Journal of Thrombosis and Haemostasis, 3: 514–521
`
`ORIGINAL ARTICLE
`
`Invitroand invivostudies of the novel antithrombotic agent
`BAY 59-7939—an oral, direct Factor Xa inhibitor
`
`E . P E R Z B O R N , J . S T R A S S B U R G E R , A . W I L M E N , J . P O H L M A N N ,   S . R O E H R I G ,   K - H . S C H L E M M E R * and
`A . S T R A U B  
`Cardiovascular Research, *Preclinical Pharmacokinetics and  Chemical Research, Bayer HealthCare AG, Wuppertal, Germany
`
`To cite this article: Perzborn E, Strassburger J, Wilmen A, Pohlmann J, Roehrig S, Schlemmer K-H, Straub A. In vitro and in vivo studies of the novel
`antithrombotic agent BAY 59-7939—an oral, direct Factor Xa inhibitor. J Thromb Haemost 2005; 3: 514–21.
`
`Summary. BAY 59-7939 is an oral, direct Factor Xa (FXa)
`inhibitor in development for the prevention and treatment of
`arterial and venous thrombosis. BAY 59-7939 competitively
`inhibits human FXa (Ki 0.4 nM) with > 10 000-fold greater
`selectivity than for other serine proteases; it also inhibited
`prothrombinase activity (IC50 2.1 nM). BAY 59-7939 inhib-
`ited endogenous FXa more potently in human and rabbit
`plasma (IC50 21 nM) than rat plasma (IC50 290 nM). It
`demonstrated anticoagulant effects in human plasma, doub-
`ling prothrombin time (PT) and activated partial thrombo-
`plastin time at 0.23 and 0.69 lM, respectively. In vivo, BAY
`59-7939 reduced venous thrombosis (fibrin-rich, platelet-poor
`)1 i.v.) in a rat
`thrombi) dose dependently (ED50 0.1 mg kg
`venous stasis model. BAY 59-7939 reduced arterial (fibrin-
`and platelet-rich) thrombus formation in an arteriovenous
`)1 p.o.) and rabbits
`(AV) shunt in rats (ED50 5.0 mg kg
`)1 p.o.). Slight inhibition of FXa (32% at
`(ED50 0.6 mg kg
`ED50) reduced thrombus formation in the venous model; to
`affect arterial thrombosis in the rat and rabbit, stronger
`inhibition of FXa (74%, 92% at ED50) was required.
`Calculated plasma levels in rabbits at the ED50 were 14-fold
`lower than in the rat AV shunt model, correlating with the
`IC50 of FXa inhibition in rabbit com-
`14-fold lower
`pared with rat plasma;
`this may suggest a correlation
`between FXa inhibition and antithrombotic activity. Bleed-
`ing times in rats and rabbits were not significantly affected
`)1 p.o., AV shunt). Based
`at antithrombotic doses (3 mg kg
`on these results, BAY 59-7939 was selected for clinical
`development.
`
`Correspondence: E. Perzborn, Cardiovascular Research, Bayer
`HealthCare AG, Building 500, Aprather Weg 18a, D-42096
`Wuppertal, Germany.
`Tel.: +49 202 36 8354; fax: +49 202 36 8009; e-mail: elisabeth.
`perzborn@bayerhealthcare.com
`
`Received 3 March 2004, accepted 3 November 2004
`
`Keywords: antithrombotic activity, Factor Xa inhibitor, oral
`anticoagulant.
`
`Introduction
`
`Anticoagulants in current clinical use comprise the vitamin K
`antagonists—such as warfarin—heparins
`(including low-
`molecular-weight heparins), and parenterally administered
`direct thrombin inhibitors. Warfarin can be administered
`orally; however, its major drawbacks include the need for
`monitoring—because of a narrow therapeutic window and
`large inter- and intraindividual variability in dose–response—a
`slow onset and offset of action, and extensive food and drug
`interactions [1–3]. Heparins have a rapid onset of action, but
`must be administered parenterally. Despite recent develop-
`ments, there is still an unmet need for safe, oral anticoagulants
`for both short- and long-term use.
`Factor Xa (FXa) has emerged as a particularly promising
`target for effective anticoagulation because it acts at the
`convergence point of the intrinsic and extrinsic coagulation
`pathways. FXa catalyzes the conversion of prothrombin to
`thrombin; one molecule of FXa results in the generation of more
`than 1000 thrombin molecules [4]. Thus, inhibiting FXa may
`block this burst of thrombin generation, thereby diminishing
`thrombin-mediated activation of coagulation and platelets.
`Recent research has focused on the identification of small-
`molecule FXa inhibitors with good oral bioavailability and
`predictable pharmacokinetics. An oral, direct FXa inhibitor
`that does not require routine coagulation monitoring would
`offer significant advantages over current therapies. BAY
`59-7939 belongs to a new class of small-molecule, active-
`site-directed FXa inhibitors. It is a non-basic compound with
`high oral bioavailability in rats and dogs (60–86%) [5].
`Currently, BAY 59-7939 is in clinical development for the
`prevention and treatment of thromboembolic disorders.
`We report the in vitro properties of BAY 59-7939,
`its
`antithrombotic efficacy in animal models of arterial and venous
`thrombosis, and its effect on hemostasis—the pharmacological
`profile on which BAY 59-7939 was chosen for clinical
`development.
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`MYLAN - EXHIBIT 1020
`
`

`

`Materials and methods
`
`Agents
`
`BAY 59-7939 (5-chloro-N-({(5S)-2-oxo-3-[4-(3-oxomorpholin-
`4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-car-
`boxamide; Mr ¼ 435.89 g mol
`)1; Fig. 1) was synthesized by
`Bayer HealthCare AG (Wuppertal, Germany). Human, rat,
`and rabbit purified FXa, thrombin, and plasmin were obtained
`from Kordia (Leiden, The Netherlands); Factor XIa (FVIIa)
`from CalbiochemÒ (Schwalbach, Germany);
`trypsin and
`urokinase from Sigma (Taufkirchen, Germany); activated
`protein C (APC) from Haemochrom Diagnostica (Essen,
`Germany); Factor VIIa (FVIIa), Factor IXab (FIXab), FX,
`and prothrombin from Enzyme Research Laboratories
`(Swansea, UK); tissue factor from American Diagnostica Inc.
`(Stanford, USA). Chromogenic substrates (chromozym TH,
`X, U, trypsin, and plasmin) were from Roche Diagnostics
`(Mannheim, Germany); S 2366TM from Chromogenix Instru-
`mentation Laboratory
`(Bubendorf,
`Switzerland);
`and
`PefachromeÒ FXa from Pentapharm (Basel, Switzerland).
`Fluorogenic substrates
`(I-1100 and H-D-Phe-Pro-Arg-6-
`amino-1-naphthalene-benzylsulfonamideÆH2O) were
`from
`Bachem (Bubendorf, Switzerland); Russell’s viper venom
`(RVV) from Pentapharm; NeoplastinÒ Plus (thromboplastin)
`and PTT-Reagent from Roche Diagnostics; hirudin (Reflu-
`danÒ) from Aventis (Strasbourg, France). Xylazine (Rom-
`punÒ) was from Bayer HealthCare, ketamine (KetavetÒ) from
`Pharmacia & Upjohn (Karlsruhe, Germany), and pentobarbi-
`tal-Na (NembutalÒ) from Richter Pharma (Wels, Austria).
`
`In vitro studies
`
`Enzyme assays
`The activity of BAY 59-7939 against
`purified serine proteases was measured using chromogenic or
`fluorogenic substrates in 96-well microtiter plates at 25 °C. The
`enzymes were incubated with BAY 59-7939 or its solvent,
`dimethyl sulfoxide (DMSO), for 10 min. The reactions were
`initiated by the addition of the substrate, and the color or
`fluorescence was monitored continuously at 405 nm using a
`Spectra Rainbow Thermo Reader
`(Tecan, Crailsheim,
`Germany), or at 630/465 nm using a SPECTRAfluor plus
`(Tecan), respectively, for 20 min (if not otherwise stated).
`Enzymatic activity was analyzed in the following buffers (final
`concentrations): human FXa (0.5 nM), rabbit FXa (2 nM), rat
`FXa (10 nM), or urokinase (4 nM) in 50 mM Tris–HCl buffer,
`
`O
`
`N
`
`O
`
`O
`
`N
`
`O
`
`Cl
`
`S
`
`H
`N
`
`O
`
`Fig. 1. Chemical structure of BAY 59-7939 (5-chloro-N-({(5S)-2-oxo-
`3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-
`2-carboxamide).
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`In vitro and in vivo studies of BAY 59-7939 515
`
`pH 8.3, 150 mM NaCl, and 0.1% bovine serum albumin (BSA);
`Pefachrome FXa (50–800 lM) or chromozym U (250 lM) with
`thrombin (0.69 nM), trypsin (2.2 nM), or plasmin (3.2 nM) in
`0.1 lM Tris–HCl, pH 8.0, and 20 mM CaCl2; chromozym TH
`(200 lM), chromozym plasmin (500 lM), or chromozym trypsin
`(500 lM) with FXIa (1 nM) or APC (10 nM) in 50 mM
`phosphate buffer, pH 7.4, 150 mM NaCl; and S 2366 (150 or
`500 lM) with FVIIa (1 nM) and tissue factor (3 nM) in 50 mM
`Tris–HCl buffer, pH 8.0, 100 mM NaCl, 5 mM CaCl2 and 0.3%
`BSA, H-D-Phe-Pro-Arg-6-amino-1-naphthalene-benzylsulfon-
`amideÆH2O (100 lM) and measured for 3 h as described
`previously [6]. The FIXab/FX assay, comprising FIXab
`(8.8 nM) and FX (9.5 nM) in 50 mM Tris–HCl buffer, pH 7.4,
`100 mM NaCl, 5 mM CaCl2 and 0.1% BSA, was started by the
`addition of I-1100 (50 lM), and measured for 60 min.
`The inhibitory constant (Ki) against FXa was calculated
`according to the Cheng–Prusoff equation (Ki ¼ IC50/1 + [S]/
`Km), where [S] is the substrate concentration, and Km is the
`Michaelis–Menten constant. Km was determined from a
`Lineweaver–Burk plot. The IC50 was the amount of inhibitor
`required to diminish the initial velocity of the control by 50%.
`Prothrombinase assay
`The effect of BAY 59-7939 on
`prothrombinase activity was measured via thrombin genera-
`tion, as described previously with some modifications [7].
`Briefly, human FXa (0.025 nM) was incubated in 10 mM
`HEPES buffer, pH 7.4, 2 mM CaCl2 and washed human
`)1) for 10 min at 37 °C. The reaction was
`platelets (1 · 107 mL
`initiated by adding prothrombin (1 lM) and BAY 59-7939 or
`DMSO. After 20 min, 20-lL aliquots were diluted with 160 lL
`buffer, and thrombin activity was measured using 20 lL
`chromozym TH (500 lM).
`FXa activity in plasma Human, rat, or rabbit plasma
`)1), 5 lL BAY
`(45 lL) was mixed with 5 lL hirudin (10 lg mL
`)1;
`59-7939 or DMSO, and 50 lL RVV (human, 0.7 mU mL
`)1), dissolved in 50 lM CaCl2 at 37 °C.
`rat/rabbit, 3.5 mU mL
`Chromozym X (50 lL; 600 lM) was added after 15 min. The
`increase in optical density was measured at 37 °C, as described
`above.
`Coagulation assays Activated partial thromboplastin
`time (aPTT) and prothrombin time (PT) were measured using
`commercially available kits. BAY 59-7939 or DMSO (3 lL)
`were added to 100 lL platelet-poor plasma (PPP) and
`incubated for 10 min at 37 °C. Clotting times were measured
`in a coagulometer (Biomatic 4000; Sarstedt, Nu¨ mbrecht,
`Germany), in accordance with the manufacturer’s instructions
`(final volume 303 lL). Anticoagulant activity was defined as
`the concentration required to double the plasma clotting times
`[CT2 (lM)].
`Plasma preparation Human blood was collected by
`venipuncture from healthy subjects who had not been
`medicated during the last 10 days. Rabbit blood was obtained
`by puncture of the A. carotis, and rat blood was withdrawn
`from the abdominal aorta under anesthesia. Blood was
`collected into plastic tubes containing 1/10 volume of 3.8%
`trisodium citrate. PPP was obtained by immediate centrifuga-
`tion at 2500 g for 10 min at 4 °C, and stored at ) 20 °C.
`
`

`

`each animal 90 and 105 min after administration of oral
`BAY 59-7939 or vehicle. Blood from the incision was
`removed with filter paper every 30 s. The time until the
`bleeding stopped was measured.
`
`Statistical analysis
`
`Student’s t-test (one-way ANOVA) was used for unpaired data,
`with a statistical significance level of P < 0.05. Data are
`expressed as mean ± SEM. IC50 values were calculated using
`Graph Pad Prism, version 3.02 (Graph Pad Software Inc., San
`Diego, CA, USA). ED50 values were calculated by linear
`regression analysis using Excel 97 (MicrosoftÒ).
`
`Results
`
`In vitro studies
`
`Enzyme assays
`BAY 59-7939 inhibited human FXa
`concentration dependently, with a Ki of 0.4 ± 0.02 nM
`(Fig. 2). It
`is a competitive inhibitor of
`the amidolytic
`activity of FXa, as demonstrated by Lineweaver–Burk
`analysis (Fig. 3). At concentrations up to 20 lM, BAY
`59-7939 did not affect related serine proteases; selectivity was
`more than 10 000-fold greater for FXa (Table 1). BAY
`59-7939 showed a similar affinity to purified human and
`rabbit FXa (IC50 0.7 ± 0.01 and 0.8 ± 0.01 nM, respect-
`ively), but was less potent against purified rat FXa (IC50
`3.4 nM; Table 2).
`To determine whether BAY
`Prothrombinase assay
`59-7939 was an effective inhibitor of FXa complexed with
`Factor Va and Ca2+ on a phospholipid membrane, we
`reconstituted the prothrombinase complex on platelets.
`The generation of thrombin was inhibited concentration-
`dependently, with an IC50 of 2.1 ± 0.4 nM, as measured in an
`amidolytic assay (Fig. 2).
`
`FXa
`Prothrombinase
`
`100
`
`80
`
`60
`
`40
`
`20
`
`FXa inhibition (%)
`
`0.1
`
`1
`BAY 59-7939 (nM)
`
`10
`
`100
`
`00
`
`.01
`
`Fig. 2. Effect of BAY 59-7939 on purified human free Factor Xa (FXa)
`using a chromogenic substrate of FXa (d), and on prothrombinase
`activity on platelet surfaces using prothrombin as substrate (measuring
`generated thrombin;
`). Each value represents the mean ± SEM of five
`measurements in triplicate.
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`516 E. Perzborn et al
`
`In vivo studies
`
`Animals and anesthetics
`Fasted, male Wistar rats
`(HsdCpb:WU) were anesthetized by intraperitoneal injection
`)1, respectively); in
`of xylazine and ketamine (12 and 50 mg kg
`)1) was
`the bleeding-time model, pentobarbital-Na (60 mg kg
`used. Fasted, female New Zealand White rabbits (Esd:NZW)
`were anesthetized by intramuscular administration of xylazine
`)1, respectively). All procedures
`and ketamine (5 and 40 mg kg
`were conducted in accordance with the German Animal
`Protection Act (Deutsches Tierschutzgesetz).
`Rat venous stasis model
`Thrombus formation was
`induced in anesthetized rats (n ¼ 10 per dose group) as
`described previously, with minor modifications [8]. The
`abdominal vena cava was exposed and two loose sutures
`(8–10 mm apart) were placed below the left renal venous
`branch. BAY 59-7939 dissolved in polyethylene glycol/H2O/
`glycerol (996 g/100 g/60 g), or vehicle was given by intraven-
`ous (i.v.) bolus injection into a tail vein 15 min before thrombus
`)1) was injected into a
`induction. Thromboplastin (0.5 mg kg
`femoral vein and, after 15 s, the proximal and distal sutures
`were tied. Fifteen minutes later, the ligated segment was
`removed,
`the thrombus withdrawn and weighed. Blood
`samples were obtained by cardiac puncture immediately before
`thrombus removal.
`in rats and rabbits An
`Arteriovenous shunt model
`arteriovenous (AV) shunt in anesthetized rats and rabbits
`was performed as described previously, with minor modifica-
`tions [8–10]. The right common carotid artery and the left
`jugular vein were cannulated with two 100-mm-long, saline-
`filled catheters. In rats (n ¼ 10 per dose group), the polyethy-
`lene catheters (PE-60; Becton Dickinson, Sparks, MD, USA)
`were connected with a 30-mm-long polyethylene tube (PE-160;
`Becton Dickinson)
`containing a rough nylon thread
`(40 · 0.15 mm), folded into a double string. In rabbits (n ¼
`6 per dose group), polyurethane vein catheters (outside
`diameter 2.1 mm; Braun, Melsungen, Germany) were connec-
`ted with a 40-mm-long polyethylene tube (PE-240; Becton
`Dickinson), containing a rough nylon thread (60 · 0.15 mm),
`folded into a double string. BAY 59-7939, dissolved in solutol/
`ethanol/H2O [40%/10%/50% (v/v/v)], or vehicle was given
`orally 90 min before the shunt was opened for 15 min. The
`nylon thread was then withdrawn and weighed. Blood samples
`were withdrawn from the carotid artery just after thrombus
`removal.
`BAY 59-7939 (n ¼ 10 per dose
`Rat tail-bleeding model
`group) or vehicle was given orally 90 min before the tails of
`anesthetized rats were transected 2 mm from the tip and
`vertically immersed in saline at 37 °C. The time until
`continuous blood flow ceased for > 30 s was measured, with
`a maximum observation time of 10 min (longer bleeding times
`were assigned a value of 10 min).
`Rabbit ear-bleeding model
`Ear-bleeding time (EBT)
`was determined in anesthetized rabbits (n ¼ 5 per dose
`group), as described previously [11]. A standardized 3-mm-
`long incision was made at different sites of the right ear in
`
`

`

`In vitro and in vivo studies of BAY 59-7939 517
`
`In vivo studies
`
`Rat venous stasis model
`In a venous thrombosis model,
`thrombi were obtained by employing a combination of stasis
`and injection of thromboplastin. BAY 59-7939, administered
`by i.v. bolus before thrombus induction, reduced thrombus
`)1), inhibited FXa, and prolonged
`formation (ED50 0.1 mg kg
`PT (Fig. 4A–C) dose dependently. PT and FXa were affected
`slightly at the ED50 (1.8-fold increase and 32% inhibition,
`)1 (dose leading to almost complete
`respectively). At 0.3 mg kg
`inhibition of thrombus formation), BAY 59-7939 moderately
`prolonged PT (3.2 ± 0.5-fold) and inhibited FXa activity
`(65 ± 3%).
`Rat AV-shunt model
`Thrombosis was induced by
`exposure of a thrombogenic surface in an AV shunt. To
`evaluate its potential oral efficacy, BAY 59-7939 was given
`orally before blood was circulated in the shunt. BAY 59-7939
`reduced thrombus
`formation dose dependently (ED50
`
`5.0 mg kg)1; Fig. 5A). It also had a dose-dependent effect on
`
`***
`
`**
`
`0.03
`
`0.10
`
`0.30
`
`***
`
`*
`
`**
`
`0.30
`0.10
`0.03
`BAY 59-7939 (mg kg–1) i.v.
`
`**
`
`0.03
`
`0.10
`
`0.30
`
`100A
`
`80
`
`60
`
`40
`
`20
`
`0
`
`formation (%)
`
`Inhibition of thrombus
`
`B
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Inhibition of FXa (%)
`
`0
`
`5 4 3 2 1 0
`
`C
`
`Prolongation of PT (X-fold)
`
`Fig. 4. Effect of BAY 59-7939 in a rat venous stasis model. BAY 59-7939
`or the appropriate vehicle was given by i.v. bolus injection 15 min before
`thrombus induction. (A) Inhibition of thrombus formation. (B) Inhibition
`of endogenous Factor Xa (FXa) after activation by Russell’s viper venom.
`(C) Prolongation of prothrombin time (PT). Blood samples were with-
`drawn by cardiac puncture immediately after removal of the thrombus.
`Results are mean ± SEM of 10 animals. *P < 0.05; **P < 0.01;
`***P < 0.001.
`
`600
`
`500
`
`400
`
`300
`
`BAY 59-7939 0.9 nmol/l
`BAY 59-7939 0.7 nmol/l
`BAY 59-7939 0.5 nmol/l
`BAY 59-7939 0.2 nmol/l
`Control
`
`/OD/min
`
`2001
`
`0.015
`0.010
`0.005
`1/chromogenic peptide (µM)
`
`0.020
`
`100
`
`00
`
`.000
`
`Fig. 3. Kinetic analysis of the inhibitory effect of BAY 59-7939 on Factor
`Xa (FXa). Lineweaver–Burk plots of the activity of 0.5 nM FXa against a
`chromogenic substrate in the absence or presence of 0.2, 0.5, 0.7, and
`0.9 nM BAY 59-7939. Results are mean ± SD.
`
`Table 1 Human protease selectivity profile of BAY 59-7939
`
`Inhibition of
`
`Factor Xa
`Factor VIIa, Factor IXa, Factor XIa, thrombin,
`activated protein C, plasmin,
`urokinase, trypsin
`
`Concentration (nM)
`Ki ¼ 0.4 ± 0.02
`IC50 > 20 000
`
`Table 2 Effect of BAY 59-7939 on inhibition of human, rabbit, and
`rat Factor Xa (FXa) in buffer, plasma FXa, and the concentrations
`required to double the prothrombin time (PT) and activated partial
`thromboplastin time (aPTT) in vitro (CT2)
`
`Species
`
`FXa (buffer)
`IC50 (nM)
`
`FXa (plasma)
`IC50 (nM)
`
`PT
`CT2 (lM)
`
`APTT
`CT2 (lM)
`
`Human
`Rabbit
`Rat
`
`0.7 ± 0.01
`0.8 ± 0.01
`3.4 ± 0.02
`
`21 ± 1.0
`21 ± 2.0
`290 ± 20.0
`
`0.23 ± 0.02
`0.12 ± 0.01
`0.30 ± 0.02
`
`0.69 ± 0.09
`1.97 ± 0.49
`2.09 ± 0.19
`
`Results expressed as mean ± SEM.
`
`In plasma, endogenous human
`FXa activity in plasma
`and rabbit FXa, generated by RVV, was inhibited to a similar
`extent by BAY 59-7939 (IC50 21 ± 0.001 and 21 ± 0.002 nM,
`respectively), whereas 14-fold higher concentrations were
`required in rat plasma (IC50 290 ± 0.02 nM; Table 2).
`Plasma clotting times
`BAY 59-7939 prolonged PT and
`aPTT concentration dependently; the PT assay was more
`sensitive than aPTT. In the PT assay, anticoagulant activity
`was greatest in the rabbit (CT2 0.12 ± 0.01 lM), followed by
`human (CT2 0.23 ± 0.02 lM), and then rat (CT2 0.30 ±
`0.02 lM; Table 2). In the aPTT assay, BAY 59-7939 was most
`potent in human plasma (CT2 0.69 ± 0.09 lM) and less
`effective in rabbit and rat plasma (CT2 1.97 ± 0.49 and
`2.09 ± 0.19 lM, respectively).
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`

`

`**
`
`3.0
`
`***
`
`*
`
`1.0
`
`
`
`***
`
`0.3
`
`**
`
`0.3
`
`1.0
`
`3.0
`
`**
`
`***
`
`3.0
`1.0
`0.3
`BAY 59-7939 (mg kg–1) p.o.
`
`100A
`
`80
`
`60
`
`40
`
`20
`
`0
`
`formation (%)
`
`Inhibition of thrombus
`
`B
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Inhibition of FXa (%)
`
`012345
`
`C
`
`Prolongation of PT (X-fold)
`
`518 E. Perzborn et al
`
`***
`
`10
`
`***
`
`*
`
`2
`
`***
`
`**
`
`3
`
`6
`
`***
`
`***
`
`2
`
`3
`
`6
`
`10
`
`***
`
`***
`
`***
`
`***
`
`10
`6
`3
`2
`BAY 59-7939 (mg kg–1) p.o.
`
`100A
`
`80
`
`60
`
`40
`
`20
`
`0
`
`formation (%)
`
`Inhibition of thrombus
`
`100B
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Inhibition of FXa (%)
`
`0123456
`
`C
`
`Prolongation of PT (X-fold)
`
`Fig. 5. Effect of BAY 59-7939 in a rat arteriovenous (AV)-shunt model.
`BAY 59-7939 or vehicle was given orally 90 min before blood was circu-
`lated in the shunt. (A) Inhibition of thrombus formation. (B) Inhibition of
`endogenous Factor Xa (FXa) after activation by Russell’s viper venom. (C)
`Prolongation of prothrombin time (PT). Blood samples were withdrawn
`from the carotid artery catheter just after thrombus removal. Results are
`mean ± SEM of six animals. *P < 0.05; **P < 0.01; ***P < 0.001.
`
`Fig. 6. Effect of BAY 59-7939 in a rabbit arteriovenous (AV)-shunt
`model. The extracorporeal circulation was opened 90 min after oral
`administration of BAY 59-7939 or vehicle. (A) Inhibition of thrombus
`formation. (B) Inhibition of endogenous Factor Xa (FXa) after activation
`by Russell’s viper venom. (C) Prolongation of prothrombin time (PT).
`Blood samples were withdrawn from the carotid artery catheter just after
`removal of the thrombus. Each value represents the mean ± SEM of six
`animals. *P < 0.05; **P < 0.01; ***P < 0.001.
`
`FXa activity and PT (Fig. 5B,C); at the ED50, BAY 59-7939
`inhibited FXa by 74% and prolonged PT 3.2-fold, as calculated
`from the dose–response curves.
`Rabbit AV-shunt model Oral BAY 59-7939, given before
`opening the shunt, inhibited thrombus formation dose depend-
`)1; Fig. 6A). It also had a dose-dependent
`ently (ED50 0.6 mg kg
`effect on FXa activity and PT (Fig. 6B,C); at the ED50, FXa was
`almost completely inhibited (92%), but PT was prolonged only
`slightly (1.2-fold), as calculated from the dose–response curves.
`Rat tail-bleeding model
`Tail-bleeding time was evalu-
`ated at the antithrombotic-effective oral dose (minimal dose
`preventing thrombus formation in AV shunt model) of
`)1 and multiples thereof. Bleeding time was not
`3 mg kg
`different from baseline at the antithrombotic-effective dose of
`BAY 59-7939 (Table 3). At doses greater than the ED50 (6 and
`)1), there was a dose-dependent, moderate prolonga-
`10 mg kg
`tion of approximately 2- and 3-fold, respectively.
`Rabbit ear-bleeding model
`EBT was assessed at 90 and
`105 min in the same animal after oral administration of BAY
`
`Table 3 Effect of BAY 59-7939 on rat tail-transection bleeding time
`and rabbit ear-bleeding time measured 90 and 105 min after oral
`administration
`
`Prolongation of bleeding time (X-fold)
`
`BAY 59-7939
`)1) p.o.
`(mg kg
`
`Tail-bleeding
`time, rat
`
`Ear-bleeding time, rabbit
`t ¼ 90 min
`t ¼ 105 min
`
`0.3
`1.0
`3.0
`6.0a
`10.0a
`
`ND
`ND
`1.0 ± 0.1
`2.1 ± 0.2*
`2.7 ± 0.2***
`
`1.4 ± 0.7
`1.7 ± 0.9
`1.6 ± 0.8
`ND
`ND
`
`1.0 ± 0.5
`1.1 ± 0.5
`1.3 ± 0.7
`ND
`ND
`
`ND, Not determined. *P < 0.05; ***P < 0.001. Results are expressed
`as mean ± SEM. aBleeding did not stop within the observation time of
`10 min in two of 10 rats.
`
`59-7939. At all doses tested, there was no significant increase of
`EBT, even at multiples of the ED50 in the AV-shunt model
`(Table 3).
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`

`

`Discussion
`
`BAY 59-7939 is a highly potent, competitive, reversible, direct
`FXa inhibitor with a Ki of 0.4 nM for purified human FXa.
`In vivo results indicate that direct inhibition of FXa with
`BAY 59-7939 is a highly effective strategy for the prevention of
`both arterial and venous thrombosis. Bleeding times in rats and
`rabbits were not significantly prolonged at antithrombotic-
`effective doses.
`BAY 59-7939 is highly selective for FXa: its inhibitory effect
`against FXa was > 10 000-fold higher than for other biolo-
`gically relevant serine proteases. In contrast to several other
`FXa inhibitors, BAY 59-7939 does not inhibit trypsin [7,12,13]
`and therefore is not expected to interfere with this digestive
`enzyme in the gastrointestinal tract.
`The potential of BAY 59-7939 to reduce prothrombinase
`activity was evaluated. BAY 59-7939, in the nanomolar range,
`effectively inhibited human FXa bound to the phospholipid
`surface of platelets (IC50 2.1 nM). In the prothrombinase
`complex, the rate of prothrombin conversion is highly accel-
`erated, approximately 280 000-fold [14]. This, in addition to the
`high concentrations of prothrombin used in our study (almost
`physiological concentrations), supports the high affinity of
`BAY 59-7939 for FXa within the prothrombinase complex.
`These data are further supported by recent results showing a
`concentration-dependent reduction in thrombin generation
`triggered by tissue factor in human platelet-rich plasma (IC50
`25 nM) [15]. Interestingly,
`in that study, almost complete
`inhibition of thrombin generation was observed with 80 nM
`BAY 59-7939. In contrast, maximum inhibition of thrombin
`generation of 60% was reported with the pentasaccharide
`fondaparinux, an antithrombin-dependent FXa inhibitor [15].
`These results suggest that BAY 59-7939, a direct FXa inhibitor,
`could access the active site of FXa within the prothrombinase
`complex more effectively than indirect FXa inhibitors.
`Because FXa is at the convergence point of the intrinsic and
`extrinsic coagulation pathways, direct inhibition of FXa by
`BAY 59-7939 is expected to prolong both PT and aPTT. The
`sensitivity of the PT and aPTT assays varies among different
`chemical classes of FXa inhibitors and may reflect differences
`in enzyme kinetics [16]. The PT assay was more sensitive to
`BAY 59-7939 than the aPTT assay. In vivo, we demonstrated a
`dose-dependent prolongation of PT in rats and rabbits. In the
`rabbit AV-shunt model, a strong correlation between PT and
`plasma concentrations of BAY 59-7939 (r ¼ 0.98) was
`observed (data not shown), suggesting that PT can be used
`to characterize the anticoagulant efficacy of BAY 59-7939 in
`humans. In clinical phase I studies, good correlation between
`plasma levels of BAY 59-7939 and prolongation of clotting
`times was observed [17,18].
`An antithrombotic effect was achieved with BAY 59-7939,
`even at low or moderate levels of anticoagulation: 1.8-, 3.2-,
`and 1.2-fold increases in PT at the ED50 in the rat venous
`model and the rat and rabbit AV shunt models, respectively.
`Other animal studies with direct FXa inhibitors have shown a
`moderate increase in PT and aPTT at antithrombotic doses
`
`Ó 2005 International Society on Thrombosis and Haemostasis
`
`In vitro and in vivo studies of BAY 59-7939 519
`
`(for review, see Leadly et al. [16]). These results suggest that the
`antithrombotic efficacy of direct FXa inhibitors can be
`achieved at doses that produced only a low to moderate
`increase in systemic anticoagulation.
`In order to speculate on the effect of BAY 59-7939 in
`humans from the in vivo thrombosis results, it was necessary to
`elucidate the species differences between rats and rabbits.
`Species differences in FXa inhibition in humans, rabbits, and
`rats are well documented. Various compounds show similar
`inhibition of human and rabbit FXa, but are less potent against
`rat FXa [13,19,20]. We showed that the affinity of BAY
`59-7939 for purified human and rabbit FXa was similar, but
`BAY 59-7939 has a 5-fold lower affinity for purified rat FXa.
`Under similar enzyme kinetic conditions, human, rabbit and
`rat plasma anti-FXa activity was significantly lower compared
`with the purified enzymes. This may be explained by non-
`specific plasma–protein binding, which is greatest in rats, and
`lowest in rabbits [5].
`As potent FXa inhibition was demonstrated in both rats
`and rabbits, these animals were chosen for investigation of
`the ability of BAY 59-7939 to prevent thrombus formation in
`established venous and arterial thrombosis. Thrombi in the
`rat stasis model are fibrin-rich, platelet-poor, red thrombi
`(mimicking venous thrombosis), whereas thrombi in the AV
`shunt in rats and rabbits are considered ÔmixedÕ thrombi,
`consisting mainly of platelets and fibrin—mimicking arterial
`thrombosis.
`BAY 59-7939 showed dose-dependent antithrombotic activ-
`ity in both venous and arterial thrombosis, with higher potency
`in the venous model. Compared with the rat arterial throm-
`bosis model, lower inhibition of FXa (32% vs. 74%) and
`PT prolongation (1.8- vs. 3.2-fold) were required to reduce
`thrombus formation by 50% in the rat venous thrombosis
`model. This corresponds to 10-fold lower plasma concentra-
`tions of BAY 59-7939 in the venous stasis model ( 0.1 lM)
`compared with the AV-shunt model ( 1.0 lM), as estimated
`from ex vivo PT and anti-FXa activity. FXa activity and PT
`were measured after thrombus removal (15 min after thrombus
`induction); however, due to the pharmacokinetic profile of
`BAY 59-7939 in rats (t½ 1–2 h), these values reflect conditions
`at thrombus induction. Higher potency in venous than arterial
`thrombosis has also been reported for other FXa inhibitors
`[20–22]. These differences probably reflect the greater platelet
`enrichment in arterial thrombosis.
`In contrast to our results with BAY 59-7939, fondaparinux
`has lower efficacy in a rat AV-shunt model, resulting in maximal
`thrombus reduction of 50% [23]. Oral BAY 59-7939 reduced
`)1. The higher plasma
`thrombus formation by 73% at 10 mg kg
`levels of BAY 59-7939 achieved after i.v. administration reduced
`thrombus formation almost completely (92%; data not shown).
`These data suggest that a direct FXa inhibitor, such as BAY
`59-7939, may be more effective against platelet-rich arterial clots
`than an antithrombin-dependent FXa inhibitor.
`In contrast to the rat model, in which PT was increased
`3.2-fold, there was only a 1.2-fold increase in PT at the ED50 in
`the rabbit AV shunt, which does not support a strong
`
`

`

`520 E. Perzborn et al
`
`correlation between anticoagulation and thrombus reduction.
`However, in both rat and rabbit AV-shunt models, strong
`inhibition of FXa activity (74% and 92%, respectively) was
`required to reduce thrombus formation by 50%. BAY 59-7939
`plasma concentrations at the ED50 were 14-fold lower in the
`rabbit compared with the rat AV-shunt model (0.070 and 1 lM,
`respectively; extrapolated from the dose–response curve at the
`ED50), which corresponded to the 14-fold lower IC50 values for
`the inhibition of FXa in rabbit vs. rat plasma in vitro. These data
`suggest that the antithrombotic efficacy may be predicted more
`precisely by the anti-FXa activity of BAY 59-7939 in plasma,
`rather than by PT.
`The antithrombotic effect of BAY 59-7939 is primarily
`attributed to the inhibition of FXa: it does not directly affect
`platelet aggregation in vitro [24,25]. However, BAY 59-7939
`may decrease platelet activation in vivo indirectly via inhibition
`of thrombin generation, and may thereby affect thrombin-
`induced aggregation [26].
`In order to distinguish between the antithrombotic and
`accompanying antihemostatic effects of BAY 59-7939, we
`investigated bleeding times in well-characterized experimental
`models that measure bleeding from small vessels. At anti-
`thrombotic-effective doses, BAY 59-7939 did not prolong rat
`tail-bleeding time or rabbit EBT. Whether the significant
`increase in tail-bleeding time, but not EBT, observed at doses
`of BAY 59-7939 above the antithrombotic dose, correlates with
`the higher PT values in rats, or depends on the animal model
`used, warrants further evaluation. Although these results may
`not be directly applicable to humans, they may provide an
`estimation of bleeding tendency. In clinical studies in healthy
`male subjects, BAY 59-7939 did not increase bleeding times or
`signs or symptoms of bleeding across a wide range of oral doses
`[17,18].
`BAY 59-7939 is a reversible inhibitor of FXa; therefore, it is
`conceivable that a minimal amount of thrombin could be
`produced even when FXa is strongly inhibited. Numerous
`studies have demonstrated antithrombotic efficacy with FXa
`inhibitors at doses that have little or no effect on template
`bleeding times, tail-bleeding time, or cuticle-bleeding times (for
`review, see Leadley et al. [16]), suggesting a relatively wide
`therapeutic window between antithrombotic efficacy and
`bleeding tendency.
`In summary, BAY 59-7939 is an oral, direct FXa inhibitor
`that inhibited thrombus formation in established rat and rabbit
`thrombosis models at doses that did not significantly increase
`bleeding times. The clinical relevance of these data needs to be
`investigated. Based on its potency, selectivity and efficacy,
`BAY 59-7939 may offer a safe and effective oral therapy for the
`prevention and treatment of arterial and venous thrombosis;
`BAY 59-7939 is currently undergoing clinical evaluation.
`
`Acknowledgements
`
`The authors thank U. Buetehorn for measuring plasma levels,
`and B. Arndt, M. Harwardt, U. Lange, A. Trabandt, and M.
`Voegler for technical assistance.
`
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