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8/12/2019 Coccidial Vaccine and Methods of Making and Using Same (Patent)
1/41
Coccidial vaccine and methods of making and using sameWO 2006113594 A1
http://www.google.com/patents/WO2006113594A1?cl=en
Nomo
!u"likasiWO2006113594 A1
#enis
!u"likasiPenerapan
Nomo
a!likasiPCT/US2006/014354
$anggal!u"likasi
26 Okt 2006
$anggal
!enga%uan14 Apr 2006
$anggal
!ioitas15 Apr 2005
#ugadi!u"likasikan
se"agai
CA2605165A1, EP1877085A1,EP1877085B1,US7354593,US20070026023
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&emohon "er*a( Lt$,4 La*nna .
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&t*pan Paten33,&t*pan %nPaten23,*r&&k %(e-1,(a**ka*6,Per*t*a :&k&;9
http://www.google.com/patents/WO2006113594A1?cl=enhttp://www.google.com/patents/CA2605165A1?hl=id&cl=enhttp://www.google.com/patents/EP1877085A1?hl=id&cl=enhttp://www.google.com/patents/EP1877085B1?hl=id&cl=enhttp://www.google.com/patents/EP1877085B1?hl=id&cl=enhttp://www.google.com/patents/US7354593?hl=id&cl=enhttp://www.google.com/patents/US7354593?hl=id&cl=enhttp://www.google.com/patents/US7354593?hl=id&cl=enhttp://www.google.com/patents/US20070026023?hl=id&cl=enhttp://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Larry+R+Mcdougald%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Alberta+Lorraine+Fuller%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Joyce+Anita+Pritchard%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Joyce+Anita+Pritchard%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=inassignee:%22Merial+Ltd%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=inassignee:%22Merial+Ltd%22http://www.google.com/patents/WO2006113594A1.bibtex?cl=enhttp://www.google.com/patents/WO2006113594A1.bibtex?cl=enhttp://www.google.com/patents/WO2006113594A1.enw?cl=enhttp://www.google.com/patents/WO2006113594A1.ris?cl=enhttp://www.google.com/patents/WO2006113594A1.ris?cl=enhttp://www.google.com/patents/WO2006113594A1?cl=en#backward-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#npl-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#npl-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#forward-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#forward-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#classificationshttp://www.google.com/patents/WO2006113594A1?cl=en#legal-eventshttp://www.google.com/patents/WO2006113594A1?cl=en#legal-eventshttp://www.google.com/patents/WO2006113594A1?cl=enhttp://www.google.com/patents/CA2605165A1?hl=id&cl=enhttp://www.google.com/patents/EP1877085A1?hl=id&cl=enhttp://www.google.com/patents/EP1877085B1?hl=id&cl=enhttp://www.google.com/patents/US7354593?hl=id&cl=enhttp://www.google.com/patents/US20070026023?hl=id&cl=enhttp://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Larry+R+Mcdougald%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Alberta+Lorraine+Fuller%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=ininventor:%22Joyce+Anita+Pritchard%22http://www.google.com/search?tbo=p&tbm=pts&hl=en&q=inassignee:%22Merial+Ltd%22http://www.google.com/patents/WO2006113594A1.bibtex?cl=enhttp://www.google.com/patents/WO2006113594A1.enw?cl=enhttp://www.google.com/patents/WO2006113594A1.ris?cl=enhttp://www.google.com/patents/WO2006113594A1?cl=en#backward-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#npl-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#forward-citationshttp://www.google.com/patents/WO2006113594A1?cl=en#classificationshttp://www.google.com/patents/WO2006113594A1?cl=en#legal-events8/12/2019 Coccidial Vaccine and Methods of Making and Using Same (Patent)
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8/12/2019 Coccidial Vaccine and Methods of Making and Using Same (Patent)
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T-* app(*#at*%n #(a*; pr*%r*t t% U>S> App(*#at*%n Ser*a( %> 11/348,084, *(e$ +e)r&ar 6, 2006 -*#- * a
#%nt*n&at*%n*npart % U>S> App(*#at*%n Ser*a( %> 11/106,780, *(e$ Apr*( 15, 2005, -*#- *&e$ a U>S>
Patent %> 6,998,127 %n +e)r&ar 14, 2006, -*#- * a #%nt*n&at*%n % U>S> App(*#at*%n Ser*a( %>
10/730,206, *(e$ e#e;)er 8, 2003, -*#- *&e$ a U>S> Patent %> 6,908,620 %n &ne 21, 2005, -*#-
#(a*; pr*%r*t t% U>S> Pr%?**%na( App(*#at*%n Ser*a( %> 60/432,298 ent*t(e$ DCOCC
8/12/2019 Coccidial Vaccine and Methods of Making and Using Same (Patent)
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tra#t, re&(t*n' *n t-e re(eae % %&r p%r%#t> T-e p%r%#t pa *nt% t-e $&%$en&; -ere t-e are
e@p%e$ t% )*(e an$ $*'et*?e enG;e re&(t*n' *n t-e re(eae % t% p%r%G*te per p%r%#t>
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re(eae$ r%; t-e ;*#r%'a;et%#te, a G'%te * %r;e$ -*#- #reate a #t a(( a)%&t *te(> T-e ne(
%r;e$ %%#t * pae$ %&t % t-e *ne#te$ #-*#ken *t- t-e e#a( $r%pp*n'>
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ee$ #%n?er*%n, #eat*%n % e'' pr%$t*%n an$, *n %;e #ae, $eat-> T-e *n#reae *n *nten*?e
pr%$t*%n % p%&(tr -a )een a##%;pan*e$ ) e?ere (%e $&e t% t-* para*te= *n$ee$, #%##*$*%*
-a )e#%;e an e#%n%;*#a(( *;p%rtant para*t*# $*eae> Pr*%r t% t-e a$?ent % #-e;%t-erape&t*# a'ent, *;pr%?e$ an*tat*%n &*n'
$**ne#tant, t%'et-er *t- t-e ;e#-an*#a( re;%?a( % (*tter, a t-e ;a*n ;et-%$ e;p(%e$= &*#*ent
%%#t, -%e?er, &&a(( re;a*ne$ t% tran;*t t-e $*eae> T-e *ntr%$t*%n % #%##*$*%tat*# a'ent *n
t-e ee$ %r $r*nk*n' ater, *n a$$*t*%n t% '%%$ ;ana'e;ent, re&(te$ *n %;e #e at $*eae #%ntr%(>
S- a'ent -a?e )een %&n$ t% &er r%; a $r%p *n ee#t*?ene %?er t-e ear, $&e part( t% t-e
$e?e(%p;ent % $r&' re*tant tra*n % #%##*$*a> +&rt-er;%re, e?era( #-e;%t-erape&t*# a'ent -a?e
)een %&n$ t% (ea?e re*$&e *n t-e ;eat, ;ak*n' *t &n&*ta)(e %r #%n&;pt*%n> U>S> Patent %>
4,438,097= 4,639,372= 4,808,404= 5,055,292= 5,068,104= 5,387,414=
5,602,033= 5,614,195= 5,635,181= 5,637,487= 5,674,484= 5,677,438= 5,709,862= 5,780,289= 5,795,741=
5,814,320= 5,843,722= 5,846,527= 5,885,568= 5,932,225= 6,001,363 an$ 6,100,241 re(ate t% #%##*$*%*
?a##*ne, *n#(&$*n' (*?e an$ re#%;)*nant ?a##*ne> :%e?er, t-ere are pr%)(e; *t- e@*t*n' #%##*$*%*
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a?a*(a)(e a pr*%r art t% t-e preent *n?ent*%n> SU""A!J O+ T:E
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T-e a$$*t*%n % %t-er E*;er*a tra*n, - a E> )r&nett*, E> ne#atr*@ an$ E> prae#%@ ;a )e
$*a$?anta'e%& *t- repe#t t% e*#a#, #r%*ne#t*%n %r pat-%'en*#*t % t-e ?a##*ne> S*n#e E>
)r&nett*, E> ne#atr*@ an$ E> prae#%@ are n%t ne#ear %r t-e e*#a# % t-e #%##*$*%* ?a##*ne $*#(%e$
-ere*n, *t %&($ )e a$?anta'e%& t% e@#(&$e t-ee tra*n r%; t-e ?a##*ne % t-e preent *n?ent*%n>
, t-e #an ;ean D*n#(&$eD, D*n#(&$e$D, D*n#(&$*n'D, an$ t-e (*ke= an$ t-at ter; - a
D#%n*t*n' eent*a(( % an$ D#%n*t eent*a(( % -a?e t-e ;ean*n' a#r*)e$ t% t-e; *n U>S> Patent
(a, e>'>, t-e a((% %r e(e;ent n%t e@p(*#*t( re#*te$, )&t e@#(&$e e(e;ent t-at are %&n$ *n t-e pr*%r art%r t-at ae#t a )a*# %r n%?e( #-ara#ter*t*# % t-e *n?ent*%n>
T-ee an$ %t-er e;)%$*;ent are $*#(%e$ %r are %)?*%& r%; an$ en#%;pae$ ), t-e %((%*n'
eta*(e$ e#r*pt*%n> ETA Pre#%#*%& tra*n are $er*?e$ r%; *e($
pe#*e are n%t pat-%'en*# -en a$;*n*tere$ at t-e r*'-t $%e> %-n%n, t-e $*#(%&re %
-*#- * *n#%rp%rate$ ) reeren#e *n *t ent*ret> "*#r%%r'an*; #an )e atten&ate$ ) t-e*r e(e#t*%n %r
pre#%#*%& $e?e(%p;ent a $e#r*)e$ a $e#r*)e$ *n A?*an Pat-%(%', 17 305314, 1988 ent*t(e$
DE*;er*a % A;er*#an C-*#ken C-ara#ter*t*# % S*@ Atten&ate$ Stra*n Pr%$e$ ) Se(e#t*%n %r
Pre#%#*%& e?e(%p;ent, P>L> L%n' an$ %#e > %-n%n, t-e $*#(%&re % -*#- * *n#%rp%rate$ )
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per*%$, *>e>, -en t-e %%#t *rt -% &p *n t-e e#e> S- ;et-%$ are e(( kn%n t% %ne % k*(( *n
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t-e art an$ #%nt*t&te r%&t*ne e@per*;entat*%n> St%#k #&(t&re % t-e E*;er*a tra*n %r t-e ee$ #&(t&re
*n#(&$e, )&t are n%t (*;*te$ t%, t-e %((%*n'> T-e parent % t-e E*;er*a a#er?&(*na, %)ta*ne$ r%; T> >
eer at :e an$ C(ark La)%rat%r*e *n 1969, a t-%&'-t t% -a?e )een *%(ate$ ) r> "> "> +arr at
USA, Be(t?*((e, ", -*#- a $er*?e$ r%; a *n'(e %%#t> T-e E*;er*a ;a@*;a #&(t&re a $er*?e$
r%; an *nter)re$ ;*@t&re % 10 p&r**e$ *%(ate %)ta*ne$ r%; e%r'*a, e(aare, "ar(an$, *r'*n*a an$
Te@a> T-e parent % t-e E*;er*a ;*t* #&(t&re a *%(ate$ r%; a*ne?*((e, e%r'*a *n &( 1978 an$
a p&r**e$ ) *n'(e %%#t *%(at*%n> T-e parent % t-e E*;er*a tene((a #&(t&re a %)ta*ne$ r%; a
#&(t&re ;a*nta*ne$ at Penn(?an*a State Un*?er*t ) r> Patten *n#e t-e ear( 1960F, an$ a
a#H&*re$ ) t-e Un*?er*t % e%r'*a *n 1982> Ot-er pre#%#*%& E*;er*a tra*n *n#(&$e LS(OO
pre#%#*%& E> A#er?&(*na *%(ate 80913, an$ LS pre#%#*%& E> ;*t*, %)ta*ne$ r%; "er#k !eear#-
La)%rat%r*e, -*#- ere %)ta*ne$ r%; r> Peter L%n'> A(ternat*?e(, atten&ate$ pre#%#*%& E*;er*a (*ne
t-at -a?e )een $ep%*te$ a p%r%#t at t-e E&r%pean C%((e#t*%n % An*;a( Ce(( C&(t&re DECACCD a
patent $ep%*t ee, e>'>, U>S> Patent %> 5,055,292, t-e $*#(%&re % -*#- * *n#%rp%rate$ ) reeren#e
*n *t ent*ret are &e&( t%#k #&(t&re t% 'enerate t-e E*;er*a ee$ #&(t&re $e#r*)e$ -ere*n>
Spe#**#a((, $ep%*t % E> a#er?&(*na $ep%*t n%> ECACC 86072203, E> ;a@*;a $ep%*t n%> ECACC
86112011 an$ 86112012, E> ;*t* $ep%*t n%> ECACC 86072206 an$ E> tene((a $ep%*t n%> ECACC
86072201 a $e#r*)e$ *n U>S> Patent %> 5,055,292 are &e&( t%#k #&(t&re %r t-e ee$ #&(t&re %
t-e preent *n?ent*%n> A$?anta'e%&(, t-e ;*#r%%r'an*; are atten&ate$ ) t-e*r e(e#t*%n %r
pre#%#*%& $e?e(%p;ent a $e#r*)e$ a)%?e> T-e t%#k #&(t&re $e#r*)e$ a)%?e are a$?anta'e%&( ;a*nta*ne$ *n t-e (*H&*$ %r ?ap%r
p-ae % (*H&*$ n*tr%'en> S- ;et-%$ are kn%n t% %ne % k*(( *n t-e art> Sp%r&(ate$ %%#t are pae$ #e*?e(,
*t-%&t (*;*tat*%n t% t-e paa'e, *n #-*#ken &nt*( t-e n&;)er % %%#t are &*#*ent t% )e &e$ a
ee$ %r pr%$t*%n> A$?anta'e%&(, t-e #&(t&re -%&($ n%t )e -e($ %r (%n'er t-an 12 ;%nt- *n %r$er
t% ;a*nta*n ?*a)*(*t/*ne#t*?*t> T-e p%r&(ate$ %%#t r%; t-e ee$ #&(t&re are *%(ate$ r%; t-e )*r$ e#e,
a$?anta'e%&( ) #entr*&'at*%n> T-e &pernatant * $*#ar$e$, an$ t-e %(*$ are re&pen$e$ *n a at&rate$ aC( 80/? %(&t*%n *n ater> T-e re&(t*n' %(&t*%n * #entr*&'e$> T-e %%#t are #%((e#te$ re;%?e$ r%; t-e
t%p % t-e (*H&*$, an$ re&pen$e$ *n ater> Opt*%na((, t-e re;a*n*n' (*H&*$ * $*(&te$ t% 20 40 aC(
*t- ater an$ #entr*&'e$> T-e pe((et * t-en re&pen$e$ *n a at&rate$ aC( %(&t*%n an$ re
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%$*&; -p%#-(%r*te %(&t*%n ) repeate$ 3 #entr*&'at*%n an$ re&pen*%n tep> T-e *na(
re&pen*%n * ;a$e *n a 2>5 aH&e%& %(&t*%n % p%ta*&; $*#-r%;ate 2CrO7> T-e %%#t ere
t-en tranerre$ t% p%r&(at*%n ?ee(> Sp%r&(at*%n * a#*(*tate$ ) par'*n' t-e &pen*%n *t- a*r %r a
per*%$ n%t t% e@#ee$ 72 -%&r at 27M30C> +%((%*n' p%r&(at*%n t-e %%#t are -e($ at 5M 30C &nt*( t-e
*na( pr%$t * pr%$e$> S-
;et-%$ *n#(&$e t-%e $e#r*)e$ *n > +> !(e at a(>, Para*t%(%' 73311326, 1976, P> L> L%n' et a(>,
+%(*a eter*nar*a Lat*na S> Patent %> 6,627,205, t-e $*#(%&re % -*#- are
*n#%rp%rate$ ) reeren#e *n t-e*r ent*ret*e> A##%r$*n' t% %ne ;et-%$, #%;;er#*a( )r%*(er #-*#ken,
appr%@*;ate( 2 eek %($, are *ne#te$ *t- t-e E*;er*a pe#*e % *nteret ) %ra( 'a?a'e % an
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57 $a p%t *ne#t*%n, )(en$e$ an$ *(tere$ t% re;%?e $e)r*, t-en #entr*&'e$ at a pee$ &*#*ent t%
pe((et t-e re;a*n*n' e#a( ;ater*a(> T-e pe((et * re&pen$e$ *n a at&rate$ a(t %(&t*%n, *n -*#- t-e
%%#t (%at an$ ;%t % t-e #%nta;*nat*n' $e)r* #an )e re;%?e$ ) #entr*&'at*%n> T-e %%#t
&pen*%n * t-en $*(&te$ t% (%er t-e a(t #%n#entrat*%n> T-e %%#t are a-e$ repeate$( t% re;%?e
t-e a(t an$ re&pen$e$ *n p%ta*&; $*#-r%;ate %(&t*%n 2>5 /?> T-e %%#t &pen*%n *
*n#&)ate$ at 29 C *t- -ak*n' e>'>, 140 rp; %r appr%@*;ate( 72 -%&r t% *n$e p%r&(at*%n % t-e
%%#t> A(ternat*?e(, t-e %%#t #an )e treate$ *t- %$*&; -p%#-(%r*te an$ t-en p%r&(ate$> T-e
n&;)er % p%r&(ate$ %%#t/;( * $eter;*ne$ ) $*re#t #%&nt &*n' a -e;%#t%;eter %r "#"ater (*$e,
an$ t-e #&(t&re * t%re$ &n$er rer*'erat*%n &nt*( nee$e$> T% prepare p%r%#t, t-e p%ta*&;
$*#-r%;ate * re;%?e$ r%; t-e %%#t &pen*%n $e#r*)e$ a)%?e ) repeate$ a-*n' % t-e %%#t,
-*#- *n?%(?e #%((e#t*%n % %%#t ) #entr*&'at*%n an$ re&pen$*n' *n $e*%n*Ge$ %r $*t*((e$ ater>
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&pen*%n * ter*(*Ge$> A$?anta'e%&(, t-e ter*(ant * )eta pr%p*%(a#t%ne BPL> +%r e@a;p(e, %%#t
#an )e )r%ken t% re(eae p%r%#t ) ;*@*n' t-e %%#t *t- '(a )ea$ % 14 ;; $*a;eter an$-ak*n' ) -an$, ?%rte@ ;*@er, %r -ak*n' *n#&)at%r, %r &*n' a -an$-e($ -%;%'en*Ger> Un)r%ken
%%#t an$ %%#t a(( #an )e eparate$ r%; t-e re(eae$ p%r%#t ) $*erent*a( #entr*&'at*%n *n
50 PE!COLL, a #%((%*$a( &pen*%n % p%(?*n( prr%(*$%ne #%ate$ *(*#a part*#(e %($ ) P-ar;a#*a
B*%te#- %r 1 " r%e a $e#r*)e$ *n &(k* et a(>, A?*an *eae, 32 235239, 1988> T-e p%r%#t
#an )e &e$ *n t-e preent ?a##*nat*%n ;et-%$ e*t-er ;*@e$ *t- %r eparate$ r%; t-e &n)r%ken %%#t
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an$ %%#t a((> A$?anta'e%&(, t-e $%e % p%r%#t * eparate$ r%; t-e %%#t an$ %%#t
a((>
, L%n' %ner, Pr%t%G%%(>
1984 %?= 314 53541 an$ S-*r(e, A#ta et :&n'> 1997= 453 33147, t-e $*#(%&re % -*#- are
*n#%rp%rate$ ) reeren#e>
T-e *%(ate$ p%r&(ate$ %%#t r%; t-e ee$ #&(t&re $e#r*)e$ -ere*n are #%;)*ne$ t% %r;&(ate a
;*@t&re % p%r&(ate$ %%#t r%; pre#%#*%& tra*n % E> a#er?&(*na, E> ;a@*;a an$ E> tene((>enera((, t-e ;*@t&re * a)%&t 10 t% a)%&t 1000 %%#t % E> #er?&(*n, a)%&t 10 t% a)%&t 100 %%#t
% E> ;a@*;a an$ a)%&t 10 t% a)%&t 1000 %%#t % E> tene((a> A$?anta'e%&(, t-e ran'e % p%r&(ate$
%%#t *n t-e ;*@t&re * a)%&t 125 t% a)%&t 500 %%#t % E> a#er?&(*na, a)%&t 25 t% a)%&t 100 %%#t
% E> ;a@*;a an$ a)%&t 25 t% a)%&t 500 %%#t % E> tene((a> a#er?&(*na, a)%&t 25 %%#t % E> ;a@*;a an$ a)%&t 25 %%#t % E> tene((a> a#er?&(*na, a)%&t 50 %%#t % E>
;a@*;a an$ a)%&t 50 %%#t % E> tene((a> ;*t*, a)%&t 125 %%#t % E> ;*t* *n a (% $%e, a)%&t 250 %%#t % E> ;*t* *n a
;e$*&; $%e an$ a)%&t 500 %%#t % E> ;*t* *n a -*'- $%e>
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125 t% a)%&t 500 %%#t % E> ;*t*, a)%&t 125 %%#t % E> ;*t* *n a (% $%e, a)%&t 250 %%#t % E>
;*t* *n a ;e$*&; $%e an$ a)%&t 500 %%#t % E> ;*t* *n a -*'- $%e>
T-e *n?ent*%n a(% re(ate t% pe#**# rat*% % p%r&(ate$ %%#t *%(ate$ r%; pre#%#*%& tra*n % E>
a#er?&(*na, E> ;a@*;a an$ E> tene((a, -ere*n t-e rat*% % E> a#er?&(*na*E> ;a@*;a E> tene((a * a)%&t 101
t% 22 t% 10 *>e>, %r e?er 10 p%r%#t % E> a#er?&(*na, t-ere are a)%&t 1 t% 2 p%r%#t % E> ;a@*;a
an$ a)%&t 2 t% 10 p%r%#t % E> tene((a> A$?anta'e%&(, t-e rat*% % E> a#er?&(*naE> ;a@*;a E>
tene((a * a)%&t 511 *>e>, 10 22>
a#er?&(*naE> ;a@*;aE> ;*t*E> tene((a * a)%&t 5151 *>e>, 10 2102>
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A$?anta'e%&(, t-e ;*@t&re * a)%&t 500 %%#t % E> a#er?&(*na, a)%&t 50 t% a)%&t 100 %%#t % E>
;a@*;a an$ a)%&t 100 t% a)%&t 500 %%#t % E> tene((a> ;*t*>
A$?anta'e%&(, t-e %%#t are &pen$e$ *n a preer?at*?e #%n*t*n' % a 0>01" p-%p-ate )&ere$
a(*ne %(&t*%n #%nta*n*n' 'enta;*#*n> A$?anta'e%&(, t-e %%#t are t%re$ *n ter*(e, )%r%*(*#ate '(a ?*a(> +%r e@a;p(e, )&t
n%t ) (*;*tat*%n, t-e %%#t are aept*#a(( *((e$ *nt% ?a##*ne ?*a( *t- a e;*a&t%;at*# %r a&t%;at*#
$*pener, t%pper are ;e#-an*#a(( %r ;an&a(( *nerte$ an$ a(&;*n&; ea( are p(a#e$ an$ #r*;pe$>'> @ant-an '&; %r '&; a#a#*a, a
#e((&(%e $er*?at*?e, e>'> #ar)%@;et-( #e((&(%e, -$r%@pr%p( ;et-( #e((&(%e %r ;*#r%#rta((*ne
#e((&(%e, #ara'eenan, %$*&; a('*nate, pe#t*n %r tar#-= a p%(pept*$e &pen$*n' a'ent - a 'e(at*n=
a nt-et*# p%(;er &pen$*n' a'ent - a p%(a#r(*# a#*$= %r a *(*#ate &pen$*n' a'ent - a
;a'ne*&; a(&;*n*&; *(*#ate ee, e>'>, U>S> Patent %> 5,055,292, t-e $*#(%&re % -*#- *
*n#%rp%rate$ ) reeren#e *n *t ent*ret>
T-e preent *n?ent*%n a(% pr%?*$e %r ?er**n' t-e p%r&(ate$ %%#t are #-ara#ter*t*# % t-e
pre#%#*%& tra*n % E> a#er?&(*na, E> ;a@*;a %r E> tene((a> A$?anta'e%&(, a(( %%#t are atten&ate$, *n
t-at t-e are pre#%#*%&> , #-*#ken> T-e #-ara#ter*t*# % t-e ?ar*%&
E*;er*a pe#*e are &(( et %&t ) L%n' P> L> an$ !e*$ W> "> 1982 A &*$e %r t-e *a'n%* %
C%##*$*%* *n C-*#ken= Un*?er*t % e%r'*a !eear#- !ep%rt 404 an$ %ner L> P> 1978
a#er?&(*na, E> ;a@*;a an$ E> tene((a> A$?anta'e%&(, t-e tet*n' re(ate t% a$;*n*ter*n' a
#-a((en'e $%e % a)%&t 100,000 t% a)%&t 500,000 %%#t % E> a#er?&(*na an$ a)%&t 10,000 t% a)%&t
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20,000 t% a)%&t 500,000 %%#t % E> ;a@*;a %r a)%&t 20,000 t% a)%&t 50,000 %%#t % E> tene((a>
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;*t*, a$?anta'e%&( a)%&t 200,000 %%#t % E> ;*t* ;a )e &e$> T-e *n?ent*%n &rt-er pr%?*$e %r
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#%;;%n re&(t * a)%&t 1>90 %r 2>00> One p%*nt *n ee$ #%n?er*%n *n #%;;%n (*n'% * >01, -*#- eH&a(
a)%&t 0>5 -a( % a per#ent>
T-e preent *n?ent*%n re(ate t% *;;&n*G*n' a #-*#ken, a$?anta'e%&( a )r%*(er #-*#ken> :%e?er,
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p%r%#t % E> tene((a> A$?anta'e%&(, t-e rat*% % E> a#er?&(*naE> ;a@*;aE> ;*t*E> tene((a * a)%&t
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*ntrt*%n %r a$;*@t&re an$/%r a$;*n*trat*%n>
'>, *na#t*?ate$ %r atten&ate$ E*;er*a, %r an *;;&n%'en %r ant*'en %r ep*t%pe t-ere%, an$
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: an$/%r +, e>'>, : an$ += %r , S %r S( an$ " an$/%r = %r CA
t-e *;;&n%'en * a$?anta'e%&( P( an$/%r P2= %r '>, en? an$ 'a'/pr% %r 'a'/pr%= %r
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a e(( a k*t t-ere%re>
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a;e t*;e> T-* a$$*t*%na( *;;&n%'en*# #%;p%nent #an )e an atten&ate$ %r *na#t*?ate$ ;*#r%%r'an*;,
a re#%;)*nant #%ntrt %r &)&n*t e>'> pr%te*n, '(#%pr%te*n, p%(pept*$e, %r ep*t%pe> Ep*t%pe
$eter;*nat*%n pr%#e$&re, - a, 'enerat*n' %?er(app*n' pept*$e (*)rar*e :e;;er et a(>, et a(>, at&re
B*%te#-n%(%', 1999, 17, 533561, #an )e &e$ *n t-e pra#t*#e % t-e *n?ent*%n, t% $eter;*ne ep*t%pe %
*;;&n%'en, ant*'en, p%(pept*$e, '(#%pr%te*n an$ t-e (*ke, *t-%&t &n$&e e@per*;entat*%n> +r%;
t-at *n%r;at*%n, %ne #an #%ntrt n(e*# a#*$ ;%(e#&(e en#%$*n' - an ep*t%pe, an$ r%; t-at
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?e#t%r %r p(a;*$ t-at e@pre *;;&n%'en, ep*t%pe %r ant*'en= a(( *t-%&t &n$&e e@per*;entat*%n>
T-e p-ar;a#e&t*#a(( %r ?eter*nar*( a##epta)(e #arr*er %r ?e-*#(e %r e@#*p*ent are e(( kn%n t% t-e
%ne k*((e$ *n t-e art> +%r e@a;p(e, a p-ar;a#e&t*#a(( %r ?eter*nar*( a##epta)(e #arr*er %r ?e-*#(e %r
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?*tr%= a$?anta'e%&(, t-e #arr*er, ?e-*#(e %r e@#*p*ent ;a a#*(*tate trane#t*%n an$/%r *;pr%?e
preer?at*%n % t-e ?e#t%r %r pr%te*n> %e an$ $%e ?%(&;e are -ere*n $*#&e$ *n t-e 'enera(
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r%; t-* $*#(%&re rea$ *n #%n&n#t*%n *t- t-e kn%(e$'e *n t-e art, *t-%&t an &n$&e e@per*;entat*%n>
T-e *;;&n%'en*# #%;p%*t*%n an$ ?a##*ne a##%r$*n' t% t-e *n?ent*%n ;a #%;pr*e %r #%n*t
eent*a(( % %ne %r ;%re a$&?ant> S&*ta)(e a$&?ant %r &e *n t-e pra#t*#e % t-e preent *n?ent*%n
are 1 p%(;er % a#r(*# %r ;et-a#r(*# a#*$, ;a(e*# an-$r*$e an$ a(ken( $er*?at*?e p%(;er, 2
*;;&n%t*;&(at*n' eH&en#e , Pr%#> at(> A#a$> S$>, USA, 1996, 93, 28792883=
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WO98/16247, 3 an %*( *n ater e;&(*%n, - a t-e SPT e;&(*%n $e#r*)e$ %n p 147 % Da##*ne
e*'n, T-e S&)&n*t an$ A$&?ant Appr%a#-D p&)(*-e$ ) "> P%e((, "> e;an, P(en&; Pre 1995,
an$ t-e e;&(*%n "+59 $e#r*)e$ %n p 183 % t-e a;e %rk, 4 #at*%n (*p*$ #%nta*n*n' a H&aternar
a;;%n*&; a(t, 5 #t%k*ne, 6 a(&;*n&; -$r%@*$e %r a(&;*n&; p-%p-ate %r 7 %t-er a$&?ant
$*#&e$ *n an $%#&;ent #*te$ an$ *n#%rp%rate$ ) reeren#e *nt% t-e *ntant app(*#at*%n, %r 8 an
#%;)*nat*%n %r ;*@t&re t-ere%>
T-e %*( *n ater e;&(*%n 3, -*#- * epe#*a(( appr%pr*ate %r ?*ra( ?e#t%r, #an )e )ae$ %n (*'-t (*H&*$
para*n %*( E&r%pean p-ar;a#%p%e*a tpe, *%pren%*$ %*( - a H&a(ane, H&a(ene, %*( re&(t*n' r%;
t-e %(*'%;er*Gat*%n % a(kene, e>'> *%)&tene %r $e#ene, eter % a#*$ %r a(#%-%( -a?*n' a tra*'-t
#-a*n a(k( 'r%&p, - a ?e'eta)(e %*(, et-( %(eate, pr%p(ene '(#%(, $*#apr(ate/#aprate, '(#er%(
tr*#apr(ate/#aprate an$ pr%p(ene '(#%( $*%(eate, %r eter % )ran#-e$, att a(#%-%( %r a#*$,
epe#*a(( *%tear*# a#*$ eter>
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p%('(#er%( %r pr%p(ene '(#%( an$ %n t-e %t-er -an$ %(e*#, *%tear*#, r*#*n%(e*# %r -$r%@tear*# a#*$,
a*$ eter )e*n' %pt*%na(( et-%@(ate$, %rp%(%@pr%p(enep%(%@et-(ene #%p%(;er )(%#k, - a
P(&r%n*#, e>'>, L121>
A;%n' t-e tpe 1 a$&?ant p%(;er, preeren#e * '*?en t% p%(;er % #r%(*nke$ a#r(*# %r
;et-a#r(*# a#*$, epe#*a(( #r%(*nke$ ) p%(a(ken( et-er % &'ar %r p%(a(#%-%(> T-ee
#%;p%&n$ are kn%n &n$er t-e na;e #ar)%;er P-ar;e&r%pa, ?%(> 8, n%> 2, &ne 1996> One k*((e$ *n
t-e art #an a(% reer t% U>S> Patent %> 2,909,462, -*#- pr%?*$e - a#r(*# p%(;er #r%(*nke$ ) a
p%(-$r%@( #%;p%&n$ -a?*n' at (eat t-ree -$r%@( 'r%&p, preera)( n% ;%re t-an e*'-t - 'r%&p,
t-e -$r%'en at%; % at (eat t-ree -$r%@( 'r%&p )e*n' rep(a#e$ ) &nat&rate$, a(*p-at*# ra$*#a(
-a?*n' at (eat t% #ar)%n at%;> T-e preerre$ ra$*#a( are t-%e #%nta*n*n' 2 t% 4 #ar)%n at%;, e>'>
?*n(, a((( an$ %t-er et-(e;F#a(( &nat&rate$ 'r%&p> T-e &nat&rate$ ra$*#a( #an a(% #%nta*n %t-er
&)t*t&ent, - a ;et-(> Pr%$t %($ &n$er t-e na;e Car)%p%( B+ %%$r*#-, O-*%, USA are
epe#*a(( &*ta)(e> T-e are #r%(*nke$ ) a((( a##-ar%e %r ) a((( pentaert-r*t%(> A;%n' t-e;,
reeren#e * ;a$e t% Car)%p%( 974P, 934P an$ 971P>
A t% t-e ;a(e*# an-$r*$ea(ken( $er*?at*?e #%p%(;er, preeren#e * '*?en t% E"A "%nant%, -*#-
are tra*'-t#-a*n %r #r%(*nke$ et-(ene;a(e*# an-$r*$e #%p%(;er an$ t-e are, %r e@a;p(e,
#r%(*nke$ ) $*?*n( et-er> !eeren#e * a(% ;a$e t% > +*e($ et a(>, at&re 186 778780, &ne 4,
1960> W*t- re'ar$ t% trt&re, t-e a#r(*# %r ;et-a#r(*# a#*$ p%(;er an$ E"A are preera)( %r;e$ )
)a*# &n*t -a?*n' t-e %((%*n' %r;&(a
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*n -*#-
!1an$ !2, -*#- #an )e t-e a;e %r $*erent, repreent : %r C:3@ V 0 %r 1 , preera)( @ V 1 V ( %r 2,
*t- @ M V 2>
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T-ee p%(;er are %(&)(e *n ater %r p-*%(%'*#a( a(t %(&t*%n 20 '/1 aC( an$ t-e p: #an )e
a$&te$ t% 7>3 t% 7>4, e>'>, ) %$a aO:, t% pr%?*$e t-e a$&?ant %(&t*%n *n -*#- t-e e@pre*%n
?e#t%r #an )e *n#%rp%rate$> T-e p%(;er #%n#entrat*%n *n t-e *na( ?a##*ne #%;p%*t*%n #an ran'e
)eteen 0>01 an$ 1>5 /?, a$?anta'e%&( 0>05 t% 1 /? an$ preera)( 0>1 t% 0>4 /?>
T-e #at*%n*# (*p*$ 4 #%nta*n*n' a H&aternar a;;%n*&; a(t -*#- are a$?anta'e%&( )&t n%t
e@#(&*?e( &*ta)(e %r p(a;*$, are preera)( t-%e -a?*n' t-e
%((%*n' %r;&(a *n -*#- !1* a at&rate$ %r &nat&rate$ tra*'-t#-a*n a(*p-at*# ra$*#a( -a?*n' 12 t% 18
#ar)%n at%;, !2* an%t-er a(*p-at*# ra$*#a( #%nta*n*n' 2 %r 3 #ar)%n at%; an$ * an a;*ne %r -$r%@(
'r%&p>
A;%n' t-ee #at*%n*# (*p*$, preeren#e * '*?en t% "! P>, 1994, B*%#%n&'ate C-e;*tr, 5, 382
389, t% %r; "!
A$?anta'e%&(, t-e ;*@t&re *t- t-e a$&?ant * %r;e$ e@te;p%rane%&( an$ preera)(
#%nte;p%rane%&( *t- a$;*n*trat*%n % t-e preparat*%n %r -%rt( )e%re a$;*n*trat*%n % t-epreparat*%n= %r *ntan#e, -%rt( )e%re %r pr*%r t% a$;*n*trat*%n, t-e p(a;*$a$&?ant ;*@t&re * %r;e$,
a$?anta'e%&( % a t% '*?e en%&'- t*;e pr*%r t% a$;*n*trat*%n %r t-e ;*@t&re t% %r; a #%;p(e@, e>'>
)eteen a)%&t 10 an$ a)%&t 60 ;*n&te pr*%r t% a$;*n*trat*%n, - a appr%@*;ate( 30 ;*n&te pr*%r t%
a$;*n*trat*%n>
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W-en OPE * preent, t-e "!, 11>
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re*t a #-a((en'e $%e % a)%&t 100,000 t% a)%&t 500,000 %%#t % E> a#er?&(*na an$ a)%&t 10,000 t%
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*n?ent*%n> T-e ?a##*ne ;a )e a$;*n*tere$ ) an % t-e ;et-%$ e(( kn%n t% t-%e k*((e$ *n t-e art,
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Penn(?an*a State Un*?er*t ) r> Patten *n#e t-e ear( 1960F, an$ a a#H&*re$ ) t-e Un*?er*t %
e%r'*a *n 1982>
Ea#- % t-e E*;er*a tra*n &e$ a a pre#%#*%& tra*n % t-e repe#t*?e ;*#r%%r'an*; a $e#r*)e$ *n
A?*an Pat-%(%', 17 305314, 1988 ent*t(e$ DE*;er*a % A;er*#an C-*#ken C-ara#ter*t*# % S*@
Atten&ate$ Stra*n Pr%$e$ ) Se(e#t*%n %r Pre#%#*%& e?e(%p;ent, P>L> L%n' an$ %#e > %-n%n,
t-e $*#(%&re % -*#- * *n#%rp%rate$ ) reeren#e *n *t ent*ret> T-e ;*#r%%r'an*; ere atten&ate$
) t-e*r e(e#t*%n %r pre#%#*%& $e?e(%p;ent a##%r$*n' t% t-* ;et-%$> C&(t&re> Ea#- E*;er*a pe#*e
&e$ *n t-e pr%$t a *$ent**e$ ) t-e*r &n*H&e ;*#r%#%p*# appearan#e, *Ge an$ -ape, an$ t-e G%ne
% t-e #-*#ken *ntet*ne %r #e#a *ne#te$, a $e#r*)e$ *n L%n', P>L> an$ W> "> !e*$, A '&*$e %r t-e
$*a'n%* % #%##*%$*%* *n #-*#ken> !e> !ep%rt 404 !ep%rt 355 re?*e$, At-en, A C%((e'e %
A'r*#&(t&re E@per*;ent Stat*%n, Un*?> % e%r'*a= 1982, t-e $*#(%&re % -*#- * *n#%rp%rate$ )
reeren#e *n *t ent*ret>
T-e ;*#r%%r'an*; ere atten&ate$ ) t-e*r e(e#t*%n %r pre#%#*%& $e?e(%p;ent a $e#r*)e$ a)%?e>Ea#- #&(t&re a ree % pat-%'en ) tet*n' &*n' t-e pr%#e$&re $e#r*)e$ *n 9 C+! 113>37> T-e
t%#k #&(t&re $e#r*)e$ a)%?e ere ;a*nta*ne$ *n t-e (*H&*$ %r ?ap%r p-ae % (*H&*$ n*tr%'en>
C-*#ken, 2 t% 8 eek % a'e, ere &e$ %r t-e pr%$t*%n % ee$ #&(t&re> T-e r%&te % a$;*n*trat*%n
a-er % *>e>, %ra((> A(( ee$ ere p%r&(ate$ %%#t an$ are pr%$e$ *n SP+ #-*#ken, 2 t% 8
eek % a'e> e$*#ate$ a#*(*t*e ere ;a*nta*ne$ %r ea#- E*;eZa pe#*e> A &*#*ent ?%(&;e %
p%r&(ate$ %%#t ee$ a ;*@e$ *t- ee$, ree % ant*#%##*$*a(, %r a a$;*n*tere$ ) %ra( 'a?a'e
t% pr%?*$e ea#- #-*#ken *t- t-e ;*n*;&; $%e (*te$ *n Ta)(e 1> Sp%r&(ate$ %%#t ere pae$
#e*?e(, *t-%&t (*;*tat*%n t% t-e paa'e, *n 2 t% 8 eek %($ SP+ #-*#ken &nt*( t-e n&;)er %
%%#t ere &*#*ent t% )e &e$ a ee$ %r pr%$t*%n> T-e #&(t&re #ann%t )e -e($ %r (%n'er t-an 12
;%nt- *n %r$er t% ;a*nta*n ?*a)*(*t/*ne#t*?*t>
Ta)(e 1 "*n*;&; $%a'e % p%r&(ate$ %%#t ee$
r%pp*n' ere #%((e#te$ $a*( r%; t-e t-*r$ t% t-e e*'-t- $a ater *n%#&(at*%n> !an$%; #-*#ken ere
a#r**#e$ an$ %)er?e$ %r #-ara#ter*t*# *ne#t*%n %r ea#- pe#*e *t- t-e e@#ept*%n % ;*t*> %
-ar?et a ;a$e * t-ere a an e?*$en#e % e@trane%& $*eae>
T-e t%#k #&(t&re %r reeren#e ere ;a*nta*ne$ *n (*H&*$ n*tr%'en> Pr%$t*%n #&(t&re ere ;a*nta*ne$ a
5M30C, an$ ere pae$ *n SP+ #-*#ken t% t% e*'-t eek % a'e> T-e preparat*%n % &pen*%n %r
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ee$*n' %r *n%#&(at*%n *n?%(?e$ er*a( paa'e % ee$ #&(t&re *n SP+ C-*#ken, t% t% e*'-t eek %
a'e, &nt*( &*#*ent %%#t ere pr%$e$ %r ;an&a#t&re>
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a$;*n*tere$ t% pr%?*$e ea#- #-*#ken *t- t-e ;*n*;&; $%e (*te$ *n Ta)(e 1> r%pp*n' ere #%((e#te$
a %ne )at- r%; t-e t-*r$ t% t-e e*'-t- $a ater *n%#&(at*%n> !an$%; #-*#ken ere a#r**#e$ an$
%)er?e$ %r #-ara#ter*t*# *ne#t*%n %r ea#- pe#*e *t- t-e e@#ept*%n %*;*t*> % -ar?et a ;a$e *
t-ere a an e?*$en#e % e@trane%& $*eae> T-e ;*#r%%r'an*; are atten&ate$ ) t-e*r e(e#t*%n %r
pre#%#*%& $e?e(%p;ent a $e#r*)e$ a)%?e> :ar?et> Pr*%r t% re;%?a( % ;*#r%%r'an*;, %r pr%$t*%n
p&rp%e, #-*#ken ere -%&e$ *n a r%%; $e*'nate$ %r t-e pr%$t*%n % *n$*?*$&a( pe#*e> T-e
#-*#ken ere ;a*nta*ne$ %n a $*et ree % ant*#%##*%$*#a(, an$ ere a((%e$ ree a##e t% ater>
r%pp*n' ere #%((e#te$ $a*( r%; t-e t-*r$ t% t-e e*'-t- $a ater *n%#&(at*%n, an$ ere -e($ at 5M 30C
&nt*( pr%#ee$> T-e te#-n*H&e % -ar?et*n' ;*#r%%r'an*; %r pr%$t*%n p&rp%e ere -ar?ete$ a
%((%> r%pp*n' ere -%;%'en*Ge$ at an appr%@*;ate rat*% % 10 /? *n ater> Lar'e part*#(e ere
re;%?e$ ) pa*n' -%;%'enate t-r%&'- #reen> S%(*$ ere eparate$ ) e*t-er #entr*&'at*%n,#reen*n' %r ) -%($*n' at 5M30C &p t% 24 -%&r> T-e &pernatant a $*#ar$e$, an$ t-e %(*$ ere re&pen$e$
*n a at&rate$ aC( 80 /? %(&t*%n *n ater> T-e re&(t*n' %(&t*%n a #entr*&'e$> T-e %%#t ere
#%((e#te$ re;%?e$ r%; t-e t%p % t-e (*H&*$, an$ re&pen$e$ *n ater> Opt*;a((, t-e re;a*n*n' (*H&*$
a $*(&te$ t% 20 40 aC( *t- ater an$ #entr*&'e$> T-e pe((et a t-en re&pen$e$ *n a at&rate$
aC( %(&t*%n an$ re#entr*&'e$, &nt*( n% a$$*t*%na( %%#t are re#%?ere$> T-e %%#t ere a-e$ n%
;%re t-an t*#e> T-e %%#t ere a-e$ ree % a(t ) repeate$, re&pen*%n #entr*&'at*%n ##(e
%((%e$ ) re&pen*%n *n a 0>5 %(&t*%n % %$*&; -p%#-(%r*te %r 10 t% 15 ;*n&te> T-e %%#t
ere t-en a-e$ ree % t-e %$*&; -p%#-(%r*te %(&t*%n ) repeate$ 3 #entr*&'at*%n an$
re&pen*%n tep> T-e *na( re&pen*%n a ;a$e *n a 2>5 aH&e%& %(&t*%n % p%ta*&;
$*#-r%;ate 2CrO7> T-e %%#t ere t-en tranerre$ t% p%r&(at*%n ?ee(> Sp%r&(at*%n a
a#*(*tate$ ) par'*n' t-e &pen*%n *t- a*r %r a per*%$ n%t t% e@#ee$ 72 -%&r at 27M30C> +%((%*n'
p%r&(at*%n t-e %%#t ere -e($ at 5M 30C &nt*( t-e *na( pr%$t a pr%$e$>
A(( %%#t ere atten&ate$, *n t-at t-e are pre#%#*%&, a $e#r*)e$ a)%?e> T-e %%#t ere
&pen$e$ *n a preer?at*?e #%n*t*n' % a 0>0
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A er*a( a ae;)(e$ a %((%> B&(k &pen*%n (%t % ea#- % t-e %&r E*;er*a pe#*e are re;%?e$
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a)%?e *n Ta)(e 2, a $eter;*ne$ ) #a(#&(at*%n )ae$ %n #%&nt % ea#- % t-e )&(k &pen*%n> S&*#*ent ter*(e, 0>0
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Sa(;%ne((a a -%n t% )e ee#t*?e( k*((e$ ) )%t- %$*&; -p%#-(%r*te an$ ) p%ta*&; $*#-r%;ate,
t-ere%re, n% a$$*t*%na( tet*n' a #%n$te$> "#%p(a;a a -%n t% )e ee#t*?e( k*((e$ ) %$*&;
-p%#-(%r*te, t-ere%re, n% a$$*t*%na( tet*n' a #%n$te$>
Ea#- er*a( a tete$ %r e@trane%& pat-%'en *n a##%r$an#e *t- 9 C+! 113>37> T% tet t-e aet %
t-e ?a##*ne, ea#- % at (eat 25 %ne$a%($, pe#**# pat-%'en ree #-*#ken a ?a##*nate$ ) pra
?a##*nat*%n *t- 10 $%e % ?a##*ne> T-e #-*#ken ere %)er?e$ ea#- $a %r 21 $a> C-*#ken t-at
$*e$ $&r*n' t-e per*%$ ere e@a;*ne$ an$ t-e #a&e % $eat- a $eter;*ne$> "*($ *ntet*%na( (e*%n #-ara#ter*t*# % t-e ?a##*ne
ere #%n*$ere$ n%r;a(, an$ are n%t #%n*$ere$ *n t-e aet e?a(&at*%n>
T-e *rt er*a( % *na( pr%$t pr%$e$ r%; ea#- ne )at#- % pr%$t*%n ee$ *(( )e tete$ %rp%ten#> T-e pr%$t *(( )e tete$ *n SP+ %r )r%*(er tpe #-*#ken 1 t% 14 $a % a'e> S&)eH&ent
er*a( pr%$e$ r%; t-e pr%$t*%n ee$ *(( )e e?a(&ate$ %r p%ten# &*n' pre%r;&(at*%n #%&nt
pe#**e$ a)%?e an$ re#%?er % %%#t r%; *n%#&(ate$ )*r$> % ;%re t-at 70 #-*#ken ere &e$> %
;%re t-an 35 ere ?a##*nate$ per % *t- %ne *e($ $%e % ?a##*ne> Tent *@ t% t-*rt $a %((%*n'
t-e *n*t*a( ?a##*nat*%n a(( #-*#ken ere *n$*?*$&a(( *$ent**e$ an$ e*'-t re#%r$e$> % ;%re t-at 10
?a##*nate an$ 10 #%ntr%( r%; ea#- % t-e 'r%&p (*te$ )e(%, ere #-a((en'e$ per %, *t- 1>0 ;( %
ea#- % t-e $%e % #-a((en'e a -%n *n Ta)(e 4> Ta)(e 4 C-a((en'e $%e
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;*n*;&; #%re % at (eat t%>
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a#r**#e$, e*'-e$, an$ t-e *ntet*ne an$ #e#a ere e@a;*ne$ an$ #%re$ %r (e*%n> S#%r*n' a *n
a##%r$an#e *t- t-e %-n%n an$ !e*$ #%##*%$*a( #%r*n' te;, a $e#r*)e$ *n E@per*;enta(
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P%t preparat%r tep> T-e ?a##*ne * ;arkete$ a a ;&(t*p(e $%e #%nta*n*n', 2000 $%e ?*a(, 5000
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'r%%&t p-ae an$ 2 ;%rta(*t> A##eptan#e #r*ter*a *n#(&$e$ eH&a( %r &per*%r e*'-t 'a*n an$ ee$
#%n?er*%n *n ?a##*nate$ )*r$ ?> )*r$ ?a##*nate$ *t- C%##*?a#B %r ;e$*#ate$ *t- t-e ant*#%##*$*a(
$r&' an$ (% ;%rta(*t *n ?a##*nate$ )*r$ ?> &n?a##*nate$ #%ntr%( )*r$, C%##*?a#B ?a##*nate$ )*r$, an$
ant*#%##*$*a(treate$ )*r$>
"ater*a(> T-e ?a##*ne are $e#r*)e$ *n Ta)(e 6> Ta)(e 6 a##*ne
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Ta)(e 7 *r&(en*/C-a((en'e Or'an*;
Ta)(e 8 An*;a(
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An*;a( ere ;ana'e$ *;*(ar( an$ *t- $&e re'ar$ %r t-e*r e(()e*n'> An*;a( ere -an$(e$ *n
#%;p(*an#e *t- a(( app(*#a)(e re'&(at*%n>
E@per*;enta( $e*'n> T-e e@per*;enta( $e*'n a a %((%> Ta)(e 9 E@per*;enta( treat;ent 'r%&p
\ 20 ;* $e(*?ere$ per 100 #-*#k>
T-e t*;e(*ne a a %((%> On a 0, )*r$ ere e*'-e$ ) pen> T-e )*r$ *n r%&p 1 an$ 2 ere
?a##*nate$ a $e#r*)e$ *n Ta)(e 9, *t- 1300 )*r$ per ?a##*ne re'*;e, $*?*$e$ )eteen 2 pen, %ne *n
ea#- -a( % t-e -%&e> T-e )*r$ *n r%&p 3 ere tarte$ %n Sa(*n%;#*n, 60'/t%n, $*?*$e$ a $e#r*)e$
%r r%&p 1 an$ 2> T-e pen ere t%#ke$> E@tra -at#-;ate ere reta*ne$ *n *re #a'e a
&n?a##*nate$ #-*#k>
On a 21, t-e ee$ r%; tarter t% 'r%er a #-an'e$ %r a(( )*r$> Sa(*n%;#*n a #%nt*n&e$ *n
r%&p 3> On a 28, t-e )*r$ t% )e #-a((en'e$ ere ;%?e$ t% *re #a'e> Sa(*n%;#*n a $*#%nt*n&e$
*n r%&p 3 )*r$ t% )e #-a((en'e$> AU )*r$ an$ ee$ e e*'-e$> +re- e#a( a;p(e ere #%((e#te$
r%; t-e (*tter r%; ea#- 'r%&p *n t-e (ar'e pen, 20 a;p(e per pen> O%#t ) H&ant**e$ ) pe#*e>On a 29, )*r$ *n t-e #a'e ere #-a((en'e$ &*n' t-e n&;)er % %%#t $e#r*)e$ *n Ta)(e 7>
On a 35, t-e #-a((en'e )*r$ ere ter;*nate$, t-e (e*%n ere #%re$, an$ e*'-t 'a*n ere taken>
On a 42, a(( 'r%&p ere #-an'e$ t% &n;e$*#ate$ *n*-er ee$ an$ t-e ee$ )a#k a e*'-e$>
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On a 49, a(( )*r$ an$ ee$ ere e*'-e$ an$ t-e tr*a( a ter;*nate$>
E@per*;enta( pr%#e$&re>
a##*nat*%n/"e$*#at*%n> T-ree treat;ent % )*r$ *$ent**e$ a r%&p 13 *n Ta)(e 9, ere rep(*#ate$ *n
-a( -%&e, t-ree pen per -%&e, 650 )*r$ per pen, %r a t%ta( % 1300 )*r$ per treat;ent> B*r$ *n
r%&p 1 t-r%&'- 3 ere ?a##*nate$ a $e#r*)e$ *n Ta)(e 9> An a$$*t*%na( treat;ent 'r%&p 4 a
;a*nta*ne$ eparate( *n #(ean #a'e a &n?a##*nate$, &ntreate$ #%ntr%(, %r &e *n t-e #-a((en'e tet
%n(>
Treat;ent r%&p 1, 2, an$ 4 ere '*?en n%n;e$*#ate$ ee$> Treat;ent r%&p 3 a '*?en Sa(*n%;#*n
60'/t%n )e'*nn*n' %n a 1 t-r%&'- a 42> T-e )*r$ *n r%&p 3 t% )e #-a((en'e$ ere '*?en n%n
;e$*#ate$ ee$ ater t-e ;%?e t% t-e #-a((en'e #a'e> Starter ee$ a '*?en &nt*( 21 $a, 'r%er &nt*(
42 $a an$ *n*-er &nt*( 49 $a> T-e ee$ *&e$ a re#%r$e$, an$ an re;a*n*n' &n#%n&;e$ ee$
a e*'-e$ )a#k at t-e t*;e % ee$ #-an'e an$ at ter;*nat*%n>
O)er?at*%n> B*r$ ere e*'-e$ ) pen at 0 an$ 28 $a an$ ) pen an$ e@ at 49 $a> ea$ )*r$
ere #%((e#te$ t*#e $a*( an$ ne#r%p per%r;e$ t% $eter;*ne #a&e % $eat-> T-e e#a( a;p(e
#%((e#te$ at a 28 ere %)er?e$ %r ?*a)(e %%#t an$ H&ant*tate$ ) pe#*e>
C-a((en'e> At a 28, 3 rep % 10 )*r$ r%; ea#- pen t%ta( % 60 per treat;ent ere e*'-e$ an$
;%?e$ t% t-e )atter #a'e %r #-a((en'e *t- *e($ tra*n % E*;er*a #%rrep%n$*n' t% t-e pe#*e &e$
*n t-e ?a##*ne> AU )*r$ ere '*?en n%n;e$*#ate$ ee$ *n t-e #a'e> On a 29, )*r$ % r%&p 14
ere #-a((en'e$ an$ t-en ter;*nate$ %n a 35> Le*%n #%re an$ e*'-t ere re#%r$e$>
T-e an*;a( ere ran$%;( a*'ne$ t% ea#- treat;ent 'r%&p ) p*#k*n' t-e; &p at ran$%; r%; t-e
#-*#k )%@e )e%re ?a##*nat*n'> T-e #*ent*t ere n%t )(*n$ t% t-e treat;ent 'r%&p *n#e tr*#t e#&r*t
a ;a*nta*ne$ )eteen t-e 'r%&p t% pre?ent #r% #%nta;*nat*%n>
C%n#&rrent "e$*#at*%n an$ "ana'e;ent % A$?ere E?ent> T-e C%##*?a# ?a##*nate$ )*r$ )e#a;e
*#k 14 $a *nt% t-e tr*a(> T-e $*a'n%* ;a$e a ne#r%t*# enter*t*> T-e rep%n*)(e a'ent a
*$ent**e$ a C(%tr*$*&; %r$e((*> AU )*r$ *n t-e tr*a( ere a$;*n*tere$ Pen*#*((*n t% $e#reae
#%nta;*nat*%n, an$ t-e ae#t t-ere%, )eteen pen an$ t% e(*;*nate an $*eren#e *n treat;ent a;%n'
t-e 'r%&p t-at #%&($ ke t-e #%##*$*%* re&(t>
ata ana(*> Cr*ter*a %r "ea&re;ent> T-e pr*;ar ?ar*a)(e %r t-e (%%r pen p-ae ere (*?e e*'-t
at 28 an$ 49 $a an$ ee$ #%n?er*%n at 49 $a> +%r t-e #-a((en'e p-ae, pr*;ar ?ar*a)(e ere
e*'-t 'a*n *@ $a ater #-a((en'e, an$ (e*%n #%re % t-e &pper, ;*$ an$ #e#a( '&t e#t*%n, a(% at
*@ $a ater #-a((en'e>
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Stat*t*#a( Ana(*> AU tat*t*#a( ana(e ere #%n$te$ &*n' SAS, ar, C er*%n 8>2> Stat*t*#a(
*'n**#an#e a )ae$ %n t%ta*(e$ tet % n&(( -p%t-ee t-at ere e@a;*ne$ *n t-* t&$, re&(t*n'
*n p?a(&e % 0>05 %r (e>
T-e *n#*$en#e an$ e?er*t #ate'%r*#a( (e*%n #%re % p%t#-a((en'e
*ntet*na( #%##*$*a( (e*%n ere ana(Ge$ &*n' a (%'*t ;%$e( *t- a#t%r % treat;ent 'r%&p an$ )(%#k
an$/%r a &r?*?a( ana(* ;%$e( *t- a#t%r % treat;ent 'r%&p an$ )(%#k $epen$*n' %n t-e nat&re % t-e
$ata>
"%rta(*t>
+(%%r Pen P-ae> W*t-*n treat;ent 'r%&p, pa*re$ ttet ) )(%#k an$ a an ent*re 'r%&p, *t-%&t#%n*$er*n' )(%#k ee#t ere #%n$te$ t% $eter;*ne * t-ere are *'n**#ant $*eren#e *n ;ean (*?e
e*'-t *t-*n ea#- treat;ent 'r%&p> Pa*re$ #%;par*%n *n#(&$e$ a 0 ?> a 28 e*'-t pre
#-a((en'e an$ a 28 ?> a 49 e*'-t p%t#-a((en'e> Pa*re$ ttet ) )(%#k an$ a an ent*re
'r%&p, *t-%&t #%n*$er*n' )(%#k ee#t ere #%n$te$ t% $eter;*ne * t-ere are *'n**#ant $*eren#e *n
;ean ee$ #%n?er*%n e*'-t *t-*n ea#- treat;ent 'r%&p> Pa*re$ #%;par*%n *n#(&$e$ a 28 ?> a
49 ee$ #%n?er*%n e*'-t p%t#-a((en'e an$ a 42 ?> a 49 ee$ #%n?er*%n e*'-t
&n;e$*#ate$ *n*-er ee$ ee#t> Beteen treat;ent 'r%&p, a repeate$ ;ea&re AOA *t- a#t%r
% treat;ent 'r%&p, $a, )(%#k, an$ 'r%&p$a *ntera#t*%n a &e$ t% $eter;*ne * *'n**#ant $*eren#e
e@*te$ *n ;ean (*?e e*'-t )eteen 'r%&p, %n a(( $a e*'-t ere re#%r$e$>
C-a((en'e P-ae> W*t-*n treat;ent 'r%&p, pa*re$ ttet ) )(%#k an$ a an ent*re 'r%&p, *t-%&t
#%n*$er*n' )(%#k ee#t ere #%n$te$ t% $eter;*ne * t-ere ere *'n**#ant $*eren#e *n ;ean (*?e
e*'-t *t-*n ea#- treat;ent 'r%&p> Pa*re$ #%;par*%n *n#(&$e$ a 28 ?> a 35 e*'-t 6 $a
p%t#-a((en'e>
Beteen treat;ent 'r%&p, ana(* % ;ean (*?e e*'-t )eteen treat;ent 'r%&p, p%t#-a((en'e,
ere #%n$te$ &*n' t-e re&(t % t-e )eteen treat;ent 'r%&p ana(* %r t-e (%%r pen p-ae,
%#&*n' %n t-e a 28 an$ a 35 #%;par*%n>
!e&(t> Ta)(e 10 28 a S&;;ar
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\S*'n**e tat*t*#a( $*eren#e ) 'r%&p*n'> Ta)(e 11 49 a S&;;ar
Ta)(e 12> C-a((en'e !e&(t
60 )*r$ ere #-a((en'e$ per treat;ent> ]S*'n**e tat*t*#a( $*eren#e ) 'r%&p*n'> a, ), # an$ $ are
&n#anF eparat*%n % ;ean> &n#an tet * a tat*t*#a( -%# tet t% eparate t-e ;ean> 1 * t%ta(
(e*%n #%re
2 * a?era'e t%ta( (e*%n #%re
*#&*%n> T-ere ere n% *'n**#ant $*eren#e *n t-e (e*%n #%re %r t-e e@per*;enta( ?a##*ne an$
C%##*?a# %r t-e E> ;a@*;a, E> a#er?&(*na an$ E> ;*t* #-a((en'e> C%##*?a# $*$ -a?e (%er (e*%n
#%re %r E> tene((a a #%;pare$ t% t-e e@per*;enta( tra*n>
C(%tr*$*a( $*eae -a )een (*nke$ t% *ne#t*%n *t- #%##*$*%* *n t-e *e($> E?en t-%&'- t-e *ne#t*%n $*$
prea$ t% t-e %t-er treat;ent, t-e C%##*?a# ?a##*nate$ )*r$ ere t-e ;%t ae#te$ -%*n' 11>77
;%rta(*t> C%n#(&*%n> T-e e@per*;enta( E*;er*a tra*n ere -%n t% )e e*#a#*%& *n t-e a#e %
?*r&(ent #-a((en'e> T-e )*r$ ?a##*nate$ *t- t-e e@per*;enta( tra*n -a$ )etter )*r$ per%r;an#e a
$e*ne$ ) ee$ #%n?er*%n rate -en #%;pare$ t% C%##*?a# an$ Sa(*n%;#*n treate$ )*r$ at 49 $a
-*#- #%rrep%n$ t% a e#%n%;*#a( a$?anta'e t% t-e p%&(tr pr%$er> E@a;p(e 3
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S*@ $a %((%*n' #-a((en'e, t-e ?a##*nate an$ #%ntr%( are a#r**#e$, an$ t-e *ntet*ne an$ #e#a are
e@a;*ne$ an$ #%re$ %r *n$*?*$&a( (e*%n a##%r$*n' t% ea#- E*;er*a pe#*e an$ t-e #%rrep%n$*n'
e#t*%n % '&t t-at #%rre(ate t% *ne#t*%n *>e>, E> a#er?&(*na *ne#t &pper *ntet*ne, E> ;a@*;a *ne#t ;*$
e#t*%n % t-e '&t an$ E> tene((a *ne#t t-e #e#a> ata are ana(Ge$ % t-at a *'n**#ant $*eren#e
)eteen ?a##*nate an$ #%ntr%( #an )e a#erta*ne$ at t-e 0>05 (e?e( % *'n**#an#e &*n' t-e "ann
W-*tne U Tet>
+%r ea#- E*;er*a pe#*e #-a((en'e$ %r p%ten#, t%ta( t-e repe#t*?e *n$*?*$&a( (e*%n #%re %r ea#- %
t-e ?a##*nate an$ #%ntr%(> Ea#- )*r$ re#e*?e a #%re %r E> ;a@*;a, a #%re %@ E> a#er?&(*na an$ a#%re %r E> tene((a>
!ank #%re % a(( an*;a( r%; t-e ;a((et t% t-e (ar'et> +%r *$ent*#a( rank, a*'n t-e a?era'e t%
ea#- an*;a(, e>'> * t-e 5t- an$ 6t- an*;a( -a?e an *$ent*#a( #%re, a*'n a rank % 5>5 t% ea#- %ne>
!eta*n t-e *$ent*t % t-e rank a t% -et-er *t * a ?a##*nate %r #%ntr%(>
T%ta( t-e rank*n' n&;)er % t-e #%ntr%( 'r%&p> Ca(( t-* t%ta( D^!#>$$D T%ta( t-e rank*n' n&;)er % t-e
?a##*nate 'r%&p> Ca(( t-* t%ta( D^!>D Up%n $eter;*n*n' t-e &; % t-e rank %r t-e #%ntr%( 'r%&p, t-e
"ann W-*tne U
Stat*t*# % t-e #%ntr%( U0 * #%;p&te$ &*n' t-e %((%*n' eH&at*%n
W-ere t*erepreent t-e n&;)er % an*;a( % t-e #%ntr%( 'r%&p an$ n?repreent t-e n&;)er %
?a##*nate>
Up%n $eter;*n*n' t-e &; % t-e rank %r t-e ?a##*nate 'r%&p, t-e "annW-*tne U Stat*t*# % t-e
#%ntr%( U? * #%;p&te$ &*n' t-e %((%*n' eH&at*%n n?rQ M (
U?V n?n#M _\?
W-ere r*#repreent t-e n&;)er % an*;a( % t-e #%ntr%( 'r%&p an$ n?repreent t-e n&;)er %
?a##*nate>
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T-e tet * *'n**#ant at t-e 0>05 (e?e( * U#* (e t-an U?, an$ U#* (e t-an %r eH&a( t% t-e #r*t*#a( ?a(&e
(*te$ *n Ta)(e 15 %r t-e appr%pr*ate 'r%&p *Ge> Ta)(e 15> Ta)(e % Cr*t*#a( ?a(&e %r "annW-*tne U
Stat*t*# T%Ta*(e$ 0>05 a(&e
a(*$*t Cr*ter*a> +%r a ?a(*$ tet, at (eat 50 % t-e n%n?a##*nate$ #-a((en'e #%ntr%( ;&t -a?e a (e*%n#%re % t% %r -*'-er> % 'r%&p % ?a##*nate %r #%ntr%( #an -a?e (e t-an e*'-t an*;a(>
Un$*?*$&a( (e*%n #%re per tra*n %r t-e ?a##*nate r%; are t%ta(e$ r%; )%t- tet e*%n> !ank
#%re % a(( an*;a( r%; t-e ;a((et t% t-e (ar'et> +%r *$ent*#a( rank, a*'n t-e a?era'e t% ea#-
an*;a(, e>'> * t-e 5t- an$ 6t- an*;a( -a?e an *$ent*a( #%re, a*'n a rank % 5>5 t% ea#- %ne> !eta*n t-e
*$ent*t % t-e rank a t% -et-er *t * r%; t-e *rt %r e#%n$ tet e*%n>
T%ta( t-e rank*n' n&;)er % t-e *rt tet e*%n> Ca(( t-* t%ta( DE!1>D
T%ta( t-e rank*n' n&;)er % t-e e#%n$ tet e*%n> Ca(( t-* t%ta( D^!2>D Up%n $eter;*n*n' t-e &; %
t-e rank %r t-e ?a##*nate r%; *rt tet e*%n, t-e
"ann W-*tne U Stat*t*# % t-e *rt tet e*%n U1 * #%;p&te$ &*n' t-e %((%*n' eH&at*%n
1 U`1 n* n((=, M < D11 M 1 2, ! 1"
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Up%n $eter;*n*n' t-e &; % t-e rank %r t-e ?a##*nate 'r%&p % t-e e#%n$ tet e*%n, t-e "ann
W-*tne U Stat*t*# % t-e #%ntr%( U? * #%;p&te$ &*n' t-e %((%*n' eH&at*%n
U b T-e tet *
*'n**#ant at t-e 0>05 05 (e?e(, t-e *n*t*a(
re&(t ;a )e #%n*$ere$ a D%TetD $&e t% %?er#-a((en'e, an$ t-e ?a##*nate an$ #%ntr%( *(( )e
#%;pare$ &*n' $ata 'enerate$ r%; t-e e#%n$ tet e*%n> T-e $ata ana(* *(( )e #%n$te$ a
pre?*%&( %&t(*ne$>
05 (e?e( * U#* (e t-an U?, an$ U#* (e t-an %r eH&a( t% t-e #r*t*#a( ?a(&e
(*te$ *n Ta)(e 17 %r t-e appr%pr*ate 'r%&p *Ge> Ta)(e 17> Ta)(e % Cr*t*#a( ?a(&e %r "annW-*tne U
Stat*t*# !etet % Ser*a(
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!eeren#e S-ek*n, a?*$ > :an$)%%k % Para;etr*# an$ %npara;etr*# Stat*t*#a( Pr%#e$&re, 3r$ e$>
2003= 423428, 1151>
\ \ \
:a?*n' t-& $e#r*)e$ *n $eta*( a$?anta'e%& e;)%$*;ent % t-e preent *n?ent*%n, *t * t% )e
&n$ert%%$ t-at t-e *n?ent*%n * n%t t% )e (*;*te$ t% part*#&(ar $eta*( et %rt- *n t-e a)%?e $e#r*pt*%n a
;an apparent ?ar*at*%n t-ere% are p%*)(e *t-%&t $epart*n' r%; t-e p*r*t %r #%pe % t-e preent
*n?ent*%n>
($+&AN &A$'N
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&aten ang
iu%uk
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WO1996034109
A111 Apr 1996 31 Ot 1996 !"cal #nc
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#nc.
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(t"l""ng cocc")"al sporoo"tes
8$5055292 9 Apr 1990 + Ot 1991
at"onal
esearch-e&elopment
orporat"on
!acc"nes 'or cocc")"os"s
compr"s"ng l"&e spor(late)oocsts 'rom stra"ns o' e"mer"a
spec"es
8$506+104 1 Ags 19++ 26 o& 1991
A. >. o"ns
ompan
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&aten ang
iu%uk
$anggal
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$anggal
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8$53+,414 , Apr 1993 , 7e 1995
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8$5566064 26 e" 1995 15 Ot 1996
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electrol(m"nescent panels
8$55+0+59 1+ ar 1994 3 -es 1996!"cal
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8$55+9466 26 an 1995 31 -es 1996 !"cal#ncorporate)
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8$5602033 2, (n 19+9 11 7e 199,Ao oel
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8$5614195 2 (n 1995 25 ar 199,Ao oel
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8$56351+1 20 -es 1994 3 (n 199,
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8$56,44+4 10 (l 1992 , Ot 199, C'"er #nc.ont"n(o(s cell l"ne an) &acc"ne
aga"nst a&"an cocc")"a
8$56,,43+ 2, ar 1990 14 Ot 199,Ao oel
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8$5693622 , (n 1995 2 -es 199,!"cal
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8$5,09+62 + o& 1993 20 an 199+ ;r"t"sh
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39/41
&aten ang
iu%uk
$anggal
!enga%uan
$anggal
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8$5,+02+9 6 (n 1995 14 (l 199+Ao oel
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8$5,95,41 10 o& 1994 1+ Ags 199+Ao oel
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8$5+14320 , (n 1995 29 $ep 199+Ao oel
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8$5+43,22 21 (n 1996 1 -es 199+Ao oel
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8$5+4652, 30 Apr 199, + -es 199+ C'"er #ncont"n(o(s cell l"ne an) &acc"ne
aga"nst a&"an cocc")"a
8$5++556+ 12 $ep 1995 23 ar 1999Ao oel
.!.occ")"os"s po(ltr &acc"ne
8$5932225 2 o& 1995 3 Ags 1999 h"lwalner!acc"ne compr"s"ng e"mer"a
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8$6001363 26 an 199+ 14 -es 1999Ao oel
.!.occ")"os"s po(ltr &acc"ne
8$6100241 3 (l 1996 + Ags 2000Ao oel
.!.occ")"os"s po(ltr &acc"ne
8$652,205 11 an 2001 4 ar 2003 ase lcAgr"c(lt(ral part"c(late mater"al
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