the external vibration. As the sample transforms from a liquid state to a gel-like state during clotting, the elastic modulus (stiffness), and therefore the resonant frequency, increases. The TEG® 6s Hemostasis System measures these variations in resonant frequency during clotting and lysis. The coagulation properties determined using the TEG® 6s technology can be used by the analyzer to perform the processes described herein to determine the amount of fibrinogen in the mixture and / or to determine the amount of thrombin inhibitor in the mixture. Additionally, in another non-limiting embodiment, the coagulation assay can be performed on the VerifyNow® system offered by Werfen® SA.

[0054] (Chromogenic and Fluorescent Assays) In some implementations, the concentration of a thrombin inhibitor in a test sample can be determined by a chromogenic or fluorogenic assay. In such assays, a thrombin inhibitor inhibits the activity of thrombin in a reaction mixture containing the test sample. The assay measures the free thrombin remaining in the mixture based on the cleavage of free thrombin with a chromogenic or fluorogenic substrate. Chromogenic assays use a colored substrate to quantify the activity of free thrombin. Fluorescent assays use a fluorescent substrate to quantify the activity of free thrombin. In each assay, one or more curves are generated based on the amount of thrombin activity, as defined by fluorescence or absorbance values, and the concentration of thrombin inhibitor in the reaction mixture.

[0055] FIG. 8C shows an exemplary process (820) that can be performed to determine the thrombin inhibitor concentration associated with a blood sample using a fluorescence assay. A test sample is received (821) and mixed with a liquid thrombin reagent and a fluorescent substrate (822). The fluorescence of the cleaved fluorescent substrate in the resulting mixture is measured (823). In this example, the fluorescence of a series of samples with known thrombin inhibitor concentrations is previously measured and used to generate a standard curve relating the fluorescence values ​​to the thrombin inhibitor concentrations obtained in operation (824). The thrombin inhibitor concentration in the blood sample is determined (825) by comparing the fluorescence concentration of the mixture to the standard curve of samples with known thrombin inhibitor concentrations. That is, the fluorescence of the mixture is identified on the standard curve, and the corresponding thrombin inhibitor concentration is obtained from the standard curve. However, determining the thrombin inhibitor concentration is not limited to processes using a standard curve. In some implementations, an additional method, such as, for example, a machine learning algorithm, uses the results of the fluorescence assay (e.g., fluorescence and / or another parameter derived from the results) to derive the concentration of a thrombin inhibitor in the test sample.

[0056] In some implementations, the fluorescent assay can be performed on a PHERAstar® FSX fluorescent microplate reader provided by BMG LABTECH® Inc. This system comprises a microplate-based system for high-throughput, multi-mode reading of test samples and liquid thrombin reagents described herein. In another non-limiting example, the fluorescent assay can be performed on a Xenius XOF fluorescent spectrometer provided by SAFAS® Ltd. This system comprises a microplate- or cuvette-based system for in situ, high-throughput reading of test samples and liquid thrombin reagents described herein.

[0057] FIG. 8D shows an exemplary process (830) that can be performed to determine the concentration of thrombin inhibitor in a test sample using a chromogenic assay. According to process (830), a test sample is received (821) and mixed (832) with a chromogenic substrate and a liquid thrombin reagent of the type described herein. The absorbance of the cleaved chromogenic substrate in the resulting mixture is measured spectrophotometrically (833). In this example, the absorbance of a series of samples with known thrombin inhibitor concentrations is pre-measured and used to generate a standard curve relating the absorbance values ​​to the thrombin inhibitor concentrations obtained in operation (834). The thrombin inhibitor concentration in the test sample is determined (835) by comparing the absorbance of the mixture to the standard curve of samples with known thrombin inhibitor concentrations. That is, the absorbance of the mixture is identified on the standard curve, and the corresponding thrombin inhibitor concentration is obtained from the standard curve. However, determining the thrombin inhibitor concentration is not limited to processes using a standard curve. In some implementations, an additional method, such as, for example, a machine learning algorithm, uses the results of the chromogenic assay (e.g., absorbance and / or another parameter derived from the results) to derive the concentration of a thrombin inhibitor in the test sample.

[0058] In some implementations, the chromogenic assay can be performed on the ACL TOP® Family Hemostasis Test System offered by Werfen® SA. This system includes a fully automated system for loading and unloading cuvettes holding a mixture of a test sample, such as blood, and a liquid thrombin reagent as described herein. In another non-limiting example, the chromogenic assay can be performed on a SPECTROstar Nano® absorbance microplate reader offered by BMG LABTECH® Inc. This system includes a microplate or cuvette-based system for reading the full range of absorbance of a test sample and a liquid thrombin reagent as described herein.

[0059] (Thrombin activity test in liquid thrombin reagent) This experiment is presented to demonstrate the stability of the liquid thrombin reagent using clotting and chromogenic assays.

[0060] For the clotting assay, liquid thrombin reagent was diluted with aqueous buffer to a final thrombin concentration within 5–15 U / mL of thrombin. Then, 20–40 microliters (μL) of the diluted liquid thrombin reagent was mixed with 80 μL of 20 mM HEPES, pH 7.4 reaction buffer containing 150 mM NaCl and 0.5% BSA (bovine serum albumin). After incubation at 37°C for 60–90 seconds, 80 μL of normal pooled plasma was added to the mixture, and the time to clot was determined using a threshold algorithm.

[0061] For the chromogenic assay, the liquid thrombin reagent was diluted with aqueous buffer to a final thrombin concentration within 1–5 U / mL of thrombin. Then, 20 μL of the diluted liquid thrombin reagent was mixed with 90 μL of the same reaction buffer used in the clotting assay. After incubation at 37°C for 60–90 seconds, 90 μL of 0.2 mM thrombin substrate S-2238 (HD-Phe-Pip-Arg-pNA) prepared in reaction buffer was added to the mixture, and the reaction rate of the liquid thrombin reagent was determined using a first-order kinetic algorithm.

[0062] To normalize thrombin activity, clotting times determined by the clotting assay, and kinetics determined by the chromogenic assay, the calculated thrombin activity was compared to assay kinetic curves of human α-thrombin or thrombin standards publicly available from the National Institute for Biological Sciences (NIBSC).

[0063] (Effect of β-thrombin on the stability of liquid thrombin reagents) β-thrombin and γ-thrombin are known to degrade α-thrombin in liquid thrombin reagents. In particular, β-thrombin in liquid thrombin reagents increases the autolytic activity of α-thrombin, leading to a decrease in α-thrombin activity, for example, when the β-thrombin content exceeds 30% of the total thrombin content.

[0064] The following experiments were performed to demonstrate that under certain conditions, β-thrombin has little or no effect on the liquid thrombin reagent described herein. The effect of β-thrombin was analyzed using clotting and chromogenic assays.

[0065] In this experiment, a liquid thrombin reagent of the type described herein containing α-thrombin was diluted with an aqueous buffer to a final thrombin concentration of 100 U / mL α-thrombin. A liquid thrombin reagent of the type described herein containing both α-thrombin and β-thrombin (α / β-thrombin) was diluted with an aqueous buffer to a final thrombin concentration of 100 U / mL α-thrombin and 50 U / mL β-thrombin. In this experiment, the aqueous buffer contained 20 mM MES, pH 6.3, 100 mM NaCl, 25 mM sodium acetate, 25 mM sodium succinate, 5 mM CaCl2, 0.5% BSA, and 0.4 g / L NaN3. The α-thrombin and α / β-thrombin liquid thrombin reagents were stored at 37°C for 0, 1, 2, 3, and 4 weeks, and thrombin activity was determined using both the clotting assay and the chromogenic assay, as described above.

[0066] The results of the clotting assay for the α-thrombin and α / β-thrombin liquid thrombin reagents are shown in Figure 2A. The results of the chromogenic assay for the α-thrombin and α / β-thrombin liquid thrombin reagents are shown in Figure 2B.

[0067] As shown in Figure 2A, the α-thrombin liquid thrombin reagent (200) and the α / β-thrombin liquid thrombin reagent (201) stored at 37°C for 0 weeks had similar thrombin activity as measured by clotting assay. Storage at 37°C for 0, 1, 2, 3, and 4 weeks affected both the α-thrombin and α / β-thrombin containing liquid thrombin reagents as measured by clotting assay.

[0068] As shown in Figure 2B, when stored at 37°C for 0 weeks, the α / β-thrombin liquid thrombin reagent (201) had 33% higher thrombin activity than the α-thrombin liquid thrombin reagent (200), as measured by a chromogenic assay. The decrease in activity for the α / β-thrombin reagent was greater than that for the α-thrombin liquid reagent after 1-2 weeks of storage at 37°C, but was comparable to that for the liquid thrombin reagent stored at 37°C for 2-4 weeks.

[0069] The test results shown in Figures 2A and 2B indicate that the thermal stability of liquid thrombin reagents in carboxylate salts is reduced, and that under these conditions, β-thrombin is more unstable (prone to cleavage) and has limited effect on α-thrombin activity.

[0070] (Effect of the factor XA inhibitor apixaban on the stability of liquid thrombin) Factor Xa inhibits or prevents the autolysis of thrombin. The following experiment was performed to demonstrate that Factor Xa improves the stability of thrombin when included in the liquid thrombin reagent described herein.

[0071] In this experiment, the direct factor Xa inhibitor apixaban was analyzed by coagulation assay. A liquid thrombin reagent containing impure bovine thrombin was diluted with an aqueous buffer containing 20 mM MES, pH 6.1, 100 mM NaCl, 25 mM sodium acetate, 25 mM sodium lactate, 5 mM CaCl, 0.5% BSA, and 0.4 g / L NaN to a final thrombin concentration of approximately 100 U / mL. Apixaban was dissolved in dimethyl sulfoxide (DMSF) and diluted to a final concentration of 500 ng / mL in the liquid thrombin reagent sample.

[0072] Liquid thrombin reagents with and without apixaban were stored at 45°C for 0, 2, 4, 7, 10, and 14 days and then tested in quadrants in a clotting assay after mixing with normal pooled plasma. The mean clotting time for the mixtures at each time point was compared to the baseline for the mixture stored at 45°C for 0 days.

[0073] As shown in Figure 3, the liquid reagent without apixaban (301) required longer to clot the test sample (due to the increased TTC) than the liquid reagent with apixaban. This indicates that apixaban contributes to the stability of the liquid thrombin reagent, as thrombin activity in the liquid reagent was not affected over time. Compared to 301, a significant decrease in thrombin activity was observed after 7 (302), 10 (303), and 14 (305) days of storage of the liquid thrombin reagent at 45°C, indicating that apixaban improves the stability of the liquid thrombin reagent over the long term.

[0074] (Effect of alkylated guanidines on factor XA activity) To demonstrate that guanidines and their derivatives described herein inhibit Factor Xa activity and increase thrombin activity in liquid thrombin reagents, the following experiments were performed.

[0075] In this experiment, guanidine sulfate and guanidine derivatives: 1-methylguanidine sulfate, 1,1-dimethylguanidine sulfate, 1,1-diethylguanidine, and N-benzyl, N-methylguanidine sulfate, were analyzed using a chromogenic assay. Serial dilutions were prepared in water with the guanidine or guanidine derivatives of the type described herein. 40 μL of each guanidine or guanidine derivative was mixed for 20–60 seconds with 40 μL of 6 nanokat / mL Factor Xa diluted in 20 mM HEPES, pH 7.5 buffer. After an incubation period of 150–180 seconds, 10 μL of Factor Xa chromogenic substrate S-2732 (1.5 mM) was added to each guanidine and Factor Xa mixture, and color development was measured after 20 seconds at milliabsorbances per minute at 405 nm (mAbs / min).

[0076] Mixtures containing guanidine derivatives, such as alkylated guanidine derivatives, inhibited Factor Xa activity. As shown in Figure 4, the greatest extent of Factor Xa inhibition was observed in mixtures containing dialkylated guanidine (401) (dimethylguanidine sulfate and diethylguanidine sulfate). Mixtures containing a guanidine derivative containing a benzyl group (403) (N-benzyl-N-methylguanidine sulfate) reduced Factor Xa inhibition compared to that in mixtures containing dialkylated guanidine (401). Mixtures containing guanidine sulfate (404) alone reduced Factor Xa inhibition the least. These results indicate that alkylated guanidines inhibit Factor Xa activity. Direct Factor Xa inhibitors, such as apixaban, support the stability of thrombin in liquid thrombin reagents by inhibiting Factor Xa activity, and this experiment demonstrates that guanidine derivatives provide a similar stabilizing effect on thrombin in liquid thrombin reagents.

[0077] (Effect of alkylated guanidine derivatives on the stability of liquid thrombin reagents) To demonstrate that alkylated guanidine derivatives stabilize thrombin activity in the liquid thrombin reagents described herein, the following experiment was performed.

[0078] The effects of guanidine derivatives were analyzed using a coagulation assay. In this experiment, a liquid thrombin reagent containing bovine thrombin was diluted with an aqueous buffer containing 20 mM MES, pH 6.3, and 0.1% ProClin™ 300, with and without 25 mM 1-methylguanidine (MMG), 25 mM 1,1-dimethylguanidine (DMG), and 25 mM 1,1-diethylguanidine (DEG), to a final concentration of approximately 70 U / mL thrombin.

[0079] Liquid thrombin reagents of the type described herein, with and without a guanidine derivative, were stored at 37°C for 0, 7, and 14 days and tested using a clotting assay after mixing with normal pooled plasma. As shown in Figure 5, liquid thrombin reagent 500 without a guanidine derivative (liquid thrombin + MES buffer alone) stored at 37°C for 7 (502) and 14 (503) days resulted in a significant reduction in thrombin activity in the reagent. However, liquid thrombin reagents with guanidine derivative (506) (1,1-methylguanidine sulfate, 1,1-dimethylguanidine sulfate, or 1,1-diethylguanidine sulfate) stored at 37°C for 7 and 14 days showed little or no reduction in thrombin activity. This relatively constant level of thrombin activity for the liquid thrombin reagent containing guanidine derivative (506) indicates that the guanidine derivative stabilizes the liquid thrombin reagent.

[0080] (Alkyl guanidine concentration on thrombin stability) The following experiment was performed to demonstrate exemplary concentrations of guanidine derivatives, such as alkylated guanidine derivatives, that stabilize thrombin activity in liquid thrombin reagents.

[0081] A series of liquid thrombin reagents containing dimethylguanidine (DMG) were analyzed using a coagulation assay. A series of liquid thrombin reagents containing 7 U / mL liquid thrombin were prepared from a buffer containing 20 mM MES, pH 6.3, 0.1% ProClin™ 300, and final concentrations of 5, 10, 15, 20, 25, or 30 mM DMG. The resulting liquid thrombin reagents were stored at 37°C for 20 days, and the residual thrombin activity was compared with that of liquid thrombin reagents containing the same concentrations of guanidine derivatives stored at 2-8°C for 20 days. As shown in Figure 6, the thrombin activity (600) in liquid thrombin reagents containing less than 20 mM DMG stored at 37°C for 20 days (601) decreased (i.e., the remaining amount of thrombin activity (605) decreased), while liquid thrombin reagents containing 20 mM or more DMG had comparable residual thrombin activity. This maintenance of thrombin activity in liquid thrombin reagents containing DMG indicated that 20 mM or more DMG in the liquid thrombin reagent is an exemplary amount of DMG to achieve thrombin stability at the temperatures and ranges considered.

[0082] (Thrombin concentration for stability of liquid thrombin reagent) The following experiment was performed to demonstrate an exemplary amount of guanidine derivative (DMG in this experiment) for stabilizing liquid thrombin.

[0083] The effect of guanidine derivatives at a range of thrombin concentrations was analyzed using a clotting assay. A series of liquid thrombin reagents containing 30 mM DMG was prepared by diluting a 100 U / mL liquid thrombin reagent in MES buffer (containing 30 mM DMG, 20 mM MES, pH 6.3, and 0.1% ProClin™ 300) to final concentrations of 70, 56, 42, 28, 14, or 7 U / mL thrombin in MES buffer. The liquid thrombin reagents were stored at 37°C for 7 days and then tested for thrombin activity using a clotting assay. As shown in Figure 7, the activity (701) of liquid thrombin reagents containing 30 mM DMG and less than approximately 20 U / mL of liquid thrombin after 7 days of storage at 37°C decreased (i.e., the remaining amount of thrombin activity decreased), while the activity of liquid thrombin reagents containing 30 mM DMG and more than approximately 20 U / mL of thrombin did not decrease significantly. This data indicates that liquid thrombin reagents containing 20 U / mL or higher thrombin and alkylated guanidine are stable.

[0084] Assays using the liquid thrombin reagents described herein can be performed manually or automatically. For example, the reagents can be activated manually or automatically, e.g., by mixing and testing the sample with a robotic or flow-through fluidic device. Software applications can be used to analyze the test results and provide or calculate parameters, such as fibrinogen concentration or thrombin inhibitor concentration. Software applications can be used to generate additional information that can be used, for example, to inform medical intervention. The use of the liquid thrombin reagents is not limited to the specific examples described in this disclosure, and applicable assays are not limited by the particular system for implementation. The liquid thrombin reagents can be packaged and stored in bulk or test volumes. The liquid thrombin reagents can be packaged and stored in any format, including in kit or cartridge form. Non-limiting exemplary systems are described below, along with exemplary uses of the reagents in these systems.

[0085] For example, the liquid thrombin reagent can be used as a Clauss fibrinogen assay reagent, a thrombin time assay reagent, or a thrombin reagent diluted for direct thrombin inhibitor use. The Clauss fibrinogen assay is a quantitative, clot-based functional assay. This assay measures the time it takes for fibrinogen in a test sample, such as plasma or whole blood, to be converted to fibrin under the influence of a thrombin reagent. Under these conditions, the fibrinogen content is rate-limiting, so TTC can be used as a measure of the fibrinogen concentration in the test sample. In particular, TTC is inversely proportional to the fibrinogen concentration in the test sample. The thrombin time assay is a test that measures the time it takes for a fibrin clot to form in the plasma of a blood sample. This time corresponds to the activity of fibrinogen.

[0086] The diagnostic testing instruments / medical analytical instruments and assays described herein can be controlled using a computer system or any suitable computing device having one or more microprocessors, one or more microcontrollers, programmable logic such as one or more field-programmable gate arrays (FPGAs), or one or more processing devices such as one or more application-specific semiconductors (ASICs). The diagnostic testing instruments / medical analytical instruments described herein can execute one or more computer program products, such as one or more computer programs tangibly embodied in one or more information carriers, such as one or more non-transitory machine-readable media, that control, for example, coagulation assays and perform all or part of the methods described herein.

[0087] Components of different implementations described herein may be combined to form other implementations not specifically described above. Components may be removed from the structures described herein without detrimentally affecting their operation. Furthermore, various separate elements may be combined with one or more individual components to perform the functions described herein.

Claims

1. A composition that is a liquid, Thrombin and a component comprising one or more guanidine derivatives; A composition comprising: The composition, wherein the one or more guanidine derivatives comprise an alkylated guanidine derivative having the chemical structure shown in the formula: 【Chemical 1】 wherein R1 is hydrogen, methyl, ethyl or benzyl; R2 is methyl or ethyl, and N is nitrogen and H is hydrogen.

2. The composition of claim 1 , wherein the one or more guanidine derivatives comprises a monoalkylated guanidine derivative.

3. The composition of claim 1 , wherein the one or more guanidine derivatives comprises a dialkylated guanidine derivative.

4. 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 (N represents a substituent on the nitrogen).

5. The composition of claim 1, wherein the one or more guanidine derivatives include sulfate.

6. The composition described in claim 1, wherein the thrombin has a concentration of 20 U / mL (units per milliliter) or more in the composition.

7. The composition of claim 1, wherein the one or more guanidine derivatives have a concentration of 20 millimolar (mM) or more in the composition.

8. forming a mixture comprising a liquid reagent and a sample, the sample comprises whole blood, a portion of whole blood, or a derivative of whole blood; The liquid reagent comprises the composition of claim 1; determining the amount of fibrinogen or the amount of anticoagulant relative to thrombin in the mixture.

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