J Thromb Thrombolysis (2011) 31:478–492
`DOI 10.1007/s11239-011-0551-3
`
`Preclinical discovery of apixaban, a direct and orally bioavailable
`factor Xa inhibitor
`
`Pancras C. Wong • Donald J. P. Pinto •
`Donglu Zhang
`
`Published online: 13 February 2011
`Ó Springer Science+Business Media, LLC 2011
`
`Abstract Apixaban (BMS-562247; 1-(4-methoxyphenyl)-
`7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-
`1H-pyrazolo[3,4-c]pyridine-3-carboxamide),
`a
`direct
`inhibitor of activated factor X (FXa), is in development for
`the prevention and treatment of various thromboembolic
`diseases. With an inhibitory constant of 0.08 nM for human
`FXa, apixaban has greater than 30,000-fold selectivity for
`FXa over other human coagulation proteases. It produces a
`rapid onset of inhibition of FXa with association rate
`constant of 20 lM
`-1/s approximately and inhibits free as
`well as prothrombinase- and clot-bound FXa activity in
`vitro. Apixaban also inhibits FXa from rabbits, rats and
`dogs, an activity which parallels its antithrombotic potency
`in these species. Although apixaban has no direct effects on
`platelet aggregation, it indirectly inhibits this process by
`reducing thrombin generation. Pre-clinical
`studies of
`apixaban in animal models have demonstrated dose-
`dependent antithrombotic efficacy at doses that preserved
`hemostasis. Apixaban improves pre-clinical antithrombotic
`activity, without excessive increases in bleeding times,
`when added on top of aspirin or aspirin plus clopidogrel at
`their
`clinically relevant doses. Apixaban has good
`
`P. C. Wong (&)
`Department of Cardiovascular Biology, Bristol-Myers Squibb
`Company, 311 Pennington-Rocky Hill Road, Pennington,
`NJ 08534, USA
`e-mail: pancras.wong@bms.com
`
`D. J. P. Pinto
`Department of Medicinal Chemistry, Bristol-Myers Squibb
`Company, Princeton, NJ, USA
`
`D. Zhang
`Department of Pharmaceutical Candidate Optimization,
`Bristol-Myers Squibb Company, Princeton, NJ, USA
`
`123
`
`bioavailability, low clearance and a small volume of dis-
`tribution in animals and humans, and a low potential for
`drug–drug interactions. Elimination pathways for apixaban
`include renal excretion, metabolism and biliary/intestinal
`excretion. Although a sulfate conjugate of O-demethyl
`apixaban (O-demethyl apixaban sulfate) has been identified
`as the major circulating metabolite of apixaban in humans,
`it is inactive against human FXa. Together, these non-
`clinical findings have established the favorable pharmaco-
`logical profile of apixaban, and support the potential use of
`apixaban in the clinic for the prevention and treatment of
`various thromboembolic diseases.
`
`Keywords Apixaban Factor Xa Anticoagulants
`Thrombosis Atrial fibrillation
`
`Introduction
`
`Thrombosis is a major cause of morbidity and mortality in
`the Western world and plays a pivotal role in the patho-
`genesis of numerous cardiovascular disorders, including
`acute coronary syndrome (ACS) (i.e. unstable angina and
`myocardial infarction), sudden cardiac death, peripheral
`arterial occlusion, ischemic stroke, deep vein thrombosis
`(DVT) and pulmonary embolism. Despite recent advances
`in interventional and drug therapy for thrombosis,
`the
`burden of thrombotic disease remains unacceptably high
`[1, 2]. There is therefore a significant need for new anti-
`thrombotic therapies that are more effective and provide
`improved safety profile compared with current treatments.
`This review focuses on the pre-clinical discovery of apix-
`aban, a promising new oral antithrombotic agent that spe-
`cifically targets activated factor X (FXa) of the blood
`coagulation cascade.
`
`BMS 2009
`MYLAN v. BMS
`IPR2018-00892
`
`

`

`Preclinical discovery of apixaban
`
`479
`
`Drug discovery strategy—targeting factor Xa
`
`As the last serine protease in the blood coagulation cas-
`cade, thrombin is the key enzyme responsible for physio-
`logical fibrin clot
`formation and platelet activation.
`Thrombin also plays a prominent role in the pathologic
`generation of occlusive thrombi in arteries or veins, a
`process that may lead to arterial or venous thrombotic
`disease. Thus, attenuation of the activity of thrombin—
`either via direct inhibition or via blockade of other prote-
`ases that lie upstream in the coagulation cascade and are
`intimately involved in thrombin generation (e.g. FXa)—
`has been intensively investigated as a novel means to
`prevent and treat thrombotic disease.
`Three key observations supported our hypothesis that
`inhibition of FXa may represent an acceptable approach for
`effective and safe antithrombotic therapy. First, as the
`process of blood coagulation involves sequential activation
`and amplification of coagulation proteins, generation of
`one molecule of FXa can lead to the activation of hundreds
`of thrombin molecules [3]. In principle, therefore, inhibi-
`tion of FXa may represent a more efficient way of reducing
`fibrin clot formation than direct inhibition of thrombin
`activity. This principle is consistent with an in vitro
`observation, suggesting that
`inhibition of FXa but not
`thrombin may result in a more effective sustained reduction
`of thrombus-associated procoagulant activity [4]. Second,
`inhibition of FXa is not thought to affect existing levels of
`thrombin. Further, reversible FXa inhibitors might not
`completely suppress the production of thrombin. These
`small amounts of thrombin might be sufficient to activate
`high affinity platelet thrombin receptors to permit physio-
`logical
`regulation of hemostasis.
`Indeed, experimental
`evidence from animal studies suggests that the antithrom-
`botic efficacy of FXa inhibitors is accompanied by a lower
`risk of bleeding when compared with thrombin inhibitors
`[5–7] (for review, see Hauptmann and Stu¨rzebecher [8] and
`Leadley [9]). Finally, the strongest evidence for FXa as an
`antithrombotic drug target is the clinical proof of concept
`studies of the indirect FXa inhibitor fondaparinux [10].
`Taken together, these observations suggest that inhibition
`of FXa is a potentially attractive antithrombotic strategy.
`We initiated a drug discovery program on small-mole-
`cule direct FXa inhibitors, with the goal of identifying
`novel oral anticoagulants not burdened by the well-known
`limitations of vitamin K antagonists such as warfarin,
`agents that remain the only oral anticoagulants approved
`for long-term use until very recently [11]. (On October 19,
`2010, FDA approved the oral direct thrombin inhibitor
`dabigatran etexilate to prevent stroke and blood clots in
`patients with non-valvular atrial fibrillation [12].) These
`new FXa inhibitors would have the following target profile.
`First, they would be direct, highly selective and reversible
`
`inhibitors of FXa, with a rapid onset of action, and would
`demonstrate a relatively wide therapeutic index and few
`food and drug interactions (thereby avoiding the need for
`frequent coagulation monitoring and dose adjustment).
`Second,
`these FXa inhibitors would have predictable
`pharmacokinetic and pharmacodynamic profiles that allow
`fixed oral dosing, accompanied by low peak-to-trough
`plasma concentrations that provide high levels of efficacy
`and low rates of bleeding. Finally, as the FXa target resides
`in the central or blood compartment, the pharmacokinetic
`profile of these agents would also feature a low volume of
`distribution (to minimize off-target risks) and low systemic
`clearance
`(to
`reduce
`the
`potential
`for
`drug-drug
`interactions).
`Based on many years of research and development, we
`have identified the potent, highly selective and direct FXa
`inhibitor, apixaban (BMS-562247) [13–15]. Apixaban is
`one of the most promising specific, single-target oral
`anticoagulants in late clinical development. In clinical tri-
`als, apixaban has been shown to provide predictable and
`consistent anticoagulation, accompanied by promising
`efficacy and safety profiles in the prevention and treatment
`of various thromboembolic diseases [16–22]. The phar-
`macological and clinical profiles of apixaban suggest that it
`has the potential to address many of the limitations of
`warfarin therapy, currently the standard of care in chronic
`oral anticoagulation. In this review, we summarize the
`chemistry and pre-clinical profile of apixaban.
`
`Chemistry
`
`Apixaban is a small-molecule, selective FXa inhibitor. It is
`chemically described as 1-(4-methoxyphenyl)-7-oxo-6-[4-
`(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyraz-
`olo[3,4-c]pyridine-3-carboxamide. The molecular formula
`for apixaban is C25H25N5O4, which corresponds to a
`molecular weight of 459.5.
`
`Discovery of apixaban
`
`In the early 1990s, DuPont scientists invested a great
`amount of effort in the development of inhibitors of gly-
`coprotein IIb/IIIa. These efforts resulted in several com-
`pounds that were advanced to clinical trials as potential
`anti-platelet agents. By the mid-1990s, scientists at DuPont
`had recognized similarities between the platelet glycopro-
`tein GPIIb/IIIa peptide sequence Arg-Gly-Asp (RGD) and
`the prothrombin substrate FXa sequence, Glu-Gly-Arg
`(EGR). Consequently, a high-throughput lead evaluation
`program was initiated to screen the IIb/IIIa library for FXa
`inhibitory activity. This effort resulted in the identification
`
`123
`
`

`

`P. C. Wong et al.
`
`H2NO2S
`
`MeO2C
`
`O
`
`NH
`
`N O
`
` 2 (SF303)
`
`fXa Ki = 6.3 nM
`
`SO2NH2
`
` 4 (SN429)
`
`fXa Ki = 0.013 nM
`
`H2N
`
`NH
`
`HN
`
`O
`
`H3C
`
`N
`
`N
`
`H2N
`
`NH
`
`Me2
`
`NN
`
`N
`
`F
`
`HN
`
`O
`
`NH2
`
`F3C
`
`N
`
`N
`
`O N
`
` 6 (Razaxaban)
`
` fXa Ki = 0.19 nM
`
`H2N
`
`O
`
`N
`
`N
`
`N
`
`O
`
`NH
`
`HOOC
`
`N
`
`O O
`
`O
`
`N
`
`1
`
`Initial Screening Lead
`fXa Ki = 38.5 μM
`
`H2N
`
`NH
`
`SO2NH2
`
`HN
`
`O
`
`O
`N
`
`H2N
`
`NH
`
` 3
`
`fXa Ki = 0.1 nM
`
`SO2Me
`
`F
`
`HN
`
`O
`
`F3C
`
`N
`
`N
`
`NH2
`
` 5 (DPC423)
`
`fXa Ki = 0.15 nM
`
`OH
`
`N
`
`F3C
`
`N
`
`N
`
`N
`
`O
`
`O N
`
`NH2
` 7 (BMS-740808)
`
` fXa Ki = 0.03 nM
`
` 8
`
`OCH3
`
`fXa Ki = 0.14 nM
`
`H2N
`
`O
`
`N
`
`N
`
`N
`
`O
`
`O
`
`N
`
`OCH3
`
` 9 (BMS-562247, Apixaban)
`
` fXa Ki = 0.08 nM
`
`
`
`480
`
`Fig. 1 The evolution of the
`pyrazole-based FXa inhibitors:
`the discovery of apixaban
`
`of a small number of isoxazoline derivatives such as 1
`(FXa Ki = 38.5 lM) (Fig. 1) [23]. Using molecular mod-
`eling and structure-based design, an optimization strategy
`resulted in the identification of a benzamidine containing
`FXa inhibitor 2 (SF303) with enhanced potency (FXa
`= 6.3 nM) and potent antithrombotic activity in an
`Ki
`experimental model of thrombosis [24–26]. Aside from the
`
`key amidine P1 and the enzyme Asp189 interaction, the
`biarylsulfonamide P4 moiety was designed to neatly stack
`in the S4 hydrophobic box of FXa, which contains the
`residues Tyr99, Phe174 and Trp215, with the terminal
`O-phenylsulfonamide ring making an edge-to-face inter-
`action with Trp215. Subsequent re-optimizations led to
`vicinally substituted isoxazole analogs such as compound
`
`123
`
`

`

`Preclinical discovery of apixaban
`
`481
`
`3, which retained anti-FXa potency (FXa Ki = 0.1 nM)
`[27] and a pyrazole analog 4 (SN429), which demonstrated
`13 pM binding affinity against FXa and good antithrom-
`botic activity in a rabbit model of thrombosis [28, 29]. The
`discovery of SN429 was tremendously important in that it
`set the stage for an optimization strategy that led to the
`important compounds, such as 5
`discovery of several
`(DPC423), a phase I clinical candidate with a long terminal
`half-life of approximately 30 h in humans [6, 28–30], and 6
`(razaxaban) [31, 32], a compound that was advanced to a
`phase II proof-of-principle clinical trial. In fact, razaxaban
`was the first small molecule FXa inhibitor to provide
`clinical validation of the effectiveness of FXa inhibition
`strategies [33].
`Development of razaxaban was quickly followed by the
`identification of a novel bicyclic tetrahydropyrazolo-pyr-
`= 0.03 nM) [34].
`idinone analog 7 (BMS-740808, FXa Ki
`The evolution of the bicyclic pyrazole template allowed for
`the incorporation of a diverse set of P1 groups, the most
`important of which was the p-methoxyphenyl analog 8
`(Ki = 0.14 nM) [13]. Compound 8 retained potent FXa
`affinity and good anticoagulant activity in vitro, was effi-
`cacious in in vivo rabbit antithrombotic models and
`showed high oral bioavailability in dogs. A significant
`breakthrough was subsequently achieved, via the incorpo-
`ration of a pendent P4 lactam group and a carboxamido
`pyrazole moiety, that led to the discovery of 9 (BMS-
`562247, FXa Ki = 0.08 nM) [13], a highly potent and
`selective FXa inhibitor with good efficacy in various ani-
`mal models of thrombosis. Importantly, compound 9 also
`showed an excellent pharmacokinetic profile in dogs, with
`low clearance, low volume of distribution and high oral
`bioavailability [13]. The superior pre-clinical profile dem-
`onstrated by 9 enabled its rapid progression into clinical
`development as apixaban [15]. Figure 2 illustrates the
`X-ray structure of apixaban bound to FXa and shows the
`
`p-methoxyphenyl P1 deeply inserted into the S1 pocket,
`with the aryllactam P4 moiety neatly stacked in the
`hydrophobic S4 pocket.
`
`In vitro pharmacology
`
`Potency, selectivity and kinetic mode of inhibition
`
`Apixaban is a highly potent, reversible, active-site inhibitor
`of human FXa, with a Ki of 0.08 nM at 25°C and 0.25 nM
`at 37°C in the FXa tripeptide substrate (N-a-benzyloxy-
`carbonyl-D-Arg-Gly-Arg-pNA) assay [35]. Analysis of
`enzyme kinetics shows that apixaban acts as a competitive
`inhibitor of FXa versus the synthetic tripeptide substrate,
`indicating that it binds in the active site. Apixaban pro-
`duces a rapid onset of inhibition under a variety of con-
`ditions with association rate constant of 20 (lM
`-1/s
`approximately, and shows competitive inhibition of FXa
`versus the synthetic tripeptide substrate. Reversibility
`of FXa inhibition is demonstrated by the recovery of FXa
`activity at 37°C upon 200-fold dilution of a pre-formed
`FXa:apixaban complex into tripeptide substrate, an effect
`associated with
`a
`dissociation
`rate
`constant
`of
`*0.0113 s
`-1. Unlike indirect inhibitors of thrombin and
`FXa, such as heparin, the low molecular weight heparins
`and fondaparinux, apixaban, a direct FXa inhibitor, does
`not require the presence of antithrombin III to inhibit FXa.
`As shown in Table 1, apixaban has greater than 30,000-
`fold selectivity for FXa relative to other human coagulation
`proteases and structurally related enzymes involved in
`digestion and fibrinolysis [13].
`In the prothrombinase assay, apixaban is an effective
`inhibitor of the action of human FXa on its physiological
`substrate, prothrombin, blocking the action of FXa on
`prothrombin within the prothrombinase complex with a Ki
`
`Table 1 In vitro Ki values for inhibition of human enzymes by
`apixaban at 25°C [13]
`
`Enzyme
`
`Factor Xa
`
`Activated protein C
`
`Chymotrypsin
`
`Factor IXa
`
`Factor VIIa
`
`Plasma kallikrein
`
`Plasmin
`
`Thrombin
`
`Tissue plasminogen activator
`
`Trypsin
`
`Ki (nM)
`
`0.08 ± 0.03
`[30,000
`3,500
`[15,000
`[15,000
`3,700
`[25,000
`3,100
`[40,000
`[20,000
`
`123
`
`Fig. 2 X-ray structure of apixaban bound to factor Xa
`
`

`

`482
`
`P. C. Wong et al.
`
`Table 2 In vitro potency (Ki) of apixaban against human, rabbit, rat
`and dog factor Xa (FXa) and the concentrations required to double
`the prothrombin time (PT), modified prothrombin time
`(EC29)
`PT EC29 (lM)
`mPT EC29 (lM)
`
`Species
`
`FXa Ki (nM)
`
`(mPT), activated partial thromboplastin time (aPTT) and HepTest
`in human, rabbit, rat or dog plasma [15]
`
`aPTT EC29 (lM)
`
`HepTest EC29 (lM)
`
`Human
`
`Rabbit
`
`Rat
`
`Dog
`
`0.081 ± 0.002
`0.16 ± 0.01
`1.3 ± 0.1
`1.7 ± 0.2
`
`3.6
`
`2.3
`
`7.9
`
`6.7
`
`n.d. not determined
`
`0.37
`
`0.6
`
`n.d.
`
`n.d.
`
`7.4
`
`4.8
`
`20
`[20
`
`0.4
`
`1.8
`
`n.d.
`
`n.d.
`
`of 0.62 nM [35]. It should be noted that when apixaban was
`evaluated as an inhibitor of FXa versus the physiological
`substrate prothrombin in its prothrombinase state, non-
`competitive inhibition was observed. This finding is con-
`sistent with prothrombin binding being dictated primarily
`by interactions at exosites of FXa [36]. Apixaban also
`inhibits thrombus-associated FXa activity with a concen-
`tration causing 50% inhibition (IC50) of 1.3 nM [37]. In
`summary, apixaban is capable of inhibiting the activity of
`free FXa, thrombus-associated FXa and FXa within the
`prothrombinase complex. Apixaban is a direct inhibitor of
`FXa from rats, rabbits and dogs, with Ki values of 1.3, 0.16
`and 1.7 nM, respectively (Table 2 [15]). Previous studies
`involving other small molecule, direct FXa inhibitors have
`also reported a species difference in FXa inhibition among
`humans, rabbits, rats and dogs [29, 38, 39].
`
`In vitro pharmacodynamic studies
`
`To evaluate the in vitro pharmacodynamic activity of
`apixaban in human plasma, studies were undertaken to
`examine [1] thrombin generation, [2] anticoagulant activity
`and [3] platelet aggregation. By inhibiting FXa, apixaban
`prevents the conversion of prothrombin to thrombin,
`resulting in decreased generation of thrombin. Using the
`thrombogram method, apixaban was shown to inhibit tissue
`factor-initiated thrombin generation in human platelet-poor
`plasma in vitro. The IC50 of the rate of thrombin generation
`was 50 nM, and the IC50 for attenuation of the peak
`thrombin concentration was 100 nM [40].
`In human
`platelet-rich plasma, apixaban inhibited tissue factor-
`induced thrombin generation, as measured by the release of
`prothrombin fragment 1 ? 2, with an IC50 of 37 nM [41].
`As expected for an inhibitor of FXa, addition of apix-
`aban to normal human plasma prolonged clotting times,
`including activated partial
`thromboplastin time (aPTT),
`prothrombin time (PT), modified PT (mPT, using diluted
`PT reagent) and HepTest. Among the three clotting time
`assays, it appears that the mPT and HepTest are 10–20
`times more sensitive than aPTT and PT in monitoring the
`in vitro anticoagulant effect of apixaban in human plasma
`(Table 2 [15]). In both the PT and aPTT assays, apixaban
`
`123
`
`had the highest potency in human and rabbit plasma, but
`was less potent in rat and dog plasma, which parallels its
`inhibitory potencies (Ki) against human, rabbit, rat and dog
`FXa (Table 2 [15]).
`In the human platelet aggregation assay, apixaban had
`no direct effects on platelet aggregation response to ADP,
`collagen, c-thrombin, a-thrombin and TRAP [15, 41].
`However,
`it
`indirectly inhibited platelet aggregation
`induced by thrombin derived from tissue factor-mediated
`coagulation pathway, with an IC50 of 4 nM [41]. The potent
`indirect antiplatelet effect of apixaban, together with its
`direct antithrombotic and anticoagulant activity, suggests
`that apixaban may possess dual mechanisms to prevent and
`treat both venous (platelet-poor and fibrin-rich) and arterial
`(platelet-rich and fibrin-poor) thrombosis. It should be
`noted that
`the in vitro tissue factor model of platelet
`aggregation is a useful tool for evaluation of the anti-
`platelet mechanisms of action of anticoagulants. However,
`caution should be exercised as in vitro antiplatelet poten-
`cies of compounds obtained in this model may not directly
`translate into antithrombotic potencies in patients in whom
`multiple prothrombotic mechanisms, complications of
`cardiovascular disease and polypharmacy are common.
`
`In vivo pharmacology
`
`The non-clinical pharmacology of apixaban has been
`studied in vivo in rats and rabbits. Its in vivo effects were
`assessed over a comprehensive dose range in various
`well-established non-clinical models of thrombosis and
`hemostasis. These non-clinical models have been well
`characterized with standard antiplatelet agents and anti-
`coagulants, making them appropriate for evaluating the
`antithrombotic potential and bleeding liability of apixaban.
`
`Antithrombotic and bleeding time effects in rats
`
`Dose-dependent effects of apixaban were examined in a
`broad range of experimental models of thrombosis and
`hemostasis in rats [42]. Efficacy was evaluated using
`established models of thrombosis, including arterial-venous
`
`

`

`Preclinical discovery of apixaban
`
`483
`
`Table 3 Potency of apixaban in multiple thrombosis models
`IC50 (lM)b
`
`ID50 (mg/kg/h)b
`
`Species
`
`Modela
`
`Ratc
`
`Rabbitd
`
`AV-ST
`
`TF-VT
`
`FeCl2-VT
`FeCl2-AT
`AV-ST
`
`pDVT
`
`ECAT
`
`1.20
`
`1.55
`
`0.39
`
`0.72
`
`0.27
`
`0.11
`
`0.07
`
`5.71
`
`7.57
`
`1.84
`
`3.23
`
`0.36
`
`0.065
`
`0.11
`
`a Experimental models included arterial-venous shunt
`thrombosis
`tissue factor-stasis venous thrombosis (TF-VT), FeCl2-
`(AV-ST),
`induced vena cava thrombosis (FeCl2-VT), carotid artery thrombosis
`(FeCl2-AT), prevention model of deep vein thrombosis (pDVT) and
`electrically induced carotid arterial thrombosis (ECAT)
`b Potency for 50% decrease in thrombus weight was determined for
`concentration (IC50) and dose (ID50)
`c Data from Schumacher et al. [42]
`d Data from Wong et al. [7, 15]
`
`tissue factor-stasis venous
`thrombosis (AV-ST),
`shunt
`thrombosis, and FeCl2-induced vena cava thrombosis and
`carotid artery thrombosis. Hemostasis was assessed in
`models of cuticle bleeding time, renal cortex bleeding time
`and mesenteric bleeding time. Apixaban was given by a
`continuous intravenous (IV) infusion 1 h prior to the
`induction of thrombosis or bleeding.
`Apixaban at 0.1, 0.3, 1 and 3 mg/kg/h IV produced
`dose-dependent increases in ex vivo PT (1.24, 1.93, 2.75
`and 3.98 times control, respectively). In the various models
`of thrombosis, doses and plasma concentrations of apix-
`aban for 50% thrombus reduction ranged from 0.39 to
`1.55 mg/kg/h and 1.84 to 7.57 lM, respectively (Table 3)
`[42]. The 3 mg/kg/h dose of apixaban increased cuticle,
`renal and mesenteric bleeding times to 1.92, 2.13 and 2.98
`times control,
`respectively. Bleeding time was not
`increased by apixaban at 0.1 and 0.3 mg/kg/h in any model.
`The 1 mg/kg/h dose produced an increase in mesenteric
`bleeding time, but showed no effect on renal or cuticle
`bleeding time. In comparison, heparin increased renal and
`cuticle bleeding times to two times those of apixaban when
`given at a dose (300 U/kg plus 10 U/kg/min IV) that
`matched the efficacy of apixaban (3 mg/kg/h IV) in arterial
`thrombosis. These studies demonstrate that in rats, apix-
`aban has broad-spectrum antithrombotic efficacy and that
`these beneficial effects can be obtained at doses that show
`limited activity in multiple models of provoked bleeding.
`
`Antithrombotic and bleeding time effects in rabbits
`
`The antithrombotic efficacy of apixaban was evaluated in
`anesthetized rabbits using established models of thrombo-
`sis, including AV-ST, electrically induced carotid arterial
`
`thrombosis (ECAT) and DVT (a thread-induced vena cava
`thrombosis model). Hemostasis was assessed in a rabbit
`model of cuticle bleeding time. Apixaban was given by a
`continuous IV infusion 1 h prior to the induction of
`thrombosis or cuticle incision.
`
`Antithrombotic studies
`
`Apixaban exhibited strong antithrombotic activity in the
`rabbit models of AV-ST, ECAT and DVT, which com-
`pared well with standard antithrombotic agents (Figs. 3, 4)
`[7, 15, 43]. For instance, apixaban, the direct FXa inhibitor
`rivaroxaban, the direct thrombin inhibitor dabigatran and
`the oral anticoagulant warfarin showed similar efficacy in
`the prevention model of DVT (Fig. 3) [7, 15]. In the pre-
`vention model of ECAT, apixaban was as efficacious as the
`antiplatelet agent clopidogrel and warfarin (Fig. 4) [7, 15,
`43]. Doses and plasma concentrations of apixaban for 50%
`thrombus reduction ranged from 0.07 to 0.27 mg/kg/h and
`0.065 to 0.36 lM, respectively (Table 3) [15]. The 1 mg/
`kg/h dose was associated with approximately 80% anti-
`thrombotic efficacy in these models. Interestingly,
`the
`potency of apixaban in arterial and venous thrombosis
`prevention models was broadly equivalent. Apixaban also
`effectively inhibited the growth of a pre-formed intravas-
`cular thrombus in a treatment model of DVT, suggesting
`that apixaban shows potential for the treatment of estab-
`lished thrombosis [7].
`
`Bleeding time studies
`
`The bleeding potential of apixaban was compared with
`those of rivaroxaban, dabigatran and warfarin in the rabbit
`cuticle bleeding time model [7, 15]. At the highest effec-
`tive doses studied (each of which caused *80% inhibition
`of thrombus formation), warfarin increased bleeding time
`almost six-fold, whereas apixaban, rivaroxaban and da-
`bigatran prolonged bleeding time 1.13-, 1.9 and 4.4-fold,
`respectively (Fig. 3) [7, 15]. As shown in Fig. 3, the anti-
`thrombotic efficacy and bleeding profiles of warfarin and
`dabigatran were less favorable than those of apixaban and
`rivaroxaban. It should be noted; however, that extrapola-
`tion of pre-clinical bleeding time data to humans requires
`caution. Provoked bleeding measured in anaesthetized
`healthy animals may not directly translate into spontaneous
`bleeding observed in the clinical setting, where complica-
`tions of cardiovascular disease and polypharmacy are often
`present. Nevertheless, pre-clinical bleeding time studies are
`still useful for generating hypotheses for clinical investi-
`gation, for example by allowing the anti-haemostatic pro-
`files of experimental agents to be ranked and compared
`with those of established agents such as warfarin. The pre-
`clinical comparison of these agents’ therapeutic windows,
`
`123
`
`

`

`484
`
`P. C. Wong et al.
`
`Fig. 3 Plots of thrombus reduction and bleeding time versus dose in
`apixaban,
`rivaroxaban, dabigatran and warfarin-treated rabbits.
`Thrombus reduction, measured in the prevention model of venous
`thrombosis, was expressed as the percentage reduction in thrombus
`weight after treatment, relative to the mean vehicle thrombus weight.
`Bleeding time effect was expressed as a ratio of treated versus the
`mean vehicle value. Data are mean ± SE (n = 6 per group for the
`thrombosis and bleeding time studies and n = 12 per dose for plasma
`concentrations) (data from Wong et al. [7, 15]; reproduced with
`
`as summarized in Fig. 3, remains a hypothesis, and head-
`to-head clinical studies are required to validate these
`results.
`
`Combination therapy
`
`therapy with clopidogrel and aspirin
`Dual antiplatelet
`currently represents the standard of care for the reduction
`of atherothrombotic events in a broad range of patients. To
`understand the benefit-risk ratio of apixaban therapy in
`combination with standard antiplatelet therapy, apixaban
`was evaluated in combination with clinically relevant doses
`of aspirin and/or clopidogrel for the prevention of arterial
`thrombosis in rabbit models [44]. These evaluations
`showed that the triple combination of apixaban, aspirin and
`clopidogrel resulted in improved antithrombotic activity
`versus mono-therapies, without excessively increasing
`bleeding time in rabbits. Such data suggest that intensive
`
`permission). Reproduced from ‘‘Favorable therapeutic index of the
`direct factor Xa inhibitors, apixaban and rivaroxaban, compared with
`the thrombin inhibitor dabigatran in rabbits’’ published in ‘‘Journal of
`Thrombosis and Haemostasis’’ (2009), John Wiley and Sons; and
`from ‘‘Apixaban, an oral, direct and highly selective factor Xa
`inhibitor:
`in vitro, antithrombotic and antihemostatic studies’’
`published in ‘‘Journal of Thrombosis and Haemostasis’’ (2008), John
`Wiley and Sons
`
`antithrombotic therapy with apixaban, aspirin and clopi-
`dogrel may be a viable option for enhancing antithrombotic
`efficacy without unacceptable increases in bleeding.
`This hypothesis was tested in a large phase III study,
`APPRAISE-2, in high-risk patients with recent ACS trea-
`ted with apixaban or placebo in addition to mono (aspirin)
`or dual antiplatelet (aspirin plus clopidogrel) therapy. Very
`recently, the trial was discontinued based on ‘‘evidence of a
`clinically important increase in bleeding among patients
`randomized to apixaban, and this increase in bleeding was
`not offset by clinically meaningful reductions in ischemic
`events’’ [45]. The investigators of the APPRAISE-2 trial
`will continue to review the available data to better under-
`stand the effects of apixaban in this ACS patient population
`and will publish the results [45].
`As discussed above, the translatability of preclinical
`bleeding models to safety in clinical settings requires
`caution. It appears that the preclinical cuticle bleeding
`
`123
`
`

`

`Preclinical discovery of apixaban
`
`485
`
`Fig. 4 Dose-dependent effects of apixaban, clopidogrel and warfarin
`on integrated blood flow in the electrically induced carotid arterial
`thrombosis rabbit model. Data are mean ± SE (n = 6 per group,
`except n = 12 for clopidogrel). *P \ 0.05 versus the corresponding
`
`vehicle (V) (data from Wong et al. [15, 43]). Reproduced from
`‘‘Apixaban, an oral, direct and highly selective factor Xa inhibitor: in
`vitro, antithrombotic and antihemostatic studies’’ published in ‘‘Jour-
`nal of Thrombosis and Haemostasis’’ (2008), John Wiley and Sons.
`
`effect of apixaban in combination with dual antiplatelet
`therapy in rabbits does not translate directly into sponta-
`neous bleeding observed in the APPRAISE-2 trial. The
`underlying causes for this disconnect are not known, but
`may be related to species differences, bleeding time versus
`spontaneous bleeding, vascular bed differences, and the
`fact that unlike animal bleeding models, the APPRAISE-2
`patients had the highest tendency to bleed due to advanced
`age, diabetes, complications of cardiovascular disease,
`other comorbidities and the additive hazards of combina-
`tion antiplatelet treatment. Finally, the APPRAISE-2 find-
`ing does not mean that apixaban cannot benefit other
`patient populations, as recent phase III clinical trials of
`apixaban have demonstrated promising results in patients
`with venous thromboembolism (ADVANCE 1, 2, 3) and
`atrial fibrillation (AVERROES) [18, 21, 22, 46].
`
`Ex vivo coagulation markers
`
`The traditional clotting time tests for adjusting anticoagu-
`lant doses of heparin (aPTT) and warfarin (PT) are not
`sensitive for specific, single-target anticoagulants such as
`the FXa inhibitors. As shown in Fig. 5, apixaban only
`prolonged ex vivo aPTT and PT modestly, even at the
`highest dose that produced 80% antithrombotic efficacy in
`rabbits [7]. As expected from its mechanism of action,
`apixaban did not prolong thrombin time (TT). Among the
`clotting time tests, mPT was the most sensitive for apix-
`aban and tracked well with the antithrombotic activity of
`apixaban. Similar mPT results were also observed with
`
`Fig. 5 Plots of thrombus reduction (bar graph) and ex vivo clotting
`times (line graph) in apixaban-treated rabbits. Thrombus reduction,
`measured in the prevention model of venous thrombosis, was
`expressed as the percentage reduction in thrombus weight after
`treatment, relative to the mean vehicle thrombus weight (data from
`Fig. 3). For clarity, only mean data for thrombus reduction and the
`bolus dose (mg/kg) are shown. Activated partial thromboplastin time
`(aPTT), prothrombin time (PT), modified prothrombin time (mPT)
`and thrombin time (TT) were expressed as the treated/control ratio.
`Data are mean ± SE (n = 6 per group for thrombus reduction and
`n = 12 per group for clotting times) (data from Wong et al. [7];
`reproduced with permission). Reproduced from ‘‘Favorable thera-
`peutic index of
`the direct
`factor Xa inhibitors, apixaban and
`rivaroxaban, compared with the thrombin inhibitor dabigatran in
`rabbits’’ published in ‘‘Journal of Thrombosis and Haemostasis’’
`(2009), John Wiley and Sons
`
`123
`
`

`

`P. C. Wong et al.
`
`486
`
`Fig. 6 Ex vivo anti-FXa and
`anti-thrombin effects of
`apixaban in arterial thrombosis
`rabbits from Fig. 4 (top) and
`correlation of ex vivo anti-FXa
`with antithrombotic effects and
`plasma concentrations of
`apixaban in arterial thrombosis
`rabbits (bottom). *P \ 0.05,
`compared with the vehicle.
`Mean ± SE and n = 6 per
`group (data from Wong et al.
`[15]; reproduced with
`permission). Reproduced from
`‘‘Apixaban, an oral, direct and
`highly selective factor Xa
`inhibitor: in vitro,
`antithrombotic and
`antihemostatic studies’’
`published in ‘‘Journal of
`Thrombosis and Haemostasis’’
`(2008), John Wiley and Sons
`
`other FXa inhibitors such as rivaroxaban [7]. Data from a
`phase II study with apixaban show that the anti-FXa assay
`is more accurate and precise than the mPT test [47].
`Indeed, we also observed that the anti-FXa assay tracked
`well with antithrombotic activity in rabbits with arterial
`thrombosis [15]. As shown in Fig. 6, apixaban produced a
`dose-dependent
`inhibition of FXa and did not
`inhibit
`thrombin activity ex vivo [15]. The ex vivo anti-FXa
`activity of apixaban correlated well with both its anti-
`thrombotic activity and plasma concentration (Fig. 6).
`Thus,
`the anti-FXa activity assay may be suitable for
`monitoring the anticoagulant and plasma levels of apixaban
`if needed in certain situations such as an overdose, acute
`bleeding or urgent surgery.
`
`Drug metabolism and pharmacokinetics
`
`The metabolism and pharmacokinetics of apixaban have
`been studied extensively in animals and humans. In these
`studies, absorption of apixaban after oral administration
`was rapid, with a time to peak plasma concentration (Tmax)
`of 1–2 h. Absolute oral bioavailability of apixaban was
`good in rats, dogs and humans [48–50]. Following IV
`administration, apixaban was slowly eliminated in rats,
`
`