Recombinant factor Xa inhibitor

antidote (PRT064445) mediates

reversal of anticoagulation

through reduction of free drug

concentration: A common

mechanism for direct factor Xa

inhibitors

A. Hutchaleelaha, G. Lu, FR. Deguzman, MJ. Karbarz, M. Inagaki, S. Yau, PB. Conley, U. Sinha, SJ. Hollenbach

Portola Pharmaceuticals, Inc,

South San Francisco, United States of America

Disclosures for Hutchaleelaha et al

All authors are employees of Portola Pharmaceuticals, Inc.

2

r-Antidote: a recombinant human fXa variant

lacking the membrane binding Gla-domain and

active site serine

PRT064445

S S

EGF1,2

S419A

fXai

S S

S419A

EGF1,2 Gla

Light Chain Heavy Chain

Inactive

Catalytic Domain

PRT064445 (r-Antidote)

• Unable to assemble into the prothrombinase complex and cleave

prothrombin

• Retained binding ability for all fXa inhibitors

3

r-Antidote has a high affinity for fXa inhibitors

Inhibitor r-Antidote

Kd (nM)

fXa

Ki (nM)

Rivaroxaban 1.53 0.40*

Apixaban 0.58 0.08*

Betrixaban 0.53 0.12

*Perzborn at al J Thromb Haemost 2005;3:514,

Pinto et al J Med Chem 2007; 50:5339

0 50 100 150 200 250 300

0

25

50

75

100

Control

2.5nM Rivaroxaban

5.0nM Rivaroxaban

7.5nM Rivaroxaban

PRT064445 (nM)

FX

a a

cti

vit

y (

mO

D/m

in)

4

r-Antidote completely reverses the activity of

rivaroxaban in vitro and ex vivo

In vitro in plasma Ex vivo in rat model

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0

25

50

75

100

125Human plasma+rivaroxaban (230nM)

Rat plasma +rivaroxaban (230nM)

PRT064445 (M)

An

ti-f

Xa

(%

)

30 35 900 6030 35 90

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Vehicle+Vehicle (n=4)

Infusion of Rivaroxaban(0.25 mg/kg/hr)

Rivaroxaban+PRT064445 (n=4)

Rivaroxaban+Vehicle (n=4)

Bolus + Infusion of PRT064445 (4 mg + 4 mg/hr)

*

*

*P-value 0.01 (PRT064445 vs. Rivaroxaban Alone)

Time (min)

Whole

Blo

od P

T (

INR

)+

SD

ISTH 2009 5

Time (min)

0 10 20 30 40 50 60 70 80 90

To

tal

an

d U

nb

ou

nd

Riv

aro

xa

ba

n

Pla

sm

a C

on

c (

ng

/mL

)

0

5

10

15

20

200

400

600

800

1000

1200

1400 Rivaroxaban Alone: Total

Rivaroxaban + (4+4) mg PRT064445: Total

Rivaroxaban Alone : Unbound

Rivaroxaban + (4+4) mg PRT064445: Unbound

Infusion ofRivaroxaban

Infusion ofPRT064445

Free drug concentration of rivaroxaban was well

correlated with reversal of PD parameter

30 35 900 6030 35 90

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Vehicle+Vehicle (n=4)

Infusion of Rivaroxaban(0.25 mg/kg/hr)

Rivaroxaban+PRT064445 (n=4)

Rivaroxaban+Vehicle (n=4)

Bolus + Infusion of PRT064445 (4 mg + 4 mg/hr)

*

*

*P-value 0.01 (PRT064445 vs. Rivaroxaban Alone)

Time (min)

Whole

Blo

od P

T (

INR

)+

SD

6

Has free drug concentration been used as a

surrogate marker for pharmacodynamic activity?

• Reversal of digoxin toxicity by decreasing free digoxin concentration with digoxin-specific Fab antibodies

7

Pharmacokinetic Aspects of Digoxin-Specific Fab

Therapy in the Management of Digitalis Toxicity

Within minutes of Fab dosing, there is a 10- to 30-fold increase in total digoxin conc and free digoxin conc decreases from 75-90% to 0-5%

When free concentration rebounds beyond 0.8 ug/mL, signs and symptoms of digoxin reintoxication return in some patients

Michael R Ujhelyi1 and Sylvie Robert2

Clin Pharmacokinet 28(6):483-493 1995

8

Apixaban

Time (min)

0 10 20 30 40 50 60 70 80 90 100 110 120

To

tal a

nd

Un

bo

un

d A

pix

ab

an

P

las

ma

Co

nc (

ng

/mL

)

0.1

1

10

100

1000

Apixaban Alone: Total

Apixaban Alone: Unbound

Apixaban + (6+6) mg PRT064445 : Total

Apixaban + (6+6) mg PRT064445: Unbound

Infusion ofApixaban

Infusion of PRT064445

30 350 60 9030 35

1.5

2.0

2.5

3.0

3.5

4.0

Apixaban + Vehicle (n=5)

Vehicle + Vehicle (n=5)

Infusion of Apixaban (0.5 mg/kg/hr)

Bolus + Infusion of PRT064445 (6 mg + 6 mg/hr)

Apixaban + PRT064445 (n=5)

*

**

* p-Value < 0.01 Apixaban + PRT064445 vs Apixaban alone

Time (min)

Wh

ole

Blo

od

PT

(IN

R)

SD

9

Time (min)

0 10 20 30 40 50 60 70 80 90 100 110 120

Tota

l an

d U

nb

oun

d B

etri

xab

an

Pla

sma

Co

nc (

ng

/mL

)

1

10

100

1000

10000

Betrixaban Alone: Total

Betrixaban Alone: Unbound

Betrixaban + (6+9) mg PRT064445: Total

Betrixaban+ (6+9) mg PRT064445: Unbound

Infusion ofBetrixaban

Infusion of PRT064445

Betrixaban

10

Is free drug concentration correlated with

correction of blood loss in animal model?

• Rabbit model of liver laceration*

• Anesthetized rabbits were treated with vehicle or rivaroxaban via IV bolus. After 30 minutes, PRT064445 or vehicle was administered as a bolus injection

• Two liver lobes were lacerated (5X each lobe 1-cm long, 3-mm deep incisions)

• Lost blood was collected on pre-weighed gauze over 15 minutes

• Non-protein bound free fraction of rivaroxaban was separated by equilibrium dialysis and quantitated by LC-MS/MS

• Anti-fXa activity was measured by modified LMWH kit

*Adapted from Godier et al, Anesthesiology 2012:116, 94

11

Free drug concentration correlated with

correction of blood loss in animal model B

loo

d L

oss (

gm

)

Ve

hic

le

Riv

a

Riv

a+

PR

T

Ve

hic

le+

PR

T0

10

20

30

40 *** *

***P (Riv a vs. Vehicle) = 0.0006 * P (Riv a+PRT vs. Riv a) = 0.0130

P (Riv a+PRT vs. Vehicle) = ns P (PRT vs. Vehicle) = ns

correction of peak anti-fXa units = 98%

0

50

100

150

200

250

30 35 50

Un

bo

un

d R

ivaro

xab

an

C

on

c (

ng

/mL

)

Time (min)

Rivaroxaban

Rivaroxaban+ PRT064445

12

Summary

• PRT064445 (r-Antidote) is a factor Xa derivative which reverses

the anticoagulant activity of fXa inhibitors

• Reversal of anticoagulation in rats correlates to reversal of pharmacodynamic markers (anti-fXa units, PT/INR)

• Reversal is mediated through reduction of free fraction of fXa inhibitor in plasma

• Rivaroxaban mediated blood loss in rabbit liver laceration model was reduced by 85% upon administration of r-Antidote

• Maximal reduction of free fraction of rivaroxaban ~ 98%

• Reduction of Anti-fXa activity = 98%

• Reversal of Prothrombin time = 74%

• r-Antidote is currently in Phase 1 studies in healthy volunteers

13

Back Up

14

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Vehicle Rivaroxaban Rivaroxaban +PRT064445

PRT064445 alone Rivaroxaban + rVIIa rVIIa alone

Blo

od

Lo

ss ±

S

D (

gm

)

P<0.001 P<0.02

P<0.01 P<0.01

Reduction of Blood Loss in Rivaroxaban

Anticoagulated Rabbits with PRT064445 but

not rfVIIa using Rabbit Liver Laceration Model

15

Time

(min)

% Free Rivaroxaban

Rivaroxaban

alone

Rivaroxaban

+

PRT064445

30 100.0 100

35 107.0 1.7

50 109.4 7.2

16