Identification of VKORC1 genotype leading to resistance to tecarfarin

Article abstract of DOI:10.1002/jcph.332

The VKORC1 gene codes for the VKORC1 enzyme that is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). The use of VKA in human medicine is difficult because of inter-individual variability in VKA requirements. This variability is partly due to basic physiological parameters but also to genetic polymorphisms in CYP2C9 involved in VKA metabolism. Tecarfarin, a new VKA, was developed to avoid the CYP2C9-dependent metabolism. This molecule is metabolized by carboxylesterases. Nevertheless, genetic variations in the VKORC1 gene are also associated with inter-individual variability in VKA requirements. This study was performed to estimate the benefit of using tecarfarin versus traditional VKA in human patient carrier for hVKORC1 mutation. Wild type hVKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to tecarfarin was assessed by the in vitro determination of inhibition constants. Among the 14 mutated VKORC1 proteins analyzed in this study, 6 mutations (i.e., A26P, A41S, V54L, H68Y, I123N, and Y139H) led to resistant to traditional VKAs, while only 4 mutations (i.e., A26P, V54L, I123N, and Y139H) led to resistant to tecarfarin. Compared to other VKAs, a clear benefit of tecarfarin is observed for the mutants A26P, A41S, H68Y, and I123N and an absence of benefit for the mutant Y139H. Finally, tecarfarin is particularly inefficient on V54L-VKORC1.

Full text of DOI:10.1002/jcph.332

Pharmacogenomics
The Journal of Clinical Pharmacology
54(8) 896–900
© 2014, The American College of
Clinical Pharmacology
Identification of VKORC1 Genotype Leading to
Resistance to Tecarfarin
DOI: 10.1002/jcph.332
Benjamin Matagrin, MD1, Adrien Montagut-Romans, MD1,2,
Marlene Damin, MD1, Marc Lemaire, PhD2, Florence Popowycz, PhD2,3,
Etienne Benoit, PhD1, and Virginie Lattard, PhD1
Abstract
The VKORC1 gene codes for the VKORC1 enzyme that is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the
target of vitamin K antagonists (VKA). The use of VKA in human medicine is difficult because of inter-individual variability in VKA requirements. This
variability is partly due to basic physiological parameters but also to genetic polymorphisms in CYP2C9 involved in VKA metabolism. Tecarfarin, a new
VKA, was developed to avoid the CYP2C9-dependent metabolism. This molecule is metabolized by carboxylesterases. Nevertheless, genetic
variations in the VKORC1 gene are also associated with inter-individual variability in VKA requirements. This study was performed to estimate the
benefit of using tecarfarin versus traditional VKA in human patient carrier for hVKORC1 mutation. Wild type hVKORC1 and its spontaneous mutants
were expressed in Pichia pastoris and susceptibility to tecarfarin was assessed by the in vitro determination of inhibition constants. Among the 14
mutated VKORC1 proteins analyzed in this study, 6 mutations (i.e., A26P, A41S, V54L, H68Y, I123N, and Y139H) led to resistant to traditional VKAs,
while only 4 mutations (i.e., A26P, V54L, I123N, and Y139H) led to resistant to tecarfarin. Compared to other VKAs, a clear benefit of tecarfarin is
observed for the mutants A26P, A41S, H68Y, and I123N and an absence of benefit for the mutant Y139H. Finally, tecarfarin is particularly inefficient on
V54L-VKORC1.
Keywords
tecarfarin, vitamin K epoxide reductase, vitamin K antagonists, inhibition, resistance
Vitamin K antagonists are widely used for patients who
are at risk of venous thrombo-embolism. At least 3.5
million patients require anticoagulation therapy in the
United States.1 Thrombotic and hemorrhagic accidents
associated with the use of these drugs represent the most
important cause of iatrogenic accidents. Around 1 in 5
patients are hospitalized for bleeding within 6 months of
starting the drugs and lethal cases account for 0.6% of all
treated patients. While the therapeutic index appears to be
within narrow limits, the inter-individual variations
responses to VKA are very important. These inter-
individual variations are due to numerous causes such as
physiological parameters, comorbidity, drug interactions,
nutritional intake of vitamin K but also to genetic
modifications. These latest comprise genetic polymor-
phisms in CYP2C9 (ie, CYP2C9*2 or *3) involved in VKA
metabolism2 and genetic polymorphisms in the promoter
of VKORC1.3,4 These 2 kinds of genetic polymorphisms
can explain up to 50% of the variability in VKA
requirements.
selective inhibitor of vitamin K epoxide reductase enzyme
(VKORC1), and presents the same mechanism of action.5
Unlike warfarin, whose metabolism is dependent upon
cytochrome P450 enzymes, tecarfarin is principally
metabolized by carboxylesterases to form an acidic
1USC 1233 INRA-Vetagro Sup “Rongeurs Sauvages, Risques
Sanitaires et Gestion des Populations” Veterinary School of Lyon,
Marcy l’Etoile, France
2Laboratoire de Catalyse, Synthèse et Environnement, Institut de
Chimie et Biochimie Moléculaires et Supramoléculaires ICBMS-
CNRS-UMR 5246, Université Claude Bernard—Lyon 1, Villeurbanne,
France
3Laboratoire de Chimie Organique et Bio-Organique, Institut National
des Sciences Appliquées (INSA-Lyon), Institut de Chimie et Biochimie
Moléculaires et Supramoléculaires ICBMS-CNRS-UMR 5246,
Villeurbanne, France
Benjamin Matagrin and Adrien Montagut-Romans contributed equally
to this article.
Submitted for publication 5 February 2014; Revised 14 May 2014;
accepted 15 May 2014.
Tecarfarin (ATI-5923) [4-(4-hydroxy-2-oxo-2H-chro-
men-3-ylmethyl)-benzoic acid 2,2,2-trifluoro-1-methyl-
1-trifluoromethyl-ethyl ester] was proposed as a new
vitamin K antagonist able to avoid inter-individual
variations responses.5 This molecule, like warfarin, is a
Corresponding Author:
Virginie Lattard, USC 1233 INRA-Vetagro Sup, 69280 Marcy l’Etoile,
France
Email: virginie.lattard@vetagro-sup.fr

Matagrin et al
897
metabolite (ATI-5900).6 Due to this alternative metabolic
pathway, tecarfarin escapes the metabolism via cyto-
chrome P450 enzymes.7 Therefore, anticoagulation
therapy using tecarfarin avoids inter-individual variations
responses due to CYP dependent drug to drug inter-
actions, food interactions or genetic polymorphisms in
CYP2C9.
Nevertheless, genetic variations in the VKORC1
sequence are also frequently associated with inter-
individual variability in VKA requirements. Indeed, up
to 26 different mutations have been described in the
coding sequence of the VKORC1 gene8–14 and at least, 6
mutations (A26P, A41S, V54L, H68Y, I123N, and
Y139H) lead to an increase in Ki for warfarin and/or
fluindione, demonstrating their involvement in the
resistance phenotype to traditional VKA used in human
medicine.15 This study was performed to estimate the
benefit of using tecarfarin versus warfarin in human
patient carrier for hVKORC1 mutation. Therefore, the
ability of tecarfarin to inhibit mutated hVKORC1 was
evaluated comparatively to wild-type hVKORC1.
Results
To evaluate the ability of tecarfarin to inhibit the recycling
of vitamin K in patients homozygous for wild-type
hVKORC1, we first determined Ki of tecarfarin and ATI-
5900 using recombinant hVKORC1. As usually observed
for VKA, tecarfarin inhibited the VKOR activity
catalyzed by recombinant hVKORC1 in a non-competi-
tive manner (data not shown) with a Ki of 0.08 mM. Its
metabolite ATI-5900 was as expected inactive as an
inhibitor of hVKORC1 (Ki > 100 mM) (Table 1).
Surprisingly, Ki (0.08 mM) reported herein using
recombinant hVKORC1 is quite lower than IC50 value
(0.67 mM) reported previously using human liver micro-
somes,6 while Ki toward warfarin obtained using
recombinant hVKORC1 (1.65 mM) and IC50 using
human liver microsomes are more similar. Such a
difference might be due to the presence of carboxylester-
ases in human liver microsomes. During pre-incubation
time, while tecarfarin is incubated in the presence of
human liver microsomes (in which carboxylesterases are
expressed) and before the VKOR reaction is triggered by
DTT, tecarfarin might be actively hydrolyzed to ATI-
5900, resulting in a decrease of the effective tecarfarin
concentration and thus in an elevated apparent Ki in liver.
On the contrary, in the presence of yeast microsomes, no
esterase activity is observed (data not shown). Conse-
quently, tecarfarin is not metabolized to ATI-5900 and
Methods
Tecarfarin and ATI-5900 Synthesis
Tecarfarin and its metabolite ATI-5900 were synthesized
according to the method described in the license WO
2005/100336 A1.16
Heterologous Expression of Recombinant Wild-Type
and Mutated hVKORC1
Recombinant hVKORC1 proteins were expressed in
Pichia pastoris and microsomal fractions containing
recombinant proteins were prepared as described
previously.15
Table 1. Ki Values of the Mutated VKORC1 Proteins Toward
Tecarfarin
O
O
F
3C CF3
O
O
O
O
O
CH3
OH
Esterase
OH
OH
Tecarfarin
ATI-5900
Inhibition of Vitamin K Epoxide Reductase (VKOR)
Activity Catalyzed by Recombinant hVKORC1 by
Tecarfarin
VKORC1
Tecarfarin (mM)
WT
0.08 Æ 0.01
0.23 Æ 0.02**
0.14 Æ 0.01
0.08 Æ 0.02
0.20 Æ 0.06
0.14 Æ 0.04
0.12 Æ 0.04
0.10 Æ 0.02
0.09 Æ 0.01
3.21 Æ 0.15***
0.11 Æ 0.01
0.16 Æ 0.04
0.09 Æ 0.02
0.18 Æ 0.02**
0.31 Æ 0.03***
A26P
A26T
L27V
H28Q
D36Y
D36G
A41S
V45A
V54L
S56F
VKOR activity was performed as described previously.17
Ki values were obtained from at least 3 separate
experiments performed on 3 different batches of protein.
The same batches of proteins were already used in the
study performed by Hodroge et al15 to determine Ki
toward warfarin and fluindione. To evaluate the inhibiting
effect of tecarfarin and ATI-5900 on VKOR activity, Ki
were determined after addition of anticoagulant to the
standard reaction [ie, 200 mM HEPES buffer (pH 7.4)
containing 150 mM KCl, 1 mM dithiothreitol, 0.75–
2 g LÀ1 of total proteins containing membrane VKORC1]
in the presence of increasing amounts of vitamin K1
epoxide (from 0.003 to 0.2 mM) using anticoagulant
concentrations from about 0.05–20 Â Ki. Data were fitted
by nonlinear regression to the non-competitive inhibition
model using the R-fit program.18
R58G
H68Y
I123N
Y139H
Inhibition parameters were assessed using anticoagulant concentrations
from about 0.05 to 20 Â Ki.
**P < 0.01 compared to Ki obtained for WT.
***P < 0.001 compared to Ki obtained for WT.

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The Journal of Clinical Pharmacology / Vol 54 No 8 (2014)
thus is more effective to inhibit VKOR activity.
Therefore, the apparent Ki observed with yeast micro-
somes is lower than that observed with human liver
microsomes.
for wild-type Vkorc1 or Y139F-Vkorc1.24 As a conse-
quence, despite theoretical controversies about the DTT-
driven VKOR assay, we consider that this approach is of
great interest to characterize the susceptibility of
VKORC1 to VKA and thus we use this validated method
in this paper to characterize the efficiency of tecarfarin to
inhibit human wild-type and mutated VKORC1 proteins.
Ki toward tecarfarin (0.08 mM) obtained for
hVKORC1 appears to be very low in comparison to the
value obtained when warfarin, acenocoumarol, or
fluindione were used (1.65, 0.33, and 0.25 mM respec-
tively). Such a low Ki is observed when second generation
rodenticides such as brodifacoum or difethialone are used
as inhibitors (0.08 and 0.04 mM, respectively). Interest-
ingly, the studies of the consequences of these second
generation rodenticides on the target mutations in rodents
demonstrate that the lower the Ki is, the less the resistance
factor is modified. In this context, besides the advantages
conferred by its specific metabolic pathway, the use of
tecarfarin might allow to by-pass the resistance phenom-
ena due to spontaneous mutations of the coding sequence
of hVKORC1.
Up to 26 mutations in the coding sequence of
hVKORC1 were described from human patients requiring
at least threefold posology of VKA used in human
medicine to obtain stable anticoagulation.8–14 All these
mutated VKORC1 proteins were already expressed in P.
pastoris.15 Among spontaneous mutations of hVKORC1,
eleven mutations lead to approximately total loss of
VKOR activity and one was not expressed successfully in
P. pastoris. As a consequence, we evaluated tecarfarin as
an inhibitor of the VKOR activity catalyzed by 1 of the 14
active mutated VKORC1 proteins. Figure 1A presents the
resistance factor of tecarfarin (ie, Ki of mutated
VKORC1/Ki of wild-type VKORC1) for the mutated
VKORC1 protein.
Six of these mutants (A26P, V54L, I123N, Y139H,
A41S, and V54L) were found to be resistant to warfarin
and/or fluindione.15 Among the 6 resistant mutated
VKORC1 proteins, only 4 lead to a significant increase
in Ki toward tecarfarin (ie, A26P, V54L, I123N, and
Y139H). Nevertheless, the ratio corresponding to the
resistance factor of one of these VKA to the resistance
factor of tecarfarin (Figure 1B) is high for all VKA tested
using A26P mutant. This argues for a low resistance to
tecarfarin of this mutant. This ratio is also high for
fluindione but not for warfarin when I123N mutant is
used. For Y139H-VKORC1, resistance to tecarfarin is
similar to resistance to warfarin, acenocoumarol, and
fluindione. On the contrary, V54L-VKORC1 presents a
particularly high resistance to tecarfarin. For A41S- and
H68Y-VKORC1, belonging to the group of resistant
mutants, Ki toward tecarfarin is not increased. A41S
mutation was reported to lead to selective resistance to
fluindione.15 Tecarfarin, as other 4-hydroxycoumarin
Using the same recombinant hVKORC1, Ki for VKA
traditionally used in human medicine were previously
reported by Hodroge et al.15 Ki for warfarin was 1.65 mM
that is ꢀ20-fold higher than that obtained with tecarfarin;
Ki for acenocoumarol and fluindione was 0.33 and
0.25 mM, that are, respectively, ꢀ4- and ꢀ3-fold higher.
Tecarfarin is thus, in this model, a more potent inhibitor of
VKOR activity than VKA already used in human
medicine. Ki for tecarfarin is similar to those obtained
for molecules such as difethialone or brodifacoum (with
Ki, respectively, of 0.04 and 0.08 mM).17
To evaluate the ability of tecarfarin to inhibit the
recycling of vitamin K in human patients carriers for
hVKORC1 mutation, Ki of tecarfarin were determined for
all the recombinant mutated hVKORC1 proteins. Tecar-
farin inhibited the VKOR activity in a non-competitive
manner for all the mutants we analyzed (data not shown).
Ki values obtained for the mutated VKORC1 are reported
in Table 1. Among the 25 mutations of the coding
sequence of hVKORC1 detected in patients clinically
resistant to VKA, only 4 mutations led to increase in Ki
toward tecarfarin (ie, A26P, V54L, I123N, and Y139H).
Discussion
Tecarfarin, a new VKA, was developed to allow a more
secure anticoagulation therapy in human medicine. The
VKOR activity is supported by both VKORC18,19 and
VKORC1L120 proteins. In the liver, the expression of
VKORC1L1 is very low and consequently VKORC1 is
the main protein involved in the VKOR activity. Two
experimental approaches were previously proposed to
characterize VKORC1 dependent-VKOR activity, the
direct approach (ie, the historic DTT-driven VKOR
assay) and the recently developed indirect approach (i.e.,
the cell-based VKOR assay).21,22 This cell-based VKOR
approach implies the overexpression of VKORC1 and a
substrate of GGCX in HEK293 cells. Moreover, in 1
variant of the proposed cell-based VKOR approach, the
endogenous VKORC1 and VKORC1L1 genes are knocked
out.22 While some results obtained by these 2 variants of
the cell-based VKOR assay appear to be conflicting,
authors nevertheless questioned the historic DTT-driven
VKOR method.21 The validation of the DTT-driven
VKOR approach is much more complete than any other
and resistance factors obtained by the DTT-driven VKOR
approach using yeast microsomes expressing VKORC1
proteins were shown to be totally coherent with resistance
factors obtained by the DTT-driven VKOR approach
using liver microsomes,17,23 but also with resistance
factors obtained in vivo using strains of rats homozygous

Matagrin et al
899
Figure 1. Efficiency of tecarfarin as VKA in human medicine. (A) Determination of the susceptibility of mutated VKORC1 proteins to tecarfarin using
recombinant yeast microsomes expressing mutated VKORC1 proteins, comparatively to the wild-type VKORC1. (B) Comparison of the efficiency of
tecarfarin as VKA with VKA already used in human medicine.
derivatives is logically still able to inhibit this mutated
VKORC1. Strictly in term of resistance, a clear benefit for
the effect of tecarfarin is observed for the mutants A26P,
A41S, H68Y, and I123N and an absence of benefit for the
mutant Y139H. Finally, tecarfarin is particularly ineffi-
cient on V54L-VKORC1.
For the 8 mutations not involved in the resistance to
VKA (ie, 26T, L27V, H28Q, D36Y, D36G, V45A, S56F,
and R58G) there are no clear modification of Ki toward
tecarfarin. Therefore, these mutants are susceptible to the
inhibition effect of warfarin, acenocoumarol, fluindione,
and tecarfarin.
5. Ellis DJ, Usman MH, Milner PG, Canafax DM, Ezekowitz MD. The
first evaluation of a novel vitamin K antagonist, tecarfarin (ATI-
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8. Rost S, Fregin A, Ivaskevicius V, et al. Mutations in VKORC1
cause warfarin resistance and multiple coagulation factor deficiency
type 2. Nature. 2004;427:537–541.
9. Bodin L, PerduJ, Diry M, HorellouMH, Loriot MA. Multiple genetic
alterations in vitamin K epoxide reductase complex subunit 1 gene
(VKORC1) can explain the high dose requirement during oral
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resistance to warfarin is associated with nucleotide substitutions in
VKORC1. J Thromb Haemost. 2008;6:1663–1670.
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mutations associated with oral anticoagulant resistance: insights
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Funding
This work was supported by grants from Agence Nationale pour
la Recherche (RODENT 2009-CESA-008-03) and by DGER.
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