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`
`The in vivo pharmacological pro®le of CS-747, a novel antiplatelet
`agent with platelet ADP receptor antagonist properties
`
`1Atsuhiro Sugidachi, *,1Fumitoshi Asai, 1Taketoshi Ogawa, 2Teruhiko Inoue & 1Hiroyuki Koike
`
`1Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo
`140-8710, Japan and 2Ube Research Laboratory, Ube Industries, Ltd., Kogushi 1978-5, Ube City, Yamaguchi 755-8633, Japan
`
`1 CS-747 is a novel antiplatelet agent that generates an active metabolite, R-99224, in vivo. CS-747
`itself was totally inactive in vitro. This study examined in vivo pharmacological pro®les of CS-747
`after single oral administration to rats.
`2 Orally administered CS-747 (0.3 ± 10 mg kg71) partially but signi®cantly decreased [3H]-2-
`methylthio-ADP binding to rat platelets. CS-747 (3 mg kg71, p.o.) treatment neutralized ADP-
`induced decreases of cyclic AMP concentrations induced by prostaglandin E1, suggesting that
`metabolites of CS-747 interfere with Gi-linked P2T receptor.
`3 CS-747 (0.3 and 3 mg kg71, p.o.) markedly inhibited ex vivo washed platelet aggregation in
`response to ADP but not to thrombin. CS-747 also exhibited a marked inhibition of ADP-induced
`ex vivo platelet aggregation in PRP with a rapid onset (50.5 h) and long duration (43 days) of
`action (ED50 at 4 h=1.2 mg kg71).
`4 R-99224 (IC50=45 mM) inhibited in vitro PRP aggregation in a concentration-related manner.
`5 CS-747 prevented thrombus formation in a dose-related manner with an ED50 value of
`(6.2 mg kg71) and ticlopidine
`0.68 mg kg71. CS-747 was more potent
`than clopidogrel
`(4300 mg kg71).
`6 CS-747, clopidogrel, and ticlopidine prolonged the bleeding time. The order of potency of these
`agents in this activity was the same as that in antiaggregatory and antithrombotic activities.
`7 These ®ndings
`indicate that CS-747 is an orally active and a potent antiplatelet and
`antithrombotic agent with a rapid onset and long duration of action, and warrants clinical
`evaluations of the agent.
`British Journal of Pharmacology (2000) 129, 1439 ± 1446
`Keywords: Platelet aggregation; thrombosis; bleeding time; CS-747; platelet ADP receptors; active metabolite; R-99224
`Abbreviations: ADP, adenosine 5'-diphosphate; BSA, bovine serum albumin; EDTA, ethylenediamine tetraacetic acid; HEPES,
`N-2-hydroxylethylpiperazine-N'-2-ethanesulphonic acid; IBMX, 3-isobutyl-1-methylxanthine; 2-MeS-ADP, 2-
`methylthio-adenosine 5'-diphosphate; PGE1, prostaglandin E1; PPP, platelet-poor plasma; PRP, platelet-rich
`plasma
`
`Introduction
`
`The platelet activation and subsequent platelet aggregation
`play an essential role in the pathogenesis of cardiovascular,
`cerebrovascular, and peripheral vascular diseases (Antiplatelet
`Trialists' Collaboration, 1988; SchroÈ r, 1995). Upon vascular
`injury, ADP, a potent platelet activator, is released into the
`bloodstream from damaged cells and activated platelets, which
`in turn acts on other platelets (Gachet et al., 1996). ADP
`induces a number of responses in platelets, including shape
`change from disc to sphere, aggregation, and secretion of
`granule contents (Gachet & Cazenave, 1991). These responses
`are considered to be mediated by ADP's interaction with
`speci®c binding sites on the platelet membrane that have been
`tentatively designated as P2T receptors (Hourani & Hall, 1994;
`Gachet et al., 1996). The P2T receptors are probably composed
`of three distinct receptors, i.e., the P2X1, ligand gated ion
`channel receptors and two distinct G-protein coupled ADP
`receptors (a Gq-linked P2Y1 receptor and a Gi-linked P2T
`receptor distinct from P2Y1) (for review see Kunapuli, 1998a,
`b).
`
`The importance of the Gi-linked P2T receptor in platelet
`aggregation has been demonstrated in recent studies using
`ARL 66096, ticlopidine and clopidogrel (Mills et al., 1992;
`
`*Author for correspondence; E-mail: toasai@shina.sankyo.co.jp
`
`Fagura et al., 1998; Daniel et al., 1998). Thienopyridine
`derivatives such as ticlopidine and clopidogrel are orally active
`inhibitors of ADP-induced platelet aggregation with a slow
`onset and long duration of action (McTavish et al., 1990;
`Coukell & Markham, 1997). Previous studies have demon-
`strated that these agents inhibit the binding of radiolabelled 2-
`methylthio-ADP (2-MeS-ADP), a stable analogue of ADP, to
`human and animal platelets ex vivo. It has been speculated that
`the mechanism of those actions involves the inhibition of the
`Gi-linked P2T receptor (Mills et al., 1992; Savi et al., 1994a;
`Gachet et al., 1995), but the putative active metabolites remain
`to be elucidated (Saltiel & Ward, 1987; Savi et al., 1994b;
`Coukell & Markham, 1997). The clinical ecacy of these
`agents was demonstrated in human clinical trials, e.g. CATS
`(Gent et al., 1989). TASS (Hass et al., 1989) and CAPRIE
`(CAPRIE Steering Committee, 1996). However, the therapeu-
`tic doses of ticlopidine are accompanied by serious side eects
`such as neutropenia and abnormal liver function. Clopidogrel
`has been reported to be as safe as aspirin, but its therapeutic
`bene®t compared to aspirin is marginal (CAPRIE Steering
`Committee, 1996).
`CS-747 (Figure 1) is a novel antiplatelet agent which
`generates an active metabolite, R-99224 (Figure 1), in vivo. In
`the present studies, we investigated the in vivo pharmacological
`pro®le of CS-747 in rats. In addition, we compared the
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`Measurement of cyclic AMP concentration
`
`To indirectly measure adenylyl cyclase activity, cyclic AMP
`levels were determined according to the method of Defreyn et
`al. (1991). PGE1 was used to elevate cyclic AMP levels. A
`mixture of 1.5 ml buer (in mM: Tris 15, NaCl 120, KCl 4,
`MgSO4 1.6, NaH2PO4×2H2O 2, glucose 10, 0.2% BSA, IBMX
`1.5; pH 7.4) and 3 ml PRP (56108 platelets ml71) was
`incubated at 378C for 1 min, and then PGE1 (10 mM) was
`added. ADP (10 mM) or saline was added to the reaction
`mixture at 3 min after PGE1 stimulation. Aliquots in a volume
`of 0.5 ml were taken from the reaction mixture before and 1, 3,
`4, and 6 min after PGE1 stimulation. These samples were
`supplemented with 50 ml of 6N HCl and 50 mM EDTA solution
`and boiled for 5 min. After rapid cooling on ice, the samples
`were centrifuged at 10,0006g for 5 min at 48C. After adding
`CaCO3 (60 mg), the supernatants (300 ml) were incubated at
`room temperature for 15 min and then centrifuged again at
`10,0006g for 5 min at 48C. The ®nal supernatants were
`assayed for cyclic AMP levels using a commercially available
`EIA kit (Amersham, Buckinghamshire, U.K.).
`
`Measurement of ex vivo platelet aggregation and shape
`change
`
`All aggregation studies were performed in Mebanix aggreg-
`ometers (model PAM-6C and PAM-8C, Tokyo, Japan). The
`washed platelets (36108 platelets ml71) or PRP (56108
`platelets ml71) in a volume of 240 ml were incubated at 378C
`for 1.5 min in the aggregometer with continuous stirring at
`1000 r.p.m. and then stimulated with 10 ml of ADP, collagen,
`or thrombin. Changes in light transmission were recorded for
`7 min (ADP) and for 10 min (collagen and thrombin) after
`stimulation with these agents. The extent of aggregation was
`expressed as a percentage of the maximum light transmittance,
`obtained with the suspension buer (washed platelet aggrega-
`tion) or PPP (PRP aggregation). Platelet shape change was
`determined using an aggregometer, PAM-6C according to the
`method by Michal & Motamed (1976), and was estimated
`quantitatively by measuring the maximum height above
`baseline level.
`
`Arterio-venous shunt thrombosis model
`
`The ability of test agents to prevent thrombus formation was
`assessed using an arterio-venous
`shunt model originally
`described by Umetsu & Sanai (1978) with slight modi®cations.
`After anaesthesia with pentobarbital sodium (40 mg kg71,
`i.p.), the jugular vein and contralateral carotid artery of rats
`were exposed and they were cannulated with a polyethylene
`cannula which contains a silk thread in its lumen and is ®lled
`with heparin solution (30 unit kg71). Blood circulation was
`started through the cannula allowing thrombus formation to
`occur on the silk thread. After a 30 min circulation, the
`cannula tube was removed and the silk thread was weighed.
`The weight of thrombus formed on the thread was calculated
`by deducting the wet weight of an equivalent length of the
`standard thread. Drugs or vehicle were administered 4 h
`before starting blood ¯ow.
`
`Bleeding time
`
`The tail transection bleeding time was determined by the
`method of Dejana et al., (1979). The test drugs were orally
`administered 4 h before the tail transection. Under anaesthesia
`(40 mg kg71,
`with pentobarbital
`i.p.),
`the rat
`tail was
`
`Figure 1 Chemical structures of CS-747 and its active metabolite,
`R-99224.
`
`antiplatelet and antithrombotic eects of single oral adminis-
`trations of CS-747 to those of clopidogrel and ticlopidine. The
`pharmacological pro®le of CS-747 revealed in the present
`study shows its potential as an antiplatelet agent.
`
`Methods
`
`Animals
`
`The experimental procedures employed in this study were in
`accordance with the guidelines of the Institutional Animal
`Care and Use Committee at Sankyo Research Laboratories
`(Tokyo, Japan). We used male Sprague-Dawley rats purchased
`from Japan SLC (Shizuoka, Japan). The animals were allowed
`free access to standard rat chow and water.
`
`Preparation of platelet-rich plasma and washed platelets
`
`sodium
`pentobarbital
`anaesthetized with
`Rats were
`(40 mg kg71,
`i.p.). Blood was drawn from the abdominal
`aorta into a plastic syringe containing 3.8% (w v71) trisodium
`citrate (1 : 9 volumes of blood) as an anticoagulant. Platelet-
`rich plasma (PRP) was prepared by centrifugation at 2306g
`for 15 min at room temperature. Platelet-poor plasma (PPP)
`was obtained by centrifugation of the remaining blood at
`20006g for 10 min. Platelet counts in PRP were adjusted to
`56108 ml71 by adding PPP.
`Washed platelets were prepared as described previously
`(Sugidachi et al., 1998) with slight modi®cations. After
`prostaglandin E1 (PGE1) at 100 nM was added to the PRP,
`the mixture was centrifuged at 13006g for 6 min, and the
`resulting platelet pellet was resuspended in a washing buer
`containing (in mM): NaCl 140, KCl 2.7, NaHCO3 12,
`NaH2PO4 0.4, MgCl2 0.8, glucose 5, HEPES 10, and
`3.5 mg ml71 fatty acid-free bovine serum albumin, pH 6.7.
`Finally this platelet suspension was further washed and
`resuspended in the suspension buer (same composition as
`the washing buer, pH 7.4). In studies on washed platelets, the
`platelet suspension was supplemented with 0.068 mg ml71
`human ®brinogen and 1 mM Ca2+.
`
`[3H]-2-MeS-ADP binding
`
`The washed platelet suspension (26108 platelets ml71) was
`incubated with 10 nM [3H]-2-MeS-ADP at room temperature.
`After 30 min, the reaction mixture was layered onto a 20%
`sucrose solution in suspension buer and the bound ligand was
`separated by centrifugation at 10,0006g for 3 min at room
`temperature. After careful aspiration of the supernatant, the
`platelet pellet was dissolved in NCS-II (Amersham, Buck-
`inghamshire, U.K.) and its radioactivity was measured by
`scintillation counting. Speci®c binding was de®ned as the
`dierence between the total binding and nonspeci®c binding
`determined by addition of unlabelled 2-MeS-ADP at 100 mM.
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`1441
`
`transected at 4 mm from the tip by a scalpel, and the tail was
`immediately immersed into warmed (378C) saline until blood
`¯ow stopped. Bleeding time was assessed as the time from the
`tail transection to the termination of blood ¯ow. Bleeding
`times beyond 1800 s were regarded as 1800 s for the purpose of
`statistical analysis. The BT2 was de®ned as the dose that
`doubled the control bleeding time.
`
`Drugs and administration
`
`CS-747 (2-acetoxy-5-(a-cyclopropylcarbonyl-2-¯uorobenzyl)-
`4,5,6,7-tetrahydrothieno [3,2-c]pyridine), R-99224 ((2Z)-[1-[2-
`cyclopropyl-1-(2-¯uorophenyl) - 2 - oxoethyl] -4-mercapto-3-pi-
`peridinylidene] acetic acid, tri¯uoroacetate), and clopidogrel
`hydrogensulphate (SR25990C) were synthesized by Ube
`Industries (Yamaguchi, Japan). Ticlopidine hydrochloride,
`ADP (sodium salt), human ®brinogen, fatty-acid-free bovine
`serum albumin (BSA), 3-isobutyl-1-methylxanthine (IBMX),
`ethylenediaminetetraacetic acid (EDTA), N-2-hydroxylethyl-
`piperazine-N'-2-ethanesulphonic acid (HEPES), apyrase and
`gum arabic were purchased from Sigma (St. Louis, MO,
`U.S.A.). Collagen was from Nycomed (Munich, Germany),
`heparin (sodium salt) was from Fuso (Osaka, Japan). PGE1
`was from Funakoshi (Tokyo, Japan), and bovine thrombin
`was from Mochida (Tokyo, Japan). [3H]-2-MeS-ADP (ammo-
`nium salt, speci®c activity 85 Ci mmol71) was obtained from
`Amersham (Buckinghamshire, U.K.), and 2-MeS-ADP (triso-
`dium salt) was obtained from Research Biochemicals
`International (Natick, MA, U.S.A.).
`CS-747, clopidogrel, and ticlopidine were suspended in 5%
`gum arabic solution at appropriate concentrations and orally
`administered to rats in a volume of 1 ml kg71 body weight. All
`experiments included the vehicle-treated control groups. In in
`vitro study. R-99224 and CS-747 were dissolved in DMSO, and
`added to the PRP; the ®nal concentration of DMSO was 0.4%.
`
`Statistics
`
`Results were expressed as mean+s.e.mean. Approximate
`estimate of the ED50 and BT2 values were calculated from
`linear-regression analysis. Dunnett's test for multiple compar-
`ison was used to determine signi®cance of dierences between
`mean values within groups. A P value of less than 0.05 was
`considered statistically signi®cant.
`
`Results
`
`[3H]-2-MeS-ADP binding
`
`Preliminary studies showed that [3H]-2-MeS-ADP binding to
`rat platelets was time-related and saturable, with an apparent
`equilibrium dissociation constant (Kd) of 2.37+0.27 nM and a
`total number of receptor sites (Bmax) of 180.2+13.8 fmol 108
`platelets71 (n=4). In vitro treatment with CS-747 (100 and
`300 mM) had no eects on [3H]-2-MeS-ADP binding to the
`washed platelets of rats (data not shown).
`Ex vivo eects of CS-747 (0.3 ± 10 mg kg71, p.o.) and
`(3 ± 100 mg kg71, p.o.) on [3H]-2-MeS-ADP
`clopidogrel
`(10 nM) binding to rat platelets were examined 4 h after
`dosing. After 30 min incubation, the speci®c [3H]-2-MeS-ADP
`binding to platelets from vehicle-treated control rats was
`134.4+15.2 fmol 108 platelets71 (n=6). As shown in Figure 2,
`signi®cantly (P50.01) de-
`[3H]-2-MeS-ADP binding was
`creased in platelets from rats given CS-747 (1 ± 10 mg kg71,
`p.o.). But the inhibition remained partial (approximately 43%)
`
`even at a dose of CS-747 as high as 10 mg kg71. Orally
`administered clopidogrel also showed similar partial inhibition
`of [3H]-2-MeS-ADP binding to rat platelets, but clopidogrel
`was about ten times less potent than CS-747 (Figure 2).
`
`Cyclic AMP levels in platelets
`
`Since ADP inhibits adenylyl cyclase via activation of Gi
`protein (Defreyn et al., 1991; Ohlmann et al., 1995), we
`examined the ex vivo eects of CS-747 on ADP-mediated
`suppression of PGE1-induced cyclic AMP elevation. An
`addition of PGE1 (10 mM) produced a progressive increase of
`intraplatelet cyclic AMP levels, indicating the activation of
`adenylyl cyclase. The elevated cyclic AMP levels were
`suppressed by ADP (10 mM) added 3 min after PGE1
`stimulation. As shown in Figure 3, the inhibitory eect of
`ADP (10 mM) on elevated cyclic AMP levels was inhibited
`substantially in platelets from CS-747 (3 mg kg71, p.o.)-
`treated rats. There was no dierence in the basal cyclic AMP
`levels between CS-747-treated (0.39+0.15 nmol 109 plate-
`lets71, n=5) and vehicle-treated platelets (0.37+0.06 nmol
`109 platelets71, n=5).
`
`Ex vivo and in vitro platelet aggregation
`
`Washed platelets To eliminate possible ®brin production
`induced by thrombin, experiments to test agonist selectivity
`
`Figure 2 Ex vivo eects of CS-747 on speci®c [3H]-2-MeS-ADP
`binding to rat washed platelets. CS-747 (0.3 ± 10 mg kg71) and
`clopidogrel (3 ± 100 mg kg71) were orally administered to rats 4 h
`before blood collection. Results are expressed as the mean+s.e.mean
`(n=6). *P<0.05, **P50.01 vs control.
`
`Figure 3 Ex vivo eects of CS-747 on ADP (10 mM)-induced cyclic
`AMP decrease in PGE1 (10 mM)-stimulated rat platelets. CS-747
`(3 mg kg71) was orally administered to rats 4 h before blood
`collection. Results are expressed as the mean+s.e.mean (n=5).
`*P50.05, **P50.01 vs control.
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`
`was performed using washed platelets. A single oral
`administration of CS-747 (0.3 and 3 mg kg71) produced a
`dose-related inhibition of ADP (0.3 ± 30 mM)-induced ex vivo
`aggregation in washed platelets (Figure 4A). Collagen (0.3 ±
`3 mg ml71)-induced aggregation was also inhibited by CS-747
`administration in a dose-related manner (Figure 4B). In
`contrast, CS-747 moderately inhibited platelet aggregation
`induced by a low concentration of thrombin (0.06 unit ml71),
`but not that by high concentrations of thrombin (0.1 and 0.3
`unit ml71) (Figure 4C). Apyrase (0.3 and 3 IU ml71) also
`inhibited platelet aggregation induced by ADP (30 mM) and
`collagen (3 mg ml71), but not by thrombin (0.3 unit ml71)
`(data not shown).
`
`Platelet-rich plasma (PRP) PRP was prepared from rats
`administered with a single dose of CS-747 (0.3 ± 3 mg kg71,
`p.o.). Figure 5 shows the ex vivo eects of CS-747 on a
`concentration-aggregation curve for ADP determined at 4 h
`post-dose. We also investigated the antiaggregatory eects of
`clopidogrel and ticlopidine, two existing platelet ADP receptor
`inhibitors. A single oral administration of clopidogrel (3 ±
`30 mg kg71) caused a dose-related inhibition of ADP-induced
`aggregation, but the inhibitory eects of ticlopidine (30 ±
`300 mg kg71) were minimal. Table 1 summarizes ED50 values
`for CS-747 (1.2 mg kg71), clopidogrel
`(16 mg kg71) and
`ticlopidine (4300 mg kg71) against ADP (3 mM)-induced
`platelet aggregation determined at 4 h post-dose. Typical
`tracing of platelet aggregation induced by ADP (3 mM) in PRP
`from rats treated with CS-747 is shown in Figure 6. As can be
`seen, CS-747 (1 and 3 mg kg71, p.o.) suppressed the maximum
`extent of platelet aggregation in a dose-related manner. Similar
`eects on platelet aggregation were obtained with clopidogrel
`and ticlopidine (data not shown).
`
`Figure 4 Ex vivo eects of single administration of CS-747 on
`washed platelet aggregation induced by ADP (A), collagen (B), and
`thrombin (C) in rats. CS-747 was orally administered once to rats at
`doses of 0.3 and 3 mg kg71. The aggregation was measured 4 h after
`the dosing. Results are presented as the mean+s.e.mean (n=6).
`**P50.01 vs control (vehicle-treated group).
`
`Figure 5 Ex vivo eects of single administration of CS-747 (A),
`clopidogrel
`(B), and ticlopidine (C) on ADP-induced platelet
`aggregation in rats. The aggregation in PRP was measured 4 h after
`the oral dosing. Results are presented as the mean+s.e.mean (n=6).
`*P50.05, **P50.01 vs control (vehicle-treated group).
`
`Table 1 Comparison of antiplatelet, antithrombotic, and antihaemostatic eects of CS-747, clopidogrel and ticlopidine
`
`Agents
`
`CS-747
`Clopidogrel
`Ticlopidine
`
`AV-shunt
`(ED50AV)
`
`0.68
`6.2
`>300
`
`Aggregation
`(ED50AGG)
`
`1.2
`16
`>300
`
`Bleeding
`(BT2)
`
`0.50
`4.6
`130
`
`(ED50AV/BT2)
`
`Ratio
`(ED50AGG/BT2)
`
`1.4
`1.3
`>2.3
`
`2.4
`3.5
`>2.3
`
`Agents were orally administered to rats 4 h before tests. In platelet aggregation (AGG) and the arterio-venous shunt thrombosis model
`(AV), the ED50 values (mg kg71) are doses at which the agents inhibit platelet aggregation and thrombus formation by 50%,
`respectively. BT2 values (mg kg71) are doses at which the agents double the control bleeding time (BT).
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`We also determined that in vitro eect of R-99224, a
`metabolite for CS-747, on ADP (10 mM)-induced platelet
`aggregation in rat PRP. Figure 6 shows typical changes in
`ADP induced aggregation tracing at dierent concentration
`of R-99224. Note that the ex vivo eects of CS-747 and in
`vitro eect of R-99224 on aggregation were similar (Figure
`6). R-99224 (7.53 ± 75.3 mM) produced a concentration-
`related inhibition of platelet aggregation with an IC50 value
`of 44.9 mM.
`In contrast, CS-747 (30 ± 300 mM)
`showed
`minimal eects on in vitro rat platelet aggregation, and the
`maximal inhibition at 300 mM was 9.0+2.7% (the IC50 value
`4300 mM, n=5).
`
`Time course study The time course of the anti-aggregatory
`eect of CS-747 (1 ± 10 mg kg71) after a single oral dosing
`were
`examined in comparison with clopidogrei
`(10 ±
`100 mg kg71). As shown in Figure 7A, a more than 80%
`
`inhibition was observed in CS-747 (10 mg kg71, p.o.)-treated
`rats at 0.5 h after dosing. At this time, clopidogrel exhibited
`minimal inhibition even at the highest dose used (100 mg kg71.
`p.o.) (Figure 7B). The inhibitions of platelet aggregation by
`CS-747 (1 ± 3 mg kg71, p.o.) and clopidogrel (10 ± 30 mg kg71,
`p.o.)
`reached a plateau within 2 and 4 h post-dose,
`respectively. The inhibition of platelet aggregation by CS-747
`was of long duration: a slight but signi®cant inhibition was
`observed at 72 h post-dose in the CS-747-treated rats (Figure
`7A). At 96 h post-dose there were no dierences in platelet
`aggregation between the control and CS-747-treated groups.
`The long duration of antiaggregatory eects was also observed
`in the clopidogrel-treated animals (Figure 7B). The ex vivo
`eects of CS-747 on ADP (0.03 ± 1 mM)-induced platelet shape
`changes were determined at 4 h post-dose. Orally administered
`CS-747 (1 and 3 mg kg71, p.o.) did not signi®cantly aect
`ADP-induced shape changes (data not shown).
`
`Figure 6 Representative aggregometer tracing showing the ex vivo eect of CS-747 (A) and in vitro eect of R-99224 (B) on ADP-
`induced platelet aggregation in rat PRP. CS-747 (1 and 3 mg kg71, p.o.) inhibited ADP (1 ± 30 mM)-induced platelet aggregation.
`R-99224 (7.53 ± 75.3 mM) inhibited 10 mM ADP-induced aggregation.
`
`Figure 7 Time courses of the antiplatelet eects of CS-747 (A) and clopidogrel (B). Ex vivo platelet aggregation in PRP was
`measured at 0.5, 1, 2, 4, 24, 48, 72, and 96 h after the single oral administration of the agents. ADP at a concentration of 3 mM was
`used as an agonist. Results are presented as the mean+s.e.mean (n=6). *P50.05, **P50.01 vs control (vehicle-treated group).
`
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`Antithrombotic eects in a rat arterio-venous shunt
`model
`
`A rat model of arterio-venous shunt (Umetsu & Sanai, 1978;
`Sugidachi et al., 1993) was used to examine the antithrombotic
`eects of CS-747 in comparison with those of clopidogrel and
`ticlopidine (Figure 8). After a 30 min circulation of blood
`through the canula, the weights of the thrombus formed on the
`silk thread were not dierent among the three control groups.
`In the CS-747 (0.1 ± 3 mg kg71, p.o.) groups,
`thrombus
`formation was decreased in a dose-related manner, and the
`ED50 value was estimated as 0.68 mg kg71. Clopidogrel (1 ±
`30 mg kg71, p.o.) also decreased thrombus weight in a dose-
`related manner, with an estimated ED50 value of 6.2 mg kg71.
`Ticlopidine (10 ± 300 mg kg71, p.o.) produced a signi®cant
`decrease of thrombus weight, but the ED50 value was not
`obtained (4300 mg kg71) since the maximum inhibition was
`less than 40%.
`
`Bleeding time
`
`transection bleeding time (Dejana et al., 1979;
`tail
`Rat
`Sugidachi et al., 1993) was measured to determine antihaemo-
`static eects of CS-747 in comparison to those of clopidogrel
`
`and ticlopidine. The bleeding time averaged 446+16 s (n=7)
`in the vehicle-treated control group. All the drugs tested
`prolonged bleeding time in a dose-related manner (Figure 9).
`The BT2 values of CS-747, clopidogrel, and ticlopidine were
`0.50, 4.6, and 130 mg kg71 (p.o.), respectively. To evaluate the
`ecacy and safety of these agents, the ratios of ED50 for
`antiplatelet (ED50AGG) and antithrombotic (ED50AV) potencies
`to BT2 values were calculated for each agent (Table 1).
`
`Discussion
`
`The results of this study demonstrated that CS-747 was a
`the Gi-linked P2T receptor ex vivo.
`potent
`inhibitor of
`Through this work, we showed that the inhibition of Gi-linked
`P2T receptor with CS-747 produced a speci®c and dose-related
`inhibition of ex vivo ADP-induced platelet aggregation which
`developed rapidly and lasted for a long period of time. CS-747
`had potent antithrombotic eects in a rat model of arterial
`thrombosis, and antihaemostatic action in the rat bleeding-
`time assay. In contrast to these potent ex vivo and in vivo
`activities, CS-747 was inactive in vitro. R-99224, an in vivo
`metabolite of CS-747, however, produced a concentration-
`related inhibition of in vitro PRP aggregation. Thus, as in the
`case of clopidogrel and ticlopidine, in vivo activities of CS-747
`are attributed to its active metabolite.
`2-MeS-ADP is a stable agonist for P2Y1 and Gi-linked P2T
`receptor (Kunapuli, 1998a,b). A single oral administration of
`CS-747 produced a signi®cant but partial inhibition (about
`43% inhibition at 10 mg kg71) of [3H]-2-MeS-ADP binding to
`platelets
`in the ex vivo study (Figure 2). Clopidogrel
`(100 mg kg71) also showed similar partial inhibition of [3H]-
`2-MeS-ADP binding. This agreed with the previous ®ndings
`that clopidogrel and ticlopidine produced partial inhibition of
`[3H]-2-MeS-ADP binding to platelets (Savi et al., 1994a;
`Gachet et al., 1995), although the extent of maximal inhibition
`(%) was greater than our present study (approximately 70% vs
`45%). This dierence in maximal
`inhibition between the
`present study and the aforementioned studies may involve the
`use of dierent gender of rats or dierent technique of
`separation of the radioligand.
`Recent studies have demonstrated that the platelet ADP
`receptor is not homogeneous. In addition to the ligand-gated
`ion channel P2X1 receptor (MacKenzie et al., 1996), two
`subclasses of G-protein-coupled ADP receptors have been
`proposed to exist on human platelet membranes: the Gq-linked
`
`Figure 8 Eects of single administration of CS-747 (A), clopidogrel
`(B), and ticlopidine (C) on the arterio-venous shunt thrombosis
`model
`in rats. Blood circulation was started 4 h after the oral
`administration of the agents. Results are presented as the mean+
`s.e.mean (n=6). *P50.05, **P50.01 vs control
`(vehicle-treated
`group).
`
`British Journal of Pharmacology, vol 129 (7)
`
`Figure 9 Eects of single administration of CS-747, clopidogrel, and
`ticlopidine on tail transection bleeding time in rats. Agents were
`orally administered to rats 4 h before the tail transection. Results are
`the mean+s.e.mean (n=7). **P50.01 vs control
`expressed as
`(vehicle-treated group).
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`Ex. 1011, p. 6 of 8
`
`
`
`A. Sugidachi et al
`
`In vivo pharmacology of CS-747
`
`1445
`
`P2Y1 receptor and a Gi-linked P2T receptor (Fagura et al.,
`1998; Daniel et al., 1998; Jantzen et al., 1998). The Gi-linked
`P2T receptor has also been suggested to exist in platelets in the
`rat and the rabbit (Defreyn et al., 1991; Savi et al., 1996).
`Indeed, our present study con®rmed that ADP lowers cyclic
`AMP concentration in rat platelets stimulated with PGE1, an
`activator of adenylyl cyclase, and that CS-747 treatment
`inhibited this eect (Figure 3). In addition, CS-747 did not
`aect P2Y1-mediated platelet shape change. These results
`suggest that metabolites of CS-747 selectively interfere with the
`Gi-linked P2T receptor on rat platelet membranes.
`Treatment with CS-747 inhibited ex vivo washed platelet
`aggregation in response to ADP but not to thrombin (Figure
`4). This is consistent with the hypothesis that the antiag-
`gregative action of CS-747 is due to its speci®c inhibition of the
`Gi-linked P2T receptor rather than its interference with the
`®brinogen receptors. Nonetheless, collagen-induced aggrega-
`tion was inhibited by CS-747, but this may be interpreted on
`the basis of previous studies demonstrating that platelet-
`derived ADP plays a major
`role
`in collagen-induced
`aggregation of rat platelets (Wey et al., 1982; Tschopp &
`Zucker, 1972; Emms & Lewis, 1986). Indeed, apyrase, an ADP
`scavenger, has similar inhibitory pro®le on rat washed platelet
`aggregation. Hence,
`the
`inhibition of
`collagen-induced
`aggregation by CS-747 is most probably attributed to
`inhibition of the eects of ADP released from the dense
`granules of collagen-stimulated platelets.
`The present
`study showed that CS-747 possesses an
`antiaggregatory ecacy approximately ten times more potent
`than that of clopidogrel (Figures 5 and 7). In sharp contrast,
`a single administration of
`ticlopidine
`caused minimal
`inhibition of platelet aggregation. This is in agreement with
`previous studies demonstrating clear antiaggregative eects of
`ticlopidine only after repeated administrations in humans
`(McTavish et al., 1990). In addition, the time course study
`showed that CS-747 (10 mg kg71) had a more rapid onset
`(50.5 h) of antiaggregatory action than clopidogrel (Figure
`7). The rapid onset of CS-747 was observed not only in rats
`but in various species including human (unpublished data).
`Although the precise mechanism responsible for the rapid
`onset of CS-747 remains to be elucidated, one possible
`explanation is that CS-747 may be more rapidly metabolized
`to its active metabolite in vivo. Ticlopidine and clopidogrel,
`which are inactive in vitro, reportedly must undergo hepatic
`metabolism to become active (Savi et al., 1992), and it has
`been suggested that the metabolic activation of clopidogrel
`involves the cytochrome P450-1A subfamily (Savi et al.,
`1994b). Further study is necessary to determine the metabolic
`pathways of CS-747, but the agent is speculated, based on its
`chemical
`structure,
`to generate active metabolites more
`readily than clopidogrel.
`The time course study also showed that CS-747 had a long
`duration of antiaggregatory action. In fact, the durations of
`inhibition of platelet aggregation by CS-747 and clopidogrel in
`this study were comparable to the life span of circulating
`platelets in the rat (Cattaneo et al., 1985; Jackson et al., 1992).
`In addition, the antiaggregatory eects of CS-747 observed in
`washed platelets were not easily reversed by washing of
`platelets. Hence,
`it is likely that CS-747 inhibits platelet
`aggregation in an irreversible manner. Taken together, these
`results suggest that the active metabolite of CS-747 exerts
`antiaggregatory action through an irreversible modi®cation of
`the Gi-linked P2T receptor on the membrane surface. Further
`studies are now underway to investigate the in vitro interaction
`between an active metabolite of CS-747 and platelet ADP
`receptors.
`
`Several lines of evidence suggest that in vivo antiplatelet
`eects of thienopyridine derivatives are due to their active
`metabolite(s) as described above. But the active metabolites
`have not yet been reported so far. In contrast, Weber et al.
`(1999) reported that incubation of washed platelets with
`clopidogrel resulted in inhibition of ADP-induced in vitro
`platelet aggregation without hepatic bioactivation. This report
`also showed that clopidogrel did not show any inhibition of in
`vitro aggregation in PRP. Our present study con®rmed that
`CS-747 did not show any inhibition of ADP-induced PRP
`aggregation. The present results, however, showed that R-
`99224, an in vivo metabolite for CS-747, inhibits ADP-induced
`in vitro platelet aggregation in the presence of plasma (Figure
`6). Our preliminary results also have shown that R-99224
`shows more potent antiaggregatory activity in washed
`platelets. To our knowledge, this is the ®rst report that
`described the active metabolite for thienopyridine derivatives.
`Taken together, the inhibitory eects of CS-747 on platelet
`aggregation are, at least in part, likely to depend on its active
`metabolite, R-99224.
`A rat model of arterio-venous shunt thrombosis has been
`extensively studied and used to examine antithrombotic eects
`of various agents (Marand et al., 1988; Herbert et al., 1996;
`Odawara et al., 1996). In this model, CS-747 showed a potent
`antithrombotic ecacy that exceeds the potencies of clopido-
`grel and ticlopidine. The order of antithrombotic potencies
`among these agents was
`the
`same as
`the order of
`antiaggregatory potencies (Figure 8). In addition, CS-747 was
`devoid of in vitro and ex vivo anticoa
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