PROCEDES D'IMAGERIE INTRAOPERATOIRE

INTRAOPERATIVE IMAGING METHODS

Abrégé français

L'invention concerne des procédés pour une imagerie intraopératoire de structures anatomiques utilisant des composés fluorescents, par exemple des composés qui émettent une fluorescence dans la région de lumière invisible (IL) du spectre, c'est-à-dire au-dessus de 670 nm. Un exemple de composé est le bleu de méthylène.

Abrégé anglais

Described are methods for intraoperative imaging of anatomical structures
using fluorescent compounds, e.g.,
compounds that fluoresce in the invisible light (IL) region of the spectrum,
i.e., above 670 nm. An exemplary compound is methylene
blue.

Revendications

WHAT IS CLAIMED IS:
1. A method of imaging an anatomical structure selected from the group
consisting of
vasculature, myocardium, parathyroid gland, a thoracic duct, a biliary tree, a
ureter,
or a portion thereof, in vivo, the method comprising:
administering to a subject a preparation comprising an invisible light (IL)
fluorophore, wherein the administration is systemic or by injection directly
into the
anatomical structure, and
obtaining an image of IL wavelength emissions from the fluorophore,
wherein said image is a representation of the anatomical structure.
2. The method of claim 1, wherein the IL fluorophore is filtered by the kidney
into
the urine stream, filtered by the liver into the bile stream, or both, and the
image is
a representation of a biliary tree, a ureter, or a portion thereof.
3. The method of claim 1, comprising:
injecting a preparation comprising an invisible light (IL) fluorophore into a
lymph node of a subject, and
obtaining an image of IL wavelength emissions, wherein said image is a
representation of the thoracic duct.
4. The method of claim 1, wherein obtaining an image comprises positioning an
electronic imaging device adjacent to the structure.
5. The method of claim 1, wherein the image comprises some portion of the
subject,
and the method includes obtaining a first image of one or more wavelengths of
visible light and obtaining a second image of IL wavelength emissions of the
IL
fluorophore.
6. The method of claim 5, wherein the visible light image and the IL
wavelength
emissions image are obtained concurrently.
7. The method of claim 1, wherein the preparation is administered
intravenously.
8. The method of claim 1, wherein the preparation is administered by direct
injection
into the anatomical structure to be imaged.
31

9. The method of claim 1, wherein the anatomical structure is a biliary tree,
and the
preparation is administered by injection into a portal vein, left hepatic
duct, or right
hepatic duct.
10. The method of claim 1, wherein the anatomical structure is a ureter, and
the
preparation is administered by injection into a renal artery, bladder, or
ureter.
11. The method of claim 1, wherein the near-infrared fluorophore has an
emission
wavelength in a range from about 670 nm to about 1,000 nm.
12. The method of claim 1, wherein the near-infrared fluorophore has a
structure of the
formula:
<IMG>
wherein, as valence and stability permit,
X represents C(R)2, S, Se, O, or NR5;
R represents H or lower alkyl, or two occurrences of R, taken together,
form a ring together with the carbon atoms through which they are connected;
R1 and R2 represent, independently, substituted or unsubstituted lower
alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl, aryl, or aralkyl,
optionally
substituted by sulfate, phosphate, sulphonate, phosphonate, halogen, hydroxyl,
amino, cyano, nitro, carboxylic acid, or amide, or a pharmaceutically
acceptable
salt thereof;
R3 represents, independently for each occurrence, one or more substituents
to the ring to which it is attached, such as a fused ring, sulfate, phosphate,
sulphonate, phosphonate, halogen, lower alkyl, hydroxyl, amino, cyano, nitro,
carboxylic acid, or amide, or a pharmaceutically acceptable salt thereof;
R4 represents H, halogen, or a substituted or unsubstituted ether or thioether
of phenol or thiophenol;
32

R5 represents, independently for each occurrence, substituted or
unsubstituted lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, or aralkyl,
optionally
substituted by sulfate, phosphate, sulphonate, phosphonate, halogen, hydroxyl,
amino, cyano, nitro, carboxylic acid, amide; or a pharmaceutically acceptable
salt
thereof.
13. The method of claim 12, wherein two occurrences of R taken together form a
six-
membered ring.
14. The method of claim 12, wherein R1, R2, and at least one R3 include
sulphonate.
15. The method of claim 1, wherein the near-infrared fluorophore is selected
from the
group consisting of IRDye78, IRDye80, IRDye38, IRDye40, IRDye41, IRDye700,
IRDye800, Cy5.5 and Cy7, or an analog thereof.
16. The method of claim 1, wherein the near-infrared fluorophore is methylene
blue
(MB).
17. The method of claim 16, wherein the preparation is a solution comprising
about
100 nM - 100 µM MB.
18. The method of claim 16, comprising administering a sufficient amount of MB
to
achieve a concentration of about 10 - 40 µM MB in the structure to be
imaged.
19. The method of claim 16, wherein the MB is administered in a total systemic
dose
of about 0.1 to 10 mg/kg of body weight.
20. The method of claim 16, wherein the MB is administered in a total systemic
dose
of about 1 mg/kg of body weight.
21. The method of claim 16, wherein the MB is administered in a total systemic
dose
of less than 7.5 mg/kg of body weight.
22. A method of imaging an anatomical structure in vivo, the method
comprising:
administering to a subject a preparation comprising methylene blue (MB)
such that it appears in the anatomical structure, wherein the preparation is
administered systemically or by direct injection into the structure, and
obtaining an image of invisible light wavelength emissions, wherein said
image is a representation of the anatomical structure.
23. The method of claim 22, wherein the preparation comprises from 0.1 to 10%
MB.
33

24. The method of claim 22, wherein the preparation comprises about 100 nM -
100
µM MB.
25. The method of claim 22, comprising administering a sufficient amount of MB
to
achieve a concentration of about 10 - 40 µM MB in the structure to be
imaged.
26. The method of claim 22, wherein the MB is administered in a total systemic
dose
of about 0.1 to 10 mg/kg of body weight.
27. The method of claim 22, wherein the MB is administered in a total systemic
dose
of about 1 mg/kg of body weight.
28. The method of claim 22, wherein the MB is administered in a total systemic
dose
of less than 7.5 mg/kg of body weight.
29. The method of claim 22, wherein the structure is vasculature, biliary
tree, thoracic
duct, ureters, or a portion thereof.
30. The method of claim 22, wherein the tissue or organ has high uptake of MB.
31. The method of claim 22, wherein the tissue or organ is myocardium or
parathryoid
gland.
32. A method of imaging first and second anatomical structures in vivo, the
method
comprising:
administering to a subject a preparation comprising methylene blue (MB)
such that it appears in the first anatomical structure, and
obtaining a first image of invisible light wavelength emissions of the MB,
wherein said image is a representation of the first anatomical structure;
administering to the subject a preparation comprising a second invisible
light fluorophore (ILF) with an emission wavelength of at least about 780 nm
such
that the second ILF appears in the second anatomical structure, and
obtaining a second image of the invisible light emissions of the second ILF,
wherein the image of the invisible light emissions of the second ILF is a
representation of a second anatomical structure.
33. The method of claim 32, wherein the anatomical structure represented by
the first
image is vasculature, biliary tree, thoracic duct, ureters, heart, parathyroid
glands,
or a portion thereof.
34

34. The method of claim 32, wherein the anatomical structure represented by
the
second image is vasculature, biliary tree, thoracic duct, ureters, heart,
parathyroid
glands, or a portion thereof, and is different from the anatomical structure
represented by the first image.
35. The method of claim 32, wherein the second ILF is indocyanine green (ICG).
36. The method of claim 32, wherein the second ILF is the carboxylic acid of
IRDye.TM.800CW.
37. The method of claim 32, further comprising obtaining a visible light image
of the
structures.
38. The method of claim 37, wherein the first image, second image, and visible
light
image, are all obtained concurrently.
39. A method of imaging the vasculature of a tissue for evaluation of flap
design
desirability, assessment of flap viability, or determination of suitability of
a failing
flap for salvage therapy with fibrinolytics, the method comprising:
administering to the subject a preparation comprising an invisible light (IL)
fluorophore, and
obtaining an image of invisible light wavelength emissions, wherein said image
is
a representation of the vasculature of the tissue, and wherein the vasculature
of the
tissue indicates the desirability of the flap design, the viability of the
flap, or the
suitability of a failing flap for salvage therapy with fibrinolytics.

Description

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
IÃ~~trat~~:_~e~ati.ve In_,.:~ in:~~ Methods
FEDERALLY SPONSORED RESEARCH OR ~EVEIyI~PINM.IsINT
11.3.s i.?#v#:fli:Ã.;.3n was mt3do wltl` GL?i'C=rinnent >:.eppo1t under
GF"i1?:t Nf},RW'CA-
, ,15296 1d~i SaÃ;dZ`d by the Nil.at'ÃL3riai In5:Ãi.l:~tes oziHmhi:3 s?nwa
<_?rai?! No. DT_.TGt,MIER6,31 Ks'
~.fE'f=~' }', Tbe Goi'L`TTixilw.nt it%hs e.'.eÃai'3 rf t,i33., ?,,3 the
`IEC'~~~ICA~ ~IEf"J-)
T.i.iv ?:1'4 +? .lt? 3 TS :hate4 to :7C'fl'3`.~dS aS.?F.'
:d7t7"<.?3peF`23.ik.Y=?Q i721c'd,~ll:g `i1s:Ilg- iiui;?`t 4uii7;
i:oÃ3"apC?Li#: dti, compounds i-hat fl`'r.?C3TosC.'.. in the ?f11'As3bzo
i.ight (;.L) region e-a :aiv
s,pu+.:i:?'utn.?.L., above 670 Fil'ia.
1 BACKGROUND
L:~in~ surgical procedures, it is often difficult: li3r i:~ie, ~"'A; g"cota :o
t(l~.~
location of s=Ãua.airws stÃ.t:b as the thoracic d'ltcÃ., UIii.3ar?^ tr:act,
wid b~~dli i.t.`v' trw:., and
_?%'. di3ac>ting i'ar:er"3;.:s.
SUNIMARY
1w ~i~ .?.. ~,,, ',..aa`.~., :II~'te`~"~'~is based, at least 1"i ~)w~l~.,
:?i~. ..+~~. discovery ~:e~~;Ã.it ~,-:.'ÃFÃ ~~.Z'i'S
_ c~,,.. t1~,.?k sahc3t $euo1esou~ in the inv`.s-1blE: light (IL) i'C'.~1C?k',
of the Sr7tw4'~?~i:~.aii are t~4G,~i~.l ~:3:~ ?Y;~.?~?4 3.Ã3;.,M
oc,t:in s nbEtoliiic:>i structures, e.w;., during surgery. Thus, pr?v1u.t"S:1
6w,.eI`a, a:'e mehoCi4 tbr
rF:'1 -.n:y sta udl-u.`es sLit h as td:i fl>mil~. .4c duT.t. urinary iTaciy
J13.Ãc2r-y tr#J:,., and i3er..:3i t3tiig
arw 1c:', using IL LaS'es,20 3ia : fi;:,f. C]k?~,'~:5:t,
3.S~k~'i.:Yk''e?i~:1.iL,'~.~ features 2.etLkSJ(5s AGI, ~~~.~2c~~ Ã~>~k:, an
c.?~1~tÃ.tt~~rci~.-'C~i
a
S':ruL.tI::`c seie'~:iS..:i fs"'ozTi the gmi,3p ,f,'..oI353.sfiASa.~,~' of a
b}.3.:<iÃy, itree, urE;1:s:.1, l.1."::3 p3.:i'L7in i ae:':."`of:
ayl v.?v(`. >71w me;ht)ds ?a~i a?li~~ zlf~ll%13~i4~~=? Ã1~S' to a s:fb~ecÃ. a
pI'cp:?`c#.t.Wi. ?ziwlWm,3.', :3r,
zn~.tsf~.'33~:< lÃJm` (IL) fl'L?o# L?p`,.i'e that Ã4 fiftE'red b''fh~.~
kIdnu~% i,it+;..3 t~iL urine sÃ_~. ? ~'...[:5r.:t~'
by the ;.~vcx ixa<r the b-iIY;:. SÃ>iai3., or both, and obÃ~aÃntng an ix~~ ge
t?_i`1L wz3t e'len',
2 5 tallas.lor7's z:i"f:Il the fil?.1or#3;3hC3xep, whGS4,1G:1 the ffla<f#?G;
is a iep1"esC;?att's:txiEf?;. of the aÃ~i:tl.oml+..al
i-mclL?Ã`S..a.
In s, i,?dm 2aspC.ct. the itÃ4S::i7tRC?tl features ~I~.offii.3d.' for
ii`Iiak;n~`..~. z3: thLS,caclc da~t or i.;
pZS ~ion tÃ.eim?f :n V'?vE3. '.f'Ai~E.'`~ m~.''`{thods Ãns?lud5v iqjt:cting,
a prepa.atZa`3i.i a.{-iC.'ll~?~E#?i;:'i;?>
w.
H ,a:~.liL?or:?phoi'i: (11.:F) Ãnto a lymph I%t7[ac t?fa sll~#f'"1' SEIC}i
fl3:3i. ?t <?ppZ.w.i 5 in the .ht3i'::cii;:
i

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
duct, e=g, any aymp;3. ttodc, tli at drains into tl-ic Ãhc.2-tacic duÃ:t, c
g., at or t~~=}s; '~`,~s level of
the di.a~ph?'ugprn; und obtaining an 1magi;~ of 1:ivis1blL z!ghia (aL.)
wavelength cnriission:a:
= y. = .. . duct. :: ~. Ã~i~taÃ,~e is a i'~. pÃ"Gs~'Inabl~111.In of tlc
iZ~iT~i~.i?;~. i.iÃ.3t.t. In st 1">i1e Lt.1bo-&Ã1?,ents= a
8,:.1ffi?:=lvÃat w.nnt#uÃ'~t~. oftime is allowed to pass ior the ILF .Sr flow
into itiL tAi<)ras:ic i.-tÃct
`? ;lcft;;'o t.:u Ãmagc is (?llÃ<i:zlle;d.
~~ ar < a.a 'v t7~ ~ ~ y r c~'tl ~s
i: :~+-,F?~S`.' ~.eia7f.9{~7Ãn?L.II~~, ".~li.= 1T?.c3.~y:. ]I34:lui~e'.~
;iS,Ã31~: ~ZZ3~2'~aE?3~ Uf ,:?a: S?ajv~:i x... and .C~ a:3e
lynxagC'% ial<...,ide's x, fir?it ii-nagv L?'..stiz3.Ãned ftGfl';r Cfilc or
3IiL?"s('_= wavelt:.=nA?t$':3 of 'v'24..?3e light and
..
soci?ind i23 riga~ i}ytai;net3 fiom. IL w:t oliingf13 emissions of the ILF. hl
4:?mo eIi3,bodimi=s -1is',
~~1~. a_I~i:~
t^ 5~~ ~= ~t visible t t the ~ ?i3f',=~ ".,~Ã,Ã`~:~~' a Y~ I'~1~3i'~'. light
image of t~1C. surgical field a#';I.`~+. m 11, w=`'vC`A'a:21gfq IÃ;:i~.g-4
of .,.3e s u, m~,c:3l 7::',.id. hi 5i?i.li einb(?Ci2'Ãi"3:a`:IIts,, fllU F'i
ilb I c :igh, l i mage and :}':Ca. .`L wzxv +`.',o:ng:h
i:~~afso are obtained L=oncw>ently,
In `mti?me ei3:bodIi3:'La,iit:^`., 41E?4c .n.Ãrs.'~.g an 1.nnage
Ãili,'.32.Ãdes positioning an 4?3::<:brC}ilii';~
v
i;.7? Ã?y.l~ j . as i;'3i;.I?:~tVd ~'i~'.Ã'i::4:~15 %'t~.~i3~:=~:Ã.i.a. to
the 4GÃ'I~~.E~,.''L`cF'~:~; 4#t..iÃ:=T' `:...;~:, structure or
~:32ic S c?eo Y`if.`i~ ] `v'ai> \ ~ l ,~_~)~)ed. so ~..`~``12~~. the
`~~?v _aww are ?n c tJlc enough prÃ3:S1mEE-y :o cIIt:bla:3 tilt;<
In so:Ã1i.o cmbC L>i>i2t;:its, the preparation is i:idx.l;;ili s14'i'ed
systeib3ica.l"4', e.g.,
iii,T, ~'G.'~,LZ uror y, ois3ltt:mi,Ci.velya by di1~ot injC:[,'.t3.t3E1. The
<?da7iin#?it?'afi43Ã. is not t.tip2cad, In
some i'irlboC,i.fi.nez3t::, thu an~to.I,~dicii~ structure i~`s a bÃfit3"'v
tree, and the pà a;~.3 t :a?:ioll ,s
adk:tf.1=steiii:$ by .i2,S ~ .1i3F1 (c.g, caaÃni.3lati#in) into a f s`3.,i.a?Z
vein, 1~:ft >iepi2t:Ã:. &,Ic$, or Ã`i:3t
r;4 h>..pcnii~ duC.i, hn sonni< vi.:.nllf-r`s~s'e$.mo,~'.i#S, tlIcaTi?
IfC1:'I724a?r. s:T`E?mit'8: I, 22.aIa~:~t.L~, Ã#'1#r 4.Enw
;`fÃ=ep<!i'aEioa.. is administered by iiWa>tIon into a renal artery,
bl:ÃdS=C:1`, fi-r In se,.Ã-nt;
~ ~d~lieae'r C lÃ~..II if.ei', t`
ennbLiYIÃ;:il(+I`?t.~~.' the ~,=nC:t.3ods IInS: 3,Ão~f:
'ct'L1i.:nillEsii[3t1f}I~ ~.Yl`ie ~ ' `~~ ,, .
aceÃazo`i:mid2; or sQ~churn, bicar;sonatc.
i:i some emlbodÃmonts, the ILF is a ncu-ini:rarecl fluorophore (NI~.~.a )
wi~~~i ar?
25 `'..:T3is4i?'o?n :i avt:iC'=ingth in a T<3a3g'l.' frE.i2'3'#. about 670 F?m
to about 1,000 .`3n,,, t:;.g.s, .tRDyC1 8,
:RIDyu~~~ jRDvu'38, xRI3ve4(,), J-R.DyU4l,. IRD-ye700, HRDvcSOO. Cv5.5, :un:
Cy7 r or an
au,u:log the-mof
In Sorn enlbodtmctat,~, t'cie Ticar-inftdÃred flfac?r~~p-hore is nneth lf tsl
2ie (_~-IB'~, h.
Yi.37?F: c.mbota?fnuÃaa:'õ'~~c: p#'opai'at'i.oai is a solution compt'iS12ig
w:.~oÃlt :1110Ãi'A .... 14,`S, +-&I N1:s.
iQ 3in V:E;frini`.= embodiments, t1h(` methods InclÃ1de c1d3nI#niS.f3i'iing_.
,_2 ,7siÃa;.nft r.ÃiIn `iz- ?; MB tci.
achieve a cor:cs.ÃnfAafic,2i t?f abota~ 10 --- 40 ~tM MB in the ti> be .` 4_d,
nn, aon-w
;'
E~:Ãni'bt3i~ÃI,?.;ntsY >1'n~. MB is ~.C~~7iE>11S'~i.'i~:d in a if)f~~Ã~.
Sl'St~:.Ãn"Ii>i: ~`~.3s~, s?s a:i3'v`ut 0.1 w :i; mL?'r>.`r.:?f
bod.f+wt;igh?.. _;I3 :ii;i3ie i:Y?`#bo#.lb3'iz..e23.ts, the M13 i,;
~?dindaÃ4t:i:=rC~S.~ Ã.3 a toUl s~y)t(=.i'?1e-c da3so f}k:
2

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
ci;ii.,?ut I m,:/kn, of body weit l3t, Id3 stI?ne embodÃI7te:`Iis, 1:iiL' MB
is admin;:JteÃ;wd ir, a tE_tva>
Syq-e.Ã"f.'k E:t?se of less than 1``~ ~ng;"kg, e,g., less than 7,5 m`4-'1kg,
of body we1ghi:
In a.ilota?C;Ã" a4peofs? the invention provides methods for imaging an
al1a`eÃx3.(:,i:l
_ ,.f v=
Js~`Ll~'.t~~.~`4~p ~v'.W~.} vasculature, ~~i~~L~.~~~ tree, t~~.~~rC.~.i.~~.Gs
duf:l:. ureters, ~~,'L,'=C~d.i ii~~`ci~L~..~~u~.~%>, w
V~ V = Y~ri V cells), ~ a5 ~~~~ .~if, ]
i~ ~~.3ext'ta.~l~?~"L i~ .f~.~~i+a, a ~';z3~E~~'('.='cl~r 3rslÃ:~, ..~..a tu
mor `~...~.., t~6Ã?>~s.f'ti {t~' ~}t3~?t ~ Ãt~ a. t)
parav}:yroid origlai;, or a portion thoreoi', in vivo. 17he rue.Ãhod,--
include systam;caI1y
"1Ã tY'i? t'Un1'3ilsly; t`.idM;I31St4'Ã"`:1g to a. Si3~:Jcct a preparation
Ix3ebutiÃÃag fn+`,'.thZ%lL~=ne 37lE~'L'.
w:Ãch that it appears in ( .c. fl:rL'S'S #Ilto, R .f ., by roÃ3`~1gi lt~e or
afl:ert?~~~Ã:iC flow) il'ie
am$.a?i~.~.~.ÃLid `t'~".~Ltule; wnd i?bia.n1n''~~`, i3.i11miÃ~^;tl
i,fz11.1f:3n'.-slrent %'.nn14sii)Ã3s I3"i3"i. .h`v' N1.13õ
`.i' ?iÃ'.Ãn i ~' Z Ã4 ~ 3'r,f'1t:i2~~.~:iC1~, i;~~: t}l``.
~.21s~ro.i~la1Ã`~:~%tl S~i~3~::i.`,.Ir~":. ln:'i3a~:tz :~:i
of ~ `s Ã~` , r , , ~
~ t'~~sli~'~3:#
~'w c`>i' S3~'M,~;t.~~x:` uptake C#: ~=IB, `'~`Ch ~Ã.;~ hi.'~:lÃ"~.
~l~"k"+~`~ea~.Sr.it~..F, i3i t `~t. p
gdti.
I:El a f1~thea' as~..tect, th`a? :_1:Ibri .aitlof3 p:C1S..`ldk:..a
rF.ieffiods~:s~> Ãas3u?>il~
aruxwt-nieal~ s;nucÃurosa.t vivo. `~~-ic =-i-ictli~.~ds .ittclLtdc
adÃninister,ng, e.g., s; r.b~.,m~.call
i4 sI'"111#?i3si.i:3i7Ã4: (C.#.;., intravenously), to a sllbjf:c~ a
pR't:paF=7.tki3 ~I3 =~~~~~~~Ã~? :i~:: 3~~`~ ~r L bi:.Ãc
M,~~~ such that it i3~3pi',a: i in (i.e., flows id7R)., t:>f`.v., by s
C.trotMÃu.deor a.iltei"45~?,~ Ãd+.~ f:+ E2 ) i.;:;sv
fiõi` anatomical ~trÃ:I?wfli$'~,', C3~.E7ti1ÃnF,ng a flIst image of
iÃlb'A53:"9lC: l:gl1t wavf3leiiL ,>? ~:lllf~~lt:> 1
t?.i `...7e ?1013, iFihe.'e:Y2 the image is Y iZ:.paC'i4=ntUti4Zn of t}2e.
first a.`Ã:`att)ÃIlical stF"`L3.cÃur':,
i1d-m:nÃsteaing :~.~ fluC`= ::1l.b;+e'Sct a preparation including a second
la]v14;blC lkg,11112i :uor~~+`pho:e
#~.~,a` s~.~i~n 3vtba~,it at least about r;` 8~~ ~?Ã'.~~.~i~, i ~ ~.~ 't .~ir
Ãt-~? +3vv ., '~~:
s.~L~ ~.4.) with C~+?~~..ÃF~ ~~rtt~i~e=.:~lS~1 1 of Y#' l s.~.L ~.., ~ ~.6 Ã~
. suC.l:.
~ x ~ i
¾~~. s~.'~;L~.l>:i.~ :LI' ~:~t ?~}~.::i:i'.'s in (Ã.i~., flows into, 4~.g., by
i~3tA'(?4~':~:Ãii:~ or <~2t~.~"~.~gÃ".`~s.a:Ã~: ~.ii~L3 t ~:i'..e
second an.ÃtL.f2n.t.:=Ãl structure; and f~~~tai:i.ÃÃ.i~t'G..,. i.'Ã cseconLf-
a~mag4 of invisible i.igFi.eeÃaõÃ~"~i>"e.`ir:~
c-
+:?f the second Tc :' ~b'}lE r`~:3a3. ~.?7te image of the invisible lI~~ it
emissions t3~3~t~1L.~ St;3..a 3.~.=F is
art'.p;eSenE.a'liii?;., of a su~.:oÃid anatomical structure.
25 In Na9m+.''.1 embC?d1~1^i4Ã1~5y lÃ~' anatomical xtrLZC tL:3 : rfi.p`t
.Jf..31t~:i3~. by ~.~.3:'.` ~~I ~t a"~l ~ xo i s a
pÃ3rof3n,ui the a biliary tree, a thoracic duct, a 1.3.Ã`.LtC.i. ""s~>`~..'
beaà i a pjrat.,.~'roiia
.O..+~'.,~.nfa_. a pancreas islet cells), a tumor (e.g., a tL1moÃ' of
pancreatic oi paÃ":#:t.~~-vmÃ.d
t3~~~~ ~ 3> +_ F' a r3sw}: lÃi,Ã,. ths.Ã`i3iÃa:, In some :3"~iW1meilt'd, the
taÃ1ek-an1ccl:z .e~ro seÃ;ied
bJ the 5coond image 15 i.1 poIliol'': of the vasculature, e.g,: bli2ar"y
t:"ea, a f1h43racic duE:;'t, a
30; Ute.el', a .wa`t. 'a pwrckth~`2'otd ,s~~'i~3d, a pancreas (< .~.~., islet
cells), a tw:3'if?F' a t,?ma:r of
kZ:.ancroatc o;" paÃ'%~~E~oyÃ'{"ai3 ongÃ.Ãi)w or :3 poi'tio:> ffiert,:olc and
is dÃfauLnt from iE.':t: aÃ':atc1l-nic:::.ll
s2'rucLuL-.e .'~iV`txie: Vi,Zted bv the IfirF.,~>t :ÃnuC`.Ãge-
3

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
Zj.i sorS.2e. eS?..ibS.+kLALkS.eR?t.3. LASW sS.i1.ro.li-iSS ILF is tF.blL\.!c-
T aa;ine gS,Ve3.C $ }CG) G#.Y!L, ci rboxZtli3..
acid k>f I1~Dye'11800Mz
I:'1 sS3Y?a:`' ~.=Ã":?bodÃ.3?Ln:s, ta?e methods fuit;.e:' include obtcÃi.-d-
u~ ..? visible light ia:::E~;;:
~ all of ~.:3e structures. The .L.ti4.i,~.lla;gt:, ueG;f).3E~?8Y7.I?:~ix and
LÃsa:~.14 light ;:lt~.ag~`:, cc..,: :Ã~i> bo
obtalaI,~:~l co.?.eur~ent;y, ~:.~d opÃiormlly superimposed to crea~o a merged
im<?go;
in i:Fa addf}lz?nal i?7;?t..i. the T3?ve?1t1ixi features "11Ã::hodsiC1> in3
iginf.~, e?. A7t3::"t.f;I"I of ~.2~;.;;
'~"i in ' Y1 ~~ 3~ F Y' 7tt ~ a 4 Y ~
t`~?tic4~lw`:.~arv Ã32 ; io L: A hrv> s~:vi.`L~~'fS a:~~'=1 aZ?C=.
<3~.~.~?~.fa~li',t~.~.`g t1~2`F.~~-e,.A..~j;:t i a pn5,`.p.'d.:ati.:nciud:ng
met;?yhmeb1ue (MB) such t~iat it appcars in (Ãx,< flows, :l?ta, O.g'~ b;
.'rwtr,?~rad4
or ani~ro:~~ade f.ow) :h. vasculature, ati.i obtkainiÃ?g ai:. imag;: of
iÃrv;siLsu: light (.;'L
;:,t w..4 eiL-'ngffi i 3?'i~~~?CaÃ? u`bLiciia iffic iit>is .Ã
Ã`ea31i:se?]taii.Ão.s.]: of ÃA7e Z <l"c?:.: ali.?m
..i:~` .~.~'~.{~i ~r.~] a
I_,` a 2 ?.aa~s~~ t'~~ ?s7 ~C~~, a~1~L Ã.Ã "E >~'e]]tit3Ã? ?~i+~ ~' ~Ãi ~
l7Ã3Ei4 s:a ~:3Ã FI?~%.Ã ~ zTla ~', ' i1Ã# vc .\ N
. k.~ i ~; I"13i~
ii.4wiu ~~~~ ovG:huat>nn of ~lzip design desirability, assessiner~t of flap
,riabffi>;, ,,;-
it_;:w>.mtÃ::%iEoi? i?Ã ;^uÃtt3b.2hi'y of a Il.i3i3.d'.ng.flCip for salvage
therapy wft2l l..ÃV'i.~zi?<AY".1cs. Tf:i:
methods ?.i?;lud+`,:: ad7?37n?stiE:#i21M to the subject a preparation 1
.?Gli?~~:1rF: an invi:iibic iig?Y.i i L:
fluorophore 3ui:h flhat Ãt appears Ãkl flows 1:3'Ãfi0, t:=,g., by
riftoi2Tc:iCae u <331ÃE'3"4?~.,ryzde fl+?v,`)
ffic v~^ws:'. 3iz iuf'e of flhc tsSue, aFid obta1nÃ4't- ag"3 ]I? i9,.xe of
t;.?'v is"INQ lT:4:~.it w:3.vGlengti7.
a::di;l-s:'s1L`}Fis, w:ioi'..Rn the f?'la4se is a rep"e4eI?.Ãat3'l?.Ã"~~. of
the vwsC:u.iatilri:, of the fi~~ ue. 3nd.
wherein \l1G vf=swulat:m~ of t};utisSL,e i1:d1ct3.c;~ the des?Ã'a:3-flTty of
the fli:Ãp design, the
~~labilztyof ta3c flap, oz dic sultabiii.yofa tailÃp~g fiap aor ;~~~lvage
therapy wi a
An lÃgr?t fluoroj5horC (IIJF) is a compound that e:Ãi't4 Iigj?Ã in E ac
.i i~. ; `
~ , of :h~' `~+uctaL7~?? (670 I?I?'z ~i? 1 ~~~~ i~00 1`f'~7) such ^ ~ ~ c 4
F:t~=eEe z3.1:I"s a C.A
:i3i'4'i,i.~3~~; ~Ã~ ~?E.Ã ; ,.+
a?F:l ti` 1000 r`.~.Ã11) to rnit infrared (1000 mn tZ) 20,000 imn) to far
infrared (20,00~~? iun: to
>.00,0V02i.2ri),\ -s any light (A~~v4= 67\11a31Lf is iIvR:3tb.(e to tj,?4=
.Ã3.Zdk.Glk human \+j Yr =~~Iw.7~~v
a;? :: ?vISible lÃgght. f1uo?"oph+.?I'Z:s {l3 not s6bsai$?'Ã'1"w'lly
0.=l?aaigG ti1e appuI'ei:i;c ci3AÃ?3o su#'g~i,:.a:
field, and because tissue aItoflE1:"LsGCÃll:eilt Ãz?[~~'wi?.iotei14:¾h;d FS
oTi%, detection
. # L S-~} q?.W~Ã`[)~?3.?tZ~~~':~
l's`~~a ~a.Ã~?3_7~~.' %?~~~.i.t3 i~.{s`~w ideal reagents 4.f~~ `.~'{.~Ãr~;?
.#.~ .;s:;
.,%'?7e3"ia:g. 3Ã1 sÃ)an'L: en?bodÃmonnt ILFs can also include
iluZ7rFphÃ3:"es that aie. v.Ã~,,Ãbl`v :o, z;,#a~
Il?keZ cZ i z?s >t9;?~,=~ i34 they ;1ls:?fluibÃ'cscG`= in the li?v2ssIt3la.
light I`f:ÃJIi7Eb.
'>h, iurf.3 "near Ãnfz'arCd fluL`_ti'ophC?re'i' rtaf"e?'`',~` tt? ;.}at-
1pt3?..Ield:5'.hcl flzfLL.3..:3f:t:-ndl the
a .5,,.`s. d :nf1a.e\f. i~eg,?on of th-c spet:i.I'ui-n.. i.e., about 680 nin
to 10t.3~.i nÃi.?, jI'l.3esC l.#F ~L ~Ã,~.f =".`i
incl.Ãdo ind.~cvaniuc ~Pc:4~ (ICG)s IRi)ye~'~,178 (Li-COR, Lincoln, NE),
1IRDve'? O,
IRDye3g, P-,',:Dv~.~0 :[R:~ve4 1. 1:RDyc7 00,1~yc"RASOt#f~~' z;L1M COIZ,
mi'ct.a:d to, ut:.:ziw a.s
4

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
CW800 of CW800-CA= ax,, th~.' cw;bt~xyliU acid form~ of the "~~R flu<>rophore
~~~DyJ~ ~04 C,115.5; Cy^?'. Cy:5, 1R-786,1_)RAQ5N'O Ã~i N-oxideniodit.~od
abr:;q13im3?:`4'i$ quantum dots, and ia.7i.~.lo~.'.J ta;iluGof, ovK'.
.}`,}'d;C.sph#i:Ec. ;.analo4 S Z::-
ti;:I~p~~o:~i3.tLi3 analogs t~7~.~'Q~_ .II'1 some ~;I7i~?[~t~1Ft-1,ill:~+,
tli~: ~''~.I~L ~t is a i:~.I~Zri?pt3t?z e i.Ee:zkà sw;d
15 in US: P?iri'i;,i,crna~ ~latUrÃApplac tr~.~n No. 60'835,3144.
T ha: ,,r~iae5ori methods h<ave a Iiti13'Ibeà of advantages. FE?: e;$S"<'3.
npzet. tl1'~.~. :yC.s usodi
l:exein can ~~ iYZi`~~~~~% ~? t~ *5' ~ z3i s3~~ ~ 1;11tCXl~a 3~1f3T1S, yet
it:'e!A.fl ab.s`te ii3 ovadc-t:1a.elleIlt
4J:s:.ai'rlut.o,3; For e-munple, fl1c doses i3finCthylenc-:.~luE~ (MB) used à -
ki tffic i~.:z3re`cL.ia:i;
~,Y..h.L,t,~~ .o h; described `i herein are ~z s2%`~D`illai~YtG~1;~l~' ~
C=tiÃ..b 'i~~.;ta ~'.t}~=~~~1 ~~]ia~z :~~~~:i+., ~3~:~.(~~=.x s.t1i. MB i.S
:t ~ a r > Ly a-+ toxicity ~ ~s;SCr t
used w:.e a ~:"$,~r~:~? ~~~#.~L.~ i~.~ t E,Y thus avoiding E~t f,'if 1
~T~$i~<:' ...~2 .Fl~. tf>\~4~ `c.LT.=xe;iti'.u W IS.h
MB. Surprisingly, even at these low d<,3scs, MB is seorC;ted or partition prt
te.eF'.fi3.illy i;'11to
ur'lz3i:`.= zw>d .33.aw; :a4 concentrations thuit.a~e optimal JtC?r
xli3C3.a<3G3: .n: ;'isua,1zatiCsf, :a:IoI`ax?;h 4ui:=a?
%tSnE:`.=vnt1". .2i~ms (+.:.ti.} a c+?I?cUnt.ra:El.o1a. in the tTskLii: of
only about 10.r40 ;i.M; cg,A about 2S,i
a~~ , M) are far below those useÃti` abr ~~ipual Ji;;lit imtaging:
defined, "~ 1). a`.it"`~7's S."tx1+~.;> yxi..4i; izall tc5:<h.l.c:tl a12d
s!i.:iE''T'zbfii= :E.n3x.'~~. :.;\f.4,t hh=xLir, x*3as L the
~:ii~:t= mi;~:iiis,t.~.~,, ws ct:rn-mt?A l4' un(iS:I'std>i9t ~. by o"tio of
ordinary skat: i1~ :?ti =:trt ._? .4'h14.h th1.3
i?3v:#'i.lot: 3Te:.oras. Methods and, materials are dL'~`.~'cribC:d hGTGa.I1
for E_ a,: in :ali: pzc~4i.:i:
;n:t'emua'S11 other, suitable mo-r1CitiS am~d materials kn<#w-?t 1,`. -ffiL
art can also be usod. Fhe
materials, mefliods, and exii1n~`+~~s:s :~.fL 1~:lL~~.~!"i.3t~:~`c% Oiiz~.
w1x not iiitt:i.i,`:~.l t~? be d,:l'.`.ttlll4, s ~8i
applications, i.~c~teÃ1tS, s"~';qL7E.'nC:e47 daia`?.tJ: entries, zi71d
.3flbo'A rt S at'AG= ~L~~
,,nktxt=c?ned her;:#::n, are incorporated by refcrence ati t~ic;.r i.=nbr~ty:
i:a case of conflict, ;I:e
p;. -zent 5pC c3 fic`e;.`iYoEa. including df,.~.:'t.3.~tions,,, will control.
i1o~~ii?g
Other f~y Ftlfeatures a~i'~~:~il~~~in~ii~g<:S of the A1 inv4'=tition i51a7 1
be _:om the : iCy;
t:vt<a1E:~.~d. ~~~ senptit?n and fi4.E.1reZa, wid from ~iai: claims.
25 .~~~SCR.1.PFIO.~ OF MtANVING~
~ !G. .EA is a I:Iic 4:?-re`2 IlluJirit.l1g the t3h-aiiwo lil
flu{.}ri..si.#;;ri:. K:ftcn-sItv w.~t'ri
inc.e.a~ing conunAraÃiors .?f:-neÃhy;ene blue (MB) Ati pha~spla te buk?cxe:d
,.x..nz:a<
~-:lied squarc::), 100% f~Ãtal bs}-vine serun ÃFBS, opez-l. triangles pointing
rt=p. .iashed :n,,;r or
,
.ini ONR~~x As open triangles pi?inti.ng down, solid llnt: f.
30 FIGs. I B and I C <3.rk- bar ~;~r.~p~~s ill~.s,vt=~.~hn~. ~ak abso=.~3~awwo
~~avQ c,.g:}: Ã z B?
PG;ak flE:o.LSi:ui:ca; ein3S;ilon wc3-ve;.41Igth (: C) for II1G:~eas1ng
c<3.i"#.Genttcltxous ox MA.~s iil AI~~~
~ ~lack ihars'. Ft3~ (g-roy biars), or MtOH (wh;:te bars).

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
~ i~ r~~) )tYty~:~ F'~" iF ]p}Sy+)t'iJpr;.~ise~.~iA~~.~\. e}.i. ynl+~~ t ~w-
eZ.rwp.`-~'~.~4 ZSL. t¾- ,~<'p l.~=~LB }
~ ~n :..~' is a ~l t3~i w~2~3~ C~~ ~ .{.S.i.~[Ya~~'t~A \. i~i.
':[~\
pl'I --- 6. M3c'li:'~c conditions, el.ilia witl1 ttit: addition i,1f Sodium
Ase, c2s:.&bic,
i3u`xd..
FIG. 21 k ah~l of n31:o images of a rat heart c.:t sequential ,'dme "Noinis
(03 zt?i:?
i] au~v.1 12 seconds (~i-iai.3df$:.= rt-.~w)T <~titb -5 miluies
(bo4t1)ir,.'ow)) after 1~ .adul.~nisLraF.iVn: 01`:i:B,
...
T ! ~~4,
t- ~r t3nz-.. x{h aue, s~'. ~FSwn in the a 1~:'l~: s . i:olunnn, near ~ (N.
Ix\,7 'Sy .l;a?Sj
:~ c3 w a~i."s4'1...xTz ~:>
~..~31,.
4e (merged) w'e ~~~i?i~'l'a tI1 t:7 , xg~fti i. ?iei I1n..
s.E3ddla' and aA3Vio1'.=NIR (3vefl.? i:ilid,
4? c i ati a s:iiit`7le ai?iage that displays both a:lafom~' (color video)
imCA function (IR:
flu'qui:JCelale), e110 M fli?ore5C.w:inct; image was peeuii#3-cE,li?s'ed in
IT.i">ii. +rt:e: wli; overlaid
Wi.ai field.
i:.~a 100"r i 3 i3i'a pilrenY y ;3n top of ihi` color vi d4=o image of the
Si?inC sur g
FrG, .3A is a p,.vir of 1~iib graphs ali-ust4-atizig sig:sa.to baQkgr13u-nd
ratio Ã;~~I() in
fonrrnom bile d#,li:i (CBD) versus pa-Ili:~rea4 (left graph) or liver (right
giaph)s -at sei.]u+'.ilt`Ll
:`Ã:E~~'~e points a-l'ter of MB.
e'IG. 3B ts <311" i# of tii?lago` of rat CBD 1171agef: al`Ee1'
ivad:71E,11stF'<7.Ã;.t1i: of N14.13.
f Color i n?c~3 ( ~ ~ ~ shown in ~~L ~G~Ii?C3lLÃ:73, i ~?<3:I' 1n3~i i'S t~
(N ~`~ ~ A71.T:t:;s i~7~i, sa;.-$3 r',~.E3 ~.1~. the
inidiil3 and color/N Lk overlay .'1'lic:l~'.,.~~in (m{:3'ged) a3'C.~ shown
in thc'`. I?'O, it s'rU:iln:x .
1, 1G. 4d~,:` a trio ol iL1dag'k. =s of rat C:Z?~~ imaggeZJ after U:i_'=,5
aduw4i.ii.;4.i~.C1o~n of iYb~~:
Color innge:o- i3..e shown in thL' ~~t-I column, near 1niT`uk'Oi~ (N1k)
urkars~ s-, anresh3wA, in tiv
7?Eddit cLaf iinis and c3lt?r%NUR overlay lmagc4 (:]`1En4?4) i3ri=.
shuw.E#.IÃ. lllu F:gIit :=i?`uI1~.;n.
2 0, FiG. 5 is a set of 'ti}L; images ltfCBD imaging ~.'itf;, L~~ ~ (top
row), as\^.~C`) "TI:.ii~.~.~ai:;
~
row), aÃ`:tt CWuO0Cr'1 Co4SEao33 row), wh'fcll are able. to pii7vId.'
:T~iLii:ii`r?"u tl+?f. {}f the bFl%~-
dÃ.cÃ. 10 :,3tg: zafl..:5 i~~~~~.~~~. ~~r ~~ ~:~ . of 1000 ~Ã:~~~ It_.~~-
.~~?~~ :~f? liI:_ of 100 .,~~ ~:E~~: :3~~.(~t x two
NIR flux3-rt3v23horU si.3aLtt2iZf''x nso po'=toi voin of the rat<3. Shi?wi-
F i1T'S: a still fri}n col.>8 i:l{:e,.=t
(RAT.). .~~R fl3:#.Z}esi;:.imi:`s'.~~ (3n3.d3.~lf:=)> c.$.I3.d mer{~~d,
images of the two Sr4_^".l.tf. CBD i:i.t.ii bi::.
~'~'~~ ~' ,~? Lw~s' no dye stacking :?
2>; 4'. ;.Ew'.iizei by a.13 ...~ ci"S.1`?.rii`3t agents (`srri3vl,hcadS),
bLit C~ :~~_ ~.la~.
~
thi. Rv<:r and ai~ijhz:s- SBR thaii Ã.he sst~iez two. N-UR Ia?~iocescent
ex,:3og.aa=~.~ time as'200in,,se::.,
~~ [G5, :;.A-D at`e line ~,~3'il~ih4 ~llu5$rtltll"ig quantification of fl;Z
ti 4a E ~~ ~ i7711xS`iC3}"
and signal :o baz.k~r.ou:~~~~ raW (SBR) kinetics for CW8,300CA. =Th;: S.13M
(rzwa:r., :1- Sxt:y=' of
thi. Lom3-ni5`e`1 i:lS; duct a`i:li1:i'4'f,'~ to the p:~.!?uvt`.5 (6A)
u$~;ad liver (Ã1B) wwI'f} qI.1.<ai{.dficd o-vo<y
..,: finw lrjicÃ~iork of'50gl.: of l.f?, 20 50, ar:i~ 100 ~;.-,M ( v~ 800CA
solution. fl.+,? SFiR tt) fl%i: Pi:l.Ili:.:G`s4 (6C) w.tid livcr (61)) wL-
`rC: al5o i:aE..fab::eF. cafle pt?.Ii'cd3
i:j,., t&.~n of 50 ~LL of 10. 20, 50, and 1 00 }iM CWSOOCA solution.
6

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
~I~:5 ~..'i. t ~ '~13 are sets c?f'fil~.~~cÃ~ Ã~~~~:3r>~:s of ~.'~3I~ ~~~~:p-
;~- ~.~~.~ .C..~iÃ~~'~~r~v~:'B`~~~~.:~
~, 7A :~t a:Za r f. images a
;di~mfii:cf~ under nonnt3~ ~~~iditioiis (i:~) mc3. after the in~ertion of 3
bead~ of 2>5mi:n and ;
bt ti: of'.5mm Ind3ainc.tcx into the C`BD f-rom p3pi11:avaÃ.er Ã;713;. '~ n-
A. ofia50PM
CW800CA (7,5 i.fg / kg total fllloiopht).:e) w#s 1njeczC'd Fn:Ã'%ivuil?a.is1
4 and #.i;i7gt:=d t3. ` ~
.`..` m#.si:~ Shown are s:1llu from cil?orb'adeo #:eaÃ~~ NTIR
flZ:#t?I'4,s',`enirG'. (i:'.lit fize), and z:3fu.a;ryf'd
i2I...3ga:;i of the two L`. fs.3`x0. Allbcldti
could bc: t t,.tccteN ~.3t:r-E.,ctxy M each tr#Ct1.
FIG. 7C is 21t:i.; of a)'$aga',:> of pig ~LID .']Ft.IL'.ea aat:r
e1G,aii_i.z3:i4Ãrate<`,E# of M13 rkil:d
ICG. .e..~`~ ll. color anagey'#niddli-:<. W X3 N I.R. fl#.iC.3-r"'SGE.'.i1Ã 1-
maggo (~.~~0i; #~7#~',, and ~ghÃ,.
I ~~ _~~~efl,Mene blu~., NIRF ~~~~~~~ (MO rm). In th:>s4 :mages. ICG
~~ghltt;~.~tsi blood vcsEo.s.,
wh#ie .%,l33 shows the CBDa
F K" 8 : a [ i'.t's of i 3:0Ã' Imagi 4 (?~ tht I`a46 1L duct An's? ~3~,3i11'uw
in a pig. Shol`,,a zir:'
sfills. famn, color vÃdeo, (left), NIR fluorescence (nuc3d:Ie), ui-i3 -
rner=,ec~ i~.na,.4es of ~ V;;o
("Ili_a h t) .
1:: 9.'IG.,7' "J a set of twvlTb': fi-nag'`.s of thoracic l~vta.L
#.na~..~~.~i#ig %3 t,: rat i-#siÃg ICGE (liY:(-s
coaunii~). I~' GH:3A. (second ccfluann), C~'~ SOOCA (third cohm-ir ), and
I3SA800
:.c3aumm), Shown are stills ~~om color video ktop row), NIR. _i" uorescencv
f~-x)zÃo,.m, ro,~m)>
-i.#id mkmc`iCl i#anarc?'iasoa` fhe vwJ(m#.ddlL~~o-w).
~ 10>, a0aÃ3d 11 i3re rll2e '~,-~mpi3s illustrating qi1;:1n~E1ficaÃ.if?n ?a
Ã,.7E fl..3oi'i:sc:=#.:E:o
2C eTni"sion and ~ipal to backgmu#>d ratio (SBR) a~ine..iis f:or CWS,;>5OCA:
11w SBR (#~~.ealn
SE .''aA) the ureter relative if_) Ãh'':`. k.kine'1+` (10) and
abC.t3a1"I3$3.:~~~_~ wall (11) 3'Yere q~i:mir.tC;i:
over 3m.-ras~enous injection of 50 p1; of 10, 20a 50, and 100 z'Nt CWHi:#i_;A
Sz?atEi5n,
1, aCf. s 14, is a scrics of t1~~~ec images of tit.,ew T-napping,n tA r':aÃ.
Sb:i5wm are qil:y
24 video ~~eff,'l3õ NIR ykoke5e#.'.i3ce i~in1ddle~1 and illf''~%;C.S.i 1'm#g~
of the two t~"I4;=zft
F:f_:s< 13 at3.d. 14 i'-1:#'c ge 11ph;i illi.15traÃlli". th'~,.,`' 1#::
a`i.ilt`'s of IAIPIYCh`ni3ss >'sP+,.c~Ãi'olllt'~~:r3,
meia,bo3aY i:.,Tatvsis of CW8\i'Li carboxylic a4.t.dIAfte3 excretion .L-itti?
u2iA4e. ?lssbiiwn, by th'.-~
:~i.,o ey:?Lnce cÃ.ro#l#uÃz3gt'i.#pi], the dye i`tppt;:aI3 as essentially ii
single pcak w~th, the ?E; FIG. 15 iS 14=2'#.e;i t;f o#gj1Ã C:SifJi `G'li~eo,
(left i%oluTI1F7; and neaa I#.,,&ar:.ÃF flEao..L_4u: -riC'e
(i'#;;~i: cLrla#an) Ã,..~t~:g,',s of ~.`s~ti.la during intravenous
IIIr'cÃ'.iC?a3. 3. 0.5 m;j. (14 I#g.I;Y;
:a:~?~n141e ,~`,.'''.11 (1CG) GIlÃ~) 3 .~'7S kg ~rE^~~'~,5h1Te pig. Shown
Fe~#'L'= pra -iII;G'ction
i3.e.tofi43#.3rG:?c#c..` ~'i5p), [1:rà eAi.#E fiffliag :it 5 ~oo p'a~t-
Y#ijvctC3n ' bC;c?nd row) and venous

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
filling at 10 se~:. post-irkõ~.~:ctÃon. Note tliag the nipple in thefie,~~
(top right) .Menr~~~~--, as aTF
additional internal ct:}.,1t3C?: -IfFi F?stQfiai v:s'.venCtiI:Is phasws.
~.. ~ and r + ~..~Y1
~`Ii.~. 16 :s a '~i}z3..a~:<l [)
f s..:~Eei%4 Tr7:l~Fgi'3 of Fl~~:.k ~l~~li.lf~= blue ?.. t:~.C_t~3 IV la?j:.
..a~i;I?s
~~~o~~=i.ig C131> a:s? CystiÃ:Atte.Ãy (`vA), as ~.Ye1l as coronary arteFy, in
an intact (top 3
rows) and : e;:cwYte~ (boÃ.o,::~ row) lioart.
FR:5: 17 is a paFts;l of Amkges sliomdng the IesuFss og parat3$4`I'S?.iid
gFaTFc. .i]uIging.
DETAILEW~~~SCRl ~.~'T:l0N
i,i:traopi :ati?<'~ im<7~,~"Vtt3g is eU.indispensable dTi3g,,?stFc, tool for
ida''ntFficat1.Ã3I1 of
noIF?'3ti:i or ~~~ eÃ~i..T?~33 anaio:nF.i.i:l~ AT`n3.ct[F-Ie5. Si3 gt C?:3s
usually F1~o flLFo: ? t Zfpt%, kt,FSo:!;?(.
i u ~~~jec}-ion oflhÃg& doses of a blue dye (e:gr, iiiUtli}{leiio blue (MBf,
L.3rrnk~h,'.:zz.rid.1~1, rir
ivan's B1~~~~, or radÃÃ:tschnfigrvFphy for intrF~opc.r.Ftive imeigiag. I'h4so
m0 th,3~~s QtF.r~..~vl
,play;igy~?fiCif.nt >s3ics, however, F:acii methC)d l;.ay stJi:ii`
~raLvbs..i.k~, sF:ii,...3..z:ti m..,:adF.;7ti0114
3"`a:iSC?viati,d wFtl1 .``+iuC:s?'GFfiC:i;..?py mId
1a:Fd1(?Sl:iIltl`'s~.ryl~.`~hy), low SpC',+.i"CÃ``..}% a.1L, lo:T.:
sei
-.s.fi.v:iv (y;,Tual biue, dye:j), lrt soai-te cases, tirr exasn>~le IMB, the.
<Ia.sos r~.:.~u::e~.~. tf)
aa.z#Fc-ve vzsuei~:l- a~~3c:11tÃtzti,atiz:371 m-t' associated w1d1 significant
t{3x1c1ty.
t>?C: mQiChods described hemin ts,3k4 iilivantiage of tl14 ~Y opeF`t.FF,'..4'
oa. fluorescent dyes,
e;g., invisible light (IL) aha(92"esct:I3t dyes, to provide &afE,4
fipa:'.i,F~; .e, a;?d Sia..`.s.fF.vS:;= raetE'f~fds-
ior l<ib';-'li.Fx4. and d5:tocthng anatomical Tht.Zo;-..ns:t3:tt'3d.4`s
11W:Wwo
thwr LFSQ of d v:: 's tha~ Fa~ are '~vltcrod aiid excreted by tr ~~. ~`
kiainey (¾c?F- ,~~~.~, ` ~l`rFg of L' L'~C. xh)i?: 120 taken up by the 3:ver
md. seL~retedin,..} bele (i`o.F= latieIffig of-th~.~ biliary .'eo). `Fho
pFcfrru.;
d~c. <b.+.4=aot si.gn'aficCiniaYV 3''Zii:Fb.]SJibod or m'rvt[2btilizf,.d ~.CS
the lqoE4y .Thf: m.od.\?d,,, t, - LS,:':
.. . .. . . . . . . ~p.+.:.:a
?3F diA'#.Fa~ a kni1LY7? dye tC3 affect its filtration and n?zt:3:?t1i:l
prL)s14~I':I~..4 ~i'r exe3.mple,
s
~'~t~T~1',:~:~~C?#.i f~3'~. a ia~{.i:;~ f;:~.3.~a bi: uh~ .?~. to
.~i~.~.~3.~i~~~.Cii;1`~tr~ z~'{f~.FeC:~ liver uptake, t.=?y
~la";.F.T~.`1i~~~r
c{y:" iiito~ the urintFrv tract.
'Mc..th;jd;
".~ie 3method5 desoibFr:d lFerc;iii can bc, practiced wÃf~i any intr;Foperat.;
e :~Ma
19i
ywte.i'F.l fl)z3E Caa dduct 8z3vÃsÃi?.(t:llga}1i. (IL) flE3t?r~scenC'.e i'yt
vivo, C.g., ihL. 'Systo.+`~F1s described
in Dc>= Gtmid :i~~d FraÃz&ioÃd, Toelmol. Cancer Res. Treat. 2(6).553-.62
(2003), U.S. P<u
App. Pub. ~:` ~ ~~t~'~i~~Zila ~~ i3 ? ~ f?, ~ ` i~.jS~). lp1.3.~. ~eÃ?.
~,?~~õ~ ",~?~"~~~3~ :`.~:?
~ ). tz ~~-r~.~~ i~~F~a~ ~. aF,~ U.S. ~Pat.
;:axangi~~ ~, U.S. . ~ , ~i3~~. Pub. Now ?:~05:/t~t~'0~~L i ;r umioF,. et : i
, mFd U3. P ;. Ap;.
Pub. 'klon ; 00:?:`ts3:''s2?2l W,FT'angii?Fli, -all of which are ln'4'~o-
,ps3raxted hC,'.i cÃI: byHi.fLT'cI':;~:;~=.
~

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
T}.> ~~-keth-ot:s described herein Lam be used : .,; pLirt of an ~TTaaRrF;.~~
S-ytsÃem" . g,
-ph1Ãn or tE?J.w:og
t^F3h?.c 2Ã27kgIng sysioÃa1> ioà high sensitivity da,"tCs%tt{.3Ã3 of flEaoI'E
thus, :llc .,>Zwi:>ztds are ideal ,t3i intraoperative in1zx~4jiraf. Mo;t c~
vc;', the n1e`h.3i,4 described
hek'E'.Yi.n L'ian be \a.E...+.d f+ pt`E'3Y%ide ci5lo1', fluC?-roscvnce. and
r.E7eEMf:fx images y ~~ keep ~ # ~ ? @' ~.{ ' YQ\ real ~'S
:~`~lfif~: :3xL;ZG'~ i~~~~4 ?`t~~ to ~v~}3 ~-~!= i of f ,3 ~ ~ , ;~f?~E
~L:vx1~~T C3~'~=a ~#~L S~;z~CS~ z~i~~~ IÃ .~..,na
iah 1Fr,f~-aT~: c:~. procedures i~3'.'=~' C:s3`~44-.',S3i.lI~r~,-3.,.
~~k.'~CI]'l~l`Ã1g on t-~:f'. RtI':.'~3:~ih of the flL`at?.c?4FvC:nS.t., and
f11c %E3SõaEaEin, ;aaid size of jthE? `it.'13`Nt`iir`.'. desired :C) be #:TIa-
gL.'.d, zaE:t3rosoi.x1i:E:= thatt~'r Isp W
C~- the L>ai >2~iI~.`i from +x}Ã. sttÃfc?t E? cvli1 be E;LaaE,~t'if 'iYÃt-h
pi.<aIi<3a : elle Z .s?Ã3cQ 3ÃÃl:xVg[
Deeper fisstxs .,aIi bL III:ag;..d .14:i1.~ t~sÃiTtiSt?t<1~:.~7;~a
2371~d#,'li'f.~ methods, such .E ~.~'~.i~ #.tL:~~~'-
adEt?;.iax33 ;bott?,s.] in"Ta':1.di??n. E?; s.Imc<,dÃ3I11a:17i t&h$1~qv::i,
wA11c?3 will likely ~.::~tc31el (t`<.`.a1tIl
dc,~7 ~ ~~ , r ~r #~~ ~
4ti..a;`'~.~1'~)Ã~ to a.,.~ rc~, i'~; 4- tC~ I i O~iwi,a ~~%~Ã?g~
(a'i'.~'ie%Z'4`+:td i~~. r~',l,;tci t:t.'~:'6i~'aL:i: ct i3.~.~. b~~i.
`l~?A'~i. ~..F.at~~~~A.
B1:5 , 6z641-3350 02) .ia3i~ Nb,.i3f r3ri.sl3s ea aL, EtIl`. R<3.cI`aO..,
13:195208 (2004).
~.~xv x:lg~:x~.if~la sf,.~lci=ted aiia3E?m9.cai: structures, a`k'`+'fi main
broad atÃ,-0:.u~ic~, can be 1:S00,
ACia' i~1tIi i ei trt}1> of ~~ie dyes Lx~:S~:~`i~r'v'E~ ~31~'I'4`,31~.
yc pass throtigat the a4guan
iu 1) hk#raLenotÃs ;Ã~Jccfion. 11-ii.s.nnelhoÃ:f roqu:itesffaa: the d,
P by the liver or #~id.''sety
2Ã:t4~thC; bod~~yfl-uiEq e :_f.ycirl,.=ul`s.:'tL in tFic bAood.~`stroarn,
b''c.:tak1:n -ti
and sZ`"4.ra4d .3vW bile or 's3riÃi'L, aFld2
2) Di~eit i,.~js>cfitrn :1l1tE3 the S$rui:tu2'E' to be 4'i: ualÃzr>~. Diref
#a.cÃ.1on also has twr',o
j`:>,;~~~~'.1i}IeS;
20 i An-tt.'wgr:ade inJG:i;'Li0}F'a, e.#,~.~'.. ÃÃ]~E~~1s3I:F w: the "tG~i`y
of t`~4 s~`F:-~S:`¾ti~Ã'~., b'ciY.o.C..a..
1-lro, fjE>c fol1.owi th~.~ normal iflow of the bcfdi~~~ BLi:Ãd.; o g-, z3fle:-
iÃ:iec;ion into à he lell
o; r`gf-: h ep.~~ic duq, the dyÃ: floY,~ s distally to ~,is,Ãaai.re C'BD, and
:~) Retrograde i~~ec:ion, e.g., caÃÃzaulation of the oz'ifioe of :; s4ru:
RIx`Q,
which involves fqjeist3ng the dZ',-. against the naaural st2've`.liTt of the
rlt.'~id.
f ~
j1?"'.~ urea0rs i:"E3sanE:c~ the kicitlexa' to the bladdf't: CU,'t1;11.1g un-
ne hadun with toxins
absorbed tom t1he blt363dti.rL'=ifln by the ktdney~S, leakage {J- which I.ntc?
:li''r`< ~.~o~..y :Mty +:an
i.?amse peÃiÃE?n:tfi;. During aibdom:Enal surgt;=ri'.s stiE:li as caosarian
hcc,oris :.Etad ut':klt3;u-:teA
t:r s~ix~ >:~,` ~r Csal .~~i~~i. ~:1c S11i'gt.+:?Ii be iiEblE.; to identify
t1he ureters, e.;.-',., to ei!''i;it;.3
damaging 3hi."':rn, or W iF'pai-F them after aatr<JME;1`~ic or ~xtc'>mal dsla-
na,,t,v;E;.,
9

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
Metb,rd: ym ama~.ing the uÃ'etefs 'iÃfcl:Ãde if~jed:yÃg wn irfvzs'Ne lig}4t
flu"o;:opho:`e
fõ #LF) into th,~'Y+i.oi.3dsU.eaÃ>.i, Y.ir CLit~.ect. cannulation, C+ithvr
':.a:[>tvsrot~..Y,rad4 oi. LeLf o.`'ty2:a.dc i} fLi Lht:
ureters 3F bliFS.iili .=?. ~,~'l.Ich that it appears :TF the F:fF":f3e
'~~TS.:i$.n.
fmagt; tbe 3,F[etvF's= F: >s desi1"-a~.~lo :'F? E,ICo an 11- fl3.Fi:F1`t.St.-
C;FAt c:.+mp+:,tild that is
~+ /; by ~y q }¾ ,l ,c ty ty [} y~
.~il~~~+F'J..~4'if~Y~ f33G~,~2F.aaiii=@=~ A.il.f.? t~3~. :#~"1#:t:. S~J
\4.~4S/<y5k Maa'.~.~1.rlAti2 ~'C~tlt ~f ~'[.+t-}.a Z' i .`A~ ;'\ [.YJ \jf~
~ have iA t.: t~2 ~a o11c4F~:_a
:a:FC.F2)e?:e.1 of sGSs, than 5 Ilfnz; are hydf'oj~hiht;; and are
TlotsTgF7.FfiCr1.F"7tly positively :.lcxi .ged.
Agents that c:}ef: i,+: used iÃ1?:.lf3.dL'NIR flu't?rC7p.lf}i=e`~' S?ic:1 as
Fa3'vlh?`lÃ:'ile i3ba,. n;y:Y,86,
CWSOO-('AQ Cu5:5; Cy"¾ C3 i.S. :(Rdz=4:`Ã'-,A~OOCW (LICOR), and IRAYb'r 8
(:_.ICOR:).
T;.ac lci`i:l 0, i hydfo :3a3i Ic1ty of a %f):11.poaFf'nd pt a5'S a role ftl
d3FC.ci.ing #.:":1E.alkc 'ÃLk :hw
kitiFii:y alaaVor liver; tho>bfo5e, t.3eh5rt..ie3pllfla:dty of -a
mE;dF.fiak3lF;=: ctgÃ,ait ca.~fy a3e zenS...FF. asw'\~~-n
s''.g.T by 3n<:=Ã'e<`F.sIng the aQ+'G'I t'sf SulphC5z123:t3f.}Ila to increase
uptake :av~=':" f.ind. a3Ã" : idlivy. .1
fsL=..ti.'t:>Fbed h'L'r5:`=ixl, c3:,:TLMs 1',:;f3:t t3re u'z3sul}:ah1'tnatGd.
ITe.?i.iCyst1lph(;}:1a-tc:i,Y or fl1si.F_pl-iotlzl:.i:d art.
g=,n<,rYiÃly fap3d`yf uequz.stc~od b-y thc lõJcõr. btit a.Fr:. nc}t. .creE;.d
Ã~~to bilu officio.3fly, .Ã.d rt;
thus not partacWhFrl}? useful iil' xnI.F]9u t13e b$h<f.T'y lF t',t<, Ageizi:w
that ;.F3'e ti7'#Sf.FaP}eO;3ZF~.S`,(l,
mulplf:mF3..'4d, Per-aa3.suEphiDne:;d, or hepi.mulphon,':3.tt`,d are mt7w''
likely to be secf'E''tLi3 bi
thà lxvxE?" fTit[3 bile, .~.f~d aF'e also x7.?oTG likely >~) be available in
the circulation s#.~.. :f?~ S3tat~z;
by the kidney into and aF''L tt311: usa:ff.ll fci1` i21,f1g'ÃI'Ãg the
u#'ct+'=f':^ a.2Cl hFl, i}'y :~oc,
Agents that can ~,"`',i~, \ ~ ;-nodafi~.f~ h F~ this rfF1~,l:1~~t include
F=~'u~i, ! J 5 i~'~~E:.:~ such 4..`~t'5 (_~ ~;rtF~..;~~.c. ~a =~\
~ ~'i'3i. P1~:~. < ;J ;)...m
BiL.3, ca1.'.nf..vs); c g. Lv, su:&ofa.ati(..}n.
'Ont:a a-f'v n[$tF.3rfiil1' taken 3.Fp by she liver wllI'.'ià injected
sytitC,mFLd:l y }S~
2 0 SoITif wg
:5efec.t.Ã'4'5:l4 liii?s.I the u1'ti irSj such agents should be modified,
e.g., by <f.d:f.a.f,3.F t,: `sF i7F ?~f f '
s:;ch~ as~ .F PEG i.;,, by p~gy1at3i;n, that p:i'uvents t.fptak:: by ti1u
A7ve.x,`to :fnp;o..='::' their
Ske ir,Ff itv zbr the ureters. Methods for pegylsitif:g ki}Fx1poF.:ta~dti
<.i.F`e knowi1 i?a ::h art.
~
25 ;.~di is~~;j' ~z ~:.' d':?'t ,'~,:Fx'$r,.
lF1~,.F'a't?pt,.rz3tFvi:, :holaÃIgogi.`e3'phy (10C) li.~-s beiia Shf3wf1
tf.~? dcC,l;:~se the risk of
ct;fs.>11or., lb1le dFEWt injury during 3:m"asbvE' S[#.YgFi;;~l procedures
such as L:holi.cysiWtfFfey.
IFlv'lsHe LFgh~ (~~~) ftut3F"esF:F<:13LL`=b.lae duct imaging offers so4'i,F'al
wdv:F.F~tages over siKtl,da?'i
IOF with x-rays including no ioF1Iz1Tigk'i3fllEFtif?f3., Ie't:Fl-iImS.'
fa"flc3wTf.g. and sf"aaf..fii.a:'i`4Q:l.i:i
31 Ã; :rnagpFng et:tia ÃtÃ~:= c,.~loi- vFdoo fimige of the surgical fxe:d.
In tl?c. moahctda described here.in. 11_.FsÃhat are. fil :oreai by the liver
into d_Ãe .?:iae
stya:<3li1 i3lLo3y V3HiFi.a:%3L<7.t3on of structures of tt1e biliary sF s,.
.('S including tlic c:;F..3FI`:o.'^: bf lc : uC i
~~~

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
(4.=BD), w.lÃcr? is vulnerable to a4'Cld`s'.mal inj~uS` (bui`iI'Ãg Si.irgei'i
as it is hidden by, c1i-ld
f.f.)l-n, otlii<,r fis:?u'L.S.
Methods fi~a firmging the biliahy tree includt. intecting, an ILF into tiw
bkxodst;vwa:
Si:idh ~'ii-lat it i3:pp~am in the bile 5:7`ei3.ETF. In 4eT:eY'i>f,
k::.iE?si^. 4:go.ilts ~l-i`ai$ w.Ãl""s label Li14 lIÃi'>i:OÃ'S
; w.ll,als:? 1abel t~e bilian t:ee themtb:e; agcaÃs t~~~~t have pZopezti : ~
~~h11:lar to ÃI ho,e
d;.=s...>zb~.~ above for labeling ureters can be used. ILFs th~.Ã ewn be used
inc:ude NIR
fl[l.fYi`i>a h{3m,~'_.+ such bi5 31i.ctl4ylunc blue, il7d'l)cyfiTl].nS',
ta~?,I'Ã?t'.:il (J~.~G)$ IR-7~~.S6,, and CZ`:+ SOO-CA>
z~y,5. ?, Cy:, iYs 7.5, mi~ IRDys;''-,~", 8,
F.51' rapid :inds.,}eL:ive Imi;3?>ng of the bilr:l#'y tice, a;-i IL4 can be
iÃ``,...ca,'d i$,'ei:.ÃIS`
iii,t{:f the portal win. Direct c<#.17i.11i:3l7C>iI in1.i'~ `.l1e i'igl1t or
li:ft .C.,pa.l:i:= duct for anterograde fiT
i'i'broÃ71'e'Ã.ixt; labeling of the b1.3iw-y`iiot.'. Incl:adlilg the CBD
C=:.'+,~. al st1 bc LasLd,
:.hor<.cÃ~ diict is an impz~~-tant partoi~ the lynnj~aatac s; ;;ten'i, ce~lice-
in:. most o'a-
Ã:1 ti~.~., ~.~~"Ã:~~~.~'3. in i`.~li; ~.~L3~4' i?Ã..'?i3 t~~';S3I]:1I3~; it
i31Ã.#3 f`l.a:: Jy4Ã(;71~~11?. Cia'E:=L2IatiC3i1. i h,.:t:>, >zzcthL?CS`u` fo:
1~`t'lcz.g,ia"ig the thf}:iloic d...d include IIIji:=i:t.ÃIi,~d.~,, e.`~.i7 IL
fluorescent compound, < if., a
E.f.?Ã'~.'SGe,.t ct9aut:1dy into a lymph n4_>dc, e..g:. the inguinal iE.
lylpht~CSLxS;, and dowE.,t.;:i3.if i;
fl
fl:aofeJcS ei.: sEpmal &om tlio dwIa.GÃ.c du4=t,
lÃa some, eT11bE3dit::~enÃ.J, flti'= i31effia}ds i?.t;ad:.l'.Ãbt:=d hÃ:.'eiIi
PoÃ" i.ma,~,'~n,? :tic ;hoi"acic duct
t
l:icliiixe tffie 3:#i:i:,' of 1~~~~th'S' C:lla`.: _hw as the ILE
"`;m 3,3~i?: rai~
'Th;. a$3314;4` to cl`~`.'~ai'iv i.dt:ilfiiv L;`is+;.'.ulatul"c in Ã'i;ia-
wmi,' during surgical õD*'f3r:t. .dum;
y Wc3lild : ., ..ÃiL'i~;sllii?bl: fii.s-? :a3G
sL~cli as 4z1rSziac surgery, neldiLx5urg<1y, and wTf'3iei'ill siS#g'__'
~ s:~ -~w..}Ã~. As described lac>.~-ci~~., it has 1~e~:t~ z~i:~~:o~~c;,r~:~~
:liat ILFs, ~..~-i~::~.c~.i i~;.~-~s~.=t~~vI..ne ~.~luw
Li~i b4 usi:ti foi "Ztaa ti3'i3.s~ li-t tÃr:gx41gr:tpaty of F)lood .'k:
sel,,', t o al Ir?'w iihs LS;,t i.Ã fx
vcs'-,~`,Cl ps` tt`Ã::i.y; 'wiwh a gt'?eid sÃgni3.l-w-backg2Ã3u;.7d ratio.
.i xx4F;..~,i 'tI~JS Ir{)t` d{!.~'f:`s:Ã3t'2tF!^t#~'sri3'i.':f'gtxtr~r
3u> :n g,::.s ii~i. ~'~;t~f~~aSÃr~lititie ~irgir4' is ~?>i~ l un
~lz'itiC~1~3.~~ of :Ã6S~:u,31..`=~~~:i11~:~`it and
tissue most ComplLx i-econ;t-riiGiivc ~,woc:edure: make use of`flwps,"
i!'hici7. are blocks of tissue that can be rT:1ob'd C]3'
ii"'t1319pl<:i:i7ts(:=d based o$r, t3#Q an: wm:2:Ã'
chaI,.3ctL=r1s11a=s. of the blood supply to ~ti>;: 3.~a;?; Si.tch fl:3pii may
be (,3:'E-ipt3St'd of skin,
I I

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
muscle, e}43?.1e, rr.,`.dt?F;1, n`~.avep b:i,.)o5"f vessel, $õ>i-('.st1Ã"1:a,
:'1i.1d vitÃi(x`L}.s combinations of Ãlw6c
3onaiiClni h.k:;
ÃypC;`s of ÃA5s1ic. These xiapS ceO.tl be moved from a "donor" site, whos1
S.#",I.i
ac-equ~~le mo:;?td?tye Ão the "reCÃp?crt" site, ~.vhkcre rÃ:.constnioÃior,
?;s, neÃ:=d,,A For uAan,xA?,e;
a l:om`',a_3{3s3to 1': `S. : flap of ~ibua r3~id overlying skin ~.`,aii b
i:se{"~ to recon: tmC.t a naandiblo
airdflooE"-:)f-3nt?uth defect fol~oit3i11g a lÃ54jor c+Ã"a. ~=~~ri~=t":.
T:.SoitiC3i'Ã. Ota?:;i' ex<3;?,:?ia'::
include IleÃa;ts~'-nl"F'wiahz fli3p --, tl?I'f%tG:lal Tlt'rve PaTalV=kda5
fibi1lF3., o5tof_lC,'ra3.fwS3us .;.2tp:~ fiuÃ'
mandible a3~d oral reci9t:sInuCtit`.+Ã2, bAm:st 2'.`L<3tlstI'Ã].'L'Ã?o11 fol>o-
wln'k< b,ei4t caslcex, lower
z. ;alxemity salvage following tra~uri<:, airÃd total penile
rof:..t?nstruction,
The s"ing.Ãw: most lth pi`,T'ta3:tt} dt::F:a .kFigv i3T1d t,lT.1:1q".aliL'
s3gI_a.t,I:Ã~~ C}fx flap Ã~ li:~
~~:,.. ...~s~~" ~~ .~~~?~'i~?~ts:~~'. if Ã~~Ã~Vascular s4Ippl}'t~~ a fla~? is
not t~~:t~~Ã: at~., Ãhe[lap =.~~;;
lxwo:ia:e and fa?3 whtin t:'atlsfQrrGd to its new PosItÃom 11-1c s= aJ::ula;'
S'~,:Ãpp.,, to a
~ 1~. ~ ,` ; .u.õ.-~it~sà be adequate ~ supply > ! ._~-Ã~: ;~l:a::l.~~,...~: t
a~~ t ~.
~?~x?~.:~ of x?~~,zc.=, ~~~3s~{,~ as a `~s.=c~:~~ÃÃf? ~:a~?~p. a ,se
>:otiie.;`ti"'~m11~~_ be eic~~'f.E;:F. .:3.c3znta1d3<:d during thr.', surgery
(pedYcl'vd _q:1p)., or if -i I'...i:yt bÃ3
iiL c :~.t: ~ ~ ~ief a',1 } ~ r~" o2 the body ' to I c.1cl`:~, y}i~. st `~.=,
I; ;-; `:
-~~~c~ ~:: ~~. à `~.~..tà ~;i.:Ãz~~, r -~~.~ ~~E
Ã"'K:.al3a5t33nto5ot:` to blood vessels in Ãt'1: ie=L.":1pLfu13.t z3.1'i:,il_
LÃs-iÃ;'g microsurgical cal Ãef h211q.3.t" c3s3d
rnicrZ?::tLat gici:l inst-rumCnt:e.Ãiti'~~~ (free fli3p)~
I F3C ,zw.rrphi:=f Ftl blood ~ sv,,pplk to the slti iÃ3 1F1 i.n35: fi c4~
~lap donor sites EÃ';aes ft,.t1.;,
Y
one of t'i ? 4 ~35 S~`S.. ]SZt. 4S~tt.. "( 1~tF"4,:i.=te ~..3I..;i~.\ A tl`k=
~Y g: j'Y
Si~:: S,tii~::C.=`=,='. :~~-'~3~Ãf~C',lEÃ:~~ ~.'t~,: which course
~s~iV'~33:1~3~,
A9eYfcli"i3tor vessels, WhÃL=h pÃ:',AictaatG }i1L3'^'1:l.'.t* C1nderh'?ni.F
:I?u s3Si;1. and emanate ele:"~,`"dd'`d ftoin
20 Li c, I-rw:'ii=lL. in dliscI"cÃ.es but wJi'1ablt 4itE:<Sõ The inconsistent
t11lat:'timj nk. fth~-- small arterial
pe~foraÃon, within free tissue Ãmui;~fers that are vital tc? Ã~io =vivai of
the key cuÃrlneous
p:?n~f? s of .I:.: fl~a_F~ has boen a 3mt#oÃ- roadblock in reconstructive
uirgi,Ãy.
I'he :-noth}adS described lieresn. provide nztt otils to: the
idell=Ã.ticatiSFn and l3c alnow:
of perlbx~t'01s., but also flw~Ã" relative size and dtbecÃtf?n of
,?:z'botif>a~on,,
25 substantial deNigi iia>p?ovÃ:menÃs wb~=I3 they arc: needed, prior to making
an =Ã:-wisiz?ra, Af,Q:-.
Fli1.p C'.lt"`=vabiin, f-?i .mE'.=thod:s cw1.1i tFr.= usei.k to confirm tha,
the key peri.3r"x..tf .i's V~rG're not
izj;..:reZi. dun$if?. the dis:iLctÃ;ta illld that itt`~.' ~ ~scuzrÃ~ supply
to the flap is adoa.,.1~ite; in
addition, L~ie. n'!effiods provide an E5e7jijcL1ve mLki7?s of determining
i:lrw, or fIa; .. dt.4t1I.3oi.`~i
?b} ;?Gi:3:osYs, so 1:h.~~~~ flap :iTtodõfi::at#on or ill#;vF'Ãkatlve
corrective i3?~,:a.sir es cs,3t1.d be taken
<sf? ~:ilttic3~?~?Ã:="~`=u.~~i.''Liy, FiFSa$v, the link3giI1g in4"Ãt7s:3dS
described he:re1;.'# ari. 1seitA in
pvswoporfi Ã?~O :11oni.oring o~.';hetlap, as flaps must L~e ccx?it:s;?uousiy
nic?nit3. ed a?i the caray
day.`s 1`611i3Z.vin#? fired` ~~aJ;l"cf.'.Ãt:iinsfer for evidence of artt;.lal
of vi:ll=>j`;3> th1"o:3h:'Sl-"~' sanC.',,`
only S' *` t ,p t C, f i 4 , T ~`~ . iT
~`~ ~.=~tv is is.Z1i~Y ~.~i~!'~: ~~.n.G~~`a2.~:f5u~.~'.~~'E4 ':~~~f. x~+.F~
~~it.'"~3 ~'~~`=~~' reoperation :t ~~3, rFap,: ~ ~ ~:~C, 14'~12

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
~ A~ a ~~~ a-iv a ~ be ar,v -~~F ` r o ~g; tY
;~ ca
:>~:~.:~ ~7~~, a ~~ a.aa>~~d;r can als~? b~, used lbr ;~ssv s ~Ã~ , i3:a~ ~ i
abia~.~7 and ~ai,ainZf ~ surif
`s lft'its at flap &Avslge7 .17.cxlidii?~ the use of fibI;Ã?(?lil3c
agi.;it`s, vvheà coiz~phcat:oll. o~.-':.x:=n
rF C r: AFii3i',.`7F':Fri1-zF3i:
D..Lziiig c&rdi;Ãe. ;.Ãlte.ry bypass g:affing t;C ABM and ~~flier cardiac sui
ge.=y it is
: 9:Ã13p<}.11iInt to .3.~.~Ss+.vs myocardial ~.r`e#'fL1s1oll. A N IR
~luCiICsLeÃ1i. >Ã'ict',' tffisi> a ..tum?:3.s ia's m t~i;:
i.'iC.a,-I after intTa-ve`,i{}u'~ injt:c~iony s`"v.~.cli a.~M-1~irhyS`fv`no
Ni~~'.vy wfJ~~ld periyiit :ioni.rvauive
ii ta,g: ig oif c'xirdz~~ perfusion.
x
r=F=~~'3'z3d.;~ i.>ts7nC.'s
%
ril?c &b:ifitv iEi ,5.i3C.c3~o the pw.c:-d1yroid :7laÃ1d'.~~" dLtÃizlgI7L4k-
,5tir`,~..Ã"4' ~:.=etn ..~c.: emci{3:
I deten-z:.nant :.~ success, wh:ffieh the pra'ihyroici g~Eand5 zAre tdi~
ini.endeci surglu<zl ia:;;et oz
:iri; ti? i -void,`i d;ng a proced:li'e. I31 some embodiments, th5.
Ti"i.i:thC}C~s c-s";F2 bc used to
f.:.~etect, pw-ah~Toid adeÃromas See, o,g, Kes.viÃ;~y Ã.Ã <i,., BriÃs <~.
Surn., 1969:
56(8)-595-5911.
and :d{ StrEr'~?":s C4nC~3 1"t wNeas.Intu{i1`7s.+tna
Wths~len:: blt i: has pTc:viousiy been tlenlonsÃ,ated to be uaeiul. as a 3
xstial dye of
mere~~ and imquhnonaaa. See, e.g.> KckÃvenv <:tal.~ B.-it-. T, Surs;, . 9'r 1
"'8(,3).2 333-
<I>e P,
. ) i. a i a ' . . . ~- y~ -a ' _. 7 ' 2 . \^ . : and
c. ~?~~`~~#~~~ c~~: ~~~~'e~~ rÃe.~:ir~ ~:;~ii ~5~. us~::~~ to identify
tl?~.,~~.Ã-~ ~..~.~, .>l 4"~~cls~~~:~r x.}<.
other Ui~..?.3i's :li:c to the teildi:.icy of MB to collect in ilshues;. ffie
M'k-3 can bt;;
,~z~ YI C k \ n i e . . ... ~~; ~ #~{ Z~...,.;
:~iil.sr#;\'v?`~::~a s* ztL.=+a~'~i~:i~.li~: Cyr`~a~"!o:~`.ti.J~ ti~ tl?S~
E7rF4i1T~. C .~.a ia?t~? the 7"t~ S 3~~~.aÃ.~ of ::~L C3ra~.t:
20 Systemic %[t3mtt;1Stratlo3 of Ã:h't'., ilyG`. C:'t3.:? z E:t3 bT:' usi:Si
$Ct#.de11tifZ`
1`ie.a~tat.i. Ã'3:n`io: S ftini tis4l:1es that have hi'~y'll uptake of NIB,
ti.g., t?.IÃI,or: of pz3nc'ea.;tic,
G.Lf , nlswinon3.w, .`.Ã~igl-D.
Any IL 1.,Lza?TZ'.=5c+.`nt i.'C7e33pS5i.ilid that is ilf?tE.'cti3b;.t; in the
djqLlL" to be imi~ged L=.r;:,
-11) i.?"'etZ;,r} thoi'aV.:=Ãc dE.ef a, b`Eii.iy tree, or s='wiclil%It:kro.
Ce:iÃ3 bL L1Si'=iI. In gi;t3t S`::.?#, thotit,.' +'õE3r"n.pÃ>I-..,dti
hm ~~uoi~.W~ o in, t1~e N I~_ range (6ft~-1~34)~~ ~~_~ ?) tire ~~seiua, iÃA
adc~itit?~~, a<wo~~Ãs fibruse in
~\ ~ 1.
~
F~.c z~r~.~~.~,=vrs s~~.~ ~: r i~d ~, i~. t ~,~:. ~`' be sÃK~nii~c~~~a~ f{w a
4z~.4~.,,~~.? F ila~~,.i. ~~a~~ by Ã;~: ~~~ ~. ~;
<" ~~~Y.Y ev :~Y ~ ~,~~~~:~~~ s~.rÃt~,~~3~~?c.~ or ~~
i:
F, it b~e t~..~. liver or k.Ãd~.L'~j'~s, i",i. }~:ui~~bi+l. ~.~A
~.+~?~~poun\~s that wohfvc fow.ial, C..rw s
imciti?i.33#:, :..e ureters e3re known ?-Fl. the art, ?i#c:ud#Ã`ig ~rgc3niS.%
filuC3-rophUrer<, L;le Ã:t::?'i
30 conui.3i?.i c3f wwhich~re poly-me4hines. One: in~~or~ant class of t1le., is
i.~lQ
hu~ta'i~~th.Ãp"o, cyanines, coT:lpÃ'Ã.5ed of bi~'3'I, oYa7ole, betvf?:.'h;.r'#
Yt?k, indolyl, 2 -qi.:ÃnZtfino,
;3

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
"qui-no8ia~e, snidlclaSTe~,. E'Xemp3arv ckFmiSFAuii.b,:51nE9=wude mv6hy~:enc.
~~lu¾; (Ma-t)j
and 4
indocyanine gri,m (ICG), IR-7 86; and ~:~~~~ SOO-CA,
It: so~ne :mbfJ~~In~enk5, t}:. nea: -i:nfrax~d fluorophore has a structure of
~onmuh.,.
#;<
. l. ~;=.;^"'..,
e.~..
=~
: .P
.\;. 2 i=~i,^=" :~ ~õ,l~ `
F3:)amulli-1 I
wh+;.rÃ.`in,,~s valence and :+tsb.ÃlIty piLÃiI7Ã.t,
X represents QR}'?, S, Si'. 0, or i'VRi;
R Ãcpmients 1'1 or li3'w"4'T alkyl, or ii~~'it i)GC:131"Ã'C'.ilces i):"~` R,
ic.Fk'Oil t<3pÃ:thcf, ?:oI,1i a ring
foyethu WÃldhthe i:aÃ';f9natoa~~~s flucn.#gf3w1dch tl~iev are (:~onnF:ewd?
R# and R ri:pmsvnx, i-nde.~~endenilyn st}bstituÃcd or Luisuhst;tu Eed :ow;.#-
aIky~S lower
~r.~c.,; ~:.~ i.~. t ~ or iÃ:~'i.l.~,~+:~r`a~ ~~~~~.Ã<~~1' ~~~'
wÃ.l`~.'3~..#~.:#~L.'`= tL=,
3 = =t~~`w } i{'}~~":~.~# '~~ `ac3c.e~.~S`i=~.Fk ~#, %Ã r~,'3. t~. d by
~~iÃaa{.~A
iÃF~~.,~Z'~, ., 4
phosphate, svdphF..Irx`.":,, phosphonate, a'{:Ã:lt3gt'.t,, ~.^ayd,Ã'i)x::yl,
al`r,:~7.t#.cs cyesno, Ã'::}~ro, c:s,:bt3xv:~>i:~
-acid, oi am1:i.'. or a ph<1,i'1Fu".`Ã.i,eL#~ica:Ã4ly acceptable salt
::hoieci3'}r
R-y rqpt S ~c i iS, 1#Ã~~~A3~'ÃiC~~il~~~' i?i` ( ~%~; ~3~ t:tt~ 3 i'Ã:wt <~11L
t3; ?~ ? ~ Lt]:~.. I~f:i:=Ãtx~ Ãf~ ~~i' '
-r#:ng t:,>'%,h{ch x: .s ;~ Ãa,.hed. sFÃC~h as a fused 1"#R3g, stlÃfa?cK
ph:?:~phate., suJphÃ3t3ate~
pfilo:,p>`sonate,h.1:ogo.i3Y lower ailqlz hyd1:(:3xya, e1II31no, 4=vs-tÃ'1C,.,
Ti,Esl"tiz, ctÃ.I':bf3x4lic acid, or
wnidt<; or a phan-na4.uu .icayly accepEab'e saft tiiGÃ'eoi;
R re-pra,seÃ?ts 111x hnlog31, ciS' a substituted (}'r u1rs4bst.1tET:e(z ether
or tM(}t =thE''.~I' of
ps3ei1ol or thiomhenff 1;
R, A4prosenis, independently for eat.l^ c}c:uuiA:n:Ce, s'ubstÃ,il-ted or
umsubs;:~~llW'
lower iizkya, :;:~~~,'C)i3iilk.L'l; t;yC;.aÃ1:1lkf lalkyl.n c3r34s t:r
ifi~.:~~kyl9 k2ptiorfi1?lL,: sE.1.?:hiltuÃei3 by si`allaw.
M.
p}lospba:i:~? `hoÃac3`.e, phosplIonato, hyÃl~~ge:-. 1.-lydI'C?xL'~lw
aÃe3..Ã.a(?. eyt3i3~?, i~AÃiT'[), ck;ra3i`xyC
-zd, &niide; or a phaÃnna.ceiat:call v acceptable salt the>eiof
xÃc
a:: s~~~~~~. ~=~~`~~~~.~Ã.~.oa~is, i~:x, two t~c;;~:Ã~vcP~.~..;. ~a~'R taken
tt?~.~..~:~` ~f~~#~A a i"f7t.TiabC:.Ã`t:ts ning. ha so'Ãi-ifi`.
C'=Ã37i7i>i"ÃÃ7?o,1ts. RI, R2, and one or both R3 ÃncliItlt~ Sulp~.lonilte.
Ã4

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
r r~ ~,g~~.{~ 7
7tdfY it~~'~iS +3~.t~1f{.` (:Yi~.i=s) Aftlii.`r~I:ÃgÃi it was pÃ"evÃL?.1sN
kÃ1own to use Ni,B as a visual dve, .he uV~e. of N1113 ::`:
fluoÃSõ'.sS;.L`Tit.fi: nnagÃnb has not bS::ti:.?.s C.g71Ifii=%$i'lfly ilppm
=xtlt('d. As described }3ii`Ã:rl
b.i%Ãi. s3as pF.opertt1':s, Tl?o LmÃas.i<3't] ,'*:sa+ <:l;nr;tb 16770 mm to
{ ...r`'.~~ "lin 4't 3.i:i a i3eak that s}:1Ã:ffs :3..5 a f:I31cti i71-i of
dye C'ons:.'Ã`>I3tÃ:ÃtaC n.) is within tt:Ss N R i't`,Ãn<.õ-i'
'c3: physÃ.t>io:sit:e~~ly .s`s:~~e co;i1coa3trcitl.ons and t~orÃ.;fo3:e
p4Ã.t>`fits hiLi
,h and high
23:Aal to baCkgron:nd citiw t`, low auiofluor~.s;:enÃ;e in huni<mS and
aninials. . his
:~'.:1a1't:cti;I'lst$ti= d1;ows MB tfr` be used as a Y'asL.4.lls:r 4Clit=iilst
agC'lZi., using fl:ist?....si:t>1`:~;w
Ã.:;i3tÃk;I Ãlg 1eci1alÃ's3og JS:lril'is~3~tf y, ~~~? is aeir~'tGC~. or
A~~Ãt;<,Ils s~t~..~I~;Ã=~%lln' into certain
ir. ~~ 1'#~~~ ~k`~~ i3~4?<1Ã~ 5 FÃ"1Ci.3z~iÃ'nw'r~lw ~t~7.t>li~LÃ:c duct,
~?llE' f~i~.~t~~~T1 g 4=j Ã:?3T ~..;t3C:t7 ~?f;3Ãe:ii :y` :rtif`i:
1Ã:6ne ('s;.llf?1. 3;:1.g. VÃS`aalÃ;e`at:Ãt?n of flic. u.Ã'i:.te1'S), liLaÃ`t.
JnyÃ7+. a:'d.ÃiaÃras vÃ?>GulatGÃc :al3.owing
iÃ:"i.Ã.gi3g va, alia, the inZ%Ã}Ãf:ir4:Ã`i1in, comonary artery, etc.), and
~.3,,Ã13c;"ec~~ (e.g., into
beta :.G;i;s; alli>t-Ong L'l-sila.:Ã.:%atÃÃ;n of tt1.at organ <Ã#d
1'`iIÃ'ilÃ}:':i nid Ã.aeÃ'.a5t,si'5 si !tf': a
i1amà $<asa:?c CMg:iil. à ,g., ln6ulÃ:sE+Ã~zis).
N-iB h-as %h~.` adva1:3.l%I~?i.: of 7euread'Lr being approved by the U.S. Food
& t. F~Ã"Ã..1;`~
,. .~..,
At1in'#.a'7sttc1iiÃ3n :3-s a blue dvL= to asSwss gaStrf?.Ã71ti..stÃna~.z tube
pl>Ãuc.n.i~::"i'Ã and a.", a tfeat'iÃ;i'wA
for z>:s.:ahmmoglc~bineÃnÃv: Howcvcr, thoracic dtieÃs can be visualized
clearly offly aftox
4~2 ÃI1~~ll~'~~2~~~:i SÃ~31t?l~' I~k.asi' I z .;
color delea .iÃ;Ã;. But ag +,~tmlonstf ali.d fieÃ"eÃti, the thC?~acIL= ducts
Ca.Ã1 bL dc:eczcs,i ~as>Ãli~ i~~.A3
20 fluorC::sC:ouZc at `mli;;h J4Aw4Ã" L:<)Il"Z'Fltl"at'ioTlsg '~wen
Ã3.sa.ng$h+..~ sr1liiC ÃT3'=fi;:1n tc cla:~iqi Ã;., FtZ =
H aliqale. WIaQTI used as a "Au.,_ dve tonaq? tho.t-~~~ic duct. MB is injic
.,~tefl dlr;.c.Ãav....
stitaà il~ i à t~tÃ:> ;> a I `;"'i, (3 1 :Tl~'~.~ ~Ã3li;tJo$1 in sterile p 'f
iCsjt si=d wat> f: Who>1 tlso-i a.s a
LIti=~roph3Jsi>., and lÃ3ieCted directly 1n:i) the Ã33Ã3T'a<'.3.C dtict. one
:1e:d--~ .a? Ãit:h:+,.v~d- a
L'=;3a?GentfatEi`n a.".Ã the :-#.sstIc of o.Ã1.~v about 3 0-40 ~M,, e;g
it.botit ?0- 30 pM (wh>ch is abo,,i`
25 a ,01,00 1b36.Ã ow(';:=r dos:'v)45f #qjL`.'~ctÃn~..~.~ a distal iiode that
would drain into ~+c 11)j a
concentration bc:wG?ei'i 3 S~ 1.~ M and 33 1 mM can be 1:.;sed. This
>F,a;;ge5t:-, that flÃ.aoreiCe.>ci.~
;:mc:~,?li'~g is n1Gm Si32sifive ffi~,.1 viSldraj inspection with h;.imaI2
c'y'cs or Qoii3r e:i:moÃ'as,.
Doses ci>` a;O-10 mg ~~r. of mefbyIz.ne ~~~~iQ tirf? widely used ,alinioa,ly
'.~~~~ the
L:~'~`~'.ktn~S`.~.:~t ly3Yxnuffi`L:niS.'~rg-lG.3binaemi"(.{, and.. Ã.~iuf.'.in
larger di3sL=s (on i..SiE~~:~o:Ã.t`ti>r of 4.0-7.5
~~~;.:='{ ::M=.
,. s >; for ~ ~g .. ,,7; g> y~ 7 t ~t>t. ~
~> <:i~> + S3~.~;~~.a.#.~~~i`.'f"~,.~:
}~iÃ~i.~.43~Z~iÃ..~'it1'S~'i~''~9t.,,~if~~..it'~, ~:;C:Z:'~3t~.~'Ã. .~.~.
~~a~: ~i~fr.kc:Ã end, z.-.g_
MB :ÃZ:i33Ãt3l:e~i<`ai1.Ã3~i xiÃ3iF1C 7.a:tl.tk causes ~i~~1~IB.'. <~,F~.~'t
i9t ":'~2.f.1 .ÃÃ)e-IS, L;~
m~~theÃl>ogli)b:'me131:a or anaphylaxis. IÃ1 addition, :hGTear% some
1:'epÃ1#tS i.ilddci:ttitfg th:a>
inilc3dt:,maE 1_i1j4 .ctiw:F's~ of MB :"..i-r1 cause :ikitl d:~11atie. For
'xu.Ilapi`r,'~,Qi.ht: hÃgr. Z3<::=...s used for

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
r.od~.1 t~~lection, ::.g;, around 4 tril of :.~~.3 mM MB, a:-cuss,.~i:i~~ted
witli reports ,y,f
iiijf.cLaon :~~temaC."4io.~.~`is. At these b.Ãgh concenaratÃon.s.4, no
;`~liof~.'sGw'3'?cL;' wo':-Ili' '.3i: visible
dE.e.o 4h~ i irÃ~:aE Ã~~Ã`E1~:,t3~ ~3`~'=~3c Ã"~# ~~~~.~ qaÃ4.1'3.cr1ÃI` g of
MB s=tlltssÃ7Ã1s, Tbnis, in K; ÃleÃ'i I the
do`'si:s::iset3 iri ':hv methods described berc:iÃ1 are abi?ut 10 F:ÃÃ1":ee4
lower, 'wf3d in Sonni::
Li'31boa:`~:12v.~+~3s 100 times lower thc"'ln tl:t3se previously used,
a3'.1+.l wrt: expected no``=.to ca::is e
Ã':lliof ti'x_ÃYi faaÃ>:a c:?r adverse .~'i:~c'~"~;:.ii3Ã~.Ã>. For example,
in s~:~Ãz'~~~ ~.F~~?S.'~{~iil'c4Ã'Et^~,. t~:+,. mcth.>:.;:
;nea3..dV:hi. %Ãdn-mlÃstft$tÃt>:Ã1 ofa. solution ;23i:"l`aidll]gat Ii'i]s`:'
U1 % M13. 3w:& about 0.03 to
ME'~' < g OAOS % to 10%, e.g: I "Xs to 3..5%. These pe;'ceTÃtages, are
weÃ:;h::`LvL~ghft,
i,e., a 10% i^s?tt~~.,lt,Z?ai is 100 mg/m.L IE"l gC.FieI"'c :a the total
do>'sc iha. `;`l~l be t3.sed ft1.?.' Yi`Ãi.5st
i~~~?l i ~3tÃt>I3 s is ~:~~i's:$t a 4 ~~~~ of body weight 8t'~34'2i
i~c~113I~: . , l5~'3iÃ:,Il^c;w1;.;t ::<.;= fox
a 70 kg l?ummi, :~.z`d a desired systeinÃi: dose of a(?ot12 I m:Ak:g, i.?IIc
woltlt~3 need 70 a:lg;
~SJ".ich is equal it? "17 3rfl -csf a, 10% ==- 100 Ã?'!g Ãil1 ;iolÃ.3tÃC3;t]:
or 70 1TtA of a I % ::>: 10 t?-1&~An I
:~i~~.'~Iti<`.i#.. I~. is desirable to achieve < concentration "ax"#.'~i.~.~..
t5s:i:a.i. tC? ~~..;~naÃ.?Ã.C< o:`el')L sLit i 0..
40 W, e.g., about '20-30 Ã.cA The coucs:at:;3Ãion e"aÃ.: wary ~ependin{} o.-ki
it:c local
envÃrcruiYcnt of the strucÃur., ca be: imagod c..g.~ the pH of tl-is or the
cÃ?Ã,,ce$~trwÃt:i.`>ii o. paS.3ic]Ã1S: IL`a some i:$Ã1b1?d1Ã2;ei1ts. <:`.
opEÃ1'f7i.3l c+.?ni;+:Ã";.F.3atiÃ.`3n can iie
s~~eTitified, based, e:g., on t1ic graphs iri [~igÃares `1A-D,
~a .<;. rZ herein
It. 3~3~:Ii2'i~2~'~~'; the ,~~:3 ~~Lt~.;Ã`es~.ai:e flfl1~Ig31I~.~
~3t+,~i~'#i~:i~:~ 1,~.:~+~t: ~e.s Ã;~ Ã'C:-alti e
~z# ~.Ã~~ ~Ã.~t:~-, ii-
s seÃ?SÃÃÃ\%1' i 'v~fltr~ lower LÃrsvs of ~'I~. Nle~~?~`lcA~e ~~z~lti= cÃI~
.hi~Ehs. as a l~`I.alp
:?i? bile duct ~3I;i1 ureter 3Ã::d.,;CaiZ?i, tit?d a vascular contrast
a;ons.; Ibosc broad 3nckwtxo3'ts
u7.7'CidE'sL:=C: I?"1e2'4 :?pv.i.;.3x3s for t,:itr:3t?~erata.lv4' ÃP7la~3.Ã1g.
In uddftÃi?n, mCrtily3i?.ne bllle can bC..
used :n cian3.bin>:Eloil with i5flle:i' "ilt.icÃ"e5eci~t ag~:`Ills, s~#:ici .l
as ICG, to pit3v.Ã& multi
),5` ,'4,cloa~g,.th., 3z">`a, AtÃ"-colo fluorescence 7.ÃIii3.g1Ã'ag.
25 N>R T'huc.Frqpf?ores: I CG; IR-r"86, and C PV80 0-C:#4
3('L_s is a d3-:suip}le)i:Ã.3lod hepfiazl2u[h1nc it]dCSi;>.%:aF3Ãf::i:= that
:s F;`,.3Amt3ps Ã'owed for
1`aÃ.d.,;ac and hepi aÃc. f3IÃ1c4:oT3. s:'udi~s. CW800vCA is a cafboxyl1L;
acid analog of
IRDyc.:hic`4.~L)0CW, es newOT hi',p1,'im1efh1ne iÃtdot :''tdÃ3Ã11c iA Ãth
higher iI_uzllltÃ:Sm. yields and
Mo.ar e:Xtax7"-:isEexa o+;1-~~ffic1crt#-s< fR-786 is a ht=ptaf"21e$hI.ne
?,ndi>r-yan3.nic with E"Ã{? ;;u3pho ibit Csn,
30 ,~~id is att .:m-zo>.`ÃeI s= hydrophobic agent. 011 the other hand, CWSOO-
CA is a tc;sr<A-
:~rt:lx~*IoÃ?atL d 1-k a3tt3ITieYhÃnÃ` indocyanine, vvh1C:h Fni IE a Cs its h
vd~~ T'~> htii+=ity
~
z WSOO-C14 ~" l ~``~' F~ Ia~c:~: '1'l:~c, e~~~;=~-~~.~x~~~ic t~c>id of
I~~.~~':vc ~:~~`
pÃ"Y:paa(:3.A :\^h}daoxy:au\.cft~,,~~"'Aidc Cst>:., Ihy
16

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
hydl'ct~c~^~<<i of ~.}:e ester 1] water at pH i>.:?. Thif.`a fis xi':.etr.:s-
.s'ux,?hoà atL.=d h.tt?'s3a'TIÃ3i~1t:~0
i.Ã1docpcai.3Fnu with eF?t;.S'sTC7ii ^ 800 ÃII7. After t11t',tvi'ni3LiS
injection it is t'ap1sel`4'
cleared by: 1) the liver at~~i oxcre¾ed. in.Ão bile wnfi ?) tht. I :.~~-kc;~s
and exerctcd
urite: Tatus., this dyc is u.sef~~ for ima.r~.ii;w t2ic biliary free aiid
uretUr..s.
w ~~'~}' ~~ Is t?A~~ 111c. . CoÃ:amcreiz-fflyr available and FDA-Ã.:ppruved
near-
:nf;ra:c'd hcpitanic.:~~ine indocyanine fluorophore that is dÃ-
sÃ.~~.~h~.~~~~~~od, >3tlt::~
3:1I)eZ;t1oJ3~ it is rapidly t lt at`eCs fT i3i1~ thG blood r? S' ~ he iiv` ;
blit is i;taly
hicffiowenÃly transported rnto bile. ICG can be used to xn<~i~e the
s#.raetwres
described herein when adm1l?.,Set'ed by direct a:"?ject"i}t" or
cadin:.tlwdE?Ãl of t`]e
i 0 ~ ~
~it.Ã':~.i:~:i.~2'~i.~..
;R ;86 (Shuna,Aldrich, lpc,): Commercially avai1abIo.non-s uIpho.t:~ed
nea_: iFttai3t'a`,.'.d hepI't3.llie~.~~."$lile ikldoi.:ywi:ine
fll,lot;'.i?pnC7Ã'e. After :I1trad`ve3tous It.~i.C;3::0:1.
".t is r~:pidiy ~xt`icii i.i. into 9n3ny tissÃ.iGs 3ii. tirc ho[ty7
especially the I..VOF', `r3.Ãat: I4
inL.f-ficik afly 2xc~n""Ported into bia'~~.'. U~'"786 cait be ~i+9ed. 4o. im:-
s. wL~~i~
ti~scrib~.~.: he'~in when admÃiiiStcre~ by direcà :~~ect..ion m: E_
znrtul.aiAon of th::.
~'Ã. ~." .onirnerciall4f available tetra-sulfonated heputÃ~ieÃh>nk:
IRD
indocyanine-type NIR. fluorophorc witb. pe,& abs~~~itic3ti at 772 tnn. and
~~oa1:
~l 790 Tim. HIDyO'S Ã~:~ar, be oso-d t~~ J."Mage ~~~e. si~..~ucturcs do-
sazzbtid heroir;
20 whe;,i administered by direct t7~ectAC~ii or cannulation of
t'hestruc.EÃ.o. Si,e, er?.,
Zaheer et wL, Mol, 1na~;iÃig, 2002; 1(4):754-~.~s4.
IC'G wid IR-786 are taken up almost exclusively by ttY.o liver and
Srwz_,et:~.~d only
~iieffic:i::naly into tli:: biic, bi:Ã CW500-CA is readilsr Ã.ak..a-i :~~i by
bot~i kidn::v ~:iid livcr, a;?<l
%2SA3t3p'ie(a >,iit:~ bhl~.`~ i1.id :ÃI:i$ie, S1::1ce flit l"a3a~Et3-r
c}.]e"micil. d.i~.~o"el'la.?Ã` aE`non~.M~'. Ãht'sf' ntoiecWoJ
s i .~~ =.:~ i~~:~ ~ i~~~~ of s:.Tl"~r~:L7t~ ~..a.~i~tn? and ~ *:IL ^. ~ ~ ~
Ã.=sG'I'~31.'iI ~ herein, it ~? t C v"k .1~-:.~~
~~r`..: t~~C, ~;i34~i:;i~ ~,`?Tl ~~3L.= I~:%,s 4 #.'~^, t~.:~,SvI`afa:
vxpC.+-'I. Un<3w, t3:i3y t't,'c3F,'=<IIl{,.:3'ed fl7.i.C?d'op.`fore will work
as b'J't,?ll or lbi3te`~.'t than CW800-("A.
r t'. . .~~~..
proti ti4 d h'2, t it =s fi?~.ior~,d b~~ ~ the kidrY<= b1lt~~'r i3 For " r
lve:E. ~t:~x`3. ],~i1"i3l~~ :, 4L3L'~< ~ :~~: :3~'t3~,~~~,, f ~ \ ~ =~.s
, , .
i:~c3~1 .~a3 Sta:iphonats:d.
i_i~,.'~;3:~.~~.4 f:='sxFl ~i:: 1.E~4~, a ~i1Y~~"'~:I:z13.`.~;`~::; ~'s3~~<'
;~ {F: tt~3e 1`C;rot.1~~4 (145~~d~1~.~; ;~~ .~~IÃ;: >~ ~5
I'oilsC3s3.''stble tct t..'2pC'.ctthclt peklt'r.t^^ :md
hi.pt~.mS3.Tlph(`slla::ed dC:T'tvstt"z'v'oti shoLildithiC~ kvC3.Ik, i?ald.
n~~~~3~ L~ z7.~'s'~ :? more ~ ~t~?#t~ t32]~L'.1~ SliÃa~e t~3t'lt' excretion
ji3~'o bile E~:tid urine iS Q ~,p.~.z'~~ to 2-3~:.
;r:tL?~>rv xt:~~ =
mo.e rapid than i1ic t,,Ãra-su5phonf~te:=, The tr.Ã-s~.~lphonat.es will
prt:r....mn :ii an
i.i:i~r?.Z.ion !Yb'iChzc~pect to di"aid tetra-su~phonates:
17

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
C3 AdA#2f 23;i1riitFi3#1
in Ã-b:; ~:~.edi-nicziI st.udics described horein, NIB h:~~ bcori fÃiL:i-izL
to be ,~s'-efu; i-v,
~ im.ij;ig :he bile-ductg uretcrs and paraffiyrord giai-W. In som.. ct~sc.s,
los:, than opkima~:
Pe? .oi-t~?ance was f?:>se.ved in the ureter. This is becauqs;, laku marrs_
other dycs, xÃ;.e
1'~~~~~re~~en::e ~.~i-operties of meffi3~Iene blite a3-e sensitive tc. the
~.>E i: of its x;?
iic..d,
'1a ccmkditzom'~' ffic abSt~rptii3n and. quantum 1 effiiit'=l~cy of tl14
fli1:i5ri!peliiie dt:c}eei~c;i
40b'Am3t'.allb''. r.,hui, i.l `f1i.33 ..a-sG4 s3.Gt?ve b1.?~fe337g \'.+z
Ãlli: ?il Si:i.~ y. p..3 43.i":h a iin.i,aiy
~ ?~ t> ~~=~~~ t r ~ ` ,~ ic- r ~_
i 3 i:l~.c>zl~c an ci~,.~Gi?t t.~ac:x rc~3SC:'.~ t i'= pil z3f the
i~fl"~E~.~~ ~ i~,.f,., E'>C ~Ft~ .. _i~}3i3.?s31:~.: +:?~
~=+yt ;~~~`5~3~.li~il~e} '~C1An =~i1st~:~'~:=~a ~i '~~,iti-i ~iG~t~.~l~ t
'>~:~C:i S. 1~~. ~ rr=' imaging
ia~:w.
Z~,~.~ is i:::3-;3[aõ~ a h~;i.~Tkz~~ aEa.71~~1~5~.~=1'"~~+=,~ for 11,?,,y
~"1C= `x.re:'ri"=~ (e.,t.x,., b~ L:,f3 I ?C'et.?7.ylZ.'`ile blE3C%).
In St?:tl?C3 iun33od.itYae.?t5, the methods C+`,'.~sc1`tbd:"li het"cif+
:Ili.,i[I+`..fL. ffi'=
~5 of ;x:-~eof mo:i`c ~~ther CoiA?pcy-und5 that ei.t1~an. c the peftumimee tff
tho dyes. :ior Lxan~~~~lo,
:
i~t some c.~~>:~z}dinnunts, a
EXAMPLES
The ].. <'entloE? i4 fUI"theT deacrib:'.=d IL3 tllc. ~~.3flowing Uxi:m(?les,
wli1L ii do nt?t lIFrk?i
the scope of the :h?v`,~`ntimnL3L>t9ci.iV'e'.7. in ~~-le i.='l<~~in':f.
This F-IX.a.,n& dese-tibe:~ experiments that evartoaÃed the ~~~~
~luc3res.,z::'tt: ~roper6t<s
of:~e-vea~~l d5<`cs >m;ud~~~.~gmcthylene blue (NtB), lCG. IR-786, ::ixi:` C
W800 CA. L~ing
t?~3~t~;.~~~1~ z''~~iii ~~~~>t?Ã=~ spect,omei'v m'Id fll1CYiG~cencf: quarmum:
y-wdd
25 `-`L?:,'s3fbaacm:= spectaC xiI2Ut15` wLiS pe:foT aned wa1:h a >-c:?:;
padl iei? k':h t _3ar"lz cw#: i=t>t: in
~ ?
a USB2000 :k~.~~.i`--Ã>pti~. ~,..~~:~:~~ot~~etc~~ #:~~ce;~..ti Optics,
Dunedin. 1 I;: . ~ ~i~c~F~~~.~ll
spd..L;=t`i;F1?~try `=A"'as pf;ra%mmeia Li'i.tli a I -%::nn paldh Icngth,
>?4pf iS.b<C: k.rw'.?l:it > yIi;1tL=
o.wett.s; (Fish:.r Sc~entitrÃ:.2 llmmpion, N1-1) .i~~ a FdR2+:100 fiber~optyc
t~.~oor:-omet<:-,, a-id
xf:mr-Way cuvette holder (Oceiiri Op~ics), iising a 655 nzn laser diz?d.=
:::g~.:_:
37 source (O}'CONA O.E'. fim,. Xiamen, ChiI?i?)tv~tb ?i:a illT7im: output lt
ks tha: mW. Data
ac.{uis.i1>f:Fii was performed with a computer us.1ng zlli 00:-Ba5e321_
sp:~{:tmmcie: t?pi,ro-Ii11:$
sc5Pn4a'e ',,'`~a'i.;~~go (Occan Oj)fics). 'I'}li; spectral riillge of the
UÃ?B2000 FT
;. ri

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
iuas fion) :. 50 to 10100 Ãini with spectral resolution of 7.6 t3m, and that
of the H R-20430
spectrometer was fi:?tn 200 to ; t OÃ) ilm ~i'ftl1 't' speC~tral resolution of
6,~.i ".tm<
FauifF~'..-u3'.oric'C ina`L =i.a`'~~aty wasnnF4'.&suF"LJ'lf.iF,~ ...~Fc.(~
soEvut (F-BS. EiBr.~.~. arai-nddmnC.;1) ai
i,ad;:h ca}F:IcSUS3trat.().?~ o: thf..' dye to dde.Ã'knzI1C= the offei~t of
the moihyl$.t`L bhx cE3ili:.CntraftFO.:1
A sC?i'b's xv on
;m
'- iÃ3ia': ~lEaE?F'es rriGb'T1C=ld:. >~ lu<324 iCi'ncs quantum ~~~ldf of m
PBS, FsS, :i:'.Fd metht1.T]ol were L;ale'LitatC'.d using t?xa.r.iut 725 in
k:"iyl'G.nw- glytvoz (d,;umÃ>t-mm
L'iela? : 19%) as a cs3l?brc?tl.ffin ;':$amFLi>Frd, LFÃ:dC=F the
cE?d3dtt1.oÃ.as of matched absc3FJ:::nC:e f-3~~
In~th~~leme blue xz:Ã Cac~~ ~~~~~~ent.
MB :;F <. ::,~~~~~~~ m.,~~~ecu(e (M.W: 31-0 I7a) caiio.Ã-aic thiazine dye that
>s.FDA aÃ~prowt:(;
d a~l i,~t s~ ~~if7: i~:~~ 1Ã,d #ex~t~?Ã dye Ã~7,~~3~'li.1F~.i In t+13i
Q:'Li1Si: ,~le, ~~~~ ~~t.F.Ãt.iFa.~ef~ h'<?i"#:F:
N,. .~ne PhtiF'ma l_..c. (I'arwnus, NJ) was diluted Ix-~ adequate:= wns~~um
of'pho=Ã?=x
salinw (PBS) :o obta:i:~i 1, 2, 5, 10, 15. 210, 30, 40, ~;uad 50 ~i-M bof<3,ca
use.
The, opt ca] pÃ"qpeÃt:45 of IMB are Sut'~lmt7..~:?.f:d in TtF,Jle `' ffio
a71oa1F L=:tLF:Ãla..li)n
ooefficient, in~ i ~.~S is ? 6.000fi aml fluorescence qua~~l-uni yield ÃÃ:Ã FB
is 151",q. >hese
>otF.iE.S` indicated i,ha`. ~~'Gq flu$;rCsi:eflef::, which has an
L.~L'~t#nc:.IiJa1 ci`. (;ffi+:,I:ri?: F3nd.
flut?xoseence quaF~~turn yield in FBS of 168,000 ~~id 9` N. respectively, is
:abmu~ 8 times 11 iam`?,t.? :~ic' ..,ii; ~~'i#i'!1G i3~L~C`.' ~sIF..F.~si"L'
aa t 33i:u p E i SF:'>
Tab~~ ~ ~ Phy~icW and Opt-ic I'z"Ã~~erti~.As 0~.~ M~thylent Bli-~e -anc~ ICG
------------
....... .:....... _____ _ --- ----- ..... ......._. __ --
Molar ~~?~.
:~~s31~ ~.xcitt~tic~ll ~.ri~~FSSIcsr: `' `}~
Q~A~C~cZ 4im
EYtFkT3C1101A ~'~ avelG1'1ghWags4leIlgtil YÃel.i. Ex, <:=r x ivL~~.~~~;. < <
M.W. ~.~~1~'.?i~ ~, `{3xISZ1u321 tc 12ti7itFli~i 0
r os ~ tt3 tt?
(:~~~) (~~-Fci~~ } ~9:1~~} (r ~~l) P~~S Ã~aAe U.;Ãne
PBS (~ ~~~) PBS (FBS) PBS (:~BS)
[Mtan] ;Mtot:11 rmtolil
~ ~ ~ ~ r ~')
M <~..th1l ~.11e ` 715~~~~ `~6.. (666) 68u~~Ã. ~5..>t ,~ . 5~ < ~e ~, (2. _
~3i::Ã~.~ ~' "w (86500) 6 5
[715Ã30] ~ [681] [{>},5;~
I i 3 .............. _ 1s.7000 779 (8~~~} ~t~~~ (811) 1 '9)
(168,000)
2 u
a.n addition, MB -flufS:t;~cmCo iN wlth'F12 thi, NIR Eim~e~ .?Ã:d has about ~i
hw3C>>w:d
::~~a:~Fa~~:te, c~.affcà e~~:. e in .xrt3v,l~:t~:~t~3 ~~()Irl ICG and 1
~Ie.Fvt:tÃi e . This
diffc-mme should pennit flae 5eparF,Ãzm of these fltÃc:>:O,cÃ;nce with. s;ur.
c;~,Y,>.1
iÃF~ck~~ik:g '~i,sa .
P9

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
C3p;d
~itnhy~ene blue is in c<~tfflit3ritu-ii wat~l Iettci~lre~lyhwn~.' blue,
tlwlatw be-Ãnf t:lewrr.
'k;.Ã it.aa4: s w.`ad `1oRi' flus3aesfvent. 1w11i. C'.S?nvii"siL3n.
Y`=.?etLi'oC:n the two is .i:i.iÃ?x and p='l^
p~..'..i':de33i, wi~i, ::.igh ptil 63 fciva3Ã'ÃÃ3g. the Il19ort~~ce?`kt
Ã?1C=ihy1iTle blue i:Zrm and h-tw:`
pA S i : r'oTillts the Gl.t,`.ur, colorless, and ni,t7.-fl 13.i?Tcs ci fi:
roi`in,
~=~eCa~3d..:~e bbue oxhibit,,~ complex fluoÃ'esce:.f.~~.e output, Wi~~l peak
;f?w;~s~~y fz :f~1 ;:In 1? ~~. ÃIja.v`~31~:t~ii:ifi' ~:C1Ã1 [iltr :tiC)~?s
li~;`~~ t!~~ uM. Ii3tCÃas1ty is Wiso h gb3 v
ik,"',z:nd::=na t?f; C,1Ã;.i71#.e>'s1 ~,`;:'vi3'o#.ii-lf'.:F'at, with
}il.=.ti~-.~.~.a output in i.Ãp1d-ikà tiÃFr'.7_iõ+~ F 3:~=f3,~. Ft3I~
~~lo, i~,Xpkmm~.~ntshown in.Figpire 1 A, p-tir~.~ m~thylenv bIre powder was
distiol;'cf; iÃ-a
ph~~sphate-bti~lered S:.2~~~ic (pi[ 7.4), 100`f~ fetal or <Ãb_.o.ute
inÃ,,,".rA ioI, a:: tli.-I
CtJneer%wa if?:.: shown, EÃni^ flui?r`4,=4cC:nce intensity (aÃ'bÃ.-ff awrf'
vs". al.Iey) aitc-iksLÃr+`"~d.
Metl?vic:Ãie blue exhibits complex optis:.a: behavior. Its wavelength c+f peak
~~tislla~:Ã:Ã:Fi3 C.;?S,:~a~Ãi;~l#:'f`3~'+.. (i.e., i~~?4E:.~Ã'+3e:..~`~,.e,~r
is C~~:"g?+.:~Ãt~~~'=."ifi. `~?Ã: ~:i?lIzt.Ã1t;t1:tiuT; and (.he:I3iZ:afl
01:vxroniaaunT (see Mg. 1 B), a;x is its wavelength of"peak flui>Ã-esc:enc..
etr:i"S:z?n (I .g, i C),
For fl`_:s C,xpei3.a34nt, }.`Ã3,.tÃ's, Metllyli;2'1e blÃic powder was d1~-
Notved i:3 PBS, 17,BSa or
T'nGthÃ.lno3, at t17z'. k:?ni,::ertttat:oT1S shown in ~~lG'4; I A-C, the
spL=CY3:u-n of .lts eabsC?;E'b.1noe
~~c~il~ of ~a~~~ ~~~~~Ãi~i~=:I.
flizorw~eu.cc w :~issic~Ã. obtained, ~~.d the
~a }
~T <-(i~t te.~l fluorescence <a ~ ~ )r. ~ "l.=>ld of ~a~t~t ¾ ~' ~'t ~ylT'~~
;~e ~~a~.~fs. ~` 3 ~ ~i:i . x~~:23ir'3i<f.l~~C.i t ~ i"3t t`A'~ w~ = t
y~~Ã.rc~k C=~t?i
}~`~= r 4~i ~w~`'":t?n ..~~~.>
``, I A) and pc's3k iÃbf3C.ZI'baFAL%t ~ ~ve_EE;Ã:?~th reI7ia]t1s sto+`_~~ (I
B) in zl'I'Ã:hydvta~,", wide ;.?.{?;;e i$f
2t; the dye ct?nccina'r3tif3n5 aD'aad pGak fliÃozeu%onve 1:1aStssIoÃ1
Wil'tielLÃig-thd{:xes IiC;( ..1cÃ::'loaie a=.
bÃ~,~.~,hLÃ' ci>n-c:.::;Ãati;3ns (IC), allowing ~'C.31' aCtjlist:znent of Ã3-w
C`i?4o of MB fo'" Fr`..Yap".i=fg t{3
i3pi'm:i'fc si
',.'ii_:a i9 background ratio.
FÃnafl~,; flic pli dependence of NI[3 wzis bv diss< _ ~~ingM.3 ,:tf. PIRBS Ãp;-
s
Ã>~, ~.5e:=a~r~~ic:;'t.sõid, x~C'~, a.-ii~i a Na~-a~'0:~..~~~~~?~e~'od
;~~3lt;tioas. As vhcz,~~~A }~~ i i~= ID, tl,z _
. . . . .. . . . . . . . .. . L~.
r~
2;; ~ 1r~3;~:s;~s~Ã1~Ã ~ :i~> no ~~ :iaC~es~=..s ~~C fi:= in acidic -a-~ the
~ StAt a~ttic3~~w= and significant ~:~t:i~.~ G st;i`,~: .of. :t. ;~iu=
s . .
:nienla, .absoz,p,i.0-a Often limit the usable dose range of fluorescent
;.oiitTas: aguc.ts: Thõu:,
the conditions aliil tJnCeI1t?'~~tion can bo optr:nÃ:zod:
_r4
:30 IN?R FiufrCi+)?'þJr'..'i: .l.
R 7 86r and 0=VY00=-"
IR^71 86 and sf."+dà [;I:.la3 i.CG (C'x::"d7o5E4% wl:."=rE:= pÃ.1rchascd
ftoi?:. Sigma (S:, Louis,
MO). HUM vv.~:.~ diluted to 100 gNM ia3 ~~hospha;e-bufferec3 salinw t13BS;i
,,tÃ,splen.ented

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
with c{ )l.1{> 2 !~#C/ '.. a~ ~ '4 A ~~:).4'rn~ZF~ ~~ 1~af:4=a~ 7 .~L
(1.s5~t.{2 ~,y j =~~Iy)5 Sraq{S >0iL5'~W ~(r~((tt.~2 }~`!t.L+~~+~~ .~A fi\ i e
L=i:~t~r5"~tF'( \ 4a \p^ }t tt-~cYiv~~ ~rr~ fi } !\q 3'.
A~t~ L~~~\x.t 1 ~ v F~:~~i ~i ;~ ~
<
e.Ii~:~i?~~Ll':-c
CW800 CA is the carboxylic acid foant ot"IRdyJ '~ 800CW NIR dye, wh:4Ii was
obtained as an NHS-ester from LI-COR (..sir;::ola_ NE) <~.~~d hydrolyzed ini
Law,i-teabL3sc d
:_... , :
:~~Ã~C'=I'~5x`,i 8.~? ~"i~T' ~>I~:i ~~}'~Er at rf3L~i~~, iC~I~1
~341'u1`;:l~ri; ~7~'.iE3:t`4.` ~#Ã~'~7z.'.a$.?ir`.is by ~IP LC i:+n a C 1.
c~f.
5;.:?l;aI711:Ã.
`Ã. ~.~~~~~ ,~dss:~: ~f?l~Ãis-~Ãa~ ~~~I~~~~ ~~~ ; ~ ~~~~1 ~:`~~FS~i# ~',~
~~r~r~= sÃ~~Ã~:~~ ~ ~;3~~~'~t.= ~;~
,<~,C in the d
:ar,- ICCI and CW800-~~':-A wer~.Y diiuted in PBS boabr::~ ~se,
I'A3L?= 55pt.ici.Ãl P"3pw..'tÃes of these contrast agents are sÃi.
`1r>.il:Ã'Ãzi,k in 'riibl~.' -2 A:;
3 0 three iflu3.3Tophvros have similar peak absorb%3ncc and C #elÃ'.Z`SIf?I`.
;3 :Ãb`+,.lÃ.i: :h4. a.:#t> flae
Ãiu`t`sÃiaml 4a~,'=sd of CWSOs~-CA w;3:s t[lf;r l3,ig}1e;it o. the t5'irce.
I'able 2 --- .~hy:~ical aaid Optic Properties of NIR Flu0rophoreS
~.._ _ < < ------- .. .......
~ ~~f 13cak Peak ~`.`,~,.Ã: n um, ` xer. R.. :.
Ieve:
NaÃ~ie M.W: Sulphcz- Absorbance Ftuore .erzce Ylskld k3 =
6 Ã.3PBS inP~:-~s Bi~~~:;;.
rr3~f.~ZZ.~~. , in 3.~ #g~ .~3S (z~'.nm)
r.~,
~'W800CA :14: 4 775 .f~`~t7 9
;a`a~.` +?8t`4 584 0 {~~~ 803 3,3
ICG7r'6..F. 779 806 :E
eifusa7~~r~ L'p 1;
s
.2:
IT,F~. An~~~~!&gjZtji'j.: LÃ.i M-voz~..._... ....... ......-,_ardif?3 P
ggu~'YDa~v1Ã:}r, 300 g zats obtained from Cb.;rles River ~ a-bo:at~.~>'ies
Ma1~ Spsa
iW:h-n:ing:c~~~, MA) w4r~. ara~~thetb,.ed with 65 zn`kg H' pci:tobafbita:. 30
k,- adi.FFt .:enltdle
1 .. & MA) = ~b~.'Ã~. > iÃ~~~~F~~:~. v-. C with : <`~ .t.~ rx.,~., };:
"? 3r~~.~it~,-~i~~ : ,~ _.~~=~. ~~~;~~~~:s s'~ Sons, ~-~:~t~~es~, ~~~~r~.,x ~
:k,s
lEs:~~F:~~iSL=~zii~~ r>#.Te'sl.4 ~ (Fon Dodge Labs, l'oÃi. D\?dt.rC'=,. IA).
Pigs wt'r4 ia3ft:bated.::tz3.d
.t:i#ÃÃai4`rÃ.:#*c-d with 1. 5 'y 't3 154,1fiuraÃlt`.,
Lq a1?L=s.h..`.Ãi.eÃ, rats, 0.1, 0,5 and l n3gskg 1013 we-re, iTljecÃ.eil
mtraveaf+ua>y .nd
~ to ~. ,=3 Ã.lltk,
+=,, ~=\
thL 'p images takei-#.d:ah'ing 15 r`a.~#.Ãl at-'ter injection. Signal tt.
~~~ik~'' ~ ratio (SBR)
(the S:liat tÃl of 1 ftaf3E:~~iii 1` ~~at S~=~^eiÃ1~ bÃw3.ch of coronary
artery (LAD)'CC:lafiv,' ~ _= :3?::i of
chest we''.:ll. and the sÃ( g~,~,al of myoca'L3Ãlm relative to that of
cl3(l'~t wcà Mw<3s q.Ã:$a:.]fii?d
20 :zslim;; t[le stiÃlle region of interest {ROD through etitire
c?bscrvtÃ.fioÃt In ar?es.h:.l::r..ed
f~Q i \ ~2' ``l : .f
: ~3: ? ;.i,J__ik,? of i;<<.L il?C.,tt1~F'3~;#(. ~.?i::E~= (I .{~?
,~1.121E7l/~i:g' was ~lY :a?~.'wa;e~. 71't%ai34'i3'tCr~z' ~slZ"iÃi.E3 l3f:t
::fl.:,
s wer~:' obsc> veduszng the imaging syst::m descr~bed hc.=r-.An, 1,~:r ~~lul.~-
Wa:Ivea .l:gffi
;iean
21

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
>.f GlorescE???ce I.Ã',.3agÃngy 0.25 ml of 100 6tM IC':C.r (75 11t.:-101
Tl,~~,I) MI` 1:e jC itOd
rats ai.ici>).0Zlntg,`kg KtG ({ ~~~no1/.'E~.g) in pigs.
neu-in-tra.red tniing ;;Y~~emus~.~l in this and txe following e-,"a.>tple5
IlaS
:;S.t;Ã'i dE:st,iii?i:d pTov3oÃF~ly in Ms3i.l (JcC;, e.g.. Do GraI1.d and
FT'at:gZ.oi Zõ .~~C;i:hÃ2ol. Cancer
Rks. T~ew., 2#0.553--62 (2003); U.S. Pat.App. :f'ub, No. 2006'0:05509to
F.anL,;i43~~i ca al.;
[J.& ~.~at App. Pub. No. 200:~,=02850_>8 if3 Frangicrn: U.S. PaL App. Pti:b.
No.
~~',005, 00,....0~Zi 4~r~ ~,., t , .. and ~,~.S U.S. Pat. :~~ ~. 7 T. PZ. 200
` r;a~'S a~ `2 E to
~.= ~~ a G'~¾~Ã~~~~i~-~~ r et a.~.i~. ~c. . ~, t
FÃ a.igit'sni) . T, i3i: hyste11: WtZS, 43i.t_j Z.tStÃ:ta tO ',Wi:i)l$.@A for
~`illc Cipticiil P1"f3_3t:F~'a.toi C3fn.wth'?riE.`:Ãi4=
b1u
i~ I_~Ticfliv t~~~ ~~f
_J-a soiirce of the orÃgÃnaI system is composed of a,~vlhaÃe laglit s:? wrc.
io a.~t ^z~z~l 7 ~. .:~"f-ha eti"rarF;.Za ~hy t ;lt source. 2. il~"'.
su1`SS1+: ali i~~ Ei~Z[i I:TF11L::;i.~ii i.~IS:;L~: S4Et~1 b`Ji ?~
: w+ Ã
:'X..N a~.Z.''.3 itaea:cQ i"citSY Ã.s 5 -niW/:.'iI~?. White light and
:xi.y:i,E 3IZfiai`E:d images were able t.? })e
t:avt..#I'ed svA02Ã.i':3tG=]Si [I.sIf3,, a dÃ.C%h1-i)ai: filter cube 4i.:::
and these F;.Zimt?F.,s cÃ3il`si be I'nfi,i-.Lii..
;,maH;es '.1Q1c captured mZd rt;~fresh~.''.=d 15 times a second, 1 3iÃS
cup:$t'.1l1:y, w1t'll 1oCtn. .I#:; c.:.:3Ã3
a\}t1_f ro`.ktJ 1.LS131.¾io1, and a cardiac gating function OYvSEt~h
prCfV=2dV4 a clear 2ma~e, of Ow
ht'aft ~,n:}Z f;vv I7Z=L?:'1i5FÃ a4'ti ftif is by 4yi':t;:blaZt]:Izit3g the
ffiZZ3kZ;Y of 3iZli:Ãge capture wiflh c.A.ti{:=Ãat%
aTid :~~~~~ias.s tFzrc.e hi)aMes at tl-Ã~.4 same tirnV), allows .4=za;-timc,
precise
Ãf.fiontific+.atior, }~'3 .`h'~v anatomical structure.
.
T}ti. S4`sI~,'Ã43 was #lx{3f~ii14'd. in 3 Ft'i'~'aaE3r i3ki."~~~']~.t1 MB. :
Ã1'.~t ¾white 'i~f~t,c x~:~.~.;.
. ,. . . . . . . . the . ,. ~_ a. 4 's r:w.. ~,
: Toclino1c.~g~A, Brattleb(.)z(s, VT). Se.ond, 4pa:irs oWf)
:-PA, 660 .ri.m, LED,, wc;rc added and fiatcA'r.~d w~~i. HQ650/45X ~e1x c~
t~~~,~ insread' of dr:te
i`lcai" il.:f e'r<ti`+:3d light source. EKi.ltii:taCi.il fluence rate w1ffi
this ~.`ow 3.1glZ.:. source is I i2:w13;,>. "..
Th3:'d.> the diihÃ'uic filter and emission {iftC'.Ã' were changed tt's
680deAi' (i,'hÃ"o:,>a.),
HQ (.)(Y':~~ ~chronia.)f respectively. T1::i5 al.czw", eil~cttve sc;paration
of white light and
fluorc~scez.t light eoaningftom methylena~ ~it.:, tistd pr;~-vidles color atid
'r'_: .` separately, and si6lows display of the iivo ;i21agCs wItl1 a
me.t'ged. image `~~`1nwh:~a.1eo..Ãslb' `,Ã;:
:'eG:]-fi3:Z.t:.
Ax3gg?:g{
~
;'~~.~tc?i~uur~.~scence oiit of rAoTrnally peÃ:Fu,~~ed rat hearts wasminim<tk.
<
.~'~ ~z"~' ~b'ca~:i~ used to Z'14i~ic~ 1t'.e coronary arteries, bi71
~3l`.'7'l"~7~Zl:T"`~.3 _,Ã"a#~~~::li.w and cE33'ÃFrli F'y
310 vÃ'#11 'svvxe d"io` dtUte-E:=t:~bi:: $i4f.iZgNIB in 1'i3t:s. I#.iti
awzc.~4a: i,~laT (aÃ'4eÃ``s;Z-l) sIaF.I"Ãi:I:k wcF'e-ni>t
dÃ:teett:d witb ltZwÃ.:i: d``~esof F21ethyleileb31dC: (OA Ir1z?+`?ig); with
lsiT'~.?C" a~l?~~:3.1i.Zis (0.5 and LO,
,n~,~=~u;¾ coronary arSer.4s c<o~~~~ be detected. Met1i~~le-tie blue is
rapidly ,<,i&ri~.~Lftcd lro~m
y~.

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
the b1oo~~~~earn, to myt3cardial t, :: iat ::;-lud;n 9 the nwoc.ardiw:Ã.,
which likely expl.~~n,",
w.~.~y #~~ic uoi~~~iarv verious return cannot be ~~~slial~zed.
A F;~igpxai f;F`ovn '~he he&rt wall was detectable in all loading doses- of
~~~~, Whii:.h iuggosÃed that MB e:~#~ be used as aii optical andicato> <}f
rxys.>cmdiai porA'V,SI:?D.
'llii; #iat::nsFly wa3 dep'4aides`~i.l on thi; loading dose of Ãi,tethyle:5e
bl1:1 .lowev+,'.z', the deer .."i:so
of the fluf?it=L.3:ce in the cardiac wall was I:Ã#1ea.Ã' and the Swt pe was
ci,F'.z.Ai#.r#f and
independent of loading i::osÃ:i
is?.1 a i1t;;tn.:iliy PC, ai.FS~':~d pIg'ti hLil:i"t, aCitol3LFi3Ã`eseF:nf<o
was :nf#ifiT3al. Fo``siow'#.ng
:~~JL~t t1i,ll of 7~~e'i}l~'l~.3~G blue via ~~[ & xai:~T?~ilj 11
"ai~~F~.F~~' b'3i~ ~l:w;~~ ~ ~;3.~~ :~?
%i? bavkgri?:i'-.i"id (S;,.skO a12aa:;''~`.s of zi3',te1Ãtil blood tl`+w kIl
the beafi.IIg , 54-aà e. 1~"cf'E'. 6b~tt3aE
t`yp r~.~,; )r .3nd. sub.w~~~~i-iily, thomo~.:~~.:~ious fluorescence O>it of
the Whc:Elehea:rt Wa1l w<ti.,
i3 _gt; SBR Za'%ibl3lit 1.5 (Figure 2, Irt'cldie). WheF7 aiv.."wta~~
cFTte.F"i;:Fz3 o4.O1;#s#.{.SF:: ~'L%a;i
FA.itF='oducvd by ligation of i.ip'l'vripS1Le2a1 diagd.1na i t>r'tFa
~Gfa't..Z~f the .i'L.ft...i';.at~.~daE~~.~ ~SL~`aE;.`.~:~~'.~t~ c~a~t.: t?
fie3 3T24th?'lun;u 37l#z.c i=4as. injected kntmi%e#3E?i.#.sly,
i:F'#yi?cFiTd1`i:FllA distal. W ths;;=t.ct.lus#.{3tih<:C3
~w fi4c, -ati ld low o'3' S>4Ã1~l ~3:~3.) the F~~`ellY~?or~"'~iS~G,E~.
j3i~.F"Ã of the .F;.Fv
z?~#iF
~~ `~.':a{. : :xe NI~~ i;~t~~g~'(F'i~?tiai ~.; ;~~rttu~':~.~:
4t~.~~.~. ~r~~
4 ~~
~ IN a3.,#~f~
Ext:tliopatiu bile duct is oftepi ditBui.It to identify .~~~~hosit conÃra&
agents in r:.~>:.-s.
20 '17hd: 'tise of N fr:ieÃhyle-Fle bz4io i'Ii1:S 4,v#?it.#:3~.:ed :#F r1h
desCr3bQd in Yi21S E4aaiplQ. The imaging St'~toI3i dCtiCrI~.?ed in Example
<.,' was used :z~ thF~,s:'
per#.mC.#3ts as AdfAhr?tf'tre B:?ui"
IrI tLil.e s tl3~~i z e d r aaI ". 10 0 ~ ii .`kg t,f lM B ( l - r#rg'k k;) w
a s i :n;o c 4c;d in a..v c?rFc) v~'l v
I5 via peall4e v6F';. .c`5. slii3:altaneo iss color 4'ideo:`NIzw
fli:#.o.F'e",i,eI:ce A3:itF`a<7pez"at: YL l.t'aa ig1i.Fg
~~~~em v'ws. ci?"a,3Ja#yed f4T# quaF1t.F~'#.5..~tIa n of flt1c+rGsGe1iF t.
images. For 1tEaftW wc1v64'tAtrffi
0.~:~5ml of I Ã~~LA~ ICG (75 nmol:1~) was i.qjo4ted. i,it;.av;<r oi~~ly in
s3aC~3 hepac x'teT#i34-rcipi;.y and pi,rti3 ~.1 3.ppd' ay t <aF7. bi'= sec#l.
111 c3:oA4.etized pigs, bilcCiiJC`=E was vFsuali.ti;F:'3 by 1MiÃ.'%FvL'aS?%Ih
t~j <'c(t(xn of
30 3 00;.#:.l~rkg of 1zi4 M.B, or ta.iÃ:ct +A:3:#aI3lY.~`F,.z''Ãi[?z? l_I-Itei
ti.S;: oaF.13llo-n b'iG;'. s:t3ct f;3x a 31ectiC1I'¾ ova`:
iO ;.M of NIB, Fii.it3T+.~icenCo Fa3teasft:e4 of bile taFli:=[, IFVer (F'Igj3t
3i?hsv), and ...:'3iom3nix,
weie nioas~`t.~~d at.>. 5, 10,f 20,an4.S 30 miFautesfoitow#atg Fn.jC:6.t#on, F
orl':ep:sfiiNc3>
` vtcI'.i~:..,.~ ~ ~ ~ta=3~1~% e T i^ 'S t'3/~t' (`~ ICG ~ ~ ~7~ ~~ is a '1=
c` s~ ri n ~4~E3~ ~:;C?#'~t~3#~~'c~~3n.a~4'. ~.~,~!~7~~,~~,~, i~fi?l?~ ~T ~?
r4C:~F:~- a~1z,'av..l.
zo"l7SiZy.
L3:

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
I n rats, flue common bile duct (CB.[~) was, cl~~ee Ãab lc;just 2-3 1.~i-nutos
after
MkÃw~~enoLis aÃ~~jecLLon ~).1 the MR Tlier<ÃÃio o.Eic~fm-moi.! bile duct
Ãant4ns:Ãty ae1.a~i've t{l
p<3iaZ:Ã"o`_';as and L.ii l:voT' rapidly r("s:chod mi3} 1iIAi:Il at around 10
minutes and wass4-L43dv' at
lea-ft 30 :i1inuj-t..s after Ãrajec:ioia (Figure 3A). No dyc s;-aiakfiq~f #.i
t~:~ liver was ,~?~_~~.. ,~:~.;.
y
The C13D coalai be detected 4'3ntv in the t
llaoI'o`E4Ãli e tl?ia , gc and .ÃI.L qgQd.1>i1 a rri , ht we'i`ei'.
n4`~ in thi c}it.eàs lliige (3" ~ gU3 ' 313).
h-i p}~~.'u, b3.lir had a faint ~~-110. Ã ~'= +~ falxv~ , il;F?d con}:tI3o37
br3i L:ui::r rxaS d,. , k,C:tiible as #.Ã i
the rat However, Ãi1c .nL'iabG=x?iÃtic btlc,'. dilCt was not tdowÃ.fit,ble by
i?i7riÃ'?ghepza.zedÃ5soci;.ol'.i. Direct reÃi'C3gi'adi'
iEdÃ"ilii3lstA33t:t?II:.,f MB ..a3to ilic CBI)
sÃxc.~sm l=r vrsutÃ~ized the CBD (Fi-;me 4). 'lliis dzroct iq;e:.tÃon of MB
ÃnÃo bile tluk.;
Z-'m,t=i1cd c40a5tL'i 'L}tit~Ii.Saldial.dZi11.~~~`~.=o52 1.ib.7..F~~ruV than
ldxdÃF~4S{.ii'.~t.Ã~L4b.` Y-~fCii~i.~25:.`ts13l.[t:i~i\ S..
~~~~:Ã~~.>i~~Ã~., if access to. the biliary systc~.Ã~~ii is z,vmlable; dirc:cÃ
.Ãn,eddo.: (1Ã' M B is 'j-11
ajt.ematIZ.. 1-nL`>i,L3d, fÃ?i v-PtEaalÃzasÃwn of the bÃa`i1,"' S"..stikm.
3+fi re~~i'} ~~; t ~ L:.Yai~' ~tr~~+-'~.~t=E
50 ~tL of 100,Al IR-7 8Ãi or CWStltx (.".A (15 ~tp`kg), or ` inM .CG (150
injected in.Ão poS.Lai vein 1ÃF:5.t2.iArs{.tAe.`ii..k+t}i.aÃi3. A A
a`ti[FSli.~[t<]n'b4iLlitcolor vidt,o. TQ3\
w}.6.xorti.=mA=k)Z.=4iSil.dL.CL/At1W.tai)vW)ÃA?agil.2i^ 4't}~stem was employed
tf3J l~t.f:[.1.if,AA~L.='CLf.~~iiw.\,f ].F_t.tZ.
~2{5 fr~.% t:.'1.=a?S.r ~1 2V w5~ ~ ;~c~. {y g 1~ i32.~x1)t2i . a.. rats, ~
S~f ~+ \', bile Fl~ t~( tdVt ~'r'1 { x ~2 ~if~ )5tfE.=l~ b$ ~`( T~ ~ -~'~
w5.4 ~q ~i2~~'~.=iTti.i t6rt.i li2:5 SCA:Z~`~ Ã~~{2{1.G':41.,~,.~:~ ~N .,1.: 1
<,r iectimn (N-16) of 50 pL of CWSOO-t~~A at . 0 20.. 50 arz~.~ Ot) a,M ;;?
20 i.`~11w?:1f~` :hc E?esi et`3ncomiat1oi1 foÃ" :?.1lc dtÃC?tfmag1#3g.
liver (:~ghà ;c;b: )~ and pancreas were mea4urcd at "a, -'S, 1(120, i.i?c~ ~0
min ~ :c?,,~ring
WIthtn i=iÃ'm m:2'xizti.n., afteÃ"pr?i'Ã.cil i'+u13:1 iÃ:e:eCtÃt7:1, all
ffiIz.e
h:-n<Ã.g,ing of the bile ducÃ. Both IC"G <~id IR-';N'6 could be vismaliF:ed in
:he, CBD, bLit Ã:{:ey
:25 s ~.c~ ~i~,Ã~~fiiar~à dye stacking to ~?f :li~J: r, which à i~ t c? bright
Ãlu , reseei? .c t:ia.n sfw
liv,.: (FigiÃ.ro. 5AwI3). CW800-(YA also shc}v,,,ed Ãttc CBD, -wzrh an
eEc;.lIeni ~3Ã~~~.ltÃ.l Ã.o
background -raz=~~ (SBR). but wit~~i no ,igrm'ficant d:yH stackffi~,Y to the
liver (~ ipa~~ 5C).
Txm~; CWSOO-CA was sellekÃed for CBD visualization.
I'a3(' ilE lxt cxnd zTnE:3t4 were 3xa"Ãz:td%fà o
p1iili~ $Y7~ Ollik:' iIose axad admii1.st:i:a37Z'S T.`i?~~e:
3:3 As ~~~~own in Fi tv~s 6A-D. in {~ ? ~.~ec.rax ~.l, ~'~~1~ could be idc
Ãi~#i~.~~ "~zita? utc.s after cit}~e Ã
~ ~ . ,.
:n.T',AvenZ?i:is or "pC??'Ãtil l-njectiC,n; blià portal vein Ã21jecÃ2on
improved tzxÃ~ SBR. .~~ pe.,ially
using 10 and 20 ~tkf CWSOi~-C:`~ ad.nffiistra1zon. Peak, p~~rfim-nance for-
CBD V:~sual.za;.<.?a::
i sp6 aeC:d i:: 's3~?~3~'~?:'~inli.i~'y~i~v' 10 1~1Ã~'SLtÃGS Ã.Ã1 ~~~:i4`
[+,?n~:E'.31t1'<$tÃt5i-i C;fC:WSOO^CA, <3:I:i~ 50 #-:N:
,24

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
injection showed ~~c- best SB:R. S.~.Itbv]00Ã.:~MCW;s00-C`r~ ~ ~ ~
~q.r~(g) adÃnin-ietraiion was iiot always better than tbat by. 50 ~im CW800-CA
Based w> >h'. results obtained Ia `~. rats, CWcZOO i..:'~ ~~siS as a: it,,'~~
<:E;ii... tFkri~y.E. ~,f a*_ Ã~ #-
. . . . . . . . . . . . : . . was chosen ~1~ euz,
~ br a lt:rges:nimal znod;l appro.:chin~,F the size of humÃ~1. In '~
~nes~~ictizeÃ~ ~~igs~, 5 mL or.
100 :Nt CWSOO-CA. 5 ~L?. kg total) was injected ir3.tt? portal N'S>:it: (.'SV
-- 3)? s.3r<` -;::i.Y.v. of 50
&'-.g 'ota.Ãrespectively) wope
:Tice C~a.~~ ,ntr<ii'`oltt3uslv.
As shown in Figure {A} tho:'3oi"7:11<Il CBD could bf:: v1Su.i.i.zeL`"a us.lng,
5 nfl. of
:
;2{. ~ ~:~ ~ ; \`. ~~ ..~"~~: O~.~:~ (i `~ Ã.~~: ~~?;.~ i ~+~~ ~~~,~li both
pi~zt~~l tt-_, .~:~~a ~~ ~ h n:~; '~~~_. ~rw.~~
~ <?zl ~'~L~ ~~~ a~z~~.c.;rc}~ ;~ of ~s'A ~:~ ~t.
10`0 CW800-CA (7,5 arici 15 i.~gikg. respectively) aiid Intra'vex~cn~~
~~~joc~jon; T. <O
rs kt:=~i), Vasualii~= 4i:~i?IIi1ft}-lÃs *. cE~Sa'i ~ ~ :1Evj possible i}"3F}
_.~"?l~~~S: c. ~.~~ia,r'~:~ ~A.~r'i Y ~iL~.;: =
CBD was :t=c ~~+i3 1ir~l+..z..
Lw
Bcad, of 2.5 cir 3,5 mm in ~~im:ct:u i-~`erc irxs';;rt,,d ir:iz} ccTZ~~inon
bile du::=; z'rr:;
p2pildF;;: icvt.r: by d;:odeii.ostL3rn;? (N=.~) to 53Iiuflaie blockage F3a
thi= CBD. As 5ht}wk, iz",
Figure 7B, iv.i.oT: th.o two types of beads weI'i:= itise.eC4dif.:o (~BD,
thC~so beads could b:;
~ietewied coa-iplet~ly in 2 of 2 pigs, wI-iich were admin;sterot~ ~inL of 50
~mki :,`WI8:,0()-CA
mtrai~enou; ly.
d+mazying
' ...,. :,..f~
~~..~ ~`Ii~9 15 t3i.''., low LTii~ S.. A 1?~C:'IR tiii~~~., t3~.'sii`x~:
~`.9~. ;'0 .;:#..luõ ci.`w SC.~ is :ils-,o
2{y !dec3.~ :fi9:`;.3:ae F.:1 Ã.+3'i:',biI..4t,i`it3I1 :i="itlINli `x91i1
fluorophores i~q?icii~ly used ioB.' guI.fll'fi,.t_ C:'^.'#.iC.h
~
:~} IC;3;~ 1... kMo--channc=i Ns_R fluorwsce<nt imaging method:4. Therefore,
MB was used to
CBD F used .
+'JS~.~.wl~~ze \_-B$ a~~>'~.n i~.~~~i.tirhGt~::4=~F ~~ ~w, while I~:'lF
'4~&~.1ya~:~f:.~i.f to ~'i :~'~.~3l~~~i ~'t~a~~s<::i:ture.
:31`s. aesial"3,. shown hl FigEue .'C, dt;ll7oifat3ate that a
F:.<)Ii'iblnat;oIl o.t MB c1atha
z~tF`kf.'i'w.`avoicõ1Lthi cinl$tln~,~ l yC% such as ICG is ffeL'ttvtr f4)i'
eaiu;s:l%'4 c3'. :ii'.nf,'..3.h .in1c.#c;it t~
~'..~
'stFlZ`wi>``s;` siYiui.itt n~,'ots ideniif.cisE#t3t:t of 7Iiu3tiptc tissue
ski"LE3::tCro4.
~~>.~C 8d~:3i:=~~
Direct tkiC:ctioI1 of bii#.i: dyes into tkSsueh ~.`,a3i iT;dzScri zninci.t#'ry
i:harliy.' E~i~w C'.-i?l3;' of
tlssEaes in ftwsuqgrc~~ field, 34`h#cli cu..i.-, lead to dif"ficu;:y in
dissecting t`9s",3t IF"ziwetif:fn of
blue dy(: Za.1ro!:%~ly the coIii9 il:i?il bile dl.lct #:i i:d3suf:.k eIat as
th5..'. chF:iagi: o`~ the bile c??<sr
3, not d.otecuabl:f ti`, the }iuman i:`s''c. I-li3wE:vei:, in our study, C:B.D
could ~c i:? ~ m.:tied, w:ta?
:ti`IR in -a~l rats and pigs, boeause NÃR f`uoroscerFce fri3m Ãahe5w z~g&nts
withiix th::.
. 4. . ?..` . .. .. . .
CBD pcneà n.te: O$:tis eIy de:,~lyÃhrz3~gli: fl;ic ti;;aue=, ulthz?t zÃ:f.~-
XIM c.iinn~:~~ bo, dQtoxi.eci: if
the ovu1)ing ":ssvw is toc# thick f'oa ti-zc fluor~sunco to ~encirate, Ir,
addition,
.yy

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
at¾;ofir,ortiseen-L:e in the aange of NIR. fyc}r{"f the tf:~sue ~n t}i,.
su:4ical. f..:~d %va, qua ~
`a'-hu15 thi Ing prc?cedure ::?r CBI) is IfÃgbl} sensitive aiid
Th.t"L`e NIR contrast i?gtvS.1i5 w4=r#.'. <.=i3?Ilpi:fl'Ã:d a6r 1.E3+`.'. in
bile df.a.`c.=t :fi.f`s.ig3ntj. WGS;
k. W.
f"W800-CA, and I_R-786. CW800-CA proved to be ~ie most selective, a4 ffiL.
~Ah{ r Ã:,,vo
co-n~xasà agonts suatcsect tAc+an lack of efficae-rit -qccr<:tion i:,r.o, bilc
~?~o~~~ the bver. The ;?;,.:}:
two agoms h~avo maa=kedl4r:laydrophobac properties, w?.z3 high affinitlt
the cell .?mnnbmne. CWSt3O .1?a4 4 -,.-Li1.phonated rcaii~ucss ~ut .~ivre is
zero in IIZ786 and
m ay I'Nvo su.is no.?aied ar:=siducs fii ICG; t1-ic nai~aber of a~lphonated
tosxdu:o's, is reI~~~cd to ,1-'.c
hydr.?phil;c properiy4?f the contrast agents. In addtf~on, CW8()0~CA was
s:tc.}wn to bÃ:
excreted into t1?o bile i , a. substantially unn?etab.3li.:ed Iba'm, wl;.ich
increases flic napid:ty
wft.i3 <vh.Eeh i-#.?Akg%..s can t?i,: obtained after ad1;slLafStrf3tÃof,,
ht7gges1z.Il;M that f> will be 5aze ti3r
z,5e in hurn&ns.
The dose of CWSOO-CA re-tjta=red f~.~r CBD cleto~~ion i-s in the ~ange oà :*_
< 5 UvI ;-
;C?~ ~3~?~~z V61, ~f?j~'>~t3~3s ti~?~~ '~-i~ ~#,~'~';~g lJC1f
:if'~tt::71;t?~. i.~>.~Y'c35~.:'IE31.ls fF~;~:f:='~fE?.~;.. ~~~A'riu goal :i3
dE`sc i:tctilistn?vnt is io zToF CW800~CA c<?i]Ct'Tltra:f(3n pf3 CBD as clw?Sw
tij 10 #.F.NI as pobsibii'~
ar ~~ ~~i:i in 3as b:ftaiiyTLrlfirt~ as low as ~7C? ifb ~.i. Sfgf1 ~7.i
c3T~t E~ 1Ief~iG~lf,~izg .}x~zflffE?:vscGx2~:-e
occurs Zbove 10 :iM C'WSÃ#O-CA (Ohnishi c:t at. Mol: bnagi-ng., 2005~ 4(31).
should be a..i~;ed that 1: is unlikely that i-l#ght` a,'.~o-n{:ef?~'f"~:r of?.
of CWSOO fnje~:~t3ts1a would
improve the CBD i:;?agzng becau~o, of this fact.
,;;~
~
Exm:?lahoraci. Duct M
- i.? p:
In ~f.~; a:he:ti ze;d, pigs, -S mlr of P"/ ~ met}4~~lerfc blue mis f~~jecUX1
~~-ito a;i
Ivnphnode, then the f1h~~racic dt:~~t was obsen.,ed. Thoracic du.tw~ ~~~~s
visuz.l:zud with a
'`da,i!r -fiai17s blue cfdli:'sl`:i?''e the color imafõe<btti was clearly
viatte`.aFft;i.:Ca in the Mor`w,',"ceTfc?;:
25 i:'.? ;g;:- (Figurc \).
Add]tio-nal L'`?>pv.#"`amG33:5 on fl3i?i'E3.t=1c dl1ct vfS#.~:alF:~:ai.Ã(?AZ
#2: t}2o rat mCd%lv"Verc Ltiw-l
p; rio ~~ed using o:he-r N IR fluoi:~~~~~t m~t~1:dts. 50 p.1v ofa 10 W
solutio`,, cif _CG,
:CG' 1:[.SA (a o$'ICG to 1?.un?an serum. a1.bumt.?), C W8MOCA, .~~:
m ~:lb1':~~'4'31Fc; was injeL=t:::Zi i.if~:o
HSAW.}~~ (a cÃ3'vaaeSet c(?fi~ugatioi3 of MN` AS'110 t> human sL*ru,
3~> meSt.nterfc l;~~p~~ iiode, and vzsmil.arc=d 5 minutes la.:ur. The aesuifts
are shown in z if,.uire 9.
The thoracic duct was 31.+'3. t visible tC? the 31.al~Cd eye. f.3the :;ola#T
maÃ.s,x:.'., bz~:it w{.s f:li: .af'ly
`viSuc#l,::ed fn the flluoms(:<.Ã:ey,"'~ image.
26

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
Ex,Lr,a ~Ã ~' E_}rox~'.It3~:i~~Ã~Tim
In znesihet_.ze~~ 3 pi>s I ni-cpIg ofnic:.fi~~lene blue was in;ected
>nCzavun.3us'iv Feadl
dhaIÃ: tiI~;. abilo.Ã-nan was opened Sti::i;gÃci4Ãxly iÃ31C Zri,'=ter wt3'~
id+,'.n`.'lfii'.G by it'n.~gli`:g< For pel; :,''
i.iztona~~i~~~3~i~
f1 Fk-i E'i2t-; ureters "'ve:re vi3`i.]Mi; ed by intravenous s31~ectICDIs of
5;) +_'r. CWSOO CA
10, 20, 50 and 100 :N l to qii 1nIl$y' the best c:?nE:eFti.$cl~.~:f371 fd:.~:r
bile duct #.naa Ãiig.
?`lu[37`e',~`.f;f,.ns;~ ?n.S,.nsia.1C:` 3i~? ureters, ~i,.S.~.i~v,;, and
cibC~.:.i.~1ini.ll wall ~.3~(:rl'.Ia2`4-`.``~:~..Ã.~ ii~y, ~, ``~
x. r~ ~' > ~
'2.0, and 30 raÃn following ina4=ctiora
As shown in Figures 10-I1, in general, ~~~~las could be 1:.c,n!,.Aied .3 Ã
ain:aE.e, aft:;r
iii'Ã.<#.vL3l'C):l's ii'.ijedid',`n. P;iik perfi31`in:i:nC=e fi?i`urCtE`.r
visualization ,app..=i:ri:Ã3 at
xdp~lio~:r11~,c1t4i~% 1`i~ :3'l*^.s`z`v':~ in C:v~::~' v3:3a (:e:.~~eri3tlt?õ:
oa`CWSOO CA" and ~~~ U.M C~'~`80i.?,.i>:A
~~ M800-CA 05 ; Ãgi1g)
(W ~.M;.kg)i4ec.tiÃ~~~~ owed t,:~~ be~~ SBI.t. SBR for 100 ~~
i3dn13211stiat:fsii w.``~'~.:in5t <sw7.ys better ttIa:i7 that ~~~ 50 P, M
M800-^~.~A i:dnaiia.s::i'iitÃf5n.
~: W~OOCA provided c.xcvl~ent vasualizkLCFn of ttret.ers as, is Sh.ovq.i in
Fir;txie 12,
~ ~amm`r1.c 6: ML, a5>o1i`k?t1 of CW800-CA
As desc.'d e'`beta h'vÃ"e13i, u:G.'.t<:r vEsi?.t3li..F.<$t;.f1Ti can usually
be obt:i.FA`3i,:,d a%q)`L>lv, oL oi: i3i, i~
few 11-~inut~s following rIlii~ suggested that CW800-CA may be oxcreted
1#z"'Ãni`= el1n1?ITc~tei as an E:nClia31ged ft:+i'.F7i, m me:ht.3lÃsatit3bl
would likely &s::o'S''
,.g . , - = i yq~ ~y
~'~~' ~,'.:~i~.~~.~' t~ a~Ei ~~`~i.Ã.t.~ an d-3i`TrC.'mxtl :if)Z;=4t..omLtry
f:i3'i;in4(:=~~.~.~.~CZ,`~+~~
.3:-fliÃ;ht (E-S-'.;'OF) rnets;iod has :?eeÃA s~~~ide;,
tjai3c ~;~>Ãpzes were c{~~~lockd. ftotn pigs (N:-z:5), and xna.y., i:=d. The
dt'Ã`. Ã-Fs17zffly C-....
b~. pinpointed w:theà fluC?rd.::sceTÃL'=~~ de-teL:=ti..~r equipped wÃtll Es-
rrOF ;'3.ass'
U`i'ÃF?esi#.Ã'i:plus, wom,' fir`t pas5al over a &000 D.,~ cEÃtoff 4el-
l"Htz:Ãt:trn oC3li:imÃ, to remos''~
~?r+~~~0'>l~s=; t3,~1~. ~:lt~2it~'.. less t~'R<1.I7. C3.,~~~~~`.~ _~:::1. was
i3l2:li~;Z(:~o ~.?I~ i3. Z_` cc3`~"3tÃ:
A3uffor A was wate.r, Bi<3F1us (Arnc Ã'ican B.~.~a.nalyficaa. Natick, MA) and
Bufffe: B~ was
Ãi~. Using <i. gnadisP;nt iif 0 tb-.s 50% ~~~for 'B ot~~~ '0 mir,, urine
s4inqp1~s were
i'e:4iki ti'ed 3.n, a 4,6 x 150 mm vey'Ã'.;~xi11etÃ"y C 18 column (~'~r
i3t^wi4) z$l a ilow rate f i~~ i ili: i:3er
minute with e~ ua',e fed iiiif3 :~ Wate:N LCT ~szi'oi`ma:~~ spe;Jt:-on:s;ter:
Ron rna.de wa,"~" sw:i
'1'v`s w^'vrC. si:t at iu?d 3 000
to 'L'.l b.trt3s;p.'ay' negative (ES ), E3rfd cone :Ãild i :qTPllzl `4''
4'i3lta
(V), ;t:spect:vel y. D::sf?ivation aiid Sotircc ien:ipurature were :.et at
'50S 140 (C):
;-cspew~-tively. DMa were amilyr s:d. wiEILi MassIynA (Waters) software,.
21

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
RLsF#hs r#3e ::how'n in F#4?;2ik..'s 13 and 14. f hS' flu{?rE'.sL.fi~'?"ici'
fhZ#n the :?r#~:53 `cimp`s.'
was ~~cte:.red aiid pinpointed at tlic identzcal mc~lectflaa weÃ~T~~ t of
C~~~~~~-CA. which
su<3<~~s4~i~ that CWSOO-CA t3vaÃS excreted into bFi 3c{ as ' z ~ :vi`'w
3a_fFtil:C:i :~i7I1~F. 1z3b= < a Ã.1.;~.<:~~ ~i
kse:~ t. an ..Ã~31`i~~. 3s
i'.o:15.ÃSÃ-Ã'71t w8^.h our tex"i.?Z'.a,:ta:b:i7-n.. This rapid chi-
nix3.3tTf2:F1 ffi?Ãl'1 Ã.i3e btif~Zy'suggi'.oted
CW800-CA ~hf3uld bt a %ate i;g.;iat for use in hum;atiis.
~: ~c~i~a a { Reion4tri#ik]ve Suj~.ti~ry
S as.Ã~..f=.co.33 i PC~~foF.mi:ng", e(:'S)nstru(:=tà w`L.. .~~.'F=:~~ycnes
;,..eods a s`aiexsÃ#a;ple, m."`.mm'w:lly
invasive means of 3m;.Ãg111L,' .3Ã?i` di:l'4nn1mI.Ã?g th ?ktlcl~i??Ãtf tÃ~
paEff:.n {?: :..3trsi key
?i? pea ~s`#'"',.iaa,; vessels ,:Ãi. r::al-t#.Ã~~it in. the operat.Ãng
Ã't?ot3i during the ~ivnzng and oxvww..Vn of
fl%:pi.5'uÃ'getar'. In i#dldEtI.C)n, IÃ;.:he pÃ:Fsl(,q-i#,'.Ail.fiv`r'
p=L:S"Ãt}da ttPo cl3_nf~~i'm ;icoil4 a ~11-n1~~iia?' F11eians
fsf._Ãssessi~g flap. 'vi.ibhlÃtiy and ~.~tddir#.~ surcsica1, efforts at flap
salvage whon i .;?mpxic-ali,<n.'s.
occar.
Al t.~it present ti.no, tlicrc is little if a~~~~ intm:iperaÃt?vo iiiiagi#3g
p;;rftmnid during
e"e,",.f?#.`hL.~,~,m#-vi:: s.8: g~i=i~?. The z'. t ~~?t)~dti fE ~T this
~311..E1 ~-3lf;.. 1.-' ~.5:, ~ ~ .~5 f.~% 3 ..:`E:t~~.~.. ~ s.. s are ?. ~f
z~'~.~, Ãj:ci3. as
i:i3mpw,c;d eJrni3gf'<Ã:?h'v (CT), T1'#.qgi.eft TesiJfic:33'aCi;:
]1"i'3a:;f7ig (F 4r~RI); single photon Lm#.SsÃ:_i'#
:.omp?.it;d ti,2'I3Ã?,.i'aphZ+ (SPECT) a?nd, positron 01 11s5ion,
i.oFni`,g,..~'iapby (:SEI) are ;nÃpnaCi.ica::
i311t3.'~C'so,~~Si~y :':`,3' intTaC~pGra#.?v`~-` use. 131t3'aSi'?uÃ4d
d5pf?rtdbto; a3 nC2p1'ob<:s a"FlS7 be %t.i.dE'?`"+:fa
. ~
::t~ i?.5:, ~~~i3~~3~t'~'C:``, it a ~1.Ii;i::,..''s L'f3`ltiact +,=~".~1 the
tlssl'Ãf% ~e.tii~{~~.1 StS~.C~y. ~7_F~:s i#.`F~.1.k:.=f..
....~:ii";.~1.3:~t~3`; >:
20 Il#. ~3#ter ; .,, tf~ '" v`+~. ~ ~, : ` ~ = i~L~~.~). <Ã.~aCa =~ ~ has
1:dT1F'~4.'ia ~ options 'r~='itx3 ~ 3"(i:~ ~.?Ci:t ii: q ~.t)aÃ+:~'e.#:2 s
~.~i.=; (F :=nC.~"i1t '~F~.)~- :
.. = ~ ~ t .i..=~i: ~,
`c~.~I#;'= only fithCGs modality avi7.l.lbfi, int7':3p+,.'I"r#.tF4'tly
x'o'I.' 14.=#.`C'.?n^itlÃ.1HfilC. s'3.Erk,:-y i\ x-ray
alllw#t ~i'~~3~,`r'. i~f'~'ti'P"~ r~Ã~~?E?gÃa}~~~~f 3~'o~.~f7$Ã'i:=`,~'s3
dedicated i::`iC~ c7xe?,Tf'I?Stt`t~ F'~t~ ~,?;~.Ã'L~F ~?:a~~'y
3'Z'ql~#ri h exposure of patient and caregivers to x-rays, and exposes fl34
pi.#t:.Ciat lf? #.i1lil:~33ae:`z;;'
wnwentraiionsof nC:~~-LrliÃ`ox'iciot3.Ãm. .Empoi;tana for tIl,is iiLudT0+,
angiography wclgh =:? all
ne, vessels ;~~~~~~~~~~i-ng a ;~lap cyLmlly, itlthun.gbin casus of
PC.rtb:ratc}r artery Ãdi.=~ififi~:atio.>, vbw
-' 4ssezs t1iF?ÃC: wiri7n'ly.
~dec~ I;i3i? Ã IEa;ti tqi3.iv L~'ou.ld w?.gi3t surface
`~ e, 4vi~luatethc: usetu~~~ ~s o: ~lR4igents i~~~ ~uchmFuthod4, 0.5 mg(14
u;.. Lg{
3ndt`+f<3'aÃ="z1 `#ti, g>e#.'.kl (IC~ ) w'as ?.i3ject[NI .Ãn~'t#'aN. S
.Ã7;Ã3usl'F' :Ã;.lti? :a 35 kg YciÃ.k5h'Ãre pig, cÃn'd l'ze
~~~~i w:~.., image<a as tac,sct'ibkd herc:Ã"it.
30 The results aÃ'e Sh(?wr: ÃI2. A'IG. 15. Shown are ;Z.ew,'r1jS.G=I7:3?i
3ut<AI?:iG3"'4;4i:i: nci; ;:topi,
;.~.Ã-wrÃ.i-~l filling at 45 see ~`.o:^'.t-1I1;'L.''9::~io.`.3 (second row) wd
venous fill1"?g at 10 ~ t poS::N
ir~tccfdcma. UsLnM, . thi::~t'= data, the exact li7CsltÃC)Il of each
pfiriC?i'.;EoF' is ,Tlsi?'!ied Z?,.1 #:iõc. <kÃ:.e
with black =T3a.f=koa'r` and the flap is `x=le1';.itel`~ (b4PEtorn T4.Z4'' ).
NaR fli?.i,;rLSt;C--'=n..+. ,#irt3<3.i.T,es havw?
12 S

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
#elon #ca-11 expoyurE' times (67 11ikhec.) and II:ClIi%13liz.#t4l>i7s. Cllie
i.T1a4}i.s were :CquÃfL=d c':='G.ry
200 ÃÃÃ5ec, TiNlEste rlitiÃt the nil?ple in t>?c field (top riojit) serves as
<in :Ã.ddzÃiondl intom.Ãa
Co#3E.p,'ol -ft) i#1te-rA41. vs, venous phases. HE',iiec, in mal-t#T#ie, zuud
w1tholf.ti .::i~ui-Izlg
lC?n.AÃ11ym6:#.t.$oFey ;hfi pt'T"x-oI ;iiiAl` r iteI#e,'.~ i3S:ita -ve1n4 can
be :dmtIfi'Ld `s.si#~g N.IaZ
ahi,,,E LsLtits indicate th3t the u4v Clf NIR agi'3:ts, x'Sid U}lf#,fz f.aM1,
methods described heseÃÃi, are useful R3Ã flap design, assessnÃent f fl ,Ã.p v
Ã.b-ility, atÃd
sc~cctioÃÃ of 111ÃtiÃÃw; flaps -ftir salvage Ãho-rapy ,A= #th
~`ibÃ.`Ãnoaytacs.
vt iÃi_WE3~~engÃh FIu.aresc.:n;e
1Ã, To f'~r`~4#:lE#.3l.ti1d :3C3?.}#bÃl7:1 '(' of 3.11:E:xlt# wa6t'i:lG?lgti#
fluorescent F nnzzg7:ng W:_. . ..i.~~~s
.rn: thvaL31L blEÃ:t,:wi.'Ã"e used.
rhe :'Gs3hs <7iv- "Ia(?wÃ: in F`#t?vii4 16. 1 }.2?g"kg m#.=th}`le# !L: ba#.ic
TV iÃEji:i.E3.=sl, wah
iollE?wt=d, 30 mIa3 tfltvT by ~},~~ Ã~1~'~Gg I~~~Xl IV 1i1j+:i:~.lCi:Ea, As
can ~~, s~ .~P`: "1:,.: ~.~:I ~s. >(`.~~4' E??
in Figure 1,65. the. orientation of ti1S C.ia#I'e$#lt?n bl:lE` duct (CBD)T
gwzlbiiodfseI. ÃiE?.fc:k
(G ~`~~), wid i:ystic. zÃ~;-teÃy (CYA) is welI -0swahzed. NIR caÃÃwra
e;~~~~~surctim.Q was 100
'll'ÃsOC.
NL.=:{t onnC% of fiha. d1aksoEii<.kl branches of the i;E?zi`11i3i'!!'
ans,+r`y' (M) waN E.3wmpvd :NWhlEe
&rÃfsw3,. and Ã;hcÃ: I nnn/kg of`rÃÃeth~,~lon4= blue was ii~j e~.M.tect
inÃsatvenÃ:fu5ly4 4c: One second
mw of Ã##3szgt': j3#stiAftE# .ÃewE1sI17g:he c3c#IYl'#3 0,03 ll3.if'~~ ICG was
#njvETtid #;??taava#aL'>#:sl5.
20 D.' vv<l:a d i.`.Ei'.L:E:E:d clearly (;.'i'.lli.3w arrow). The
ri',t~F`[Yos# image in. fl.-w sk:c+`.`+i.i: iwx was
created by #i1orr?:i~~ the i:mi:lg~.~ of the coronary aT¾Ca'1ogii3phy
E#.sf.#ig .ÃI`c1h~1L,nc ~.?13:#t. at
chur, pi-na D2 aaid t?:~e image ÃÃsing I~ G after declamping D'~..
After iakÃ,E, the 1.CG was evenly distributed over ffilC: E:min
:?'#4'oc i`s'(o.lE##i~ ~se = third row of Ã:n's3 Y=sN tlIC red pyet1dC?Loli3Ã
the mer ~ed # > .ag~.'= of 'C:.ahi:E.=Ãc:E:
p^".xfl35ioÃI;, however, a pf;I`:'EiSio2t defect cvE`sld still be detected in
7 00 ÃÃÃi1 fEltllG
;.-lak3.Qseence ÃÃ-alage. (gÃ`ceÃÃ ps,:odoCo3or) and in ilic Ã'i-ieÃgCl image.
11.7bk. area w}::Ãc bÃ?:h,
i34:y c3.Tf: :~is''LIibi]ti'.d was a wt'%k: t);'zu#gE' pseudocolor.
15 >:T}#.i3:i,.te4 afti.~i' 1CO 511;ji'=c`s1JI'!, the :ieaft, was resected and
."ATF#nei:i3;tE.ly vxwr,`#?ue.,;
~i~i~ IL'=~Ia~~~ .e i: a~1C2~~'.Ã~ zY2 ~ki Ãi~.1`: gf'.6 #Il -d:w}?C:+ftibm
row. 3L> :`,t327fir.: ~l+. c{. N-Ir, E:arne_t=.3 exposure 'Ui.ÃaTe was
3`ziSee,
,9

CA 02692923 2010-01-04
WO 2008/076467 PCT/US2007/072800
Ex~~~~;n3w ~~:Parasl;~rorc~ G1a ~~~~ r.~~~.WJsI&' ""Sir~(" Njcthylet ca~~lue
(1\4~)
T(,, d~~~~1-1irla.r wbothe. ~~B fluorescence emissiom could be iasM to
~rnag:~ie
para1h~ro~~ ~lar,d.2 i-f-,g'kg :~~~B,,~-=as ad-miniyteroci over 1.5 minutes;
along witti
p,rif)r :^u~w=Q:Y3t. As cm be see-i-k in Fi~ire 171 ihC+ pc3raLhyrL22(a was
i.ndwcd vAsib3.t`.
OT 8 3.~L.{i~t EMBODE.M.8.'.. Nf i=S
IE is to b~~, understood tha} while the 1nveat:t.on has bt.i'.n rc+'eticr, bvd
33i Q-C~:+zlÃl.a.actli'Ã':t
,v.th the dc:ata.ilod dosc~i.ptiz?D UICa~.~f the fbxC&Wlty- dcscrip:i.ort _is
intended to iifiust_z:~te and
not lIn"It the Sc~ipe of t}3L 'ii`evi:ntl'.+n, whff:h 1S de<IiiE>=d by the
scope of the appended t.iutMs,
Otl3'Lvr i3..to-pS.c`.S xidS':antc3.+,es, i323d modifications a1`'e w4t-h1n
txa4< ,.cL7pe o. t-he -fLdk?w#~g ch3i`.'Im
C~ t3