Method for determining factor Xa inhibitor dosage

Abstract

A method of detecting Factor VIII level in a subject, particularly in a subject in need of treatment with at least one Factor Xa inhibitor. The method comprises (a) selecting at least one subject in need of treatment with at least one Factor Xa inhibitor; and (b) detecting Factor VIII level in a sample obtained from the at least one subject with the aim to determine an appropriate dosage of the at least one Factor Xa inhibitor. Preferably, the method comprises a further step of administering at least one Factor Xa inhibitor to the subject.

Claims

1. A method of treating a subject having thrombosis or thrombosis risk in need of treatment with apixaban, the method comprising: detecting Factor VIII level in a sample obtained from the subject to determine an appropriate dosage of the apixaban, and administering to the subject: a dose of 5 mg of apixaban per day if the Factor VIII level detected in the sample is below at least one reference value; or a dose of 20 mg of apixaban per day if the Factor VIII level detected in the sample is above at least one reference value, wherein the reference value is a Factor VIII level of between 100% and 200% of Factor VIII level in standard plasma.

2. The method according to claim 1, wherein the apixaban is to be administered to the subject once or twice daily.

3. The method according to claim 1, wherein detecting the Factor VIII level is performed before and/or during treatment of the at least one subject with the apixaban.

4. The method according to claim 1, wherein the sample is a body fluid sample selected from the group consisting of a blood sample and a blood plasma sample.

5. The method according to claim 1, wherein detecting the Factor VIII level is repeated at least once.

6. A method of treating a subject having thrombosis or thrombosis risk with apixaban, the method comprising: administering to the subject: a dose of 5 mg of apixaban per day when a Factor VIII level detected in a blood sample and/or a blood plasma sample taken from the subject is less than a Factor VIII level of between 100% and 200% of Factor VIII level in standard plasma; or a dose of 20 mg of apixaban per day when a Factor VIII level detected in a blood sample and/or a blood plasma sample taken from the subject is greater than a Factor VIII level of between 100% and 200% of Factor VIII level in standard plasma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: For five different Factor VIII concentrations, i.e., 50%, 75%, 100%, 150% or 200%, five bars are shown representing subsamples to which apixaban was added to a different total concentration, i.e., 0 ?M, 0.1 ?M, 0.2 ?M, 0.35 ?M, or 0.5 ?M, respectively.

(2) FIG. 2: Edoxaban and rivaroxaban both closely mimic the effects of apixaban in the clot growth rate assay.

(3) FIGS. 3 and 4: Clot size was measured for each different Factor VIII concentration (i.e., 50%, 75%, 100%, 150% or 200%) and apixaban concentration (0 ?M, 0.1 ?M, 0.2 ?M, 0.35 ?M, or 0.35 ?M).

DETAILED DESCRIPTION

(4) It can be observed that for a Factor VIII level of below 100%, the lowest apixaban concentration already achieves close to maximal inhibition of clot growth rate. Here, treatment with a low apixaban concentration is indicated (in plasma: 0.1 ?M) to not create a bleeding risk. For factor VIII levels above 100%, 0.1 ?M apixaban will not be very effective and 0.35 ?M in plasma is suggested for substantial inhibition. Here, the treatment is aiming at reducing thrombosis risk.

(5) Experimental Section

(6) Clot Growth Rate Assay

(7) The clot growth rate assay monitors over time fibrin clot development in a nonstirred thin layer of platelet-free plasma activated by immobilized tissue factor. For this, the Thrombodynamics Basic Kit can be used as an in vitro diagnostic kit used to perform measurements of spatiotemporal dynamics of fibrin clot formation in blood plasma samples. The kit consists of measurement cuvettes, activating inserts and reagents for samples treatment. The kit is obtainable from HemaCore intended for professional use in the laboratory on the Thrombodynamics Analyzer T-2. See, for further details, Soshitova et al. (Blood Coagul. Fibrinolysis, 2012 September; 23(6):498-507).

(8) Factor VIII Measurement Assays

(9) Rapid ELISA Assay

(10) The VisuLize? FVIII Antigen Kit (Affinity Biologicals Inc.) can be used to quantitatively measure Factor VIII in human plasma. It has the following features: Rapid sandwich ELISA to measure FVIII (F8) antigen (FVIII:Ag) 1 hour 55 minutes total incubation time FVIII:Ag reported as International Units/ml traceable to WHO standard for FVIII antigen Detection limit to 0.008 IU/ml FVIII:Ag (0.8%) Includes normal and low controls Shelf-life: 18 months Semin. Thromb. Hemost. 2002 June; 28(3):247-56.

(11) This rapid ELISA assay is described extensively by O'Donnell et al. (Thromb. Haemost. 1997; 77:825-828). Alternatively, the Human Factor VIII total antigen assay ELISA kit can be used (Molecular Innovations).

(12) One-Stage Clotting Assay

(13) The one-stage clotting assay is originally described in 1953 (Langdell et al., Translational Research, 1953, Vol. 41, Issue 4, pages 637-647) and later modified into the activated partial thromboplastin time (APTT). The principle is to measure by what extent a plasma sample corrects the prolonged coagulation time of FVIII-deficient plasma in an APTT-based assay.

(14) In the one-stage assay, FVIII-deficient plasma is added to test plasma and the APTT reagent, mixed and incubated for 3-5 minutes. This is the contact activation phase during which factor XI (FXI) is activated, but little happens to FVIII. The mixture is then recalcified, and the coagulation time is recorded. This takes approximately 40-140 seconds depending on the APTT assay and the FVIII content. To determine the FVIII:C, the clotting time is compared to a standard curve, which is constructed by plotting the clotting times of serial dilutions of standard plasma (FVIII 0-200%) vs. factor VIII activity on logarithmic/linear scale graph paper. The World Federation of Haemophilia (WFH) recommends a parallel line analysis where identical dilutions of the test and standard plasma are compared (see S. Kitchen, A. McCraw, M. Echenagucia, on behalf of the WFH Laboratory Sciences Committee, Diagnosis of Hemophilia and Other Bleeding Disorders: A Laboratory Manual, 2nd edn. Montreal, Qu?bec, Canada: World Federation of Hemophilia, 2010). The parallel line analysis decreases variability and increases precision. The one-stage clotting assay is extensively described in Laboratory Techniques in Thrombosisa Manual (Jespersen, ed., Bertina, and Haverkate; Springer Science+Business Media Dordrecht 1999).

(15) Chromogenic Assay

(16) This assay involves a first incubation stage wherein FVIII activity is rate limiting to generate FXa and a second stage to determine the amount of FXa produced. A reagent containing purified coagulation factors (FIXa, FX and thrombin) in optimal concentrations is used in the first stage, which does not rely on the extrinsic or intrinsic initiation pathways as the thrombin activates FVIII. The amount of FXa generated in the first stage is measured by its action on a specific chromogenic substrate, which releases a chromophore upon cleavage that absorbs light of a certain wavelength. The color intensity produced is directly proportional to the amount of FXa, which, in turn, is directly proportional to the FVIII activity in the sample (see also S. Rosen, M. Chiarion Casoni, Chromogenic determination of factor VIII activity in plasma and factor VIII concentrates. Chromogenic Monograph Series). The chromogenic assay is extensively described in Laboratory Techniques in Thrombosisa Manual (Jespersen, ed., Bertina, and Haverkate; Springer Science+Business Media Dordrecht 1999).

(17) Plasma Level of Factor VIII Indicates the Appropriate Dose for any Factor Xa Inhibitor

(18) Five plasma samples were prepared containing 50%, 75%, 100%, 150%, or 200% Factor VIII, respectively. The samples were prepared using FVIII-deficient plasma, normal plasma from healthy subjects, and FVIII concentrate and Factor VIII levels were measured and confirmed by the one-stage clotting assay. Each of these five plasma samples was subdivided into five subsamples to which apixaban was added to a total concentration of 0 ?M, 0.1 ?M, 0.2 ?M, 0.35 ?M, or 0.5 ?M in plasma, respectively. The 25 subsamples were subjected to a clot growth assay as described above, and the clot growth rate was measured at 2500 seconds after start of growth.

(19) Results are shown in FIG. 1. For each different Factor VIII concentration (i.e., 50%, 75%, 100%, 150% or 200%), five bars are shown representing subsamples to which apixaban was added to a different total concentration, i.e., 0 ?M, 0.1 ?M, 0.2 ?M, 0.35 ?M, or 0.35 ?M, respectively.

(20) Similarly, for each different Factor VIII concentration (i.e., 50%, 75%, 100%, 150% or 200%) and apixaban concentration (0 ?M, 0.1 ?M, 0.2 ?M, 0.35 ?M, or 0.35 ?M), clot size was measured (FIGS. 3 and 4).

(21) It can be observed that for a Factor VIII level of below 100%, the lowest apixaban concentration already achieves close to maximal inhibition of clot rate. Here, treatment with a low apixaban concentration is indicated (in plasma: 0.1 ?M) to not create a bleeding risk. For factor VIII levels above 100%, 0.1 ?M apixaban will not be very effective and 0.35 ?M is suggested for substantial inhibition. Here, the treatment is aiming at reducing thrombosis risk.

(22) The results will be the same in case of other Factor Xa inhibitors than apixaban. Apixaban and other Factor Xa inhibitors (such as edoxaban and rivaroxaban) share the same mechanism of action. As can be seen in FIG. 2, edoxaban and rivaroxaban both closely mimic the effects of apixaban in the clot growth rate assay.

(23) Different Dosages of Factor Xa for Patients with Different Factor VIII Levels

(24) Four subjects in need of Factor Xa treatment are selected, including three patients with thrombosis, and one patient with coronary artery disease. Measurement of Factor VIII (by the rapid ELISA assay as described herein) in the subjects reveals:

(25) TABLE-US-00001 Subject for Factor Xa treatment Factor VIII level Patient 1 (thrombosis) 80% of Factor VIII level in standard plasma* Patient 2 (thrombosis) 95% of Factor VIII level in standard plasma* Patient 3 (thrombosis) 220% of Factor VIII level in standard plasma* Patient 4 (coronary artery 240% of Factor VIII level in standard plasma* disease) *WHO International Standard plasma (07/316)

(26) To verify that plasma levels of Factor VIII indeed indicate the appropriate dose for any Factor Xa inhibitor as proposed herein (in this case by using a reference value of 150% of Factor VIII level in standard plasma), the following Factor Xa dosage is prescribed, either according to the disclosure or not according to the disclosure:

(27) Dosage According to the Present Disclosure

(28) TABLE-US-00002 Subject for Factor Xa treatment Factor Xa dosage per day Patient 1 (thrombosis) between 1-10 mg Apixaban (in this case 5 mg) Patient 3 (thrombosis) between 10 mg and 30 mg Edoxaban (in this case 20 mg)
Dosage not According to the Present Disclosure

(29) TABLE-US-00003 Subject for Factor Xa treatment Factor Xa dosage per day Patient 2 (thrombosis) between 10 mg and 30 mg Edoxaban (in this case 20 mg) Patient 4 (coronary artery between 1 mg and 10 mg Apixaban disease) (in this case 5 mg)

(30) Patients 1 and 3 thus receive a Factor Xa dosage in accordance with the present disclosure, while patients 2 and 4 do not. Coagulation status of patients 1-4 is determined by a routine coagulation test (activated partial thromboplastin time, APTT).

(31) Coagulation status of patients 1 and 3 reveals no increased risk of blood clotting or bleeding events. However, coagulation status of Patient 2 reveals an increased risk of bleeding events and coagulation status of Patient 4 reveals an increased risk of blood clotting events.

(32) It can be concluded that plasma levels of Factor VIII indeed indicate the appropriate dose for any Factor Xa inhibitor as proposed herein.