The discovery and development of rivaroxaban, an oral, direct factor Xa inhibitor

Abstract

The activated serine protease factor Xa is a promising target for new anticoagulants. After studies on naturally occurring factor Xa inhibitors indicated that such agents could be effective and safe, research focused on small-molecule direct inhibitors of factor Xa that might address the major clinical need for improved oral anticoagulants. In 2008, rivaroxaban (Xarelto; Bayer HealthCare) became the first such compound to be approved for clinical use. This article presents the history of rivaroxaban's development, from the structure-activity relationship studies that led to its discovery to the preclinical and clinical studies, and also provides a brief overview of other oral anticoagulants in advanced clinical development.

Full text

Anticoagulants are used for the prevention and treatment
of venous and arterial thromboembolic disorders. Many
approaches have been explored in the development of
antithrombotic drugs that inhibit enzymes in the coagu-
lation pathways. However, most currently approved drugs
for the prevention and treatment of thromboembolic dis-
orders have been on the market for a long time, in some
cases for decades. Unfractionated heparin (UFH), which
was discovered in 1916 (REF. 1) targets multiple factors in
the coagulation cascade2, but has a number of limitations,
including a parenteral route of administration, frequent
laboratory monitoring of coagulation activity and the
risk for patients of developing potentially life-threatening
heparin-induced thrombocytopaenia2,3. Low-molecular-weight
heparins (LMWHs), which were developed in the 1980s,
promote the inactivation of both thrombin (factor IIa)
and, to a greater extent, factor Xa2.
LMWHs have largely replaced UFH owing to their
lower risk of causing bleeding, lower levels of binding to
plasma proteins and endothelium, good bioavailability,
longer half-life and superior pharmacokinetic properties
compared with UFH2. However, their use remains lim-
ited because of the need for parenteral administration2,
which can be inconvenient, especially in an outpatient
setting, with patients needing to be trained to self-inject
after discharge, and nurse visits required for those unable
to do so4. Both UFH and LMWHs are indirect inhibitors
of coagulation, and their activity is mediated by plasma
cofactors, principally antithrombin and, to a lesser extent
for UFH, heparin cofactor II (REF. 2).
Warfarin, the prototype vitamin K antagonist (VKA),
was originally discovered in 1941 (REF. 5). Until recently,
the VKAs were the only available oral anticoagulants, as
well as the most frequently prescribed. However, VKAs
have numerous well-documented drawbacks, includ-
ing unpredictable pharmacokinetics and pharmaco-
dynamics, a slow onset and offset of action, a narrow
therapeutic window, multiple food–drug and drug–drug
interactions6, and considerable inter-individual and
intra-individual variability in dose response. In addition,
regular coagulation monitoring and dose adjustment are
required to keep patients within the target international
normalized ratio (INR) range, usually 2.0–3.0, which can
be costly6. Furthermore, establishing the optimal dose
of warfarin is complicated by variations in warfarin
sensitivity due to common genetic polymorphisms,
particularly in CYP2C9 and VKORC1 (REF. 7). Attempts
have been made to use pharmacogenetics to estimate
dose, although the clinical value of such assessments
is debatable8. Nonetheless, LMWHs and VKAs are still
the basis of contemporary thromboprophylaxis and treat-
ment. However, the difficulties and shortcomings that
surround the practicalities and clinical management
of these established anticoagulants — particularly
parenteral administration, the need for monitoring
and the lack of predictable response — has spurred the
development of new agents that are less burdensome for
the patient and health-care system, and address both
patients’ and physicians’ unmet needs. TABLE 1 con-
trasts the characteristics of the VKAs with those of a
*Global Therapeutic
Research, ‡Global Lead
Generation and Optimization,
§Global Clinical
Pharmacology,
||Global Clinical Development,
Cardiovascular
Pharmacology, Pharma R&D,
Bayer HealthCare, Aprather
Weg 18a, D‑42096
Wuppertal, Germany.
Correspondence to E.P.
e‑mail: elisabeth.perzborn@
bayer.com
doi:10.1038/nrd3185
Published online
10 December 2010
Thromboembolic disorders
A group of conditions
characterized by an increased
incidence of thrombi in the
vasculature, such as deep-vein
thrombosis, pulmonary
embolism, systemic embolism
or coronary and cerebral
ischaemia.
Unfractionated heparin
(UFH). An anticoagulant
administered intravenously or
subcutaneously. It binds to
antithrombin, greatly
increasing its activity and
resulting in the inhibition of
factors Xa, IXa, XIa, XIIa and
thrombin (factor IIa).
The discovery and development
of rivaroxaban, an oral, direct
factor Xa inhibitor
Elisabeth Perzborn*, Susanne Roehrig‡, Alexander Straub‡, Dagmar Kubitza§ and
Frank Misselwitz||
Abstract | The activated serine protease factor Xa is a promising target for new anticoagulants.
After studies on naturally occurring factor Xa inhibitors indicated that such agents could be
effective and safe, research focused on small-molecule direct inhibitors of factor Xa that
might address the major clinical need for improved oral anticoagulants. In 2008, rivaroxaban
(Xarelto; Bayer HealthCare) became the first such compound to be approved for clinical use.
This article presents the history of rivaroxaban’s development, from the structure–activity
relationship studies that led to its discovery to the preclinical and clinical studies, and also
provides a brief overview of other oral anticoagulants in advanced clinical development.
CASE HISTORY
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Heparin-induced
thrombocytopaenia
The process by which
antibodies against the complex
of heparin and platelet factor 4
activate platelets, resulting in a
decrease in platelet numbers
of more than 50%.
Low-molecular-weight
heparins
(LMWHs). A class of
anticoagulants derived from
unfractionated heparin by
chemical or enzymatic
degradation. They induce a
conformational change in
antithrombin that greatly
increases its anticoagulant
activity.
Thrombin
Thrombin (also known as factor
IIa) is the terminal enzyme of
the coagulation cascade and
converts fibrinogen into fibrin,
which forms clot fibres.
Thrombin also activates several
other coagulation factors, as
well as protein C.
hypothetical ‘ideal’ anticoagulant, and FIG. 1 chronicles
the historical development of anticoagulants.
Despite the accumulated knowledge on the coag-
ulation system (FIG. 2), its complexity has presented
numerous obstacles to the discovery and development
of potent anticoagulants that are both effective and safe.
In recent years, research has focused on new classes of
anticoagulants that target a specific coagulation enzyme
or step in the coagulation cascade9,10, including inhibi-
tors of the factor VIIa–tissue factor complex10, inhibi-
tors of factor IXa11,12 and factor XIa13,14, direct thrombin
inhibitors15,16, synthetic indirect and direct inhibitors
of factor Xa (activated factor X)17–20, and recombinant
soluble thrombomodulin21,22. In addition, recombinant
activated protein C (APC) mitigates the procoagulant
state associated with sepsis23,24.
In the search for new anticoagulant drugs, the acti-
vated serine protease factor Xa is a particularly promising
target and has attracted great interest in recent years18,25–27.
This article describes the discovery and development of
the first oral, direct factor Xa inhibitor to be approved for
clinical use — rivaroxaban (Xarelto; Bayer HealthCare).
Rivaroxaban was approved by the European authorities
in 2008 for the prevention of venous thromboembolism
(VTE; comprising deep-vein thrombosis (DVT) and pul-
monary embolism) after elective hip or knee replacement.
The article then briefly considers the future clinical
potential of rivaroxaban and other factor Xa inhibitors
currently in advanced clinical development.
Factor Xa: function and biology
Factor X has long been known to have a key role in hae-
mostasis28 and factor Xa plays a central part in the blood
coagulation pathway by catalysing the production of
thrombin, which leads to clot formation and wound
closure20 (FIG. 2). Conversely, deficiency of factor Xa may
disturb haemostasis. In the very rare factor X deficiency
disorder (for which 1 in 500,000 is homozygous and
1 in 500 heterozygous), very low plasma and activity levels
of factor Xa manifest as severe bleeding tendencies29–31.
Studies of variants of factor X deficiency indicate that fac-
tor X plasma activity levels must be as low as 6–10% of
the normal range (approximately 50–150% of the popu-
lation average) to be considered a mild deficiency; cases
with factor X activity levels below 1% are considered to
be severe29,32. Thus it seems that factor X activity can be
markedly suppressed without affecting haemostasis. An
ideal anticoagulant would prevent thrombosis without
inducing systemic hypocoagulation, and would thereby
avoid unintended bleeding complications. Therefore, a
factor Xa inhibitor could potentially have the properties
of a desirable anticoagulant.
Validating factor Xa as a drug target
The first factor Xa inhibitors. Although factor Xa was
identified as a promising target for the development of
new anticoagulants in the early 1980s, the viability of
factor Xa inhibition was not tested before the end of that
decade. In 1987, the first factor Xa inhibitor, the natu-
rally occurring compound antistasin, was isolated from
the salivary glands of the Mexican leech Haementeria
officinalis33,34. Antistasin is a 119 amino-acid polypep-
tide; kinetic studies revealed that it is a slow, tight-bind-
ing, potent factor Xa inhibitor (inhibition constant (Ki)
of 0.3–0.6 nM) that also inhibits trypsin (half maximal
inhibitory concentration (IC50) is 5 nM in the presence
of 1 nM trypsin)35. Another naturally occurring factor
Xa inhibitor, the tick anticoagulant peptide (TAP), a sin-
gle-chain, 60 amino-acid peptide, was isolated in 1990
from extracts of the soft tick Ornithodoros moubata36.
Similarly to antistasin, TAP is a slow, tight-binding
inhibitor of factor Xa (Ki of ~0.6 nM).
TAP 37 and recombinant forms of antistasin38 and
TAP 38–40 were used to validate factor Xa as a viable drug
target and to improve understanding of the role of fac-
tor Xa in thrombosis. The antithrombotic effects of
these compounds were compared with those of direct
thrombin inhibitors and of indirect thrombin and fac-
tor Xa inhibitors (that is, UFH) in animal models of
thrombosis. These studies suggested that direct factor
Xa inhibitors might be a more effective approach to
anticoagulation37,39, and might also offer a wider thera-
peutic window, particularly with regard to primary
haemostasis40,41.
In vitro and in vivo studies. Clot-bound factor Xa was
shown to be enzymatically active in v itro and able to acti-
vate prothrombin to thrombin42. In addition, factor Xa was
found to be an important contributor to clot-associated
procoagulant activity in vitro43. Clot-bound factor Xa
activity was resistant to inhibition by antithrombin42, sug-
gesting that the ability to directly inhibit clot-associated
factor Xa, with no requirement for a cofactor, could
provide an effective and highly localized approach to
the prevention of thrombus growth. The clot-associated
Table 1 | Comparison of an ideal anticoagulant and a vitamin K antagonist
Property Ideal anticoagulant Vitamin K antagonist
Administration Oral Oral
Onset/offset of
action
Rapid (several hours) Slow (several days)
Therapeutic window Wide Narrow
Variability in dose
response
Little or no
inter-individual or
intra-individual
variability
Considerable inter-individual and
intra-individual variability
Interactions Little or no interaction
with food or other drugs
Multiple interactions with food
and other drugs
PK/PD Predictable Unpredictable and variable
Coagulation
monitoring
No routine monitoring
required
Regular monitoring required
Dose adjustment None required Required
Efficacy Highly effective
in reducing
thromboembolic events
Effective in reducing
thromboembolic events when
properly controlled
Safety profile Good, especially with
regard to bleeding
Difficulties in maintaining patients
within the target therapeutic
range (INR 2.0–3.0); contributes to
an increased risk of bleeding
INR, international normalized ratio; PK/PD, pharmacokinetics/pharmacodynamics.
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Parenteral
Oral
Parenteral
Parenteral
Parenteral
Oral
Oral
Unfractionated heparins: antithrombin-dependent
inhibition of factor Xa and IIa in a 1:1 ratio
Vitamin K antagonists: indirectly affect
synthesis of multiple coagulation factors
LMWH: antithrombin-dependent inhibition
of factor Xa > inhibition of factor IIa (2:1 to 4:1)
Direct factor IIa inhibitors
Indirect factor Xa inhibitors
Direct factor
IIa inhibitors
Direct factor
Xa inhibitors
1930s
1940s
1980s
1990s
2000s
2008
Nature Reviews | Drug Discovery
Antithrombin
An endogenous glycoprotein
that binds covalently to
thrombin and other
coagulation factors, resulting in
their inhibition. Antithrombin
functions as a natural
anticoagulant, and its
inhibitory action is accelerated
by heparin.
Warfarin
A vitamin K antagonist that is
currently the most commonly
used oral anticoagulant.
Vitamin K antagonist
A class of compounds that
inhibit the vitamin K-dependent
carboxylation of specific
coagulation factors, resulting in
decreased levels of the
affected coagulation factors,
leading to anticoagulation.
Therapeutic window
The interval between the
lowest dose of a drug that is
sufficient for clinical
effectiveness and a higher dose
at which adverse events or
toxicity become unacceptable.
generation of fibrinopeptide A (a byproduct of fibrino-
gen cleavage by thrombin) was inhibited to the same
extent by both recombinant TAP and hirudin, a direct
thrombin inhibitor. This observation suggested that
procoagulant activity in the clot was due to de novo acti-
vation of prothrombin to thrombin, rather than to the
activity of pre-existing thrombin43. It was subsequently
shown that inhibition of factor Xa by recombinant TAP
provided sustained in vitro44 and in vivo45 inhibition
of clot-associated procoagulant activity, which may, in
turn, protect against ongoing coagulation after cessation
of anticoagulant treatment.
Overall, these data suggested that direct factor Xa
inhibitors might not be linked to the phenomenon of
‘rebound’ thrombosis that was associated with the direct
and indirect thrombin inhibitors previously under inves-
tigation46,47. Rebound thrombosis can be thought of as
a transient increase in thromboembolic events occur-
ring shortly after the withdrawal of an antithrombotic
medication46,48. Furthermore, low concentrations of a
direct thrombin inhibitor may partly suppress the nega-
tive feedback on coagulation by APC, in contrast to fac-
tor Xa inhibition, which does not seem to measurably
affect the thrombin–thrombomodulin–APC system49.
However, whether these experimental observations have
any clinical relevance remains to be determined.
The first synthetic factor Xa inhibitors
Although antistasin and TAP provided support for the
concept of factor Xa inhibition, development of these
compounds was discontinued. The reasons were never
disclosed. Nonetheless, the encouraging results from
studies using recombinant versions of the natural fac-
tor Xa inhibitors prompted several pharmaceutical
companies to initiate chemistry programmes to develop
selective, small-molecule, direct inhibitors of factor Xa,
such as DX-9065a50 and YM-60828 (REFS 51,52) (FIG. 3).
Both these compounds contain a highly basic amidine
residue, designed as mimics for the arginine of the
natural substrate prothrombin.
DX-9065a, a widely studied non-peptidic small
molecule, shows rapid, direct and reversible binding
kinetics for factor Xa (Ki of 41 nM)53. At physiologi-
cal pH it is a zwitterion with high water solubility and
low lipophilicity54. However, human oral bioavailability
was only 2–3%54. A small Phase II study was conducted
in patients with non-ST-elevation acute coronary
syndrome (ACS) who were randomized to low- or
high-dose intravenous DX-9065a or UFH55. A non-
significant trend was observed towards a reduction in
ischaemic events and bleeding for DX-9065a compared
with UFH55.
Because of the success of indirect dual factor Xa and
thrombin inhibitors, such as LMWHs, indirect inhibi-
tors of factor Xa with greater selectivity, such as fonda-
parinux (Arixtra; GlaxoSmithKline)56,57, were developed
in parallel with direct factor Xa inhibitors.
Both UFH and LMWHs contain a unique pen-
tasaccharide sequence that mediates binding to anti-
thrombin. Binding induces a conformational change
in antithrombin that potentiates its ability to inhibit
coagulation factors. Inhibition of thrombin occurs
through heparin chains of sufficient length to bridge
antithrombin to thrombin in a ternary complex, after
which antithrombin binds covalently to thrombin and
the heparin chain dissociates2. However, such bridging
is not necessary for antithrombin to be able to inhibit
factor Xa. Most LMWH chains are too short to cata-
lyse thrombin inhibition, but can nonetheless promote
the inhibition of factor Xa. Thus, UFH has an anti-Xa
to anti-IIa ratio of 1:1, LMWHs have anti-Xa to anti-
IIa ratios from 2:1 to 4:1, depending on the molecular
weight distribution of the preparation2.
Fondaparinux is an analogue of the pentasaccha-
ride sequence required to promote the binding of anti-
thrombin to factor Xa2. The pentasaccharide structure is
too short to enable bridging between antithrombin and
thrombin. As a result, fondaparinux exclusively potenti-
ates the anti-factor Xa activity of antithrombin and has
no effect on thrombin2. The efficacy and safety of fon-
daparinux for the prevention of VTE after major ortho-
paedic surgery were investigated in four randomized,
Phase III trials in patients undergoing surgery for hip
fracture58, hip replacement59,60 and knee replacement61.
A meta-analysis of these trials showed the superior
efficacy of fondaparinux over the LMWH enoxaparin
(Lovenox/Clexane; Sanofi–Aventis) in reducing the
incidence of VTE. However, major bleeding occurred
more frequently in the fondaparinux-treated group
(P=0.008), although the incidence of clinically relevant
bleeding (bleeding leading to death, reoperation or in
a critical organ) did not differ between the treatment
groups62. Fondaparinux provided the proof of princi-
ple that selective inhibition of factor Xa could provide
clinically effective anticoagulation.
Figure 1 | Development of anticoagulants over the past century. The timeframe
starts with the discovery of the heparins and the vitamin K antagonists, followed by
the low-molecular-weight heparins. These were followed, in turn, by the discovery
of the parenteral direct thrombin inhibitors, the development of the indirect factor Xa
inhibitor, fondaparinux, and the work of the current decade that has resulted in oral
direct inhibitors of both thrombin (factor IIa) and factor Xa. The inverted triangle
reflects the narrowing of action and increasing specificity of the anticoagulants.
LMWH, low-molecular-weight heparin.
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FXIIFXIIa
FXI FXIa
FIXFVII
FIXa
FVIII FVIIIa
FVIIa
TF
FX FXa
Intrinsic
pathway
Extrinsic
pathway
FV
FIIFIIa
Fibrinogen Fibrin
FVa
Unfractionated heparin
(+ antithrombin)
Vitamin K antagonists
Direct factor Xa inhibitors
Fondaparinux (+ antithrombin)
LMWHs (+ antithrombin)
Direct thrombin inhibitors
Nature Reviews | Drug Discovery
Numerous investigations, performed with both direct
and indirect factor Xa inhibitors, showed that inhibi-
tion of factor Xa produces antithrombotic effects by
decreasing the generation of thrombin, thus diminish-
ing thrombin-mediated activation of both coagulation
and platelets without affecting the activity of existing
thrombin. However, the residual thrombin generated
seems to be sufficient to ensure normal systemic haem-
ostasis, possibly because thrombin has a very high
affinity for platelet receptors and minimal amounts of
thrombin can provide sufficient platelet activation, thus
contributing to a favourable efficacy/safety ratio20. On
the basis of these findings, in the mid-1990s, it seemed
that small-molecule, direct factor Xa inhibitors could
potentially provide an advantage over the antithrom-
botic therapies available at that time. Several new factor
Xa inhibitors are now in clinical development. These
compounds, which include rivaroxaban and the other
direct factor Xa inhibitors discussed later, represent
new chemical entities with similar binding modes at the
active site of factor Xa.
The discovery of rivaroxaban
From the first screening hit to the oxazolidinone lead.
When we initiated the factor Xa programme at Bayer
HealthCare in 1998, no orally active factor Xa inhibi-
tors with sufficient antithrombotic activity were known.
All known potent inhibitors that had previously been
investigated contained an amidine group or other highly
basic residues, which were designed to act as mimics
for an arginine present in the natural substrate, pro-
thrombin. For a long time, these mimics were thought
to be a prerequisite for high binding affinity63. However,
we found that strongly basic mimics contribute to poor
oral absorption, an observation also later published by
others64,65.
High-throughput screening of approximately 200,000
compounds revealed several hits that selectively inhib-
ited the cleavage of a chromogenic substrate by human
factor Xa63. The most potent of these hits was on a minor
impurity in a combinatorial library — a phosphonium
salt with an IC50 of 70 nM (compound 1 in FIG. 4). We
proposed that this positively charged phosphonium
moiety might serve as an arginine mimic and could be
interchangeable with an amidine group. This resulted in
the synthesis of lead compound 2 with similar potency
(IC50 of 120 nM)63. Further optimization resulted in
the synthesis of compounds of the isoindolinone class,
among which imidazoline 3 (FIG. 4) was the most potent
(IC50 of 2 nM).
To achieve oral bioavailability, we explored less basic
or non-basic amidine replacements. Aminopyridines,
such as compound 4 (IC50 of 8 nM; FIG. 4), showed IC50
values in the low nanomolar range; however, although
they were less basic, oral absorption remained insuf-
ficient. The less potent pyridylpiperazine derivative 5
(IC50 of 48 nM), meanwhile, revealed a significantly
improved oral bioavailability of 38% in rats. Although
we had demonstrated that improved oral bioavailability
could be achieved using less basic amidine replacements,
we were not able to meet our target of identifying factor
Xa inhibitors with both high potency and sufficient oral
bioavailability in the isoindolinone class of compounds.
However, from these studies we did learn that broad
variations in the benzylamidine part were permissible,
but found that there was a very steep structure–activity
relationship (SAR) for the chlorothiophene carboxamide
moiety, which was already present in the lead structure.
Figure 2 | Simplified schematic for the blood coagulation cascade. The figure
identifies the target points of various anticoagulants and illustrates that factor X (FX) can
be activated through either the intrinsic or the extrinsic pathway. The factor VIIa–tissue
factor (TF) complex (extrinsic tenase) activates both factor IX and factor X, as well as
factor VII itself (dashed arrow)150,151. Initiation of either pathway activates the inactive
precursor, factor X, to factor Xa. This makes factor Xa a desirable intervention point for
novel anticoagulants, because it occupies a central role in the blood-coagulation
pathway20. Furthermore, in addition to initiation, both the intrinsic and extrinsic
pathways lead to the propagation and amplification of coagulation through the
activation of factor X. During the initiation phase of coagulation, the factor Xa produced
generates some thrombin (factor IIa). This initial thrombin activates factor XI, and factors
V and VIII, to factor XIa and the activated cofactors, factor Va and VIIIa, respectively. It
also activates platelets (not shown), which are required for the formation of the intrinsic
tenase (factor VIIIa–factor IXa) and the prothrombinase (factor Va–factor Xa) complexes.
The prothrombinase complex, on the platelet surface, is substantially more efficient than
free factor Xa at activating prothrombin to thrombin; the rate of thrombin formation is
increased by approximately 300,000-fold over the rate with factor Xa alone152. Thrombin
is the principal enzyme involved in the formation, growth and stabilization of thrombi.
Thrombin mediates the conversion of fibrinogen to fibrin, the activation of factor XIII
(which crosslinks and stabilizes fibrin), the activation of platelets and the
above-mentioned feedback-activation of upstream coagulation factors, factor V, factor
VIII and factor XI151,153, resulting in the amplification of its own formation150,154. Because
one molecule of factor Xa catalyses the formation of approximately 1,000 thrombin
molecules25,154, this amplification can be substantial. Compared with thrombin, factor Xa
is thought to have fewer effects outside coagulation and, together with factor Va (as the
prothrombinase complex), acts mainly to convert prothrombin to thrombin. Specific
inhibition of factor Xa does not affect pre-existing thrombin but does inhibit thrombin
generation155. Conversely, although thrombin generation is delayed in the presence of a
thrombin inhibitor, the amount of thrombin generated is only reduced at higher inhibitor
concentrations, albeit in a concentration-dependent manner156. LMWH, low-molecular-
weight heparin.
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N
N
H3C
O O
OH
NH2
HN
FF
N
N
H3C
OOH
N NH2
NH
S
O
O
HO O
O
N
H3C
NH
N
O
O
N
O
O
H
N
O
S
Cl
N N
NN
NH2
O
O
O
O
CH3
N
H3C
NH
CH3
H
N
O
H
N
ON
Cl
O
CH3
Fidexaban
(ZK-807834, CI-1031)
Otamixaban
(FXV673, RPR130673)
DX-9065a YM-60828
Rivaroxaban
(BAY 59-7939)
Apixaban
(BMS 562247)
Betrixaban
(PRT054021)
Edoxaban
(DU-176b)
Fondaparinux
N
H
O
CH3
O CH3O
HN NH2
N+
O–
NH3C
NH
O
HO O
NH2
NH
NN
S
H3C
N
H
O
O N
CH3
CH3
HN
O
O
HN
N
Cl
O
NaO3SO
NaO3SO
NaO3SO
O
NHSO3Na
NHSO3Na
NHSO3Na
HO
HO
O
NaO2C
NaO2C
O
OH
HO
O
O
NaO3SO
O
HO OSO3Na
O
O
O
CH3
HO
Nature Reviews | Drug Discovery
International normalized
ratio
(INR). Because prothrombin
time-test results vary according
to the activity of the
thromboplastin used, the INR
conversion is used to normalize
results for any thromboplastin
preparation. It is valid only with
vitamin K antagonists.
Thromboprophylaxis
A measure taken to prevent
the development of a
thrombus. It can be
pharmaceutical or mechanical.
Tissue factor
A cell-membrane-bound
receptor protein that is
exposed to the circulating
blood during vessel injury.
Pre-existing factor VIIa in the
blood binds to tissue factor,
initiating the coagulation
cascade.
Direct thrombin inhibitors
A class of anticoagulants that
bind directly to thrombin and
block the interaction with its
substrate, fibrinogen, thereby
inhibiting the generation of
fibrin and clot formation.
Thrombomodulin
A membrane-bound thrombin
receptor that, when bound to
thrombin, functions as a
cofactor in the
thrombin-induced activation of
protein C.
Protein C
The inactive precursor of
activated protein C (APC). APC,
with its cofactor protein S,
inactivates factor Va and factor
VIIIa, thus providing an
important anticoagulant
feedback function.
Factor Xa inhibitor
A class of anticoagulants that
inhibit factor Xa in the
coagulation cascade, either by
binding directly, or indirectly
through antithrombin.
Inhibition of factor Xa reduces
the production of thrombin.
Venous thromboembolism
(VTE). A condition in which a
blood clot (thrombus) that has
formed in the venous system
breaks free (becoming an
embolus) and migrates through
the circulation to lodge in and
block another blood vessel.
The failure to find a compound with sufficient
potency and bioavailability could have ended the
project. However, we decided instead to re-evaluate
the weaker screening hits. The oxazolidinone (com-
pound 6; FIG. 4) was a weak factor Xa inhibitor, with
an IC50 of 20,000 nM. Considering the importance of
the chlorothiophene residue in the class of compounds
studied previously, we replaced the thiophene moiety of
compound 6 with a 5-chlorothiophene group, thereby
creating lead compound 7 (FIG. 4). This had a >200-fold
higher potency (IC50 of 90 nM) and did not include any
basic group63.
Figure 3 | Structures of various factor Xa inhibitors. Fidexaban, otamixaban, DX-9065a, YM-60828, rivaroxaban
(Xarelto; Bayer HealthCare), apixaban (Pfizer/Bristol-Myers Squibb), betrixaban and edoxaban are shown. The structure
of YM150 has not been published. Not all of these compounds are still in clinical development (for example, fidexaban
and DX-9065a) but study of their structures contributed to our understanding of the structure–activity relationships
and pharmacology of factor Xa inhibitors. Key features to note are the highly basic arginine mimetic amidine groups as
P1 moieties in fidexaban, otamixaban, DX-9065a and YM-60828, which contribute to poor oral bioavailability. These
can be contrasted with the non-basic P1 moieties: the chlorothiophene moiety in rivaroxaban, the methoxyaryl group
in apixaban and the chloro-substituted pyridine rings in edoxaban and betrixaban, all of which allow improved oral
bioavailability. Other synthetic factor Xa inhibitors have also been developed26. The structure of the synthetic indirect
factor Xa inhibitor, fondaparinux (Arixtra; GlaxoSmithKline), is also shown. This is an analogue of the heparin
pentasaccharide sequence required to mediate the conformational change in antithrombin and subsequent binding
to factor Xa2.
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6 R = H IC50 = 20,000 nM
7 R = Cl IC50 = 90 nM
NS N
O
HN
O
S
R
O
F
R N
O
HN
O
S
Cl
O
F
*
8 R = IC50 = 32 nM
Oral bioavailability: 94%
NO
*
9 R = IC50 = 40 nM
N
R N
O
HN
O
S
Cl
O
*
10 R = IC50 = 4 nM
Oral bioavailability: 65%
N
O
*
11 R = IC50 = 0.7 nM
Oral bioavailability: 60%
NO
O
Oxazolidinones
O
R
N
HN
O
O
O
S
Cl
NP *
+
N
N
CF3COO–
1 R = IC50 = 70 nM
5
IC50 = 48 nM
Oral bioavailability: 38%
*
2 R = IC50 = 120 nM
H2N
NH
R
N
HN
O
O
S
Cl
*
3 R =
IC50 = 2 nM
Oral bioavailability: <1%
N
HN
*
4 R = IC50 = 8 nM
Oral bioavailability: <1%
N
HN
N N N
O
N
OHN
O
S
Cl
Isoindolinones
Nature Reviews | Drug Discovery
Deep-vein thrombosis
(DVT). A blood clot in a deep
vein, usually in the leg. Distal
DVT occurs in the calf, whereas
proximal DVT occurs above the
knee.
Pulmonary embolism
A blood clot or
thromboembolus in a
pulmonary blood vessel. Such
emboli generally originate from
a deep-vein thrombosis and
can cause permanent lung
damage, chronic pulmonary
hypertension and death.
Haemostasis
The complex process that leads
to the formation of a blood clot,
causing bleeding to stop.
On the basis of this promising lead, a medicinal
chemistry programme was followed that focused on fur-
ther improving the potency of the oxazolidinone class
without compromising its pharmacokinetic profile.
The optimization programme leading to rivaroxa-
ban. The starting point of these investigations was the
thiomorpholine group; morpholine and pyrrolidine
derivatives (compounds 8 and 9; FIG. 4) showed some
improvement in potency (IC50 values of 32 nM and
40 nM, respectively). Although not sufficiently potent,
compound 8 was the first compound to show a favour-
able pharmacokinetic profile, with a high oral bioavail-
ability of 94% in rats. An ortho-substitution led to the
pyrrolidinone derivative 10, with significantly improved
potency (IC50 of 4 nM) and an oral bioavailability of
65%. However, the in vivo antithrombotic potency of
this compound, evaluated in an arteriovenous shunt
model in anaesthetized rats, was estimated to be too
low63. Our knowledge of the SAR then led us to design
the morpholinone residue, resulting in compound 11
(rivaroxaban; FIG. 4), for which binding to factor Xa
depends on the (S)-configuration at the oxazolidinone
core. Rivaroxaban showed increased potency in vitro
(IC50 of 0.7 nM) and in vivo after oral administration in
rats63. This compound also showed favourable oral bio-
availability (60% in rats and 60–86% in dogs)63.
The optimization programme was continued in
order to gain a more in-depth understanding of the
SAR. However, further variations evaluated at this time,
such as substitution of the aryl ring or less lipophilic
replacements for the chlorothiophene carboxamide
moiety, did not result in further improvements63, and
the morpholinone derivative 11 (rivaroxaban) remained
the most attractive candidate. The X-ray crystal struc-
ture of rivaroxaban in complex with human factor Xa63
(BOX 1) helped us to understand its binding mode and to
explain the steep SAR observed earlier. A similar bind-
ing mode has been reported for several other factor Xa
inhibitors66–70 (BOX 1).
With its combination of high binding affinity and
good oral bioavailability, rivaroxaban was identified as
the drug candidate for further development.
Preclinical studies
Rivaroxaban is a direct, specific factor Xa inhibitor that,
unlike indirect agents such as fondaparinux, does not
require a cofactor71. In vitro kinetic studies showed that
the inhibition of human factor Xa by rivaroxaban was
competitive (Ki of 0.4 nM), with >10,000-fold greater
selectivity for factor Xa than for other serine proteases71.
Rivaroxaban inhibits prothrombinase (IC50 of 2.1 nM)71
and initial data suggest that it also inhibits clot-bound
factor Xa activity (IC50 of 75 nM)72. In human plasma,
Figure 4 | Optimization of oxazolidinone factor Xa inhibitors. Optimization of oxazolidinone factor Xa inhibitors
resulted in the discovery of rivaroxaban (compound 11)63. Half-maximal inhibitory concentration (IC50) values are for the
inhibition of factor Xa activity; oral bioavailability is shown for rats.
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Tyr99
Asp102
His57
Tyr228
Ser195
S1
Asp189
Gly219
Phe174
S4
Trp215
Nature Reviews | Drug Discovery
Fibrinogen
A soluble plasma protein that,
in the final phase of the
coagulation process, is
converted to fibrin by
thrombin. Fibrin then
polymerizes and forms the
fibrous network base of a clot.
Oral bioavailability
The total proportion of
pharmacologically active drug
that enters the systemic
circulation after oral
administration. It is affected by
both absorption and local
metabolic inactivation.
Prothrombin time
A laboratory test that
measures clotting time in the
presence of tissue factor
(thromboplastin). It is used to
assess the activity of the
extrinsic coagulation pathway.
Activated partial
thromboplastin time
A laboratory test that measures
the clotting time of plasma
after contact activation. It
assesses the function of the
intrinsic coagulation pathway.
rivaroxaban inhibited thrombin generation — and there-
fore the amplification processes of coagulation — through
the inhibition of factor Xa generated by either the intrin-
sic or the extrinsic coagulation pathways73,74. Thrombin
generation was almost completely inhibited in platelet-
rich plasma at physiologically relevant concentrations
(80–100 nM) of rivaroxaban73. This and other studies dem-
onstrated that rivaroxaban prolonged the initiation phase
of thrombin generation, potently inhibited the physiologi-
cally relevant prothrombinase complex-bound factor Xa
on the surface of activated platelets71 and reduced the
thrombin burst produced in the propagation phase73.
In addition, preliminary work has shown that rivar-
oxaban inhibits thrombin generation in the presence
and absence of thrombomodulin in human plasma in a
concentration-dependent manner. This suggests that it
does not interfere with the thrombin–thrombomodulin–
APC system and, therefore, probably does not suppress
APC-mediated negative feedback75, as was shown for
DX-9065a49. Further preliminary data suggested that
rivaroxaban did not directly affect platelet aggregation
in platelet-rich plasma induced by thrombin, adeno-
sine diphosphate or collagen76, but potently inhibited
tissue-factor-induced platelet aggregation in an indirect
manner, by inhibiting thrombin generation77.
Rivaroxaban demonstrated anticoagulant effects in
human plasma, with the prothrombin time being more
sensitive than the activated partial thromboplastin time71, a
finding also observed with other direct factor Xa inhibi-
tors in clinical development78,79. This may be because
direct factor Xa inhibitors, including rivaroxaban71,
are highly effective inhibitors of the prothrombinase
complex, although differences in enzyme kinetics may
also be responsible20. A dose-dependent prolongation
of prothrombin time was demonstrated in vivo in rat
and rabbit models, with a strong correlation observed
between prothrombin time and plasma concentrations
of rivaroxaban (r=0.98)71.
In vivo, rivaroxaban given prophylactically had potent
and consistent antithrombotic effects in venous71,80 and
arterial71 thrombosis models in rats and rabbits. In a rab-
bit treatment model, rivaroxaban reduced the accretion
of radiolabelled fibrinogen into preformed clots in the
jugular vein, relative to untreated controls80. Bleeding
times in rats and rabbits were not significantly affected at
antithrombotic-effective doses71, indicating a favourable
efficacy/bleeding ratio.
Although rivaroxaban has a short half-life in
humans81,82 (see the Xarelto Summary of Product
Characteristics (SMPC) from the European Medicines
Box 1 | Binding mode of rivaroxaban
The figure shows the X‑ray crystal structure
of rivaroxaban (carbons coloured orange)
in complex with human factor Xa63.
Essential amino acids and binding pockets
(S1 and S4) are indicated; hydrogen bonds
are shown as dotted lines.
Two hydrogen bonds are formed between
rivaroxaban and the amino acid Gly219 of
factor Xa. The first, a strong interaction
(2.0 Å), is from the carbonyl oxygen of the
oxazolidinone core. The second, weaker
interaction (3.3 Å), is from the amino group
of the chlorothiophene carboxamide
moiety. These two hydrogen bonds support
the oxazolidinone core in directing its
substituents into the S1 and the S4 subsites
of factor Xa. This results in rivaroxaban
forming an L‑shape, a binding mode typical
of synthetic, direct factor Xa inhibitors63.
The aromatic rings of Tyr99, Phe174 and
Trp215 in factor Xa define a narrow
hydrophobic channel that comprises the S4
pocket. The morpholinone moiety of rivaroxaban is ‘sandwiched’ between Tyr99 and Phe174, while its aryl ring is oriented
perpendicularly, extending across the face of Trp215. There is no direct interaction between the morpholinone carbonyl
group and the factor Xa backbone; rather, this carbonyl group contributes to a planarization of the morpholinone ring,
further supporting the sandwich‑like arrangement63. Using the morpholinone moiety, as well as other six‑membered rings
such as lactames or pyridinones, we found new, non‑basic P4 residues (see REF. 146 for patent application), yielding
strongly increased binding to factor Xa. Such residues have since been used successfully for several other factor Xa
inhibitors69,147, including apixaban148 and PD0348292 (eribaxaban)149.
In the S1 pocket of factor Xa, the key interaction is between the chlorine substituent of the thiophene moiety and the
aromatic ring of Tyr228 at the bottom of the S1 pocket. This novel interaction obviates the need for strongly basic groups,
such as amidines, to achieve high factor Xa affinity, and therefore enables non‑basic rivaroxaban to achieve both high
potency and good oral bioavailability63. A similar binding mode has been found and reported for several other factor Xa
inhibitors66–70.
Figure modified, with permission, from REF. 63  (2005) American Chemical Society.
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CYP450 isoforms
Many therapeutic drugs are
metabolized by cytochrome
P450 (CYP450) enzymes, a
‘superfamily’ of related but
distinct enzymes that differ in
their substrate specificity.
Creatinine clearance
The rate at which the kidney
clears the blood of creatinine (a
waste product from muscles
that is excreted at a fairly
constant rate). Creatinine
clearance is used as an
approximation of the
glomerular filtration rate.
Agency (EMA); Further information), it may be of
interest to determine whether the anticoagulant effect of
rivaroxaban can be reversed, because there might be rare
instances in which this would be of use — for example,
a need for emergency surgery. However, it is worth not-
ing that several established anticoagulants also lack full
antidotes. For example, the anticoagulant effect of the
LMWHs is only partly reversed by protamine sulphate2
and there is no available antidote for fondaparinux (see
Arixtra’s prescribing information; Further information).
Preliminary studies in primates and rats have been con-
ducted to evaluate possible antidotes for rivaroxaban.
These studies suggested that recombinant factor VIIa,
activated prothrombin complex concentrate (FEIBA
(factor VIII inhibitor bypassing activity); Baxter)83 and
prothrombin complex concentrate84 can partially reverse,
in a dose-dependent manner, the effects of high-dose
rivaroxaban on bleeding time. However, it is important to
note that there is no clinical experience with any of these
reversal strategies (see the EMA’s SMPC on rivaroxaban;
Further information).
Clinical pharmacology
In Phase I and Phase II studies, rivaroxaban exhibited
predictable pharmacokinetic and pharmacodynamic
profiles, as follows. It is rapidly absorbed, with maximum
plasma concentrations (Cmax) occurring 2–4 hours after
tablet intake82,85. Oral bioavailability of rivaroxaban is
decreased at higher doses, possibly owing to poor solu-
bility. However, at the currently approved 10 mg dose,
oral bioavailability is 80–100%85.
Food (a high-fat, high-calorie meal) did not affect the
area under the plasma concentration–time curve (AUC)
or Cmax for the 10 mg tablet (EMA’s SMPC on rivaroxa-
ban). Rivaroxaban was safe and well tolerated across a
wide dose range, with dose-proportional pharmacoki-
netics and pharmacodynamics. No relevant accumula-
tion was observed at any dose level after multiple dosing
in healthy subjects at steady state82.
Rivaroxaban is metabolized by CYP450 isoforms, par-
ticularly CYP3A4, which is strongly inhibited by ketoco-
nazole and ritonavir (see prescribing information from
Janssen Pharmaceutica (ketoconazole) and Abbott (riton-
avir), and the EMA’s Committee of Medicinal Products
for Human Use (CHMP) report for rivaroxaban; Further
information). These drugs were, therefore, predicted
to affect the metabolism of rivaroxaban and, because
both drugs are also strong inhibitors of P-glycoprotein
(P-gp) and CYP3A4 (REFS 86,87), may increase plasma
concentrations of rivaroxaban. This expectation was
confirmed in subsequent Phase I studies that showed a
strong interaction between rivaroxaban and these two
drugs. However, there was no clinically relevant inter-
action with clarithromycin, a strong CYP3A4 inhibitor
but only a moderate inhibitor of P-gp, indicating that
rivaroxaban may be used with substances that strongly
inhibit only one of the two elimination pathways (see
the EMA’s SMPC on rivaroxaban and prescribing infor-
mation from Janssen Pharmaceutica (ketoconazole) and
Abbott (ritonavir)). Conversely, these findings show that
concomitant use of rivaroxaban with strong inhibitors
of both CYP3A4 and P-gp such as ketoconazole or HIV
protease inhibitors such as ritonavir is not recommended.
In addition, rivaroxaban should be used with caution if
strong CYP3A4 inducers are administered concomitantly
(EMA’s CHMP report for rivaroxaban).
Rivaroxaban has a low propensity for drug–drug inter-
actions with frequently used concomitant medications
such as naproxen88 and asprin89, and no interaction with
the cardiac glycoside digoxin (Lanoxin; GlaxoSmithKline)
(D. Kubitza, unpublished observations).
Furthermore, dietary restrictions are not necessary
at the 10 mg once-daily dose; rivaroxaban was given
with and without food in the Phase III VTE-prevention
studies (RECORD studies 1–4)90–93 (EMA’s SMPC on
rivaroxaban).
Rivaroxaban is distributed heterogeneously to tissues
and organs, and exhibits only moderate tissue affinity in
rats; importantly, it does not substantially penetrate the
blood–brain barrier94. However, like many small mol-
ecules, rivaroxaban is expected to be able to cross the pla-
centa, although specific studies have not been published.
In vitro and Phase I studies showed that rivaroxaban has
a dual mode of elimination, with one-third eliminated
unchanged by the kidneys and two-thirds metabolized by
the liver to inactive metabolites, with no major or active
circulating metabolites detected in plasma95,96. Elimination
of rivaroxaban from plasma occurs with a mean terminal
half-life of 7–11 hours81,82 (EMA’s SMPC on rivaroxaban).
With a systemic clearance rate of approximately 10 L h–1,
rivaroxaban can be classified as a low-clearance drug97,98.
Low intra-individual and moderate inter-individual phar-
macokinetic variability have been observed97,98. In Pha se I
studies, the coefficient of variation for AUC ranged from
18% to 33%, and for Cmax from 16% to 39%, for inter-
individual variability. Median intra-individual variability
was only 14% and 19% for AUC and Cmax, respectively
(EMA’s CHMP report for rivaroxaban).
Rivaroxaban inhibited factor Xa activity in a dose-
dependent manner after single dosing in healthy sub-
jects. Rivaroxaban also inhibited thrombin generation
in healthy subjects, and some parameters of thrombin
generation remained inhibited for 24 hours after admin-
istration of a 30 mg dose74. These results offered the first
indication that once-daily dosing might be feasible.
There were close correlations between pharmacoki-
netic and pharmacodynamic parameters82. Rivaroxaban
plasma concentrations correlated closely with prolonga-
tion of prothrombin time and inhibition of factor Xa.
Plasma concentrations correlated with prothrombin
time both in healthy volunteers82 and in patients under-
going either total hip replacement (THR) or total knee
replacement (TKR) in Phase II studies97.
Various Phase I and II studies, using total daily doses
ranging from 5 mg to 80 mg, indicated that rivaroxa-
ban could be given irrespective of age, gender81, body
weight99 and mild (creatinine clearance: 50–79 ml min–1 )
to moderate (creatinine clearance: 30–49 ml min–1) renal
impairment100. A further preliminary clinical study sug-
gested that rivaroxaban can also be given irrespective
of mild hepatic impairment (classified as Child–Pugh
class A)101. Fixed doses of rivaroxaban were administered
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Chromogenic assay
An enzymatic assay in which a
colour develops during the
course of the reaction, which
can then be measured spectro-
photometrically. Colour
development is reduced in the
presence of an inhibitor.
Venography
Radiography of the veins after
intravenous injection of a
radioactive isotope or contrast
dye. This can be used to
confirm the presence of
deep-vein thromboses.
in Phase II studies investigating rivaroxaban for the pre-
vention or treatment of VTE, with no restrictions on
gender, or mild or moderate renal impairment102–105.
These parameters were shown to have no clinically
relevant effect on the pharmacokinetics and pharma-
codynamics of rivaroxaban97. In addition, rivaroxaban
had no effect on heart-rate-corrected QT interval pro-
longation106. Owing to its pharmacokinetic and phar-
macodynamic profiles, rivaroxaban can be given at
fixed doses to adult patients with no requirement for
routine coagulation monitoring. However, there may be
rare occasions when monitoring is of use — for exam-
ple, where poor compliance is suspected or emergency
surgery is required, or to confirm a possible overdose.
This concern has led to the evaluation of a number of
different assay types. Initial results suggest that, in light
of an apparent strong correlation between the extent of
factor Xa inhibition and rivaroxaban plasma concen-
tration, chromogenic assays for factor Xa may be par-
ticularly suited for the quantification of rivaroxaban in
plasma107,108.
Clinical development of rivaroxaban
Rivaroxaban is approved for the prevention of VTE after
elective hip or knee replacement in approximately 100
countries, including member states of the European
Union (as of 30 September 2008) and Canada (as of
16 September 2008). Clinical development is ongoing
for the prevention and treatment of thromboembolic
disorders in other conditions, including the treatment
of VTE, the prevention of VTE in hospitalized, medi-
cally ill patients (for example, patients hospitalized with
an acute medical condition, such as cancer, heart failure
or respiratory failure, or requiring intensive care), as well
as the prevention of stroke in patients with atrial fibril-
lation and secondary prevention in patients with ACS.
The TIMELINE highlights the development of rivaroxaban
during the past decade.
VTE prophylaxis after total hip or knee replacement.
A large, comprehensive Phase II programme involving
about 2,900 patients assessed both once-daily and twice-
daily dosing regimens102–105. Collectively, these studies
demonstrated an optimal dose range of 5–20 mg per
day109, and indicated that rivaroxaban 10 mg once daily
had the optimum balance of efficacy and safety, relative
to enoxaparin 40 mg once daily103 (FIG. 5).
On the basis of the results of the Phase II studies,
rivaroxaban 10 mg once daily was selected for investiga-
tion in the Phase III RECORD programme, which com-
prised four large studies involving a total of more than
12,500 patients undergoing elective THR or TKR90–93
(TABLE 2). In all four studies, the primary efficacy end
point was the composite of any DVT, as detected by
mandatory, bilateral venography, non-fatal pulmonary
embolism and all-cause mortality. The primary safety
end point was major bleeding, which in the RECORD
programme did not include surgical-site bleeding90–93.
The RECORD1 and 3 studies were designed to compare
rivaroxaban 10 mg once daily with the standard of care —
enoxaparin 40 mg once daily — both given for 31–39 days
(extended prophylaxis) after THR (RECORD1)90 or for
10–14 days after TKR (RECORD3)92. In both studies,
rivaroxaban was more effective than enoxaparin for the
prevention of VTE90,92 (TABLE 2). Furthermore, the inci-
dence of major bleeding was comparable and not signifi-
cantly different between treatment groups90,92. RECORD3
also showed a significant reduction in symptomatic VTE
for rivaroxaban92.
RECORD2 investigated the efficacy and safety
of extended thromboprophylaxis with rivaroxaban
(35 days; range 31–39 days) compared with short-term
enoxaparin treatment (10–14 days) followed by placebo
in patients undergoing THR91. The results demonstrated
that extended prophylaxis with rivaroxaban (10 mg
once daily) was superior to short-term prophylaxis with
enoxaparin (40 mg once daily) followed by placebo for
Timeline | Rivaroxaban: more than a decade of progress
1998 1999 2000 2002 2003 2004 2005 2007 2008 2009 2010
A new class of
compounds, the
oxazolidinones, is
identified and
optimized, leading
to the discovery of
rivaroxaban
Bayer HealthCare
initiates the factor
Xa programme
Preclinical studies
on rivaroxaban
are initiated
Phase II studies
initiated
The RECORD
clinical trial
programme begins
Rivaroxaban approved and
launched in the EU and Canada
for VTE prophylaxis after hip or
knee replacement
Results of the RECORD1, 2 and
3, and EINSTEIN DVT Phase II
studies are published90–92, 117
ROCKET AF clinical trial data
presented (http://sciencenews.
myamericanheart.org/pdfs/
ROCKET_AF_pslides.pdf)
EINSTEIN DVT and EINSTEIN EXT
Phase III results published119
Rivaroxaban shows
favourable pharmacokinetic
and pharmacodynamic
profiles in Phase I studies82,85
Phase I studies show
rivaroxaban to be
well tolerated81,82,85
Phase II studies
suggest a favourable
benefit–risk
profile103–105
RECORD1 and 3 show
rivaroxaban to be more
effective than enoxaparin in
the prevention of VTE, with
a comparable safety profile.
RECORD2 shows benefit of
extended prophylaxis with
rivaroxaban90–92
Regulatory file submitted
for approval to the EMA
FDA Advisory Committee
meeting
RECORD4 and ATLAS ACS
TIMI 46 data published93,114
DVT, deep-vein thrombosis; EMA, European Medicines Agency; EU, European Union; FDA, US Food and Drug Administration; VTE, venous thromboembolism.
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Incidence–efficacy (%)
Incidence–safety (%)
40
30
20
10
0
30
20
10
0
10
520 30 40 Enoxaparin
Total daily dose of rivaroxaban (mg)
0
DVT, PE and all-cause death
Major postoperative bleeding
Nature Reviews | Drug Discover
y
Index event
The acute event that leads to a
patient’s initial presentation.
The term can also refer to the
initial event resulting in a
patient’s inclusion in a
follow-up study, such as a
survey of recurrent strokes.
the prevention of VTE, including symptomatic events91
(TABLE 2). Despite rivaroxaban being given for 3 weeks
longer than enoxaparin, the incidence of treatment-
emergent major bleeding (that is, up to 2 days after the
last dose of study medication) was <0.1% in both treat-
ment groups. Guidelines recommend a minimum dura-
tion for prophylaxis of 10 days, and also recommend that
prophylaxis be extended for up to 35 days for patients
undergoing THR110. The RECORD2 study provided
additional confirmation of the benefits of extended
prophylaxis over short-term prophylaxis after THR.
RECORD4 compared the efficacy and safety of oral
rivaroxaban 10 mg once daily with the commonly used
North American regimen of enoxaparin 30 mg twice
daily in patients undergoing TKR93. Rivaroxaban was sig-
nificantly superior to enoxaparin for the primary efficacy
end point, with no significant difference in the rates of
major bleeding between the two groups (TABLE 2). So far,
rivaroxaban is the only new oral anticoagulant to dem-
onstrate superior efficacy over the greater dose intensity
of the North American enoxaparin regimen93,111,112.
A comparison of rivaroxaban with enoxaparin in
these four studies showed the efficacy and safety of rivar-
oxaban in the prevention of VTE in patients undergoing
elective THR or TKR. The superiority of rivaroxaban
for the primary efficacy end point was demonstrated in
all four studies. Rivaroxaban also showed a good safety
profile, with a low incidence of major bleeding, com-
parable to that observed with enoxaparin90–93, and no
evidence of compromised liver function attributable to
rivaroxaban113. Furthermore, the incidence of haemor-
rhagic wound complications (composite of excessive
wound haematoma and reported surgical-site bleeding)
was similar in both treatment groups113. Liver safety is
also an important consideration in regulatory review. In
a Phase II trial of rivaroxaban in patients with ACS, no
patient who had received rivaroxaban for 6 months had
an alanine-amino-transferase level greater than three
times the upper limit of normal or total bilirubin greater
than twice the upper limit of normal114.
These studies were also conducted with no routine
coagulation monitoring and no dose adjustment for
demographic variables, consistent with preliminary
results from pooled subgroup analyses115.
Treatment of VTE. The efficacy and safety of rivaroxaban
for the treatment of VTE were assessed in two Phase IIb
dose-ranging studies116,117. These studies suggested
that rivaroxaban had good efficacy and a similar safety
profile, compared with standard therapy, for the treat-
ment of acute symptomatic DVT. An initial intensified
twice-daily regimen (rivaroxaban 15 mg twice daily for
3 weeks) followed by convenient 20 mg once-daily dos-
ing for 3, 6 or 12 months was selected for investigation
in Phase III studies. The efficacy and safety of rivaroxa-
ban for the treatment of VTE are being assessed in three
Phase III studies involving approximately 9,000 patients
in total— EINSTEIN DVT (ClinicalTrials.gov identifier:
NCT00440193), EINSTEIN PE (NCT00439777) and
EINSTEIN EXT (NCT00439725).
EINSTEIN PE (pulmonary embolism) is still ongo-
ing. However, data for EINSTEIN DVT (presented at
the 2010 American Society of Hematology meeting118
and recently published119) showed that rivaroxaban
(15 mg twice daily for 21 days followed by 20 mg once
daily) was non-inferior for the prevention of recurrent
symptomatic VTE in comparison to the current standard
of care (enoxaparin 1.0 mg kg–1 twice daily for ≥5 days
followed by VKA titrated to INR 2.0–3.0). First symp-
tomatic recurrent VTEs occurred in 2.1% of patients
receiving rivaroxaban compared with 3.0% of those in
the enoxaparin/VKA treatment arm. Major or non-major
clinically relevant bleeding occurred in 8.1% of patients
in each treatment arm119.
In the EINSTEIN EXT trial, 1,196 patients (intention-
to-treat population) who had completed 6–12 months
of anticoagulant therapy for the acute index event (VTE)
were randomized to an additional 6–12 months of therapy
with either rivaroxaban, 20 mg once daily, or placebo119.
Study medication was administered for a mean period
of 190 days in each treatment group. Recurrent sympto-
matic VTE events were observed in 7.1% and 1.3% of the
placebo and rivaroxaban treatment groups, respectively
(hazard ratio 0.18, P<0.0001), a relative risk reduction of
82% with rivaroxaban119 (hazard ratio, 0.68; 95% confi-
dence interval, 0.44–11.04; P<0.001 for non-inferiority).
Major bleeding did not occur in any placebo-treated
patients and occurred in four (0.7%) rivaroxaban-treated
patients, although none of these occurred in a critical
location or proved fatal119.
Thromboprophylaxis in medically ill patients. A
Phase III study (NCT00571649) has been initiated to
investigate the efficacy and safety of VTE prophylaxis
Figure 5 | Efficacy and safety dose–response relationships. The figure shows the
dose–response relationships between rivaroxaban total daily dose and the primary
efficacy end point (any deep-vein thrombosis (DVT); non-fatal pulmonary embolism (PE)
and all-cause death) and primary safety end point (major bleeding) in the once-daily
study investigating rivaroxaban for the prevention of venous thromboembolism after
total hip replacement103. Solid lines show the dose–response curves (logistic regression),
blue hatched lines represent 95% confidence intervals for efficacy and red hatched
lines represent 95% confidence intervals for safety. Details for 40 mg enoxaparin once
daily, the current European standard of care, are shown for comparison. Figure
reproduced, with permission, from REF.103  (2006) Lippincott Williams & Wilkins.
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with rivaroxaban 10 mg once daily (for 35 ± 4 days),
compared with short-term 40 mg once-daily enoxaparin
(administered for 10 ± 4 days followed by placebo), in
hospitalized, medically ill patients.
Stroke prevention in non-valvular atrial fibrillation.
Rivaroxaban 20 mg once daily has been compared with
warfarin for the prevention of stroke and non-central-
nervous-system systemic embolism in approximately
14,000 patients with non-valvular atrial fibrillation in a
Phase III study (NCT00403767)120. Patients with moderate
renal impairment (creatinine clearance: 30–49 ml min–1)
received a fixed dose of 15 mg once daily. It was recently
reported at the 2010 American Heart Association
meeting that, in the ROCKET AF study, rivaroxaban
significantly reduced the risk of stroke and non-central-
nervous-system thromboembolism in patients with atrial
fibrillation compared to warfarin, with comparable rates
of bleeding (http://sciencenews.myamericanheart.org/
pdfs/ROCKET_AF_pslides.pdf).
Secondary prevention in ACS. A Phase III study inves-
tigating secondary prevention of ischaemic events in
patients with ACS (NCT00809965) was started in late
2008, and is expected to enrol up to 16,000 patients. Two
doses of rivaroxaban, 2.5 mg and 5 mg twice daily, are
being investigated on the basis of the results of a Phase IIb
study that assessed safety and efficacy in approximately
3,500 patients with recent, non-ST-elevation myocar-
dial infarction, ST-elevation myocardial infarction or
unstable angina114. As noted above, this trial also showed
no evidence of compromised liver function in patients
receiving rivaroxaban for up to 6 months.
Other direct factor Xa inhibitors in development
Below, we discuss other direct factor Xa inhibitors that
are in advanced clinical development (Phase III).
Apixaban. Apixaban (developed by Pfizer/Bristol-Myers
Squibb) is a small-molecule, oral, direct factor Xa inhibi-
tor (FIG. 3) that selectively and reversibly inhibits both free
factor Xa (Ki of 0.08 nM) and prothrombinase activity79,121.
Another study reported that apixaban reacts rapidly with
factor Xa (kon 2 × 107 M–1 s–1) with high-affinity binding
(Ki of 0.3 nM at 37 °C)122. Preclinical studies have shown
that apixaban was well absorbed in chimpanzees, dogs
and rats; mean oral bioavailability was 51% (chimpan-
zees), 88% (dogs) and 34% (rats)79. The drug is currently
Table 2 | Incidence of venous thromboembolism and bleeding events across the four RECORD* studies
End points‡Efficacy end point (% (n/N)) Safety end points (patients with bleeding events) (% (n/N))
Total VTE Major VTE Symptomatic
VTE
Major bleeding Major and non-major
clinically relevant bleeding
RECORD1 (THR: prophylaxis administered for 35 days)
Enoxaparin (40 mg once
daily)
3.7 (58/1,558) 2.0 (33/1,678) 0.5 (11/2,206) 0.1 (2/2,224) 2.5 (56/2,224)
Rivaroxaban (10 mg
once daily)
1.1 (18/1,595) 0.2 (4/1,686) 0.3 (6/2,193) 0.3 (6/2,209) 3.2 (70/2,209)
P value <0.001 <0.001 0.22 0.18 NS§
RECORD2 (THR: extended rivaroxaban prophylaxis, 35 days, versus short-term enoxaparin, 14 days, followed by placebo)
Enoxaparin (40 mg once
daily/placebo)
9.3 (81/869) 5.1 (49/962) 1.2 (15/1,207) <0.1 (1/1,229) 2.8 (34/1,229)
Rivaroxaban (10 mg
once daily)
2.0 (17/864) 0.6 (6/961) 0.2 (3/1,212) <0.1 (1/1,228) 3.3 (41/1,228)
P value <0.0001 <0.0001 0.004 0.98 (REF. 157) NR
RECORD3 (TKR: prophylaxis administered for 14 days)
Enoxaparin (40 mg once
daily)
18.9 (166/878) 2.6 (24/925) 2.0 (24/1,217) 0.5 (6/1,239) 2.7 (34/1,239)
Rivaroxaban (10 mg
once daily)
9.6 (79/824) 1.0 (9/908) 0.7 (8/1,201) 0.6 (7/1,220) 3.3 (40/1,220)
P value <0.001 0.01 0.005 0.77 0.44
RECORD4 (TKR: prophylaxis administered for 14 days)
Enoxaparin (30 mg
twice daily)
10.1 (97/959) 2.0 (22/1,112) 1.2 (18/1,508) 0.3 (4/1,508) 2.3 (34/1,508)
Rivaroxaban (10 mg
once daily)
6.9 (67/965) 1.2 (13/1,112) 0.7 (11/1,526) 0.7 (10/1,526) 3.0 (46/1,526)
P value 0.012 0.124 0.187 0.11 0.18
N, total number of patients evaluable in each treatment group; NR, not reported; NS, not significant; THR, total hip replacement; TKR, total knee replacement;
VTE, venous thromboembolism. *The four RECORD studies90–93 compared rivaroxaban regimens with enoxaparin regimens (the current standard of care); summary
results are shown in the table. ‡Results shown as number (n) of patients experiencing an event; patients could have more than one event. §P value not reported, but
95% confidence interval for risk difference includes zero.
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being evaluated in a number of thromboembolic indica-
tions, including the prevention and treatment of VTE, the
prevention of stroke in patients with atrial fibrillation and
the prevention of cardiovascular events in ACS.
In the Phase III programme for the prevention of VTE
after major orthopaedic surgery, apixaban did not meet
the prespecified criteria for non-inferiority compared
with enoxaparin 30 mg twice daily (NCT00371683) for
VTE prevention after TKR111. However, a second study
(NCT00452530) conducted in patients undergoing TKR
demonstrated superior efficacy for apixaban against
enoxaparin 40 mg once daily. Clinically relevant bleeding
(major or non-major) occurred in fewer patients given
apixaban, although the differences were not signifi-
cant123. A third study (NCT00423319), presented as an
abstract, assessed extended prophylaxis with apixaban
versus enoxaparin (40 mg once daily) in patients under-
going THR and also demonstrated superior efficacy for
apixaban with similar rates of major and non-major
clinically relevant bleeding124.
A Phase II trial in patients with ACS (NCT00313300)
assessed efficacy and safety in patients taking apixa-
ban compared with placebo125. The developing compa-
nies recently reported that a subsequent Phase III trial
(NCT00831441) in patients with ACS investigating
whether apixaban (5 mg twice daily) is superior to pla-
cebo has been halted (press release, Bristol-Myers Squibb;
Further information). In addition, other apixaban trials are
ongoing for VTE prevention in patients with acute medical
illness (NCT00457002), or recently completed for malig-
nant disease (NCT00320255), as well as Phase III trials
for the treatment of VTE (NCT00633893, NCT00643201)
and trials for the prevention of stroke in patients with
atrial fibrillation (NCT00412984, NCT00496769)126,127.
Data for the AVERROES trial (NCT00496769) were
recently presented at the European Society of Cardiology
Congress. Patients with atrial fibrillation who have either
been demonstrated to be or were expected to be unsuita-
ble for treatment with VKAs received apixaban 5 mg twice
daily or aspirin 81–324 mg per day. Preliminary results
showed that the primary end point of stroke or systemic
embolism occurred at a significantly lower rate in patients
receiving apixaban, with an absolute risk reduction of
approximately 2% versus aspirin128.
Edoxaban. Edoxaban (developed by Daiichi Sankyo) is
an oral, direct and specific factor Xa inhibitor with a Ki
of 0.56 nM (FIG. 3); Ki values for other coagulation factors
are >10,000-fold higher78. In healthy volunteers, peak
plasma levels of edoxaban were observed at 1.5 hours
after a single oral dose, corresponding to the maximum
inhibition of factor Xa activity; and ex vivo thrombus
formation was reduced at 1.5 hours and 5 hours after
administration129.
Phase II trials in patients undergoing TKR130 or
THR131 have been completed, as well as a Phase II trial
for stroke prevention in atrial fibrillation132, and two
Phase III trials are now under way. One is designed to
compare two different doses of edoxaban with warfarin
for stroke prevention in patients with atrial fibrillation
(NCT00781391) and another is evaluating edoxaban for
the secondary prevention of recurrent VTE in patients
with acute symptomatic proximal DVT or pulmonary
embolism (NCT00986154).
YM150. YM150 (developed by Astellas) is also an oral,
direct factor Xa inhibitor (Ki of 31 nM). Preliminary
data show that both YM150 and its major metabolite
YM-222714 (Ki of 20 nM) have antithrombotic effects
at doses that do not prolong template bleeding time in
animal models of venous and arterial thrombosis133.
Phase II trials for VTE prevention after THR134 or TKR
(NCT00408239) have been completed, as has a warfarin-
controlled Phase II trial for stroke prevention in patients
with atrial fibrillation (NCT00448214). Three Phase III
trials are now in progress. One will assess once-daily and
twice-daily doses of YM150 against enoxaparin for VTE
prevention in patients undergoing elective hip replace-
ment (NCT00902928). In another Phase III trial, YM150
is being compared with mechanical prophylaxis for
the prevention of VTE after major abdominal surgery
(NCT00942435). A third study is being conducted in
Japan to evaluate YM150 for the prevention of VTE in
the 28 days following hospitalization for an acute medical
illness (NCT01028950).
Outlook
For more than 65 years, VKAs have been the only avail-
able oral anticoagulants, and although effective, the need
for dose adjustment and periodic coagulation monitoring
considerably complicates their clinical management. New
and improved anticoagulants could potentially address
the shortcomings associated with the current standard of
care and it should be noted that other approaches, besides
the development of oral, direct factor Xa inhibitors, are
also being evaluated. For example, the parenteral direct
factor Xa inhibitor, otamixaban (developed by Sanofi–
Aventis), has completed Phase II trials for short-term use
in non-ST-elevation ACS135 and in percutaneous coronary
intervention136. The encouraging results obtained in these
studies have been followed by an ongoing Phase III trial in
patients with unstable angina or non-ST elevation myo-
cardial infarction undergoing an invasive intervention
(NCT01076764). Conversely, idraparinux, and its succes-
sor idrabiotaparinux137 (Sanofi–Aventis; both now discon-
tinued) are parenterally administered pentasaccharides
(indirect factor Xa inhibitors) with very long half-lives138
that were developed to permit once-weekly dosing in
indications requiring long-term or chronic therapy139,140.
In addition, direct thrombin inhibitors are also in
development, most notably dabigatran (Pradaxa/Pradax;
Boehringer Ingelheim), which, like rivaroxaban, has
completed several Phase III thrombosis prevention and
treatment studies141–144 and has been approved in mem-
ber states of the European Union and other countries for
the prevention of VTE after elective hip or knee replace-
ment. Dabigatran is also in trials for a number of other
indications, as is AZD0837 (developed by AstraZeneca),
which is in Phase II145. Although indirect comparisons,
based on meta-analyses, can be conducted, direct com-
parative trials are required for a comprehensive evalua-
tion of one particular drug regimen versus another.
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In the future, it is anticipated that long-term antico-
agulant therapy will favour oral agents with a wide thera-
peutic window and a predictable anticoagulant response
that do not require routine coagulation monitoring or
dose adjustment. Emerging data suggest that direct fac-
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for short-term use and promising agents for long-term
usage. As they have shown a predictable pharmacological
profile, are given orally and do not require routine
coagulation monitoring, these new agents may also
facilitate patient compliance and adherence to clinical
guidelines. Thus, they are likely to improve anticoagula-
tion treatment in thromboembolic disorders and reduce
the burden associated with long-term therapy, thereby
providing important alternative options for the manage-
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Acknowledgements
The authors would like to acknowledge S. Salaria and S.
McMillan, who provided medical writing services, with
funding from Bayer HealthCare and Johnson & Johnson
Pharmaceutical Research & Development. We would also like
to thank Proteros Biostructures, Planegg-Martinsried,
Germany, for performing the X-ray crystal structure work on
rivaroxaban.
Competing interests statement
The authors declare competing financial interests: see web
version for details.
FURTHER INFORMATION
ClinicalTrials.gov: http://clinicaltrials.gov
Bayer HealthCare Xarelto (rivaroxaban) Summary of
Product Characteristics: http://www.ema.europa.eu/docs/
en_GB/document_library/EPAR_-_Product_Information/
human/000944/WC500057108.pdf
GlaxoSmithKline, Arixtra (fondaparinux) prescribing
information: http://us.gsk.com/products/assets/us_arixtra.pdf
Janssen Pharmaceutica Products, Nizoral (ketoconazole)
tablets prescribing information: https://www.ortho-mcneil.
com/ortho-mcneil/shared/pi/nizoral_tablets.pdf
Abbott Laboratories, Norvir (ritonavir) prescribing
information: http://www.rxabbott.com/pdf/norvirtab_pi.pdf
EMA CHMP Assessment Report for Xarelto (rivaroxaban):
http://www.ema.europa.eu/docs/en_GB/document_library/
EPAR_-_Public_assessment_report/human/000944/
WC500057122.pdf
Bristol-Myers Squibb press release 18 Nov 2010
(APPRAISE-2 study with investigational compound
apixaban in acute coronary syndrome discontinued):
http://www.bms.com/news/press_releases/pages/default.
aspx?RSSLink=http://www.businesswire.com/news/
bms/20101118007161/en&t=634257431381752059
Rocket AF study: http://sciencenews.myamericanheart.org/
pdfs/ROCKET_AF_pslides.pdf
ALL LINKS ARE ACTIVE IN THE ONLINE PDF
REVIEWS
NATURE R EVIEWS
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DRUG DISCOVERY VOLUME 10
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JANUARY 2011
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