Method and device for the determination of platelet function under flow conditions

Abstract

The invention lies in the area of platelet function diagnostics and relates to a method for the determination of platelet function under flow conditions as well as a device for the implementation of this method. The method is particularly suitable for the determination of the effect of clopidogrel and of other P2Y(12) antagonists with antithrombotic activity as well as the determination of P2Y(1) antagonists with antithrombotic activity.

Claims

1. A method for the determination of an antithrombotic effect of a P2Y(12) antagonist or a P2Y(1) antagonist in a whole blood sample, the method comprising the following steps: a) passing the blood through a capillary and then through an opening of a partition member; and b) measuring the time that is required for the formation of a thrombus at the opening of the partition member up to closure of the opening; wherein the partition member comprises: i) an activator of purinergic receptors; ii) an activator of intracellular adenylate cyclases; and iii) calcium ions; wherein the whole blood sample comprises one or more of the P2Y(12) antagonist and the P2Y(1) antagonist, and optionally an acetylsalicylic acid, and wherein the time measured in step (b) indicates the antithrombotic effect of the P2Y(12) antagonist or the P2Y(1) antagonist in the whole blood sample, and wherein the time measured in step (b) does not indicate the antithrombotic effect of the acetylsalicylic acid in the whole blood sample.

2. The method of claim 1, wherein the partition member used comprises an activator of purinergic receptors from the group adenosine-5′-diphosphate, 2-methylthioadenosine-5′-diphosphate, and their derivatives.

3. The method of claim 1, wherein the partition member comprises an activator of intracellular adenylate cyclases from the group prostaglandin E1, forskolin and their derivatives, prostaglandin I2 and its stable derivatives, illoprost, and cicaprost.

4. The method of claim 1, wherein the whole blood sample is anticoagulated with a direct thrombin inhibitor.

5. The method of claim 1, wherein the whole blood sample is anticoagulated with a direct Factor Xa inhibitor.

6. The method of claim 1, wherein the whole blood sample is anticoagulated with citrate.

7. The method of claim 1, wherein the P2Y(12) antagonist is selected from clopidogrel, ticlopidine, prasugrel, AR-C67085MX, cangrelor, C1330-7, MRS 2395, and 2-methylthioadenosine-5′-monophosphate.

8. The method of claim 1, wherein the P2Y(1) antagonist is selected from MRS 2179, MRS 2279, MRS 2500, A2P5P, A3P5P, and A3P5PS.

9. The method of claim 3, wherein the activator of intracellular adenylate cyclases is prostaglandin E1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1

(2) FIG. 1 shows by way of example how a device for the determination of platelet function according to the invention can be constructed. Shown is a test cartridge in accordance with WO 97/34698 in longitudinal section that is placed in a suitable apparatus for implementing the method according to the invention and into which extends a vacuum apparatus (15) that is responsible for the generation of the partial vacuum The vacuum apparatus (15) has a ring gasket (27) which is located as a seal on the circumferential edge (12) of the sample container (10). The test cartridge has a housing that forms a reservoir (61) and a test chamber (63). The test chamber (63) is constructed to accept a sample container (10) the cavity of which can also be referred to as measurement chamber. The sample container (10) supports a partition member (6) treated with reagents and with a central opening (aperture) and a capillary attachment (30, 31) that connects the capillary (40) with the sample container (10). Reservoir (61) and test chamber (63) are separated by a penetrable element (70). The figure shows a phase of the test cycle after the vacuum apparatus (15) is in contact with sample container (10) and has moved downwards so that the base of the sample container (10) is in contact with the support (71) and the capillary (40) has penetrated the penetrable element (70) and penetrated into the sample (11). The apparatus produces a partial vacuum in the sample container (10) by means of which the sample (11) is pulled through the capillary (40) into the first compartment (18) of the measurement chamber and then through the opening in the partition member (6).

(3) FIG. 2

(4) Diagram for the illustration of closure times (in seconds [s]) for normal untreated whole blood samples (control) and for whole blood samples that had been treated with the P2Y(12) antagonist MRS 2395 and/or the COX-1 inhibitor acetylsalicylic acid (ASA) in vitro (see Example 2). Whole blood samples from 11 healthy donors anticoagulated with sodium citrate were used. On the left of the diagram are shown the mean values and the standard deviations of the closure times determined with the ADP/PGE1/calcium test cartridge according to the invention (cut-off: 81 seconds). On the right of the diagram are shown the mean values and the standard deviations of the closure times that were determined for comparison with conventional Col/Epi test cartridges (cut-off: 158 seconds). A comparison of the two types of test cartridge shows that with use of an ADP/PGE1 test cartridge according to the invention, closure times that lie significantly above the upper reference value (cut-off) were measured with samples that were treated with the P2Y(12) antagonist MRS 2395, whereas the same samples with the use of a Col/Epi test cartridge lie to a greater extent below the upper reference value (cut-off). That means that the method for determination of platelet function according to the invention allows a more sensitive determination of platelet dysfunction induced by a P2Y(12) antagonist than the comparison method from the prior art.

(5) FIG. 3

(6) Diagram for illustration of the closure times (in seconds [s]) for normal untreated whole blood samples (control) and for whole blood samples treated in vitro with the P2Y(12) antagonist MRS 2395, the P2Y(1) antagonist MRS 2179 or the COX-1 inhibitor acetylsalicylic acid (ASA) (see example 4). Whole blood samples from 10 healthy donors anticoagulated with P PACK were used. In the diagram are shown the mean values and the standard deviations of the closure times that were determined with the ADP/PGE1 test cartridges according to the invention (cut-off: 90 seconds). The performance evaluation shows that with use of an ADP/PGE1 test cartridge according to the invention, closure times that lie significantly above the reference value (cut-off) were measured with samples that were treated with the P2Y(12) antagonist MRS 2395 or the P2Y(1) antagonist MRS 2179, whereas the samples treated with COX-1 inhibitor acetylsalicylic acid show no prolongation of closure times and thus lie below the cut-off.

EXAMPLES

Example 1: Preparation of an ADP/PGE1/Calcium Test Cartridge According to the Invention

(7) For the preparation of a partition member for a test cartridge according to the invention a polyether sulfone filter membrane (Supor® membrane, Pall GmbH, Dreieich, Germany) was cut into strips. 1 μL of a solution comprising 7 μg/μL ADP (adenosine-5′-diphosphate potassium salt.2H.sub.2O, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 5 ng/μL PGE1 (prostaglandin E1, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 367.5 μg/μL CaCl.sub.2.2H.sub.2O (equivalent to 100 μg/μL Ca.sup.2+ ions) were pipetted punctiform onto the membrane and the membrane was dried. Next a circular opening (aperture) with a diameter of 100 μm was punched out of the middle of the region of the membrane treated with the reagents. The membrane thus prepared was used as partition member in the measurement chamber of a PFA-100® test cartridge (Dade Behring Marburg GmbH, Marburg, Germany).

Example 2: Use of an ADP/PGE1/Calcium Test Cartridge According to the Invention for the Determination of the Antithrombotic Effect of a P2Y(12) Antagonist In Vitro

(8) 2a) Sample Preparation

(9) Venous blood was taken from 11 healthy donors and anticoagulated with sodium citrate (3.2% buffered Na citrate).

(10) Aliquots of the citrated whole blood sample were treated in vitro with the P2Y(12) antagonist MRS 2395 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose an ethanolic MRS 2395 stock solution (15 mg/mL) was mixed with the whole blood samples so that an end concentration of 100 μmol/L was obtained.

(11) Further aliquots of the citrated whole blood samples were treated in vitro with the COX-1 inhibitor acetylsalicylic acid (abbr.: ASA; Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose an aqueous ASA stock solution (1 mg/mL) was mixed with whole blood samples so that an end concentration of 30 μmol/L was obtained.

(12) Further aliquots of the citrated whole blood samples were treated in vitro with MRS 2395 and with ASA so that the previously stated end concentrations were achieved.

(13) After addition of the reagents the blood samples were incubated at room temperature for 5 minutes.

(14) 2b) Determination of the Antithrombotic Effect of MRS 2395 by ADP Induced Light Transmission Aggregometry (According to Born)

(15) In order to check whether the samples treated with MRS 2395 actually show a reduced platelet aggregation, platelet rich (PRP) and platelet poor (PPP) plasma was prepared from aliquots of the untreated and MRS 2395-treated whole blood samples described under Example 2a), and the samples were then treated with 2 μM ADP. The PPP samples were used as blank controls. The photometric measurement of the aggregation reaction was carried out in the automated coagulation apparatus BCT® (Dade Behring Marburg GmbH, Marburg, Germany) under continuous stirring (600 rpm). The platelet aggregation of the samples treated with MRS 2395 was reduced by a mean of 27% compared with the platelet aggregation of the untreated samples.

(16) 2c) Determination of the Antithrombotic Effect of MRS 2395 by the Method According to the Invention Under Flow Conditions

(17) To determine the closure time as a measure of platelet function the whole blood samples described under Example 2a) were investigated with the aid of the ADP/PGE1/calcium test cartridge according to the invention described in Example 1 in a PFA-100® apparatus (Platelet Function Analyzer-100, Dade Behring Marburg GmbH, Marburg, Germany). For this purpose 700 μL of a blood sample were placed in the reservoir of the temperature equilibrated test cartridge (+37° C.) and incubated at +37° C. for 3 minutes. Next a partial vacuum of −40 mbar was generated by the apparatus by which means the blood was sucked through a capillary from the reservoir (diameter 200 μm) and finally through an opening (aperture) of the partition member in the measurement chamber. The time required up to the closure of the aperture by formation of a blood clot was determined as closure time. Every sample investigated was determined in duplicate and the mean value of a duplicate determination was used as the measurement value.

(18) For comparison purposes the whole blood samples described under Example 2a) were investigated in parallel with a known Col/Epi PFA-100® test cartridge (2 μg collagen and 10 μg epinephrine on the membrane; 150 μm aperture diameter; Dade Behring Marburg GmbH, Marburg, Germany) in the PFA-100® apparatus.

(19) The results of the investigations are summarized in FIG. 2 and the relevant figure description.

(20) In Table 1 details are reported for how many of the respective 11 MRS 2395- and/or acetylsalicylic acid-treated samples a closure time above the cut-off was measured with the aid of the ADP/PGE1 test cartridge according to the invention and with the conventional Col/Epi test cartridge. In 9 of the 11 samples treated with MRS 2395 an abnormally reduced platelet aggregation was measured with the aid of the method according to the invention, whereas only 4 of 11 samples were classified as abnormal with the aid of the conventional method. That means that the method according to the invention has an increased sensitivity for a platelet dysfunction induced by a P2Y(12) antagonist. Moreover, it is of advantage that in the presence of free calcium ions the method according to the invention has a very low sensitivity for acetylsalicylic acid. In only one of 11 acetylsalicylic acid-treated samples, an abnormally reduced platelet aggregation is measured with the aid of the method according to the invention, whereas, in contrast, with the conventional Col/Epi test cartridge 8 of the 11 samples treated with acetylsalicylic acid were determined as abnormal. Samples that are treated with MRS 2395 and acetylsalicylic acid are classified 100% as abnormal with the aid of the conventional method, whereas only 9 of the 11 samples (as with sole addition of MRS 2395) are classified as abnormal with the method according to the invention. Thus on the basis of its high sensitivity for platelet dysfunction induced by P2Y(12) antagonists and its low sensitivity to platelet dysfunction induced by acetylsalicylic acid, the method according to the invention is suitable for differentiation of the two classes of antithrombotics.

(21) TABLE-US-00001 TABLE 1 Number of samples with closure times above cut-off (n = 11) Sample MRS 2395 (P2Y(12) Acetylsalicylic acid MRS 2395 + Test cartridge antagonist) (COX-1 inhibitor) Acetylsalicylic acid ADP/PGE1 9 1 9 Col/Epi 4 8 11

(22) 2d) Determination of the Reference Range for Col/Epi and ADP/PGE1 Test Cartridges

(23) Venous blood was taken from healthy donors and anticoagulated with sodium citrate (3.2% buffered Na citrate). The closure time determination was carried out for each whole blood sample in the PFA-100® apparatus. Samples from 186 donors were determined in duplicate with a Col/Epi PFA-100® test cartridge [see Example 2c)]. Samples from 159 donors were determined in duplicate with an ADP/PGE1/calcium test cartridge according to the invention [see Examples 1 and 2c)].

(24) The reference ranges (normal range) for the Col/Epi closure time and the ADP/PGE1 closure time were established in that the measurement value ranges in which 90% of the measurement values found for the healthy subjects lay were determined (90% central interval of the normal distribution of all measurements). This gave the following reference ranges for the closure times:

(25) TABLE-US-00002 Col/Epi 70-158 seconds ADP/PGE1   46-81 seconds.

(26) The upper reference limit of the reference range was defined as cut-off, i.e. as threshold value, for a platelet dysfunction. If the closure time of a patient sample deviates from the reference range it can indicate a platelet dysfunction. This means Col/Epi closure times that are greater than 158 seconds and ADP/PGE1 closure times that are greater than 81 seconds indicate the presence of a platelet dysfunction within the sense of a reduced aggregation efficiency.

Example 3: Use of an ADP/PGE1 Test Cartridge According to the Invention for the Determination of the Antithrombotic Effect of the P2Y(12) Antagonist Clopidogrel Ex Vivo

(27) Venous blood was taken from 13 patients suffering from peripheral arterial obstructive disease and who had been treated with a daily dose of 75 mg clopidogrel (Piavix®, Sanofi-Aventis) as sole antithrombotic for a period of at least 4 weeks and the blood was anticoagulated with sodium citrate (3.8 buffered Na citrate). The samples were investigated with the aid of different methods to determine platelet function. 1. according to the invention under flow conditions and use of an ADP/PGE1/calcium PFA-100® test cartridge (see Examples 1 and 2c), cut-off: >81 seconds; 2. under flow conditions and use of a PFA-100® test cartridge that differed from the test cartridge according to the invention in accordance with Example 1 in that it comprised no PGE1, cut-off: >78 seconds; 3. with ADP-induced light transmission aggregometry (according to Born) with addition of 2 μM ADP (see Example 2b), cut-off: <40% light transmission at end of test; 4. with ADP-induced light transmission aggregometry (according to Born) with the addition of 5 μM ADP (see example 2b), cut-off: <40% light transmission at end of test. This method is recommended by Sanofi-Aventis, the manufacturer of the clopidogrel preparation Plavix®, for the determination of the antithrombotic effect of the medicament.

(28) The cut-offs for the individual test procedures were determined by preliminary investigations with whole blood and plasma samples of normal donors.

(29) Table 2 presents in detail with which of the four methods in which of the 13 patient samples an antithrombotic effect of clopidogrel could be detected. “+” means an antithrombotic effect could be detected. “−” means no antithrombotic effect could be detected. “0” means that the duplicate determination gave contradictory results, i.e. one value above and one value below the cut-off.

(30) TABLE-US-00003 TABLE 2 Method 1 2 3 4 5 6 7 8 9 10 11 12 13 Sensitivity 1.) ADP/PGE1 − 0 + + + − + + + + + + + 77% 2.) ADP − − + + + − − − + − + − + 46% 3.) Aggregometry − + − + + − − + + + + + + 69% 2 μM ADP 4.) Aggregometry − − − + + − − + + + + + + 62% 5 μM ADP

(31) With the aid of the method of the invention the antithrombotic effect of clopidogrel intake could be detected in 10 of the 13 patients (77%). The duplicate determination gave contradictory results with one patient (patient no. 2), whilst with 2 patients (patient no. 1 and 6) no reduced platelet aggregation could be detected. However, in these two patients an effect of dosage of clopidogrel could not be detected in any of the methods used.

(32) The method according to the invention is more sensitive towards the platelet dysfunction induced by clopidogrel than the standard method of ADP-induced light transmission aggregometry according to Born and more sensitive than the method with which a test cartridge is used that comprises ADP but no PGE1.

Example 4: Use of an ADP/PGE1 Test Cartridge According to the Invention for the Determination of the Antithrombotic Effect of a P2Y(12) and a P2Y(1) Antagonist In Vitro

(33) 4a) Sample Preparation

(34) Venous blood was taken from 10 healthy donors and anticoagulated with 75 μM PPACK.

(35) Aliquots of the whole blood samples were treated in vitro with the P2Y(12) antagonist MRS 2395 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose an ethanolic MRS 2395 stock solution (15 mg/mL) was mixed with the whole blood samples so that an end concentration of 150 μmol/L was obtained.

(36) Further aliquots of the whole blood samples were treated in vitro with the COX-1 inhibitor acetylsalicylic acid (abbr. ASA; Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose an aqueous ASA stock solution (1 mg/mL) was mixed with the whole blood samples so that an end concentration of 30 μmol/L was obtained.

(37) Further aliquots of the whole blood samples were treated in vitro with the P2Y(1) antagonist MRS 2179 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose an aqueous MRS 2179 stock solution (1 mg/ml) was mixed with the whole blood samples so that an end concentration of 75 μmol/L was obtained.

(38) After addition of the reagents the blood samples were incubated at room temperature for 5 minutes.

(39) 4b) Determination of the Antithrombotic Effect of MRS 2395 and MRS 2179 with the Aid of the Method According to the Invention Under Flow Conditions

(40) For the determination of the closure time as a measure of the platelet function the whole blood samples described under Example 4a) were investigated with the aid of a ADP/PGE1 test cartridge according to the invention in a PFA-100® apparatus (Platelet Function Analyzer-100, Dade Behring Marburg GmbH, Marburg, Germany). The ADP/PGE1 test cartridge according to the invention used was prepared essentially as described in Example 1 but without the partition member having been treated with CaCl.sub.2.2H.sub.2O. The test cartridge thus comprised 7 μg ADP and 5 ng PGE1 but no calcium ions.

(41) 700 μL of a blood sample was added to the reservoir of the temperature equilibrated test cartridge (+37° C.) and incubated at +37° C. in the apparatus for 3 minutes. Next a partial vacuum of −40 mbar was applied by the apparatus when the blood was sucked through a capillary (diameter 200 μm) from the reservoir and finally through an opening (aperture) of the partition member in the measurement chamber. The time that was required up to the closure of the aperture by formation of a blood clot was determined as closure time. Every sample investigated was determined in duplicate and the mean value of a duplicate determination was used as the measurement value.

(42) The results of the investigations are summarized in FIG. 3 and the relevant figure description.

(43) In Table 3 details are given for how many of the respective 10 MRS 2395-, MRS 2179- or acetylsalicylic acid-treated samples a closure time above the cut-off was measured with the aid of the ADP/PGE1 test cartridge according to the invention. In 9 of the 10 samples treated with MRS 2395 and in 10 of the 10 samples treated with MRS 2179 an abnormally reduced platelet aggregation was measured with the aid of the method of the invention, whereas none of the samples treated with acetylsalicylic acid was classified as abnormal. This means that the method according to the invention has a high sensitivity for a platelet dysfunction induced both by a P2Y(12) antagonist and by a P2Y(1) antagonist. Moreover, it is of advantage that the method according to the invention has a very low sensitivity for acetylsalicylic acid in the presence of free calcium ions.

(44) Thus on the basis of its high sensitivity for platelet dysfunction induced by ADP receptor antagonists and its low sensitivity towards platelet dysfunction induced by acetylsalicylic acid the method according to the invention is suitable for differentiation of the two classes of antithrombotics.

(45) TABLE-US-00004 TABLE 3 Number of samples with closure times above cut-off (n = 10) Sample MRS 2395 Acetylsalicylic MRS 2179 (P2Y(12) acid (P2Y(1) Test cartridge antagonist) (COX-1 inhibitor) antagonist) ADP/PGE1 9 0 10

(46) 4c) Determination of the Cut-Off for ADP/PGE Test Cartridges

(47) Owing to the use of PPACK as anticoagulant the cut-off determined with citrated whole blood in the above-described examples cannot be used. Therefore, the reference range for the ADP/PGE1 closure time from the 10 samples of the healthy donors treated with PPACK was calculated by the determination of the 90% central interval of the normal distribution of the mean values of the duplicate determinations. This gave the following reference ranges for the closure times:

(48) ADP/PGE1 51-90 seconds.

(49) The upper limit of the 90% central interval was defined as cut-off, i.e. as threshold value for a platelet dysfunction.