Note: Descriptions are shown in the official language in which they were submitted.
SEP 21 '90 IZ,3Z FROM FINNEGRN HENDERSON PRGÉ.005
-` ' 202~907
.~ ~, ' -
~'. There are ~any in~tanc~s in which d~ ery of thera~Qutic
'"agents, ~or ~ampl~ drug~, or diagnostlc ~gents to the bra~n aro
"de~ired. However, it ha~ b4en v~y d~fficult to deliver tho~e
.!a~ ~t~ ~o the bra~n directly from th~ ~y~Qmic circulation becaus-
¦of ~he ~loo~-braln b~rrier.~
ll ~hi~ ~rri~r consi~ o~ un~for~ tiqht ~un¢t~on~ ~etw2en
.!ad~a~ n~ ~ndothell~l cell~ lining braln ~apilla~ . Th ~e
!~unc~on- p~0v~nt ~e pe~et~tion ~nt~ th brain of many
jlwat r-~ol~ble molecule-. In add~tion, ther~ i8 a relativ~ ab~enc-
lof th~ ~ntr~llular bulk tFan~gort ve~lcle~ that ~huttle
!~molocule~ acro~ endotholial coll~ ~n other organ~ The~e
,i~ar~le~ ~rev nt ~he nt~y into tho bra~n o~ a ~ide variety of
! ~otentially ther~peutlc compound~ a.~ni~torod to tho ~ystemic : .
c~rculation.
.1 A~ an e~am~le, ~h blood-brain ba~r~er pr~vonts certa~n
l!nerotran~ittor~ ~uch ~ dopa~ino~ and mo~t macro~oleculos, auch
¦a~ nerv~ growth f o~o~-, f~o~ ont ring tho brain from tho
c~raul~tion. ~h ~8Ck O~ g notration of t~-e compound~ into the
~¦brain upon sy~t~mic a~m~ni~tration ~v rely limlt~ th~ir u~e ~n
trc~s~nt o~ n~rode~oner~tiv~ dl~ , AUCh t~ Par ~ ~on~
!! and ~l~ho~ di~ , ro-p~ctlv~ly.
For tho~- r-~on~ thod- h~vo boo~ ~ought ~o ~enotrato th~
~blood-b~ain ~arrl-~ and deliv~ therap ~tlc agent~ dlr ctly to th
~CW.~D~W~ i br~ln from the ci~c~l~tion- one method propo-~d for d~liver~ng
o~ "non-p~n~trat~ng d~u~ tO ~ho bra~n i~ to c~-mically link ~h drug
"0~
... ~ . ~ , .. ....... . .
SEP 21 190 IZ: 33 FRO~`1 F INNEGRN HENDERSON PRGE . 006
',1 202~907
~o ~ ca~rier co~pou~ that i~ cap~ble of penetrating tho
blood-brain barr~er.. The psnetrat~ng c~mpound~ that ha~e b-en
Ipropo~ed for use ~9 carr~0r~ include ~mall, lipid 801Ub1~
,~molecul~o, ~uch as modif~ed dlhydrop~r dine~ (~odor, 1987, Ann.
!~ Y. Ac~d Sci 507: 289-306)~ or compound~ that enter the brain
through a speclfic transport sy~tem in brain ndothelial c-115,
such as the tran~port ~y~te~ for tran~forrin, in~ulin, an~
¦~n~ulln-lik~ growth factor~ I and II ~Pardridgo, 1988, Ann. N.Y.
¦IAcad. 5cl~ ~.29~ 50-60).
I ~hi~ propo~d mothod i~ sub~ect to two ~erious drawbac~ that
¦maY preY nt d~ ry to the brain of a wid ranqe of th rapeuti¢
¦agents Fir~t, ~lth-r th drug mu~t retain itJ a-ti~ity wh~n
ohemically aoupl~d ~o the carri-r o~ thoro mu~t b ~n ndog~nou~
and acce~lbl~ ~rain en~yme able to uncoupl~ the drug and carrier ;
'~onc~ they ~r~ in~ido th brain Socondly, l~rgo drag~, for
jle~ampl- macromol cul-- ~uch a- nerv~ growth factorJ, aro ~ry
likely to dom~nat tho ch d cal prop rtie- of tho drug_carrier
, co~bin~tion ~nd prev n~ p netr~tion of tho comb~natlon into the
bs~in
¦ An ~dditional potential drawback to thi~ propo~ed doli~ ry
~y~ten 1- th inabllity to proto~t sen~itive drug~ from being
ln~ativat~d ~n th blood For esompl-, many potentlally
therap~utic p~ptid~ uCh ~ analg ~ic p- ndorph~n-, ara rapidly
¦d grad d in th blood (Houghten u~ al , 1980, 2~cC~ NAtl acad
~Sci t U S A 77 4588-4591)
~w O~ Ce~
F~ C~ H~D~ i The pre~nt ~nvontor- ha~ overco~e the-- probl m~ through
e~ DU~
'o'~.'c,~owoo. ith u-- 0~ liposom-- tnrgeted to ~ndog-nou~ brain tran8port
~o~ a i l
. ~ -- 2
~1' cl 'g0 1~:33 FRO~1 FINNEG~IN HENDERSON PI~GE.007
-: 1 202~
¦~ ~y~tem~ that will be u~d to encapsulate an~ delivex nor~ally non-
penetrating therapeutic agent~ to the brain. ~ince the
ther~peutic agen~ will be encap~ulated in lipo~o~, they will be
'prot~cted rom enzymatic inactivation in the blood. ~oreover,
~h~re will be no need to chemically couple the~e ther~peutic
lagento to th~ carrier and chemically uncouple ~he~ in the brain.
"In additio~, liposom ~ are capable of delivering large
"macromolecule~, 8uch a~ nerve grewth factors, that would be
i unlikely to penetratc the brain when ch~mically coupled to a
c~rri~r molecul-.
ThQ lipo80mes of the invention are targ~tsd ~y the add~t~on
~to th~ outside layor of the lipo~ome of one-or more molecule~ th~t-
jar~ normally tran~ported acro~s the blood-brain barrier. Such
I.~ran~ported molecul-~ include, but ~re not limited to,
}'tran~errin, insulin, ~nd insulin~ e groweh factors I and II as
,Idescribed ~y Pishman et al., l987, J._ N Y~Ui~L Reis~. l8:2g9-304:
,,and Frank ~t al., ~98C, Diab~te~ 35-654-661, each of which i~
I ~p~if~c~lly inco~por~t ~ ~-rein by ri3iference. ~ach endogenous
;',transport y,~tem con~ist~ of ~peici~ic membr~ne r~ceptor~ on the
endothelial c~ll surface to ~hich the tran~portsd molecule binds,
~ollowed by ~echani~m~ for in~ernationaligation of the molecule
into an intraceillul~r ~sicl-, and expulsion of the con~en~-s of
t21e vesicla into tho brain aJ d-~cribed by D~utry-Var~at et al.,
,l987, ;r. No~ 8sZ99-304s ~lau~ner ~t al., 1983, ~I.
'B~Q~ . 258s ~71S-4724t and Duffy ot al., 1981, each of ~hich
F~ e~ H~iD~
F.~ O~ R~t ..isi~pcaifically incorporat-d by referenc~ herein. ~he liposome~
~ Dl iU
~ TO~ e~..cwO. ar- at~ached to the transiport sy~it~m~ by coupling to the~r
0 ~ .
-- 3 --
SE P 21 ' 90 12: ~24 F R~M F I NNE GRN HE NDE RSON PFIGE . 008
i~ 202~9~
.,. I' , .
extRrnal surface either one of the tran~ported molecule~ m ntioned~
¦~boYe ~tr~naferrin, in~ulin, or in~ulin-llke grcwth factors) er
an~ibodi~s direct~d to the ~pec~fic brain endothe7ial cell
receptor~ for the~e tran~ported molecule~. Su~h coupling methodc
are do~$bed, for oxampl~, by Schneider et al., 1984, Nature 311s
~675-678 specifically incorporated horein by re~erence.
¦ S~M~ARY O~ TH~ INVE~TION
Th in~ntio~ consl~ts of method~ for d-livering thcrapeutic
and diagnostlc aqen~s to th- brain acro~ tha blood brain b~rrier.
Such ag~nt- are dellver~d to th~ brain by encap~ulating them in
lipo~om~s targeted to any of a numbor of endo~enous brain
¦¦tran~port sy~tem~ that transport ~p cific ligand~ acros~ ~he blo~d~
,bra~n ~drrier. The lipo60me~ ar- targeted to ~uch endogenouR
~ransport ~y~tems by coupling to their outer ~urface ei~her the
~p~cifLcally-tr~nsported liga~d or ~ntlbodie~ tO the ~rain
'-ndothelial c-ll recepto~ for the lig~nd. Example~ of such
lipo~ome-targeting ~olecule~ are t~e ~ecificnlly-tr~nspo~t d
~rot-in- transf-rrin, in-ulin, or in~ulin-like gr~wth factors I
,iand T~ and antlbodi~,~ to tho rec-ptors for tran~fer~n, insulin, :~
ior ins~lln llke ~ro~th factor- I or II.. Advantag ~ ~f the~
~ethods includ the ability to dcliver any molecule, ~sv,an
~ac~o~oloculo-~ that can b incorporat-d into l~o-ome~, the,
llab~ y to prot-ct en,slti~e ~ol-c~los durlng d-liv-ry by
¦0na~psulating tho~ in~ida A protectlvo l~id bilayerr and the
l!ab~lity to dellver ~oloculiis ~thout chemical ~odiflcAtion.
~ o~r~
F~ O~C~R~t ¦; Tho acc~mpanying drawing~, whlch ar~ incorporated ho~ein and
5 D~~N~5~ .
,con~titute ~ part of t~i~ appl~cation, illu~trate varlous
W~lrlNOtON~l.C ~00~ !
.O.
i ~ -- S
.
j 202~07
embodlm~nt~ of this in~ention and, togeth~r with ~he de~cript~on, !
i sezYa~ to explain the prineipl~s of the Lnvention.
¦¦ P~S~RIP~ION OF THE_DRA~INGS
~, Figure 1 illuætrate~ ~he ~esult8 of a compet~tion bind~ng
experi~ænt ~escribed in Exampl- 1,
TAI~ED_~S~RIP~I~N OF_T~E PR~F~R~D EMBQDI~ENTS
Re~e~ence will now be made ln detail to the presently
iprofe~r~d.embodiments of the ~nvention, which, tog~ther with the
lloll~wing oxamples, sQrve to oxplaln the principlo~ o tho
¦¦in~ontion.
~ noted previouely, the present invention relates to th~ ~se.
¦¦o targeted liposomeæ to deliver therapeutic and/or d~agno~ti~
¦jage~t~ a~ro~ the blood-brain barrier. Tho liposomes may be
, prepar-d by any of a wlde variety of 3tandar~ method~ for
,Ipri~duc~ng stable, unilamellar l~pos~s of uniform internal
diameter. In a preferrcd e~bodimRnt, lipo80me- are composed of
~aturated pho~pholipid~ and cholest-ro~ in relatlv~ propo~tion~
i,suitabl0 to p~oduc~ ~table lipo~o~es. The llpo80~e8 alco contain
i a modified lipid t~t i8 capable o* cova}ently link~n~ a var~ety
of targeting molecule~ to the external lipo-o~e ~urf~c~. In a
~referr-d ~mbodi~ont, the co~alont-link-r llpid i6 MPB-P~. In a
prof-rred ~mbodiment, ~he lipo~ome~ are composed o diRt~aroyl
. pho~phatidylcholine, chole-terol, and ~PB-P~ 4-(p
mal~imidophcnyl) ~utryl] pho~phatidyleth~nol.~mine3 in the ratio
!!mol~r of 1:1;0.06~, a--us~ng th~ final compo~iticn ~eflects the
~,~ ot~c~
F~uow. c~nr j
i~ ~i'JN~
tn ~t ~crt. N. ~
o-o-~.o.~--oo~
..O..~ o !'
.
i. ~ 5
, ' .
~ ~5! ~ ¦
SEP 21 '90 12:35 FROM FINNEGRN HENDERSON PRGE.010
'I 202~907
ll In pre~erred e~kodiments, l~posome~ are generated ~y pas~ago
; oither through ~ microporo membrane or through a mlarofluidlzer to
I gene~ate unilamellar lipo~omes of uniform diameter. Exa~ple~ of
~ hese method~ may be found in ~limchak and Lank, 1988
,310~armac~utic~ ~EB:lB~ nd Olsen et al., 1979, Biochem4
' BioDhY8~_Act~ 557:9-23, each o~ which i8 ~pecifically lncorporated~
j,herein by reference.
,I The lipo~om 5 ar- tsrgat~d for paasage thxough the blood-
¦~lbrain barrier ~y th~ coupling, to tho out~ide of the liposo~e, of ;:
¦jmolecules which ara ~ct vely transported acro-s the blood-brain
I barri~r. These transported molecule~ are added to the out~lde of
, intact lipo~omo~ and bocome covalently linked under ~pproprlate
reaetion condition- to a su~t~bly modifi~d lipid incorporated into .
, the liposomo. Exampl~ of euitabl~ transport sub~tanco~ include,
,but a~e not limited to, tran~ferrin, insulin, insulin-like growth
, f~ator 1 (IGF-I), insulin-l ke ~rowth factor II (IG~-~I), and
i! antibodie~ against sp ciflc brain endotheli~l cell receptors for
j tran~forrin, ~n~ul~n, IGF-I, and IGF~
~ In a preferrod embod~ n~, lipo~ome~ are co~al4ntly coupled
¦¦th~ou~h tho ~odif~ d llpid N-~4-p-~aleimidophenyl) butyrl~
pho~hatidylet~hanolamine ~PB-PE) to iron-saturate~ tran~ferrin on
¦¦tho out~id ur~c-. In on ex~mpl- of uch a pr fe~r~d
¦ ~ odi~nent, tran~ ~rrin-coated lipo-o~ competed eff~ct~vely with
,fr~e tran~ferrin for the t~ansferrin rec-ptor~ on human cells
ample 1, Figure 1). Such ~ran~ferrln-coat~d l~po~ome~ were
..~ o~e~
F~R~DOW,G~TT .,~l~o abl- tO increa~- tho penetratlon into the brain of a
R . 1
'O~O'N^'~N0O. '¦radiol~beled trac-r (Example 2, Table 1).
u~ 0 ~ j
. - 6 -
SEP Zl '90 lZ:35 FROM FINNEGRN HENDERSON PQGE.011
.1 2025907
.i
A ~ide va~i~ty o~ therapeutic agents are envisioned for ,
! enc~p~ula~ion w$thin ~he lipo~o~e~ o~ thi~ in~ ntion. ~h 80
¦include- ;
(for example, ne~ve growth
,jfactor~ to tr~a~ b~ain in~ur~ and ne~rodogen~ra~iv~ disea~s~;
!1 g~y~e~ to r-placo snzy~atic act~vit~e~ lo~t through ~en tic
lldefect~ whe~e tho 10~8 cau~e~ er~ metabolic toxage di~-a~e~
¦I-uah a~ ~ay-Sach~ di~ea~o;
uch as dopamine and
llp--ndo~phin, that ~ould ~e u~oful ~or treating Pa~k~n~onJs
; ¦ disea~- *nd intractabls ~ain, r~spocti~ely, or conditions
includ~ng disord~rs of mo~ent, cognition, and ~ehav~or.
for tre~tinq inf~ctiou~ disea~es, ~uch as
uro~yphili~ or AID3, who~o pen~tration into th~ brain of
y~te~aally admini~ter~d .sntib~otic~ i~ pre~ently a bloc~ to
't~Q~t~en~;
' for treatlng brain tu~or~ ~ith agent-:
.!that do not ~eac~ th~ tumor ~n sufficient amnunts when tolerable
do~e~ aro adm~n~-ter-~ y~te~ica~ly and
~ uch a~ spocific contra~t media for brain
aging, that are curr-ntly not U8ed becau~o of poor pen~tration
into th~ br~ln ~po~ ~y~t~c adminlstration.
ollowing Bs~mpl-~ orve to illuxtrate certain of tho
pr~f-rr~d embodiment~ of th~ presan~ in~ n~ion. All art~c~e~ and
C~ 6~D~RX~ ~ patont~ rof~rr~d to in the-e Ex~mple- are ~p-cifically
F~h~cw. G~R6lr
s &ut~
In~ . W.
WA~ OT--~, O~ 0~ ~ ~
~lOi~ O jj _ 7 _
,.`,c~5. : ~
UI'I r 1 I~lNEGliN HENDER~;ON PRGE . 01Z
`' i1
'I 202~j907 ,, ,;,
incorporated herein by r~ferenc~.
l ~XA~PL~ 1 - ME~5LD FOR ~QEY_I~Ç LIPOSOM~
! Tho foll~wing llpids are di~solved in 25ml chloroform and .
.'evapsratet to drynes~ a~ a ~h~n film in the bot~o~ of a round-
.lbottom~d fla3ks 30~mmol dfso~royl phosphat~ylchol~ne, 300m~o1
.'~hole~tarol, a~d ~0.4 mmol N-t4~(p-maleimidophenyl) ~utyrl3
,phosphatidylethanolam1ne (~PB-P~). The ~P8-PE contains a reactiv~
,l~roup capable of coupling to a wid- var~esy of protoin~. ~artin
'l~nd P~pnhad~opoulo-, 1982, ~. Biol. Chem. 257s286-288). Th~
,Imaterial to b~ encapsulated i8 dis~olved in Buffer I con~l~tin~ of.
¦!108~M N~Cl, 35m~ Na2 W 4, 20~ ci~ric acid, lmM EDTA, pH ~.5. ~hei
Illow F~ prevent~ premature hydroly~i~ of the covalent linking group,
i.o~ ~PB-P~
,'1 Lip~d~ are ~wollon ln this ~olutlon for 3 hours, then the
.,solution i~ pa6s~d sev n tim ~ through a ~icrofluidiser ~110 a~
.ide~cribed by To~arden t_pl. in Pharmacoutical Re~ 482-487
"(1988) a~ a f~nal nitrogen pres~ure of 10,000 p~i ~o cseato
, un~lamollar lipGAoms~ ~n a narro~ ~ise range with ~n avera~
"diameter of approx~m~s~ly lOOnm. ~he unilam~llar n~ture of the
lipo80m~ w~S conflrm~d by X-ray dif~raction and the ~se
di~trlbuelon determin~d by light sratt~rlng.
To -paxato lipo~omo~ fr~m uni~corporated mat rials, tho
¦¦~ixturo i~ pa~d o~ r a S-phado~ G150 column egullibrat~d with
,jbuffer I. Ih liposom s om rge from the colu~n in th~ void
~wo~-let~ ;jvolume. rrhQ lipo-ome- are dea~r~ted undor argon ~or 2 hours. Ten
F~ , H~t~DWON
F~UOW~ TT ''mg of ~ron-satur~t-d tran~f-rrin is added dissolv~d in RlngQr~s
~ OO~ alt olution. The.pH i ralsed to 7.0 at whic~ pH thR coupling
,l - 8 -
~ ~, ..
SEP ;~1 190 12:36 FROM FINNEG~N HENDERSON PF/GE.013
. 2Q2~907
group on MPB-PE i8 activated, and the r~ction allowed to ~tand
~ under argon overnight at ~C. Reactive group~ on MP~-P~ that do
¦¦not couple to tran~ferrin during this -tep, including those
'Imolecules of MP3-PE who~e reactive gro~ps face th~ interior of the
,lipo~ome, a~ hydrolyzod and inactivated when oxyg~n i5
,¦r~in~roduced duri~g sub~equent proce~ing. Tran.~errin-coupled
lipo~ome~ are geparat~d from free, unreacted transferr~n by a
lla~cond pa8-age of the reaction mixture throug~ a G-150 column
¦lequil~brated with Ring~r'~ salt ~olutlon. The amount of
¦trans~errin is mea~ured in each raction em~rging from the column ~ :
~by ~tandard protein a~8ay in order to calculate the amount of
unincorporat~d ~ran6ferrin and the number of coupl6d trans~errin
~molecules per liposomo.
! To ~eter~in wh-ther tran~ferrin and encapsulated drug are
~stably agsoclated with the l~posome~, an aliguot of each lipo~ome
~pr~par~tion can be ~tored at 4C for var$ou~ length~ of time, then
i,pa~d over a G-150 ~i~lng column to determin~ what proportlon of
,ithe tran~ferrin aAd ncapsulat-d drug emergQ in th0 lipo80me ~.
jth~ ~on-llpo80me ~ractions.
To det~swin~ whether the tran~errin is present at the
li~oJowe ~urface ~n a form that i~ still cap~ble o~ reactins w~th
it- specific C-ll surface tran~port-mediatlng receptor, a
comp tit~on a~s~y can ~ run Comparing the ability of
tranYfesrin-coupled li~osome~ to c~mpete with free tran~ferrin for
~wO,.,~ the traA~ferrin rec-ptor. Figur~ 1 illustrates the results of one
FlN~ N, H~NW~ N ¦
F~O~ r ~ ~UCh co~p-tition a~ay- rhe exp~r~m~nt con~i~t~ of introducing ~
5 S~
w~ eito~ e~i~o~ ,jfi~ed amount (lnM) o~ ~25I-labeled tran~ferrin into tube~ ~a~h of
~O~ A ----~O ~ I ,
! 9
SEP ~1 '90 IZ:37 FROM FINNEGRN HENDER~ON PRGE.014
,' ~ 6 2025907
wh~h contain8 2xlO ~562 human erytholeukemic cQll~ that pO88Q88 .
a high density of tr~n~ferr~n receptors (Rlau~ner ~t ~l., 1983, J.
¦ BioL. Chem. 258:471~-4724~. Ta~t wells also contain lncrea~ing
: I concentration~ o one of tha followin~: free unlabel~d
~transferrln, tran~ferri~-coupled liposome~, or lipoeomes wi~hout
tran8f~rrin coupled to thoir out~r surface. The reduction in the
amount of 125I-la~e~ed tran~errin bound wa~ m~a~ured fn
t~iplicat~ tube~.
Figure l plots ~he l25I-lab~lsd tran~ferrin ~ound in each
¦ tuhe (a~erage + standard d-viation~ vs. ths log of the final ~ol~r~
. concentration o$ ither lipo~omss or unlabeled tran~ferr~n. The
rQsult~ indicate that tran~ferrln-coupled lipow~e8 can b~nd well
¦ to ths ~anferr~n rec-p~or. ~ndeedt the ro~ult~ ind$cate that, on'
¦a molar ba6i~, tran~forrin-coupled llpo~omes co~pete more
effecti~ely than fr e~tran~f~rrin for the tran~ferrin receptor.
Liposome~ that had no tran~ferrin coupl~d to their surface did not.
reduce ths a~Gunt of 125I-label d t~ansf~r~n bound to the cells,
! indicating that ~uch lipo8om~ failed to compete for th-
tran~f-rrln receptox.
E$~PI~L2 - ~FCq~YI~FQR LI~QsoM~-pER~usIoNs TO TEST DELrvERy
Per~u8ion and a8~--~ment of drug doliv~ry acro~s the
. blood-brain barrior are p rform~d a~ da~crib d in Pishman et al.,
. 1987 ~J. N~urc~ci~_R~. 18:29~-304). Briefly, male ~200g ~-
Sprague-Dawley rat~ are anae~to~iz~d with N-mbut~l and cl-ared of
blood by perf w ~on ~hrough an aortic cannula with Buf~-r II
~w ~Ir~loe- ~
~F~C~o~G~Rn~ con~ ing of Rin~er~ lt ~olution ~cnta~ning 0.2% ~o~ine.serum
UNN~
oo~.o~e~ j a}bum~n. ~h~ subcla~ian arter~e4 are tied off and ~he pe~fusion
1~;121 ~ -0
I - 10 - '
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~,EP cl '~0 12:37 FROM FINNEGRN HEN~ERSON P~GE.015
: -'.. ii
1! 202~9~7
i ~ontinued to the re~a~nder o~ th~ upper half of the body. ~ha
jjper~usate i~ circulated at 6-8ml/min. The P02 i-S ~aintained at
¦~210 + 20 ~nd 16~ + 2mm Rg ln the infusate and exfu~ate,
resp~cti~ely. The PC32 typically range~ from approx~ately 12
.,12 ~n the infu~a~ to 22 + 4mm Hg in the exfusate. The 2
con~umption and C02 p~oduction are monitor~d continuously and do
,not change apprec~ably during the cour~e of perfus~on. ~ach
expqrimQntal condit~on i~ r~n in triplicate on tkree rats.
,~ ~ran~err$n-coupled llposomss ar- prepar~d with elther
,¦rad~olakeled t~acer, such as an 125I-lDb-led peptlde, or a
histocheMical tracer, ~uch a~ a blotinylat~d poptide, encap~ul~ted,
,~n~ide th~m. The lipo omes are su~p~nded ln ~uffer II. After the~
,blood is cl~ared ~rom an animal, typically l~min af~r perfusion
}ha~ begun, the lipo~om~ are add~ and perfusion continued for
~arious time~ up to 60min At the ~nd Or each e~perimental
,interval, fre~h Buff-r II $~ ~ubstituted for the
' ~posome-containing ~erf w ato and perfus~on continu~d untll none
iof the encapoulated tracer i~ d toctable in the circulation,
,typ~cally lOmin
il To m asur d-livory to the brain bioch d cally, the brain
~-culature i8 i801at~d by th~ method of ~rand-l ~Fi-h~an et
~al , lg87, J Neuro~ci RQs 18 299-304; ~rand l et al , 1984,
Sc~e~cR 185~9S3-95~j at roo~ tQ~perature Briefly, a~ter
~',p rfusion, brain~ ar rQmo~ed an~ homogen1z~d The homogenate i8
~,pas~-d through nylon meshe~ of d crea~ing pora diameter The
~ o~-lee~ ,
FlF~C~ G~e~6Tr~ ',m~terlal capSured on Sh~ final ~ylon filter ~onsists 801ely of ~DU~R
~,n~ T~OT~eO~e~,0~O. ~ thin ~rain v~scular elements, devoid of s~ooth mu~cle cells
120~ 0
., " . i
~ c r c ~ c . ~ ~ r r~ r I I~ lY C J l-~ l`l n c l~/ ~ c ~ a ~ r ~ u c . ~ 1 o
,.'~ ~.i 202~907
;' ,i .
¦!(Fi~h~an et al., 1987, J. ~urosci. Re5. 18:29g-304; ~randol et
!lal., 1974, ~ç~ 185~g53-955). ~he amount of lipos~e
¦~.encapsulated rad~olab~led tracer th~t appears in this va~cular
fraction i~ mult~plied by a predetermin~d correction factor in
,, .
, ordsr ~o calculate the total a~ount of encapsulated r~diolab~led
,.tracer in the br~in v~s~ulature. The correction factor
,~cor~eopond~ to the percentago of thc total braln ~asculature
"ty~cally recoY~red in the inal f~action reta~nYd on nylon me~h
¦j.and i~ determined as de~cri~d in Fishman et al., 1987 (J.
."Neuro4¢~. ~e~. 18:299-304).
,I The fra~tion of the brain homogenate that i8 not retained on
;,~he final paszage thxougn nylon mesh is the b.rain parenchymal
~f~tion. The total amount of lipo~ome-encapsulats~ radiolabeled
,Itracex in this fraction ~ea~urefi tho amount deli~er~d tO the
"brain. By thi~ ~ethod ene ~an determine ~eparately the a~o~nt of .
',encapsul~ted ~at~rial dellvered to t~ bra~n parenchym~ and the
,~brAin va~cul~ture.
,~ Th~ tran~port of lipo~omQ-encapsulated matorial into the .:~
~,b~a~n 1~ al~o demon~trated by dlrect localization of tran~porte~
¦¦material ln brain ectionc u-ing hi~toch~mic~try. After perfu4ion
Ija~ abcve with tran-ferrin-coupled lipo~ome~ containing a
¦¦b~otlnylat-d peptid (La Roch lle and Froehn-r, 1986, J~ Biol.
261~5270-5274), the brain i~ fixed ~y continued p~fusion
l~w~h 4~ ~arafolmalde~yde for light micro~copy ~ 4~
,paraformaldehydo and 0.25~ glu~arald-hyde for olectron microsco~y.
~ or--c~
F~ c~ Variou~ ~rain r~gions ar~ dissec~6d, Qmbedded in F.pon-Araldit- an~ .
D~ gP.
,'0-O'~'Oc'.~O. . ~ect~on~d on an ul~ramicroto~ for ~lectron microscopy. Ssctions
~o~
~ 12 -
SEP 21 '90 IZ:38 FRO~l FINNEGRN HENDERSON Pf:lGE.017
.
202~907
I are eXpO8~d to avidin-horse ~adi5h p roxidaso ~avidin-HRP~ which
,ibind~ exclu~ively to t~e slte~ where biotinylated pept4d~ has
localized. The complex of a~idin-ERP and biotinylated peptide i~
vtæualized by the production of a ~visible" H~P reaction p~oduct
in the light or electron micro~cope aa de~cribed ~Connor ~nt Flne,
1986, ~ain Res.~ 368; 319-328 ) .
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IPOSOMES ~ V~SCUL~R PE~ET PA-~ENCkYMAL SUP
! TR~N5FERRIN 2 1,095 ~ 6l cprn 1.S4S ~ 38g cpn~
OVAL8U~IIN 2 3~;- tt9cp~ 4~1 _ 167c~m
Il_4E~ ELIVERr OF ~AC~10~'~0 TRACE2 6r I R~NSr E.~RIN
¦¦ VS. OVAI 8UMIN COATD l.IPOSO~lES
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SEP 21 '90 12:39 FRO~ FINNEG~N HENDERSON PRGE.019
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.~ I 202~907
~ nble 1 illu~trate- th8 re~ult~ of an exp~rLmen~ in whLch
2.3~1014 lipo80m~ taverag- d$am tor = 45.2nm) contaln~ng
2.0x106cp~ of 32P-oligonucl~otide tracer and C3Upled tO 48g
~ran~ferrin~ or 5gO ovalbumin~ per lipo~ome ~ere p~rfu~ed through
the cerebral circulation o anae~th tLz~d rats for 4S ~inu~e~.
~Perfu~ion ~a8 contlnued for another 10 minute~ in Ringo~'~ without
li2o~o~e8, then the brain was remo~ed and fractionat~d into a
repre~ent~ive vs~cular compartment and a brain parenchymal
¦¦compartment, a~ de~crib~d above. ~wo rat- wer perfu~d with
lipo~mo~ coat-d with iro~-8aturated rat tran-f-rr~n, ~hile two
rats wer~ perfu~ed wi~h lipo~ome- coated with a control
~nontra~ported prote~n, ovalbumln. .
Th r~8ult8 indicat- that t~o brain par~nchy~al and va~cular
compartments cont~ined at least 3-4 fold more radloact~ve tracer
~h~n tr~n8ferrin-~oated lipo60~e~ wore ~d. Th~8~ re~ult~ .
~8u~ge8t that d~ ery to th- brain occurred and wa~ tran~e~rin
~dopen~ent. Th~ amount of radio~ctivity in th paronchym~l
:co~E~Irub nt corr~-pond~ to the delivery of the tracer in~id~
~gr~ter than 1 ~n 2,000 lipo~omes.
5hl- amount o~ tran-port, althou~h by no mean~ the ~aximum
that ~hould be achiov~blQ with thi~ t-chni~ue when optimized, is
ph~r~colog$c~11y 8~gn$icnnt. ~ran~port of the contont~ of 1 in
2,000 llpo~o~ ~ would allo~ deliv-ry o~ about lOOng of
p-endorphin to tho brain, makinq thsii Con~r~t~ve a~Jumption that
¦¦200pq had been anca~ulated ln a do~- of tXlol5 lipo80~e-. lOOng
~ O-~le~ ,
IC~H~N~U~NI of p-~ndorphin ~rodu~-Y dotectablo analg--ia wh n ~n~ected into
~;~UN~ j
~n~ cT,
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SEP 21 '90 IZ:40 FRO~ FINNEGRN HENDERSON PRGE.0Z0
. - - ;l
.: 1 202~907
¦¦the brain ve~tricles of rat~ (Ti~eo et al., 1988, J. ~kprm~L
',~b~h~ kcL 246:44~_4s3~.
I! It would be apparent to tho-- akilled in th- ar~ that various
~'modlfication~ and variationR can b~ made to the process~ and
'product~ of the pr~nt inv-n~ion. Thu~, it i~ intend~d that tho
~r~se~t invention cover the modif~ation~ and varlation~ of thi~
~j~nvention provided th~y co~e within tho ~cope of tho app~.nd~d
clYi~ ~d th~lr eq~ nt-,
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