`(Sankyo Kenkyusho Nenpo)
`
`Review
`
`CS-747, a New PlateletADP Receptor Antagonist
`
`Fumitoshi AsAJ*, Tomonori KoNSB'1, Atsuhiro SuomAcm:, Toshihiko IKEDA~>~,
`Atsushi SANBUJSSHo<1 and Talcashi HlROTA41
`
`Pharmacology and Molecular Biology Research Laboratories, Product Development
`Labaratories,>l Analytical and Metabolic Research Laboratories, b) and Laboratory Animal
`Sciense and Toxicology Laboratories,<) SANKYO CO., LTD.
`2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, JAPAN
`Planning and Promotion Department,.., SANKYO CO., LTD.
`7-12, Ginza 2-chome, Chuo-ku, Tokyo 104-8113, JAPAN
`
`(Received September 29, 1999)
`
`I. Preface ............................................................................................................. 3
`II. Physicochemical Properties
`1. Physicochemistry. ... .. ....... ...... . ...... .... ... . ..... .. . ... ..... . ..... .... .... ..... .. .... .... . ... .. .. 4
`2. Stability ...................................................................................................... 6
`3. Racemization and Deacetylation Reactions of CS-747 in
`Aqueous Solution...................................................................................... 7
`ill. Pharmacology
`1. Inhibitory Activity of CS-747 on Platelet Aggregation ............................ .lO
`2. Antitbrombotic Effects ofCS-747 ............................................................. 13
`3. Effects of CS-747 on Bleeding Time ......................................................... l6
`4. Mode of Antiplatelet Action of CS-747 .................................................... 17
`5. Conclusion ................................................................................................. 22
`
`MftQ.J!niJ~ADP~~14>~CS-747
`~#~!it·. ~ilt1fti.o, ~ro&:t:. 7t!!m¥:~.,. =:?t-m.IJ'PJ<l, !1lm*'ffl'~
`=~~~~:a ~-~4tm~m-
`"~-~.mt.im~~m. •• ~.fff1tjMlilf~m • .,~~lVfMr • ., lfi~~~~~i!4~
`*The author to whom inquiries concerning this paper should be addressed.
`This paper is available on line at btrp://www .sankyo.co.jp
`
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`Asai, F. et lll.: CS-747, a New Platelet ADP Receptor Antagonist
`
`IV. Absorption, Distribution. Metabolism, and Excretion in Animals
`1. Absorption ................................................................................................. 22
`2. Distribution ................................................................................................ 24
`3. Metabolism ................................................................................................ 25
`4. Excretion .................................................................................................... 27
`5. Conclusion ................................................................................................. 28
`V. Safety Evaluations in Animals
`1. Single-dose and Escalating-dose Sntdies .................................................. 28
`2. Repeated-dose Studies ............................................................................... 29
`3. Mutagenicity .............................................................................................. 31
`4. Antigenicity ............................................................................................... 31
`5. Reproductive Toxicity Studies ................................................................... 32
`6. Comparative Toxicity Studies .................................................................... 33
`7. Conclusion ................................................................................................. 35
`VI. Clinical Evaluation (Clinical pharmacology studies)
`1. Phamtacokinetics ....................................................................................... 35
`2. Phannacodynatnics .................................................................................... 38
`3. Safety and Tolerability ............................................................................... 42
`4. Conclusion ................................................................................................. 42
`
`I.
`
`p
`c
`d
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`J
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`Annu. Rep. Sankyo Rts. Lilb. 51, 1-44 (1999)
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`3
`
`I. Preface
`Platelet activation and subsequent
`platelet aggregation play a central role in
`coronary artery diseases such as myocar(cid:173)
`dial infarction and unstable angina, and
`cerebrovascular diseases such as stroke
`and transient ischemic attack •.z> Although
`platelets are activated by a variety of
`endogenous agonists, ADP is the earliest
`described and the most important platelet
`aggregation agonist. ADP induces primary
`aggregation.3~ In addition, ADP released
`from aggregating platelets induces sec(cid:173)
`ondary aggregation via the feedback
`process amplifying and propagating
`platelet activation induced by other ago(cid:173)
`nists.u' ADP activates platelets via ADP
`receptors, which have been tentatively
`designated as P2T receptorsP> The P2T
`receptors are probably composed of three
`distinct receptors: the P2X,, the ligand(cid:173)
`gated ion chan.nel, and two distinct G-pro(cid:173)
`tein-coupled ADP receptors (a Gq-1in.ked
`P2Y, receptor and a G,-linked P2TAc
`receptor)!'
`The importance of ADP in the patho(cid:173)
`genesis of arterial thrombosis is supported
`by the recent d~monstration that ADP-spe(cid:173)
`cific inhibitors of thienopyridine deriva(cid:173)
`tives, such as ticlopidine and clopidogrel,
`are effective in reducing the risk of athero(cid:173)
`sclerotic vascular disease.9
`0J However,
`·'
`these drugs are by no means satisfactory
`in terms of their adverse effects and effica(cid:173)
`cy. Ticlopidine can have significant
`adverse effects at common dosage levels.111
`Indeed, the use of ticlopidine is discour(cid:173)
`aged in patients with severe organ failure,
`and the blood cell count should be moni-
`
`tored regularly during the first 3 months of
`ticlopidine administration because 1% of
`patients receiving ticlopidine may e>.1Jeri(cid:173)
`encc severe agranulocytosis.91 Clopidogrel
`has been demonstrated to be safer than
`aspirin, the gold standard of antiplatelet
`agents at present. However, the clinical
`efficacy of clopidogrel over aspirin has
`proven just marginal. 10'
`TI1e above-mentioned research and our
`interest to develop a novel antithrombotic
`agent prompted us to search for a more
`active antiplatelet drug with ADP-specific
`action. but with fewer side effects com(cid:173)
`pared to ticlopidine and clopidogrel. In
`1989, we began a collaborative study with
`Ube Industries (Yamaguchi, Japan). We
`performed in vivo screening tests on many
`compounds synthesized in Ube Industries.
`Among these compounds, CS-747 (2-ace>
`toxy-5-(a-cyclopropylcarbonyl-2-fluo(cid:173)
`robenzyl)-4,5,6,7-tetrahydrothieno[3,2-
`c]pyridine) was finally selected (Fig. 1)
`for further development. CS-747 was
`found to be an orally effective platelet
`aggregation inhibitor with high potency,
`fast onset and long dul'ation of action. For
`example, CS-747 was 100-times more
`potent than ticlopidine in various experi(cid:173)
`mental animals. Antiaggregatory effects of
`CS-747 were evident as early as 0.5 hr
`post-dose, and maintained up to 72 hr in
`
`"·Y'(()
`
`0
`
`Fig. 1. Chemical sttucture ofCS-747
`
`• ~ *
`
`-··
`
`0
`
`, '
`
`•
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`Asai, F. eta/.: CS-747, a New P/(IJe/et ADP Receptor Amagolllst
`
`}
`
`~ HS
`
`i'lg. 2. Chemicnl structure of R-99224, an active
`metabolite of CS-747
`
`animals. In contrast to this high pharmaco(cid:173)
`logical potency, CS-747 was revealed to
`exhibit minimum activity in general phar(cid:173)
`macological and toxicological studies.
`The metabolism and pharmacokinetics
`of CS-747 were examined in various ani(cid:173)
`mals. These studies revealed that CS-747
`was rapidly and well absorbed after oral
`administration and extensively metabo(cid:173)
`lized in the liver, most of it being excreted
`in the feces. CS-747, along with ticlopi(cid:173)
`dine and clopidogrer,m was inactive in
`vitro, whereas these agents are all very
`active after oral administration. This raises
`the possibility of in vivo generation of
`active metabolites of thienopyridines, but
`they remain to be identified. R-99224 iso(cid:173)
`lated from the incubation mixture of CS-
`747 and rat liver microsomes was found to
`show potent in vitro antiaggregatory activ(cid:173)
`ity. Its chemical structure (Fig. 2) was elu(cid:173)
`cidated in 1994; R-99224 was detected in
`the blood of various animals that received
`oral administration of CS-747. The phar(cid:173)
`macological profile of R-99224 matches
`that of CS-747, except for the high oral
`absorption of CS-747, with potent, selec(cid:173)
`tive and irreversible in vitro antagonistic
`activities against P2T AC receptors. The
`results suggest that R-99224 is an active
`metabolite of CS-747 and contlibutes to
`
`the pharmacological activities of CS-747
`after its oral administration.
`These pre-clinical studies on CS-747
`have suggested its potential as a novel
`antiplatclct agent with a wide safety mar(cid:173)
`gin. In particular. comparative toxicologic
`studies with clopidogrel revealed that CS-
`747 was less toxic than clopidogrel. Thus,
`Phase I clinical studies were struted in the
`U.K. in 1997. Single oral administration
`of CS-747 at 30 mg produced potent inhi(cid:173)
`bition of platelet aggregation with a fast
`onset and a long duration in healthy vol(cid:173)
`unteers. This is noteworthy, since maximal
`effects were observed only after multiple
`administrations of ticlopidine (500
`mg/day)tl and clopidogrel (75 mg/day). 131
`A multiple dose of CS-747 administered
`once daily for 10 days was tolerable, and
`produced a significant and steady-state
`inhibition of platelet aggregation. There
`were no abnonnal changes in the safety
`parameters in any dose groups.
`In this review, we will examine the pre(cid:173)
`clinical and clinical evidence that suggests
`the clinical usefulness of CS -747, a novel
`antiplatclct agent with P2T..,c antagonistic
`action, for patients with occlusive vascular
`diseases.
`
`(Fumitoshi Asai)
`
`II. Physicochemical Properties
`The physicochemical properties of CS-
`747, including the appearance, melting
`point, solubility, partition coefficient, ele(cid:173)
`mental analysis, spectroscopic properties,
`and acid dissociation constant, were inves(cid:173)
`tigated. The stability of CS-747 in
`
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`Sl
`
`7
`
`7
`
`c
`
`I ·-,,
`
`e
`n
`t-
`;t
`t-
`tl
`c
`0
`3)
`
`d
`d
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`:1
`c
`r
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`
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`
`A1111u. Rep. Sonkyo Res. Lab. 51, 1-44 (1999)
`
`5
`
`buffered solutions, and in the solid state,
`was also examined. Th confirm the ratio~
`nale for developing CS-747 as a racemic
`mixture, racemization and deacetylation
`reactions of CS-747 in aqueous solutions
`were studied.
`
`1. Physicochemistry
`1) Appearance
`CS-747 occurs as a white to pale-yellow
`crystalline powder containing masses.
`
`2) Melting point
`The melting point of CS-747 is
`121 °C-122°C.
`
`3) Solubility and partition coefficient
`Solubility data of CS-747 in various
`orgamc solvents and aqueous solutions at
`20°C are given in Table 1 and Table 2,
`respectively. CS-747 is freely soluble in
`benzene, chloroform, and acetone, and
`slightly soluble in methanol and ethanol;
`however, it is practically insoluble in
`water and neutral buffered splution. With
`decreasing pH, CS-747 becomes slightly
`soluble. It is assumed that the solubility of
`CS-747 in aqueous solution depends on its
`dissociation constant (pKa) of 5.40 (see
`
`Table 1. Solubility of CS-747 in organic solvents
`
`Sol vent (20°C)
`Berv~ne
`Chloroform
`Acetone
`Ethyl acetate
`Acetonitrile
`Methanol
`Ethanol
`n-Hcxane
`
`Solubility (mglmL)
`>100
`>100
`>100
`82.5
`67.0
`9.01
`5.91
`0.65
`
`Table Z. Solubility of CS-747 in aqueous
`soluuons
`
`pH(200C}
`3.57
`4.38
`4.85
`5.25
`5.47
`6.00
`7.02
`7.12
`
`Solubility {J.lg/mL)
`59.8
`37.5
`14.2
`5.54
`5.82
`4.01
`0.08
`0.15
`
`Table 3. Blemental analysis (Batch No. 5)
`c
`S
`F
`N
`H
`Element
`64.40 5.57 3.75 5.23 8.78
`Found(%)
`Calcu1ated (%) 64.33 5.40 3.75 5.09 8.59
`
`below 6) Dissociation constant). The parti(cid:173)
`tion coefficient (Log P) of CS-747 was
`determined to be 3.7 between n-octanol
`and phosphate buffer (pH 7.0) using the
`flask-shaking method.
`
`4) Elemental analysis
`The results of the elemental analysis are
`presented in Table 3. The data conformed
`to the calculated values (C, 64.33; H, 5.40;
`N, 3.75; F, 5.09; S, 8.59) .
`
`5) Spectrophotometric analysis
`The UV spectrum of CS-747 in ethanol
`is presented in Fig. 1. It exhibited no
`absorption maximum in the range of 200
`nm to 350 nm, and showed three shoul(cid:173)
`ders at around 218,238, and 258 run. Tbe
`corresponding molar extinction coeffi(cid:173)
`cients of these peaks were 1.28 x 10', 7.68
`x 10', and 6.44 x 1Qlcm· 1mol-'L, respec(cid:173)
`tively.
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`Asai, F. et aL: CS-747. a New Platelet ADP Receptor Antagonist
`
`Table 4. Tho m band assignment~ forCS-747
`
`Wave number (cm-l)
`Assignment
`30J3, 3085 A!ornalic C-H stretchinz
`2718-2944" Aliphatic C-H stretching
`C=O stzetcbing of ester
`1757
`0:0 stretching of ketone
`1703
`1489
`C-H deformation
`1193
`C-O asymmentrical stretching
`Cli out-of-plane defonnation
`758
`of the disubstiluted benzene
`ring
`
`Thblc s. Stability ofCS-747 in aqueous solutions
`ar various pH levels
`
`p1{
`1 hour 3 hours 6 hours 24 hours
`1.2 (J.P. I) 76.6
`74.9
`11.5
`33.7
`2.0
`76.7
`73.4
`71.0
`59.6
`4.0
`95.1
`94.7
`91.7
`77.6
`6.8 (J.P.2)
`98.S
`88.1
`81.4
`49.0
`8.0
`93.3
`80.7
`67.0
`29.0
`The concentration of CS-747 wa~ J 00 Jtg/mL in 20%
`CH3CN/buffu.
`
`J
`
`01~~~~~~~~~=:~~~~
`320
`200
`240
`280
`350
`Wavelength (nm)
`
`Flg. 1. UV spectrum ofCS-747 in ethanol
`
`lOS
`
`15
`
`01+--r~~~~--~~~--~~
`4000
`3200
`2400
`1600
`800 400
`Wave number(cm~')
`Fig. 2. IR spectrum of CS-74 7
`
`The IR spectrum of CS~ 747, determined
`by the potassium bromide disc method, is
`shown in Fig. 2. The assignments of the
`characteristic absorption bands in the IR
`spectrum are listed in Table 4.
`
`6) Dissociation constant
`The pKa for CS-747, determined spec(cid:173)
`trophotometricaUy, was found to be 5.40 ±
`0.05 at 25°C. This pKa is assumed to cor(cid:173)
`respond to the protonation of the tertiary
`amine stmcturc in the piperidine moiety.
`
`2. Stability
`The stability of CS-747 in aqueous
`solutions was examined under various
`conditions from pH 1.2 to pH 8.0 at 37°C
`over a period of 24 hr. The results are
`summarized in Table 5. It was found that
`CS-747 is rather stable at pH 4, giving
`78% as the residuru percentage after 24 he
`but it was unstabJe in solutions having a
`pH lower or higher thao 4.
`Long-term and accelerated storage tests
`of CS-747 in the solid state were per(cid:173)
`formed, as shown in Table 6. CS-747 was
`confirm~d to be stable at 25°C-60% RH
`(relative humidity) for 18 months, and at
`40°C for 12 months when stored in closed
`glass bottles.
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`Annu. Rtp. Sankyo Res. Lab. Sl, 1-44 (1999)
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`7
`
`Table 6. Stability of CS.747 in tile solid state
`
`Stocage condit.ions
`2S0 c-609& RH
`40°C
`
`Initial
`101.0
`100.1
`
`1 month
`100.5
`99.8
`
`3months
`100.7
`100.2
`
`6month.~ 9 months
`100.6
`100.6
`100.6
`100.4
`
`12months
`100.7
`100.2
`
`lSmonths
`100.0
`
`Table 7. Photostability of CS-747 exposed to a cool white fluorescent lamp
`
`Test
`Assay(%)
`
`Initial
`100.0
`
`600000
`99.3
`
`Lux-hours
`1200000
`98.9
`
`2400000
`98.3
`
`Reference
`99.8
`
`Table 8. Photostability of CS-747 exposed to a near-ultraviolet lamp
`
`Test
`Assay (9'0)
`
`Initlal
`100.0
`
`120
`99.8
`
`240
`99.6
`
`480
`99.0
`
`Reference
`99.5
`
`The photostability of CS-747 was tested
`under light exposure of a cool white fluo(cid:173)
`rescent lamp and a near-ultraviolet lamp.
`The results are presented in Table 7 and
`Table 8, respectively. It was found that
`CS-747 is stable in both light exposures.
`
`~0
`
`R Conn ofCS-747
`
`3. Racemization and Deacetylation
`Reactions of CS-747 in Aqueous
`Solution
`CS-747 has one cbiral center, and thus
`consists of two optical isomers, as shown
`in Fig. 3. The kinetic properties of racem(cid:173)
`ization and deacetylation of each optical
`isomer were studied in aqueous solutions
`at various pHs at 37°C. The enantiomer
`ratios of CS-747 as a function of time,
`starting from the R- or S-form in pH 7.4
`aqueous solution at 37°C, are shown in
`Fig. 4. The kinetic plots for the racemiza- .
`tion are presented in Fig. 5. The rate con-
`
`S Conn ofCS.747
`
`Fig. 3. Chemical structures of optical isomers of
`CS-747
`
`stants and half-lives (T11J of the racemiza(cid:173)
`tion are swnmarized in Table 9. The pH
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`8
`
`) A
`
`100
`
`It
`
`80
`
`60
`
`40
`
`20
`
`~
`
`0
`
`0
`
`) B
`
`100
`
`80
`
`60
`
`40
`
`*-
`
`I .I
`
`20
`
`0 • 0
`
`50
`
`150
`
`200
`
`100
`Tim.e(m.i.n)
`Fig. 4. Racemi%ntion of the R form and S form (A
`and B, respectively) in pH 7.4 aqueous
`solution at37°C
`
`dependence of the T 112 in the racemization
`of each optical isomer of CS-747ls shown
`in Pig. 6. The racemization occurred
`rapidly, with a TIll of about 1 hr at pH of
`no lower than 4.
`The deacetylation of CS-747 was also
`found to proceed similarly under the same
`
`conditions. The kinetic plots of the
`dcacetyJation of each optical isomer in pH
`7.4 aqueous solution at 37°C ace shown in
`Fig. 7. The rate constants and TIn values
`for the deacetylation are summarized in
`Thble 10. The pH dependence ofT In in the
`deacetylation of each optical isomer of
`CS-747 is plotted in Fig. 8. The deacetyJa-
`
`A.sai, F. et al.: CS-747, aN~ Platelel ADP Receptor Antagonist
`
`r:
`
`R(%)
`S(%)
`
`4
`
`•
`
`•
`
`•
`•
`
`• •
`
`50
`
`100
`Time(min)
`
`' 150
`
`200
`
`f:
`
`R(%)
`8(96)
`
`I
`
`4
`
`•
`
`•
`
`•
`•
`
`•
`•
`
`I• (R-$)/(R+S)
`~ 1---t---..
`
`...........
`
`A)
`
`100
`
`10
`~
`~ 1
`5 .....
`~ O.l
`~
`
`- 0
`
`0.01
`
`0.001
`
`
`
`50
`
`100
`Tlme(m.in)
`
`150
`
`200
`
`I· (S-R)/CR+S) I
`
`---1---. t---t---..
`
`B)
`
`100
`
`10
`~
`~ 1
`~ .....
`~Ol
`I
`~
`
`O.Ql
`
`0.001
`
`0
`
`50
`
`100
`Time (min)
`Fig. S. Kinetic plots for the racemization of the R
`form and S form (A and B, respectively) in
`pH 7.4 aqueous solution at 37°C
`
`150
`
`200
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`AMJL Rep. San kyo Res. Lab. 51, 1-44 (1999)
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`9
`
`- - R-2-oxo
`--&- s-ZGw
`
`100
`
`10
`
`l
`
`3
`
`5
`pH
`
`7
`
`9
`
`3
`
`5
`pH
`
`7
`
`9
`
`Fig. 6. T w for racemization of each optical isomer
`of CS-747 at 37"C with respect to the pH.
`Error bars indicate standard deviation
`(mean± S.D., n=3).
`
`A)
`LOXIO"
`
`• S+R
`
`jL_.
`
`--
`
`50
`
`100
`
`150
`
`200
`
`LOX 10'
`0
`
`B)
`LOXIO"
`
`-..
`
`lOXIO'
`0
`
`50
`
`100
`Time (min)
`
`)50
`
`200
`
`Flg. 7. Dcacetylalion of the R form and S form (A
`nnd B, respectively) in pH 7.4 aqueous
`solution at 37°C
`
`Fig. 8. T111 for deacctylation of eacb optical iso(cid:173)
`mer of CS-747 at 37"C with respect to the
`pH. Brror bars indicate standard deviation
`(mean± S.D., n=3).
`
`tion reaction was found to be relatively
`slow, with a T 112 of over 3.9 hr, compared
`with the racemization.
`It is predicted that each optical isomer
`of CS-747 will be stable under the acidic
`conditions of the stomach after oral
`administration. However, after reaching
`the intestinal tract, which is maintained at
`a neutral pH, the compound will rapidly
`undergo racemization before absorption.
`These data support the concept that the
`development of CS-747 as a single enan(cid:173)
`tiomer has no rationale. The deacetylation
`reaction gives the 2-oxo form, which is the
`relatively stable intermediate leading to
`the pharmacologically active metabolite,
`as will be described later, and the reaction
`is considered to proceed enzymatically in
`the in vivo situation, rather than the non(cid:173)
`enzymatic mechanism shown in this chap(cid:173)
`ter.
`
`(Tomonori Konse)
`
`R
`n
`
`e
`f
`n
`s
`n
`
`f
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 9 of 44
`
`
`
`10
`
`Asai, F. eJ aL; CS-747, a Ntw Platelet ADP Receptor Antagonist
`
`Table 9. Kinetic paramet~rs of racemization of CS-747 optical isomers
`
`R~S
`
`s~R
`
`pH
`1.2 (J.P. I)
`2.0
`3.0
`4.0
`7.4
`9.0
`
`k (xl0-2h-1)
`1.6± 0.4
`1.4± 0.7
`0.9± 0.4
`25.0± 2.7
`68.3±24.6
`47.1 ± 12.6
`
`Tll2 (h)
`54.7:1:23.6
`62.0±26.6
`83.0±22.3
`2.8± 0.2
`1.1 ± 0.3
`1.5± 0.3
`
`k (xl0-2h- 1)
`2.1± 1.3
`2.8± 3.3
`1.3± 0.1
`29.7± 1.2
`76.0±21.0
`37.5± 9.7
`
`Tuz (h)
`43.1±20.0
`56.6±30.7
`53.5± 2.8
`2.3 ± 0.1
`1.0± 0.2
`2.0± 0.5
`
`Table 10. Kinetic parameters of deacetylation ofCS-747 optical isomers
`
`pH
`1.2 (J.P.1)
`2.0
`3.0
`4.0
`7.4
`9.0
`
`R ~ 2-oxo form
`k (xl0-2h-1)
`6.3± 0.7
`3.3± 0.9
`3.9± 0.6
`4.S± 1.8
`17.7± J.2
`374.0±42.2
`
`Ttn(h)
`11.1 ± 1.1
`33.4± 6.9
`17.9± 6.5
`17.1 ± 0.02
`3.9± 0.3
`0.2± 0.02
`
`S-? 2-oxo fonn
`k (xlo-2h-1)
`T,n(h)
`. 8.6± 1.5
`8.2± 1.3
`12.8 ± 1.7
`5.5± 0.7
`2.4± O.o7
`28.8± 0.8
`3.3 ± 0.7
`21.4± 5.0
`18.5± 8.2
`4.2± 1.6
`332.0 ±43.5
`0.2± 0.03
`
`ill. Pharmacology
`1. Inhibitory Activity of CS-747 on
`Platelet Aggregation
`1) Single oral administration
`Tlllte courses of the ex vivo effects of
`CS-747 {1-10 mglkg, p.o.) and clopido(cid:173)
`grel (10-100 mg/kg, p.o.) on ADP (3
`~)-induced platelet'aggregation in
`platelet-rich plasma (PRP) were examined
`after a single administration of each agent
`to SD rats (Figs. 1 and 2). At 0.5 hr after
`dosing, more than 50% inhibition was
`observed in CS-747-treated SD rats, while
`clopidogrel had minimal effect, suggesting
`an early onset of the antiplatelet action of
`CS-747 (Fig. 1). Although the precise
`
`mechanism responsible for the rapid onset
`of CS-747's effect remains to be elucidat(cid:173)
`ed, one possible explanation is that CS-
`747 may be more rapidly metabolized to
`its active metabolite in vivo (see ADME).
`Maximum effects of both agents were
`observed 4 hr after the dosing in SD rats,
`but the effect of CS-747 was more potent
`than that of clopidogrel (Figs. 1 and 2).
`The inhibitory effects of CS-747 (1 and 3
`mg/kg) and clopidogrel (10 and 30 mg/kg)
`were long-lasting, and were still signifi(cid:173)
`cant 72 hr after dosing (Fig. 2). These
`similar effects of both agents had disap(cid:173)
`peared by 96 hr after dosing. The dura(cid:173)
`tions of inhibition of platelet aggregation
`by CS-747 and clopidogrel were compara(cid:173)
`ble to the life span of circulating platelets
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 10 of 44
`
`
`
`Annu. Rtp. Sankyo Res. Lab. 51, 1-44 (1999)
`
`11
`
`2) Three-day orahdministration
`CS-747, clopidogrel, and ticlopidine
`were orally administered to SD rats once a
`day for 3 days. Blood was collected 4 hr
`after the final administration of the agents,
`and effects of the agents on ADP (0.3-30
`JJ.M)-induced pl atelet aggregation were
`
`measured using PRP. CS- 747 (0.3-3
`mglkg/day, p.o.) inhibited platelet aggre(cid:173)
`gation in a dose-dependent manner (Fig.
`3). Clopidogrel (3-30 mg/kg/day, p.o.)
`also inhibited platelet aggregation, but the
`effect of clopidogrel was 10-fold less
`potent than that of CS-7 4 7. Ticlopidine
`(30-300 mglkglday, p.o.) showed minimal
`effect. CS-747, given orally, .also showed
`
`(A) CS-747
`100 ..-- - - -- -------,
`
`-o- 1 mg.ll(g(p,<>,)
`(A) CS-747
`-3
`100~------------------~
`!!< ..
`
`....... 80
`~
`c: 60
`,g
`;§ 40
`.s::.
`c: 20
`
`0
`
`....,80
`*' -;; 60
`0
`~ 40
`:2
`..: 20
`
`4
`3
`2
`0 0.5 1
`Time after single dosing (hr)
`
`96
`72
`48
`24
`04
`Time after single dosing (hr)
`
`(B) Clopidogrel =!:!~(p.<>J -o-100
`100..-------------------~
`
`-o- 10mg/kg(p.o.)
`(B) Clopidogrel
`-30
`100~------------------~
`
`....... 80
`
`e 60
`c:
`0 ;e 40
`.c -= 20
`
`,9
`
`0
`
`..... 80
`
`c. 60
`5 :::>
`;§ 40
`.c -= 20
`0
`
`3
`2
`0 0.5 1
`4
`Time after single dosing (hr)
`
`98
`72
`46
`24
`04
`Time after single dosing (hr)
`
`Fig. 1. EffeciS of single oral administrations of
`CS-747 (A) and clopidogrel (B) on platelet
`aggregation up to 4 hr post-dose in rats.
`ADP at a concentralion of 3 J1M was used
`as the agonist in PRP aggregation. ResuiiS
`are represented as the mean± S.E.M.
`(n=6). ••p<O.Ol vs. control (vehicle-treat·
`edgroup) •
`
`Fig. 2. Effects of single oral administrations of
`CS-747 (A) and clopidogtcl (B) on platelet
`aggregation 4-96 ht post-dose in rats. ADP
`at a concentration of 3 J.LM was used as the
`agonist in PRP aggregation. Results are
`represented as the mean ± S.E.M.
`(n=5.,.6). •p<O.OS. ·~<O.Ol vs. control
`(vehicle-treated group}.
`
`. '
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 11 of 44
`
`
`
`12
`
`Asai, F. et al.: CS-747, a New Platelet ADP Receptor Antagonist
`
`d
`el
`rt
`Ct
`p
`tl
`a
`ll
`c
`s
`a
`d
`l•
`a
`
`2
`1
`
`more potent inhibition of platelet aggrega(cid:173)
`tion than clopidogrel and ticlopidine in
`Hartley guinea pigs, beagle dogs, and
`cynomolgus monkeys (data not shown).
`These results clearly show that CS-747 is
`a more potent antiplatelet agent in com(cid:173)
`parison with clopidogrel and ticlopidine.
`
`3) Two-week oral administration
`Inhibitory effects of CS-747, adminis(cid:173)
`tered repeatedly over 2 weeks, on ex vivo
`platelet aggregation in cynomolgus mon(cid:173)
`keys were investigated. ADP (10 J.LM)(cid:173)
`induced platelet aggregation was mea(cid:173)
`sured on Days 0 (pre), 1, 3, 5, 7, 14, 17,
`21, and 28 during the experimental period.
`CS-747 (0.1 and 0.3 mglkg/day, p.o.)
`inhibited platelet aggregation in a dose-
`
`-o- Control
`- - O.t.g/day(p,o.J
`00.-----~~--------------~
`14 days
`
`-o-l).,'j
`
`0
`
`21
`14
`7
`Experimental day
`
`28
`
`F.ig. 4. Effects of repeated administrntions of CS-
`747 on ADP (lO J.LM)-induced platelet
`aggregation in cynomolgus monkeys. cs.
`747 was orally admiaistercd to monkeys
`once a day for 14 days. Platelet aggrega(cid:173)
`tion was measured 4 hr after dosiag on
`each day. Data are repre.~cnted as lhe mean
`± S.B.M. (n=5). *p<O.OS, **p<O.Ol vs.
`control (veb.Jcle-treated group) •
`
`(A) CS-747
`
`-o - '
`-<>-Cor.wl
`-o.JIIUJ(kg(p,o.J -3
`
`60
`
`£:
`c: 40
`,g
`~
`S! 20
`"' ~
`
`0
`
`0.3
`
`10
`3
`1
`ADP(!IM)
`
`30
`
`-o- 10
`-o- Conlrol
`-
`ao
`3n:!lllcg<,..,.> -
`
`(B) Clopldogrel
`60
`
`...... e
`c: 40
`,g
`"' ~ 20
`~
`
`0
`
`0.3
`
`10 30
`3
`1
`AOP(pM}
`
`-o-100
`--<>-Control
`(C) Tlclopidine
`-
`30mg/lcg(p.oJ- 300
`60;-----------------~
`
`0.3
`
`10
`3
`1
`ADP(J.IM)
`
`30
`
`Fig. 3. Ex vivo effects of 3-day repeated adminis(cid:173)
`trations of CS-747 (A), clopidogrel (B),
`and ticlopidine (C) on ADP-ioduced
`platelet aggregation in rats. Agents were
`orally admJnistered.to ~ts once a day for 3
`daY,s. The aggregation in ·PRP was-mea(cid:173)
`sured 4 hr after the fmal dosing. Results
`are represented 'as the mean± S.E.M.
`(n=6). *p<O.OS, up<O.Ol v~. control
`(vehicle-treated group).
`
`.. • ' ., : .. :: .. · .. '~~-y~;:~~~;;;~~ff~i~~q~_:-;,i}_.)~-~
`:_,_,_ .. _ .. : ________ : ........ ___ ;. ___ ._. -· -·· . ,..:._....;.:.;......;
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 12 of 44
`
`
`
`fOnist
`
`-ega(cid:173)
`.e in
`and
`wn).
`~7 is
`om(cid:173)
`.e.
`
`.Iris(cid:173)
`vivo
`100-
`M)-
`\ea-
`17,
`iod.
`.o.)
`>se-
`
`.s
`
`:s(cid:173)
`~Iec
`:::S(cid:173)
`eys
`
`on
`
`VS.
`
`Annu. Rep. Sankyo Res. Lab. 51, l-44 (1999)
`
`13
`
`dependent manner (Fig. 4). Inhibitory
`effects of CS-747 (0.1 and 0.3 mg/kg/day)
`reached a plateau from Day 3 to 5. After
`completion of the CS-747 dosing period,
`platelet aggregation gradually returned to
`the pre-treatment level on Day 21 (7 days
`after the :final dosing), suggesting long(cid:173)
`lasting action of CS-747. In beagle dogs,
`CS-747 (0.03-0.3 mg/kg/day, p.o.) also
`showed a similar time course of
`antiplatelet action (data not shown). These
`data indicate that CS-747 has potent and
`long-lasting inhibitory effects on platelet
`aggregation.
`
`2. Antithrombotic Effects of CS-747
`1) Arteriovenous shunt thrombosis
`model
`Efficacy of CS-747 in an arteriovenous
`shunt thrombosis model, originally de(cid:173)
`scribed by Umetsu and Sanai,'6l was deter(cid:173)
`mined in SD rats (Fig. 5). Thrombus for(cid:173)
`mation was assessed by weighing the wet
`weight of the thrombus formed in the
`shunt tube. CS-747 and clopidogrel were
`orally administered once to SD rats, while
`ticlopidine was orally administered once
`daily for 3 days. Blood circulation was re(cid:173)
`started 4 hr after the single or the final
`dosing. In the control groups, the throm(cid:173)
`bus weights after 30 min of blood circula(cid:173)
`tion were 48.8-55.1 mg (n=7-10). CS-747
`(0.1-3 mglkg, p.o.) prevented thro~bus
`formation in a dose-dependent manner,
`with an BD.!Q value of 0.65 mglkg. In con(cid:173)
`trast, clopidogrel and ticlopidine were less
`potent than CS-747 in this model; the BD.!Q
`values for clopidogrel and ticlopidine
`were 7.0 mg/kg and >300 mg/kg/day,
`respectively.
`
`(A)CS-747
`
`0
`
`O.:l
`0.1
`Dose (mglkg, p.o.}
`
`3
`
`0
`
`3
`Dose (mglkg, p.o.)
`
`10
`
`(C) llclopldlne
`~--------------·
`80
`
`l 50
`
`300
`100
`30
`0
`Ooce (m~d..y. p.o., x3 days)
`
`Fig. 5. Antithrombotic effects of CS-747 (A),
`clopidogrel (B), and ticlopidine (C) in an
`arteriovenous shunt thrombosis model in
`rats. CS-747 and clopidogrel were admin(cid:173)
`istered once orally to rats 4 hr before
`blood circulation. Ticlopidine was orally
`administered daily for 3 days to rats.
`Blood circulation in ticlopidine-treated
`rats was started 4 hr after the last dosing.
`Results are represented as the mean ±
`S.B.M. (n=7- 10). *p<0.05, **p<O.Ol vs.
`control (vehicle· treated group).
`
`2) Photochemically induced arterial
`thrombosis (PIT) model
`
`' i
`i
`j:
`!
`I·
`
`1 ..
`I.
`
`I~
`I; .,
`
`·~ ,.
`
`,.
`h • l
`I
`I'
`I
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 13 of 44
`
`
`
`.:::]
`
`AIV
`
`2.~
`pr<
`it,
`Ti
`efl
`W<
`co
`
`3)
`
`en
`(F
`ti<
`th
`ar
`lit
`(4
`n<
`ra
`bJ
`st
`re
`it
`
`'}
`
`14
`
`Asai, F. e1 of.: CS·747, a New Plaulel ADP Rea:pror Antagonist
`
`Thble 1. Effects of CS-747, clopidogrel, and ticlopidine on a photochemically induced thrombosis model
`in rats
`
`Treatment
`Control
`CS-747
`
`Clopidogrel
`Ticlopidine
`
`Dose
`(mglkg,p.o.)
`
`0.3
`1
`3
`10
`100
`
`N
`10
`·8
`8
`8
`8
`8
`
`1ime to occlusion
`(min)
`6.80±0.47
`9.33± 1.73
`10.03±2.42
`13.54 ± 2.52*
`11.92±2.81
`6.89±0.90
`
`Incidence of
`non-occlusion
`0/10
`118
`2/8
`418*
`318
`0/8
`
`Experiments wete performed 4 hr after p.o. ad.ministration of the agents. Data for time to occlusion are
`expre.ssed as the mean ± S.E.M. *p<O.OS vs. control.
`
`(A) Total number
`
`12
`
`...
`1l 6
`E
`:::1
`c: 4
`
`~ 2
`
`0
`
`1
`0.3
`3
`Dose (mglkg, p.o.)
`
`10
`
`(B) Total area
`8
`7
`~
`.§.
`6
`0 ...
`s
`~
`4
`'E
`3
`CD
`~
`!
`
`0Conltol
`~ CS-747
`
`-
`
`CJopldogcel
`
`0
`
`3
`0.3
`Dose (mg/kg, p.o.)
`
`10
`
`tD
`
`2
`
`1
`0
`
`Fig. 6. Effects of CS-747 and clopidogrel on cerebral infarction in a rat PIT model. (A) Total number of
`infarcts and (B) total infarct area (mm•). Results are expressed as the mean± S.E.M. (n=16).
`"'p<0.05, **p<O.Ol vs. control.
`
`We compared CS-747, clopidogrel, and
`ticlopidine for their antithrombotic effects
`jn a femoral arterial thrombosis model in
`SD rats (Table 1). Thrombosis was in(cid:173)
`duced in the femoral artery of anesthetized
`SD rats by endothelial damage due to the
`photochemical reaction between rose ben(cid:173)
`gal (20 mg/kg, i.v.) transilluminated with
`green light (540 nm), according to the
`method by Takiguchi et al.,m with slight
`
`modification. The agents, or vehicle, were
`orally administered 4 hr before the induc(cid:173)
`tion of thrombosis. Blood circulation in
`the artery was obstructed at 6.80 ± 0.47
`min (n=lO) after initiation of the photo(cid:173)
`chemical reaction in the control rats. CS-
`747 prolonged the time to occlusion in a
`dose-dependent manner. A statistically
`significant (p<0.05) prolongation was
`observed at 3 mg/kg of CS-747 (1354 ±
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1010, p. 14 of 44
`
`
`
`Annu. Rq~. Sankyo Re.r.lob. 51, 1-44 (1999)
`
`15
`
`2.52 min, n=8). Clopidogrel caused a mild
`prolongation of the time to occlusion, but
`it was not statistically significant (p>().05).
`Ticlopidine (100 mg/kg, p.o.) had no
`effects in this model even when the agent
`was administered at a very high dose for 3
`consecutive days.
`
`3) Embolic cerebral infarction model
`Effects of CS-747 were examined on
`embolic cerebral infarction in a rat model
`(Fig. 6). Pour hours after oral administra(cid:173)
`tion of the agents, SD rats were anes(cid:173)
`thetized and the rigbt common carotid
`artery was transilluminated with green
`light (wavelength: 540 nm); rose bengal
`(40 mglkg, i.v.) was injected to induce a
`non-occlusive thrombus f
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