Note: Descriptions are shown in the official language in which they were submitted.
7 J
-- 1 --
PR~CE:8~ FOR T~IE P~ClDUCq! I:~N O~ A~VEOtJ~ O~ 3~3PEN~3IO~
CO~l!Al:lilIN~ AC~ N~ )IENq!B
.
~ y Q~ ~h~ l~v~io~
The invention relate~ to ~ process for the production
o~ aqueous liposome suspensions containing active
ingredient~ from a ~olution o~ a substancQ or. a mixture of
substance ~also desi~nat~d b~low ~ lipid or lipid
mix~ure), which ~onm liposomes in a lower al~ohol with at
most 3 aarhon atom~ and an aqueou~ phase, the acti~e
in~rediant or the active ingredi~nt mlxture is d~'s~lved or
~u6pe~ded i~ ~he lipid ~olution andJor the a~ue~us pha~,
characteri~ed in tha~ ~he a~ueous pha~ is added to the
al~oholic solution with homogenizati~n, and the l~wer
alcohol is removed by vacuum distillation.
As is ~nown, liposomes are sel~-contained spherical or
e~liptical lipid ve~iale~, whi¢h enclose an ~qu~ou9 phase.
Depe~ding on the numb~r of lipid double layeræ pre~ent,
large and small unilamellar liposome~ (LW and S W) are
distin~ui~he~ from multil~mell~r l~pos~ tM~V).
The fir~t lipo30mes wer~ pro~uced by ~ispersion of
lipid films i~ agueous ~hases. The MLV di~per~ions ~hu~
obtained aan b~ aon~erted to S W ~y u~ing ~uitabla
homogeniza~ion proce~s~s, ~uch a~, e.g., ul~rasonic
irr~diation or high-pres~ure homogeniz~tlon. But M~V and
S W exhibit only a small inaluslon volume (volum~ o~
F~ lr,~ .t, ;~ EI ~ r~ 122 P~
7 rJ
2 --
enclosec~ aqueou~ pha~3e [ lit~,r/mol lipid] ), 30 that ~hey are
not suitabl@ ~or ef~ective inclu~ion of hydrophilia
sub~:tances ~
In contr~6t, LW exhibit a clearly in~rea;ed inclu~ion
volume and thu~ an .tncreased ~n~lusion capacity ~meaf~:ured on
the ~asi~ o~ the ~6 of th~ total active ingredien~ that can
possibly be aontain~d in the inclu8ion vslume) . LW ' s hav~ a
partlale ~l~e of 100-10, 000 nm, preferably 200-2, 000 nm.
Tha most common method~;, up to this day, ~or production
of LW C:~n be ~ummarize~ under the generic tex~n o~ olvent
e~rapora~ion" methods .
The method prol~ably ~c~st known and beæt wit~ re~;pec:t to
the ~ nqlu6ions obtair~ed i~ the RE~ (rever~;e pha~e
evaporatiorl) method o~ Papahadjopoulo~ ~U.S. paten~ no.
4,23~,871). In this ~::a~3~3i th~ lipid or lipid mixtur~ i~
fir~t di~;~olved in a ~olv~nt whi~h is not, ~r only 31ightly,
w~ter-miscible (g~ner~lly, diethyl ether or chlorofor~
After additic~n to the aqueous phase containing
pharmaceutical ~ub~tanc~e~, the mixture is then cor.verted to
a W/0 emulsion ~water-~n oil emul~iun) by ultrasonic:
irradiaticn. The solvent i~ then remov4d ~rom th~ latter
u~ing a rotary e~apora~or, and a ~1 is fir~t ~o~ d which
is converted to a lipo~ome suspenRion by increa~ing ~h~
vacuum or add ing water ~
In contra~t to thi~, in the 'lether in~ection" method
introduced by D~am6!r ancl Bangham [13iochf3m. ~3iophy5. Acta,
443 ~1976) 62~-~34], the lipid, also di~olved in diethyl
~ther, is injecte~l in~o a warm aqueouR solutian (50-60C)
under partial vac:uum. ~he lipo~omes oh~ain~d a ter
filtration through ~ 1. 2 micron filter ar~ heterogen~ous~ and
exhibit ~ize i; ~etween 150 250 n~a~
~he solver~t~ u~ad ~n the:e ~Aethods are to b~ viewed
critically Por tox~ aologlcal and ~af~ty xea~3on~c .
/ u ~ ~3
-- 3
But, if ethanol is used a~ a solvent in ~ proce~
preoedlng ~he "ether inj~3ction" method/ greatly diluted SW
d.ispersion~ result which ~re concentrate:d by ultr~filtration
~Bioahem~ Biophy6~ Act~, 298 (1973) 1015-1019~. The ~vera~e
$ ~l~e of th~3 liposom~3 thu~2 oktain~d is ~learly under 50 nm
and the MLV portion i9 lndicatecl a~ 6%.
An e~odiment of thi~ proc~ i descri~ed in
~5P-A 0 253 ~l9. In ~hiY proce~ or exampl¢, an ethanolic
lipid and ph~rma~eu~iaal ag~n~ solul;$on, which constitute~ a
tot~l of 10% o~ the to~al formulation, ~re inject~a under
pre~ura o~ 1,000 to 3,000,~00 h Pa (heato Pas~als) in an
agueou~ pha~e, wh~.ch is agitated by a homogeniz~x.
According to this proa~s~, ~7hi~h $~ very ~xp~n~ive, sm~l~
unilamell~r llposomes r~ult. ~hi3 proce ~ i~ unsuitabl~
fo~ production o~ liposome ~uspensions whi~h cc~nt~in
hydrophili~ active ingredien~ .
Also workh m~ntioning i8 a method for the p~oduction of
liposome~ with water-~olu~le ~olv~nt~;, whi ::h include~ the
production of a "monophase" ~Wo 85/00751). Thi~ ona-pha~o
2~ mixture ic gen6~rally produa~d ~xom 5 ml o~ th~3 lipid-
conta ~ nlng solv~nt ( ~ . ~ ., ethanol ) and 0 . ~ ml o~ the ~ ous
component. ~h~ ~olvent i~; then removed by introducing an
inert ~a~ duxin~ ~he ~imultaneous ultrasoni ;: irradiation o~
the mixture, and a ~ilm r~sults. ThQ latter i~ then
~5 re~;u~pended with an a4ueous ph~ e. In thi~ ca~e, so-calle~l
~: MPV (monophasic ve~i41e~) ~esult, which exhibit ~ever~l
lipld doubl~ layers. R~la~ive to the ~t~ndard MPV, the
MPVs produced by the ~bov~-d6~scribed process exhi~it
incr~a~ed tabil~y in buffers a~ well as incr~3a~ed
3 Q incluxions .
Final}y, the proaeo~ de~cribed in EP-A O 349 429, in
whlah an ~thanolic lipid solution optionally ~on~ln~l~g the
active ingredient i~ in~roduc~d into an ~queou~ pha~e w~ th
7^~
- 4 -
light ~t~rring, is al~o to be mentioned. A~ our tesk~ ~how,
conslderably l~w~r inclu~ion c~p~cit~ are a~hle~ed ln this
prev~ously known proce~s than by the proce3~ aacor~ing to
the invent~on. Moreover, the process acaording to the
invQntion has ths advantage th~t with it~ help, even with
lipid co~centration o~ over 10% rel~ive to the e~hanoli~
phas~, high inclu ion capacitieæ or even a ~urther increase
o~ ~he inclusion capacities ~an b~ achi~v~d.
In comparison wlth these previously known methods, the
proces~ ~ccord~ng ~o the invention has ~he advant~g~ ~hat it
i~ technically feaci~le on a large s~a~e in a simple wayA
Th~ u~ of strongly toxic or very ea~ily ~lammabls -~olvents
i~ avoided. ~The ~ery ~ood reproducibility o~ tha pro~s~
according to the inven~ion i~ also ~o ~e empha~ized relative
to the obtained a¢ti~e ingredient in~lu~ions ~nd li~osome
-~izes, as well as the very hi~h active ingredient inclusion
in the liposomes, up to over 50~, and the unu~ually high
~ctive in~redient ooncen~ra~ions, obtained ~y ~he proce~s
accord~nq to ~he ~nven~ion, in the liposome 6usp~n~ion~ ~up
to 800 mg/ml). Active ingredient inclusion means the
percentage of the total active ingredient ~mployed which is
contained in the inclusion ~olume of the lipids. ~ur~her,
it is worth m~ntioning that it i~ possible to produae
liposQme su~pensions, which have only a very l~w r~idual
solvent cont~nt and which hav~ a good shelf li~e, with the
help of the proc2s~ aocording to the invention. The proces~
ac~ording to the invention can al~o ~e per~ormed ~robl~m-
fre~ under a~ptic conditions.
To perfor~ the proce~ according t~ th~ invention, ~he
same substances for forming liposom~s can be used ~s in the
previou~ly lcnown proce~-~es~
Sub~tanc~s ~orming ~uitable lipos4mes are u~ually
amphiphatic lipid~ oX lipid mlx~ure~, ~uch a~, ~or ~xample,
2 ~ 7 ~
pho~pholipid~, sphin~omyel~nY or eth~r pho~phollpids. ~hese
lipids aan h~ve straight-oh~ln or branched, ~aturat~d or
un~aturated, similar or dif~erent acyl ~id~ chains.
Sult~ble phospholipld~ are, ~or example, the
pho~phatldylchol~ne~, the phosphatidylethanolamine~, the
phosphatidylserines, the pho~phatidylinosikol~, the
pho~phat~dylglycerols or ~he pho~phatidia ~cids~ Sultable
ether pho~pholipid3 are, for example, the plasmalogens
(Dr. Otto~Albert Neumuell~r: Roempp~ C~2mi~-Lexikon;
Franak~che Verlagshandlung, Stu~tgart ~D~) 2665, 159,
3g20 and 4045).
For the production o~ liposom~s, lipids or mixtures
thereo~ and in particular al~o mixtures of the~e ~ipid~ with
cholesterol, chole~terol hemi~uooina~e, alpha-tvcoph~rol,
alpha-tocopherol h~misuccinate and/or charg~ aarriers, such
a~, for example, stearyl amine, ~teario ~aid, diethyl
phosphate, oleic acid~ palmitic acid, bile acid, Cuch a~,
for QXample, choli~ a~ld, glycocholi~ a~id, ~an ~e us~d.
Suitabl~ mixture~ can contain about up to ~0 mol~ parc~nt o~
cholester~l an~ up ~o ~0 mole peroent ~f charge c~rri~r. As
solvent ~or the phospholipid3 or miX~ureR~ pre~erably
isopropanol or in particular ethanol is ussd.
In principl~ the proa~ss aacording to the invention
should al~o be feasible with water-~oluble low-boiling
solvents o~her than lower a~cohols: for exampl~
t~trahydrofuran is suitable. But, performance of thi~
prooes~ embodiment i5 ~onsiderably ~ore expensiv~.
T~ perform the proce~ ~ccording to the invention,
alcoholic ~olutt~n~ are pr~Qrably u~d which contain 0.
to 30 g of lipo~ome-forming ub~tance or su~ance mix~ure
per lOO ml o~ ~olvent~ I~ nece6~ary~ the~e ~olutions are
produ~ed by he~ti~g the components.
HPF~ i r~ .. lr~ I r~ _E! ,i~ l rEI_ 1 0-l:, i~ P0-~
~ ~ ,1, s
-- 6 --
Th~ proQass accerding to the invPn~ion i~ p~r~ormed, a~
wa~ already menti~ned, in such a way that the aqueous phase
i~ added to ~he alcoholic solution with mlxin~ (such ~, for
ex~mple, vigorou~ ~irrlng) and t~e lower alaoh~ re-
moved, ~or exampl~, by vacuum di~tl llation or by introducingnitrogen. The mixing rat~ is g~ne~ally 50-1000 rpm~, pre-
~e~a~ly 100-500 rp~.
Optlonally, th~ ~ispersion ob~ained after ~ombining the
a~u~ou-~ pha~e and the al~ohollc 6ulution i~ mixed ~or so~e
10time ~about 10 to 1~0 minutes~ at a temperatura o~ about 20
to 90C, be~ore the solven~ is compl~tely or parti~lly sepa
rate~. The volumstric ra~io of a~ueou~ phase ~o lipid pha~e
is generally abol~ 50-1000 ml, pre~erably 100-500 ml o~
aqueou6 pha~e per lQ0 ml o~ lipid phase.
15The temp~ratur~s uit~ble ~or ~he proceY accordi~g to
th~ invention are depend~nt on ~he solubility ~ th~ lipo-
~ome-forming sub~tances ~ well a~ ~heir pha~e tran~itl~n
temperature ~t~ he h~a~ stability of the activa ingr~-
dient or a~tive ingr~dient mixtu~e ~nd ~h~ vapor pre~sure o~
~he alcohol to be ~epa~ated. ThP tempera~ure i6 pr~f~rably
~etween 20C and 90~C ~nd in partiaul~r b~tw~n 40~C and
70C. In general, it will be su~ ient to oper~te w~th a
v~cuum of 10 h Pa to 100 h Pa.
The aqueou~ pha~e used ~or the pro¢es~ ac~ording to the
in~ention can op~ionally ~ontain bu~er substan~e~ and/or
i~otonizin~ add~ti~R~ Suitabl~ additl~es are, for example,
inorganic or organiC ~alts or bu~fer subs~an¢e6, such ac
60dium chloride, ~ris buf~er, pho~pha~s buf~er, a~tr~t~
~uff er, glycine ~uf~r, citrate-pho~pha~e buffer, male~te
bu~r, etc. Mono~ ~ di~ charides, ~uah as glu~ose,
la~to~e, s~harose or trehalose, ~ugar alcohol~ ~u~h ~6
manni~ol, or~itol, xylltol or ylycerine or wate~-~oluble
polymer~, ~uch as dextran or polyethlyene glycol.
Sinc~ th~ lipid~ and ~lso s~veral activ~ in~redi~n~s
are se~itive to oxidation, th8 a~ue~u~ phas~ Gan be mixe~
-- 7 --
with antioxldan~s, suoh s sodium a~cor~ate, tocophQ~ol or
sodium bisul~ite.
The a~ueous l~posome susp~nsions produced aaco~d~ng to
the proaess o~ the invention are used preferably ~or
encap~ulaking water-~oluble actiVQ in~redi~nt3.
Suah water-soluble aa~ive ingr~dient~ are, ~or example,
diagnostic agent~, such as the X-ray contr~t ~dl~
- lotrolan, iopromide, ioh~xol, iosimide, motriz~mide,
s~lt~ of ami~oa~etic acid, iotroxic zcid, lop~midol,
5-hydroxyacetamido-2,4,6-triiodo-i~ophthalic acid-(2,3-
dihydroxy-~-methylpropyl)-t~hydroxyethyl)-di~mide (=ZK
119095) and 3-carbamoyl-5-~N-(2~hydroxyethyl)-acetamido~-
2,4,~-triiodo-benzo~ acid-~(lRS,2$~ ,3-dihydroXy-l~
hydro~ym~thylpropyl]-~mide (-ZX 13~129) or NM~ contra
msdia, ~uch a~ gadolinium DTPA, yadolinium ~OTA and the
gadolinium complex o~ 10-~1-hydroxymethyl-2,3-
dihydroxypropy~ 4~7-~rls-t(carboxy~thy~ 4~7
tetraazacyclodecane~
Suitable ther~peutic aetive ingredients are, among
others, antibiotio agents; such as gentamyoin or kanamycin,
aytostatic ag~nt~, such a~ doxorubiain hydroohloride or
cyclopho~pham~de and virustat~c ~ge~t~, such as ~idarabin~
or active ingredients, suah as mitoxantrone hydro~hloride~
These water-soluble active ingredi~nt~ ar~ dissolved in
th~ a~ueous phase b~or~ per~o~mance o~ he proce~
according to the invention.
Furth~r, the ~queous liposoma susp~nsions aan ~l~o ~e
US~d to encapsulata sli~h~ly soluble act~e ~ngr~dients in
water.
Such active ingredient~ are, ~or example, plant
protecting agents, such a~ ~lightl~ soluble inR~cticides or
herbicid~s ~nd in ~ icular ~lightly oluble pharm~eutia~l
active ingredient~.
,3~
Sligh~ly water-soluble or insoluble pharmaceutical
active ~ n~redlents of the :~ollowing actlve ingredient group~3
are suitablet ~or example, for production o~ aqueous
su~pen~;ion~ according to the proae~s cs~ the invention.
Ge~agenlcally a~:t$~e 6terc~id hormone~ uch a6, for
~xa~ple, 13-ekh~ 7~eta-hydroxy~ s-d1nor-l7alpha-pr~gn-
4-en-20~ 3~ne ~levonorgestrel)~ 13-e~h~1-17be~a-hydroxy-
18,19-dinor-17alpha-pregna-4,15-dien-20-yn-3-one (-ge~to~en)
or 13-ethyl-17~e~a-hydroxy-11-methylene-18,1g-dinor-17alpha-
1~ preqn-4-~n~20~yn (=de~oge~trel).
Estrogenally a~tive steroid hormones, such a~ 3-
hydr~xy-1,3,5-(lo~-es~ra~rlen~17-on~ (-e~one~ or l,9-nor-
17alpha-pregna-1,3,5~10~-trien-20-yn-3,17beta-diol
~thinyle tradiol).
Andro~ni~ally aati~ ~teroid hormone~, ~u~h a6, for
example, 17~eta-hydroxy-4-andros~en-3-on~ (-t~sto~terone)
and i~ e~ter or ~7b~ta-hydroxy-lalpha-methyl-5Alpha-
androsten-3-on~ ~-me~terolone).
Antian~rogeni~ally ~c~i~e ~tero~d hormo~e~, such as,
~or example, 17alpha-aaetoxy-~-chloro-lbeta~2~eta-dihydro-
3H-ayclopropa~l~2]-pregna-l~4~6-triene-3~2o-diono
~cypot~ronac~tat~)~
Corticoids, ~uch a~, for example, llbeta,17alpha,21-
trihydroxy-4-pregnene-3,20-~ione (=hydroaortison~),
llbe~a,17alpha,21-trihydroxy-1,4-pragnadi~ne-3,2~-dinne
t=prednisolone), llbeta,17~1pha,21~trihydroxy~6alpha-methyl-
1,4-pr~gnatriene-3,20-dione (-methylprednisolone) and
6~1pha-fluoro-llbeta,21 dihydroxy-l~alpha-methyl~1,4-
prqgnadien2-3,2Q-dione (~ifluoortolone~ and their est~r~.
Ergolines, such as, ~or example, 3-~9,10-dihydro-6-
methyl~8alpha-er~olinyl)-1,1-diethylure~ (=ergoline~
~romo-g,10 dihydro-6-mqthyl-8alpha-~rgolinyl~
3 ~
_ g ~
diethylurea ~=bromergoline) or 3-(6-methyl-8~1pha-
ergolinyl)~ die~hylurea (=ter~ride).
Antihyper~e~sive agents, such as, for example, 7alpha-
~cetylthio-17~1pha-hydroxy-3-oxo-4-pregnen~-21-carboxylic
acid-gamma-laotone (aspironolactone) or 7alpha-ac~tylthio-
15beta~1~bet~methylene-3-oxo-17alpha-pregna-1,4-diane-
21,17-carbolactQns (~m~spirenone).
Antiaoagul~nt~, ~uch a~, f~r example, 5-[hexahydro-5-
hydroxy-4-~3-hydroxy-4-methyl-1-octen-~-ynyl)-2(1H)~
1~ pentalenyliden~ pentanoi~ acid ~-iloprost~.
P~ychopharmacological ayents, suah a~, ~or exampl~,
4-(3-oyalopen~yl~xy-4-~ethQxy-phenyl-2-pyrrolidone
~rolipram) ând 7-chloro-1,3-dihydro-1-m~thyl-5-phenyl-2H~
1,4-bQnzodiazepin-2-one ~-diazepam).
Carotinoid~, auch a~, for example, ~lpha-oarotin and
bata-carotin .
Fat-solubl~ vitamin~, ~uch a~, ~or example, vitamlns
the vitamin A, vitamin ~, ~itamin E and vitamin K group.
Beta-oarbolines, as they ar~ de~cribad, ~or ~xample, in
European patent appl~cation~ 234,173 ~nd 23~ 7, aro
another group. A~ beta-carboline~, ~or ~xampl~, there can
be mentloned 6~be~zoyloxy-4-m~thoxymethyl-b~ta-~rboline-3-
carboxylic ~cid-isopropyl ester ~=b~carnil) and 5-~4-
chloroph~oxy)-4-methoxymethyl-~eta-carboline~3~aar~0x~
a~id-1 opropyl est~r ~=Cl-P~O~IP).
Sligh~ly ~olubl~ or lnsoluble oon~ra~t m~dia are al80
worth mentioning, such a~ the X-ray contra~t medlu~
iodlpaml~e ethyl ester or NMR contrast media, such a~ the
iron or manganes~ porphyrin chelates or ~l~o the ~gnetit~s.
As ~uitable active ingr~dients, salic~lic ~id,
re~noic ~cld and azalaic acid further can be mentionqd~
Anti~y~otin~, such a~ the amphotericin B, ~conacol or
meconazol ~an al~o be used.
-- 10 -- .
Before par~or~ance o~ the proce~3 aca6rding to the
invention, th~:e aotive in~redient~3 ara disE;olved or
su~:pended in the a}coholic solut:ion or ln the aqueous pha~e.
In the proces~ ~ccording to th~ invention, generally
0 . 05 to lo g and preferably 1 ~o 3 g o~ ac:tlve ingredient ls
used per g o~ ~ubsta~nc~3 ~subs~anc6 mlxture) for forming
liposom~. S~coril~ liposome suspensions can ~e produced in
a ~i~nple way by the prooess ~coording to the inv~n~ion by
both solutions being sterili7.ed by :Eiltration be~ore
10 per~ormanc~ o~ ~he proc~ss and all ~ubse~uent prooess 8~ep6
being per~ormed under a~eptic conditions.
~h~ inclu~ione~ which are achievable in the ll:posome
~u~pensions are dependent, o~ aourss, on tha typf3 o~ active
in~redient and the substance ~ub tance mixture3 ~or ~orming
15 lipo om~a~a a~ w~ll a~ their ratio to one another.
Optionally, the unencap~ulated ~ctive ingredlen'c can ~e
r~mo~rad, ~or examplo, by ul.trafiltxa~ion, dialy~
micro~iltration or ~entri~uging.
Tha lipo~ome ~uæpension obt~lned c:an be dl.rectly
20 stored. Alternatively, the llpo~ome su~pension can be
converted by freeze-drying ~o a stable fo~m o~ ~orag~.
Be~ore the freeze- drying, if neoessary, among ~ther~, ~or
example, additi~res, suah as mannitol or ~orbit~l and/or
elec~rolytes and vis~osi~y-ln~luencing a~e~t~ su~h as sodium
25 chloride aan be added to the material~ to ~e fr~ez~-dried.
The lyophilizate~ can b~ re3usp~nded with bidistilled
water or a~ueou~ phases, which ~an con~ain the same
a~ditives aa th~ aqueouæ pha~e~ u~ed ~or lipo~ome
prsduation.
Ths amount of r~suspQnsion m~dium can in this c~se b~
O.S t~ 20, but preferably 1 to 6 ml per g ~f lyophiliza~.
The ~u~pen~lon~ obtained can be uaed dlreatly or after
~iltration through filters of ~uitable pore ize.
2 ~ 7~
~ 11 ~
The lipo~ome~ produced by the proc~ss aecordlng to the
ln~en~io~ ~.an, for example, b~ used i~ they contain ~ontr~3t
media for N~R or X-ray dia~nosis in various diagno23~ic
proc~s~s. ~hs~e include, for example, the di~gnosis of
tumors in organs of the reticulo-endo~hellal ~ystem (~or
example, liver and ~pleen~ or other i~trava3cul~rly
acc~ible ti~3ue6 as well as the arthrography. The
extrava~cular admini~ra~.lon o~ su~h lipo60~e ~usp~n~ion~
(such a~, ~or sxample, subcu~aneou~, intramuscular or
intraperitoneal) ~an al~o be use~ ~or dia~no~is purpo3~s,
~uch as, for example, the indirect and ~irea~ lymphography,
or for ther~pautl~ purpose~ ~such a~ intramu~cular depot
prepara~lo~. Of c~ur~e, it i~ al~o po~sible to admin~ter
tha preparations orally, and the prPparations opti~nally ~an
be filled in aoat2d cap~ule~ rasist~nt ~o ga~tric jUiC~5.
Further, the liposom~ su~pensions containing active
ingredients produaed ~y ~he pro~ aoco~ding to the
invention can be applied in the ca~e o~ suita~le lipid
compo~ition as blood pool ag~nt~ or a~ ~arget-directed
pharm~c~utical vehicles.
Without further ~la~oration, it 1~ bel~eved ~hat one
~killed in the ~rt can, u~ing th~ pr~a~ding d~scription,
utiliza the pr~sent invention o its ~ulle~t extent~ The
~ollowlng pre~erred speci~ic emhodiments are, ther~ore, ~o
~5 be construed a~ merely ~llu~tr~tlve, and not limitative o~
the remaind~r of the disclosure in any way whatsoever.
In the ~oregoing as~d in th~ ~ollowing ex~mples, al~
temperature~ are ~et ~o~th uncorrected in degree~ Celsiu~
and unle~s otherwiæe indioate~, all parts and peraentage3
are by weight.
The entir~ disclo3ure~ o~ all appli~ations, pat~nt~ ~d
pu~lication~ ted ab~e and below~ and o~ corr~ponding
2 ~ 7 .j
~ 12 ~
appliaation Federal ~epublic o~ Germ~ny P 4~ 13 580.2, ~iled
~p~il 24, 19~0, are her~by incorporated by re~r~nce.
In the ~ollowing exampl~, th~ foll4wing ~breviation~
are u~ed:
PC - Ph~sphatidylcholine S 100 o~ the Lipoid KG
comp~ny
CH = Powd~red ~hol~terol ~JP) ~ ~he Merck AG
company
SA V~ry pure st~aric acid oP the Flu~a AG
company
~PPG = Dipalmitoylphosph~tidylylyaerol DPPG o~ th~
Lipoid KG company
HSPC =~ Hy~rogenated 60yabean lQcithin ~Phospholipon
90H~ of the Na~t~r~ann AG company
DCP ~ etyl phosphat~ of th~ ~igma company
.
:
` `~
9.2~ g of lipid ~nixture (PC/~H/SA -- 4:5:1 mol:mc~ mol)
is di~olved in loO ml ~ eth~nol and ~t~rllizad by
~iltrativrl at 70C. Th~n, th~ ~olu~iorl i~ trz~ns~rr~d to a
reactiorl ve~6el he~ted moder~tely to 55~ and it i~ mixed
with f3tirring (300 rp~ wi~h an ac~ive lngredient solution
~terlliæed by flltration (~ontaining ~7 . 75 g c~ ~ opromid~
~nd 200 ml o~ aqueou~ 20 mmol t:ri~ Cl b~fers pH 7~5~
lo T~en, th~ alcohol i~ ~listill~d of~ at 554C~ a vacuum
~I 5, 000 Pa and the re~ulting liposome su3pen~ion is
axamined ~or its propertie3.
Then, the lipo~;ome su~pension i~ bottled dir~atly in
portion~ of ~0 y in 50 ml gla~s in~usion bottl~ and it i8
~r2~zQ-drisd. Th~ lyophillzates obtairled are re~usp~3nded 50
that an ~c:t~ve ingredient soncentration o~ a~out 2 oo mg/ml
re~ults and al~o examined.
~L:L
Parformance o~ the te~;t talce~ place a; de3ar~ bed in
~0 exzlmpl~ xcept tha~ 18 . 5 g o~ iopromide is us~d.
Per~ormanc2 ot~ the ~ect takes pîac~ a: des¢ribe~ in
exampls 2, except that 13 . 9 g of lipid mix1;ure i~ us~3d.
~xample 4
2~ P~arformancs of the te3t take~ placa as ds~cribed in
ex~mple ~, except th~t 18 . 5 g af lipid mixture is u~ed~
-- 14 -~
~Ia S
P~rfor~anoe o~ ~h~ test took pl ace as desaribed in
exam~pl Q 2, ~xcept that iotrol an i~; u~ed in~te~d of
iopromide .
5 ~AP~ 5
Performance C)f the ~est takes placa a~; d~scri}:~ed in
example 2, f3xc~pt that iopamidol is u~ed in ~cead o~
iopromide.
a~m~l~ .7
PerformanGe of tne te~t ta~es place as d~cribed in
Rxample 2, eXc~pt that iohexal i~ used in~tead ~f i~promide.
~ca~pl ~3 ~
Per~o~ma~ce of ths t~st ~akes place a descri~ed in
ex~mpl~ 2, ~xc~pt that the nonionic contra~t medium
5-hydroxyacstamide-~,4,~-tr~iod~ oph~h~lia acid-~,3-
dihydrQ~y-N-methylpr~pyl~(2~hydroxy~thyl)-di~:ide ~ used
in~tead of iopro~ide.
~x~mpl~ g
Performa~ce of the ~e~t took pl~ce a~ desaribe~ in
~0 example 2, eXa~pt tha~ ~ lipid mixture of PC~C~/S~ 4:~
mol :mol :mol is used~
Per~ormanae o~ the taBt t~kes plac~ as des~ribed i~
example 2, except that a l~pid mixture of PC~C~/~C o~ 4:5:1
mol:mol:mol is u6ed.
J
-- 15 --
~P~
Performance o~ the ~e~3t takes plaae ~ describ~d in
exampl~ 2, exoept that a lipid mixture of PC/CH/SA o~ 6: 3 :1
mol: mol: mol i8 u~ed .
~ 2
Per~ormanc~ oi~ tha te~t take~ place a de~cri~ed in
example 2, exc~pt that a lipid mixture~ o~ PC~C~/D~P of 4: 5 :1
i8 used.
Per~ormance o~ the te~t takes place as desc:ril:~e~ in
example 2, exsept tha~ a lipid mixture o~ PC~C~I 1 s 1 is uaed .
Per~ormance o~ ~he te~ ~akes plaae as de2;crib~d in
example 2, exc:ept that a lipid mixture of Pt::~HSPC/C~I/SA o~
2:2:5:1 i3 used.
Performance of the te~t take~ place as descri~ed in
example 2, except that the batch size was ill~rea~d tenfold~
2 0 Per~ormanc:e o~ the test ~ake~ plac:e as descrihed in
example ~, exc:ept that ~ one hunclred and ~ifty~old large~
batch 18 UBed.
~he followin~ table ~how~ the result~3 re~a~ed in
examples 1 ~o 1~.
,, n, i7 ~.j
-- 16 --
. .
~;~X~
v ~ v ~ ~ ~ ~ ~ .
n ,. ~ <~ n ~ . g~ ~.
<~ ~ ~ ~ ~ ~ n ~ ~ ~ . ~ 1~
~ I ~ X ~ O 1 ~-
V~ o ~ -o u~ ~ ~n ~ ~ ~ ~ ~ ~n . ~ .
V~ ~ ~ 4~ ''O ~ ~ ~ IA ~ V~ '.'~ ~ ~n . y, ' .
.... ~ ~-~
~ _ _
..
H H H H H 1-1 H H 1:3 f~ 1 H H H H
~ ~ ~ ~-
O O O O O O O O ~ X 1'- 0 0 C) O O ¢
O P )~- ~- 1' ~'-
. rt
~ ~ O ~ I"' ~. ~.
r~ W ~ ~ ~ ~ o ~o
O~ .J ~ ~ C O
~n o ~ o v~
Ul 3 0
C7 07
., O-
~ ~ ~ ~'--H ~ ~1
C:~'. '~.... U~. .~O~D........ ,~J
_ _ _ ~ t~ ~?
v~ C _ ~ O
. ~ ' ~ C ~ ~ ..
~ o~ ", "~ n ~ n ~ ~ .
~ C~ - ~ W ~ ~
c.~ ~ ~
. . t~
n ~ _
. ~.. . ~
~ O ~ O ~ ~ 1~ ~J
- ' O ~ -J
C~ . . .
~ ,".~ ~'
` : :
mpl~. ~7
The residual ethanol c:ontent o~ the liposomQ
~u~p~nsions, produced according to exampl~ 2, b~or~ ~nd
a.~tar ~reeze-dryinS~ i8 determined on 8 13atche~: by gas
ahromat4~raphy. The ekh~nol content~ are det~rrnined in the
original l~po~om~ ~uspen~ion with 0 . 5~ + 0 . 010% (m/V) .
x~lmPl~ 1~
Liposome ~u pen~ions and lyophilizate~ p~oduced
according to example 2 ar~ stored at 4, 25 and 40~C. To
~udge th~ ~t~bility, app~aran~e, ~ize o~ in~ ion ~nd pH
-- in the c~ e o~ the lyophili2a~es a~ter resuspen~ion --
~re determined at the re~pee~ve tim~.
A~tex 6evqral months o~ ~orage, the resuspended
lyophilizate~ s}~owed no significant deviationæ relativ~3 to
the ~x~mined ~3ize~ .
In pharma~:ological t~t~, the lipvsome susperla:ions thu~
produ-ed show th6~ ~ollowing properti~s:
~e~t ;~
Re uspended lyophilizates produced aGcording to example
2 ~re ~xarained relati~te to their aau~e toxic:ity after a
~ngle ~dmini~tration ~o a mou~ and rat.
'rh~ respeati~e LD50 is d~termined to b~:
.8 g o~ I/Rg body weight mou~e ~total iodin~) or
. 0 ~ of I/kg body weight rat.
~5 ~
For re~uspended lyophiliz~'ces produs~ed aaaordin5~ to
exampl~ ~, the ~ubac:u~ toxicity ~ ~: de'ce~min~d in rat~ ~n =
6) .
- 18 -
A~ter rep~ating the dv~e of l g of I/kg body weight
~total iodine) ~ time~, eaoh ~ an intsrval q~ 3 day~, no
chang~ o~ hi~t~pathologioal or ~li~ic~l p~r~meter~ ~r~
de~rmined.
T~t ~
R~ u~pend~d lyophilizate~ ob~ain~d a~oording to ex~mple
Z are examin~d rel~tive ~o ~heir organ connecti~n a~ter
intravenou~ adminis~rat~on in rat~.
1 hour a~er th~ admin~ s~rati~n o~ 100 mg o~ ~/kg body
weight (to~al iodine), 0.54 I~g o~ w~t weight corre~ponding
to 24~ o~ the admini~ter~d do~ is ~etectad in th~ liver.
t
Re~u pended lyophiliza~e~ obtained acaor~ o exampl~
2 ~re admini~tered intrav~nously in a do~ o~ lOO mg of
total iodine/kg body weigh~ ~o rabbit~ With liYer tumors~
Th~ r~ulting differen~e in den~lty o~ ~iver/tumor i6 35
Houn~ied units (~U) after 1 hour.
:` Resu~pended lyophiliz~t~ o~ained a~cordlng to example
~0 2 are ex~min~d on do~s relative ~o their suita~ility ~or
indirect C~ lymphography.
3 hours after in~er~igltal admini~tration ~$ 200 mg o~
total iodin~ ~in 3 ml of liposome suspension), at mo~t 200
~U ~ density i~crea~e ~ 5 measured in ~ in the popllt~al
~5 and iliac lymph nodes.
~ e p~ceding examples C~ be repeated wit~ similar
success b~ ~ubstituting the generic~lly or sp~ci~i~ally
d~cribe~ reactant~ ~nd/or operating conditions o~ thi~
inv~ntion for tho~e us~d in the preceding axamples~
.,~ .
~ ~ L,- ~ ~t
-- 19 --
~rom t;h~ ~oregoing de~orip~ion, one ~killed ~n the art
can Rasily as~rtain ~he~ e sent~ al charac~rletics o~ thi~
inv~ntion, and withQut departir~g ~rom the spirit and sao~e
thereo~, oan make variou~ changes and modiica~ion~ o~ the
5 inven~ion to adapt i~ to v~;rious u~ges and con~ition~.
