CA2025907A1

CA2025907A1 - Method of transporting compositions across the blood brain barrier

Info

Publication number: CA2025907A1
Application number: CA 2025907
Authority: CA
Inventors: Franklin D. Collins; Robert C. Thompson; Michael J. Yarus
Original assignee: Individual
Current assignee: Amgen Boulder Inc
Priority date: 1989-09-21
Filing date: 1990-09-21
Publication date: 1991-03-22
Also published as: AU6501390A; WO1991004014A1

Abstract

ABSTRACT
A method for the delivery of diagnostic or therapeutic agents across the blood-brain barrier is disclosed. The method comprises:
(a) encapsulation of the therapeutic or diagnostic agent to be delivered in a liposome;
(b) targeting the liposome by attachment to its external surface of either (i) a molecule which is actively transported across the blood-brain barrier; or (ii) antibodies to the specific brain endothelial cell receptors for molecules which are actively transported across the blood-brain barrier;
(c) administering to a mammal of the targeted liposomes containing the diagnostic or therapeutic agent to be delivered.

Description

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 ~ .

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 - ' ~! .

~,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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_ 14 SEP 21 '90 12:39 FRO~ FINNEG~N HENDERSON PRGE.019 ,~
.~ 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, ~ro~o-a~ . c. ~ooO- j~
o . ~ - lS - ~
11 ' ~ ........ .. .. .... . ... ...... . .

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-, .11 ; .

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Claims

1. A method for the delivery of diagnostic or therapeutic agents across the blood-brain barrier comprising:
(a) encapsulation of the therapeutic or diagnostic agent to be delivered in a liposome;
(b) targeting the liposome by attachment to its external surface of either (i) a molecule which is actively transported across the blood-brain barrier, or (ii) antibodies to the specific brain endothelial cell receptors for molecules which are actively transported across the blood-brain barrier;
(c) administering to a mammal of the targeted liposomes containing the diagnostic or therapeutic agent to be delivered.

2. The targeted liposomes of claim 1 wherein the targeting molecule is selected from the group consisting of transferrin, insulin or insulin-like growth factors I and II, and/or antibodies to the brain endothelial cell receptors for transferrin, insulin, and/or insulin-like growth factors I and II.

3. The target liposomes of claim 1 wherein the therapeutic agent is nerve growth factor.

4. The targeted liposomes of claim 1 wherein the therapeutic agent is .beta.-endorphin.

5. The targeted liposomes of claim 1 wherein the therapeutic agent is dopamine.