readers writing the first stone media event: letters to the editor
TRANSCRIPT
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
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
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