Note: Descriptions are shown in the official language in which they were submitted.
- 1~29~48
LIPOSOME PREPARATION AND ANTIBIOTIC
FIELD CF THE_INVENTION
This invention relates to aminoglycosides, analogs and derivatives
thereof, in the form of phosphate and other salts as well as the process
for making and utilizing same. Aminoglycoside phosphate liposomes and
nonguanidino aminoglycoside liposomes, their preparation and use, are
particularly described. Nonguanidino nonphosphate aminoglycoside lipo-
somes with drug to lipid ratios of greater than about 3:50 (eq. wt.) and
guanidino aminoglycoside liposomes with drug to lipid ratios (w/w) of
greater than about 9:25 (e~. wt.) are also disclosed.
BACKGROUND OF THE INVENT
Aminoglycosides are a class of compounds characterized by the
ability to interfere ~Jith protein synthesis in micro-organisms.
Aminoglycosides consist of two or more amino sugars joined in a glycoside
linkage to a hexose (or aminocyclitol) nucleus. The hexose nuclei thus
far known are either streptidine or 2-deoxystreptaminP, though others may
be anticipated. aminoglycoside families are distinguished by the amino
1329~8
sugar attached to the aminocyclitol. For example, the neomycin family
comprises three amino sugars attached to the central 2-deoxystreptamine.
The kanamycin and glutamicin ~amilies have only two amino sugars attached
to the aminocyclitol.
Aminoglycosides include: neomycin 8, paromomycin, ribostamycin,
lividomycin, kanamycin A, kanamycin B, amikacin, tobramycin, viomycin,
gentamicin C1, gentamicin C1a, (gentamicin C2, C1, Cla and analogs and
derivatives thereof collectively "gentamicin"), sisomicin, netilmicin,
streptomycin and dihydrostreptomycin. Streptomycin and dihydrostrepto-
mycin characterized by the presence of a guanidino group are understood to
be unique in associating with liposomes in higher drug to lipid ratios
than the nonguanidino aminoglycosides. The term "nonguanidino" aminogly-
cosides will include aminoglycosides other than aminoglycosides bearing a
guanidino group.
Unfortunately, use of these compounds has been limited by several
factors. Often directed to use in preventing protein synthesis in
bacteria, bacteria have demonstrated a remarkable capacity to resist the
inhibitory effect of aminoglyeosides. Resistance of an organism to
aminoglyeoside action occurs with a broad range of aminoglycosides. A
further problem of aminoglycoside use has been characteristically poor
gastric absorption and rapid excretion. Injection of aminoglycosides
results in rapid peak plasma concentration often in the neighborhood of 30
to 90 minutes following intramuscular injection which is associated with
toxicity. Another limitation is that the aminoglycosides fail to enter
the CNS or the eye.
In the therapeutic use of aminoglycosides in animals, including
humans, serious problems of toxicity have been noted. For example,
. ~
1329S48
therapeutic use in higher animals may be accompanied by ototogicity
poten~ially involving both auditory and ~estibular functions as well as
nephroto~lcity, and neuromuscular blockade culmlnatlng ln resplratory
distress.
It is an ob~ect of thls lnventlon to provide an amlnoglycoside in
the form of a phosphate salt. It is another ob~ect of thls ln~entlon to
provlde for aminoglycosldes with improved llposomal assoclatlon. I~ is a
further object of thls inventlon to provide a method of manufacture of
liposomes assoclated with aminoglycoside includlng phosphate salts
thereof. It is another ob~ect of thls in~ention that sald llpo60mes
substantially assoclate wieh said aminoglycoslde. It ls an addltloual
ob~ect of this inventlon ~hat the llpo80me8 of this inventlon provide a
high amlnoglycoslde to lIpld ratlo pa~ticularly as to nongu~nidino
ami~oglysocides. It i8 a further obJect of this l~ve~tio2 to provlde
such lipo80me8 ln a pharmaceutical dosage fon~ ~or therapeutic treatment
of 8n anl~21 lncludln~ a hu~an.
It 18 another ob~ect of this ln~ention to provide a~ intra~enously
administrable form of amlno~lycoside wlthout conco~mitant immedlate
a~allabillty of unbound or unassoclated amlnoglycoslde at high plasma
levels.
SUM~ARY OF THE INV _
It ha~ now been dlscovered that amlnoglycosites, a~alog8 and
deriYatlves the~eof9 ln the forD of phosphate salt~ and ~ulfa~e and other
salts, have ~urprislngly useful therapeutlc propertles. Amlnoglyco~ide
3alts are fou~t to be psrticusrly adapted to as~oclatlo~ wlth llposomes.
The phosphate 8alt8 of a fnoglycoaldes further may haYo reduced acute
to~lcity. The term aminoglycosite wlll be u~derstood to lnclude analogs
and derl~atives thereof.
In the past nonguanidino aminoglycosides were found to be in rather
llmlted association wlth lipo80m28~ For e~ample, Morgan et al.
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132~5~
'Preparation and Propertles of Llposome-Assoclated Gentamicin"
Antimlcroblal A~ents and Chemoeherapy, 17:544-548 (1980) reports about 4
mg of gentamicin or less assoclating wlth 100 mg of lipld. I~ the
present lnvention liposomes are "associated" with enhanced levels of a
S nonguanidino a~l~oglycoside frequently at least about 40~ of avallable
aminoglycoslde. Furthermore, the use of aminoglycoslde a~ a phosphate in
maklng liposome~ with enhanced loading efflcleacy is h~rein disclosed.
Thls i5 true for both guanidino and nonguanidino amlnoglycosldesO The
term "associated" shall be under6tood ~o be l~mobillzed on or in a
liposome, within the aqueous phase of a llposome or within the llpld
phase of a liposome.
The enhanced associaeion of lipid to nonguanitlnQ ~mino~lycoside
phosphate~ has further enabled the protuctlou of llpo80me8 ~lth greaSer
than about 10 milligrs~s (base equvslent) of nonguanidino aminoglycoælde
lS phosphate per 100 milligrams of llpid and in ~ preferred embodlment,
greater than about 30 m~ of nongusnidlno amlnoglycoslde phosphate ~base
equivalent) per 100 mg of lipld. Thi~ permlt~ aml~oglycoslde pho6phate
liposomes ant preparation~ containing ~uch liposomes to be manufactured
at hlgher potencies.
The enhanced assoclatlo~ of lipid to nonguanidino aminoglyco~ide has
further enabled the productlon of ~ongu~nidlno no~phosphate
aminiglycoslde lipo80me8 at about 1:10 ratloc (w~w) wleh greate~ tha~
about 10 mllllg~308 (actual welght) of nonguanidlno smino~lycoslde
(~ul~ate salt) per lOn milll&rs~s of llpit and i~ ~ pre~erred e~bodimen ,
a ratio over about 1:5 ~/w) greater than about 20 mg (sctual weight) of
nonguanidl~o amlnoglyco lde (~ulfate salt) per 100 mg of lipit. ~eights
af amlnoglycoslde ~y be e~presaed as actual ~elght of the aminoglycoside
(actual weight "act.wt.n) or the equlvalent welght or base equlvalent of
the trug molecule not i~cludlng the weight o~ the counterlo~ ~equivalent
wei8ht "eq. wt."). The process of thi3 invention pèr~lts the preparation
o~ aminoglyco~ide liposomes (includlng ~trepto~ycln or
dihytrostreptomycln) and preparation~ conta~nlng such liposomes to be
4--
~329~48
manufactured in a wide range oE potencies higher than obtained without
this process. (Guanidino aminoglycoside liposomes by this process attain
drug to lipid equivalent weight ratios of greater than about 9:25 (w/w);
for example, with greater than about 60 mg streptomycin sulfate/100 mg EPC
(act. wt.)).
It is a particular advantage of this invention that enhanced drug to
lipid ratio preparations require reduced administration of lipid per drug
dosage thus avoiding or reducing toxicity associated with lipid adminis-
tration.
The enhanced association of available drug with liposomes in the
preparation of the liposomes reduces the need for drug and lipid starting
materials.
Finally, high potency pharmaceutical preparations are consequently of
smaller volume and thus cause less tissue insult upon administration.
This is particularly true as to intramuscular administration.
Methods of preparing and utilizing aminoglycoside phosphate and
aminoglycoside phosphate liposomes are described more fully below.
Methods of preparing and utilizing aminoglycoside liposomes by the
modified SPLV process of, in the most preferred embodiment, requiring both
drying liposomes to powder and stabilizing liposomes, are also described
more fully below.
: ., ,~,
This invention includes liposomes comprising at least one lipid and
at least one phosphate salt of an aminoglycoside. This further includes
unilamellar and multilamellar vesicles associated with aminoglycosides
phosphates such as neomycin B, paromomycin, ribostamycin, lividomycin,
kanamycin A, kanamycin B, amikacin, tobramycin, gentamicin Cl, genta-
micin Cla, gentamicin C2, netilmicin, streptomycin, dihydrostreptomy-
cin, and sisomicin and phospholipids such as phosphatidylinositol,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine
and phosphatidylglycerol alone or in combination with
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~329~8
other llpids. This invention includes the methods of making
aminoglycoside phosphate associated liposomes as descrlbed below and
partlcularly the liposomes substa~tlally associatl~g with a~allable
aminoglycoslde phosphate. Also lncluded are nonguanidino ami~oglycoside
phosphate liposomes ~f greatsr than about 10 mg and preferably greater
than about 30 mg of aminoglycoslde phosphate per lO0 mg of lipid.
Further included ln th~s in~ent~on ls the me~hod ~f therapeutlc
treatment of ani~al lncludlng humans with therapeutl~lly effectlve
amounts of aml~oglyco~lde phosphate, liposomally asoclated aminoglycoside
phoQphate, and said phosphate in assoclatiou wlth suitable pharmaceutlcal
carr~er. Further lncluded i8 the preparation of the phosphates of
a~inoglyco~ides, and particularly the phosphates of neo~ycl~ B,
psro~omycl~, rlboAtamycin, livldo~yci~, kanamycln A, kanamycin B~
amlkaclu, tobramycin, gentamlci~ Cl, genta~lcin Cla, gentamlcin C2,
netllmicin, str~ptomyc~D, dihydrostreptomyci~, and ~l~omicln.
Adtltionally lncluded ln thls in~ention iB a~lnoglycoalte phosphate ant
llposomally a~soclated aminoglycosld~ in the treatment o$ gram-~egatl~e
pneumonla.
The 11POBOme8 of th1s In~enelo~ ~nclude liposome3 and especi~lly
SPLV lipo~o~es comprising at least one lipid ant at least one
nonguanitl~o aminoglycoside, preferably in the form of a sulfate salt.
Furthe~ lncluded ~ro ~ultllamellar ~esicle-type llposoDes ~ocia~ed with
a~inoglycoslde~ 8uch as noo~ycln a, paromomycfu, ribostamycic,
livltomyci~g ka~a~ycln ~, kana~ycin B, amlkacln, tobra~yci~, gentamIcln
Cl, gents~icln Cla, genta~lcin C2, netll~dcln, and ~i~o~lcin and
phospholipids ~uch as phosphatltylinositol, phosphatldylcholiDe,
phosphatld~lethanolamlne~ phosphseidyl~erine ant phosphaeldylglycerol.
Thi8 inventlo~ includes the methots of makl~ inoglycoside aæ~ociatet
liposomes and particu7arly aminoglyco~ide sulf&te as20ciated llpo~omes as
de~cribed below, and particularly the liposomes substantlally aGsociating
~ith a~allable aml~oglycoside sulfate. A1BO included are nonguanidino
a~ino~lyco~ide liposomes Of Breater than about 10 m8 and preerably
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~3295~8
~reater than about 20 mg of nonguanidino aminoglycoside per loo ~g of
lipid, (actual weight) correspondlng to drug ~o lipid ratios of about
l:lO and 1:5 respectl~ely.
Additionally l~cluded are guanidlno aminoglycoside liposomes of drug
to lipid ratios of greater ~han about 3:5 (actual weight/weight of lipld).
Further included in this invention iq the method of treating an
animal lncludlng a huma~ wlth therapeutlc doses of amllloglycoside
llposomes, and further ln assoclatlon wlth a sultable pharmaceu~lcal
carrier. Particu1~rly noted ls ~he treatm nt of gram-~egatlve pneumo~ia.
Thls lnventlon includes lipo~ome~ compri~l~g at lea~t one
nonguanidino amlnoglycoslde prese~ in at lesst 6.2 mg eq. wt per 100 mg
lipid and preferably at least ~bout 12.4 mg eq. wt. nonguanidino
aminoglycoslde per lO0 mg llpido Thi9 further lncludes such nonguanidino
amlnoglycosldes a~ neomycln B, paromomycin, rlbo~tamycln, livltomyci~,
kana~ycln A" kanamyci~ B, ~mikacln, tobramycIn9 vlomycln, gentamlcln
Cl, genta~icin Cla (gentamlcln C2, Cl, Cla a~alogs and
derivatlveA thereof collecti~ely ge~tamlclnn), sisomicin, netllmicin,
and preferably nonguanidl~o amlnoglyco~ltes i~ the form of sul~ate salts.
Thl~ ention lnclutes llposome~ co~prIsl~ at leas~ one
a~phipa~hlc lipld. Thi8 i~ventio~ ~nelutes the proce~ of eDhanclng
amlucglycoslte to lipld drug ratlos ln llpo80me~ of the 5PLV proce~ by
varlously drying th~ llpld-organlc ~ol~ent ~inoglycoslde-aqueou~ phase
mlYture eo powder, pe~ltti~g the rehydra~ed powder to stabillze by
standing at sbout 4C for at least about 8 hour~ a~d preferably for
about one tay. ThiA in~e~tloD further includes having dried the
llpit-org~nic ~olve~t-amiDoglycoaite aqueou~ phaoe ~i~ture over a three
to ei8h hour perlod and preferably a flve hour perlot. Al~2rnatlvel~,
the lipid-organic solvent-a~ino~lycoslte-aqueou~ phase ~sy be dryed o~ly
to a sIurry or pa~te.
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1329~8
Thls lnventlon further includes removal of liposomally unassociated
aminoglycoside from aminoglycoside liposome preparatlon by low energy
sepratory methods such as dlalysi or chromotography belng careful to
avold high energy 6epratory methods such as centrlfugatlon.
BRIEF DESCRIP?ION OF THE FIGURES
FIG. 1 is a graph of genta~icin pho~phate entrapped (mg)/100 mg egg
phosphatidylchollne ("EPC") versu3 gentamicin phosphate (mg~ available
for entrapment.
FIG.2 is a graph of the percentage trapplng efflciency versus
gentamicin phosphate (~g) avaIlable for entrapme~t.
PIG. 3 is a graph of genta~lcln phosphate ~ntrapp~d (mg)~100 mg EPC
~ersus EPC tmg) a~allable ~o entrap.
FIC. 4 is a graph of the percentage trapplng ef~lclency ~ersus EPC
(mg) avallable to entrap.
FIG. 5 i8 a graph of the comparatlve llpo~omsl entrapment of
aQlnoglycoslde sulfate and phosphate.
,' ~ .
The utlllt3v of a~lnogl~eoside contai~ liposo~e~ i~ descrlbed i~
conne~tio~ ~lth the trest3e~t of disea~e ln anl~als 1~ ~.S. Patent No.
4,552,803 to Lenk et al. The surprisi~gly increased efficiency o~
assoclatio~ of aml~oglyco~lde pho~pha~e by liposo~es ~akes the3e
prepsratious partlcularly effective end efflcient.
It ha~ now been discovered that the aminoglyco~de phouphate
assoclatlon with lipoao~es i~ enhancet o~er that of noupho~pha~e
amluoglycosides. For ~onguanidl~o anl~oglyco~ide by ~ubat~ntlally
assoclated it lq to be understoot that no ~ore than about 60% of the
~329~8
nonguanidino aminoglycoside present in a liposomal preparation remains
free in solution hence unassociated with the liposomes.
Liposomes are vesicles comprising closed biiayer membranes containing
an entrapped aqueous phase. Liposomes may be any variety of unilamellar
j vesicles (possessing a singie membrane bilayer) or multilamellar vesicles
(e.g. onion-like structures characterized by concentric membrane bilayers,
each separated from the next by an aqueous layer).
The liposomes of this inveneion may be prepared so as to associate
with nonguanidino aminoglycoside in ratios equal to or greater than about
10 mg aminoglycoside per 100 mg lipid and as high as about 30 mg nonguan-
idino aminoglycoside per 100 m8 Of lipid or higher. The use of aminogly-
coside phosphate results in a more concentrated and hence more potent
aminoglycoside liposome preparation than would be available wieh other
forms of nonguanidino aminoglycoside.
The aminoglycoside phosphate liposomes of this inventlon are formed
by methods well known in the art. The origlnal liposome preparation of
Bangham et al. (1965, J. Mol Biol. 13:238-252) Involves suspending
phosphollpids in an organlc solvent which is then evaporated to dryness
leaving a phospholipid film on the reaceion vessel. Then an appropriate,
amount of aqueous phase is added, ehe mixture is allowed to "swell't, and
the resulting liposomes which consist of multilamellar vesicles (herein-
after referred to as MLVs) are dispersed by mechanical means. The struc-
ture of the resulting membrane bilayer is such that the hydrophobic (non-
polar) "tails" of the lipld orient eoward ehe center of the bilayer while
the hydrophiLic (polar) "heads" orient towards the aqueous phase. This
technique provides ehe basis for the development of the small sonlcated
unllamellar vesicles thereinafter referred to as SUVs~ described by
Papahadjapoulos and Mlller (1967, Biochim. Biophys. Acta. 135:624 638) and
large unllamellar veslcles (hereinafter referred to as LUVs).
,
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In the practice of this invention as to aminoglycoside phosphates, a
class of liposomes characterized as having substantially equal interlamel-
lar solute distribution is preferred. This preferred class of liposomes
is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S.
Patent No. 4,522,303 to Lenk et al. and includes monophasic vesicles as
described in U.S. Patent No. 4,538,578 to Fountain et al. and frozen and
thawed multilamellar vesicles (FATMLV) as described in "Solute Distribu-
tions and Trapping Efficiencies Observed in Freeze-Thawed Multilamellar
Vesicles", Mayer ee al., Biochima et Biophysica Acta. 817: 193-196 (1985).
Large unilamellar vesicles may be modified using an extrusion
apparatus by a method described in Cullis et al., Canadian Patent No.
1,264,668 issued January 24, 1990 entitled "Extrusion Technique for
Producing Unilamellar Vesicles", (LUVETs). To make LUVET vesicles by this
technique, MLVs are extruded under pressures of up to about 700 psi
through a membrane filter. These vesicles may be exposed to at least one
freeze and thaw cycle prior to the extrusion technlque; this procedure Ls
described in Bally et al., Canadian Patent Application Serial No. 520,029,
filed October 7, 1986, entitled "Multilamellar Liposomes Having Improved
Trapping Efficiencies".
Another technique that is used to prepare vesicles is one which forms
reverse-phase evaporation vesicles (REV), Papahadjapoulos et al., U.S.
Patent No. 4,235,871.
The term lipid as used herein shall mean any suitable material
resulting in a bilayer such that a hydrophobic portion of the lipid
material orients toward the bilayer while a hydrophilic portion orients
toward the aqueous phase.
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1329548
Two general classes of lipid compounds are useful in the present
invention as to aminoglycoside phosphaces. The most prominent members are
highly hydrophobic compounds, such as triglycerides. Corn oil serves as a
convenient and economical source of mixed triglycerides, but other
vegetable oiLs, including but not limited to palm kernel oil, coconut oil,
soybean oil, sunflower oil, safflower oil, cocoa butter, and the like may
be used. Specific molecular species might be employed as well. Such
species may include, but are not limited to, trilaurin, trimyristin,
tripalmitin and tristearin, or other glyceryl esters in which the fatty
acyl chains of these compounds as well as other fatty acids are incor-
porated in a non-homogeneous fashion. Other broad classes of long chain
hydrophobic compounds such as the wide range of cholesterol esters may be
used. It has even been found that long chain organic mixtures such as
petroleum jelly are acceptable lipid materials.
Further a variety of cholesterols and other sterols and their water
soluble derivatives have been used to form aminoglycoside phosphate
liposomes; see speciflcally Janoff et al., Canadian Patent No. 1,262,334,
issued October 17, 1989, entitled "Steroidal Liposomes". Mayhew et al.,
WO 85/00968, published March 14, 1985, describes a method for reducing the
toxicity of drugs by encapsulating them in liposomes comprising alpha-
tocopherol and certain derivatives thereof. Also, a variety of
tocopherols and their water soluble derivatives have been used to form
liposomes, see Janoff et al., Canadian Patent Application Serial No.
519,854, filed October 6, 1986, entitled "Alpha-Tocopherol- Based
Vesicles". Preferred of this group are cholesterol hemisuccinate and
tocopherol hemisuccinate. The only constraint appears to be that the
hydrophobic compounds selected should, when uncomplexed with the other
components of this invention, be soluble in a particular organic solvent
chosen for use in the manufacture of the liposomes.
The second broad class o lipid materials used in this invention as
to aminoglycoside phosphate are amphipathic in character. Hydrophilic
character-could be imparced to the molecule through the presence of
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32~548
phosphatol c~rbox~rlic, ~ulp~o, amino, sulrhydryl~ rlltro? ~nd o~her lLke
3roupQ. HydrQphol~lelty could he co~4er~d by ~ht?. incluston of grt~ups
that include, but are not ll~ited ~o, lon~ chaln !i~turated and
un~a~ura~ al~ph~Ltl.o hydroc~,on ~roupa and ~uch ,3roup~ 6ub~titu~ed by
on~ or mor~ ero~a~ic, cyclc~llphatlc or hetorocyclic grcup. ~te
pr~ferred ~ phipash~lc compourlda Ar~ pho~pho~lycerlde0, r~pr~entatlve
t~xA~ple~ o~ ch ~nc~.ude pho~ph~ldylcholine, phosph~tldYlethan41~1n~,
ly,~ophosphatidylcholine, l~opho~phatldy~hanolamint3,
pho~phat~dylser~e, phogpha~ldylinoo~o~ pho3phatldic arid~
10 di~yrl~toyLphoophatidyl~lycerol and t~ipho~ph~tldylglyc~rol alon0 or tn
ca~ki~.a~lor~ with other llpid~. S~nthetic 3a~ursted 00~4pound3 ~uch l39
dimyrlstoy'phoap~at~dyloholine, d~palmltoylpho~ph~ldylcholine~ or
diotearoyLphoaph~tldylcholine or u~atur~ted 8pe~ie~ aucn ~c
dioleoylphosph~tidylchollne o~ dlllnol~oylphoophatidylcholin0 ~ight also
15 be uaable. Other compounds lackln~ pho~phorou~ such as member~ o the -
~phlngollpi~ and ~lycosphinnollpld ~ 9, arè ~l~o within the g~oup
de~i~n~ted a~ lipid.
A~phipathic li21d~ are necea~ry aa the primsry llpo~c~al ~tructural
~le~ent for ~mI~o~lycosldes m~de by the ~odfied SPLV preparatlon
20 d~3cribed belo~. In ~orming ~odlfied 5P~V pcep~ratlon ~inoglyeo3ide
liposo~s3, these ~p~lp~thlc llplds ~ay ~e ad~iYed with other llpld~
including trlglyceride~ and ~ta~ols.
A~ to pho~phAte a~$~0glyco8ide~, ~ method ~or preparin~ the 3terol
co~ainlng llpoaome~ inYol~e~ ~dding to ~n ~queou~ bu~fer B ~alt orm of
25 ~n or~ui ~ ~id de~i~at~ve of ~ ~terol cAp~ble of for~ng clo~d bil~yer8
$n a~ e~fflcle~t to ~or~ co~pletely clQsed bllayer~ which entr~p
an ~queou~ compart~ner~t, A susp nsion o ~u1tll~mellar ~re~icle~ 1~ for~ed
by ~h~klng the ~i~turo. The fcrm~tion o~ ve~icle~ 1~ facilltated i~ the
a~eoua bu ~er ~lao co~ai~s the coun~er~o~ of ~he ~lt in eolution.
~,
The applic~tion o~ en~r8y to l:he suspeneloa, e.~.. 7 s~n~catlon, or
extru~lor c7f ~h~ ~e~icles thr~u~h ~
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13295~
French pressure cell (French Press) or through a porous filte~ of the
appropriate pore size, wlll convert the multilamellar 5 terol vesicles to
unilamellar vesicles.
Liposomes entrap an aqueous mediu~ which is e~closed by the lipid
bilayers. The aqueoufi medium can be for example, water or water
containing a dissolved salt or buffer. Examples of such salts or buffers
can be sodium chloride and phosphate buffered saline (PBS). Other
buffers include but are not limited to borate, citrate,
Tris-HCl(Tris-(hydroxymethyl)-aminomethane hydrochloride), and ~EPES
(N-2-hydro~yethyl piperazine-~ -2-ethane sulfonic acid). Buffer may
be in the pH range of bet~een about 2.0 and sbout 14Ø In ~he preferred
embodiment as to amiaoglyco6ide phosphate, the preparatlonq are hydrated
w~th HEPES buffer (150 mM NaCl, 20mM HEPES~, pH 7.0, bora~e buffer (lOO
mM Na2HC03, 50 mM ~3B03, pH 8.5, or citrate buffer tl50 mM
Na-citrate), pH 8.5.
In a liposome-drug delivery system, the therapeutic agent, here
aminoglycoside, i8 entrapped in the llposome and then admlnl~tered to the
patient to be treated. For example, see Rahman et al.) U.S. Patent No.
3,993,754; Sears, U.S. Pateut No. 4,145,410; Papahad~opoulos et al., U.S.
Patent No. 4,2359871; Schneider, U.S. Patent No. 4,224,179, Lenk, et al.,
U.S. Patent No. 4,5~2,803, and Fountaln et al., U.S. Pateht No. 4,588,57B.
Pharmsceutlcal llposomal preparatlons are, ~n the preferred
e~bodlment, delivered in physiological sallne or water buffered uith
phosphate or citrate appropria~e for in~ectlonO
Optionally, the aminoglyco~ide phosphate liposomes can be
dehydrated, thereby enabli~g storage for e~tended perlods of time until
use. Standard freeze-drylng equlpment or equivalent apparatu~ may be
u~ed to dehydrate the liposomes. Lipo~omes may also be dehydrated 6imply
by placing them under reduced pre~sure. Alterns~ively~ the liposomes and
their surroundlng medium can be frozen in liquld nitrogen prior to
dehydrationO Dehydraeion with prior freezing may be performed ia the
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:~3295~8
presence of one or more protective sugars in the preparation, according to
the process of Janoff et aL., Canadian Patent Application Serial No.
488,oo4, filed August 2, 1985, entitled "Dehydrated Liposomes". Examples
of protective sugars that may be used include, but are not limited to,
trehalose, maLtose, sucrose, glucose, lactose and dextran. Alternative
multilamellar vesicles may be dehydrated with prior freezing without
protective sugars. When the dehydrated liposomes are to be used,
rehydration is accomplished by methods which include simply adding an
aqueous solution, e.g., distilled water, to the liposomes and allowing
them to rehydrate.
The aminoglycosides of this invention are administered associated
with liposomes, and if desired, in admixture with a pharmaceutically-
acceptable carrier (such as physiological saline or phosphate buffer)
selected with regard to the intended route of administration and standard
pharmaceutical practice. Dosages for aminoglycosides when associated with
liposomes will often be about that of the aminoglycoside alone; dosages
will be set by the prescribing medical professional considering many
factors including the age, weight and condition of the patient. The ratio
of active ingredient to carrier will naturally depend on the chemical
nature, solubility and stability of the aminoglycoside, as well as the
dosage contemplated. For parenteral administration or injection via such
routes as intravenous, intraperitoneal, intramuscular, subcutaneous, or
intra-mammary route, sterile solutions of the liposome composition are
prepared. For intravenous use, the total concentration of solutes should
be controlled to render the preparation isotonic.
In another example of their use, liposomal associated aminoglycosides
may be incorporated into a broad range of topical dosage forms including
but not limited to gels, oils, emulsions and the like. For instance, the
suspension containing the liposomal associated aminoglycoside may be added
to the aqueous phase as an ingredient in the liposome preparation. Such
preparations may be administered as topical creams, pastes, ointments,
gels, lotions and the like for direct application.
_14-
.
1329548
,
l. Phosphate salts o~ aminoglycosides.
The aminoglycosides each contain one or more amino sugars
linked by glycosidic linkages to a basic six-membered carbon ring.
Various phosphate salts may be ~ormed by titration with acids having
phosphate groups. In general it is easier to form a phosphate salt o~ an
aminoglycoside than link a phosphate to an aminoglycoside covalently.
For example, gentamicin base contains ~ive titratable amino
groups.
R NH Gentamicin R
C 1 (~
NH2 ¦ CH3
r I C2 H2N-C-H
k~)~ c 14 CH 2 NH 2
Depending on a number of factors including the choice o~
titrant, specific aminoglycoside, solvent and temperature, a number of
phosphate salts are theoretically possible.
Titrant Sa
H3Y04 Gent3(H3Po4)s
NaH2P04 Gent2(H2Po4)5Na5
Na2HP04 Gent(HP04)5NalO
*Gent = gentamicin base
Clearly, aminoglycosides, having the ability to associate with
a number of phosphate moieties, may be utilized in degrees of phosphate
asssciation. However, in the practice of this invention, the preferred
aminoglycosides will have a ratio of from about 1:1.6 to about a ratio of
-15-
.. ' f '
.
.~ ~
.
1~29~48
1:5 molecules of aminoglycoside to phosphate. As used herein
aminoglycoside phosphate will refer to an amlnoglycoside associated with
at least one phosphate.
2. Preparation of Phosphate Form of Gentamicin
S Gentamicin phosphate is a preferred aminoglycoside phosphate of this
invention. Gentamicin base was prepared from gentamicin sulfate as
described below, and subsequently converted to a phosphate by tltration
with phosphoric acid to a pH suf~icien~ly low to cause the phosphate to
associate with the aminoglycoside. Usually a pH of about 2.5 will be
suitable.
In general am~noglycoslde phosphate may be prepared with any solvent
that adequately solubilizes both amlnoglycoside and phospha~e but is not
appreclably acidic. Aqueous solven~sJ particularly water, are
preferred. The pH pre3ent will be characteristic of the source of
phosphate titrant and the amlnoglycoside. Source~ of phosphate are
phosphoric scids and metal phosphate salts such as sodlum or potassium
phosphate. The temperatur~ and pressure are not crltical and standard
temperature and pxessure are often most convenlent. The temperature
should not e~ceed a temperature at which the aminoglycoside remains
stable.
3. Preparation of AmiDoglycoside Phosphate Lipo~omes
Liposomes may be prepared by any of a number of the method~
disclosed i~ the above lncorporated referencas. Alternatively a method
of maklng liposomes by the process of mixing an aqueous phase with llpid
free of organlc solvent may be employed. Monophaslc vesicles (MPVB) as
described in U.S. Pa~ent No. 49588,578 are formed by ehe ge~eral method
of (a) forming a dispersion of lipld ln an orgauic solvent, (b) combining
the dispersion -~ith an aminoglycoslde phosphate in an aqueous phase to
form a biphasic mlx~ure in which ~he aqueous phase can be encapsulated,
and (c) remoYing the organic solvent.
-16-
,~ :
- ~L3295~8
More specifically, a lipid or a mixture of lipids and an aqueous
component are added to an organic solvent or a combination of organic
solvents in amounts sufficient to form a monophase. The solvent or
solvents are evaporated until a film forms~ Then an approprlate amount
of aqueous component i8 added, and the film i8 resuspended and agitated
in order to form the MPVs.
The organic solvent or combination of solvents used in the process
must be miscible wi~h water and once mixed with water should ~olubilize
the lipids used to make the MPVs.
Por e~ample, an organic sol~ent or mi~ture of solvent~ which
satisfies ~he followin~ criteria may be used in the process: (1) 5 ml of
the organic solvent forms a ~onophase with 0.2 ml of aqueous componen~
and (2) the llpid or mi~ture of lipids is soluble i~ the monophaEe.
Solvents which may be used in the process of the present invention
include but are not limited to ethanol, acetone, 2-propanol, methanol,
tetrahydrofuran, glyme, dioxane, wridine, diglyme,
l-methyl-2-pyrrolidone, butanol-2, butanol-l, iaoamyl alcohol,
isopropanol, 2-metho~yethfinol, or a combination of chlorfor~ and methanol
(e.g., in a 1:1 ratio v/v)~
The evaporation of solvent should be accomplished at ~uitable
temperatures and pre~sures which maintain the monophase and facilitate
the evaporatio~ of the sol~ents. In fact~ the temperatures and pre~sure6
chosen are not dependent upoa the phase-transition temperature of the
lipid used to form the MPV8. The advantage of this latter point i8 that
heat labile aminoglycosides which have desirable properties can be
incorporated in MPV8 prepared from phospholiplds such as
diatearoylphosphatidylchol~ne, which can be formed lnto conventional
liposomes oaly at temperatures above the phase-transition temperature of
~he phospholipids.
-17-
.
:
, ~ .
.
132954~
Stable plurilamellar vesicles of aminoglycoside phosphate, SPLVs~
are prepared as follow~: An amph~pathic lipid or mixture of lipids is
dissolved in an organic solven~. Many organic solvents are suitable, but
diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated
hydrocarbons and ether are preferred. To this solution are added an
aqueous phase and the aminoglycoside to be entrapped~ This biphasic
mixture is converted to SPLVs by emulsifying the aqueous material within
the solvent while evaporatin~ the solvent. Evaporatlon can be
accomplished during sonicstion by any evaporative technique, e.g~,
evaporation by passing a stream of lnert gas over the mi~ture, by
heatingJ or by vacuum. The volume of solvent used must e~ceed the
aqueous volume by a sufficient amount so that the aqueous material can be
completely emulsified in the mi~ture. In practlce, a minimum of roughly
3 volumes of solvent to 1 Yolume of aqueous phase may be used. In fact
the ratio of 801vent to aqueou~ phase can vary to up to 100 or more
volumes of solvent to 1 volume aqueoua phase. The amount of lipid must
be 6ufflcient 80 as to e~ceed that amount needed to coat the emulsion
droplets (about 40 mg of lipld per ml of aqueous phase). The upper
boundary i8 limited only by the practicallty and efficlency, as for
e~ample, SPLVs can be made with 15 gm of lipid per ml of aqueous phase.
~he process produces lipo60mes with differene supermolecular
organization than conven~ion~l llposomes. According to the present
invention, the en~ire process can be performed at a temperature range of
about 4 - 60C. regardless of the phase transition temperature of
the lipid used. The ~dvantage of this la~ter polnt i~ that hea~ labile
aminoglycosides w~ich have de~lrable properties can be incorporated i~
SPLVs prepared fro~ phospholipid ~uch as distearoylphosphatidylcholine,
but can be formed into conventlonal liposomes only at temperatures above
their phase-transition ~empersture.
To form FATMLVs one eaample of a suitable process is as follows:
one or more selected lipids are deposited on the inside wallR of a
suitable vessel by dissolving the lipids in sn organlc solvent ~uch as
chloroform and ~hen evaporating the organic ~olvent, adding an aqueous
-18-
,.
~- 1329548
pha~e containing aminoglycoside phosphate which is to be encapsulated to
the vessel, allowing ~he aqueous phase to hydrate the lipid, and
mechanlcally agitating (for e~ample, by swirling or vorte~ing) the
resulting lipid suspension to produce the liposomes which are then
sub~ected to a freeze-thaw process.
Alternatively, one or more selected llpids can be dispersed by
employing ~echanical agitatio~ ln an aqueous phase to produce
multilamellar vesicles ~NLVs) which also may be subjected to the
freeze-thaw process. The process requires about 1-10 minutes at a
temperature above the gel/liquid cry6talllne transition temperature.
The lipid co~centration for producl~g MLYs i8 at least about 50
mg/ml aqUeOUB solvent. At lower concentrationsJ multilamellar vesicles
having a high trapping efficiency are more difficult to form. A
preferred llpid concentrat$on i8 between about 100 and 1000 mg/ml aqueous
solvent, more preferably 100-600 mg/ml, and still more preferably 100-400
mg/ml. Nelther detergent nor organlc solven~ is required.
The freeze-thaw cycle that results in FATMLVs requires rapid
freezing of the dispersed liposome mi~ture and then warming the frozen
mi~ture ln a confitant temperature bath, to a temperature whlch will cause
the aqueous phase to melt. The temperature employed iR gener~lly above
the transition te~perature for the gel/liquid crystalline transition. A
constant temperature bath of sb~ut 25-50C, preferably a~out 40C, is
generally effertive.
Liquid nitrogen bath~ have been found to be particularly effective
for the freezing step. The number of freeze-thaw cycles affects the
propert~es of the resulting FATMLV. Generally, three or more preferrably
about five or more freeze-~haw cycles are required to obtain an
equilibrium interlamellar osmotlc balance. About five freeze-thaw cycles
in liquid nitrogen and a 40C conatant ~emperature bath, resul~ in
preferred FATMLV' 8 .
-19-
'
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.
,
~3295~8
4. har~acological Use of Aminoglycoside
The aminoglycosldes may be cla~sified as broad spectrum
antibiotic~. With respect to antibacterial spectrum, there are
similarities among them, but there are alRo considerable dlfferencesJ
hence generali~ations should be avoided. The aminoglycosldes of thiB
invention and liposomally associated aminoglycosides are useful in
therapeutically-effective dosea in the treatment of gram-negative
pneumonia.
Aminoglycosides may be administered in combination with one or more
pharmaceutlcally acceptable carriers. Such carriers are well known in
the art. Aminoglycosides are preferably administered ~ntramuscularly or
lntravenou~ly. Ophthalmic solutions and oin~men~s are also available for
topical ophthalmic application~. Creams are avallable in ~ome cases for
topical application.
Gentamicin (base equlvalent) for example, may be adminiRtered IM or
IV at about 1 to 1.7 mg/kg of body weight about every elght hours or 0.75
to 1.25 mg/kg every six hours for about seven ~o ten days. However, many
considerations are involved in determing ~n actual dosage including
incidence of renal failure9 the amlnoglycoside in use, the animal and i~s
presenting conditlon. A therapeutically effec~ive dose of an
ami~oglycoside will be tha~ dosage which, in view of the speciflcs of the
application, produce the desired result. IM or IV preparations of
a~inoglycoside phosphate associat~d liposomes are preferably administered
suspended in a sallne solution.
5. Modified SPLV Preparation of Aminoglycoside Enhanced Drug to Lipid
Ratio Li~osomeq
SP~Vs produced by a novel method are prepared as follows: An
amphipathic lipid or mixture of lipids i8 dissolved in an organic
solvent. This constltutes the first mixture. Many organic 801vent8 are
suitsble, but diethyl ether9 halogeDated hydrocarbon~ and mixtures of
halogenated hydrocarbons and e~her are preferred with methylene chloride
-20-
'I ' : ~ , ..................... .
', .'' . '' .
',
13295~8
~o~t prei~orred. To thls ~olutio~ are ~dded fln ~qU~oU~ phaae ~nd the
a~inoglycoolde to be llposo~lly aasocl~ed. A~lnoglyco~ite sul~at~ 13
moat preferred at thI~ ~t~ge. Thia biphaslc Dlis;ture 1~ conve~ted eo
SPI~Vs by e:~u~ ylllg the aqu~oua mstgri~l within ~he org~rlic ~olvent
w~lle e~poratlng the ~olve~t R~ld tzl the pr6!ferred e~obid~e.t eY~por~tIn~
to dryne~. Evaporation c~ be ~ccomplishqt by any ev~por~tive
technique, e.g. 9 evapo$atiarl by p~lng ~ stre~ of lne~t gaa ov~r -~he
mlxture, by heatin~, or by ~racuum~ Dryin~3, #nd psrticuldrl~r drylng to
powdar, ~n th~ pre~erred e~bodlment i~ acco~pltshed ove~ About 3 to 8
hour~ and pre~er~bly ov~r aboul 5 hour~ altd prefer~b:Ly while s~lrrin~ and
~oat preferQbly stlrrlng B~ hi~h speed. Drylng temp~ral:ure ia betwee~
~bout 25C ~o b,5~, me te~p~r~ture ~Uf~t rlot be at or abo~e the
bollillg pol~t o~ the solvent in u~e whlch of course ~rlll ~ary with the
pre3aure o4 the ~ysten~. Wl~h methyl~n~ chloriae Qbout 40C i~
pref~rret. I~ not dryln~ to powder~ than dryln to A pa~te or ~ rry i8
aceept~qble. T~e volu~e of or~nic aolvent uaed mu~t be proportionAte to
the ~queous volume ~o tha~ ~he n~ueous ~terial can be completely
~mul~ified ln the mi~cture. In pract1cel a ~inin~um of roughly 1 ~olume of
~o1vent to 1 vo1u~e o~ Qqueou~ ph~e mBy b~ u~ed. In fact the r~t10 of
~e1~erlt to ~queou~ pha~e can vary to up to 100 or ~ore ~olu~e~ o~ aolY~
to 1 volum~ aqueot-~ pha3e~ ~e amount of lipid mu~t be ~uff1c1ent ~o ~B
~o ea~ceed Ph~t am~uat neaded to co~t ~h~ e3~u1~io~ droplet~ ~bout 40 m~
o~ 11pld per ml o~ aqueou~ p~se). Th~ ùpper bour~ y 18 11m~tad o~y by
tho practice~1~tr ~nd ef~lc1en~yt bue SP~VD c~n be ~d~ wi~ch 15 ~ oi~
~1pid p~r ~ o~ ~ueou~ pha~
haa b~en formed, the re~ulti~g prep~r~1On iq ehe~
rehydroti ~ ~t i~ a li~t~tlon o~ thla proc~a thst ehe ~3atarl~1 be
p~rmitte~ ~ ~Y"~ta~illz~" for A period of ti~e. The ~e~peratur~ ~or
~tsbili~ ia ~at cr~t~eal but cold~r ~e~per~urea ~sbo~e ree~i~g)
30. ~n~oy le~ omal dagr~d~tio~. ~hu3 ~tabilizi~3 a~ perform~t
p~e~er~bly at Q~out 4co The p~riot of ~to~ t~o~ ia at ~lnin~
~bout a hour~ and pr~ersbly at le~st ~bout 0~1& t3r. A~t~r ~t~billÆing
u~a~oel4te!d s~no~glyco~id~ ~Ray be re~oYed~ wllich i8 done in the
. prePerred embodilQent.
--21--
' .r
, .~ ~ : ,
1329548
The removal of unassociated aminogLycoside must be done in a manner
that does not adversely affect liposomal integrity. Dialysing against a
saline solution is such a low energy type non-adverse process. Centrifu-
gation is a high energy process that could adversely affect liposomal
integrity and should not be utilized. Other low energy type processes
known in the art such as chromatography may also be used.
Prior to the removal of unassociated aminoglycoside, the liposomes
may be sized. If for intravenous use in humans these are preferably sized
to about 3 to 5 um - the size beyond which capillary blockage can occur.
Sizing can be performed by any method including homogenization and
extrusion such as by steel mesh, straight path or tortuous path filtra-
tion, including membrane filters of polycarbonate and other polymeric
substances.
Optimal results require that the liposome mixture be dried to powder
prior to rehydration. A further requirement Eor optimal results is that
the liposomes stand to stabilize overnight prior to removal of unassoci-
ated aminoglycoside. Further the most pre~erred results by this process
arise from utilizing the sulfate salt form o~ aminoglycoside. This is
particularly true of gentamicin. However, aminoglycoside salts including
phosphate, chloride, and tartrate are contemplated as well as aminoglyco-
side free base. Aminoglycoside salts formed with hydrophobic moieties are
also contemplated within this invention. Such hydrophobic moieties are
fatty acids, for example, palmitate, myristate, and stearate.
By the foregoing method of preparing liposomes of this invention the
aminoglycoside is associated with liposomes at enhanced levels does not
remain free in solution. By substantially associated it is to be
understood that no more than about 60% of the nonguanidino aminoglyco-
side present in a preparation is not associated with the liposomes.
.
-22-
.
:. .
: ' ~ ` ` . . .
.
~329~8
In some instances, liposome dispersions having a nonguanidino
nonphosphate aminoglycoside concentration oE greater than about 200 mg
(eq. wt.) per 8m of EPC were obtained and in some instances as high as
360 mg (eq.wt.) were obtained.
Aminoglycoside associated liposomes and particularly nonphosphate
aminoglycosides of this invention made by the modified SPLV preparation
have enhanced drug to lipid ratios compared to previous techniques. The
liposomes of this invention may be prepared so as to associate with
nonguanidino aminoglycoside in ratios equal to or greater than about 3:50
(w/w) which is about 6.2 mg nonguanidino aminoglycoside (eq. wt.) per lO0
mg lipid and as high as about 3:25 which is about 12.4 mg nonguanidino
nonphosphate aminoglycoside (eq. wt.) per 100 mg of lipid or higher. This
results in a more concentrated and thus potent nonguanidino aminoglycoside
liposome preparation. This preparation results in the enhancement of
liposomal association for guanidino aminoglycoside liposomes prepared by
the process of this invention, such guanidino aminoglycoside liposomes
have yielded drug to lipid ratios of greater than about 9:25 (equivalent
weight/weight) with over about 60 mg of streptomycin sulfate (act. wt.)
associating with 100 mg EPC.
Example 1
G ntamicin Phosphate
In this conversion, 200 mg of gentamicin base was dissolved in 1 ml
water. This was then titrated to the equivalence point. For H3P04 85%
(weight:volume) the equivalence point was pH 2.5. The reaction was
performed at standard temperature and pressure.
If required, gentamicin base can be prepared from gentamicin sulfate
by ion exchange chromatography. The anion exchanger resins such as
AGl-XB (hydroxide form) (BioRad) is slurried in distilled, deionized
water (dH20). A column, conveniently 2.6 cm ID x 33 cm, was poured
according to the manufacturerls instructions and washed with sufficient
-23-
:
.
...::: ~
1329~8
,
H20. In the current example, two column volumes of dH20 was
~ufficient, but each apparatus will have unique requirements well known
by those skilled in the art. Gentamicin sulfate in dH20 was applied to
the column at a moderate flow rate. One hundred ml of a 200 mg/ml
~olution of gentamicin sulfate and a flow rate of 50 ml/hr i8
convenlent~ The column wa~ then washed with dH20. Fractio~s were
collected, and those containlng gentamicln were pooled and lyophilized.
The potency of the base may be determined by any of a number of
technique~ including by bioassay and by spectrophoto~etric determination
of trinitrobenzyl adduct(s) of the drug substance. The base ~as
converted to a phosphate by aqueous titration with phosphor$c acid and
sodium phosphate buffer.
Egample 2
.
Preparatlon of Aminoglycoside Phosphate Liposomes
Preparation of Precursor Llposomes at Var$ou~ Gentamicin
GoDcentrations. 16 roundbottom flasks were set up in groups of four.
Each group con~ained four flasks containing 50, 100, 200, and 300 mg of
lipid such as eg8 phosphatldylcholine tEPC), elther as a thin film or in
powdered form. The ves$cles ln Group 1 were made wlth 50 mg/ml
aminoglycoside (in all groups here, gentamicin). Flrst, 1.0 ml aliquots
of 50 mg/ml amdnoglycoside were pipetted into he four separa~e flasks
that made up this group and they were vorte~ed vigorousl~. Complete
mlxing yielded MlV preparations that were homogeneous a~d had a mllky
consistency. These samples were tran~ferred to pla6tlc cryo~ial~. The
samples then uuderwent the freeze-thaw process. This proces~ wa~
repeated for the three remaluing groups.
The ~amples in Group 2 were made with 100 mg/ml of aminoglycoside9
and the same four starting weight~ of EPG. Likewise, ~he four ~amples in
Group 3 were made with 200 mg/ml aminoglycoslde, and the four ~amples in
Group 4 were made wlth 400 mg/ml of aminoglycosideO The re~ulting ~et of
16 sample~ were Arranged in groups of four and esch contained a different
starting proportion of lipid and aminoglyco~ide.
-24-
132g5~8
Freeze-thaw cycle. ~le aminoglycoside:lipld mlxtures were
transferred to the cryovials, whlch were then capped. It was, however,
helpful that ~he cryovlal seal allow for the expanding and contractlng
gasses to vent during freezinæ and thawlng. Each vial wa~ aecured on a
metal e~ender used to dip ~he Ylal in a liquid nitrogen tank.
Each sample was vorteYed ~igorously to miY the lipld with the
aqueous amlnoglycoside. The vial was im~edlately plunged lnto ~ llquld
nitrogen container. To enhance drug-lipit lnteraction~, the samples were
thoroughly mlxed and had A homogeneouq, milky consistency upon freezing.
When a sa~ple was completely frozen (approslmately one mlnute), the
vlal was transferred to a 40C water bath, aud allowed to thaw
completely. After thawing, the Yials were vorte~ed vlgorously and then
Immediately st~rted in the ne~t freeze-thaw cycle by plunglag the vlal
back into the liquit nltroge~ before the phaseG had a chance to eeparate.
A minimum of about five freeze-thaw cycles wa~ required for best
results; howe~er, the entrapment of ~ertain compounds has been shown to
lncrease by lncreasing the number of freeze-thaw cycles to abou~ ten.
The re3ults of the foregoing procedure u~ing gentamicln phoaphate
are ~ho~c ln Figures 1 through 4. For Yariou0 aminoglycoside phosphate~,
thls procedure may be u~eful ln determiuin8 optimal concentraelons of
aminoglyco~ide ~nd optimal aminoglycosld~:llpld ratios. The welghts o~
gentamicin pho~phate are reportet in ~aa~ of actl~e a8ent wlthout
reference to welght of the pho~phate counterion unle~s otherwlse noteta
~
A~inoglycoslde Sulfate ant Amino~cosld2 Phosphaee
.
-25-
- 1329S~8
~ ~ A~ 3~y be 3ee~ ~ro~ IG. j~ lipoaoP~al a~soc1at~orL vf
tobram~rcln in ~he for~ o~ a pno~pha~e WAR 'S~l~ more efflclent thdn
~br~ycin il~ the i~or~ of ~ s~la~e. Tobraulyc~rl Yample~ ~100 m8 ill A
total volume o~ 0.5 ~1) were ~d~u~ted to ~he ~pp~opri~te pH with
pho9phoric acid ~Flg. 5J Isarked wl~h aolid clrcles~ or ~ulf-lric acld
(Fi~. 5, marked wlth aolid ~qua~e~i). The t~br~mycitl w~ then added ~o
egg phosphatldylchvline ~cco:rdi~ to the freeze-~haw l~ethod or E~all~ple ~
utlll~ 3 10 freeze-thaw cycle~. me re~ul~ clearly lndlcs~e tha~ LC ;a
not ~erely the pH ef the ~m~no~lycQ~lde co~t~Inln~ aquqou~ pha~e t~.~t
rP.~uL~s ~r~ greater ~aoclate effieierlcy. Tobr~mlci~ phoophate over the
entire pH range shown in Fig. 5 is mo~e lipo~o~nally a~fiociated than
t~obramyc~n 3ulfa~e. Aa the tobr~lcln ~e titra~ed w~h pho~phori~ ~cld
~ho~pha~e asaociction incre~s3 ~ doe~ l~po~omal a~10~1ation. At pH o~
2.5, ~rappln~ ef~iciencle~ for tobrs~ycin pho~ph~t~ e~ceeded or ar ~:
15 exceeded ~S~ yleldin~ lip~ome di~per~ions havlng c ~obramyci~ phosph~te -
conce~tration of about 0.35 mg per m8 of EPC.
B) ~m~kaci.~ A co~parative te~t of the lfposom~l a030clatlo~ of
~mikacia-S04 ~nd a~ik~cin-P04 ~t pH 2.0 was performed. To form ~he .!
~ulfa~e, the free ba~e of amlkacln in phoephAe~ bu~erea s~llne W~8
tltr~ted ~o pH 2.0 wlth ~2S04. To ~orm che phocphate, amlkaci~ ln
pho~phate buffered ~aline wa~ tltrated to pH 2.0 with ~S~ ~3P94
~wet~ht/~olume). ~mlkacin ~ocla~lcn w~c co~p~ret at ~bout 10, 37, S0
a~d 80 ~8- ~iposomes were then prepared by the SPLV ~e~hod o~ ~enk et
al. U.8. Patent No. 4,522,803. A~lkdcin/lipo~o~l a~oci~t~on W~6
determln~d by ope~trophoto~etrlc aa~ay a~d was ~n each ~n~t~noe at le~st
~bou~ li ~ ter for the ~ikscln phosphate sal~ or~ ~h~n ~or the
a~lkacin ~ te form.
Ex~m~
~ C~'~
8 to L~id R~tlo Llpo60me~
.,
-26-- ~
,.
. .
'
'
-`` 1329~8
Preparation_of Precursor Li~osomes
One g of lipid (egg phosphatidyl choline "EPC-') was dried to a film
in 500 ml round bottom flask. The lipid was resuspended using about 50
ml of methylene chlorlde.
An aqueous solution of aminoglycoside was added to the ml~ture. The
aqueous solu~ion was 0.5 gm gentamicin sulfate (act. ~t.~ in 9 ml of 0.9%
saline (weight/volume). The resulting mixture was agitated by stirring.
This process created precursor llposomes.
Drying, Rehydration, and Standing Pro_ess
The lipo80mal ml~ure was stirred under nltrogen atmosphere. '~he
pressure was reduced but maintalned below bolling until the sample was
dried to a powder. Thi8 proce~6 took about 4 to 5 hours at 40~C. The
mixture was rehydrated with about 9 ml of distilled water and 41 ml of
O.9Z saline ~weight/volume) under nitrogen. The mixture was stirred
until a milky color wa~ achie~ed with no aggregates or clumps of
materlal. Thls procedure took 2 hours at about 40C. The mixture was
left standing or stsbilizing at 4C for one day.
Dialyzin~
The ~ample was then sized to about 3um by extrusion at pressures of
up to about 700 psi through ~ polycarbonate straight path ~embrane
filter. After flltration the material was dialyzed about 2 day~ against
0.9% saline (welght/volume). The dialysis removed subet~ntlally all
aminoglycoside not lipo803ally associaeed. The result~ of this proce~s
are ~hown in Table~ 1 and 2~ Table 1 ~ho~s the enhanced eficiency of
association of the lipid with the amlnoglycoside by this procedure versu3
not drying the liposomes to powder and not dlalyzing but centrifuging off
unassociated aminoglyco~ide.
Table 2 shows the enhanced aminoglycoside to lipid ratio attained by
the use of the procedure versus not drying the liposomes to powder and
not dlalyzing but centrifuging off the unassociated aminoglycoside.
:
Table 1 1 3 2 g 5 4 8
Comparative Efficiency of Liposomal Association
of Gentamicin Sulfate
X of Association of Available Genta~icin
. _ ~
First mixture not drled to powder First mix~ure dried to powder
Unassociated gentamicln removed Unassociated gentamicin removed
by Cen~rifuge by Dialysis
27.03 35-3
24.58 72.6
17.27 51.2
22.02 62.0
32.71 41.0
Table 2
Comparative Amount of Gentamicin Sulfate Liposomal
Association in mg Drug/mg EPG (eq. wt.)
mg Gentamicin/100 mg li~id
irst mi~ture i8 not dried to powder Fir~t mixture ifl drled to powder
Unas~ociated gentamicin i8 removed Una~sociated gentamicin i8 re~oved
by Centrifu~e by Dialysis
12.5 17.5
11.5 36.1
11.3 13.0
8.4 23.1
10.8 2~.5
,
- 1329~8
The foregoing examples are merely illustrative of the invention and in
no way limiting. Other examples will be immediately obvious to those
skilled in the art. The i~vention will be limited only by the claims.
The foregoing examples are merely illustrative of the invention and 1~ no
way llmiting. Other examples will be i~mediately ob~ious to those skilled
in the art. The invention will be limited only by the elaims.
-29-
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