CA2056435A1 - Accumulation of drugs into liposomes by a proton gradient - Google Patents

Abstract

The present invention relates to pharmaceutical compositions and methods of making liposome containing compositions exhibiting characteristics of great uptake. This uptake may be greater than what would be expected by the relationship defined by the Henderson-Hasselbach equation. The present invention also relates to lipososmal compositions wherein the liposome comprises in part a membrane-stabilizing component, for example, cholesterol, which exhibits favorable characteristics in preventing rapid release of a pharmaceutical agent selected from the group consisting of quinine, quinidine and diphenhydramine after it has been formulated in liposomes. The present invention also relates to novel liposomal compositions comprising the bronchodilators metaproterenol, isoproterenol and terbutaline. The present invention also relates to minimum buffering capacity required to achieve liposomal encapsulation of pharmaceutical agents with maintenance of a major portion of the initial pH gradient.

Description

WO ~/14~05 PCT/US90~02736 2 ~

Açç~ul~ion o~ ~r~q~ lnt~ ~iDoso~e~ a~Pro~n Gr~ient Fiçld Qf ~e~Inven~iqn The pra~nt inv~ntion relates to ph~rmaceutlcal compoqi-tions and methods of making liposome containing compositions which exhibit chara~teristics o~ uptake which may be qreater than expected by the relationship de~'ined by the Henderson-Hasselbach equation.
The present invention al50 relates to liposomal composi-tions wherein the liposome comprises in part a mem~rane-stabilizing component, for example, cholesterol, which exhibits favorable characteristics in preventing rapid release o~ a pharmaceutical agent after it ha~ been ~or~ulated in liposomes.
The present invention also relates to novel sustained release lipo~omal composition~ comprising the bronchodilators metaproterenol, isoproterenol an~ terbutaline.
The pre~ent invention also relates to minimum ~ufferin~
capacity re~uired to achiPve liposomal encapsulation of phrmaceutical agents with maintenance of a major portlon of the initial pH gradient.
aackq~Qund o~' thç Invention The th~rapeutic properties of many drugs may he dramati-cally i~p~o~d ~y th~ ad~inistration in ~ lipo~omally encapsu-lated fo~ ~S~Q, for ex~mple P.N. Shek and R.F. ~arber, Mod. ~ed.
~ , 41, 31~-332, (1986)]. In certain ca~es, for example, in th~ ad~inistration of amphotericin B and doxorubicin ~Lopez-Bernstein, et al., ~ Li_, 151, 70~-710, (198~) and ~ah-man, et al., Ç3l~a~ ~, 40, 1532 (1980)] toxicity i5 reduced while e~'~'icacy is maintained or even incr~as~d. The Senefit wV~/1~
2 ~

obtain~d ~rom liposomally encapsulated ag~
and liXely results from the altered pharmc biodistribution of th~ entrapp~d drug [Ost o~. Phanml, in pr~q.
The pharmacokinetics and biodistr:
drug will largely depend on the character Optimization of a lipsomal drug requires c ber of variables, including vesiclQ SiZQ, drug to lipid ratio. Most drug loading p2 not permit the independent variation o~ t~
which are passively trapped in liposomes drug to lipid ratios as the liposome sizq change~ in the trapped volu~e.
Several biogenic amines and antin~
been shown to acc~ulate in liposomes in proton gradient known as "remote loading"
Mayer, et al., ~ :=SI~L .KLLL1L ~ S ~ ~5~
et al., ~iochemistrv, 27, 2053, ~l988) anc C~e~ PhYs. LiPids, 4~, 97, ~l9~)]. Thi~
allows independent variation of any of the :
Much highQr drug to lipid ratioC can be ach:
conv~ntio~l t~chniqu~s [~ayer, et al- S~
333 (l986)~. In ~ddltion, the transr~e~brz drug is generally d~tarmined by the protor lates drug l~akag~ by ch~nges in the buff~
intrav~sicular ~ediu~. Th~ USQ 0~ pro1:on to trap drugs which are non-zwitterionic shown to b~ practic~l for adriamycin, thQ

WO90/14105 PCT/US90/027~
- 2 ~ ^3 dibucaine and dopamine and other drugs. Advantages o~ this system include ef~icient drug trapping, slower rate~ o~ drug releas~ than passively trapped drug, and higher drug to lipid ratios than can otherwise be achieved. In addition, because the liposomes can be prepared in the absence of the drug, problems with drug release during storage, or drug degradation during liposo~al preparation can be avoided.
Intraliposomal drug accumulation in respons~ to pH gradi-ents is believed to occur in a manner similar to that of other weak bases, for example, the pH gradient probe methylamine.
Methylamine equilibrates across liposomal membranes in the uncharged foru, and re-ionizes according to the Henderson-Hasselbach relationship of the pH o~ its environment. The equi-librium distribution reflects the transmembrane pH gradient, and its redistribution can be used to measure these gradients.
However, not all pharmaceutical agents which possess the capacity to be ionized according to Hend~rson-Hasselbach rela-tionships a~cumulate in liposomes according to this relationship.
In fact, certain agents do not seem to accumulate at all. In addition, certain agents which may accumulate according to this relationship immediately undergo release, resulting in unworkable pharmaceutical formulations which must b~ used i~metiately after production and ~hich are virtually unusable a~ ~ustained release product~.
LiposomQs are completely clo~ed lipid bilayer membranes which contain entrapped aqueous volume. Lipo omes are vesicles which may be unilamellar (~ingle membrane) or multilammelar (onion-liXe structures characterized by multiple me~brane WO ~/141~ PCT/US~/027~

2 ~ ~ CJ .
bilay~r~, each separated ~rom the next by an aqu~ou~ layer). The bilayer is co~posed o~ two lipid monolayers having a hydrophobic "tail n region and a hydrophilic "head" rQgion. In the membrane bilayer, the hydrophobic (nonpolar) "tails" o~ the lipid ~onolayers orient toward the c~nter o~ th~ bilayer, whereas th~
hydrophilic (polar) "heads" orient toward the aqu~ous phase. The ~asic structur~ of liposomes may b~ made by a variety o~ techni-qu~ known in th2 art.
Liposomal encap~ulation could pot~ntially provid~
numerous beneficial e~f~cts ~or a wide variety o~ pharmaoeutical agents and the remote loading technique should prov~ instrumental in realizing th~ potential of liposomally en~apsulate~ agents.
In addition, a high trapping e~ficiency ~or loading liposomes r2sults in v~ry little drug being lost during the encapsulation process, an advantagu that prov~3 to be i~portant wh~n dealing with expen~ive drug~. How~vQr, th~ us~ o~ liposom~ to administer drug~ has rai~ed problem~ with r~g~rd both to drug encapsulation and drug rQlQas~ during thQrapy. For example, even with the present us~ of ~remote loading" syste~s, there is a con-tinuing need to incr~ase trapping e~iciQncies so as to minimize the lipid load pr*~nt~d to thG patient. S~condly, eY~n with increa~d-trapping ~iciencie~, th~ro i8 no guarante~ that the releas~ ch~ract~ri~tic~ o~ the load~d liposome wlll re~lect acc~ptablQ sustain~d rQlease characteristics. Many drugs have b~en ~ound to bc r~pidly rel~aY~d ~rom lipo~o~es a~t~r encapsula-tion. Such rele~so r~duces th~ b~neSiclal a~octs of liposomal encapsulation.

WO 90/1~05 PCr/US90/02736 2~ ?
Oblects Oe the P~esent In~ention It is an object of the present invention to provide novel liposomal compositions and general methods for maXing such co~-positions which are designed to maximize the uptake of a pharma~eutical agent into the liposome, thereby increasin~ the amount of drug which can be loaded into liposomes and deoreasing the lipid load presented to the patient du-ing administration o~
liposomes.
It is a further object o~ the present invention to pro-vide novel liposomal compositions and methods for ma~ins such compositions which prevent the rapid disadvantageous release of pharmaceutical agent before administration of the liposomes.
It is a further object of the present invention to pro-vide novel brochodilator liposomal compositions.
8rief Dçscri~tion o~ the FLqurç~
Figure 1 shows the accumulation of mitoxantrone by EPC
vesicles exhibiting a proton gradient with an internal aqueous buffer system comprising 300 mM citrate, pH 4.0 and an external buffer system comprising 300 mM NaCl, 20 mM HEPES, pH 7.5.
Accumulation wa~ rapid and complete and evidenced no release over several hours.
Figure 2A shows the response o~ timolol uptake in EPC
vesicles (partial accumulation, uptaXe stable). ~he level of uptake is about 100 nmoles/umole (about S0% of available drug).
Figu~e 29 shows thP response of quinamrine, which is similar to ~i~olol in EPC vesicles. The level of uptake is about 80 nmoles/umole lipid after 30 minutes.
Figur~ 3A shows the response of quinidine uptake in EPC

WO ~l4l~ PCT/US~/027~

2~6~?~i vesicles ~co~pl~te accumulatian, rapid r~ s~3. Within 30 minutes about 50% o~ the druy leak~ back out o~ the ve~icles.
Figure 3B shows the e~ect o~ added chole~t~rol to the uptake of quinidins and the stability oX th~ pH gradient.
Figure 4 shows the ef~ect of phy~ostigmine tq transmem-brane pH gradient. Under the conditions used to assess drug uptake 5200 uM physostigmine) only a small decrease in the measured pH is observed.
Figure 5 shows the entrapment o~ metaprot~renol, ter-butaline and isoproterenol in response to p~ gradients using egg phosphatidylcholine 200 nm extruded liposom~s.
Figure 6 de~onstrates tXe ef~ect of drug uptake on the residual pH gradi~nt as measured by mQthylamine redistribution in the pre enc~ and absenco o~ isoproterznol. A~ shown, when the inte~nal ~nd ext rn~l pH i9 7.4 or 4.0 (no gradient), the methylamine doe3 not detect any pH gradiQnt.
Figure 7 shows the effect of temp~rature on the Entrap-ment of Metaproterenol in response to pH gradients at 21'C, 37 C
and 60-C.
Figur~ 8 ~hows th~ influenc~ of choleqterol on the accu~ulation Gf ~t~proterenol in respons~ to a p~ gradient.
Figur~ 9 -~ho~ ~ho influenco of varying the external druq conc~ntration on thQ lQvel of ~etaproterenol uptake.
FigurQ 10 shows th~ ef~ect o~ intarnal buffering capacity on drug uptake.

The present invention relates to liposo~al compositions ha~ing a p~ gradient whic~ exhibit markedly increased accumula-WO ~/14l0~ PCT/US90/02736 tion of pharmaceutical agents abov~ that expec~ed ~rom t~Henderson-Hasselbach relationship by ~ormulating the liposomes utilizin~ a ~irst internal aqueoua burfer ~nd a sacond external aqueous bu~fer wherein the conc~ntration of the pharmaceutical agent exceeds its solubility product in the internal bu~fer fol-lowing uptake. Therefore, pre~erably, the pharmaceutical agent exhibits a solubility within th~ liposome which is less than the final concentration of agent within the liposome. Preferably, the solubility of the phar~aceutical agent is less than about 20 mM and most preferably less than about lO mM. In addition, the internal buffer solution has a buffer strength of at least about 50 mMol, preferably about lO0 to about 300 mMol and ~ost preferably about 300.
The present invention also relates to liposomal composi-tions comprising in part, mem~rane-stabilizing components, for exa~ple, cholesterol, among other lipids, to prevent the rapid release of certain pharmaceutical agents from liposomes which do not contain the membrane-stabilizin~ components. SUch liposomes preferably comprise a miXtUrQ of phosphatidylcholine and cholesterol in a molar weight ratio of about 55:45.
The present invention also relates to liposomal composi-tions having a pH gradient containing bronchodilators selected from tha group consisting of metaproterenol, terbutalinD and i~oproteren~l. It has be~n shown that the above agents, which heretofore hav~ not be~n formulated in such liposo~es, will accumulate into liposomes to an apprecia~le extent to produce effective, stable liposomal composition~ us~ful ~or trea~ing con-dition~ re~uiring sustained releaso of bronchodilators. Such WO ~/1410~ PcT/Us9o/o27 203~ ,.?.~

liposo~al compo~itions comprising bronchodilator ~or~ulations may be usaful ~or treating a nu~ber o~ condition~, includin~ asthma.
Such compo~itions are exp~cted to hav~ a lonq~r re~idence tim~ in the lung than the same ~ree drug, thus obtaining concentrations of bronchodilator at the site o~ activity within the lung for a period longer than for compositions pre~ently a~ailable. Such compo~itions may be formulated a~ ae~osols within a pharmaceuti-cally acceptable solution ~or ad~inistration o~ bronchodilators dir~ctly into the lungs for tr~atment o~ acute asthma attacks.
The brochodilator compo~ieions o~ th~ present invention have b~en shown to ef~ectively accumulate in lipo~omes comprisin~
egyphosphatidylcholine (EPC) as well a~ a ~ixtuse of phosphatidylcholine ~nd cholesterol t55:45, w:w). The liposomes comprising the mixture of phosphatidylcholine and cholesterol, accu~ulate thQ bronchodilator to about th~ s~ma relativ~ extent as the EPC liposom~s, although the time need ~or accu~ulation is longer for ~he cholesterol containing lipo50~s.
In g~n~ral, the liposo~e composition~ o~ the present invention have a drug to lipid molar ratio r~nging fro~ about 0.5~ up to about 50~. Tha liposome3 o~ th2 present in~ention may compris~ phospholipids such as egg pho~phatidylcholine (EPC), hydrog~t~d ~oy phoaphatidylcholine, dist~aroylphospha~idyl-cholin~, di~yri~toylpho~phatldylcholinQ, or diarachidonoyl-pho~ph~tidylcholins, a~ong oth~rs, and m~y additionally comprise a nuDber o~ st~roidal compositions, as w~ll as other co~posi-tion~.
In gen~ral, th~ liposo~e~ range in 5iZ8 fro~ about 0.05 to gr~at~r than 2 ~icron~, with a prefQrred ran~e being about W~90/1~105 PCT/US90/0273~

O. 05 to about O. 3 microns. Mo~t pr~2~raoly, th~ lipo~om~3 ar~
unilamellar and range in size from about 0.1 to about 0.3 microns. These unilamellar llposomes may be homogeneous or unimodal with re~ard to 5iZ~ distribution.
Tha liposomes of the present invention may be administered via oral, pare~teral, buccal, topical, and trans-dermal routes of administra~ion, among other routes of adminis-tration.
Qetailed ~çscr~ption o~ ~he InventiQn The presant inve~tion utilize~ ef~icient trapping of phar~ac~utical agents in liposomes exhibiting a transmembran~
ionic gradient, preferably a trans~ambrane p~ gradient, which can result in an accumulation o~ the agent in an amount signi~icantly higher than otherwise expected ~rom the Hend~rson-Has~elbach relationship. Liposom~ compositions Or the present in~ention comprise at least one lipid, a phrmaceutical agent accumulated therein, an internal buffer solution wherein the solubility of the pharmaceutical agent within the bu~fer solution is less than the concentration o~ the agen within the liposome and an ex~ernal buffer solution wherein the solubility of th~
pharmaceutical ag~nt is pre~erably at least about 0.2 ~M. As used throughout tho specification, the terms pharmaceutical agen and drug are synonymous.
~ he liposom~s o~ th~ pre~en~ invention may be formed by any o~ the ~ethod~ known in the art, but pre~erably they are for~d according to t~e procedures disclosed in Ball~y, et al., PCT Application No. US86/01102, published February 27, 1986 and Mayer, et al. PC~ Application No. US88/00646, published Sep-WO ~/~4105 PCT/US90/0~7 1 0 ' ' --2 ~
temb~r 7, 1988. These techniques allow the loading o~ liposomes with ionizable pharmac~utical aqents to achieve intarior con-ce~trations con~id~rably gr~ater than othorwise sxpected ~rom the dru~s' solubility in aqueous solution at neutral pH and/or con~
centration~ greater than can he obtained by passive entrapment techniquQs. In this technique, a transmembrane ion (pH) gradiens is created between the internal and external membranes o~ the liposomes and the pharmaceutical agent is loaded into the liposomes by means o~ the ion (p~) gradient, which drives the up~ake. The transmembrane gradient is generated by creating a concentration gradi~nt for one or mors charged species, ~or exam-pl~ Na+, C1-, R+, Li+, OH- and preferably H+, across the liposome me~brane~, such that the ion gradi2nt drive~ t~a uptake of ionizable phar~aceutical agQnts across the membranes. In the present invention, tran~embrane ion (~+) gradients are prefQrably employed to produce the ion gradient and load t~e pharmaceutical agents, which tend to haYe weakly basic nitrogen groups, into the liposomes.
In the present invention, liposo~ea are preferably -st for~ed in an aquQous buffer solution. The first solution is ~ither acidic or basic, depending UpQn whether th~ pharmaceutical agant to ~ load~d produces a charg~d sp~cie~ at ba~ic or acidic pH~ For example, in the case of loading weakly basic pharmac~utical ag~nt~, a charged specieC is produced at low pH, i.e., a pH of about 2.0 to 5.0, preferably a pH o~ about 4Ø
After formatio~ o~ lipo~omes having an acidic int~rnal aqueous buf~er solution, thQ bu~fer solution external to the liposomes is thRn ~odi~ed to ~ pH significantly abov~ th~ pH o~ the internal Wo ~/14105 PCT/US9~/02736 2 Q ~37~ 3 bu~fer solution, preferably at least about 3.0 to 4.0 pH units abov~ the internal buffer solution. Th~ modification o~ the axternal bu~f2r results in a pH gradient which drives the accumulation of pharmaceutical agent within the liposome. The internal buffer solution may differ from the external buffer solution only in the difference in pH. In general, uncharged pharmaceutical agent will pass through the lipid layer(s) of the liposome much more readily than will charged (protonat~d, in the case of weakly basic pharmaceutical aqents) agent. Thus, uncharged phar~aceutical agent in the external buffer will readily pass through the liposome into the internal buffer, become protonatedt and remain wi~hin the liposome as a ~trapped~
protonated molecule which does not readily pass throush the liposome layer(s). Pharmaceutical agent will thus concentrate in the liposom~ as a function of the pH gradient b~tween the inter-nal and external buf~er solutions.
Such loading according to the above procedure, while effective for certain pharmaceutical agents, often does not result in maximum loading. ~ven if it is assumed that the pharmaceutical agent is maximally soluble in the internal and external buffer solutions and will readily pass through the liposomal layer(s) (an assumption not always borne out by reali~y), the maxi~u~ loading will generally reflect the rela-tionship de ined by the Henderson-Hasselbach equation [HA+]in/[HA+]out~ [H+]in/[H+]out- However, a number of fac~ors are belie~ed to effect the ability o~ a pharmaceutical agent to accumulate. These factors include the partitioning of the unprotonated agent within the lipid layers(s), the difference in WO ~/141~5 PcT/US9o/o2~

20~S.,~ 2~j P~a b~twH~n protonated spoci~s that exist in the bu~er solution and specie~ associatod wit~ th~ membrano, ~hs bu~er capacity o~
tha internal buffer solution and the solubility of the protonated species within the internal buffer.
It ha~ now been deter~ined that th~ moct important ~actor influencing th~ accumulation o~ phar~aceutical agent within a liposome abovQ what is expected ~rom the ~enderson-Hacselbac~
equation is the solubility o~ th~ protonated species o~ the agent within the internal bu~er solution. It has been determined that th2 solubility o~ tha protonated species o~ thc pharmaceutical agent to be accu~ulated will influence thP level o~ uptake and accumulation w~ich may be substantially greater than that predicted by tha Henderson-Hasselbach equation. ~iposo~e com-positions which are formulated using an internal buf~er solution in which an ioni~ed pharmacautical agant i~ minimally soluble and which preferably praCipitatQs the ionizad ag~nt, will drive the accumulation o~ the pharmaceutical ag~nt within the liposome ~eyond what would otherwisQ be expected to produc~ liposomes which consistently should have high trapping efficiencies approaching 100%.
In th~ prQ ent invention, it ha~ surprisingly been dis-coY~red ~hat th- ~ol~bility o~ th~ pharuac~utical agent in th~
internal buffer ~ay ultimately control ~he ability of the pharoac~utical agent to lo~d into th~ liposo~ to an extent grea~er than that pr~dicted by the Henderson/Hasselbach relation-ship. Th~s, in th~ present invention, liposome compositions are pre~err~d which are ~ormed utilizing a first internal bu~fer solution of either basic (pH about 8 to lO) or acidic (pH about ~ u~ PCT/US90/027~

2 ~ 3 3.0 to 5.0) charact~r and a second ext~rnal buffer solution, the pH of which i~ preferably b~tween about 6.5 and A.0, preferably 7.4. The high or low pH of the int~rnal bu~rer relative to a neutral pH of the ext~rnal bu~er produc~ a transmeMbrane gradi-ent which acts to drive the accumulation of the agent in the liposome. It has surprisingly been discovered that the most i~portant factor in det~r~ining the ultimate amount of agent which may be loaded into liposom2s using the transmembrane gradi-ent to dri~e the accu~ulation of the~agent into the liposomes above that expected by the H~nderso~-Hasselbach equation is the solubility o~ the agent in the internal buffer solution.
In general, internal buffer solutions useful in embodi-~ents of the present in~ention are chosen so that ths phar~aceutical ag~nt to be accumulated has a solubility within the internal bu~fer solution which is les~ than the total agent to be accu~ulated in th~ liposo~e. Generally, the solubility o~
the phar~aceutical agent in the internal bu~fer solution is no greater than about 65 ~M, pr~ferably no greater than about 20 mM
and most preferably no ~reater than about lO mM.
The internal buffer solution is also chosen to maximize the buffer strangth of 'the internal solution. It is believed that the bu~fer strength of the internal buffer solution is also i~portant to the total accumulation o~ agent within the liposome and internal bu~er solutions are chosen to maximize this strength. 0~ course, the solubility of ~h~ a~ent within the internal buffer solution is also a most important factor in determining accumulation. Therefore, wherQ a bu~rer solution is to be chosen, it is both the solubility ~actor and the buffer W~ ~/l4l~ PCT/US90/02736 strength factor which should be maximizad in choo~ing useful bu~fer solutions. In the present invention, it has been determined that the bu~fer strength o~ the internal bu~r solu-tion should be at least about S0 mM, preferably about lO0 mM to about 300 mM and most preferably about 300 ~M.
Lipids which can be used in the liposome ~ormulations of the prQSent inYention includ~ ~ynthetic or natural phospholipids and ~ay include phosphatidylcholine ~PC), phosphatidylethanolamine (PE?, phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol(PI), sphingomyelin (SPM) and cardiolipin, among others, either alon~ or in combination. The phospholipids useful in tho present inv~ntion may also include dimyristoyl-phosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). In other e~bodiments, distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), or hydrngenated.
soy phosphatidylcholine (HSPC) ~ay al90 be used. Dimyristoyl-phosphatidylcholine (DMPC) and diarachidonoylphosphatidylcholine (DAPC) may similarly be used. Due to the elevated transition temperaturres (Tc) of lipida such as DSPC (Tc of about 65-C), DPPC (Tc of abol~t 45-C) and DAPC (Tc ~ about 85-C), such lipids are pref~r~bly h~ate~ to about their Tc or te~peratures slightly higher, e.g., up to about 5'C higher than tho Tc, in order to maXe theso lipo~om~. In pr~ferred ~bodim~nts, egg phosphatidylcholin~ i~ uced.
In a ~umb~r o~ e~bodi~ents oP the pr~ont invention, a steroidal componant may bQ added to th~ liposome. Any o~ the a~ove-mentioned phospholipids may be used in combination with at W~ ~/14l~ PCT/US~/027 205~'~?,5 least ona additional component selected ~rom ~he group consisting o~ chole~tarol, cholestanol, coprostanol or cholestane. In addi-tion, polyethylene glycol derivatives o~ cholesterol (PEG-cholesterols), a~ well a organic acid d~rivativ~s o~ sterol-~, fo~ example cholesterol hemisuccinate tC~S) may also be us~d in combination with any o~ the above-mentioned phospholipids.
Organic acid derivatives of alpha-tocopherol hemisuccinate, (THS) may also be used. CHS- and THS-containing liposomes and their tris salt forms may generally be prepared by any method known in the art ror preparing lipsomes containin~ sterols Any o~ the above-mentioned sterols ~ay be used in liposomes, 50 long as the rasultant phospholipid-sterol mixture yields stable liposomes.
In particular, sae the procedures of Janoff, et al., U.S. ~atent No. 4,721,612, issued January 26, 1988, entitled "Steroidal Liposomes", and Janoff, et al., PCT Pu~lication No. 87/02219, published April 23, 1987, entitled "Alpha Tocopherol-8ased Vehicles", relevant portions o~ which are incoporated by reference herein.
In cPrtain embodiments in which the liposomes are designed to prevent rapid release of the pharmaceutical agent, cholesterol in an amount equal to about 30 mole% tu about 45 mole~ by waight o~ the lipid comprising the liposome is preferably used in combination with any of the above named phospholipids or phospholipid/steroid co~binations. Such com-position should, in general, prev~nt the undesired rapid release of accumulated ph~rmaceutica} agent f rom the liposome. Any com-bination o~ ~embrane-stabilizing component and lipid may be used which prevents rapid release o~ pharmaceutical agents ~rom the WO ~tl4l05 PCT/US~/021~
2 o 5 fi L~ 3 liposome, and one o~ ordinary skill in the art will be able to modify the membrane-~tabilizing component and th~ phospholipid to ~or~ulat~ liposomas which prev~nt rapid ralsas~ Or th~
pharmaceutical agent. Mos~ pre~erably, liposomes comprising a mixture o~ about 45 mol~ ~ by wei~ht choles~erol and about 55 mole % by weight phosphatidylcholinQ are usQd in this aspect o~
the present invention. Althou~h a~y number of pha~maceutical agents which show a proclivity to r~lease rapidly ~rom liposomes may be used in thiC aspect of the present invention, it has beer determined that th~ agents quinine, diphenhydramine and quinidir.a are ~specially prone to rapidly release from liposomes and thus liposomal formulations comprising these agents preferably com-prise cholesterol in an amount equal to about 30 to 45 mole ~ and preferably about 45 mol~ % of the lipid plu8 me~brane-stabilizing componen~. Although it is di~icult to deter~lnQ, strictly on th~ basis of chemical structur~, that a phar~aceutical agent will rapidly release from a liposomal for~ulation, one o~ ordinary skill in the art will be able to assess the degree of release of the agent and formulate a liposomal product consistent with the teachings of the present invention. As in other embodiments of the present invention, any buffer solution may ~e used ~or the internal a~d external bu~f er solutions in this a~pact of the pr~ont inYention regardless of the solubility of the phar~æc~utical ag~nt ther2in. However, tha pro~erred bu~er solution~ are choc~n 80 that tha solubility of the pharmaceutical agent is less ~han the concentration of t~ agent within th~ .
liposome, pre~erably is less than 20mM and ~o~t pre~erably is less th~n 10 mM a~ is the case with other embodiments o~ th~

WO ~/14105 PCT~US90/02~
.t7 pr~sent invention.
Several methods may be used to ~or~ th~ liposomes of the present invention. For exa~ple, ~ultilamQllar ve~icies (MLVs), stable plurilamellar vesicles (SPI.Vs), or rev~rse phase evapora-tion vesicles (REVs) may be used. Pref~rably, however, MLVs are extruded through filter~ forming large unilamellar vesicle~
(LW s) of sizes dependent upon the filter size utilized. In gen-eral, polycarb~nate filters of 30, 50, 60, lO0, 200 or 800 nm por~s may b~ used. In this method, disclosed in Cullis, et al,, PCT Publication ~o. W0 86~000238, ~anuary 15, 1986, relevant por-tions of which are incorporated by reference herein, the liposome suspension may be repeatedly passed through the extrusion device resulting in a population of liposome~ o~ homog~neous size dis-tribution. For example, the filtering may be performed through a straight-throuqh msmbrane filter (a Nucleopore polycarbonate fil-ter) or a tortuous path filter (e.g. a Nucleopore filter mem-brafil filter (mix~d Gellulose esters) of O.l u~ size), or by alternative size reduction techniques such as homogenization.
Tha size of the liposomes may vary from about 0.03 to above about 2 micron~ in dia~eter; pre~erably about O.OS to 0.3 microns and most preferably abo~t O.l to about 0.2 microns. The size range include~ liposom~ t~at are MLVs, SPLVs, or LW s. In the present invention, the preferr2d liposomes are those which are unilam-ellar liposom~s o~ about O.l to about 0.2 microns.
As described hereinahove, a nu~ber of lipid~ may ~ used to form liposomes having a gel to li~uid crystalline Tc above ambient te~perature. In such case~, an extruder having a heating barrel or thermojacket may b~ employed. Such a device serves to .

WO ~/1~105 PCT/US~/02~
1 ~3 2 ~ 5 increa~ the liposome suspension temperature allowing extrusion of the LUVs. The lipid~ which are us~d with the thermoj~cXeted extruder ars, for axample, DSPC, DPPC, DMPC and D~PC or mixtures thereof, which may include cholasterol in certain embodi~ents ~or preventing the rapid relea~a o~ pharmaceutical agents ~rom the liposome. ~iposomos containing DSPC are generally extruded at about 65-C, DPPC at about 45-C and DAPC at about 85-C (about S C
above the lipid Tc).
As indicated, the preferred liposome for us~ in the pres-ent invention are LUYs o~ about 0.06 to about O.3 microns in size. A~ defined in the present application, a homogeneous popu-lation of vesicles is one comprising substantially the same size liposome~, and ~ay ha~ a Gaus~ian distribution o~ particle 5iZ~. Such a population is said to be of uniSor~ size distribu-tion, and may b~ unimodal with respect to -~iZQ. The term "uni~odal~ referY to a population having a narrow polydisper~ity of particle sizes, and the particles are of a single "mode".
A liposomal population is unimodal if, when measured by quasi elastic light scattering methods, the population approxi-mates to a Gaussian distribution, and if a second order poly-nomial will fit the natural loga-ithm of the autocorrelation function o~ a ~a~pl~ (Xoppel, 19~2, J~_~he~ P~., 57:4814).
~he clo3er this ~it, the better the measura of unimodality. The clo~enes~ o~ thi~ ~it ~ay be deter~ined by how clo3e the chi sguare (chi2) Yalu~ of the sample is to unity. A chi2 value of 2.l) or less is indicative of a unimodal population.
Other size reduction techniques may be employed in prac-tiing the present in~ention. For eXampl2, ho~genization or mill-W090/141~ PCT/US90/~t7~
~9 2 ~

ing technique5 may success~ully be employed. Such techni~ues mayyield liposome~ that are homogeneous or uni~odal with regard to size distribution.
Liposomes ~ay be prepared which encapsulate the first aqueous buf~er solution having the characteristics described hereinabove. For a typical liposome preparation technique as fully described hereina~ove, this ~irst aqueous buffer solution will surround the liposomes as they are formed, resulting in the buffer solution being internal and external to the liposomes. To create the concentration gradien~, the original external buf~er solution ~ay ~e acidified or basified so that the concentration of charged species differs ~rom the internal bu~fer, or alterna-tively, the external bu~fer may be replaced by a new external mediu~ having different charge species. The replacement of the external medium can be accomplished by various techniques, such as, by passing tha liposome preparation through a gel filtration column, e.g., a Sephadex column, which has been equilibrated with the new medium, or by dialysis or related techniques.
During preparation of the liposo~es, oryanic solvents may also be used to suspend the lipid~. Suitable organic solvents for USQ in the present invention include those with a variety of polaritie~ and di~lectric properties, which solubilize the lipids, for exampl~, chloroform, methanol, et~anol, dimethylsul-foxide (DMSO), u~thylene ~holo~ide, and solv~nt mixtures such as benzene:methanol ~70:30), a~ong others. As a result, solutions (mixture in which ths lipids and other components are uniformly distri~u~ed throughout) containing the lipids are formed. Sol-vents are generally chosen on the basis of their W~ ~/l41~5 YCT/US90/027~

~3~ ~33 biocomp~tabllity, low toxicity, and solubilization abilities.
One pre~erred e~bodiment o~ th~ present inv~ntion is a 3 co~ponent liposomal-pharmaceutical agent tr~atment sy~te~ which allows for highly ef~ici~nt entrap~ont Or the agent at the clini-cal site. When the pharmaceutical agant i~ one that loads in r~sponse to a transmambrane pH gradient wh~r~ the interior o~ the liposome is acid, the ~irst component o~ thQ system (Vial 1) com-prises liposo~es in an acidic buffer solution, in whi~h for exam-ple, citric acid bu~fer (300 mmoi., pH about 3.8 to 4.2, preferably 4.0) or another bu~fer in which the ionized ~orm of the pharmaceutical agent to be trapped is only marginally soluble (solubility less than the final concentration of agent within the liposome, preferably no greater than about 20 mM and most preferably no grnater than about 10 mM). ThOE sQcond component o~
the system (Vial 2) comprises a ba~ic buffer solution, ~or exam-ple, a sodium carbonate or sodium bisphosphate solution at about 0.5 M, pH about 10 to 12, preferably abcut pH ll.S, which serves to become part oS th~ external bu~fer solution of the liposome formulation. For purposes of maximizing the loading of the pharmaceutical agent within the liposomes it is preferable that the phar~aceutical agent ha~ a solubility within the external bu~Ser solution o~ at least about 0.2 ~ol. The t~ird component (Vial 3) is the phar~aceutical agent. The above-described treat-ment syste~ may bo provided as a 3-vial system, th~ ~irst vial containing the liposomes in acidic medium, the secon~ vial con-taining th~ bas~, and ths third vial containing the pharmaceuti-cal agent as described hereinabove. A ~i~ilar treatment system may be providPd for a pharmaceutical agent that loads in response W090/14105 2 1 PCT/US~O/Ot736 2 ~

to a transmembrane gradient wheraln the internal bu~er of the liposome~ i3 r~latively ba~ic i.e., has a pH about 8.5-l1.5. The ~irst component comprises liposome~ in a relatively basic bu~fer, for example, sodium carbonate or sodium bisphosphate, at a pH o~
about 8.0-ll.O, proferably about lO. The second co~ponent com-prises a relatively acidic or neutral solution as the external buf~er for the liposomQs, ~or exampla, lSO mM NaCl bu~er/150 m~
HEPES buffer at a pH of about 7.4. The third component comprises the phar~aceutical agant which i~ less ionized at the pH of the external buf~er and is ionized at the p~ of the intPrnal bu~fer.
Following the formation of th~ pH gradient acro~s the liposo~es (by admixing the first and second vials), the liposomes may be heated prior to admixing with the drug. Under certain circumstances, and in cases where the pharmaceutical agent is to be loaded into liposomes comprising at least about 30 mole %
cholesterol to mini~ize the rapid release o~ the agent, it may be advantageous to heat the llposomes up to about 60-C to facilitate loading. To load the pharmaceutical agents into ~he liposomes utilizing the above-described treatment systems, the methods described in Mayer, et al. PCT Publication No. WO 88/06442, Sep-te~ber 7, 1988, relvant portions of which are incorporated by reSerance, h~r~in may b~ modi~ied for USQ with the ag~nts of the present in~ention.
In a lipo30m~-drug delivery system, the pharmaceutical agent is entrapped in or associated with the liposome and then administered to the patient to be treated. As used throughout the specification, pharmaceutical agent, drug and agent are used interchangably. For example, see RaAman et al., U.S. Patent No.

WO90/1~1~ PCT/US90/027~

2 ~ ~ ~ r c~ ..3 3,993,754; Sears, U.S. Pate~t No. 4,145,410: Papahadjopoulos et al., U.S. Patent No. 4,235,871; Schneider, U.S. Patent No.

4,114,179; Lenk ~t al., u.S. Pat nt No. 4, 522, 803; a~d Fountain et al., U.S. Patent No. 4,588,578. In ~he present invention, any number of dif~er~nt pharmaceutical agents and different pharmaceutical types may be entrapped in or associated with liposomes. For example, pharmaceutical agents useful in the present invention may includa any agont which readily passes throush a liposomal layer(s) and ~xhibits limited solubility in a buffer solutian internal to.the liposome at an ion concentration or pH at which the pharmaceutical agent is in an ionized form.
Such agents may include antineoplastics: for example mitoxantrone, local anaesthetics; for example, lidocain~, dibucaine and chlorpromazina, ~ronchodilators; for example, metaproterenol, ter~utaline and isoproterenol, beta-adrenergic blockers, for example propanolol, timolol and labetolol;
antihypertensive agents, for example olonidine and hydralazine;
anti-depressants, for example, imipr.. ~e, amitryptyline and doxepim, anti-convulsants, for exampl6, phenytoin, anti-emetics, for example, procainamide and prochlorperazine; antihistamines, for example, diphenhydramine, chlorpheniramlne and promethazine:
anti-arrhyth~lc agents, for example, quinidine and disopyramide, anti-malarial agents, for example, chloroquine, quinacrine and ~uinine: and an~lqesics, among a number of additional phar~ac~utical aqents.
In general, internal bufSers to be used in the liposomal composition~ of th~ present inv~ntion are chosen using ~everal criteria, the most important of which, after buf~er strength, is W090~1410~ PCT/U~90/027~

the solubility characteristics of the pharmaceutical agent to be loaded in the buffer solution, as described hereinabove. It is preferrQd that th~ bu~r system usQd as the intornal bu~er has a buffer stran~th of at least about 50 mM, preferably within the range of about 100 mM to about 300 mM, and most preferably about 300 mM. The most preferred buf~er solution~ for use as the internal buffer system o~ the present invention are there~ore characterized by their inability to ~olubilize the ionized, preferably protonated pharmaceutical agent, i.e., the ionized pharmaceutical agent is generally soluble in the buffer solution to an extent no greater than about 65 ~Mol, preferably no greater than about 20mMol and most preferably no greater than about 10 mMol and which also have bu~fer strengths of at least about 50 mM, preferably about 100 to about 300 mM, most pre~erably about 300 mM. Mo~t preferably, the internal buf~er solution precipitates the ionized species of the pharmaceutical agent out of solution.
The choice of buffer to use as the internal buffer solu-tion will vary depending upon the pharmaceutical agent chosen for loading. One of ordinary skill in the art will be able to assess the relative solubilities of ionized species of a phar~aceutical agent ~nd the bu~fer strength to deter~ina the bu~er solution to be used a~ the internal buffer solution.
Any buffer solution having the characteristics generally described hereinaboYe may be used in the present invention, pro-vided that ~h~ solution is pharmaceutically compatible, i.e., the solution may be administered to the patient without deleterious a~fects. Typical internal buffer solutions include citric acid, WO ~/1~105 PCT/US~/02736 2 ~ '~ 6 oxalic acid, succinic acid and other organic acid salts being preferred, among othsrs. Citric acid in a concentration ranging from about lOo ~M to about 300 ~M iq preferred. Most preferably, the citric acid buffer solution has a concentration ranging ~rom about 100 mM to about 300 mM. Typical external bu~fer solutions may include NaCl, XCl, potassium phosphate, sodium bicarbonate, sodium carbonate, sodium bisphosphate, potassium sulfate and ~EPES, and mixturs~ the ~of, among othar~.
Loading efficiencies of p~ smaceutical ag~nts utiliziny the present invention generally range from ~bo~t 20~ up to about 100%, preferably at least about 50S. In general, the loading efficiencies for pharmaceutical agents according to the present invention are greater than expected from the Henderson-Hasselbach relationship. 0~ course, not all agents readily accumulate in liposomes according to the Henderson-Hasselbach relationship, and certain agents (see Table 1, Example 1) appear, in certain cases, not to accumulate at all. This phenomenon may be the result of the pharmaceutical agent being too polar for penetration of the liposomes, or other factors. 0~ course, ona of ordinary skill in the art will recognize that to maximize the loading o~ a phar~aceutical agænt into liposo~es, it ~ay be n~cessary to chango ths lipid constituents o~ the liposomes, or, in certain cases, to utilize ionophor2s or other agents which may enhance th~ p2n~tration o~ the liposome by the agent in practicing the present invention.
The liposo~es ~ormed by the procedures o~ the present invention may be lyophilized or dehydrated at variou~ stages of formation. For example, the lipid film may be lyophilized after WO90~l4l0~ PCr/US90/02736 removing the solvent and prior to addinq the drug. Alterna-tively, the lipid-dru~ film may ~e lyophilized prior to hydrating the liposome~. Such dehydration may bQ carried out by exposure of the lipid or liposome to reduced pressure thereby removing all suspending solvent. The liposomes may be dehydrated in the presence of a hydrophilic agent accordinq to the procedures of Bally et al, PCT Publication No. 86/01102, published February 27, 1985, entitled "Encapsulation of Antineoplastic Agents in ~iposomes", Janoff et al., PCT Publication No. 86/01103, pub-lished February 27, 1986, entitled "Dehydrated Liposomes", Schnelder et al., in U.S. Patent No. 4,229,360, issued October 29, 1980 and Mayer, et al. PCT Publication No. 88/06442, pub-lished September 7, 1988, relevant portions of which are incor-porated by reference herein. Alternatively or additionally, the hydrated lipsome preparation may also be dehydrated by placing it in surrounding medium in liquid nitrogen and freezing it prior to the dehydration step. Dehydration with prior freezing may be performed in the presence of one or more protective agents, such as sugars in the preparation according to the techniques of Bally, et al., PCT Application No. 8S/01103 published February 27, 1986, releYant portions of which ara also incorporated by r~ference herein. Such techniques enhance the long-term storage and stability of hte preparations. For example, the pharmaceuti-cal agent may be ~ixed with a sugar solution in a suqar: lipid weight/weight ratio ranging from about 0.5:1 to about 100:1, preferably about ~0:1, without affecting ~he ability of the liposome to retain loaded agent during rehydration. In this aspect of the present invention, the liposomes preferably range WO ~/1~1~ PCT/US9~/0~736 in size from about 0.1 to about 0.2 mlcrons.
In one pr~ferred ~bodiment, th~ sugar is mannitol, or ~annitol:glu~ose:lactose in a 2:1:1 w/w/w ratlo. Following rehydration in distillod watsr, tho preparation is preferably heated for ten minut~s at an elevated temperature, for example 60-C. Other suitabl~ methods may be used in the dehydration o~
the above- disclosed liposome preparations. The liposo~es may also be dehydrat~d without prior freezlng.
once the liposomes have been dehydrated, they can be stored for extended periods of ti~e until they are to be used.
The appropriate temperature for stora~a will depend on the lipid for~ulation of the liposomes and the temperature sensitivity of encapsulated ~aterials. For example, various antineoplastic agents are heat labile, and thus dehydrated liposo~es containing such agents should bQ stored under refrigerated conditions e.g.
a~ a~out 4'C, so that the pot0ncy o~ th~ agent is not lost.
~lso, for such agents, the dehydration process is preferably carried out at reduced te~peratures, rather than at room temperature.
When ~ dehydrated liposome~ are to be used, rehydrAtion is accomplished by simply adding an aqueous solution, e.g., dis-tilled water or an appropriate buf~er, to the liposomes and allowing th~ to rehydrate. The liposo~es can be resuspended into the aqueous solution by gentle swirling o~ th~ solution.
The rehydration can b~ performed at room te~perature or at other te~peratuses apprspriate to the composition o~ the liposomes and their int~rnal contents. I~ the antineoplastic agent which is t_ be administered was incorporated into th~ high drug to lipid WO90~14105 PCT/US90~27~

2 i~ 3 ratio liposomes prior to dehydration, and no further compositien changes are desired, the reAydrated liposomes can be used directly in the cancer therapy ~ollowing ~nown procedures for administering liposome encapsulated drug5. Alternatively, using the transmembrane pH gradient procedure~ described above, ionizable antineoplastic agents can be incorporated into the rehydrated liposomes just prior to administration. In connection with this approach, the concentration gradient used to generate the transmembrane pH gradient can be created either before dehydration or after rehydration using the external medium exchange techniques described above. For example, the high drug to lipid ratio liposo~es may be dehydrated prior to establishing the transmembrane pH gradient, for examplQ, dehydrated from their first external medium. Upon rehydration, the pH gradient can be established ~y adcixing the liposomes with the second external medium of relatively acidic or basic pH. The antineoplastic agent can be admixed with the liposomes simultaneously with or following the establishment of the pH gradient.
In the case where the liposomes are dehydrated after having a transm~brane pH gradient, the liposomes may be rehydrated by ad~ixing them with an aqueous solution of neutral pH.
For exa~ple, in the above-mentioned case where liposomes containing citric acid buffer as the first medium are used, the rehydration step would proceed by adding sodium carbonate and the pharmaceutical agent, for example, propanolol. Where the liposomes already contain the base (e.g. sodium carbonate), and therefore already have ~he transmembrane pH gradient are WO 90tl4105 PCT/US~/027~

2Q~ ?

rehydrated, water or another neutral a~ueous solutlon, and doxaru~icin are adted. Finally, in the case where liposomes having a transmem~rane pH gradient and contalning the pharmac~utical aqent have been dehydrated, rehydration proceeds using water or another a~leous solution. Alternatively, a second phar~aceutical agent may be added, if d~sired.
Liposomes containing the pharmaceutical formulations of the present invantion may be used therapeutically in mammals, especially hurans, in the treatment of a number of disease states or pharmacological conditions which require sustained release formulations as well as repeated administration.
The mode of administration of the liposomes containing the pharm~ceutical agents of the present invention may determine th~ sites and cells in the organism to which the compound may be d~livered. The liposomes of the present invention may be administered alone but will generally be ad~inistered in admix-ture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intravenou~ly. For parenteral administration, they can be used, for exampl~, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or gluco3Q to maXQ the solution isotonic. The liposo~es of the present invention may also be employed subcutaneously or intra~uscularly. Other uses, depending upon the particular properties of tha preparation, may be envisioned by those skilled in the art.
For the oral mode of administration, the liposomal for-W0 ~14105 2 9 PCT/US90/02736 2~5~3~5 mulations of the present invention ca~ he used in the ~orm Oetablets, capsules, losenges, troches, powders, syrups, elixirs, aqu~ous solution~ and suspensions, and ths liXe. In the case of tablets, carriers which can be used includa lacto~e, sodium citrate and salts o~ phosphoric acid. Various disintegrants such as starch, lubricating agents, and talc are commonly used i~
tablets. For oral administration in capsule form, useful diluents are lactosa and high mol~cular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is co~bined with emulsifying and suspending agents. If desired, certain swestening and~or ~lavoring agents can be added.
For the topical mode of ad~inistration, the liposomal formulations of the present invention may bç incorporated into dosage form~ such as g~ls, oils, emulsions, and the like. These formulations may be administered by direct application as a cream, paste, ointment, qel, lotion or the like.
For ad~inistration to humans in the treatment of disease stat~s or pharmacological conditions, the prescribing physician will ultimately deter~ine the appropriate dosage of the neoplastic drug ~or a giYen hu~an subject, and this c~n be expQcted to ~ary according to th~ age, weight and re~ponse of the individual as w~ll as the phaxmacokinetics o~ the agent used.
Also the nature and severity of the patient' 5 disease stat~ or pharmacological condition will influence the dosage regimen.
While it is e~pected that, in general, the dosage of the drug in liposomal for~ will be about that employed for ~he free drug, in som~ cases, it may ~ necessary to administer dosages outside WO ~14105 PC~/US9~/027~

2~5~ 5 these limits.
The following examples are givan for purposes of illustration only and are not to he viewed aq a limitation o~ the scope o~ the invention.

PreParation o~ a~d Li~osomes T~e following pharmaceutical agents were loaded or attempted to be loaded into liposomes comprising egg phosphatidylcholine (EPC, from Avanti Polar Lipids, lnc., Birmingham, Alabama) or EPC and cholesterol 55:45 (molar ratio) (cholesterol from Sigma Chemicals, St. Louis, MO.): propanolol, timolol, di~ucaine, chlorpromazine, lidocaine, quinidine, pilocarpine, phy~ostigmine, dopamine, imipramine, diphen-hydramine, quinine, chloroquine, quinacrine, dauorubicin, vin-cristine and vinblastine (obtained from Sigma Chemicals, St.
Louis, MO.), doxorubicin, epirubicin (o~tained fro~ Adria Laboratories, ~ississauga, Ont. Canada), mitoxantrone (obtained from Cyanamid Canada Inc., Montreal Que. (Canada), codeine and pethidine (obtained ~rou Abbott Laboratories, Ltd. Downsview, Ont. Canada). Th~ radiolabels, 3~-dipal~ oylpho~;phatidylcholinP, 14C-dipaluitoylphosphatidylcholine, l4C-dopamine and l4C-Imipramine were obtained fro~ Amersham while 3H-c~lorpromazine, 3H-propranolol, l4C-pilocarpine, 14C-chlorpromazine, 14C-methylamine and l4C-lidocaine were obtained ~rom ~ew England Nuclear. The Liposome Company, Inc. ~Princeton, N.J.) kindly pravid~d l4C-timolol. Salts and rea~ents used were of analytical grade.
All loading o~ pharmaceutical agents were into EPC

WO ~/141~ PC~/US~/~t7~

2 ~ 3 ,~
vesicles Icontaining 3H-Dipalmitoylphosphatidylcholine) or EPC:cholesterol mixtures (55:45 molar ratio). EPC:cholesterol ~ixture3 were prepared by colyophilization from benzene:methanol (95:5 v/v). The dry lipid waq hydrated with 300 mM citrate p~
4.0 as internal bu~fer solution and the resultant MLVs were sub-jected to five freeze-thaw cycles employing liquid nitrogen to enhance solute distribution according to Mayer, et al. ~iochim.
3io~hyal_~ç~, 817, 193 (1986). ~arg~ unilamellar vesicles were then prepared using an Extruder (Lipex 3iomembranes, vancouver, Canada) employing the LUVET procedure as described by Hope, et al. ~iohim. ~iophy~, Acta, 812, 55, (1935) with 100 nm pore size polycarbonate filters (Nucleopore, Inc.). To establish a pH gra-dient the vesicle~ were then pa sed down a Sephadex G-50 (fine) colume ~1.5 X lOcm) preequilibrated with 300 ~M NaCl, 20 mM
HEPES, pH 7.5.
Large unilammelar vesicles (approximately 1 mM lipid) were incubated with the agent (0.2 mM) in 300 ~M NaCl, Z0 mM
HEPES, pH 7.5 at 25-C. At various times up to 2 hours, aliquots (100 ul) of the mixture were taken and vesicles separated from unentrapped drug by centrifugation thxough a 1 ml "uinicolumn" o~
Sephadex G-50 (mQdium) a~ described by Pick, AL~h~ 3iochem.
~io~h~. 212, 186, 1981. Lipid and drug were quantiÇied by the following procedure.
Lipid concentrations were deter~ined ~y liquid scintilla-tion counting o~ 3H-DPPC or 14C-DPPC using a Pac~ard 2000 CA
instrument. Si~ilarlyl pilocarpine, chlorpromazine, timolol, propranolol, imipramine, lidocaine and dopamine were quanti~ied ~sing tracer quantities of 3H- or 14C-radiolabel.

WO90~l43~ PCT/USgO~02~

2 8 ~

Physostigmine was assayed by ~luorescenc~ spectroscopy following solubilization of vesicles in 60% ethanol (v/v). The exc~tation and emission of wavelengths used were 305 and 350 nm, respectively. Quinacrine, chloroquine and quinine were also quantified from their ~luorescence using excitation and emission wavelenqths of 420 nm, 505 nm; 335 nm, 375 nm; and 335 nm, 365 nm; respectiv~ly.
Vinblastine and ~incristine were assayed ~y U.V. spec-troscopy from their absorbances at 262 nm and 297 nm, respec~
tively, following solubilization of the vesicles in 80% ethanol.
Codeine was also ~easured by U~V. ~pectroscopy at 220 n~ in this case after solubilization in 40 mM octyl-beta-D-glucopyr~noside.
Mitoxantrone was quantified fro~ its absorbance at 670 nm follow-ing solubilization of the vesicles in 2% Triton-X100.
Diphenhydramine was assayed by gas-liquid chromatography using a HP 9850 gas chromatoqraph fitted with a Chromatographic Specialties DB-225 (25% cyanopropylphenyl) capillary column. The helium carrier flow rate was 1 ml/min and detection was by flame ionization. An internal standard, methylpentadecanoate, was used to quantify diphenhydramin~ following its extraction from the aqueous 5a~pl~ in di~thylether and its separation fro~ EPC by thin lay~r chromatography. Transbilayer pH gradients were quantified e~ploying the weak base methyla~ine (14C-labelled) as previously described by Bally, et al-, 5~Y~L 3~Y .~ 47 97, (1988).
The results o the loading experiment appear in Table l, below. 3asically, the loading of liposo~es with the agents described above may ~ efined on the basis of their uptaXe char-WO ~/l4105 P~T/US90/02736 2l~5~ i~ 3~;
acteris~ics. Four drug categorles may be defined based upontheir uptake characteristics.
The rirst cat~gory o~ phar~ac~utical agents exhlbLted completR, stablo uptake. Propanolol, dopamine, dauonorubicin, epirubicin, dibucaine, imipramine and doxorubicin exhibited the characteristics of this drug category. All o~ the drugs within this categary exhibitud uptakQ greater than predicted from the Henderson/Hasselbach equation. The accumulation o~ mitoxantrone by EPC liposome~ exhibiting a proton gradient i~ shown in Figure 1. .
The second category showed partial, but s~able up~ake.
Timolol, lidocaine, chlorpromazine, serotonin and chloroquine exhikited the characteristics of this drug category. Timolol was loaded to the extent o~ about 100 nmoles/umole lipid (about 50~
o~ available drug, see ~iqure 2A) and quinacrine was loaded to a level of about 80 nmoles/umole lipid after 30 ~inutes (see figure 2B). While accumulation is lower than in the first group of agents, nevertheless uptake is quite substantial. In the case of timolol, an internal concentration of about 65 m~ is achieved against an external concentration of 109 uM.
Tha third category shows a partial uptake followed by a rapid relea~e of agent rom the lipusom~. Figure 3 indi~ates a rapid virtually complete accumulation o~ quinidine into the vesicles and within 30 ~inutes about 50~ of the agent has leaked back out of the vesicles (Figure 3A). Other agents whi~h leak back out of EPC vesicles include quinine, diphenhydramine, vin-blastine and vincristine. The leakage rates vary considerably with vincristine and vinblastine loaded vesicl~s losing only 27%

WO9~/14l05 PCT/US90/027~

2 0 ~
o~ initially seyuestered d~g over two-hours. This loss is asso-cia~ed ~ith a corresponding reduction in re~idual change i~ pH as detQrmined using ~thylamina. A simllar d~crease in proton gra-dient is obs~rv~d a~ quinin~ and diph~nhydrami~ ar~ released from EPC v~sicles.
The fourth category of phar~aceutical agents, physostig-mine, codeinR and pilocarpine exhibited no ~asurabl~ r~sponse t~
the transmembrane pH gradient. Tha suggestion that these agents causQ a ma~or increa ~ in me~brane permeability resulting in loss of ion gradient is not borne by the data from physostlgmine (Fig-ure 4). Und~r th~ conditions us~d to as~ess uptake of physostig-~ine (200 u~) only a~small d~creasQ in m~asured change in pH was o~servad.
~kL~L
Extent and Stability of accumulatian of Various Drugs Ve~icl2J Exhibiting a p~ Gradient ,, Drug Class Uptake 15 Minutes Uptake 2 Hours (nmoles/umoles lipid) ~nm/um lipid) S~
Mitoxantrone Antineoplastic 200 198 Epirubicin Antin~oplastic 201 200 Daunorubicin AntinRoplastic 200 204 Doxorubi~ln ~ntinQopla~tic 202 203 Dibucaine LoG~l Ana~sthetic~ 194 176 Propanalol Adr6n~rgic 198 187 Dopa~inQ Biogenic Amine 190l 177 cateqo~t Z
Timolol Adrenergic gs 97 Lidocain~ Lccal Anaesthetic 87 87 WO 91)/11105 PCI/US90/02736 Chlorpromazine Local Anaesthetic 98 96 Serotonin Biogenic Amine 802 78 Chloroquine Antimalarial 1043 88 Quinacrine Antiprotozoal 731 71 Cateqory ~
Quinidine Antiarrhythmic 203 74 Quinine Antimalarial 1483 81 Diphenhydramine Antihistamine 1~63 87 Vinblastine Antineoplastic 1753 127 Vincristine Antineoplastic 178 130 categor~ 4 Codeille Analgesic <1 ~1 -Pilocarpine Cholinergic <1 ~1 Physostigmine Cholinergic ~2 <1 , ~
Footnote: L ~ maxlmum uptake at 30 mlnutes, ~, maxlmum uptake at 90 minutes, 3, .maximum uptake at 5 minutes Examplç ~
Com~arisQn ~e~ween I~evel of ~rua U~take and th~D~ua's Octanol: Water Partition Coefficient Levels of drug uptake were co~pared to their octanol/water partition coefficients to determine the extent that partition coefficient and the possibility that an agent was partitioning into the liposome bilayer raight deter;nine the extent of uptake o~ a pharmaceutical agent. Fros~ Table 2, below, it appears that no clear relat onship exists between drug uptake and its partition coe~ficient. Although the values ~iven may not accurately reflect mem~rane/water partition coefficients, they are ~erely being used for a comparative basis. Chlorpromazine and doxorubicin, for example, have si~ilar partition coefficients yet display very di~fer~ont uptake levels (98 vs 202). On the WO ~/14105 PCT/US90/027~

20~S~

other hand, timolol and chlorpromazin~ are accumulated by vesicles to a similar extent despite a large di~ference in their partition coe~icients. While partition coef~icient for a ionized drug may in~luence dru~ up~ake, it can not be taken to explain the di~erences between the agents studied.

A comparison Bet~een the L~vel of Drug Uptake and Its Octanol: Water Partition Coef~icient Drug Maximum Uptake Log Octanol:Water nmoles/umole lipid Partition Coefficient Daunorubicin 2Q0 3.5 Doxorubicin202 . l.l Vincristine178 2.8 Chlorpromazine 98 l.5 Dibucaine 194 4.4 Propranolol2 198 l.3 Timolol2 95 . -0.l Physostigmine 0 O.2 Imipramine 182 4.6 Diphenhydramine 176 3.4 Quinine l48 l.7 Codein~ 0 l.2 FootnoteY ~- All data taken from Leo, A., e~ al. Chem. Rev., 7l, 525~ (l971) except for propranolol and timolol.
- Fro~ Merbate, L. 5., et al-, ~igLhY~ 49, 9l, (1986).

WO ~141~ PCT/US~/027~

2 ~ 3 ~xam~le 3 cQm~ri~Q~ ~e~e~ ~vql Q~ IL. -th~ prya'~ ~o~ t~ i~ 9u~ o1y~Q~
~ esidea bu~er strength, the factor that in~luences the level o~ druq uptake to the greatest extent is the solubility 5 the protonated spRcies in the internal bu~er. Wh~n the con-cQntration of protonated drug inside the vQsiclQ exceeds its solubility product and precipitation occur3 thie will e~fectively reduc~ the transm~mbrane concentration gradiQnt ~or the remaining solube fraction thus allowing further accumulation by the vesicles. In table 3 is show~ the maximum apparent solubilities in 300mM citrate buffer, p~ 5.O for most o~ the drugs whose proton gradient dependent uptake was examined. Drugs such as mitoxantrone, epirubicin, doxorubicin and daunorubicin which exhibit complete and stable uptak~ are relatively insoluble in the intravesicular medium. This indicates that most of the accumulated drug is in the form of a precipitata and does not contribute to the concentration gradient of the soluble protonated species, thu~ accounting for the high levels o~ uptake observed. In addition, if most Or the intravesicular drug is precipitat~d the concentration of free dru~ available to parti-tion into the mc~brans is correspondingly reduced which will con-tribute to the obsQrv~d stability o~ th~ transme~brane proton gradisnt. As ~xpect~d, agents such as timolol, lidocaine, quinacrin~ and chloroquine which exhibit uptake in good agreement with the Hender~on-Hasselbach equation have apparent solubilities which are in excess of the intravesicular concentrations achieved (See table 3, below). Without being bound by any theory, the solubility data may explain most of the observed di~ferences in Wo ~1410~ RCT/US~/02736 2 ~

uptaX~ characteristic~ ~or the various druqs examined. The data indicate~ that solubility data is most important in determinin~
upta~e o~ drugs into liposomes.
We note that dibucaine, propranolol and dopamine ~ay also ~e loaded in liposomes in an amount significantly greater tha~
predicted by the ~enderson-Hasselbach equation. T~is is a sur-prising result considering that the three agents' apparent solubility is greater than the final internal concentration of the agent in the liposome.

~a~le 3 Appar~nt Maximum Drug Solubility in 300 mM
Citrat~ Buffer, pH 5.0 Drug Apparent ~axlmu~ Solubility (mM) Mltoxantrone <O.01 Epirubicin 0.26 Daunorubicin .9.10 Doxorubicin 0.24 Vincristine >35 Vinbla~tine 19.1 Lidocaine 240 Dibucaine >700 Propanolol Timolol 135 Quinidin~
Dopa~ine 1400 Quinine 1.05 Chloroquine 585 Quinacrine 90 .

Wo ~/1~1~ PCT/U~/027~

2~ 3 Exa~DlQ ~
Isopr~terenol. MetaDroterenol and Ter~utaline E~g phosphatidylcholine (EPC) purohased from Avanti Polar Lipids (8irmingham, Alabama), and 14C-methylamine was purchas2d from N~w England Nuclear. All other chemicals and buf ~ers were purchased from Sigma (St. ~ouis, M0.) and were used without puri~ication.
Large Unilamellar Vesicles (LW s) were produced by extru-sion according to the method of Hope, et al., 3i9C.LU~_.3~D~h~_ Ac~a, 817, 193 (1985) Briefly, LUVs were pro~uced by extrusion o~ ~rozen and ~hawed lipid dispersions prepared in 300 ~M
citrate, pH 4.0, through 0.1 or 0.2 um polycarbonate filters (Nucleopore) employing an extrusion device (Lipex 8iomembranes, Vancouver, Canada~. Vesicles prepared by this t~chnique employ-ing 0.~ um filt~rs have trapped volum~s of 1.5 uL/umole phospholipid as deter~ined using 14C or 22Na and have an averag~
diameter of 90 n~. Phospholipid concentrations wzre determined by assay of lipid phosphorous as previously described by Fiske and Subbarrou, 2, Biol. Ch~., 66, 375, tl925). Transmem~rane pH
gradient~ wer~ a~tablishQd according to Exampla 1, and untrapped int~rnal bu~er re~o~ed by passing tha LW s down a Sephadex G-S0 column equilibrated with tha external bu~er ~150 mM NaCl, 20 mM
HEPES, p~ 7.4]. Induced p~ gradients w~re detQr~ined by measur-ing the transme~brane distributions of 14C-methylamine as described by HOPQ , et al., ~EE~. In short, methylamine was added to the ve3icle system to a final concentration of 0.5 uCi/~L. At appropriate time~, aliquots (100 uL~wer~ removed and WO ~/14105 P~T/U~90/027 2 ~ C~

passed down 1 mL sephadex G-50 mini-columns as previously described. The trapp~d probe was determined by liquid scintilla-tion counting employing a Packard 2000CA liquid scintillation counter, and phospholipid concentrations w~ra determined. Trans-membrane pH gradients were calculatad according to the equation change in pH~ log[MeAm]i/~MeA~]O.
The bronchodilators, isoproterenol, metaproterenol and terbutaline were incubated with liposomes with a transmem~ranepH
gradient prepared as above at the indicted temperatures in a 150 mM NaCl, 20 mM HEPES, pH 7.4 buffer containing 500 uM o~ the bronchodila~or and 6 mM of the phospholipid. Control samples without a trans~embrane pH gradient were incubated at pH 4.0 or 7.4 (both insida and outside the vesicles) to determine the degree of gradient-indepemdent membrane binding. The pH 4.0 con-trol consisted o~ tha vesicles prepared as above, but the external buffer was 150 mM NaCl, 20 m~ citrate, pH 4Ø For the pH 7.4 control, the vesicles were prepared with 150 ~M NaCl, 20 mM HE~ES pH 7.4, both internally and externally.
Free drug was separated from vesicles using 1 mL Sephadex G-50 minicolumns as described above and assayed ~photometri-cally). Figur~ 5 ~hows the entrapment of tho bronchodilators metaproterenol, terbutalin~ and isoproterenol in response to pH
gradients using ( PC) 200 nm extruded liposomes. Vesicles con-taining 300 m~ citrate, pH 4.0 were incubated with a 500 uM drug solution at pH 7.4. Th~ liposomes accu~ulate the drug to levels o2 greater than 60 nmoles/umole lipid. This is roughly 70~ trap-ping ef~iciency, and corresponds to an inside:outside drug con-centration ratio oS about 190:1, which assume~ an internal volume .

wo 90/l~lûS PCr/US90~01736 2 ~ 3 S ~ 3 3 o~ 2.2 uL/umole lipid. I~ all drug i5. in the aqueous space of the vesicles, an internal drug concentration of about 30 mM is calculated. Uptake i5 stable for at least 4 hours and is com plete within ~inutes. In the absence o~ the transmembrane pH
gradient, background binding of these drugs is less than 15 nmoles/umole lipid for both pH 4.0 (inside and outside the vesicles) and pH 7.4 (in and out), indicating that non-specific binding to the llpid is not responsible for the ;sociation o~
thesa drugs with the liposomes, and that the associ~tion is a function of the proton gradi~nt rather than the absolute pH.
Exa~ 5 Effect of Dru~ U~ake on ResiduaL ~H Gradie~t As M~surç~k~MethYL3~nç ûi$t~ib~lt~on The loqarithm of the ratios of the internal and external concentrations ~f the radioactive methylamine can be used to measure the transmembrane change in p~, because the methylamine probe does not dissipate the internal proton poool at these con-centrations. When the internal and external pH is 7.4 or 4.0 (no gradient) the methylamine does not datect any gradient (Figure .
6). When vesicle~ with an internal pH of 4.0 are incubated in a pH sf 7.4, th~ m~thylamine distribution indicates a 3.0 unit pH
gradient, in good agreem~nt with th~ 3.4 pH unit gradient. When ~etaproter~nol is added to the ~xternal buffer to a final con-centration of 500 u~, the gradient dissipates to about 2.3 pH
units as the drug i5 accumulated. The date indicates that the 190 fold achieved by the drug approximates the residual proton gradient (pH o~ 2.3 units).

W090/141U~ PCT/US90/027~

2 ~ 3 ~

~R~ 6-E~ec~ o~.H~at o.~ E~r~m~nt o~ ~e~a~rQt~ereno1 The rate of metaproterenol entrapment is increased by increased temperature (See Fiqure 7), reachiny steady-state levels arter 2 hours at 21-C, but ~aster than 15 minutes when incubatQd at 60 C. The extent of drug uptake is not dramatically affected by the temperature o~ incubation.
Exam~Q 7 Effeçt~ o~ Choles~rol on ~ntraD~ent o~ 8ronchodilators The inclusion of cholesterol in liposomes on the.uptake of bronchodilators was in~estigated. Cholesterol decreased both the rate o~ uptaXe, and ths total am~unt o~ trapped drug per u~le of lipid. 5inc~ the internal trapped volu~e o~
EPC:cholesterol (55:45 mole %) determined using l~C inulin and 22Na is about 30% lower than vesicles compos~d of EPCalone, the actual cancentration gradients of metapro~erenol achiaved is similar in both ca~es. See Figure 8.
xa~ple_8 Effect of Dru~ to Li~id Ratio on the Amount o~ T~a~Ped Druq The amount of entrapped drug also depends on the initial drug to lipid ratio (see Figure 9). At low drug to lipid ratios,.
the a~ount oS drug entrapped by the vesicle~ reflects the imposed ~ransmembrane pH gradient greater than 3 units. At sufficiently high drug concentration, the reionization of the drug in th~
vesicle interior causes a decrease in the internal pH (Figure 6).
Equilibrium levels o~ drug entrapment would there~ore be expected to relect the final change in pH rather than the initial chan~e in pH as the ionized drug overwhelms the internal buffering capa-city o the vesicle~.

WO ~/~410~ P~/US90tO27~

Example g E~ec~ o2 ~5fer S~ren~t~ Qn E~traDment Extent o~ the buf~er capacity (buf~er strength) is a fac-tor which affect~ the ability of the liposome to trap ionized drug . Low internal buf~r capacity affects the extent of drug accumulation. ~elow 100 mM citrate, the extent of drug accumula-tion is indeed af~ected by the internal citrate concentration (Figure 10). Under theqe conditions, re-ionization of the drug ov~rwhelms the internal bu~fering capacity of the vesicle inte-rior, raising the pH of the vesioles and dissipating the change in pH (inset). Including greater levels o~ citrate than 300 mM
in the vesicles does not dramatically increa~e the levels of trapped drug, since there is still a large residual pH gradient in these cases and the internal ~ufferin~ capacity does not limit drug uptake.

It will be understood by those skilled in the art that the foregoing description and examples are illustrative o~ practicing the present invention, but are in no way limiting. Variations of the detail presonted herein may be made without departing fro~
the splri~ and scop~ o~ the pres2nt invention.

Claims (46)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A liposome composition comprising:
a liposome having a transmembrane ion gradient compris-ing at least one ionizable pharmaceutical agent;
at least one lipid; and a first internal aqueous buffer solution;
said pharmaceutical agent having a solubility less than a con-centration of said agent in said internal buffer solution to produce an amount of pharmaceutical agent in said liposome greater than expected from the transmembrane ion gradient.

2. The composition according to claim 1 wherein said ion gradient is a pH gradient.

3. The composition according to claim 1 wherein said pharmaceutical agent has a solubility in said internal buffer solution of less than about 20 mM and said agent has a solubil-ity in said external buffer solution of at least about 0.2 mM.

4. The composition according to claim 3 wherein said pharmaceutical agent has a solubility in said internal buffer solution of less than about 10 mM.

5. The composition according to claim 2 wherein said pharmaceutical agent is selected from the group consisting of dopamine, dibucaine, chlorpromazine, lidocaine, serotonin, quinacrine, metaproterenol, terbutaline, isoproterenol, quini-dine, quinine, diphenhydramine and chloroquine.

6. The composition according to claim 3 wherein said pharmaceutical agent is selected from the group consisting of dibucaine, dopamine, quinidine, imipramine and diphenhydramine.

7. The composition according to claim 2 wherein said lipid is selected from the group consisting of phosphatidylcho-line, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin and a mixture of phosphatidylethanolamine and phos-phatidylcholine in a weight ratio of phosphatidylethanolamine to phosphatidylcholine of about 30:70 to about 45:55.

8. The liposome composition according to claim 1 which is dehydrated.

9. A pharmaceutical composition comprising the lipo-some composition according to claim 1 and a pharmaceutically acceptable carrier or diluent.

10. A liposome composition comprising:
a liposome having a transmembrane pH gradient compris-ing at least one ionizable pharmaceutical agent selected from the group consisting of metaproterenol, terbutaline and isopro-terenol;
at least one lipid; and at least one aqueous buffer solution;
wherein said pharmaceutical agent accumulates in said liposome in an amount equal to at least about 70% entrapment efficiency.

11. The composition according to claim 10 wherein said lipid is selected from the group consisting of phosphatidylcho-line, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin and a mixture of phosphatidylethanolamine and phos-phatidylcholine in a weight ratio of phosphatidylethanolamine to phosphatidylcholine of about 30:70 to about 45:55.

12. The composition according to claim 10 wherein said lipid is phosphatidylcholine.

13. The composition according to claim 10 wherein said buffer solution is a buffer combination comprising an internal aqueous buffer solution and an external aqueous buffer solu-tion.

14. The composition according to claim 10 wherein said first buffer solution is selected from one or more of the group consisting of citric acid, oxalic acid, succinic acid and salts of organic acids and wherein said second buffer solution is selected from the group consisting of sodium chloride, potas-sium chloride, potassium phosphate, sodium bicarbonate, sodium carbonate, sodium bisphosphate, potassium phosphate, potassium sulfate and HEPES.

15. The composition according to claim 10 wherein said buffer solution is a buffer combination comprising a first in-ternal buffer solution comprising citrate buffer and a second external buffer solution comprising a mixture of NaCl and HEPES.

16. The composition according to claim 15 wherein said first internal buffer solution is a citrate buffer of concen-tration ranging from about 100 mM to about 300 mM and said sec-ond buffer contains NaCl and HEPES, said NaCl ranging in con-centration from about 100 mM to 400 mM and said HEPES ranging in concentration from about 10 mM to about 30 mM.

17. The liposome composition according to claim 10 which is dehydrated.

18. A pharmaceutical composition comprising the lipo-some composition according to claim 10 and a pharmaceutically acceptable carrier or diluent.

19. A liposome composition comprising:
a liposome having a transmembrane pH gradient compris-ing at least one ionizable pharmaceutical agent selected from the group consisting of quinine, diphenhydramine and quinidine which rapidly releases from said liposome after accumulation;

at least one lipid which prevents said pharmaceutical agent from rapidly releasing from said liposome after accumula-tion; and an aqueous buffer solution.

20. The composition according to claim 19 wherein said ion gradient is a pH gradient.

21. The composition according to claim 19 wherein said lipid is a lipid combination comprising at, least one first lip-id selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin, car-diolipin and a mixture of phosphatidylet.hanolamine and phospha-tidylcholine in a weight ratio of phosphatidylethanolamine to phosphatidylcholine of about 30:70 to about 45:55 and at least one second lipid comprising a membrane-stabilizing lipid compo-nent.

22. The composition according to claim 21 wherein said membrane-stabilizing component is cholesterol.

23. The composition according to claim 22 wherein said first lipid is phosphatidylcholine and said second lipid is cholesterol and wherein said weight ratio of phosphatidylcho-line to cholesterol is about 70:30 to about 55:45.

24. The composition according to claim 23 wherein said weight ratio of phosphatidylcholine to cholesterol is about 55:45.

25. The liposome composition according to claim 19 which is dehydrated.

26. A pharmaceutical composition comprising the lipo-some composition according to claim 19 and a pharmaceutically acceptable carrier or diluent.

27. A liposome composition comprising:
a liposome having a transmembrane ion gradient compris-ing at least one ionizable pharmaceutical agent selected from the group consisting of quinine, diphenhydramine and quinidine which rapidly releases from said liposome after accumulation;
at least one lipid which prevents said pharmaceutical agent from rapidly releasing from said liposome after accumula-tion;
a first internal aqueous buffer solution; and a second external aqueous buffer solution;
said pharmaceutical agent having a solubility less than a con-centration of said agent in said internal buffer solution to -produce an amount of agent in said liposome greater than ex-pected from the transmembrane ion gradient.

28. The composition according to claim 27 wherein said ion gradient is a pH gradient.

29. The composition according to claim 27 wherein said lipid is a lipid combination comprising at least one lipid sel-ected from the group consisting of phosphatidylcholine, phos-phatidylserine, phosphatidylinositol, sphingomyelin, cardiolip-in and a mixture of phosphatidylethanolamine and phosphatidyl-choline in a weight ratio of phosphatidylethanolamine to phos-phatidylcholine of about 30:70 to about 45:55 and at least one membrane-stabilizing lipid component.

30. The composition according to claim 28 wherein said membrane-stabilizing lipid component is cholesterol.

31. The composition according to claim 28 wherein said first lipid is phosphatidylcholine and said membrane-stabiliz-ing lipid component is cholesterol wherein said weight ratio of phosphatidylcholine to cholesterol is about 70:30 to about 55:45.

32. The composition according to claim 29 wherein said weight ratio of phosphatidylcholine to cholesterol is about 55:45.

33. The composition according to claim 27 wherein said pharmaceutical agent has a solubility in said internal buffer solution of less than about 20 mM and said agent has a solubil-ity in said external buffer solution of at least about 0.2 mM.

34. The composition according to claim 27 wherein said pharmaceutical agent has a solubility in said internal buffer solution of less than about 10 mM.

35. The composition according to claim 27 wherein said internal buffer solution has a buffer strength of at least about 50 mM.

36. The composition according to claim 27 wherein said internal buffer solution has a buffer strength ranging from about 100 mM to about 300 mM.

37. The composition according to claim 27 wherein said internal buffer solution has a buffer strength of about 300 mM.

38. The liposome composition according to claim 27 which is dehydrated.

39. A pharmaceutical composition comprising the lipo-some composition according to claim 27 and a pharmaceutically acceptable carrier or diluent.

40. A liposome composition comprising:
a liposome having a transmembrane ion gradient compris-ing at least one ionizable pharmaceutical agent selected from the group consisting of dibucaine, propranolol and dopamine;

at least one lipid;
a first internal aqueous buffer solution; and a second external aqueous buffer solution;
said pharmaceutical agent accumulating in said liposome in an amount greater than expected from the transmembrane ion gradi-ent.

41. The liposomal composition according to claim 40 wherein said lipid is egg phosphatidylcholine.

42. The liposomal composition according to claim 41 wherein said internal aqueous buffer is citric acid buffer ranging in concentration between about 100 mM and 300 mM.

43. The liposome composition of claim 1 wherein the ionizable pharmaceutical agent is selected from the group con-sisting of antineoplastics, local anesthetics, bronchodilators, beta-adrenergic blockers, anti-hypertensive agents, anti-depressants, anti-convulsants, anti-emetic agents, anti-hista-mines, anti-arrhythmic agents, anti-malarial agents and analge-sics.

44. The liposome composition of claim 43 wherein the antineoplastic is an anthracycline or a vinca alkaloid.

45. The liposome composition of claim 43 wherein the anthracycline is doxorubicin.

46. The liposome composition wherein the vinca alka-loid is vinblastine or vincristine.