Iron chelator-containing prothrombin time reagent

Abstract

The present invention pertains to the field of coagulation diagnostics and relates to a prothrombin time reagent on the basis of recombinant or native tissue factor protein and phospholipids, which reagent can be stabilized by the addition of an iron chelator from the groups of siderophores and 3,5-diphenyl-1,2,4-triazoles.

Claims

1. A prothrombin time reagent comprising tissue factor protein and phospholipids, wherein the reagent further comprises at least one iron chelator, wherein the at least one iron chelator is a 3,5-diphenyl-1,2,4-triazole selected from the group consisting of deferasirox, ethyl [3,5-bis(2-hydroxyphenyl)-[1,2,4]triazol-1-yl]acetate, and 3,5-bis(2-hydroxyphenyl)-1-(2,2,2-trifluoroethyl)-1H-[1,2,4]triazole.

2. The prothrombin time reagent according to claim 1, comprising the at least one iron chelator at a concentration of 0.007 to 2.5 mmol/L.

3. The prothrombin time reagent according to claim 1, further comprising Ca.sup.2+ ions.

4. The prothrombin time reagent according to claim 3 comprising 5 to 500 mmol/L calcium chloride.

5. The prothrombin time reagent according to claim 1, wherein the tissue factor protein is selected from the group consisting of human or animal recombinant tissue factor protein and natural human or animal tissue factor protein from a tissue extract.

6. A method for preparing a liquid prothrombin time reagent comprising tissue factor protein and phospholipids, the method comprising filling a reagent vial with a liquid comprising tissue factor protein, phospholipids and at least one iron chelator without lyophilization, wherein the at least one iron chelator is a 3,5-diphenyl-1,2,4-triazole selected from the group consisting of deferasirox, ethyl [3,5-bis(2-hydroxyphenyl)-[1,2,4]triazol-1-yl]acetate, and 3,5-bis(2-hydroxyphenyl)-1-(2,2,2-trifluoroethyl)-1H-[1,2,4]triazole.

7. A method for stabilizing a prothrombin time reagent comprising tissue factor protein and phospholipids, the method comprising adding at least one iron chelator the reagent, wherein the at least one iron chelator is a 3,5-diphenyl-1,2,4-triazole selected from the group consisting of deferasirox, ethyl [3,5-bis(2-hydroxyphenyl)-[1,2,4]triazol-1-yl]acetate, and 3,5-bis(2-hydroxyphenyl)-1-(2,2,2-trifluoroethyl)-1H-[1,2,4]triazole.

8. The method according to claim 7, wherein the at least one iron chelator is added in an amount such that the reagent comprises the at least one iron chelator at a concentration of 0.007 to 2.5 mmol/L.

9. A method for determining a coagulation parameter in a plasma sample, the method comprising mixing the plasma sample with the prothrombin time reagent according to claim 1 to give a reaction mixture and determining the coagulation parameter in the reaction mixture.

10. The method according to claim 9, wherein the coagulation parameter is prothrombin time, derived fibrinogen, or endogenous thrombin potential.

11. The method according to claim 9, wherein a change in absorption of the reaction mixture is measured photometrically and a time from the time point of mixing of the prothrombin time reagent with the plasma sample until reaching a threshold value is determined.

12. The method according to claim 7, wherein the at least one iron chelator is added in an amount such that the reagent comprises the at least one iron chelator at a concentration of 0.01 to 0.5 mmol/L.

13. The prothrombin time reagent according to claim 1, comprising the at least one iron chelator at a concentration of 0.01 to 0.5 mmol/L.

14. The prothrombin time reagent of claim 1, wherein the prothrombin time reagent is a liquid.

15. The method of claim 6, wherein the liquid prothrombin time reagent comprises Ca.sup.2+ ions.

16. The method of claim 7, wherein the prothrombin time reagent comprises Ca.sup.2+ ions.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the change in prothrombin times in seconds (PT [s]) of the normal control plasma Ci-Trol 1 with the deferoxamine-stabilized PT reagents (9=9 mg/L=0.0137 mM deferoxamine; 12=12 mg/L=0.0183 mM deferoxamine; 15=15 mg/L=0.0228 mM deferoxamine) compared to the unstabilized PT reagent without deferoxamine (0=0 mg/L=0 mM deferoxamine) and the reference reagent (R) stored lyophilized and then freshly dissolved over time (t) in weeks. The reagents were stored at a temperature of 37° C.

(2) FIG. 2 shows the change in prothrombin times in seconds (PT [s]) of the abnormal control plasma Ci-Trol 2 with the deferoxamine-stabilized PT reagents (9=9 mg/L=0.0137 mM deferoxamine; 12=12 mg/L=0.0183 mM deferoxamine; 15=15 mg/L=0.0228 mM deferoxamine) compared to the unstabilized PT reagent without deferoxamine (0=0 mg/L=0 mM deferoxamine) and the reference reagent (R) stored lyophilized and then freshly dissolved over time (t) in weeks. The reagents were stored at a temperature of 37° C.

(3) FIG. 3 shows the change in prothrombin times in seconds (PT [s]) of the normal control plasma Ci-Trol 1 with the deferoamine-stabilized PT reagents (15=15 mg/L=0.0228 mM deferoxamine; 75=75 mg/L=0.114 mM deferoxamine; 150=150 mg/L=0.228 mM deferoxamine) compared to the unstabilized PT reagent without deferoxamine (0=0 mg/L=0 mM deferoxamine) over time (t) in weeks. The reagents were stored at a temperature of 37° C.

(4) FIG. 4 shows the change in prothrombin times in seconds (PT [s]) of the abnormal control plasma Ci-Trol 2 with the deferoxamine-stabilized PT reagents (15=15 mg/L=0.0228 mM deferoxamine; 75=75 mg/L=0.114 deferoxamine; 150=150 mg/L=0.223 mM deferoxamine) compared to the unstabilized PT reagent without deferoxamine (0=0 mg/L=0 mM deferoxamine) over time (t) in weeks. The reagents were stored at a temperature of 37° C.

(5) The following exemplary embodiments serve the purpose of clarifying the present invention and are not to be understood as limitative.

Example 1: Determination of the Stability of a Liquid Deferoxamine-Stabilized Prothrombin Time Reagent

(6) At time to, deferoxamine (Deferoxamine mesylate salt, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was added in various batches to a liquid prothrombin time reagent comprising recombinant human tissue factor protein, synthetic phospholipids, and calcium ions in the final concentrations indicated: 1. 0 mM deferoxamine, 2. 0.0137 mM (9 mg/L) deferoxamine, 3. 0.0183 mM (12 mg/L) deferoxamine, 4. 0.0228 mM (15 mg/L) deferoxamine, 5. 0.114 mM (75 mg/L) deferoxamine, 6. 0.228 mM (150 mg/L) deferoxamine.

(7) The various reagents were used in an automatic prothrombin time test (PT test) in a Sysmex® CA-1500 analyser (Sysmex Corp., Kobe, Japan). The following defined control plasmas were used as samples: Dade® Ci-Trol® Coagulation Control Level 1 (Ci-Trol 1) control plasma; normal control for determination of prothrombin time; the results are comparable to those with fresh normal citrate plasma; Dade® Ci-Trol® Coagulation Control Level 2 (Ci-Trol 2) control plasma; abnormal control for determination of prothrombin time as observed in clotting disorders; the clotting times are prolonged compared to those with fresh normal citrate plasma.

(8) The samples and reagents respectively were pre-heated to 37° C. and then mixed. The addition of the reagent triggered the clotting process, and the time until formation of the fibrin clot was measured (prothrombin time in seconds).

(9) In order to determine the long-term stability of the various reagents, the reagents were stored in a liquid state in stoppered glass vials at +37° C. over a period of 16-18 weeks. About every two weeks, samples of the reagents were taken, and the prothrombin time of the same reference plasma was determined. At each point in time, as a reference reagent, a lyophilized prothrombin time reagent (Dade® Innovino reagent, Siemens Healthcare Diagnostics Products GmbH, Germany) was freshly reconstituted with distilled water and measured.

(10) In FIG. 1 (Ci-Trol 1 as a sample) and FIG. 2 (Ci-Trol 2 as a sample), the prothrombin times of the various prothrombin time reagents (0, 12, 15 mg/L deferoxamine) over a period of 16 weeks at +37° C. are shown. Stable prothrombin times over a period of at least 10 weeks with deviations of less than 20% relative to the initial value at time to were achieved only with the lyophilized and in each case freshly dissolved reference reagent (R) and with the deferoxamine-stabilized prothrombin time reagents according to the invention. The reagent stored in a liquid state without deferoxamine yielded only sharply deviating prothrombin times at the latest after 4 weeks.

(11) In FIG. 3 (Ci-Trol 1 as a sample) and FIG. 4 (Ci-Trol 2 as a sample), the prothrombin times of the various prothrombin time reagents (15, 75, 150 mg/L deferoxamine) over a period of 18 weeks at +37° C. are shown. Stable prothrombin times over a period of at least 10 weeks with deviations of less than 20% relative to the baseline at time to were achieved only with the deferoxamine-stabilized prothrombin time reagents according to the invention. The reagent stored in a liquid state without deferoxamine yielded only sharply deviating prothrombin times at the latest after 4 weeks.

Example 2: Determination of the Stability of Liquid Prothrombin Time Reagents Stabilized with Various Iron Chelators

(12) At time to, 0.0228 mmol/L each of the following metal ion chelators was added in various batches to a liquid prothrombin time reagent comprising recombinant human tissue factor protein, synthetic phospholipids, and calcium ions: i. Pyoverdine (Pyoverdines, Sigma-Aldrich Chemie GmbH, Steinheim, Germany); ii. Ferrichrome (Ferrichrome Iron-free from Ustilago sphaerogena, Sigma-Aldrich Chemie GmbH, Steinheim, Germany); iii. Deferoxamine (Deferoxamine mesylate salt, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) Deferoxamine (Deferoxamine mesylate salt, Sigma-Aldrich Chemie GmbH, Steinheim, Germany); iv. Deferasirox (Combi-Blocks, Inc., San Diego, USA); v. DTPA (diethylenetriaminepentaacetic acid); vi. EDTA (ethylenediaminetetraacetic acid); vii. BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid).

(13) The various reagents were used as described in example 1 in an automatic prothrombin time test (PT test) in a Sysmexf CA-1500 analyser (Sysmex Corp., Kobe, Japan). The control plasmas Ci-Trol 1 and Ci-Trol 2 described in further detail in example 1 were used as samples.

(14) In order to determine the long-term stability of the various reagents, the reagents were stored in a liquid state in stoppered glass vials at +37° C. over a period of 16-18 weeks. Samples of the reagents were taken approximately monthly, and the prothrombin time of the same reference plasma was determined.

(15) The various measurement results (prothrombin time in seconds, PT [s]) at time to, after two- and four-month storage (2 M, 4 M) and the corresponding deviation in % from the prothrombin time measurement at time to are shown in Table 1 (Ci-Trol 1 control plasma as a sample) and in Table 2 (Ci-Trol 2 control plasma as a sample).

(16) TABLE-US-00001 TABLE 1 t.sub.0 PT 2 M PT 4 M PT Chelator [s] [s] [s] 2 M % 4 M % Pyoverdine 12.0 11.8 13.4 −1.7 11.7 Ferrichrome 12.1 11.9 12.9 −1.7 6.6 Deferoxamine 11.6 11.5 13.1 −0.9 12.9 Deferasirox 11.6 11.4 13.8 −1.7 19.0 DTPA 11.6 12.5 22.4 7.8 93.1 EDTA 11.8 35.9 71.5 204.2 505.9 BAPTA 11.6 40.2 72.8 246.6 527.6 No chelator 11.6 36.7 71.7 216.4 512.9

(17) TABLE-US-00002 TABLE 2 t.sub.0 PT 2 M PT 4 M PT Chelator [s] [s] [s] 2 M % 4 M % Pyoverdine 43.5 37.6 38.7 −13.6 −11.0 Ferrichrome 44.4 38.8 40.0 −12.6 −9.9 Deferoxamine 39.5 36.3 37.1 −8.1 −6.1 Deferasirox 38.9 32.8 43.5 −15.7 11.8 DTPA 39.0 39.1 82.0 0.3 110.3 EDTA 40.2 100.0 100.0 148.8 148.8 BAPTA 39.1 100.0 100.0 155.8 155.8 No chelator 38.8 100.0 100.0 157.7 157.7

(18) Stable prothrombin times over a period of four months (i.e. at least 16 weeks) with deviations of less than 20% relative to the baseline at time to were achieved only with the iron chelators according to the invention, i.e. prothrombin time reagents stabilized with pyoverdine, ferrichrome, deferoxamine or deferasirox. Both the reagent stored in liquid form without a chelator and the reagents stored in liquid form to which the metal ion chelators DTPA, EDTA or BAPTA known from the prior art (US 2017/0234895 A1) were added showed only strongly deviating prothrombin times at the latest after 4 months.

Example 3: Determination of the Stability of Liquid Prothrombin Time Reagents Stabilized with Various Iron Chelators in the Presence of Elevated Fe.SUP.3 .Ion Concentrations

(19) At time to, 0.00308 mM (0.5 mg/L) FeCl.sub.3 was added to a liquid prothrombin time reagent comprising recombinant human tissue factor protein, synthetic phospholipids, and calcium ions, and then 0.0228 mM each of the metal ion chelators mentioned in example 2 was added in various batches.

(20) The various reagents were used as described in example 1 in an automatic prothrombin time test (PT test) in a Sysmexf CA-1500 analyser (Sysmex Corp., Kobe, Japan). The control plasmas Ci-Trol 1 and Ci-Trol 2 described in further detail in example 1 were used as samples.

(21) In order to determine the long-term stability of the various reagents in the presence of elevated Fe.sup.3+ ion concentrations, the reagents were stored in a liquid state in stoppered glass vials at +37° C. over a period of 6 weeks. Samples of the reagents were taken weekly, and the prothrombin time of the same reference plasma was determined.

(22) The various measurement results (prothrombin time in seconds, PT [s]) at time to, after one-, two-, four- and six-week storage (1 W, 2 W, 4 W, 6 W) and the corresponding deviation in % from the prothrombin time measurement at time to are shown in Table 3 (Ci-Trol 1 control plasma as a sample) and in Table 4 (Ci-Trol 2 control plasma as a sample).

(23) TABLE-US-00003 TABLE 3 t.sub.0 1 W 2 W 4 W 6 W PT PT PT PT PT Chelator [s] [s] [s] [s] [s] 1 W % 2 W % 4 W % 6 W % Pyoverdine 12 11.2 11.4 11.5 11.4 −6.7 −5 −4.2 −5 Ferrichrome 12.1 11.2 11.4 11.6 11.6 −7.4 −5.8 −4.1 −4.1 Deferoxamine 11.4 10.7 11 11.1 11.2 −6.1 −3.5 −2.6 −1.8 Deferasirox 11.6 10.9 10.9 11.1 11.4 −6 −6 −4.3 −1.7 DTPA 11.8 13 13.4 14.9 17.7 10.2 13.6 26.3 50 EDTA 11.6 13 15.9 30.2 49.6 12.1 37.1 160.3 327.6 BAPTA 11.6 13.2 17.7 38.3 60.8 13.8 52.6 230.2 424.1 No chelator 11.4 13.1 17.9 48.9 59.5 14.9 57 328.9 421.9

(24) TABLE-US-00004 TABLE 4 t.sub.0 1 W 2 W 4 W 6 W PT PT PT PT PT Chelator [s] [s] [s] [s] [s] 1 W % 2 W % 4 W % 6 W % Pyoverdine 44 39.6 40 40.1 35.5 −10 −9.1 −8.9 −19.3 Ferrichrome 44.4 39.1 39.5 40.3 38.9 −11.9 −11 −9.2 −12.4 Deferoxamine 38 32.7 35.4 35.2 35.9 −13.9 −6.8 −7.4 −5.5 Deferasirox 39 35.1 33.5 31.2 32 −10 −14.1 −20.0 −17.9 DTPA 39.6 28.8 30.4 39.2 55.7 −27.3 −23.2 −1 40.7 EDTA 38.6 30.6 50 100 100 −20.7 29.5 159.1 159.1 BAPTA 38.6 32.7 61.8 100 100 −15.3 60.1 159.1 159.1 No chelator 38.4 32.1 64.8 100 100 −16.4 68.8 160.4 160.4

(25) Stable prothrombin times over a period of six weeks with deviations of less than 20% relative to the baseline at time to were achieved in the presence of elevated Fe.sup.31 ion concentrations only with the iron chelators according to the invention, i.e. with the prothrombin time reagents stabilized with pyoverdine, ferrichrome, deferoxamine or deferasirox. Both the reagent stored in liquid form without a chelator and the reagents stored in liquid form to which the metal ion chelators DTPA, EDTA or BAPTA known from the prior art (US 2017/0234895 A1) were added showed only strongly deviating prothrombin times at the latest after 2 weeks.