`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~082/099
`
`Pharmacology of CS-747 (Prasugrel,
`LY640315), a Novel, Potent Antiplatelet
`Agent with in Vivo P2Y12 Receptor
`Antagonist Activity
`
`Yoichi Niitsu. Ph.D..' Jose~h A. Jakubowski. Ph.D..3
`Atsuhiro Sugidachi. Ph.D.. and Fumitoshi Asai. Ph.D.1
`
`,2
`
`ABSTRACT
`.._-_._._._. __ .._---_ ..._-_ .._--_._---
`---------------------------------------_._._-----_._.---_._
`class of oral
`is a member of the rhicnopyridinc
`CS-747
`(prasllgrcl. LY6tl031S)
`inhibitors
`that
`riclopidine
`and clopidogrcl, A single orul
`aggregation
`includes
`platelet
`,)1"pl:H<.,kl: "gp\'l:i:tt"i,)n
`administration
`ofCSM74'1 produced
`a dose-related
`inllibi\i,.lIl
`il) !':t1".~
`INao .~pPI'{I)(i1))'IH,ly 10-
`100-{c)kl more
`that
`.!nd
`poreur
`than
`ihar
`o( dopidogl'd
`',lnd
`lid"pidin",
`!''''p(;ctivcly.
`The
`:tnliagt;l't:g'llOI,),
`d'f'CCl o( CS-747 was evident
`at ]0 minutes
`and lasted until 72 hours
`after dosing.
`indicating
`fasr onset
`and long duration
`of action,
`CS-747
`showed more
`potenl'
`anrirhromboric
`activit}'
`compared
`with
`clopidogrci
`ilnd
`ticlopidinc with the same rank order
`as rhc anliaggregawry
`potencies, Combined
`admin
`or hOII!
`of CS"'!47 with aspirin
`istrarion
`to rats produced
`~\lb~l"a!\tiHH)' gre ..rcr i.Ili>ibilion
`platelet
`agg1'l'garlllr\
`and
`thrombus
`Iormation
`t:()J))pa"l~d with
`I::!ch
`:1!~l~IlI alouc.
`'l'hl~
`or CS-747
`antiplnrclcr
`:winIl
`is due
`to irreversible
`and selective) blockade
`of platelet
`P2Y 1), adenosine
`(ADP) recl':ptors by irs active metabolite
`diphosphate
`R-:IJS727,
`In phase
`(:10 and 7S m)!;) produced>
`I studies,
`a single oral dose ofCS-747
`50% inhibition
`ofADl)w
`(:> 48 11()\I)',) ol
`platelet
`induced
`am:\rC/~ati()n. with rapid onset
`(1 hour)
`and long dunuion
`o:' 10 1111:; CS-747
`ilu·io!)_
`111 1,,;•.Ith), v(lhllm'.I."'~,
`'.lnl'l,-(hily
`'H.llYlinl,l1·:ll"i(l!l
`1(,1' 10 day"
`Irom 2 days :If'l.l!l' the firHt
`shower] sig;nilt'~'<Inr cUJ)\lllalive
`()( platelel' 'lggmgaliol\
`inhibiril)1\
`dose until nrlensi 2 cby" 'lflCI'
`the Iinnl dose, Studies
`conducted
`1"0 date
`indicate
`that CS-
`7117 is a highly effective
`antiplareler
`and anrithromboric
`agent
`and is anticipated
`to be
`effective
`in the treatment
`of athcrorhrornbotic
`and other
`ischemic
`vascular
`diseases,
`
`I(EVWORDS: CS,,'7<1],prusuorel, LYBIW315, fl,1387?'J,
`r-199221J, pintelnt (lll9ICI)Dti{)ll,
`antiplattllc)(,
`antithrornhotic, P2Y I ~ receptor
`(-lntiJ\)ol1i~;1.,ADP
`
`Objectives:
`thl< f(·!:.ldC·!f!;llI)llld b(.;abhllf'
`;lflicl(~,
`011 CIJI)\I.lllllil\1I ()lll\i!;
`1.)I:l(W(·~1·2nCS-Jt17 find
`()lopieloHtl:1l.
`!lilln
`COI)'PjJfiJllVl-l
`rl'l.JSM);, ~cc":'dilf.'d by Ih,:,1\<.:<.:r(:,(iiWli(mCOI.ln"';l for C'lrltinllinn M':'<:ii(:;:III·::dl.IC'lIi()n
`ll.lft~;t)niv(:!f!;ity
`;:>(;h(IOI01 Medidne
`Acc,edltatlon:
`to provide eOl"\linuinq'w'clic,ll education tor phvstctans.
`
`(:1.)
`
`li!;l
`
`("1) d<-!:;cribll tll(·~phHllfl:l(;()IIl()y
`
`,Ind Iwlcti(JII!; of CS·o;'t/
`
`:11\1]
`
`The PI;\tdt~~' Pi Rt~t:qHI)f~~ Bk)L'heml!irry, Phy~;')\')gy,Ph:'ftn~\(~d,}~~)',;\I}(l Clir)il:;ll A!'tPI~I~I~~I,:d:()(
`ill Chid', J,:IH:t'lI;tfd F.1'I1;1II\IIII:n,
`(;IIC~;I
`f\,1.iJ.;
`(;1l("1".'i", M ,I)" 1'1>,)) .. HIl(! Marco Cntranco, M.n,
`S,'lIIill,II)'
`ill n'I"MII/)Q.<i.<
`and 111'/1/(,.(/lI.<i.<,volume 31. IHIIl1her 2. ;lOOS, I\ddr('~,
`I<:(lil"or:;,Chri~liH"
`feqlt(~~b: FUII\jIt'J~hi A~al, Ph.n'J Plrilfn)it\~()I()g}'
`l~)( correspondence
`t.~o., I ,Id.,
`r\'1IJh:l~lI1af Biology 1{1:~C:jlrl~hl.i\hi)I';\lIH'il:~,
`'1-
`i)lld l'l~Pfilll'
`illlt!
`:-)imi{ytl
`2-·58 Hiromachi,
`Shill;l!_\i'Wi,··I"" Tokyo
`J")'ill ...
`r'>",,,il;
`t')i"iM~'iH1k)'(),('I,.ip,
`'Ph!lrn·li)("1."W and
`Bj,)i.>I.IY i{"'(i;"·.,r,
`!\1"lc"llb,.
`l4(J-8iJO,
`I,!<.I"'I'<>ky<.',,1<'lIi'I1; 'Oin'("«)r;
`J ,at.""·I1I<..ri",;, Si,"ky',
`(;').,
`J\"~('.:Ir(:h,
`lind C<HlIl'HIl)"
`lndinnu,
`'Bi<.d'edlJ1<.>i<)I!:)'
`i)i:;c,)very
`I-:li
`J ,illy
`J!ldi:'II~II',-,Ji:;,
`'1'1:1, <·IULl) SH4·A(,(,;L O()'J-I..
`C,,),yrigln
`b)' Thieme M"dic,,1 Puhlishcr«,
`1,,,,.,
`:lTl Seventh AV"J\l":, New V,),'It, NY WOOl, USA.
`«;)
`200.)
`I,02, '18(1,l<)",lh,,,,,;.~ht)l
`fl<16,.
`(,17('.1';2005)
`
`184
`
`:01. This page 82 of 99 was completed at 24.12.2010 14:13:35
`Duration: 24.12.2010 13:40:37 - 24.12.201014:21
`Received at the EPO on Dec 24, 2010 14:21 :01. Page 82 of 99
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`
`Ex. 1044, p. 1 of 11
`
`
`
`24/12
`
`2010 FRI 13:15
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~083/099
`
`Credit: TUSM do:;iqni,lIC1:; Ihi:, 1,)dl.ll)i,llionaj iJClivily Ior u maximum of 1 CiltW10IY 'j 1;1~lIjitIOWi:l!d the AMA l)hy~;iCii)l1~;H,"(;l)ljnitlon J\Willlj.
`tl1(1thl~hhe
`activitv,
`F,:",11 pily,;k:i(Hl
`l;I1()\,ld cliJil'll ,lilly 111o,:,,,,(:",dit,
`i1ewi;llly spent
`ill Ihe _,dl'Cilliolli,j
`
`PHARMACOLOGY
`
`OF CS-'147/NIII!;U
`
`1"1111.
`
`1B5
`
`aggrega-
`platelet
`including
`activation,
`l~latclet
`of acute
`plays a crucial
`role in the pathogenesis
`tion,
`accidents,
`co]'()n:uy
`syndromes
`and
`cerebrovascular
`Ahhough
`arc acrivared by a variety O! cndo-
`platelets
`W,"1(11I5 :lWllli~I'''', ADP
`\h~: <,'arli<"~I' dCij('.I'ih<"d
`:uld
`w;(~
`is one of
`the most
`importunr
`ag;onisf's
`(or plareler
`activation,
`l,1 l n additioll
`to illduci;lg
`primary
`platelet'
`aggregation,
`ADP released from activated
`platelets
`can
`induce secondary platelet
`aggrq2;ation via the feedback
`process, ampii()'illj.!; and propagating
`platelet
`actlvati.on
`l11du(:(:d hy otlll:!'
`,lW)lIi~I~, AD»
`;~~:IIval\~splalc:kt$
`via
`le:-t:->[ rwo distinct C
`A 1)]' 1\~\:\~pt())'~,;lnd
`lhcl\~ are
`Itt
`PI'()t\~ln-ro\lplt=d AI)!.' I'Ct:epIOI'S, Cq-linkcd
`recep-
`P2Yl
`tors and Gi-linked
`r(.~ccpl'Or~,:H
`P2Yll
`importance of P2Yl2 receptors ill the purho-
`The
`f!;cllesi,s of arterial
`thrombosis
`i~ ,UPI,I\ll'\,<.;d by a series or
`il'lhlbil'I»)'S or thil:'Jl(lpyl'ldllH~
`in VIVO P2YI).
`reports
`that
`(ki'iv',1\'iv(;$,
`tidl)pldllll~(i
`and dnpidng;rcl/
`nrc cf'fcC.1'ive in
`I't;d.,l(,:ing; rhc
`"isk 1)( :Irh(:msd(lrol'ic:'
`V~ISCld:u' disc:1se,7'-',)
`r"low(;vt~l',
`rhesc
`:Igerll's are hy no means
`S:lI'i:;f~ICtOJ'y in
`terms
`of
`adverse
`cffocrs
`and
`anriplarclcr
`efficacy, At
`commonly
`used doses,
`riclopidinc may cause agranulo--
`cyrosis II)
`and
`thrombotic
`thrombocytopenic
`purpura
`(TTP)
`,11,12 In addition,
`the use of riclopidinc
`is dis-
`in patients with renal or hcpuric dY$f\IlKti<)Il,C;,')
`couraged
`Clopidogrcl
`has been
`dcmonstrutcd
`It) b<~ $}\f~~l' rhun
`ir
`riclopidinc,
`also cause
`'ITP,I:l,lo1
`:\Jl-hOllgh
`l)fIY
`have c,;\llsed
`concern rc,gardil1R
`H(1Wt'V'~I',
`t'(;l:\~lIl, slildit~s
`"I
`id
`I
`'
`'" I~J(,
`f
`P l<~JlOtl1CI)()t1
`1
`I
`resistance.":
`,
`1 II'!
`C 0Pl{ ogre.
`0
`Furthermore,
`Lau ct all';,I:1 recently reported that clopi-
`is less ,'ITenive
`pl:tl<jl(~1:'lggl'C!!;alion
`dowel
`ill
`iJlhibiJ"illg
`when
`wu h ntorvastruiu, which
`cl):1d1T)iJli~I'<'I'\:d
`may
`of' clopidogrcl via c)'to-
`IlJhibil'
`tlw 1l1l;I:I!lnlic
`uct ivarion
`jA,'9 alrhollgh more recently a
`('.lI1'<)tnl~ P'150 (CYP)
`c.!opidogrcl--alOrvastalin
`interaction
`has
`been
`<]IIC5'
`'rhe~e
`racl~ and ollr
`intl,n:6{' 1'0 ,kVl~l(lp Hovd
`lioned,).()
`ami,thrombotic
`ageIJ16 PI'oU'lpll:d \I~ ('0 $~at'(~h f('JI'
`:t
`llt('J1't:
`'tlll.iplitld"t
`;1{!;Cnlwith
`grcolWl' P2Yq inhihi-
`atil':tt:liVI:!
`than
`and cl()pid()J~reL
`Inr), activity
`\'Jut of tidopidinc,
`CS-747
`(2"acetoxy,-5"'(CI.··c)'dopropylcarbonyl
`..2..
`flu()roben~)' 1)..4,5,6,'1"1','\'1'\1
`hyd 1'<.1thil,:nll
`[3,2-<:,jpyri.( Ii l'J<.:)
`l) i, a lh, ")1(.1)1yt'idyl pl'c.1dl'ug
`Ihu' I' 11'll,t;iholi'l,(:d
`(F,ip;,
`
`C$ ..l41
`FiOllro 1 Chomic(,1 t;li'ucW'-OGof CS,.'j'l'I and ilfo activo motabo'
`Il!J!J?7.4!H-'1 ;il:ll/.7, 1'1-1:ll:l1Tl I:; (I lrlixI\Jre of fc.)\11 i~C)I1'l1,!I~, H-
`liW,
`!)D:t.:!4I:) Q ll'IixlulD of {rI, S). (Jfld {S, RJ-itlOrnC(::; (a (;ornpo'K:nl of
`1'l-'13H7?71
`
`rc,
`
`moraholi
`plarclcr-inhihirory
`active
`the
`in vivo to
`antagonistic
`P2Yl?
`receptor
`1), with
`(Fig,
`R-13g727
`activity, Our previous reports showed that CS·-747
`is ,HI
`effective
`orally active platelet
`am~regati()n
`inhibitor
`with
`high
`J<.lnl~duration
`pcm:IKY,
`fa~(' OIl,~I;i',
`;Ind
`;Ktic.111
`(If
`:->luclies?:1 ;),~ and in ,I clinical
`Sll1dy/1
`borh
`in preclinical
`In this article, we will SUI\1I\1Hizt!
`the evidence
`lor
`the
`potential
`clinical
`usc ofCS-7'17,
`a novel P2Y12 inhibitor
`nuriplarclcr
`agent,
`In
`the
`treatment
`of
`thrombotic
`disorders.
`
`PRECLINICAL PHARMACOLOGY
`
`Inhibitory Effects on Platelet Aggregation
`
`ANTIAGGR!!GA TORY ACTION
`pOlEN!
`is a
`CS-,74'1
`ro clopidogrcl
`and riclopidinc,
`Similar
`a~say~
`and is
`inactive
`in platelet
`aggrep;ation
`prodmg
`added
`in vitro, When
`to
`administered
`orally
`when
`experimental
`animals,
`CS-747
`h:l~ xliown
`}WWt'V\:).',
`or platele! ag)';n:gfl-
`dose-rclurcd
`and pOI\~n\' inhibition
`is appJ'oxim:II<;ly 10- and 1OO-J()ld more potent
`rion rhut
`I'han that
`of d()r,d()g('t~l
`riclopidine,
`respectively,
`,tnd
`ex vivo anriplarclcr
`Previous
`rt~p()rts~,1 comparinj,
`the
`pnt\~l1t:)' of CS-'l'17 with
`riclopidinc
`and clopidogrcl
`(OJ to 3 mg/kg,j'J))
`mrs showed that
`(:5,·747
`inhi])il(;d
`ADP"ind\l(:ed
`platelet
`agg-n:.g;\tioll
`in
`dO:->l!-l'd:tl~!d
`by SO%
`manner with a dose
`1,1,;11' 1'(:.<.IIIC(:'<.1rile I'(:sponse
`(Ej)~I)) o{ 1,2 mt~/kr~and thar
`clopidogrcl
`and ticlopi-
`activity, with EDso
`dine showed
`less potent
`anriplarclcr
`0/',0,) and :-:-,300 IT,p.;lkg
`(/),1),),
`values
`of 16 mg/kg
`respectively
`(Table 1), The potent
`antiplat(,l(:1
`:((,~Iivily
`of CS'·,(4'1 was ah<.l ",<.l,,(il'll'll,:d in
`study,
`clinical
`:1
`summarized
`later.
`
`ill
`
`!(
`
`RAPID ONSET AND LONG DURATION OF ACTION
`of platekI' aggregation wi,th lidopidine
`] nhibition
`,ttld
`:,1 1'<.15 da)'~/I
`i~maintailll'd
`Ihis
`clopidogrcl
`OV(:r
`'Ind
`lIr acti(1) by both ag\!"I~ i~:illl"ICliw
`in
`long dlll'ati.on
`time 1'0 peak pl:\l'clci
`('.linl<:al sil'\1rttion~,
`I-Iowevel',
`Ihtl
`r,low2';,?,(,; CS-7117 shows
`inhihition is rehllivoiy
`not on I)'
`activity hilt also a more rapid
`long-hs('inl~;
`:ll1l'iphtdct
`onset of action ('han c1opidogrel docs in rat antiplntdCl
`The time COIII,'~e ~I'lldy on the antiaggn:p;atory
`studies,
`df(;(;('~ aft'er adrninim:nion
`of CS"'14'1 (1 to 10 1llg/kg,
`lInd dopidogrd
`(10 to 100 mg/kg,
`/" ()_) "how"
`jJ,rJ.)
`th}ll
`than
`!l()% inhibition
`wa::
`()b~crvcd
`in
`l'nol'C
`CS-747-trc,llcd
`(10 mg/kg)
`1'<11', 30 rninllte,;
`an'cr
`the
`dOoing, Wh':I'l:a~, at
`lhc ~:m:I(: limc point,
`dopidog)'(:.l
`exhibil'e<1
`rnillilt'ial
`inhibil'i(HI
`<~Vl;t'i al
`\'he
`hig-Iw~t
`do~c Hs,:d (Fii~' 2), sllmr,cst'ing
`c;\r]iel' on:,et' oj'
`:tcl'i01\ oj'
`CS-747
`compared wit'h dopidogreL
`The mcchani:lln
`
`:01, This page 83 of 99 was completed at 24,12,2010 14:14:07
`Duration: 24,12,2010 13:40:37 - 24,12,201014:21
`Received at the EPO on Dec 24, 2010 14:21 :01, Page 83 of 99
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`
`Ex. 1044, p. 2 of 11
`
`
`
`24/12 2010 FRI 13:15
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~084/099
`
`186
`
`S"MINARS IN THROMBOSIS AND I'I"MOS11I$ISIVOWME
`
`of Antiplat(.IM.
`"lIble 1 Comp,lr;son
`Clopidogrcl.
`and Ticlopidinc
`
`----------------------------
`::n. NI)MBEH 2 2(105
`
`Antithl'ombotic::,
`
`and Antihamostatlc
`
`Effects
`
`of CS·747.
`
`Agorlt
`-"
`CS-747
`Cloplucqrel
`'i"i<'llopi(illll"
`
`AV·Shunt
`(ED"ollvl
`_;__.::;:::='-- __
`0,6f3
`6,2
`>:lO()
`
`A9greglltion
`Bleeding
`_;_-"'==- __ ,~..'",_,._(B_T_,L
`(EOo"I\",,1
`1,2
`0.50
`Hi
`4.(j
`>:3(lO
`1:10
`
`R!ltio
`J§pl<OI\v/BT_o"')__
`1.4
`1.:3
`> ~!.:j
`
`1..11
`:Ui
`.,. 2,3
`
`___'_(E_D...:!.",·Ohc:;Cl;;_;,.Cl;:_/_B_r=,1
`
`by CS"74'l may rellccr
`this mccliauism.
`
`in.\,v'::l'~ibk
`
`1"2Yl~ bl()(~k:tdl~ vi:\
`
`ilOlll SIIIJHj,lClli (ll al"
`Dal'l
`I\V shul1l
`th(,
`IN,,()I anrl
`In pl{lt~l(lt
`to 1M'; II hours before' eV~".lllliQn.
`ArJ0"'H~wore orally administered
`flflWf.lllfl\lon
`(1(I(.lil1()!lli()/\find Ihl()l\\bu~;
`ill willeh II),) (lgOI)I" 1I\llIl)iwd pilltelol
`Ih'roIllIJO!li, ",oil", I, 11>,·,LD"fl vill"(-)}I IIl~~)/k9) .uo (lo,;",;
`[oll't1i'ilio(j by bO'ro.
`l3T;> v(liueG ("I1(J/k\J) (11'0dosos (II whkh Ihe ~ql,)')15 (Iollbl/)
`lime (tn).
`Iht3 l)a;,(,!liIW 1)I(:~(;~r.lin~l
`the rapid onset or CS··74T~ effect
`remains
`underlying
`1'0 be
`elucidated
`but
`rnig;ht
`reflect
`either more
`rapid
`aIJ"'I'jlt'i(.ltl
`<.),' ":Ipid llll;taboli:.;m 1'0 it~ a('.tiVl~md;lhnlit(,
`ill vivo,
`compared with c1opid()l~r()l. CS-747
`showed
`clfccrs at 4- hours post dose, with
`maximal antiplatclct
`anriplatclct
`cfiicacy
`as
`Ion)!;
`lasting
`as
`clopidogrcl
`inhibitory
`(Fi.g, 2), At 96 hours
`post dose,
`the platelet
`eH(.',ct's of CS','/4'/
`largely disuppeured. This prolougecl
`o( pl:l(dl~1 :1!:\,gl'l~glJ.li()l} by CS-747 and clopi-
`inhihilioJl
`cloy-no] S(:l!mS to be
`comparable
`to the
`spall
`of
`life
`."
`.
`",2H
`.
`.
`circulating
`platelets
`tn the
`rar.:":"
`Recently,
`It has
`been proposed that
`the action of rhicnopyridyl
`prodrugs
`reflects
`the
`forrnurion
`of a disulfide
`bl'idg<:.
`between
`rhe
`reactive
`..hiol
`g!'()UP oj'
`('il<.: <\\:I'iw ll)(:t·a])olit·(,
`and
`of' (ill: pbldet
`I'lY1;). ADP
`(:Y~I'('.iJl'"
`I'~~i<.hw($)
`recept()rs.:",,~11) Therefore,
`this
`long duration
`of action
`
`and Antihemostatic Effects
`Antithrombotic
`The
`IWO most widely
`used oral
`nntlplarclct
`:l~~ents,
`p,'od",(,,: ~igllifi(,':1111din;":lllwn,~-
`nspirin and d')pid')g,'(d,
`(it~, but
`('lIl,:i.1' 1'(~lll.liv(: w(,;\k ll!l(ilhl'ombnlic
`l~n(!d,; aI",!
`considered to be a major
`The potent
`litl1it,lIi()n,~\l
`anti-
`aggrcgatory activity of CS ..7'17 provides
`improved an-
`rirhrombotic
`effect over existing nnriplarclcr
`;IW'·lJI~,In 1I
`(AV) shunt
`thrf.lr)'lhf.l~i~ rnodd,:n,:n oral
`rat arteriovenous
`:Idminist'l'ali(ll)
`of' CS-,/4';
`(0.1 1.<'>
`:3 mg/kg)
`4 hours
`hd(lt'(~';1':lI'Iin1-';:l
`]O-minlll.e
`circulation
`of'blood
`through
`or thrombus
`rhc A V shunt
`resulted
`in inhibition
`forma-
`an ED so or
`tion in a dose ..related manner,
`exhibiting
`
`(A) 60
`
`10
`o
`
`(8)
`
`GO -
`
`"-o~'" C()l1tr't)I
`___
`1 mglkg (p,o,)
`-(]-3
`-.-10
`
`72
`48
`41224
`Time after (iMino (IIr)
`
`96
`
`< ,
`
`\
`
`\
`
`lid,
`
`....
`
`*;
`,
`"'*
`"-0
`
`4
`
`i-Q-~-{)--------O
`"'n~. - ~,- .t.
`1\ 1t*
`."----
`...-
`-8
`'O~
`"*'
`--
`I
`2
`0,5 1
`J
`72
`46
`41224
`TilnO ..ft(>rdosing (tlr)
`lim!;! atter dosing (hr)
`illl"I'W wa,; 11'Will'L1rG(i~I O,G,
`Til)l(;) course oj sntiplatelet ettects of (A) C8-747 and (8) clopidojrel. Exvivo platelet ('09reg~lii(ln
`Figura:?
`4f:j, T~, iHH,1 m511(l(W; ilfl~!r ,I ~,;ill(ll~~ (Hill r.jo:;(:~oi (;)ilCh 'lfWfll. A(.)P ,1\ ~ G(lIl(:(~Il\ri'!ir.lI\of
`ll~,;I,~(lil~ IIw iHJ(ll1i~I, Hr.·!:;(JI\:;
`:3 11MWil!;
`1,7,,4,74,
`'p .::0.05,
`., p « 0.01 vornun control
`(vchicirH(();,ted (jrollpl by
`the moan (SEMI (1) .... 6).
`aro pr():;(I(lkd
`(1:1 Iho rnce» "I, ,ltandnrd error of
`\(,,~~t.(l:re)!)l ~;unid"IGhi /\, e\ ,,1,81,1 Pharl1ll1col 2000;1/.~l;'I443, H('printed by p,lImi~'~;i(ln,)
`tbe i)1.lnIWt\
`
`- -0 - CMtl'OI
`- '•.
`- 10 mg/kg (p.o.)
`-0- 30
`,.,..
`-100
`
`96
`
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`IPR2015-01492
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`
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`
`
`Ex. 1044, p. 3 of 11
`
`
`
`24/12 2010 FRI 13:16
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`-----·------··--···-·--···-·-·-,···-··,.",,,."w.,,,,.,,"_.w_.~_,
`
`~085/099
`
`187
`
`1.1 AI.
`
`---------.-
`
`(A)
`
`(8)
`...-
`~ 60
`'-'.:l: 50
`t.f)
`'Cj) 40
`;:
`tn 30
`::::I
`~ 20
`o.E 10
`
`I-
`
`(C)
`
`o
`
`1
`0.1 0.3
`CS~747(mg/kg, p,o.)
`
`3
`
`o
`3
`1
`Clopidogret
`
`30
`10
`(mg/kg, p.o.)
`
`I'HAflMACOLOGY 01' CS-141fNIII":;t)
`..,__.._.._ _.._ _._.._.._ _._.._._.
`_
`!idnpidi Elf)(10 I'() 300 1))g/l<);,
`of (j,2 mg/kg, Ahhollgh
`1',0,) produced
`a statistically
`signifimnl
`inhibitioEl
`ol
`ED,o
`p..;rcatel' rlian
`the
`was
`thrombus
`formation,
`300 Iflg/kg
`(maximum inhibition
`of
`less than 110%),
`These
`results d~~;ll'l)'indicate
`(h;\! CS·-747 has more
`,",:livity
`(:I)lnj);\1.'cd with clopido-
`potent
`antirhromboric
`and riclopidinc. The rank order of the
`grcl
`:11"1(;1.1".1\1111-
`boric potencies
`<lmong these
`three aR.:nl:s is Ihl~ S:llll(, :l~
`dw !';Il)k order
`for ADP Olnti"p;g;-cgatory
`potencies,
`lit.lgg!:litillg that CS·-'!4'! exerts
`its in vivo unrirhromboric
`dl(:cl$ by ;ntcr!(;l'ing
`between ADP
`with the interaction
`to inhibition
`and AD1'
`1't!(:epr()I's Of) pl:Hd(~llj,leading
`oj"
`the plarelcr
`:l~~gmv.;:ll"ion component
`o( the thrombotic
`of- urreriul
`mass,
`Furrhc»
`evaluation
`in orhcr m()dd~
`thrombosis
`also has demonstrated
`potent
`an lilhl'(),-nbMic
`of CS·:14'1:'" Taken
`efficucy
`rogcrhcr
`these
`results
`indirak
`that
`the autirhromboric
`efficacy of CS-7'l7
`is
`'IPP"l)ximiltdy 10 and 100 times
`that of
`gn:atn
`than
`l'~~'p(~,:(ivdy,
`clopidogrel
`'lnd l;d()pidin(~,
`dTi;(\"~
`To
`determine
`p()~,ibh~
`'IT)tih':lTIl)~(ilti(:
`or CS··747,
`rat
`rail
`transccrion
`hleeding
`1:;1Il,,~:1~; wen:
`measured 4 hours after dosing, and die cfi~C1S ofCS-7<17
`WI":<; compared wid,
`those obtained with clopidogrcl
`and
`rng-/kg, p,o,)
`:1
`riclopidiuo.
`CS-74'/
`(0.3
`prolonged
`il)
`bleeding
`rimes
`in >l dose-reluu-d
`manner wirh
`dw d(1~t:.
`required
`to double
`bleeding
`ti1J)(~~, :1 B'l';~ V:.JIl(~, of
`0.50 mg/kg
`(Table
`1). Clopidogl'cl
`(.1 I() 10 nlg/kg,
`jJ.o,) and riclopidinc (30 to 300 mg/k),;,/"o.) also showed
`of bleeding times, with B'I2 values or 'fJi
`prolongation
`:111dno Il1g/kg
`(j),(),), 1'(:~pcctiy(:ly,The rank order oft-he
`anrihernostnric
`pnrt~ll'~;t:,
`amnllg
`tIH~~(~t-!IT'(:<::lg('M~is the
`as the order of the anriplatelet
`same
`and antirhrombotu:
`potencies,
`slIggcsting
`the antihcmosratic
`that
`effects
`CS-'/4'!
`arc (hie to its plarcler
`inhibitory activity,
`
`of'
`
`o
`
`100 300
`30
`10
`Ticlopidine (mg/kg, p.o.)
`1·:·11(lcH; of sin(ll" crat adminisuatio»
`of (AI C5:/117,
`Figur" 3
`(0)
`clopiclO(JI"OI,(Hid (C) li(;i(>pi<lin!)on II1(ombl.J~ [ormation in th~~AV
`in rats. 13100clcirculntiol' we,,;
`:;h\/rlt
`,ltml~1d II IlOur:; Hfl,,! (l!~1
`<j():~il)fJ of \1"1" il!J(-JIIH;. H~lwlt", ~Ie preseoted as the mean (.I: SEMI
`thrornbus w()iqhl
`:,0 I))illl/l():;
`(n·,·, G).• p «, 0.05,
`·~/J<O,Ol
`(II
`(From f;WJidachi A. ut (II. Br.J Phnnnncol
`V!:)!!;LJScontrol
`(0 1T1!.1/kq).
`znoo: 129: l/l4/1. Ikpi
`illlHd by P!~Il1Iissi()n.)
`
`of
`1). Oral administration
`O,()8 mg/kg (Fig, 3,
`'r;thle
`clopidogrcl
`(1 to 30 mg/kg') also deCl'(~;iSed t·hl'fllnbuij
`w(~igh(· ill a dlm~··rclatcd
`rnannerwith
`an est"im,lI"ed ED~{)
`
`Combined Effects of CS·747 with Aspirin
`Our data, as described
`in the pl'(~c,)diJ\g ~1)!~("i(Jn~of' !"Ilis
`that CS-7117 has more pOlCn!"
`article,
`clearly
`indicate
`and anrirhrombotic
`antiaggregatofY
`activities
`than ()dH~I'
`;tvailablc::rhicnopyridyl
`have, namely,
`currently
`prodrugs
`the most widely
`ridopidinc:
`alld
`clopidogrcl. Aspirin,
`lIs(~d an(.iplat"(~lc;("a);<::J1(",is a cyclooxygcnasc
`inhibitor,
`and
`its 'lIHi;lgg'-l~!',;<lOl')' d'f~~(:li~~p\,\~ifi(~againot
`thrornboxanc
`A2-mcdi,\l:cd
`platelet
`distinct
`agl~I'~W\li()n,
`:1 ruecluuustn
`from that of rhicnopyridyl
`prodnl!~,~_Clinic:llly,
`C<)IYI""
`bined treatment
`of aspirin
`plus dopidogrd
`Ius shown
`\·Jlica(:)'
`thrombotic
`'lddirivI:.
`ill preventing
`disordcrs,:\f,
`of C:S··'/4? included the study
`()f
`Th(~l'e!ill'e, evulunrion
`the effects of CS-7t17 in eOJlli)iTlali()l) with
`a~pir:in on
`platelet
`aggregatioll,
`thrombus
`formation,
`;llld bk(~diTlg
`times ill experimental
`animal models,
`In ex vivo I'M pluteler
`neither
`studies,
`aggregation
`CS-747 (0,(' and 1 rnglkg,/'''.)
`:ti(l!W (ll' aopirin (l() mg/
`kg, p,o,) alone showed any !;tatisticilily significllllt
`)nhibi-
`by collal?~l\
`finn
`1)1" I'at platdet
`ag).(J.'cgation induced
`
`:01. This page 85 of 99 was completed at 24.12.2010 14:14:59
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`
`IPR2015-01492
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`
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`
`
`Ex. 1044, p. 4 of 11
`
`
`
`24/12
`
`2010 FRI 13:16
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~086/099
`
`SEMINAIIS IN THROMBOSIS AND HEMOSlASISIVOLUME
`
`31. NUMBER 2
`
`2005
`
`*:¥
`
`D Control
`• Aspirin
`IlIJll] CS-747
`~ CS·747+Aspirin
`
`80
`
`60-r:
`40
`C) 20
`
`~~Q
`
`0
`
`~ ~
`
`1::1'1
`..:(
`
`188
`
`..
`
`0
`0.6
`1 mg/kg (p.o.)
`CS-747
`10
`10
`10 mg/kg (p.o.)
`Aspirin
`(Jf CS··l~/ and Qf;pirin Of! platelet <l~Jgr(;~JQlionin 1'<11:;whon adminitH(;[(:lIlllonc
`or in combination, Aoenw were ori11ly
`[ffHw;
`I"igura 4
`,lcil1linislered \0 rats 2110llfll before blood collection.
`l'l,llelol ~19(Jr':'!ylti(JnW<15induced by H) I'D/ml. colli.'O'~'L 11",!;\oIb ')1"1"
`\'Xr)(,~,;s(,"f ilS
`tI),~D\J/\n(!!lll-l:;l.
`moan
`.l: ~;I~M (n ......1)).•.•. p<:; 0.01 VI.!I,'U:;'.!(lnlf(lll)y
`
`0.6
`
`1
`
`2 h(lIl1"~,Ilk,. d<),~ing. 11(lw(,w.~I',CS-747 (0.6 and 1 mgl
`kg, 1'.(/.) when
`/'-0.)
`'~'J1nbin~~d wilL aspirin
`(10 mg/kg,
`showed marked inhibitiou
`of collagen-induced
`platelet
`'I).\F;rc!('l1:ion (Fig.
`rl),·11 indicating
`additive
`or synergistic
`cfTc:.~I:;of CS,,747 ';'ill1 aspirin, Combined
`a;ltiplatelct
`cffccrs Oil collagen-induced
`platelet
`aggl·l'g:ll·joll
`h:lv(,
`been d()(\1)n'.'/)I.~,d In in vitro ~1\J(.Ii(;s with human
`plate-
`'!'ill'
`/)\(~I,lholil"(~ Dr CS-74'!
`lets.
`(R-1.1R727,
`ilUIVl.:
`30 pl\ll)
`111cOlllblll,tl1(1)
`with :I~pi"in (300 pM)
`showed
`compared with
`nn additive
`antiplarclct
`activity
`either
`agent" alone.
`In contrast,
`additive
`effects were
`nut
`us tilt'. ag<.lIli~\
`obs(;l"ved when
`using ADP
`in rat ex vivo
`platelet ag).lfegatioll mllli(:~.;rl
`I.he in vitro results could
`To d('I'~~l'lI)i(l(:whether
`anrirhromboric
`cfficncy,
`ill vivo
`b(,
`a
`Il':\n~ht"l~d into
`and aspirin was
`examined
`combination
`of CS·747
`in a rat AV shunt" thrombosis
`after oral dosing
`2 hours
`model and on rat mil. transection
`bleeding t"i)l)(;~.Both
`CS"-'l4'1 (OJ
`and 0.6 I~lg/kg,
`ulone :lt1d :Ispil'in
`jl.O.)
`(lO mg/kg,
`/).{/.) ;\I(.)lll~ t\)()d'~I·:-lI.dy inhibited thrombus
`in file AV shun I', whereas
`aRent alone
`formarion
`neither
`showed
`:1l1)' si(~niiic<lnt prolongation
`of the tail rranscc
`S). Thl:
`rion
`blccdinjr
`times
`ar
`similar
`doses
`(Fig.
`/>.0.) with
`combination
`of CS··'747 (0.3 and 0.6 Il\g/kg',
`aspirin (10 rug/kg,
`j>,().) resulted ill gn.:at"n· aflti,ll"''>lilbo-
`tic d1~(~t$,~()mp:ll·,~dwilh either
`In conrrast,
`,Igt!rlt:ulonc.
`or CS-7'17 (0.3 and 0.6 t\1/!,/kg, /,.0.) with
`c()mhinalin\"!
`/,.0.) did not result
`in a statistically
`aspirin (1.0 mg/kg,
`prolongation
`of bkeding
`rimes, alt-hough a
`significant
`trend W,\~ ob~l::rVt:~d.'1'11(:,,:: /'(;Hlllt~ S\lgg<.:~tI"h:\I" :, combi-
`nuriou
`of CS-'/47 with :I~pi"in m~)' provide
`(1 gl't\;\I"CI'
`anrirhromboric
`eHCCI" than either agent "lone wirhour
`a
`concomitant
`increase in bleeding I"lmes.
`As dC5nib<.:d pr<.:vioudy, CS·· 747 dlOWS carlier
`d()pid()g!'<::1
`inhihil.ion
`c()rnp~lr't;d wilh
`()fl~(!t or plald,~1.
`'l'IHls, we fUfther
`compared I"ht! com-
`~l(teroral d()~il\g.
`bined dTcct ofCS ..74'1 plu~ aspjJ'in to that of dopidogrd
`phi/;
`"opil'in
`:111(:'.ll'ly 11111<.:POI Iii" :lrtt."·l"he dOhing.
`[1) I"hi~
`"I'
`
`induced by collagen
`agF;regal"ioll
`ex vivo platelet
`study
`(20 ~lt~/I11L) was measured
`30 mi nutcs
`alrcr dosing
`in
`/,,(1.) ulonc ()ill'
`rats (Fig. 6). Neither CS·,747 (3 mg/kg,
`l.r)..) alone ~how(:.d ,\Ily inili])ln),'y
`(:lO mg/kg,
`aspirin
`dli.:u:;
`:.lOIniTl\l(,~~nth,,· d()~illg. CS-747
`(6 mg/kg,
`/,.().)
`:;ll<)w(:d ~Iighl'anriplarcler
`aloft,;
`ctfccrs.
`In COl1ITa:;t:, CS-
`747 (3 and (j mg/k,:~,/,.0.) ill combination with :topirin
`
`(AI
`Oi
`
`60
`
`i:~ 1111.
`. i..
`
`J ~...............
`
`OCOtllrol
`.Allpirin
`~ CS-747
`!:] CS·74'r+Atl.pll'lll
`
`08·747
`AIIIII(III
`
`(I
`I)
`
`(I
`10
`
`0.3 0.6
`o
`0
`
`(l.3 (l.G '11(J/kO(p.o.)
`10 10 rnlJlkg (p.o.)
`
`(0)
`
`i.e
`
`OConlrol
`• ASI)irin
`~CS·747
`§!.'l CS-747+Asplrln
`
`0.3 1l.6 0.3 0.6 m(Jlkg Ip.o.)
`(I
`10 10 mg/kg (f)·o.)
`10
`A,;pitirl 0
`Q
`Q
`l'igu(I) 5 (A) Ellccts of C3··1~} and aspirin alone cmel in conlbi·
`nation on thrombus torrn;~ti()n in tile, AV (;1\\111\
`in ril(,;. A!l',"II~
`ildmilli:;l~ll(·!d
`I)ow:. bd()/(·!
`Wf,!le tlfillly
`I,) 1;11:; /.
`(;if(";ul'ilini"l
`Ihel shont. Re;,lIll:; arc presented
`an tho moan .1. SEM
`throuqh
`/J -:: 0.01 V{,'rSl.l~; C(1n(rol by (h~, 1)'.ll"In(,!ll t<,,~;,
`• f.l':: 0.0':; .••
`·6),
`(Il'
`<)nd u!;pirin
`(1.1) Eff~~cI!;of CS,/rll
`"I)«l.()"t
`by Iht·) Tuhly
`ltd.
`~I()ne and in combil1lHion on wil
`\wn~eclioll
`lirn("'; ill
`blt!(e(lil1(l
`rat):;. AH(~n1.~W(~l(~ ()lallV rJdrnilHstHI
`(.)1.1 1(.> rilts /. l1our·~.;tXJfOI'B (;"liI
`i"l,l:illll;, aft, PfO;,(:nl(;d
`Irl(';~:lI1,I. SCM (II
`lI"i.)l)S(lClion.
`:I;) thfi
`(3)
`There ~[l' no slllt;,;Ii("lt dillef('n(:~!s ,ll ~I"IYp()if1~cr.lmpi",,,t w.~h
`(;OnlfOl (Ihn Dunnett lOra).
`
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`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`
`Ex. 1044, p. 5 of 11
`
`
`
`24/12 2010 FRI 13:17
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~087/099
`
`o (:Or\If(l1
`• Aspltl,\
`IZICS·747
`L\\l c5·747"
`
`A~,>lrin
`
`o C,,,,lwl
`• Aspirin
`I!:l CIc.pldO(Jrol
`1IiIl CIDpldDDrol+ Mpiril\
`
`(H)
`_ 100
`£ 60
`c:
`..g 60~,
`~ 'II)
`.£' 20
`o
`1(10 1.lglkU (P,o,)
`100
`()
`Clopldl}(trlH 0
`1Q
`'ngl~g (p,Q.)
`0
`Anplf!l\
`10
`(l
`Figuro 6 Combined effects of (AI CS··i47 or fB) clopidogrel witll
`;l.,pil'ill
`
`(l111)1;1I<:ll<:)I."'~Hl'f:lcWiil)n il1c.iI.ll)c:ll.ll)yGI,lIl;IWln ill r~t~. i\H<:lnl~;
`wOI'O orallv <"dl'ninir;tol'edto I"t:l
`:lO minutu,l
`bolero blood collcc
`tion Platelet <i(.JI)I'80ation was
`indllced by 20 I'n/ml.. collagen,
`• n-: o.w';,
`':1:SI'.M
`(/l: ......OJ.
`H():;ulls
`illf:l
`('lXPlf:!:;~HCJ iI~
`IIHjilfl
`•• p '::0.01 \<01',;",; control by tho Dun,.,ott U»;\.
`
`(IO mg/kg,
`pronounced
`showed
`/,,0,)
`inhibition
`()f
`collagen-induced
`ap"
`indicating
`aggrc.;gation,
`platelet
`d'f.i~,(;t~of CS·-
`p;ll'l;nt addi,iw or oY'Wrgistlc Cl)lnbined
`the dr(;n~o(
`'/4'/ with
`:\~pirin, W(~ j',.,,'th<;)'
`c;v:\ll.l"t<~d
`('.1()pidogl'(~i (10() lng/kg,
`jU).) plus
`:\spil'in (10 lng/kg,
`!,.(i.) under
`the S;lI11e conditions
`and ('c)llne! only minimal,
`although statistically significant,
`inhibition. Taken
`to-
`the observed potent combined efficacy and early
`gether,
`of Hrl'i.I.JI)clf C5 ...'/47 plus aspirin
`11101)'huvc clinical
`onset
`relevance.
`
`Mechanisms of Antiplatelet Action
`
`ACflVE METABOllT!::
`Similar
`1:0 clopidogrel and riclopidine, CS-747 is innc-
`assays when
`rive in platelet
`aggregation
`added in vitro
`but, as described earlier, shows potent
`anriplarclcr
`and
`aurirhromboric
`effccrs ill vivo alter oral. dosing. suggest·'
`the; pl'\'~('Il\'{' ()f an active metabolirc
`of CS- '147,
`ing
`J\.nlc)llr~ tht! hepatic metabolites o( CS-747, W\~illiti"lIy
`identified R-~n2(·1 as a metabolire ",ilh
`phll.elc~('-inhibi-
`tory ],ropcrticti,2l,n
`Based on the chemical
`structure of
`this active mctubolirc, which contains two chiral carbons
`possible.
`in
`it·~ ,tt'll(l'Ill'("
`'t(,)'\",is'>IlI('r~
`are
`1~.Hll'
`I 1owev C:! 1', ahhollgh
`studies
`have b(;..,,) p"l'li.l)'lIl\'e,l
`several
`with R-')C)224, structural analysis has shown it
`I.(J be a
`mixture of only two srcrcoisomcrs,
`(R, S)..isomer and
`(8, R)'''i~onl(:i', R..:n:ntly, we identified
`:tnd characterized
`,1~ th(~ <\c:!.iVt~ nWI::.\b()lil:~!of' CS-'/47, ~:()n('aill'"
`R- 1:18727
`jn!~ the
`full c:()mph~rl'lcnt of
`the
`i'i'l111' optic:\]
`isonwl'S.
`
`P"IMMACO~OOYOf'CS'747/Nllll~U
`
`1'.'1AI
`
`189
`
`studies
`AggJ'\Wttion
`rhar
`it has p"opel't'ie5
`R-99224.:)~
`
`shown
`have
`R"138'!27
`with
`a1i:dogou~ ro those described li)l'
`
`AGONIST SELECTIVITY
`To determine
`the selectivity of CS-7A7 and irs active
`mcrabolire
`for
`diffcrent·
`awmi:;t:;, washed
`platelet
`plnrelcrs were used \'0 eliminate
`fibrin
`fonnution
`till'
`is u${:d in
`pb"I);\,
`I·hal'
`Ih"()I)IJ,i"
`As
`J\:~lllt~ when
`"I, R-99224
`shown
`in Figul'('
`(0.03 10 1. f.lg;/ml .) inhihi\<:d
`by
`in virro washed
`human
`platelet
`inducl:d
`'Im~l'l:g:llion
`ADP, The inhibition was concentration
`related, demon-
`that: reduced response by S()'N,
`srrarinj,
`il concentration
`(Jeso) of O,ll ~lg/mL. R,·99224 did not
`:dJ(_'ci:ADp ..
`induced shape change even at rhe highest COllu'l1trmio)'l
`aggl'(~g:\t'i()n ili<ll!(~(~d hy {:nl1:1g"'l
`used.
`i)l:\I(~kl
`and
`low conccntrarions
`o( thrombin was ulso p:tl'li:llly
`in-
`hy the highest
`hibited
`C01Kt:t1tT,ltiOI\ of R-99224 used
`
`(A)-~80 ,.
`tc 60sIII 40
`
`1:71
`
`~
`I:fl 20
`rn
`<
`0
`
`(8)
`
`100
`~ 80 -
`t_
`c:
`0 60
`-;;;
`40·
`~~.20
`C)~ ()
`
`0)
`
`(C)
`_100
`
`~ 80-c
`0 60
`:tl
`III:? 40·
`....
`Cl 20s o
`
`0.03
`
`0,3
`0,01
`COllagen (~glml'
`
`-o- Control
`--
`(1.03 'Jo/mi
`-0-0.'
`
`-0-- Control
`-+- 0.03 Ilo/rn I
`-0-0.-1
`-+O,l
`-s- 1
`
`0,1
`0.Q3
`0.01
`Thrombin (wllt1ml)
`In vitro c·)[[c·)CI:;of n-HOn'i
`(O.O:l--l
`Figura'
`!((J/III!..) Oil plillul(~1
`<lg!.jl'eoo\iol1 induced by (N ADP, (B) collaqen, MId (C) thrombin in
`!)UI'rk'lI) pliJ(l·~IBt:.~.H(1~ull:) (oH('~f~XPIH~.~:)(~da:i
`Wi)~I)({d
`Ih(·:
`IrH~ilt\
`'I
`•p< O.Oli,
`"p '::0.01
`corm:;po"dil1()
`SEM (n .5-·,6).
`VW,lU:;
`by
`llll:' 1_)ljflnc~llV~~;l.(From S"Oid~,-,hi A,
`I~t ,.1, 131' J
`conlrc)i
`by p':!lIlli:;,~io".1
`PhilrmtlGol 2001 ;132:bO.
`fj')j)lilltwl
`
`:01. This page 87 of 99 was completed at 24.12.2010 14:15:53
`Duration: 24.12.2010 13:40:37 - 24.12.201014:21
`Received at the EPO on Dec 24, 2010 14:21 :01. Page 87 of 99
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`
`Ex. 1044, p. 6 of 11
`
`
`
`24/12
`
`2010 FRI 13:17
`
`FAX 01992561934
`
`~~~ EPO Munioh
`
`~088/099
`
`190
`
`SI:MINARS IN THROMBOSIS AND HEMOSTASISIVOlUME
`
`~1, NUMBER ~ 2005
`
`the
`
`(l 1.lg/mL), However,
`I~-
`~cliYilX of
`anriplareler
`m-
`thrombin-induced
`99224
`and
`against
`collagen-
`against
`compared
`with
`that
`wus minimal
`SpOrlS(;S
`In rat platelets,
`the inhibitory
`ADl'-indu(C(,d
`responses.
`profile or plnrelet
`ag~~!,,'gat"il)J)observed was similar
`to the
`:dh,,' oral dosing.
`ex vivo p[''''fil~ I,ll' C$-747
`Further-
`more,
`intravenous
`administrution
`nf R-99224 (0.1 t·,)
`:)
`of
`mg/kg)
`to rats
`resulted
`in d()~c-!'d:H~,d inhihi(·ioll
`AJ)P:
`induced
`platelet
`<l!mregation.
`'Fhesc pliarmacolo-
`giutl
`results
`support
`the contention
`that R-99224 is an
`active metabolite ofCS··''147 responsible for inhibition of
`pl;m.:kt
`;\ggr(;galJ.e.lJ), We also note
`of
`that
`the inhibition
`by R..99224 persisted
`in vitro
`1':1\ pla\~'.h:1 aggrcg:ni.on
`;\(1t11' wr",hing tl\(: pl:\!·d(:t~ lip to rb)'~~ctimes to eliminate
`free R-9'J224 from rhe sysl(:n\. Similarly,
`inliibition
`of(;x
`aggree;ario!1 by CS-747
`(1 mg/kg, JUl.)
`vivo rat platelet
`was not diminished
`ann
`the platelets were washed three
`times, Sl1I9~estinl~ that
`the ultimate
`anriplatcler
`actions o(
`CS···'14·/
`and H,·992:?4
`arc similarly
`irreversible.
`These
`results arc consistent with the 10111-\ duration
`of action of
`CS-747 :I~ d(~,;rl'ib(,d curlier.
`
`P2Y1l BECEPTOR SELECTIVITY
`ro several
`Stimulation
`of platelets wirh ADP leads
`physiological.
`n·.~p()nses,
`iJ1l.:1mlinl~shape
`change,
`almre-
`or granule
`g:\1iOII, and secretion
`contents.
`These plarclcr
`l'('.~p(>ll~(:;; a)'<~.\'.ol1;;idt;)'(~d to be mediated
`by at
`leaw two
`distinct
`1'1I1'illt:l'gic (P2)
`1'(!(~epl·OI'';:J>2Yj nnd
`J>2Yj/..:1'1
`We examined
`the effects of R-')9224
`on rhe binding
`oj'
`CJ 1]-2-mcrhylthio-ADP
`(11I 1I-2-McS-ADP),
`a st:;blc
`liRall(l for both the P2Y I and P2Y l2 ADP receptors,·!()·,11
`to washed
`human
`plarclcrs to determine
`receptor spcci ..
`ficity. Trcarmcnt
`1(.,99224
`(0.1
`100 ~IM)
`with
`to
`I)f [:lUll·
`1'J'<)(hn:(:d a cnnccutratiou-rclatcd
`inhibirion
`
`tl_'
`
`Md)-·A.DP
`:( pl:JI~::,(l
`rc,\chillg
`binding, with inhibition
`l.lg/m1.,) H.··9922'L It should
`:3 pM.
`at
`Iw llnled,
`(1.4
`by 1<.,,·99224was subrnax-
`however,
`fh:ll: rliis inhibition
`(84% or C()[iI",'()l) <;V';ll at
`imul
`the highcsr
`concentration
`inhibited n r]-2-M"S-AIW
`used (100 ~lM). In ex vivo (,)1p(~!'inH:nl.$, CS-'14';
`also
`partially
`bindillg
`characterize
`To further
`rat platelets.
`washed
`the hindill!!;
`properties
`of H-99224, we used 0.3 pM ARL-(,(,O%,
`a
`and 300 ~1M.AJPSPS,
`s(,kCfiv~: P2YI~ :1I'tag()lJist,·u,~:I
`1.>2)\
`:\
`selcc(ive.'
`all1"ag()lli~t.4;1"H The
`couccnrrarions
`inhibition
`employed
`ShO'Wl~d maximum :11.:J,i\:vahk
`of
`1.:1Jn-2-McS-ADP
`binding.
`As
`shown
`in
`Figure.:
`8,
`10 ~1M.(/1.6 ~lr~/mL) of R-9922<1 in combination
`with
`C:;lIm.l no additional
`of [\lll-2-
`A.RL"M09()
`inhibition
`Mt.:.S-AI)P
`binding,
`In contrast,
`H··99224
`(10 pM)
`in
`abolished e'II}·
`(:(jnlbill~l!i()n with A3P5PS
`compl(~lely
`2-MeS-ADj)
`binding.
`I.ikewis«, A HI ,-660')6 combined
`wirh A3PSJ>S
`'lbolished
`C~ll]-2-Md)-ADP
`bil)(ling,
`These
`results
`clearly
`indicate
`rhar R-9<J221.{ Ius inhibi-
`wry activity
`to rhat of AH!..-
`toward
`P2Y 12 similar
`66096,
`is ;\ selective P2Y\2
`suggesting
`rhat R,99224
`ADP receptor aurugcnisr.
`To li.ll,th~~[' d(!\"Cl'lnill<; wh<:!thel' H.-992:'.4 il\hibit~
`we invcsligfltl:!d
`(h.., d'I~\'.I~()J"
`P2Y,,~ Iuncrion,
`plarelcr
`on
`ADP-induccd
`intracellular
`,;ignali.ng
`R-99224
`c';
`cvt·.nrs,7.2 P2Y1 receptors
`arc linked
`to hctC!"odin:lcric
`(G,) that
`proteins
`stimulate
`phospholipase
`C,
`leading
`to
`(1r intruu-Ilulnr
`Ca2 I .;) However,
`mobiJil',:II·i()ll
`R-99224
`hnd no em:CIS on ADP-iIH.loc(,d
`Ca~ I
`tnohili'l.:lIio!)
`in
`suggesting
`no activity
`of R-SI9224 on
`human
`platelets,
`In contrast, R..99224 neutralized
`P2Y lAD]>
`receplOrs,
`(PC) E.·
`ADP-medialed
`inhibition
`of prostagl:llldin
`induced
`udcnylyl
`cyclase
`ucrivaiion
`ill human
`pl:lll~kt~
`9), whid\
`is J)1(,di;l(~:d by P2Y)2 AI)!'
`(Fig,
`I'(:r~pt(_'l'~,
`
`(A)
`
`(B)
`
`(J)
`
`100
`
`'"'5
`t!
`:0 _
`o..Y.l
`.iI!
`CJ
`<f~
`III 60
`Ole.
`~~
`~~ 40
`~E
`u:::.
`.[
`;:=
`
`())
`
`80
`
`20
`
`r----,
`NS
`,@.,
`
`"'*
`
`120
`
`100
`80
`
`60
`40
`
`ZU
`
`fI'I
`
`0
`R-99224
`ARL·B6096
`
`+
`
`+
`+
`
`...
`
`0
`R·99224
`ARL-G6096
`+
`+
`+
`A3P5PS
`'1-
`Figure 8 (AI In vitro erlect~ of fHl9224 (10 ~IMI combined with ABL·66W6 (0.3 IlM) on I'HI·2-M(:!S·ADI)
`!)lndill\110 W<lslwd Il\HT,,1I1
`('ombined wilh IH~9224 (10 I.lM) or AHL·6G096 (0,3 ~lMI on ('HI··2··MeS·-ADP billdil1D to
`platelets. (13)In vitro dfl:,ct of A:lPljl'S (:.l(l(lI\Ml
`w:d,ed
`hlHT\,lll platelets. Hesults am OXP{(·J(;f,(·ld as lilt> ]\)(<<)I\:l:- SLM (II zr., (j). •• 1)< 0.0·', {\J~;,Iwl ~;i9niJi(';.lnt by H\e Tukev \o~t.
`(Frorn
`SU(jid!l(;i