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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, Benjamin
