CA2079450A1 - Interaction between bioadhesive liposomes and target sites - Google Patents
Interaction between bioadhesive liposomes and target sitesInfo
- Publication number
- CA2079450A1 CA2079450A1 CA002079450A CA2079450A CA2079450A1 CA 2079450 A1 CA2079450 A1 CA 2079450A1 CA 002079450 A CA002079450 A CA 002079450A CA 2079450 A CA2079450 A CA 2079450A CA 2079450 A1 CA2079450 A1 CA 2079450A1
- Authority
- CA
- Canada
- Prior art keywords
- component
- administration
- liposome
- recognizing substance
- target site
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
Abstract
Recognizing substances, epidermal growth factor, gelatin, collagen and hyaluronic acid, have been covalently bound to liposomal surfaces and utilized to attach liposomes onto a cellular target site. These "bioadhesive" liposomes offer several advantages in the area of topically and locally administered free drug. These advantages include the mutual protection of both the drug and biological environment; an increase in drug bioavailability and retention at the target site; and improved adherence or adhesion to the designated target site.
Description
WO 92/14446 PCr/US91/08112 .~, .
. .
. .
2~7945~
INTE~ACTION BET~E-~N BIOA~HESIVE LIPO~OMES AND TA~ET SIIi~.S
- BACKGROUND OF ~H~ INV~NTION
The present lnventlon relates to a novel drug dellvery system, partlcularly to mlcroscoplc drug dellvery systems ~MDDS) utlllzlng drug-encapsulatlng lbloadheslve llposomes for toplcal and local drug adm1nlstratlon.
Currently, the toplcal and local admlnlstratlon o~ a drug can be 1n lts free form, dlssolved or dtspersed ln a sultable dlluent, or tn a vehltle such as a cream~ gel or olntment.
Examples of therapeutlc or deslgnated targets for toplcal or local drug admln1stratlon lnclude burns; wounds; bone ln~urles;
ocular, skln, lntranasal and buccal lnfectlons; ocular chronlc sltuatlons such as glaucoma; and toplcally and locally accessed tumors. Several dlfflculttes exlst wlth elther the toplcal or local admlnlstratlon of a drug ln lts free form. For example, short retentton of the drug at the destgnated s~te of admlnlstratlon reduces the effltacy of the treatment and : 2D requlres frequent doslng. Exposure of the free form drug to :~ the blologlcal env1ronment tn the toplcal or local reglon can result ln drug degradatlon, transformat~on lnto lnactlve entltles and nondlscr~mlnatlng and uncontrollable dlstrtbutlon of the drug. Such degradatlon and uncontrollable dlstrlbutlon of.the drug can result ln toxlclty ~ssues, undeslrable slde effects and loss of efflcacy.
Mlcroscoplc drug dellvery systems ~MDDS) have been developed to overcome some of the d~fflcultles assoclated w~th ~ free drug admlnlstratton. MDDS ls divlded lnto two baslc -~ 30 classes: partlculate systems, such as cells, mlcrospheres, viral envelopes and llposomes; or nonpartlculate systems whlch are macromolecules such as protetns or synthetlc polymers.
Uslng these spectflc systems. drug-loaded MDDS can perform as , . .
.~ .
WO 92/14446 ` PCI/US91/081t2 2~79~
sustalned or controlled release drug depots. By provldlng a mutual protectlon of the drug and the blologlcal envlronment MDDS reduces drug degradatlon or Inactlvatlon As a system for controlled release of the drug. MDDS improves drug efflcacy and allo~s reductlon ln the frequencv of doslng. Slnce the pharmacoklnetlcs of free drug release from depots of MDDS are dlfferent than from dlrectly-admlntstered drug MDDS provldes an addltlonal measure to reduce toxlclty and undeslrable slde . effects.
Llposomes offer a range of advantages relatlve to other MDDS systems. Llposomes are lipld veslcles composed of membrane-llke llpld layers surroundlng aqueous compartments.
Composed of naturally-occurrtng materlals whlch are : blocompatlble and blodegradable~ liposomes are used to lS encapsulate blologlcally act~ve materlals for a varlety of purposes. Havlng a varlety of layers slzes surface charges and composltlons numerous procedures for llposomal preparatlon and for drug encapsulatlon wlthln them have been developed some of whlch have been scaled up to lndustrlal levels.
Through approprlate selectlon of liposome type and slze the encapsulated drug can also range in slze. Llposomes can accommodate llpld-soluble drugs aqueous soluble drugs and drugs wlth both hydrophlllc and hydrophoblc resldues.
Llposomes can be deslgned to act as sustalned release drug depots and ln certaln appllcatlons. ald drug access across cell membranes. Thelr ablllty to protect encapsulated drugs and other characterlstlcs ma~e llpo50me5 a popular cholce ln ; developlng MDDS wlth respect to the prevlous practlces of free ; drug admtn~stratlon.
Desplte the advantages offered. utlllzatlon of drug-encapsulatlng llposomes does pose some dlfflculties. For e~ample llposomes as MDDS have llmltDd targetlng abll~tles llmlted retentlon and stablllty In clrculatlon potentlal .',' . ' ' .
.~
.
.
WO 92/14~46 PCI/U~91/08112 . .
. . ~ .
2 ~7 ~ ~5 0 toxlclty upon chronlc admlnlstratlon and lnablllty to extravasate. In recent years attempts have been made to couple dl~ferent recognizlng substances with llposomes to confer target speclflclty to the llposomes namely antlbodies glycoprotelns and lectlns. Although the bondlng of these recognlzlng substances to ltposomes occurred the resultlng modlfled llposomes dld not perform as hoped partlcularly durlng 1n vlvo studies. Other difficultles are presented when utlllzlng these recognizing substances. For example antlbodies can be patlent speclfic and therefore add cost to the drug therapy.
Several cell-assoclated entltles can partlclpate ln the blndlng between cells and recognlzlng substances. These are generally dlvlded lnto three ma~or types: receptors and lS non-receptor components of the cellular system and extracellular matrlx. Receptors can be present ln several specles or states dlfferlng ln populatlons per cell and tn blndlng afflnlty. Blndlng to such receptor entltles ls usually referred to as speclf~c blndlng . Non-receptor cell membrane components also dlffer ln populatlons and in a~flnlty. Blndlng to such non-receptor entltles ls usually referred to as non-speclflc blndlng .
To perform effectlvely the toplcal or local admlnlstratlon of drug-encapsulatlng liposomes shouid have : 25 speclflc1ty for and the ablllty to adhere to the deslgnated target area and should facilltate drug access to lntracellular sltes. Currently avallable llposomes and other MD~S systems do not meet these performance requlrements of toplcal and local drug admlnlstratlon.
RY OF INVENTIOH
It has been learned that mod~fylng regular llposomes by covalently anchorlng certain recognlzlng substances to the '. '~
WO 92/14446 PCr/US91/08112 20~9~ 4 llposomal surface creates a bloadheslve llposome wlth target speclflclty and retentlon. The recognlzlng substances are molecules whlch can be utlllzed as an adhes1ve or glue attachlng a drug-encapsulatlng llposome onto a therapeutlc target slte. These bloadheslve recognlztng substances can - perform elther through receptor mechan1sms or through assoclatlons ~lth components wlthln the extracellular matrlx.
Regardless of the speclflc mechanlsm of adheslon these substances are referred to as bloadhes1ve recognlztng substances based on thelr common end result.
Through covalent anchorlng the bloadheslve recognlzlng substances become an lntegral part of the llposome yet remaln accesslble to the lnteractlon counterpart at the target slte.
They endow the llposome and encapsulated drug wlth the ablllty to adhere to the target slte. Hence bloadhes1ve llposomes .~ have been deve~oped whlch are target adherent sustalned release drug depots. The ldentlflcatlon of recognlzlng substances and the methodologles of modlfylng llposomes has been dlsclosed ln concurrently flled appllcatlons. These bloadheslve llposomes offer several advantages over prevlous pract~ces of toplcally or locally admlnlstered free drug and other MDDS whether wlth regular llposomes or other MDDS
systems. These advantages lnclude the mutual protectlon of both the drug and blologlcal envlronment; an lncrease ln drug bloavallablllty and retentlon at the target slte; and lmproved adherence or adheslon to the deslgnated target slte. These advantages result ln the potentlal reductlon of undeslrable blologlcal slde-effects of the drug belng admlnlstered.
BRIEF ~ESCRIPTI0N OF THE ~RAHING~
FIG. l shows the blndlng of bloadheslve llposomes (EGF-modlfled; open double trlangle) and regular llposomes (asterlsk) of the LUVET type to A431 ce)ls ln culture (in ~' , ' . .
., .
WO 92/14446 P~/US91/08112 , . . .
` :~
2~79~5~
monolayers), as dependent upon llposome concentratlon. Bound llposomes, denoted as B, are ln units of ng EGF per 106 cells.
Free llgand concentratlon, denoted as L, are ln unlts of ng EGF
per lo6 cells for bloadheslve llposome ~flrst row of L values) and ~n unlts of umoles llpld per 106 cells for the regular . llposomes (second row of L values).
`~ FIG. 2 shows a tlme course of the blndlng of bloadheslve llposomes (collagen-modlfled) of the MLV type to A431 cells ln culture ~ln monolayers). Collagen ls trltlum-labeled. The fractlon of llposomes relatlve to the amount present ln the lnltlal react10n mlxture at zero-tlme whlch ls cell-assoclated ls determlned over tlme.
FIG. 3 shows the blndlng of bloadheslve llposomes (collagen-modlfled) and regular llposomes of the MLV type to A431 cells ln culture (ln monolayers). Collagen ls ,j trltlum-labeled ~3 H) and llposomes are 14-C labeled. 80und llposomes, denoted as B, are ln unlts of 3-H DPM per 105cells (left scale) and ln unlts of '4-C ~PM per lOS cells (rlght scale). Free llgand concentratlon, denoted as L, are ln unlts of 3-H or 14-C DPM per 105 cells. Bloadheslve llposome wlth collagen labeled ls deplcted ~lth open double trlangles;
bloadheslve llposome w1th the llposome labeled ls deplcted wlth crosses; and, regular llposome ls deplcted wlth asterlsks.
~ U~_~LS~
~ Accordlng to the present lnventlon, bloadheslve llposomes .~ have bound to cell cultures havlng receptors or extracellular : matrlx whlch accommodate the recognlzlng substance bonded to : the llposome. Llposomes, ln partlcular, multllamellar veslcles - 30 tMLV), mlcroemulslfled llposomes (M~L) or large unllamellarveslcles (LUVET), each contatnlng phosphatldylethanolamlne - (PE), have been prepared by establlshed procedures.
Recognlzlng substances, each of whlch have been accepted for . . .
. :
.
'~ . ,, ' ' ' WO 92/14446 PCl'/US91/08112 , 9D~jO 6 human use lnclude epldermal ~rowth factor (EGF) hyaluronlc acld (HA) gelatln and collagen. Each of these recognlzlng substances have a blologlcal origin and are blodegradable and blocompatlble. Further these recognlzlng substances have functlonal resldues whlch can be utillzed ln covalent anchorlng to the regular ltposomal surfaces.
The methodologles of preparlng the spec~flc bloadheslve llposomes have been dlsclosed ln separate appllcatlons concurrently flled ~lth thls dlsclosure and wlll not be ~ 19 repeated here.
A complete accountlng of blndlng entltles has been determlned by the prevlously known multl-term Langmulr Isotherm equatlon as applled for the quantltatlve descrlptlon of the relatlonshlp between the free and dependent varlables:
n Bmax~ ~L~
1~1 Kd~ + [L~
where n is the number of dlfferent cell-assoc~ated blnding entltles that a cellular system has for a speclfic recognlzlng substance; tL] ls the concentratlon of free llgand whlch can be recognlzlng substance free llposomes or bioadheslve l~posomes; B ls the total quantlty of bound recognlzlng substance per glven number of cells~ at a given [L~; and Bmaxl and Kd~ are the total number of sltes of a glven entlty and the correspondlng equlllbrlum dlssoclatlon constant. B and ~max are normallzed for the same number of cells.
For cases ln whlch receptors and non-receptor cell membrane components partlclpate in the recognlzing substance blndlng and ln whlch the d~ssoclation constant of the non-speclflc blndlng is sufflciently large wlth respect to the free ligand concentration equation 1 can take the form:
'.
.
.
, WO 92/14446 P~T/US91/08tl2 --` 2~7~50 n-l Bmax~ ~L]
B= --------- ~ Kns "~ (2) 1~1 Kd~ ~ ~Ll ~ 5 where the last term, Kns [L), ls the contributlon of the .~ non-speclflc blndlng to B and r~nS ~s the ratlo of Bmax to Kd correspondlng to the non-speclflc blndlng.
Best-flt values for parameters n, Bmax~ and Kdl are obtalned by computer-alded data analysls, aecordlng to equatlons ~1) and/or (2) above, applylng nonllnear regresslon procedures.
The lnteractlon of the bloadheslve EGF-modlfled llposomes has been éstabllshed wlth cultures of A431 cells, ln monolayers, as a blologlcal model. Thls well-establlshed cell llne, orlglnatlng from human epldermold carclnoma, ls enrlched wlth EGF receptors. A431 cells have been repeatedly used for study of the lnteractlon of free EGF and lts receptor.
A431 cells have been shown to have three classes of EGF
receptors, dlfferlng ln thelr afflnltles and populatlons. The flrst of these classes ls the ultra-hlgh afflnlty sltes wlth an equll1brlum dlssoclatlon constant of 0.07 nM and a populatlon of 150-4000 sltes per cell. The next class ls the hlgh afflnlty sltes wlth an equlllbrlum dlssoclatlon constant of 0.7 nM and a populatlon of 1.5 x 105 sltes per cell. The flnal class ls the low afflnlty sltes ~lth an equlllbrlum dlssoclatlon constant of 5.9 nM and a populatlon of 2 x 106 sltes per cell.
Examule One To compare the blndlng a~llit~ of regular llposomes and bloadheslve llposomes, A431 cell cultures were grown ln monolayers, ln flasks, applvin~ u;lJ~l procedures for thls cell ., .
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. . ,-, ..... . ..
.... . .
: ' '` ' . ' ' . '' ,, WO 92/14446 PC~/US91/0811~
2 ~ 5 - 8 -: llne. Two to three days prlor to an experlment the cells were seeded lnto multl~ell culture plates and the experlments were done when the systems were confluent.
For purposes of assaylng the modlfled llposomes the EGF-recognlzlng substance was labeled wlth a generally known radloactlve marker. Preparatlon of EGF-modlfled LUVET was completed as dlsclosed ln the concurrently flled appllcatlons.
Prlor to the addltlon of a reactlon mixture of EGF-~odlfled .11posomes free llposomes or free EGF medla was lo removed from the A431 cells and the cells were washed wlth a blndlng buffer. The reactlon mlxture and cells were lncubated for 1-2 hours at room temperature. Upon dllutlon and wlthdrawal of the reactlon mlxture at the end of lncubatlon 2-3 successlve washlngs wlth a blndlng buffer of the wells were completed. Lysls of cells or detachment of cells from the weils was then followed by wlthdrawal and collectlon of the ~ell content denoted as the cell fractlon. Assays of thQ cell fractlon were completed by label countlng of the fractlon as compared wlth the countlng of the Immedlate products created through the preparatlon process.
A comparlson bet~een the blndlng of free llposomes and EGF-modlfled llposc~s to the A431 cells ls lllustrated ln Flgure 1. The EGF-modlfled l~posomes adhere to the A431 cells conslderably better than free llposomes as no free llposomes we.re found at cell fractlon. It ls speculated that lf free llposomes do assoclate wlth the cells the dllutlon brought by the ~ashlngs ls sufflclent to cause quantltatlve dlssoclatlon.
ExamDle Two Blndlng studles of EGF-modlfled llposomes to A431 cells were carrled out as descrlbed In example 1 and the data were processed accordlng to equatton ~l) above. The experl~ental condltlons were such that the ontributlon of non-speclflc ' WO 92/14446 PCr/US91/0811.2 ; 207~0 g `~' ~ blndlng was negllglble. Indeed the data were found to flt - unamblguously w~th a slngle type of blndlng slte for each llposome system studled. Results for several systems are `~ llsted ln Table 1.
BINDING PARAMETERS OF BIOADHESIVE LIPOSGMES
~ TO A431 CELLS IN CULTURE
~ 10 BIOADHESIVE K~ SITES PE~ CELL
LIPOSO~E SYSTEM (a) ~n~ 10- ) - EGF-MLV 0.60 + 0.017 0.17 + 0.03 EGF-MLV 5.03 + 1.9 1.07 + 0.03 EGF-LUVET 2.91 + 0.003 O.lB 0.001 EGF-MEL 0 04 + 0.007 0.042 ~ 0.0042 EGF-MEL 0.40 + 0.13 3.7 + 0.90 EGF-MEL 0.48 + 0.05 0.28 + 0.01 ~, .
- (a) Each bloadheslve llposome system ls a dlfferent preparatlon; recognlzlng substance ln each system ls EGF.
An EGF-modlfled llposome ls conslderably larger than and dlfferent from free EGF whlch ls expected to affect the blndlng parameters. For a glven class of receptors the magnltudes of the dlssoclatlon constants for EGF-modlfled llposome systems are expected to be slmllar to or hlgher than those of free EGF. For a glven class of receptors the number of receptors per cell that are avallable for the EGF-modifled llpo50me5 ls expected to be equal to or lower than the number :- of avallable for free EGF. Based on these conslderatlons the .
, , '.
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.~''',' i' ~ ~ ' .
.~.................................. , . . .
.
WO 92/14446 PCr/US91/08112 h~9~3 blnding data of the present example flt wlth the receptor classes of ultra-hlgh and hlgh af~lnltles.
Regardless of the specl~lc cell-assoclated blndlng entlty lnvolYed the blndlng data llsted ln Table 1 sho~ that 5 EGF-modlfled ltposomes blnd to thls cellular system wlth hlgh aff~nlty and w1th a sufflc1ent number of sltes for these modlfled llposomes to perform as the deslred bloadheslve llposomes. -, lo Exam~le Three Blndlng collagen-modlfled llposomes to A431 cells ~as carrled out essentlally accordlng to the procedures detalled above. The A431 cell llne ls not ~nown to contaln receptors for collagen. The lnteractlon of elther free collagen or llposomally bound collagen wlth the A431 cell llne ls expected to result from assoclatlon of collagen ~tth components wlthln the extracellular matrlx. Referrlng to Flgure 2 Incubatlon perlods up to 4 hours were completed ~lth 3 hours belng the opttmal perlod for blndlng and collagen-llposome concentratlons.
Quantltatlve evaluatlons of blndlng of collagen-modlfled ; llposomes to A431 ce11s ln culture are compared to regular llposome and exempllf1ed ln Flgure 3. The data were processed accordlng to equatlon (1) above. Through double label~ng 3-H-collagen and 14-C-cholesterol ~t was posslble to monltor the collagen and 11posome slmultaneously. The blndlng of the collagen-modlfled llposomes to the cells ls greater than the blndlng of the correspondlng regular llposomes.
For frée and collagen-modlfled l~posomes the blndlng entltles are of the extracellular matrlx type of cell-assoc~ated entlty. As ln the case of EGF-modlfled llposomes dlscussed in example 2 the dlssoclatlon constant for collagen-modlfled llposomes is expected to be slmllar to or hlgher than those of free collagen. Llkewlse the number of ' ' .
~,:
. .
.' ~ .
' - :
:` ~ 2~7~4~0 avallable sltes ln the extrac411ular matrlx avallable for collagen-~odlfled llposomes ls expected to be slmllar to or lower than free collagen. The example glven ln Table 2 flts wlth these conslderatlons. The data for free collagen demonstrate that blndlng of thls bloadheslve recognlz1ng substance to thls cellular system does occur and ls a rea5urable phenomena, whlch can be processed to yleld quantltatlve and meanlngful parameters. Moreover, the data ln Table 2 show qulte clearly that the blndSng of eollagen-modlfled llposomes to thls cellular system ls of sufflclently hlgh afflnlty and wlth a large enough number of sltes, ~or the collagen-modlfled llposomes to perform as the deslred bloadheslve llposomes.
~ABLE 2 BINDING PARAMETERS OF FREE RECOGNIZING SUBSTANCES AND
BIOADHESIVE LIPOSOME TO A431 CE~LS IN CULTURE
BIOADHESIVE K NUMBER 0~ SITE5 LIPOSOME SYS~EM _(u~) lxlO-FREE COLLAGEN 8.5 t 2.3 179 t 11 COLLAGEN-MLV 67.6 , 31.35 548 ~ 160 Whlle the preferred embodlments have been descrlbed, varlous modlflcatlons and substltutlons may be made wlthout departlng from the scope of the lnventlon. For example, the mouse EGF
and human urogasterone used ln the dlsclosed examples could be ; substltuted wlth EGF from other natural or synthetlc sources.
Slmllarly, the collagen, gelatln and HA could come from other natural or synthetlc sources. Accordlngly, lt ls to be understood that the lnventlon has been descrlbed by way of ~llustratlon and not ltmltatton.
-', ' '`, .
. ' ,
INTE~ACTION BET~E-~N BIOA~HESIVE LIPO~OMES AND TA~ET SIIi~.S
- BACKGROUND OF ~H~ INV~NTION
The present lnventlon relates to a novel drug dellvery system, partlcularly to mlcroscoplc drug dellvery systems ~MDDS) utlllzlng drug-encapsulatlng lbloadheslve llposomes for toplcal and local drug adm1nlstratlon.
Currently, the toplcal and local admlnlstratlon o~ a drug can be 1n lts free form, dlssolved or dtspersed ln a sultable dlluent, or tn a vehltle such as a cream~ gel or olntment.
Examples of therapeutlc or deslgnated targets for toplcal or local drug admln1stratlon lnclude burns; wounds; bone ln~urles;
ocular, skln, lntranasal and buccal lnfectlons; ocular chronlc sltuatlons such as glaucoma; and toplcally and locally accessed tumors. Several dlfflculttes exlst wlth elther the toplcal or local admlnlstratlon of a drug ln lts free form. For example, short retentton of the drug at the destgnated s~te of admlnlstratlon reduces the effltacy of the treatment and : 2D requlres frequent doslng. Exposure of the free form drug to :~ the blologlcal env1ronment tn the toplcal or local reglon can result ln drug degradatlon, transformat~on lnto lnactlve entltles and nondlscr~mlnatlng and uncontrollable dlstrtbutlon of the drug. Such degradatlon and uncontrollable dlstrlbutlon of.the drug can result ln toxlclty ~ssues, undeslrable slde effects and loss of efflcacy.
Mlcroscoplc drug dellvery systems ~MDDS) have been developed to overcome some of the d~fflcultles assoclated w~th ~ free drug admlnlstratton. MDDS ls divlded lnto two baslc -~ 30 classes: partlculate systems, such as cells, mlcrospheres, viral envelopes and llposomes; or nonpartlculate systems whlch are macromolecules such as protetns or synthetlc polymers.
Uslng these spectflc systems. drug-loaded MDDS can perform as , . .
.~ .
WO 92/14446 ` PCI/US91/081t2 2~79~
sustalned or controlled release drug depots. By provldlng a mutual protectlon of the drug and the blologlcal envlronment MDDS reduces drug degradatlon or Inactlvatlon As a system for controlled release of the drug. MDDS improves drug efflcacy and allo~s reductlon ln the frequencv of doslng. Slnce the pharmacoklnetlcs of free drug release from depots of MDDS are dlfferent than from dlrectly-admlntstered drug MDDS provldes an addltlonal measure to reduce toxlclty and undeslrable slde . effects.
Llposomes offer a range of advantages relatlve to other MDDS systems. Llposomes are lipld veslcles composed of membrane-llke llpld layers surroundlng aqueous compartments.
Composed of naturally-occurrtng materlals whlch are : blocompatlble and blodegradable~ liposomes are used to lS encapsulate blologlcally act~ve materlals for a varlety of purposes. Havlng a varlety of layers slzes surface charges and composltlons numerous procedures for llposomal preparatlon and for drug encapsulatlon wlthln them have been developed some of whlch have been scaled up to lndustrlal levels.
Through approprlate selectlon of liposome type and slze the encapsulated drug can also range in slze. Llposomes can accommodate llpld-soluble drugs aqueous soluble drugs and drugs wlth both hydrophlllc and hydrophoblc resldues.
Llposomes can be deslgned to act as sustalned release drug depots and ln certaln appllcatlons. ald drug access across cell membranes. Thelr ablllty to protect encapsulated drugs and other characterlstlcs ma~e llpo50me5 a popular cholce ln ; developlng MDDS wlth respect to the prevlous practlces of free ; drug admtn~stratlon.
Desplte the advantages offered. utlllzatlon of drug-encapsulatlng llposomes does pose some dlfflculties. For e~ample llposomes as MDDS have llmltDd targetlng abll~tles llmlted retentlon and stablllty In clrculatlon potentlal .',' . ' ' .
.~
.
.
WO 92/14~46 PCI/U~91/08112 . .
. . ~ .
2 ~7 ~ ~5 0 toxlclty upon chronlc admlnlstratlon and lnablllty to extravasate. In recent years attempts have been made to couple dl~ferent recognizlng substances with llposomes to confer target speclflclty to the llposomes namely antlbodies glycoprotelns and lectlns. Although the bondlng of these recognlzlng substances to ltposomes occurred the resultlng modlfled llposomes dld not perform as hoped partlcularly durlng 1n vlvo studies. Other difficultles are presented when utlllzlng these recognizing substances. For example antlbodies can be patlent speclfic and therefore add cost to the drug therapy.
Several cell-assoclated entltles can partlclpate ln the blndlng between cells and recognlzlng substances. These are generally dlvlded lnto three ma~or types: receptors and lS non-receptor components of the cellular system and extracellular matrlx. Receptors can be present ln several specles or states dlfferlng ln populatlons per cell and tn blndlng afflnlty. Blndlng to such receptor entltles ls usually referred to as speclf~c blndlng . Non-receptor cell membrane components also dlffer ln populatlons and in a~flnlty. Blndlng to such non-receptor entltles ls usually referred to as non-speclflc blndlng .
To perform effectlvely the toplcal or local admlnlstratlon of drug-encapsulatlng liposomes shouid have : 25 speclflc1ty for and the ablllty to adhere to the deslgnated target area and should facilltate drug access to lntracellular sltes. Currently avallable llposomes and other MD~S systems do not meet these performance requlrements of toplcal and local drug admlnlstratlon.
RY OF INVENTIOH
It has been learned that mod~fylng regular llposomes by covalently anchorlng certain recognlzlng substances to the '. '~
WO 92/14446 PCr/US91/08112 20~9~ 4 llposomal surface creates a bloadheslve llposome wlth target speclflclty and retentlon. The recognlzlng substances are molecules whlch can be utlllzed as an adhes1ve or glue attachlng a drug-encapsulatlng llposome onto a therapeutlc target slte. These bloadheslve recognlztng substances can - perform elther through receptor mechan1sms or through assoclatlons ~lth components wlthln the extracellular matrlx.
Regardless of the speclflc mechanlsm of adheslon these substances are referred to as bloadhes1ve recognlztng substances based on thelr common end result.
Through covalent anchorlng the bloadheslve recognlzlng substances become an lntegral part of the llposome yet remaln accesslble to the lnteractlon counterpart at the target slte.
They endow the llposome and encapsulated drug wlth the ablllty to adhere to the target slte. Hence bloadhes1ve llposomes .~ have been deve~oped whlch are target adherent sustalned release drug depots. The ldentlflcatlon of recognlzlng substances and the methodologles of modlfylng llposomes has been dlsclosed ln concurrently flled appllcatlons. These bloadheslve llposomes offer several advantages over prevlous pract~ces of toplcally or locally admlnlstered free drug and other MDDS whether wlth regular llposomes or other MDDS
systems. These advantages lnclude the mutual protectlon of both the drug and blologlcal envlronment; an lncrease ln drug bloavallablllty and retentlon at the target slte; and lmproved adherence or adheslon to the deslgnated target slte. These advantages result ln the potentlal reductlon of undeslrable blologlcal slde-effects of the drug belng admlnlstered.
BRIEF ~ESCRIPTI0N OF THE ~RAHING~
FIG. l shows the blndlng of bloadheslve llposomes (EGF-modlfled; open double trlangle) and regular llposomes (asterlsk) of the LUVET type to A431 ce)ls ln culture (in ~' , ' . .
., .
WO 92/14446 P~/US91/08112 , . . .
` :~
2~79~5~
monolayers), as dependent upon llposome concentratlon. Bound llposomes, denoted as B, are ln units of ng EGF per 106 cells.
Free llgand concentratlon, denoted as L, are ln unlts of ng EGF
per lo6 cells for bloadheslve llposome ~flrst row of L values) and ~n unlts of umoles llpld per 106 cells for the regular . llposomes (second row of L values).
`~ FIG. 2 shows a tlme course of the blndlng of bloadheslve llposomes (collagen-modlfled) of the MLV type to A431 cells ln culture ~ln monolayers). Collagen ls trltlum-labeled. The fractlon of llposomes relatlve to the amount present ln the lnltlal react10n mlxture at zero-tlme whlch ls cell-assoclated ls determlned over tlme.
FIG. 3 shows the blndlng of bloadheslve llposomes (collagen-modlfled) and regular llposomes of the MLV type to A431 cells ln culture (ln monolayers). Collagen ls ,j trltlum-labeled ~3 H) and llposomes are 14-C labeled. 80und llposomes, denoted as B, are ln unlts of 3-H DPM per 105cells (left scale) and ln unlts of '4-C ~PM per lOS cells (rlght scale). Free llgand concentratlon, denoted as L, are ln unlts of 3-H or 14-C DPM per 105 cells. Bloadheslve llposome wlth collagen labeled ls deplcted ~lth open double trlangles;
bloadheslve llposome w1th the llposome labeled ls deplcted wlth crosses; and, regular llposome ls deplcted wlth asterlsks.
~ U~_~LS~
~ Accordlng to the present lnventlon, bloadheslve llposomes .~ have bound to cell cultures havlng receptors or extracellular : matrlx whlch accommodate the recognlzlng substance bonded to : the llposome. Llposomes, ln partlcular, multllamellar veslcles - 30 tMLV), mlcroemulslfled llposomes (M~L) or large unllamellarveslcles (LUVET), each contatnlng phosphatldylethanolamlne - (PE), have been prepared by establlshed procedures.
Recognlzlng substances, each of whlch have been accepted for . . .
. :
.
'~ . ,, ' ' ' WO 92/14446 PCl'/US91/08112 , 9D~jO 6 human use lnclude epldermal ~rowth factor (EGF) hyaluronlc acld (HA) gelatln and collagen. Each of these recognlzlng substances have a blologlcal origin and are blodegradable and blocompatlble. Further these recognlzlng substances have functlonal resldues whlch can be utillzed ln covalent anchorlng to the regular ltposomal surfaces.
The methodologles of preparlng the spec~flc bloadheslve llposomes have been dlsclosed ln separate appllcatlons concurrently flled ~lth thls dlsclosure and wlll not be ~ 19 repeated here.
A complete accountlng of blndlng entltles has been determlned by the prevlously known multl-term Langmulr Isotherm equatlon as applled for the quantltatlve descrlptlon of the relatlonshlp between the free and dependent varlables:
n Bmax~ ~L~
1~1 Kd~ + [L~
where n is the number of dlfferent cell-assoc~ated blnding entltles that a cellular system has for a speclfic recognlzlng substance; tL] ls the concentratlon of free llgand whlch can be recognlzlng substance free llposomes or bioadheslve l~posomes; B ls the total quantlty of bound recognlzlng substance per glven number of cells~ at a given [L~; and Bmaxl and Kd~ are the total number of sltes of a glven entlty and the correspondlng equlllbrlum dlssoclatlon constant. B and ~max are normallzed for the same number of cells.
For cases ln whlch receptors and non-receptor cell membrane components partlclpate in the recognlzing substance blndlng and ln whlch the d~ssoclation constant of the non-speclflc blndlng is sufflciently large wlth respect to the free ligand concentration equation 1 can take the form:
'.
.
.
, WO 92/14446 P~T/US91/08tl2 --` 2~7~50 n-l Bmax~ ~L]
B= --------- ~ Kns "~ (2) 1~1 Kd~ ~ ~Ll ~ 5 where the last term, Kns [L), ls the contributlon of the .~ non-speclflc blndlng to B and r~nS ~s the ratlo of Bmax to Kd correspondlng to the non-speclflc blndlng.
Best-flt values for parameters n, Bmax~ and Kdl are obtalned by computer-alded data analysls, aecordlng to equatlons ~1) and/or (2) above, applylng nonllnear regresslon procedures.
The lnteractlon of the bloadheslve EGF-modlfled llposomes has been éstabllshed wlth cultures of A431 cells, ln monolayers, as a blologlcal model. Thls well-establlshed cell llne, orlglnatlng from human epldermold carclnoma, ls enrlched wlth EGF receptors. A431 cells have been repeatedly used for study of the lnteractlon of free EGF and lts receptor.
A431 cells have been shown to have three classes of EGF
receptors, dlfferlng ln thelr afflnltles and populatlons. The flrst of these classes ls the ultra-hlgh afflnlty sltes wlth an equll1brlum dlssoclatlon constant of 0.07 nM and a populatlon of 150-4000 sltes per cell. The next class ls the hlgh afflnlty sltes wlth an equlllbrlum dlssoclatlon constant of 0.7 nM and a populatlon of 1.5 x 105 sltes per cell. The flnal class ls the low afflnlty sltes ~lth an equlllbrlum dlssoclatlon constant of 5.9 nM and a populatlon of 2 x 106 sltes per cell.
Examule One To compare the blndlng a~llit~ of regular llposomes and bloadheslve llposomes, A431 cell cultures were grown ln monolayers, ln flasks, applvin~ u;lJ~l procedures for thls cell ., .
. , .. ;~ .......... ~ .
, , . . .
. . ,-, ..... . ..
.... . .
: ' '` ' . ' ' . '' ,, WO 92/14446 PC~/US91/0811~
2 ~ 5 - 8 -: llne. Two to three days prlor to an experlment the cells were seeded lnto multl~ell culture plates and the experlments were done when the systems were confluent.
For purposes of assaylng the modlfled llposomes the EGF-recognlzlng substance was labeled wlth a generally known radloactlve marker. Preparatlon of EGF-modlfled LUVET was completed as dlsclosed ln the concurrently flled appllcatlons.
Prlor to the addltlon of a reactlon mixture of EGF-~odlfled .11posomes free llposomes or free EGF medla was lo removed from the A431 cells and the cells were washed wlth a blndlng buffer. The reactlon mlxture and cells were lncubated for 1-2 hours at room temperature. Upon dllutlon and wlthdrawal of the reactlon mlxture at the end of lncubatlon 2-3 successlve washlngs wlth a blndlng buffer of the wells were completed. Lysls of cells or detachment of cells from the weils was then followed by wlthdrawal and collectlon of the ~ell content denoted as the cell fractlon. Assays of thQ cell fractlon were completed by label countlng of the fractlon as compared wlth the countlng of the Immedlate products created through the preparatlon process.
A comparlson bet~een the blndlng of free llposomes and EGF-modlfled llposc~s to the A431 cells ls lllustrated ln Flgure 1. The EGF-modlfled l~posomes adhere to the A431 cells conslderably better than free llposomes as no free llposomes we.re found at cell fractlon. It ls speculated that lf free llposomes do assoclate wlth the cells the dllutlon brought by the ~ashlngs ls sufflclent to cause quantltatlve dlssoclatlon.
ExamDle Two Blndlng studles of EGF-modlfled llposomes to A431 cells were carrled out as descrlbed In example 1 and the data were processed accordlng to equatton ~l) above. The experl~ental condltlons were such that the ontributlon of non-speclflc ' WO 92/14446 PCr/US91/0811.2 ; 207~0 g `~' ~ blndlng was negllglble. Indeed the data were found to flt - unamblguously w~th a slngle type of blndlng slte for each llposome system studled. Results for several systems are `~ llsted ln Table 1.
BINDING PARAMETERS OF BIOADHESIVE LIPOSGMES
~ TO A431 CELLS IN CULTURE
~ 10 BIOADHESIVE K~ SITES PE~ CELL
LIPOSO~E SYSTEM (a) ~n~ 10- ) - EGF-MLV 0.60 + 0.017 0.17 + 0.03 EGF-MLV 5.03 + 1.9 1.07 + 0.03 EGF-LUVET 2.91 + 0.003 O.lB 0.001 EGF-MEL 0 04 + 0.007 0.042 ~ 0.0042 EGF-MEL 0.40 + 0.13 3.7 + 0.90 EGF-MEL 0.48 + 0.05 0.28 + 0.01 ~, .
- (a) Each bloadheslve llposome system ls a dlfferent preparatlon; recognlzlng substance ln each system ls EGF.
An EGF-modlfled llposome ls conslderably larger than and dlfferent from free EGF whlch ls expected to affect the blndlng parameters. For a glven class of receptors the magnltudes of the dlssoclatlon constants for EGF-modlfled llposome systems are expected to be slmllar to or hlgher than those of free EGF. For a glven class of receptors the number of receptors per cell that are avallable for the EGF-modifled llpo50me5 ls expected to be equal to or lower than the number :- of avallable for free EGF. Based on these conslderatlons the .
, , '.
:.
'~"
. ", .
.~''',' i' ~ ~ ' .
.~.................................. , . . .
.
WO 92/14446 PCr/US91/08112 h~9~3 blnding data of the present example flt wlth the receptor classes of ultra-hlgh and hlgh af~lnltles.
Regardless of the specl~lc cell-assoclated blndlng entlty lnvolYed the blndlng data llsted ln Table 1 sho~ that 5 EGF-modlfled ltposomes blnd to thls cellular system wlth hlgh aff~nlty and w1th a sufflc1ent number of sltes for these modlfled llposomes to perform as the deslred bloadheslve llposomes. -, lo Exam~le Three Blndlng collagen-modlfled llposomes to A431 cells ~as carrled out essentlally accordlng to the procedures detalled above. The A431 cell llne ls not ~nown to contaln receptors for collagen. The lnteractlon of elther free collagen or llposomally bound collagen wlth the A431 cell llne ls expected to result from assoclatlon of collagen ~tth components wlthln the extracellular matrlx. Referrlng to Flgure 2 Incubatlon perlods up to 4 hours were completed ~lth 3 hours belng the opttmal perlod for blndlng and collagen-llposome concentratlons.
Quantltatlve evaluatlons of blndlng of collagen-modlfled ; llposomes to A431 ce11s ln culture are compared to regular llposome and exempllf1ed ln Flgure 3. The data were processed accordlng to equatlon (1) above. Through double label~ng 3-H-collagen and 14-C-cholesterol ~t was posslble to monltor the collagen and 11posome slmultaneously. The blndlng of the collagen-modlfled llposomes to the cells ls greater than the blndlng of the correspondlng regular llposomes.
For frée and collagen-modlfled l~posomes the blndlng entltles are of the extracellular matrlx type of cell-assoc~ated entlty. As ln the case of EGF-modlfled llposomes dlscussed in example 2 the dlssoclatlon constant for collagen-modlfled llposomes is expected to be slmllar to or hlgher than those of free collagen. Llkewlse the number of ' ' .
~,:
. .
.' ~ .
' - :
:` ~ 2~7~4~0 avallable sltes ln the extrac411ular matrlx avallable for collagen-~odlfled llposomes ls expected to be slmllar to or lower than free collagen. The example glven ln Table 2 flts wlth these conslderatlons. The data for free collagen demonstrate that blndlng of thls bloadheslve recognlz1ng substance to thls cellular system does occur and ls a rea5urable phenomena, whlch can be processed to yleld quantltatlve and meanlngful parameters. Moreover, the data ln Table 2 show qulte clearly that the blndSng of eollagen-modlfled llposomes to thls cellular system ls of sufflclently hlgh afflnlty and wlth a large enough number of sltes, ~or the collagen-modlfled llposomes to perform as the deslred bloadheslve llposomes.
~ABLE 2 BINDING PARAMETERS OF FREE RECOGNIZING SUBSTANCES AND
BIOADHESIVE LIPOSOME TO A431 CE~LS IN CULTURE
BIOADHESIVE K NUMBER 0~ SITE5 LIPOSOME SYS~EM _(u~) lxlO-FREE COLLAGEN 8.5 t 2.3 179 t 11 COLLAGEN-MLV 67.6 , 31.35 548 ~ 160 Whlle the preferred embodlments have been descrlbed, varlous modlflcatlons and substltutlons may be made wlthout departlng from the scope of the lnventlon. For example, the mouse EGF
and human urogasterone used ln the dlsclosed examples could be ; substltuted wlth EGF from other natural or synthetlc sources.
Slmllarly, the collagen, gelatln and HA could come from other natural or synthetlc sources. Accordlngly, lt ls to be understood that the lnventlon has been descrlbed by way of ~llustratlon and not ltmltatton.
-', ' '`, .
. ' ,
Claims (24)
1. A method for topical substance administration for a designated cellular target site comprises the topical application of a encapsulating liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.
2. The method of administration of claim 1 wherein the liposome component is selected from the group consisting of multilamellar vesicles microemulsified liposomes and large unilamellar vesicles.
3. The method of administration of claim 1 wherein the liposome component includes phosphatidylethanolamine.
4. The method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor mechanisms at the cellular target site.
5. The method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through associations with components within an extracellular matrix at the cellular target site.
6. The method of administration of claim 1 wherein the recognizing substance component is selected from the group consisting of gelatin collagen hyaluronic acid and epidermal growth factor.
7. The method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked.
8. The method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked by a crosslinking reagent.
9. A method of local substance administration for a designated cellular target site comprises the local application of a liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.
10. The method of administration of claim 9 wherein the liposome component is selected from the group consisting of multilamellar vesicles, microemulsified liposomes and large unilamellar vesicles.
11. The method of administration of claim 9 wherein the liposome component includes phosphatidylethanolamine.
12. The method of administration of claim 9 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor mechanisms at the cellular target site.
13. The method of administration of claim 9 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through associations with components within an extracellular matrix at the cellular target site.
14. The method of administration of claim 9 wherein the recognizing substance component is selected from the group consisting of gelatin collagen hyaluronic acid and epidermal growth factor.
15. The method of administration of claim 9 where the liposome component and the recognizing substance component are covalently linked by a crosslinking reagent.
16. The method of administration of claim 9 where the liposome component and the recognizing substance component are covalently linked by a crosslinking reagent.
17. A liposome for substance administration for a designated cellular target site comprising a liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.
18. A liposome of claim 17 wherein the method of administration of claim 1 wherein the liposome component is selected from the group consisting of multilamellar vesicles microemulsified liposomes and large unilamellar vesicles.
19. A liposome of claim 17 wherein the method of administration of claim 1 wherein the liposome component includes phosphatidylethanolamine.
20. A liposome of claim 17 wherein the method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor mechanisms at the cellular target site.
21. A liposome of claim 17 wherein the method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through associations with components within an extracellular matrix at the cellular target site.
22. A liposome of claim 17 wherein the method of administration of claim 1 wherein the recognizing substance component is selected from the group consisting of gelatin collagen, hyaluronic acid and epidermal growth factor.
23. A liposome of claim 17 wherein the method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked.
24. A liposome of claim 17 wherein the method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked by a crosslinking reagent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US65501391A | 1991-02-14 | 1991-02-14 | |
| US7/655,013 | 1991-02-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2079450A1 true CA2079450A1 (en) | 1992-08-15 |
Family
ID=24627144
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002079450A Abandoned CA2079450A1 (en) | 1991-02-14 | 1991-10-30 | Interaction between bioadhesive liposomes and target sites |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0525154A1 (en) |
| JP (1) | JPH05506040A (en) |
| AU (1) | AU656173B2 (en) |
| CA (1) | CA2079450A1 (en) |
| WO (1) | WO1992014446A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9208339D0 (en) * | 1992-04-15 | 1992-06-03 | Unilever Plc | Treatment composition |
| JPH08507070A (en) * | 1993-02-22 | 1996-07-30 | アルザ・コーポレーション | Compositions for oral administration of active substances |
| US6096863A (en) * | 1996-08-23 | 2000-08-01 | Regents Of The University Of Minnesota | Self-assembling amphiphiles for construction of peptide secondary structures |
| CA2266622C (en) | 1996-09-27 | 2006-08-15 | Hyal Pharmaceutical Corporation | Hyaluronic drug delivery system |
| US20030180348A1 (en) * | 2002-03-22 | 2003-09-25 | Levinson R. Saul | Transcellular drug delivery system |
| JP5695308B2 (en) * | 2009-10-02 | 2015-04-01 | 株式会社フェース | Cosmetic base comprising collagen-modified liposome and skin cosmetic containing the same |
| JPWO2011083845A1 (en) * | 2010-01-08 | 2013-05-16 | 富士フイルム株式会社 | Targeting agent for tumor site |
| JP5756602B2 (en) * | 2010-04-16 | 2015-07-29 | 株式会社フェース | Cosmetic base comprising liposome modified with gelatin and / or elastin-constituting polypeptide and skin cosmetic containing the same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5000960A (en) * | 1987-03-13 | 1991-03-19 | Micro-Pak, Inc. | Protein coupling to lipid vesicles |
| GB8713747D0 (en) * | 1987-06-12 | 1987-07-15 | Unilever Plc | Skin treatment composition |
| US5064655A (en) * | 1989-02-24 | 1991-11-12 | Liposome Technology, Inc. | Liposome gel composition and method |
| AU652778B2 (en) * | 1990-10-15 | 1994-09-08 | Quest International B.V. | Treatment composition |
| DE69113036T2 (en) * | 1991-02-14 | 1996-05-02 | Baxter Int | DELAYED DRUG DELIVERY THROUGH TOPICAL APPLICATION OF BIOADHESIVE LIPOSOMES. |
-
1991
- 1991-10-30 AU AU12592/92A patent/AU656173B2/en not_active Ceased
- 1991-10-30 EP EP19920904871 patent/EP0525154A1/en not_active Withdrawn
- 1991-10-30 CA CA002079450A patent/CA2079450A1/en not_active Abandoned
- 1991-10-30 WO PCT/US1991/008112 patent/WO1992014446A1/en not_active Application Discontinuation
- 1991-10-30 JP JP92505165A patent/JPH05506040A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU1259292A (en) | 1992-09-15 |
| AU656173B2 (en) | 1995-01-27 |
| EP0525154A1 (en) | 1993-02-03 |
| JPH05506040A (en) | 1993-09-02 |
| WO1992014446A1 (en) | 1992-09-03 |
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