Andexanet Alfa:Reversal agent for FXaInhibitors

Grace Gilmore,Prof Ross Baker, Dr Jim Tiao, Dr Quintin Hughes, Dr Jasmine Tay, Scott McGregor

WA Centre for Thrombosis and Haemostasis, Murdoch University, Murdoch, Western Australia.Perth Blood Institute, Nedlands, Western Australia

Introduction

• The main point of the oral Xa inhibitors was:

• No monitoring.

• More predictable pharmacokinetics

• No food interactions

• Therefore: No reversal agent

Introduction

• Clinical trials v real life• Bleeds

• Urgent surgery

• Available products• PCC

• Charcoal

• dialysis

Andexanet Alpha

• Modified recombinant protein derived from human FX

• Approx 41kDa

• Lacks a membrane-binding ƴ-carboxyglutamic acid (GLA) domain

• Is not catalytically active due to a serine→alanine residue mutation in the protease catalytic triad.

• Binds to FXa inhibitor in their active site

• 1:1 Stoichiometry

Andexanet Alfa

AMSRJ Spring2014 Volume1,Number11

Aim and Method

• Aim• Determine the effect of Andexanet Alfa on routine clotting tests.

• Methods• PT, aPTT, TCT and DRVVT were performed using Siemens reagents on the

Sysmex CS 5100 Coagulation Analyser.

• Anti FXa assay was performed using the STAGO kit.

Results• Adding Andexanet alfa to normal plasma.

0

5

10

15

20

25

30

35

40

45

0 50 100 150 200 250

Tim

e (s

eco

nd

s)

Concentration of Andexanet Alfa (μg/mL)

PT

aPTT

TCT

Results

• Drug reversal

• 100% ~ 300ng/ml

• ~85-90%

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350 400

Ch

rom

oge

nic

An

ti-X

aA

ctiv

ity

Ass

ay (

% o

f B

asel

ine)

Andexanet alfa concentration (μg/mL)

Rivaroxaban

Apixaban

Lupus Anticoagulant

• Detection a challenge due to:• Antibody heterogeneity

• Reagent and analyser variability

• Differing interpretation strategies

• Mixing studies• Not definite

• Multiple factor deficiency due to VKA are corrected, There is usually no underlying factor deficiency with FXa inhibitors.

Lupus Anticoagulant Testing

“Lupus-anticoagulant testing at NOAC trough levels” Ratzinger et al

Coagulation and Fibrinolysis 2016

“Frequent False positive results of lupus anticoagulant tests in plasma of patients receiving the new oral anticoagulants and enoxaparin” Martinuzz et al. IJLH 2014

“Interactions between rivaroxaban and antiphospholipid antibodies in thrombotic antiphospholipid syndrome” Arachchillage et al. JTH 2015

“Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti FXa assays” Hillarp et al. JTH 2014

Lupus anticoagulant

0

25

50

75

100

125

150

0 100 200 300 400 500 600 700 800 900 1000

Tim

e (S

eco

nd

s)

FXa Inhibitor Concentration (ng/mL)

Rivaroxaban Rivaroxaban + Phospholipids

Apixaban Apixaban + Phospholipids

Lupus Anticoagulant

0

20

40

60

80

100

0 50 100 150 200 250

Tim

e (S

eco

nd

s)

Andexanet alfa Concentration (μg/mL )

Minimal phospholipids Excess phospholipids

0 40 80 120 160 200 240 280

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

0 200 400 600 800 1000 1200 1400

Andexanet Alfa Concentration (μg/mL )

No

rmal

ised

dR

VV

T R

atio

No

rmal

ised

dR

VV

T R

atio

Direct FXa Inhibitor Concentration (ng/mL)

Apixaban Rivaroxaban Andexanet alfa

Figure 5. Influence of rivaroxaban, apixaban and andexanet alfa on normalised dRVVT ratios (normal phospholipid:excess phospholipid ratio) using drug diluted in commercial normal plasma.

Influence of rivaroxaban, apixaban and andexanet alfa on normalised dRVVT ratios

ResultsSample FXa Inhibitor

Level ng/mlBaselinedRVVTRatio

R1 409.6 2.73

R2 99.5 2.51

R3 143.6 1.99

A1 95.3 2.50

A2 107.4 2.45

A3 17.9 1.31

ResultsSample FXa Inhibitor

level ng/mlBaseline DRVVT Ratio

R1 115.0 1.79

R2 309.7 1.59

R3 37.7 1.34

A1 12.5 1.10

A2 46.9 1.09

A3 90.5 1.07

Summary• Andexanet alfa prolongs aPTT in a concentration-dependent manner in-vitro, with negligible effects on PT or TCT

• Andexanet alfa (250μg/mL) neutralises rivaroxaban and apixaban by ~85-90%, based on anti-Xa assays

• Rivaroxaban induces a false-positive LA test via dRVVT in a concentration-dependent manner. Andexanet alfa corrects this without affecting the true interpretation in LA-positive patients.

• Apixaban and andexanet alfa proportionately prolong the dRVVTwith and without phospholipid, so the LA ratio and interpretation remains unaffected

Acknowledgments

•Thanks To:• Portola Pharmaceuticals

• Prof Ross Baker

• Scott McGregor

• Dr Quintin Hughes

• Dr Jim Tiao

• Dr Jasmine Tay

• Rene Mamode