METHODS FOR TESTING ANDEXANET POTENCY
The present disclosure relates kits and methods for measuring the potency of an andexanet sample in neutralizing a factor Xa inhibitor and restoring the activity of a wild-type factor Xa.
1 . A method for determining the activity of an andexanet sample, comprising:
admixing a test sample comprising andexanet with a mixture comprising a human factor Xa (HFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of the fXa to the fXa inhibitor is from 0.2:1 to 0.3:1;
adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate is able to release a chromophore upon reaction with the HFXa;
detecting the amount of released chromophore; and
calculating, from the amount of the released chromophore, the activity of the andexanet sample in releasing the HFXa from the direct fXa inhibitor.
2 . The method of claim 1 , wherein the molar ratio of the HFXa to the fXa inhibitor is from 0.22 to 0.28.
3 . The method of claim 1 , wherein the molar ratio of the HFXa to the fXa inhibitor is from 0.24 to 0.26.
4 . The method of any preceding claim, wherein the HFXa, after the test sample is added, has a concentration of about 8 to 14 nM.
5 . The method of claim 4 , wherein the HFXa, after the test sample is added, has a concentration of about 10 to 12 nM.
6 . The method of any preceding claim, wherein the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, letaxaban and eribaxaban.
7 . The method of claim 6 , wherein the direct fXa inhibitor is betrixaban.
8 . The method of any preceding claim, wherein the chromogenic fXa substrate is spectrozyme-Xa.
9 . The method of any preceding claim, further comprising validating the activity of the andexanet sample with a standard curve generated with a reference andexanet sample.
10 . A method for determining the activity of an andexanet sample, comprising:
admixing a test sample comprising andexanet with a mixture comprising a bovine factor Xa (BFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of the BFXa to the fXa inhibitor is from 0.15:1 to 0.25:1;
adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate is able to release a chromophore upon reaction with the BFXa;
detecting the amount of released chromophore; and
calculating, from the amount of the released chromophore, the activity of the andexanet sample in releasing the BFXa from the direct fXa inhibitor.
11 . The method of claim 10 , wherein the molar ratio of the BFXa to the fXa inhibitor is from 0.16 to 0.20.
12 . The method of claim 10 , wherein the molar ratio of the BFXa to the fXa inhibitor is from 0.17 to 0.19.
13 . The method of any one of claims 10 - 12 , wherein the BFXa, after the test sample is added, has a concentration of about 10 to 20 nM.
14 . The method of claim 13 , wherein the BFXa, after the test sample is added, has a concentration of about 14 to 18 nM.
15 . The method of any one of claims 10 - 14 , wherein the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, letaxaban and eribaxaban.
16 . The method of claim 15 , wherein the direct fXa inhibitor is betrixaban.
17 . The method of any one of claims 10 - 16 , wherein the chromogenic fXa substrate is Spectrozyme-Xa.