IDENTIFICATION OF ANTICOAGULANTS IN A SAMPLE
20190302132 ยท 2019-10-03
Inventors
Cpc classification
International classification
Abstract
The present invention is directed to a novel method of determining inhibitors of proteolytically active coagulation factors, referred to herein as anticoagulants, in a sample, in particular the qualitative detection of direct thrombin and factor Xa inhibitors in a sample. The method of the present invention allows a qualitative determination of the nature anticoagulants present in a sample. This can be achieved with only one coagulation-based test. The method can be used in a test kit, including a point-of-care (POC) system.
Claims
1-11. (canceled)
12. A method for identification of inhibitors of factor IIa and/or factor Xa in a sample selected from blood or plasma, said method comprising: (a) establishing reference ranges (RR) using reference samples with increasing concentrations of known anti-coagulants, (b) measuring clot formation in the sample, (c) comparing the clotting times of the sample to reference ranges, wherein the clotting times are measured via prothrombinase-induced clotting time (PiCT) according to PiCT standard and/or PiCT inverted, and wherein: (i) in PiCT standard, the activator is added to the sample, the mixture allowed to proceed according to the manufacturer's instructions followed by addition of the start reagent, (ii) in PiCT inverted, the start reagent is added to the sample, the mixture allowed to proceed according to the manufacturer's instructions followed by addition of the activator; and wherein in the presence of an inhibitor of factor IIa and/or factor Xa in the sample clotting times are outside the RR when applying PiCT inverted and are either outside or within RR when applying PiCT standard.
13. The method according to claim 12, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from rivaroxaban and/or apixaban in the sample clotting times are within RR when applying PiCT standard.
14. The method according to claim 12, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from dabigatran, hirudin, argatroban, fondaparinux, unfractionated heparins (UFH) and/or low molecular weight heparins (LMWH) in the sample clotting times are outside RR when applying PiCT standard.
15. The method according to claim 12 further comprising measurement of clot formation via prothrombin time (PT) and/or activated partial thromboplastin time (aPTT) in the sample, and comparing the clotting times of the sample to reference ranges; wherein the clotting times in the sample are either outside or within RR when applying PT and/or aPTT.
16. The method according to claim 13 further comprising measurement of clot formation via prothrombin time (PT) and/or activated partial thromboplastin time (aPTT) in the sample, and comparing the clotting times of the sample to reference ranges; wherein the clotting times in the sample are either outside or within RR when applying PT and/or aPTT.
17. The method according to claim 14 further comprising measurement of clot formation via prothrombin time (PT) and/or activated partial thromboplastin time (aPTT) in the sample, and comparing the clotting times of the sample to reference ranges; wherein the clotting times in the sample are either outside or within RR when applying PT and/or aPTT.
18. The method according to claim 15, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from dabigatran, hirudin and/or argatroban in the sample clotting times are outside RR when applying PT.
19. The method according to claim 15, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from UFH, LMWH and/or fondaparinux in the sample clotting times are within RR when applying PT.
20. The method according to claim 15, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from dabigatran, hirudin, argatroban, UFH and/or LMWH in the sample clotting times are outside RR when applying aPTT.
21. The method according to claim 15, wherein in the presence of an inhibitor of factor IIa and/or factor Xa selected from fondaparinux in the sample clotting times are within RR when applying aPTT.
22. The method according to claim 12, further comprising: (d) addition of polybrene and (e) comparing the clotting times before and after step (d).
23. The method of claim 22 for detection of heparins selected from UFH or LMWH in the sample, wherein clotting times outside RR according to step (c)(i) are changed into clotting times within RR after addition of polybrene.
24. The method of claim 22 for detection of low concentration of dabigatran and/or hirudin in the sample, wherein clotting times outside RR according to step (c)(i) are not changed after addition of polybrene.
25. The method of claim 22 for detection of low concentration of rivaroxaban or apixaban in the sample, wherein clotting times within RR according to step (c)(i) are not changed after addition of polybrene.
26. The method according to claim 12 used in a point-of-care system.
27. The method according to claim 15 used in a point-of-care system.
28. The method according to claim 16 used in a point-of-care system.
29. The method according to claim 17 used in a point-of-care system.
30. The method according to claim 22 used in a point-of-care system.
Description
FIGURES
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[0070] The following examples are illustrative only and are not intended to limit the scope of the invention in any way.
EXAMPLES
[0071] Stock solutions of anticoagulants were prepared in deionized water for UFH (250 IU/mL, WHO standard 07/328, NIBSC), LMWH (250 IU/mL, Fragmin, Pfizer), and lepirudin (0.5 mg/mL, (Refludan, Bayer HealthCare Pharmaceuticals Inc) or in DMSO for rivaroxaban (0.5 mg/mL, Alsachim, France) and apixaban (2 mg/mL, Alsachim) or in 0.1M HCl for dabigatran (Alsachim), and were stored at 20 C. Fondaparinux (5 mg/mL, injectable solution, Arixtra, GlaxoSmithKlines) was stored at 4 C. Anticoagulants were further diluted with deionized water and spiked in NPP prepared from 24 healthy donors (Universittsklinikum Wrzburg, Germany) for PiCT measurements. The final concentrations of DMSO or HCl in NPP did not exceed 0.15% and 0.4 mM respectively, and did not affect coagulation.
Example 1: PiCT Adaptations and Determination of Reference Ranges (RR)
[0072] As a starting point, the RR for four different types of anticoagulants had to be established as described above. These have to be adapted to the testing system and also to the kind of drug, as e.g. in the case of fondaparinux (PiCT Method 2) or dabigatran/argatroban (PiCT Method 3). The results are shown in Table 2 wherein the results are given using fresh plasma from 17 healthy donors.
TABLE-US-00002 TABLE 2 Establishing the RR on the ACL TOP 500. The clotting time (RR, borderline, out of range) is given in seconds, indicating the mean and the lower and upper ranges (in parentheses). Method RR PiCT Method 1 27.6 (24.4-30.7) PiCT Method 2 40.0 (31.9-48.1) PiCT Method 3 44.7 (38.1-51.3) PiCT Method 4 29.6 (21.4-37.9) HemosIL aPTT SP 25.4-36.9 HemosIL PT recombiplastin 9.4-12.5
[0073] Protocols and pipetting schemes designed for ACL TOP 500 are described in Table 3. To assess the potential of Pefakit PiCT to evaluate anticoagulant effects of most common anticoagulant drugs, DRCs were performed using NPP spiked with increasing levels of UFH, LMWH, fondaparinux, hirudin, rivaroxaban, apixaban or dabigatran. The details of the different methods, i.e. PiCT Method 1 to 4, are described in the text.
TABLE-US-00003 TABLE 3 pipetting scheme for the 4 different PiCT Methods described in the text, wherein P means plasma in l, A means activator reagent in l, S means start reagent in l, and I means incubation time in seconds. PiCT Protocol for ACL Best DRC methods TOP 500 fit for Method 1 P-50/A-50/I-180/S-50 UFH, LMWH, hirudin Method 2 P-75/A-65/I-180/S-35 Fondaparinux Method 3 P-90/A-70/I-180/S-40 Argatroban, dabigatran Method 4 P-140/S-45/I-160/A-40 Rivaroxaban, apixaban
[0074] PiCT Method 4 was identified to offer best results. It consists of inverting the order of reagents' addition to plasma. PiCT is very sensitive to low levels of NOACs.
Example 2: Pefakit PiCT as a Tool to Identify Whether a Plasma Sample Contains an Anticoagulant
[0075] PiCT Method 4, which is an inverted PiCT, was developed as new method to achieve best DRCs for rivaroxaban and apixaban. After calculation of the RR for the different kind of anticoagulants (see Example 1) samples of NPP spiked with incremental amounts of anticoagulants were used in the ACL TOP 500, wherein 45 l start reagent was incubated with 140 l plasma for 60 sec. The time to clot was recorded after addition of 40 l of activator (i.e. so-called inverted PiCT). The result is shown in Table 4, wherein the number of samples (NPP) spiked with incremental amounts of anticoagulants (AC) is indicated (n), i.e. indicated as treatment. Every sample with a result starting from a certain threshold above the RR contains an anticoagulant, which is true for all drugs listed in the Table.
TABLE-US-00004 TABLE 4 identification of anticoagulants in plasma samples using PiCT Method 4. The baseline means the RR in a sample without the addition of any drug and is shown as mean and as whole range (in brackets). Conc. means concentration of the anticoagulant per ml of sample. Numbers in bold indicates a clotting time out of range, i.e. presence of a drug in the sample, wherein the negative control, i.e. within RR is underlined. Clotting time with AC present Treatment Conc. PiCT Method 4 [sec] yes/no Baseline 0 29.6 (21.4-37.9) no n = 6 UFH 0.3 IU 146 (n = 2) yes LMWH 0.4 IU 129 (n = 2) hirudin 0.4 g 300 (n = 2) fondaparinux 0.3 g 57 (n = 2) argatroban 0.3 g 147 (n = 2) dabigatran 20 ng 60 (n = 3) 35 ng 80 (n = 3) rivaroxaban 20 ng 70 (n = 3) 35 ng 80 (n = 3) apixaban 20 ng 45 (n = 3) 35 ng 54 (n = 3)
Using this method, low amounts of all NOACs, UFH, LMWH, hirudin, fondaparinux or argatroban could be detected as shown in Table 4.
Example 3: Pefakit PiCT as a Tool to Determine Which NOAC is Present in a Sample
[0076] With the normal PiCT, i.e. PiCT Method 1, wherein 50 l of plasma is mixed with the same amount of reagent 1 (PiCT activator), incubated for 180 sec and mixed with the same amount of reagent 2 (PiCT start reagent) in a ACL TOP 500 analyzer, the presence of NOACs cannot be determined in a sample. This is true even at high levels of said drugs in the sample, i.e. in the range of 300 ng/ml. Thus, in order to determine which NOAC is present in the sample, a combination of PiCT Method 1 and PiCT method 4 has to be used (see above for detailed description). To confirm this, spiked plasma samples with different levels of NOACs were prepared and the clotting time measured using first PiCT Method 1 followed by PiCT Method 4. The result is shown in Table 5. In the case of rivaroxaban or apixaban present in a tested sample, PiCT Method 1 is within the range (indicated by the grey background color) whereas PiCT Method 4 is out of range (indicated in bold). In case of dabigatran present in the sample, both PiCT Method 1 (which had to be adapted, i.e. PiCT Method 3) and 4 are out of range as indicated in bold.
TABLE-US-00005 TABLE 5 distinction between different NOACs present in a sample determined by a subsequent performance of PiCT Method 1 (PiCT Method 3 for dabigatran) and PiCT Method 4. Results within the RR are indicated by underline, results above the RR are marked in bold. For more details see text or legend to Table 4. Clotting time with Clotting time with PiCT Method 1 PiCT Method 4 NOAC Treatment Conc. [sec] [sec] present Baseline 0 27.6 (24.4-30.7) 29.6 (21.4-37.9) no n = 17 n = 17 rivaroxaban 20 ng 17 (n = 3) 70 (n = 3) rivaroxaban or 35 ng 18 (n = 3) 80 (n = 3) apixaban 300 ng 29.3 (n = 2) 162.1 (n = 2) apixaban 20 ng 18 (n = 3) 45 (n = 3) 35 ng 18 (n = 3) 54 (n = 3) 300 ng 24.9 (n = 2) 124.3 (n = 2) dabigatran 20 ng 37 (n = 3) 60 (n = 3) dabigatran 35 ng 45 (n = 3) 80 (n = 3) 300 ng 117.5 (n = 2) 278.1 (n = 2)
Example 4: Pefakit PiCT as a Tool to Further Determine the Nature of an Anticoagulant in a Plasma Sample
[0077] In order to address the above question, i.e. to determine the nature of an anticoagulant in a sample, a combination of several methods has to be performed, including PiCT Method 1, PiCT Method 4, aPTT (HemosIL aPTT SP), PT (HemosIL PT recombiplastin), and sometimes even a modified PiCT Method 1 wherein polybrene has been added. In order to simulate this, plasma samples spiked with different levels of anticoagulants were analyzed using the ACL TOP 500 analyzer. The results are shown in Table 6. For each drug, a specific pattern was obtained, i.e. clotting times within the RR and clotting times above the RR, depending on the used testing method, which is unique at least for the class of anticoagulant or even for a specific anticoagulant.
TABLE-US-00006 TABLE 6 scheme for determination of what kind of anticoagulant is present in spiked plasma samples. Only mean values are given. The n refers to the number of healthy donors. For more details see text and legend to Tables 4 or 5. Clotting time with Clotting time with Clotting time Clotting time Clotting time with Concen- PiCT Method 1 PiCT Method 4 with aPTT with PT PiCT Method 1 + Treatment tration [sec] [sec] [sec] [sec] polybrene [sec] Baseline 0 27.0 34.4 28.7 10.9 n = 6 n = 6 n = 6 n = 6 UFH 0.3 IU 78.5 146.0 66.9 11.2 32.0 n = 2 n = 2 n = 2 n = 2 n = 2 0.8 IU 119.1 300 195.8 11.4 32.0 n = 2 n = 2 n = 2 n = 2 n = 2 LMWH 0.4 IU 81.2 128.9 48.0 10.9 31.1 n = 2 n = 2 n = 2 n = 2 n = 2 1.0 IU 123.3 300 81.9 11.1 32.4 n = 2 n = 2 n = 2 n = 2 n = 2 Hirudin 0.4 g 88.9 300.0 60.6 11.8 89.5 n = 2 n = 2 n = 2 n = 2 n = 2 2.5 g 264.5 300.0 108.8 14.6 258.0 n = 2 n = 2 n = 2 n = 2 n = 2 Fondaparinux 0.3 mg 53.8 56.6 33.2 11.0 n = 2 n = 2 n = 2 n = 2 1.3 mg 84.7 138.9 35.1 11.4 n = 2 n = 2 n = 2 n = 2 Argatroban 0.3 g 98.5 147.4 61.7 15.0 85.6 n = 2 n = 2 n = 2 n = 2 n = 2 1.5 g 150.3 300.0 106.2 31.2 126.4 n = 2 n = 2 n = 2 n = 2 n = 2 Dabigatran 50 ng 56.2 100.7 39.6 11.4 59.7 n = 5 n = 5 n = 4 n = 4 n = 2 300 ng 117.5 278.1 75.9 14.4 103.8 n = 2 n = 2 n = 2 n = 2 n = 2 Rivaroxaban 50 ng 19.0 83.6 34.5 12.2 n = 5 n = 9 n = 4 n = 4 200 ng 25.5 132.8 49.6 20.3 n = 3 n = 5 n = 2 n = 2 Apixaban 50 ng 18.9 54.4 31.0 11.2 n = 5 n = 11 n = 4 n = 4 200 ng 22.3 151.0 36.4 14.5 n = 2 n = 8 n = 2 n = 2
[0078] In case of a measurement using PiCT Method 1 and PiCT Method 4, wherein the clotting time determined with the first method is within the RR but the time measured with the second method is above the RR, the anticoagulant present in the sample is either apixaban or rivaroxaban. Further tests have been performed, i.e. the aPTT or PT in order to distinguish between both anticoagulants: in case of rivaroxaban, with both tests the clotting times were above the RRbut only at higher doses, such as in the range of 200 ng or more (see Table above). If, for example, both the results from PiCT Method 1 and 4 are above the RR but the aPTT and PT revealed clotting times within the RR, the drug present in the sample is fondaparinux.
Example 5: Pefakit PiCT as a Tool to Quantify the Effect of an Anticoagulant
[0079] Depending on the drug used, the protocols to determine the quantitative effect of an anticoagulant might differ, i.e., using different testing methods, including the ones listed in Table 1 showing the RRs for the various methods. If the effect of heparins or hirudins, such as e.g. UFH, LMWH and hirudin, is to be quantified, Pefakit PiCT with PiCT Method 1 turned out to be useful, which is shown in Table 7A-C showing DRC in NPP samples spiked with UFH (Table 7A), LMWH (Table 7B) or hirudin (Table 7C) as measured with PiCT Method 1 and ACL TOP 500 analyzer. Only mean values are given. For the baseline, i.e. no anticoagulant was present in the sample, the number of measurements was n=6 with a clotting time of 24 seconds measured with PiCT Method 1.
TABLE-US-00007 TABLE 7A PiCT Method 1 using NPP spiked with UFH and n = 4. For more details see text and legend to Table 4. Concentration [IU/ml] 0.1 0.2 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.6 Clotting time 30 45 62 86 102 113 123 132 143 147 [sec]
TABLE-US-00008 TABLE 7B PiCT Method 1 using NPP spiked with LMWH and n = 4. For more details see text and legend to Table 4. Concentration [IU/ml] 0.1 0.2 0.3 0.4 0.5 0.7 0.9 1.0 1.2 1.4 Clotting time 37 47 57 67 76 92 108 114 124 132 [sec]
TABLE-US-00009 TABLE 7C PiCT Method 1 using NPP spiked with hirudin and n = 4. For more details see text and legend to Table 4. Concentration [g/ml] 0.2 0.4 0.6 0.9 1.3 1.7 2.0 2.4 2.8 3.0 Clotting time 41 55 69 86 113 142 158 183 209 219 [sec]
For fondaparinux, PiCT Method 2 was determined to be useful. Examples of results obtained with NPP samples on ACL TOP 500 analyzer are shown in Table 8.
TABLE-US-00010 TABLE 8 DRC in NPP samples spiked with fondaparinux as measured with PiCT Method 2. Only mean values are given. The number of measurement was n = 4. Clotting time for the baseline, i.e. no anticoagulant present in the sample, was 39 seconds measured with PiCT Method 2. Concentration [g/ml] 0.2 0.4 0.5 0.6 0.9 1.1 1.3 1.5 1.8 2.0 Clotting 78 92 98 103 120 122 133 139 148 155 time [sec]
[0080] With regards to dabigatran and argatroban, the DRC was established using PiCT Method 3, the relevant clotting times were extrapolated with the particular conditions as described herein. For all measurements, the ACL TOP 500 analyzer was used making use of NPP samples spiked with incremental amounts of anticoagulant covering both average peak or trough (see Stangier J, Clin Pharmacokinet 2008, 47:47-59; van Ryn J, Thromb Haemost 2010, 103:1116-1127; Baglin T, J Thromb Haemost. 2013 Jan. 24). Examples of results obtained with NPP samples spiked with either dabigatran (Table 9A) or argatroban (Table 9B) are presented below.
TABLE-US-00011 TABLE 9A DRC in NPP samples spiked with dabigatran as measured with PiCT Method 3. Only mean values are given. The number of measurements was n = 4. Clotting time for the baseline, i.e. no anticoagulant present in the sample, was 45 seconds measured with PiCT Method 3. Concentration [ng/ml] 5 10 50 100 200 300 400 500 600 750 Clotting time 56 70 122 153 190 207 220 234 245 253 [sec]
TABLE-US-00012 TABLE 8B DRC in NPP samples spiked with argatroban as measured with PiCT Method 3. Only mean values are given. The number of measurements (n) was n = 4. Clotting time for the baseline, i.e. no anticoagulant present in the sample, was 45 seconds measured with PiCT Method 3. Concentration [g/ml] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Clotting time [sec] 113 145 162 179 198 204 210 232 234 240
[0081] Regarding rivaroxaban or apixaban, DRC was established making use of PiCT Method 4 with extrapolation of relevant clotting times associated with the particular conditions. The following are examples of clotting times obtained in ACL TOP 500 analyzer making use of NPP samples spiked with incremental amounts of anticoagulant levels covering both average peak and trough concentrations of rivaroxaban (see Mueck W, Thromb Haemost 2008, 100:453-461; Xu X S, Br J Clin Pharmacol 2012, 74:86-97; Baglin T, J Thromb Haemost. 2013 Jan 24) as shown in Table 10A or apixaban (see Raghavan N, Drug Metab Dispos 2009, 37:74-81; Frost C, Br J Clin Pharmacol 2012, 75:476-487; Frost C, Br J Clin Pharmacol 2013, 76:776-786) as shown in Table 10B. Only mean values are given. For the baseline, i.e. no anticoagulant was present in the sample, the number of measurements was n=4 with a clotting time of 54 seconds measured with PiCT Method 4.
TABLE-US-00013 TABLE 10A PiCT Method 4 using NPP spiked with rivaroxaban and n = 4. For more details see text. Concentration 5 10 50 100 200 300 400 500 600 [ng/ml] Clotting time 71 83 118 149 191 220 248 270 278 [sec]
TABLE-US-00014 TABLE 10B PiCT Method 4 using NPP spiked with apixaban and n = 4. For more details see text. Con- 5 10 50 100 200 300 400 500 600 750 centration [ng/ml] Clotting 65 74 109 136 167 197 210 233 259 268 time [sec]
Example 6: Pefakit PiCT as a Tool to Evaluate if NOAC Levels are Low Enough
[0082] For answering this question with regards to rivaroxaban or apixaban, DRC was generated making use of PiCT Method 4 (see above). The relevant clotting times associated with the particular conditions were extrapolated. All data were obtained using the ACL TOP 500 analyzer and plasma samples spiked with anticoagulant levels corresponding to concentration in the plasma reported as wash off levels. The result is shown in Table 11.
TABLE-US-00015 TABLE 11 use of PiCT Method 4 and plasma samples spiked with different low concentrations of two anticoagulants, i.e. rivaroxaban and apixaban. For more details see text. PiCT method 4 clotting time Rivaroxaban: 20 ng/mL 70 sec (n = 3) 35 ng/mL 80 sec (n = 3) Apixaban: 20 ng/mL 45 sec (n = 3) 35 ng/mL 54 sec (n = 3)
[0083] In order to evaluate the level of dabigatran in a sample, DRC was generated making use of PiCT Method 3, the relevant clotting times associated with the particular conditions were extrapolated. All data were obtained using the ACL TOP 500 analyzer and plasma samples spiked with anticoagulant levels corresponding to concentration in the plasma reported as wash off levels. The result is shown in Table 12.
TABLE-US-00016 TABLE 12 use of PiCT Method 3 and plasma samples spiked with different low concentrations of dabigatran. For more details see text. PiCT method 3 clotting time Dabigatran: 20 ng/mL 84 sec (n = 11) 40 ng/mL 109 sec (n = 11)