WO2007049278A2 - A method for preparing liposomes and uses thereof - Google Patents

Abstract

The present invention provides a simplified and cost effective method preparing liposomes, the method comprising providing a dry liposome forming lipid or a dry mixture comprising a liposome forming lipid; dissolving the liposome forming lipid or said mixture comprising a liposome forming lipid of step (a) with a protic organic solvent to form a solution or dispersion of said lipid; and adding the solution or dispersion of step (b) to ions containing aqueous solution to form a liposome suspension; wherein the liposomes are capable of encapsulating an agent at a ratio between the agent and the liposome forming lipid being greater than 1.0. The method is characterized in that it does not involve drying the solution or dispersion to form a dry lipid film; and/or down-sizing liposomes in said liposome suspension to form small unilamellar vesicles (SUV). The thus formed liposomes may then be used for loading thereto an active agent, such as amphiphatic weak acid/base drugs.

Description

A METHOD FOR PREPARING LIPOSOMES AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to liposome technology and in particular to methods of preparing liposomes.

LIST OF PRIOR ART The following is a list of prior art, which is considered to be pertinent for describing the state of the art in the field of the invention.

1. WO00/09089 - Liposomal bupivacaine compositions prepared using an ammonium sulfate gradient (Bolotin EM et al.);

2. US 4,532,089 - Method of preparing giant size liposomes (MACDONALD ROBERT C);

3. US 5,192,549 - Method of amphiphatic drug loading in liposomes by pH gradient (Barenholz et al.)

4. US 5,807,572 - Multivesicular liposomes having a biologically active substance encapsulated therein in the presence of a hydrochloride (Kim S. et al.);

5. Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-9 (1981)i

6. US 5,723,147 - Multivesicular liposomes having a biologically active substance encapsulated therein in the presence of a hydrochloride (Kim S. et al.).

BACKGROUND OF THE INVENTION Liposomes are one of the most potential drug carriers available currently.

Liposomes contain both a hydrophobic bilayer, which may encapsulate hydrophobic substances, and an aqueous core, which may encapsulate other substances (e.g. hydrophilic or amphiphatic compounds). Liposomal encapsulation of therapeutic compounds has shown significant promise in controlled drug delivery. For example, some lipid-based formulations provide a longer half-life in vivo, superior tissue targeting, or decreased toxicity. In efforts to develop more effective therapeutic treatments, attempts have been made to encapsulate a variety of therapeutic compounds in liposomes. For example, many anticancer or antineoplastic drugs have been encapsulated in liposomes. These include alkylating agents, nitrosoureas, cisplatin, antimetabolites, vinca alkaloids, camptothecins, taxanes and anthracyclines. Studies with liposomes containing anthracycline antibiotics have clearly shown reduction of cardiotoxicity. Liposomal formulations of drugs modify the drug pharmacokinetics of the free drug counterpart, which is not liposome-encapsulated. For a liposomal drug formulation, drug pharmacokinetics is largely determined by the rate at which the carrier is cleared from the blood and the rate at which the drug is released from the carrier. Considerable efforts have been made to identify liposomal carrier compositions that show slow clearance from the blood, and long-circulation carriers have been described in numerous scientific publications and patents. Efforts have also been made to control drug leakage or release rates from liposomal carriers, using for example, various lipid components or a transmembrane potential to control release.

Liposomes are prepared by many methods and the obtained vesicles may vary significantly in terms of diameter and number of bilayers. Liposomes may be classified as small or large unilamellar vesicles (SUV₅ LUV), multilamellar vesicles (MLV) and multivesicular vesicles (MVV) or large multivesicular vesicles (LMVV)₅ which contain several vesicles and, consequently, several separate aqueous phases [Kulkarni, S.B., et al. J. Microencapsul 12:229-246 (1995)]. The vesicles-in- vesicles are formed during the preparation of multivesicular vesicles (MVV) [Szoka, F. and Papahadjopoulos, D. Proc.

Natl. Acad. Sci. USA 75:4194-4198(1978)] and the conversion of MLVs into freeze- thawed vesicles (FT MLV) [Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-9

(1981)], which are structurally similar to MVVs [Kramer, J.M.H., et al. Biochemistry

17:3932-3935 (1997)]. MVVs and FT MLVs encapsulate far more aqueous phase volume than SUVs and MLVs, but the structures of MVVs and FT MLVs (LMVV) are large, i.e. 0.5-15 μm in diameter. The majority of the liposome preparation methods are based on either dry lipid hydration or the evaporation of an organic solvent in which the thus dried lipids are added into an aqueous solution. The methods based on dry lipid hydration are typically multi-step processes (organic solvent evaporation from lipid solution, lipid drying, hydration, calibration, and possibly other steps). The methods based on the injection of ethanol or ether lipid solution into the buffer results in small vesicles, useful primarily only as model membranes [Batzri, S. and Korn, E., Biochim. Biophys. Acta_298:1015- 1019 (1973); Kramer, J.M.H., et al. Biochemistry 17:3932-3935 (1997)]. There are several multi-step methods based on the aqueous phase dispergation in organic, lipid solubilizing solvents, and on the organic solvent evaporation directly during vesicle formation. The reverse-phase evaporation method is the oldest of these [Szoka, F. and Papahadjopoulos, D. Proc. Natl. Acad. Sci. USA 75:4194-4198 (1978)]. In accordance with the original procedure, an aqueous phase is dispergated in an organic solvent (ethyl ether, halothane, chloroform, methylene chloride or other) to form a water-in-oil emulsion by sonicating the mixture of both of these phases. Next, the emulsion is transferred to a rotary evaporator, and the solvent is removed under reduced pressure. At this stage, some of the aqueous phase droplets combine and form the environment where buffer droplets, enveloped in lipid membrane, are suspended. Then, the aggregates are centrifuged and the supernatant is filtered to obtain LUVs, smaller than the defined sizes of a filter's pores. Another method using double emulsion characteristics is based on aqueous emulsion formation in chloroform and ethyl ether solutions of liposomal lipids by mechanical agitation with a shaker. Constant bubbling of nitrogen agitates the combined emulsions, and consequently unilamellar or multivesicullar vesicles are formed [Kim S. and Martin, G.M. Biochim. Biophys. Acta 646:1-9 (1981), and US Patent No. 5,723,147].

Disclosed in US Patent No. 5,723,147 are multivesicular liposomes containing biologically active substances, the multivesicular liposomes having a defined size distribution, adjustable average size, adjustable internal chamber size and number, and a modulated rate of the biologically active substance. The process used to form such multivesicular liposomes comprises dissolving a lipid component in volatile organic solvents, adding an immiscible aqueous component containing at least one biologically active substance to be encapsulated, and adding a hydrochloride effective to control the - A -

release rate of the biologically active substance from the multivesicular liposome to either or both of the organic solvents and the lipid component, making a water-in-oil emulsion from the two components, immersing the emulsion into a second aqueous component, dividing the emulsion into small solvent spherules which contain even smaller aqueous chambers, and then removing the solvents to give an aqueous suspension of multivesicular liposomes encapsulating biologically active substances.

A further method of preparing LMVV is also described in WO00/09089. According to this publication liposomal bupivacaine compositions are prepared using an ammonium salt (e.g. sulfate) gradient loading procedure, at a pH which prevents precipitation of the drug from the loading solution. The liposomes may be large multivesicular liposomes (referred to as GMV) which are prepared by vortexing a dry lipid film with an aqueous solution of ammonium salt, homogenizing the resulting suspension to form a suspension of SUV and repeatedly (at least 5 times) freeze- thawing the suspension of SUV in liquid nitrogen followed by water.

SUMMARY OF THE INVENTION

The present invention is aimed at providing a simplified and more cost-effective method for preparing liposomes.

In accordance with a first of its aspects, there is provided by the present invention a method for preparing liposomes comprising: (a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid;

(b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid of step (a) with a protic organic solvent to form a solution or dispersion of the lipid or mixture of lipids; (c) adding the solution or dispersion to an ion-containing aqueous solution to form a liposome suspension; wherein the liposomes thus formed are capable of encapsulating an agent at a mole/mole ratio between the agent and the liposome-forming lipid of greater than 1.0. The invention also provides a method for preparing liposomes consisting of:

(a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid; (b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid with a protic organic solvent to form a solution or dispersion of the lipid or mixture of lipids;

(c) adding the solution or dispersion to an ion-containing aqueous solution to form a liposome suspension; wherein the liposomes thus formed are capable of encapsulating an agent at a mole/mole ratio between the agent and the liposome-forming lipid of greater than 1.0.

Further, the invention provides a method for preparing liposomes comprising: (a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid;

(b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid with a protic organic solvent to form a solution or dispersion of the lipid or mixture of lipids; (c) adding the solution or dispersion to an ion-containing aqueous solution to form a liposome suspension; wherein the liposomes thus formed are capable of encapsulating an agent at a ratio between the agent and the liposome-forming lipid of greater than 1.0; with the proviso that said method does not comprise one or more of the following steps: drying said solution or dispersion to form a dry lipid film; and/or down-sizing liposomes in said liposome suspension to form small unilamellar vesicles (SUV).

The liposomes may be used to carry active agents such as drugs. Thus, in accordance with a further aspect, the invention provides a method for preparing agent- carrying liposomes comprising or consisting of:

(i) providing a liposome suspension by the method of the invention, and (ii) incubating said liposome suspension with a solution comprising said agent to form agent-carrying liposomes, wherein said agent-carrying liposomes comprise a mole/mole ratio between said agent and said liposome-forming lipid of greater than 1.0. In a preferred embodiment the method of the invention for preparing agent carrying liposomes excludes drying said solution or dispersion to form a dry lipid film; and/or down-sizing liposomes in said liposome suspension to form small unilamellar vesicles (SUV).

The invention also provides pharmaceutical compositions comprising a physiologically acceptable carrier and agent-carrying liposomes whenever prepared by any of the methods of the invention.

Further, the invention provides a method for treating a subject in need comprising administering to said subject an amount of agent-carrying liposomes whenever prepared by the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for preparing liposomes with a high agent-to-lipid ratio (by amount in moles of each), which, as appreciated by those versed in the art, may have benefits in terms of prolonged delivery, safety and other pharmacological and pharmacokinetic parameters. The simplicity of the proposed approach resides, inter alia, in the elimination of several steps which hitherto have been considered essential components in the preparation of liposomes of similar characteristics and loading capabilities.

The terms "liposome" and "vesicle" are well known in the art and are used interchangeably herein, except where otherwise specifically stated or required by context.

The method of the present invention comprises: providing a dry liposome- forming lipid or a dry mixture comprising one or more liposome-forming lipids; dissolving the lipid or the dry mixture comprising the same with a protic organic solvent to form a solution or dispersion of the lipid(s); and adding the solution or dispersion thus formed to an ion-containing aqueous solution, resulting in the formation of a liposomal suspension. It has been found that such liposomes are capable of loading an agent at a high agent-to-lipid mole/mole ratio, a high ratio being greater than 1.0. As further discussed below, the active agent may be loaded into the liposome by different methods, such as incubation of the liposome suspension with an aqueous solution of the agent.

In accordance with the invention, a "high agent-to-lipid ratio" is determined by the mole/mole or weight/weight ratio of the agent to the liposome-forming lipid(s). In the following description, unless otherwise stated, the term "ratio" denotes a mole/mole ratio. A high agent-to-lipid (agent/lipid) ratio in accordance with the invention is to be understood as any ratio being at least greater than 1.0, preferably greater than 1.5, more preferably greater than 1.8. It has been found that under suitable conditions which are within the scope of the present invention the ratio may even be greater than 2.0.

It is noted that conventional methods for preparing liposomes loaded with an agent (e.g., a drug) with relatively high agent/lipid ratio (i.e., greater than 1.0) comprise the following features, which had previously been considered to be essential components in the preparation of liposomes of similar characteristics and loading capabilities: the liposome-forming lipid needs to be dried and then rehydrated prior to the formation of liposomes therefrom; and/or - the liposomes need to be down-sized, e.g. by vortexing, by ultrasonication, by extrusion and/or by like processes, to form small unilamellar vesicles (SUV), before obtaining (following a suitable manipulation of the SUV) the final liposomes into which the agent is to be loaded; and/or the SUVs require at least five freezing and thawing (F&T) cycles in order to obtain liposomes capable of exhibiting a desirably high agent-to-lipid ratio; the selection of specific (and thus limiting) organic solvents which do not freeze under lyophilization conditions (temperatures of O⁰C or higher).

As shown herein, the method of the present invention provides agent-carrying liposomes with a high agent/lipid ratio without the need to perform any of the above steps in order to achieve the desired high loading. In other words, by eliminating the need to dry the lipids prior to liposome formation, and/or the need to down-size the liposomes, and/or the need to perform multiple (more than 1) F&T cycles, a much simpler, cost-effective method is provided. Such a simplified method may have many advantages, in particular with respect to large-scale production of agent-carrying liposomes.

The liposomes of the present invention are formed from liposome-forming lipids. "Liposome-forming lipids" (or "vesicle-forming lipids") are amphiphilic molecules essentially characterized by a packing parameter of 0.74 - 1.0, inclusive, or by a lipid mixture having an additive packing parameter (the sum of the packing parameters of each component of the liposome multiplied by the mole fraction of each component) in the range between 0.74 and 1, inclusive.

Further, "liposome-forming lipids" in accordance with the invention are lipids having a glycerol backbone wherein at least one, preferably two, of the hydroxyl groups at the head group is substituted by one or more of an acyl, an alkyl or alkenyl group, a phosphate group, preferably an acyl chain (to form an acyl or diacyl derivative), a combination of any of the above, and/or derivatives of the above, and may contain a chemically reactive group (such as an amine, acid, ester, aldehyde or alcohol) at the headgroup, thereby providing a polar head group. Sphingolipids, and especially sphingomyelins, are good alternatives to glycerophospholipids.

Typically, a substituting chain, e.g. an acyl, alkyl and/or alkenyl chain, is between about 14 to about 24 carbon atoms in length, and has varying degrees of saturation, thus resulting in fully, partially or non-hydrogenated liposome-forming lipids. Further, the liposome-forming lipid may be of a natural source, semi-synthetic or a fully synthetic lipid, and may be neutral, negatively or positively charged.

There are a variety of synthetic liposome-forming lipids and naturally occurring liposome-forming lipids, including the phospholipids (which are preferred lipids in accordance with the invention), such as phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidyl glycerol (PG), dimyristoyl phosphatidyl glycerol (DMPG), egg yolk phosphatidylcholine (EPC), l-palmitoyl-2-oleoylphosphatidyl choline (POPC), distearoylphosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidic acid (PA), phosphatidylserine (PS), l-palmitoyl-2-oleoylphosphatidyl choline (POPC)₅ and sphingophospholipids such as sphingomyelins (SM) having 12- to 24-carbon-atoni acyl or alkyl chains. The above-described lipids and phospholipids, whose hydrocarbon chain (e.g., acyl/alkyl/alkenyl chains) have varying degrees of saturation, can be obtained commercially or prepared according to published methods. Other suitable lipids which may be included in the liposomes are glyceroglycolipids, . sphingoglycolipids and sterols (such as cholesterol or plant sterol). The liposomes formed in accordance with the invention may comprise a mixture of lipids. The above- described list of lipids for use in accordance with the invention is not exhaustive and non-limiting, and thus, other lipids not disclosed herein may be used in accordance with the invention.

In accordance with one embodiment, the liposome-forming lipids are selected from those having a T_m (gel to liquid crystalline phase transition temperatures), above 45°C, such as, without being limited thereto, phosphatidylcholine (PC) and derivatives thereof having an acyl chain with 16 or more carbon atoms. One preferred example of a PC derivative is hydrogenated soy PC (HSPC) having a Tm of 52°C. The liposome- forming lipids may additionally or alternatively comprise sphingomyelins of various N- acyl chains, such as N-stearoyl sphingomyelin.

Cationic lipids (mono- and polycationic) are also suitable for use in the liposomes of the invention, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component. Such cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and the lipid typically has an overall net positive charge. Preferably, the head group of the lipid carries the positive charge. Monocationic lipids may include, for example: 1,2- dimyristoyl-3-trrmethylammonium propane (DMTAP); l,2-dioleyloxy-3-

(trimethylamino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-

N-hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethyl ammonium bromide (DORIE); N-[I -(2,3 -dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3β[N-(N',N'- dimethylaminoethane) carbamoyl] cholesterol (DC-Choi); and dimethyl-dioctadecylammonium (DDAB). Examples of polycationic lipids may include a lipophilic moiety similar to those described for monocationic lipids, to which the polycationic moiety is attached. Exemplary polycationic moieties include spermine or spermidine (as exemplified by DOSPA and DOSPER), or a peptide, such as polylysine or other polyamine lipids. For example, the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid. Polycationic lipids include, without being limited thereto, N-[2-[[2,5-bis[3- aminopropyl)amino] - 1 -oxopentyl] amino] ethyl] -N,N-dimethyl-2,3 -bis [( 1 -oxo-9- octadecenyl)oxy]-l-propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS). Further, the liposomes may also include a lipid derivatized with a hydrophilic polymer to form new entities known by the term lipopolymers. Lipopolymers preferably comprise lipids modified at their head group with a polymer having a molecular weight equal to or above 750 Da. The head group may be polar or apolar; however it is preferably a polar head group to which a large (>750 Da), highly hydrated (at least 60 molecules of water per head group), flexible polymer is attached. The attachment of the hydrophilic polymer head group to the lipid region may be a covalent or non-covalent attachment; however it is preferably via the formation of a covalent bond (optionally via a linker). The outermost surface coating of hydrophilic polymer chains is effective to provide a liposome with a long blood circulation lifetime in vivo. The lipopolymer may be introduced into the liposome in two different ways either by: (a) adding the lipopolymer to a lipid mixture, thereby forming the liposome, where the lipopolymer will be incorporated and exposed at the inner and outer leaflets of the liposome bilayer [Uster P.S. et al. FEBBS Letters 386:243 (1996)]; or (b) first preparing the liposome, and then incorporating the lipopolymers into the external leaflet of the pre-formed liposome either by incubation at a temperature above the Tm of the lipopolymer and liposome-forming lipids, or by short-term exposure to microwave irradiation.

Preparation of vesicles composed of liposome-forming lipids and lipids such as phosphatidylethanolamines (which are not liposome-forming lipids) and derivatization of such lipids with hydrophilic polymers (thereby forming lipopolymers) which in most cases are not liposome-forming lipids. Examples have been described, for example, by Tirosh et al. [Tirosh et al., Biopys. J., 74(3):1371-1379 (1998)] and in U.S. Patent Nos. 5,013,556; 5,395,619; 5,817,856; 6,043,094; and 6,165,501; incorporated herein by reference; and in WO 98/07409. The lipopolymers may be non-ionic lipopolymers (also referred to at times as neutral lipopolymers or uncharged lipopolymers) or lipopolymers having a net negative or a net positive charge.

There are numerous polymers which may be attached to lipids. Polymers typically used as lipid modifiers include, without being limited thereto, polyethylene glycol (PEG), polysialic acid, polylactic acid (also termed polylactide), polyglycolic acid (also termed polyglycolide), apolylactic-polyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. The polymers may be employed as honiopolymers or as block or random copolymers.

While the lipids derivatized into lipopolymers may be neutral, negatively charged, or positively charged, i.e. there is no restriction regarding a specific (or no) charge, the most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually distearylphosphatidylethanolamine (DSPE).

A specific family of lipopolymers which may be employed by the invention includes monomethylated PEG attached to DSPE (with different lengths of PEG chains, the methylated PEG referred to herein by the abbreviation PEG), in which the PEG polymer is linked to the lipid via a carbamate linkage resulting in a negatively charged lipopolymer. Other lipopolymers are the neutral methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the neutral methyl polyethyleneglycol oxycarbonyl-3 -amino- 1,2-propanediol distearoylester (mPEG-DS) [Garbuzenko O. et al, Langmuir. 21:2560-2568 (2005)]. The PEG moiety preferably has a molecular weight of the PEG head group from about 750 Da to about 20,000 Da. More preferably, the molecular weight of the headgroup is from about 750 Da to about 12,000 Da, and it is most preferably between about 1,000 Da to about 5,000 Da. One specific PEG-DSPE employed herein is a PEG moiety with a molecular weight of 2,000 Da, designated herein ²⁰⁰⁰PEG-DSPE or ^2kPEG-DSPE. Preparation of liposomes including such derivatized lipids has also been described where typically between 1-20 mole percent of such a derivatized lipid is included in the liposome formulation.

In addition to liposome-forming lipids (such as PCs and sphingomyelins), cholesterol and phopshatidylethanolamines can be included in the liposomal formulation (e.g. to decrease a membrane's free volume and thereby permeability and leakage of an agent encapsulated therein). In accordance with one embodiment, the liposomes comprise cholesterol. In accordance with a further embodiment, the lipid/cholesterol mole/mole ratio is within the range of between about 80:20 to about 50:50. A more specific mole/mole ratio is about 60:40.

The liposome may include other constituents. For example, charge-inducing lipids such as phosphatidyl glycerol may also be incorporated into the liposome bilayer to decrease vesicle-vesicle fusion, and to increase interaction of the liposome with cells. Buffers at a pH suitable to make the pH of the surface of the liposomes close to neutral can decrease hydrolysis. Addition of an antioxidant, such as vitamin E, or chelating agents, such as Desferal or DTPA, may be used.

The liposome-forming lipid and other lipid and non-lipid components (if used) are dissolved in a protic organic solvent, hi the context of the present invention, a protic organic solvent is typically an alcohol, preferably C2 to C4 alcohols. The solvent is preferably miscible in water. Non-limiting examples of protic organic solvents include methanol, ethanol, and tertiary butanol (tert-butanol). Ethanol is a preferred solvent.

The solvent dissolves the lipid or mixture of lipids to form a solution or dispersion. By the terms "dissolves" or "solution" it is to be understood that the lipid is preferably homogeneously mixed within the solvent; nonetheless, non-homogenous mixtures may be formed and used in the context of the present invention (in a dispersion).

The formed solution or dispersion is then added to an aqueous solution comprising ions. The term "ions-comprising aqueous solution" is used herein to denote an aqueous solution which comprises a salt, such as ammonium sulfate, calcium acetate, etc., dissolved therein. The different types of ions and salts are discussed in detail below with respect to pH or ion gradient formation. ^• The method of the invention is also characterized in that a high lipid concentration may be employed. It has been found that a high agent/lipid ratio (above 1.0) may be achieved even with lipid concentration in the formed liposomes being above 20 mM, above 30 mM, above 50 mM, above 90 mM and even up to 97-100 mM, inclusive. In accordance with one embodiment, the lipid concentration is between 30 mM and 100 mM, inclusive.

Variations in ratios between these liposome constituents dictate the pharmacological properties of the liposome. For example, stability of the liposomes, which is a major concern for various types of vesicular applications, may be dictated by selecting specific liposome constituents. Evidently, the stability of liposomes should meet the same standards as conventional pharmaceuticals. Chemical stability involves prevention of both the hydrolysis of ester bonds in the phospholipid bilayer and the oxidation of unsaturated sites in the lipid chain. Chemical instability can lead to physical instability or leakage of encapsulated drug from the bilayer and fusion and aggregation of vesicles. Chemical instability also results in short blood circulation time of the liposome, which affects the effective access to and interaction with the target.

Different types of liposomes may be prepared by the method of the present invention, including, without being limited thereto, multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multivesicular vesicles (MVV), and large multivesicular vesicles (LMVV). The different types of liposomes may be obtained by applying one or more additional treatment steps to liposomes formed by the above-described steps. For example, LMVV may be obtained by performing one, and optionally more, F&T cycles on the liposomes suspension. SUV may be obtained by any known technique to down- size liposomes (such as vortexing, ultrasonication, extrusion, etc.). Those versed in the art will know how to select the appropriate additional treatment step(s) in order to convert the liposomes formed by the method of the present invention to other liposomal forms and structures.

In accordance with one embodiment, the method of the invention preferably provides MLVs. Following one F&T cycle, the MLVs are converted to LMVVs. Additional F&T cycles may be performed. In accordance with an embodiment, at least one F&T cycle is performed. In accordance with another embodiment, between 5 to 9 F&T steps are performed. The F&T cycle(s) convert(s) the MLV to LMVV which are then incubated with the agent to be loaded therein. The liposome suspension thus formed comprises an amount of the protic organic solvent. The amount may vary, depending on the solvent and the type of the liposomes thus formed. The amount of the organic solvent will be such that the liposomes are not converted to micelles (or micellae). For example, when using ethanol as the organic solvent, transformation from liposomes to micelles will typically occur at 30% solvent (by volume). Thus, ethanol levels in the liposomal suspension may be as high as about 25% by volume. In accordance with one embodiment, ethanol level is about 10% by volume.

An active agent to be loaded into the liposomes may be any substance, e.g., a low or high molecular weight compound, having a utility in therapy or diagnostics. In accordance with one embodiment, the active substance is an amphiphatic weak acid or an amphiphatic weak base. In accordance with a preferred embodiment, the agent is an amphiphatic weak acid drug or amphipathic weak base drug.

Amphiphatic weak base drugs include, among others, the following non-limiting list: tempamine (TMN) doxorubicin, epirubicin, daunorubicin, carcinomycin, N- acetyladriamycin, rubidazone, 5-imidodaunomycin, N-acetyldaunomycine, all anthracyline drugs, daunoryline, topotecan, irinotecan propranolol, pentamidine, dibucaine, bupivacaine, tetracaine, procaine, chlorpromazine, vinblastine, vincristine, mitomycin C, pilocarpine, physostigmine, neostigmine, chloroquine, amodiaquine, chloroguanide, primaquine, mefloquine, quinine, pridinol, prodipine, benztropinemesylate, trihexyphenidyl hydrochloride, propranolol, timolol, pindolol, quinacrine, benadryl, promethazine, dopamine, L-DOPA serotonin, epinephrine, codeine, meperidine, methadone, morphine, atropine, decyclomine, methixene, propantheline, imipramine, amitriptyline, doxepin, desipramine, quinidine, propranolol, lidocaine, bupivacaine, chlorpromazine, promethazine, perphenazine, acridine orange, opiates such as morphine, and others. In accordance with one embodiment, the amphiphatic weak base is an analgesic drug. Some analgesic drugs are listed above and include lidocaine and bupivacaine. These drugs are also specifically exemplified herein below.

Amphiphatic weak acid drugs include, without being limited thereto, ibuprofen, toluetin, indomethacin, phenylbutazone, mecloferamic acid, piroxicam, citrofloxacin, prostaglandins, fluoresgein, carboxyfluorescein, methyl perdnisolone hemisuccinate

(MPS), paracetamol (acetaminophen), aspirin (acetyl salicylic acid) and other NSAIDs, and nalidixic acid.

Further, there may be interest in having glucocorticosteroids as an agent loaded in liposomes and treating these liposomes prior to administration with empty liposomes. A non-limiting list of glucocorticoids may be found at the internet site http://www.steraloids.com/, incorporated herein in its entirety by reference. Non- limiting examples include: prednisolone hemisuccinate, methylprednisolone hemisuccinate, dexamethasone hemisuccinate, allopregnanolone hemisuccinate; beclomethasone 21 -hemisuccinate; betamethasone 21 -hemisuccinate; boldenone hemisuccinate; prednisolone hemisuccinate; sodium salt; prednisolone 21- hemisuccinate; nandrolone hemisuccinate; 19-nortestosterone hemisuccinate; deoxycorticosterone 21 -hemisuccinate; dexamethasone hemisuccinate; dexamethasone hemisuccinate: spermine; corticosterone hemisuccinate; cortexolone hemisuccinate. In general, there is a variety of loading methods available for preparing liposomes with entrapped active agents, including passive entrapment and active (remote) loading. The term "entrapment" as used herein denotes any form of loading of the agent onto the liposomes, such that at least a substantial part of the agent is encapsulated within the interior aqueous core of the liposomes. Within the interior core the agent may be free or associated to the inner surface of the lipid bilayer. Thus, in the context of the present invention the term "entrapment" may at times be used interchangeably with the terms "encapsulation" or "carrying" or "loading".

The passive entrapment method is most suited for entrapment of lipophilic drugs in the liposome membrane and for entrapment of agents having high water solubility. In the case of ionizable hydrophilic or amphiphatic agents, even greater agent- loading efficiency can be achieved by loading the agent into liposomes against a transmembrane pH or ion gradient [Nichols, J.W., et al., Biochim. Biophys. Acta 455:269-271 (1976); Cramer, J., et al., Biochemical and Biophysical Research Communications 75(2):295-301 (1977)]. This loading method, generally referred to as remote loading, typically involves an agent that is amphiphatic in nature and has an ionizable group which is loaded by adding it to a suspension of liposomes having a higher inside/lower outside H⁺ and/or ion gradient.

The liposomes employed in the context of the present invention are preferably loaded by the remote loading principle. Remote loading occurs due to pH or ion gradient, such as ammonium or ammonium-like (with non-organic, organic or polymeric anions, e.g. alkylamine) gradient aggregation due to a high intra-liposome concentration of the agent and the formation of an agent-counter-ion salt within the liposome. Excess of the counter-ion occurs when the NH₃ is released from the liposomes. Remote loading via an ammonium salt is based on the large difference in permeability of the neutral ammonia gas molecule (1.3XlO^"1 cm/s) and the charged anion (<10^"10cm/s). Typically, the pH of the intra-liposome aqueous phase composed of an ammonium salt solution may be decreased by lowering the external concentration of ammonium and ammonia [Haran G., et al., Biochim Biophys. Acta 1151:201-215, (1993)]. The decrease of intra-liposomal pH results from the release from the liposome of the unprotonated ammonia compound (NH₃) leaving within the liposome protons (H⁺) and the counter-ion (e.g. HSO₄ ^" _, SO₄ ^"2); thereby an excess of the counter-anions over NH₄ ⁺ is created within the liposome.

Reduction of the pH inhibits ammonia formation and thereby inhibits its release from the liposome. When adding to the external medium of the liposome an agent, e.g., an amphiphatic weak base, it freely crosses the lipid bilayer in its uncharged form and accumulates in its charged (having low permeability) form in the internal aqueous compartment (after being protonated by the free H⁺) [Schuldiner, et al., Eur. J. Bichem 25:64-70 (1972); Nicolas and Deamer, Biochem. Biophys Acta 455:269-271 (1976)]. Evidently, this accumulation raises the internal pH and thus ammonia is again formed and released from the liposome, resulting in the reduction of internal pH and so forth, until an effective loading of the agent is accomplished. It is thus noted that for loading of amphiphatic weak bases, it is preferable that the liposome have a low mter-liposomal/high intra-liposomal trans-membrane gradient, such as ammonium salt gradient (e.g. ammonium sulfate).

When the agent is a weak amphiphatic acid, it is preferable that the liposomes have a high inter-liposome/low intra-liposome transmembrane gradient. Such a gradient may be achieved using an aqueous solution of acetate salt such as calcium acetate. In this case the acetate ion gradient is the driving force while the Ca⁺² ions, which have very low permeability through the liposome membrane, act as counter-ions of the weak amphiphatic acid within the aqueous phase, thereby stabilizing the loading and enabling better control over the release rate of the loaded weak amphiphatic acid. [Clerc, S. and Barenholz, Y., Loading of amphiphatic weak acids into liposomes in response to transmembrane calcium acetate gradients. Biochim. Biophys. Acta 1240:257-265 (1995)].

The equilibrium between charged (protonated) and uncharged agents enables the slow leakage of the uncharged weak base from the liposomes at a rate which is dependent on the permeability coefficient. Shifting the equilibrium via formation of aggregates (formed between the loaded charged agent and the counter-ion within the liposome) further improves the retention of the agent inside the liposome, and as now being disclosed, may function as a tool for controlling the release of the agent from the liposome.

In accordance with the present invention, the H⁺ and/or ion gradient is formed by dissolving the liposome-forrning lipid or the mixture comprising a liposome-forming lipid and other lipids (not necessarily liposome-forming lipids, e.g., cholesterol) with a protic organic solvent to form a solution or suspension of said lipid and then adding the lipid solution to an ion-containing aqueous solution to form a liposome suspension. The liposome suspension is then incubated with a solution comprising the agent to be loaded and an H⁺ or ion concentration suitable for achieving a respective H⁺ or ion gradient between the inter-liposomal compartment and the intraliposomal surrounding. In the case of amphiphatic weak acids or amphiphatic weak bases (as the active agent/drug) there is an ionizable group, and thus the agent is loaded by adding it to a suspension of liposomes prepared so as to have an inside/outside pH gradient. As a result, the uncharged species of amphiphatic weak acid or amphiphatic weak base diffuses trough the liposome membrane.

However, due to the different pH inside the interliposomal aqueous phase, being more acidic (for weak bases) or more alkaline (for weak amphiphatic acids) with respect to the intraliposomal surrounding, the agent is protonated or deprotonated, respectively, to become a charged species.

In accordance with one embodiment, the liposomes formed by the method of the invention may be prepared by using an aqueous buffer containing an ammonium salt, such as ammonium sulfate, ammonium phosphate, ammonium citrate, etc., typically about 0.1 M to 0.3 M ammonium salt, at a suitable pH, e.g., about 5.5 to 7.5. The gradient can also be produced by including sulfated polymers in the aqueous solution added to the lipid solution. For example, such sulfated polymers may include dextran sulfate ammonium salt, heparin sulfate ammonium salt or sucralfate. After liposome formation, the external medium may be exchanged for one lacking ammonium ions. In this approach, during loading, the amphiphatic weak base is exchanged with ammonium ion.

An H⁺ZiOn gradient may also be achieved by including in the liposomes with a selected ionophore. To illustrate, liposomes prepared to contain valinomycin in the liposome bilayer are prepared in a potassium buffer, after which the external medium is then exchanged with a sodium buffer, creating a potassium inside/sodium outside gradient. Movement of potassium ions in an inside-to-outside direction in turn generates a lower inside/higher outside H⁺ or ion gradient, presumably due to movement of protons into the liposomes in response to the net electronegative charge across the liposome membranes [Deamer, D. W., et al., Biochim. et Biophys. Acta 274:323

(1972)].

A similar approach is to add the lipid to an aqueous solution having a high concentration of magnesium sulfate. The magnesium sulfate gradient is created by dialysis against 20 mM HEPES buffer, pH 7.4, in sucrose. Then, an A23187 ionophore is added, resulting in outwards transport of the magnesium ion in exchange for two protons for each magnesium ion, plus establishing a inner liposome high/outer liposome low proton gradient [Senske DB et al. Biochim. Biophys. Acta 1414: 188-204 (1998)].

Yet another approach is described in US 5,939,096, incorporated herein by reference. In brief, that method employs a proton shuttle mechanism involving the salt of a weak acid, such as acetic acid, of which the protonated form translocates across the liposome membrane to generate a higher inside/lower outside H⁺ or ion gradient. An arnphiphatic weak acid compound is then added to the medium to the pre-formed liposomes. This amphiphatic weak acid accumulates in liposomes in response to this gradient, and may be retained in the liposomes by cation (i.e. calcium ions)-promoted precipitation or low permeability across the liposome membrane; namely, the amphiphatic weak acid is exchanged with the acetic acid.

In the case of a weak base, the H⁺/ ion gradient may be formed by using salts having a counter-ion selected from, without being limited thereto: hydroxide; sulfate; phosphate; glucuronate; citrate; carbonate; bicarbonate; nitrate; cyanate; acetate; benzoate; bromide; chloride; other inorganic or organic anions; an anionic polymer such as dextran sulfate, dextran phosphate, dextran borate, carboxymethyl dextran and the like; as well as polyphosphates. In the case of a weak acid the counter-ion may be calcium, magnesium, sodium, ammonium and other inorganic and organic cations, or a cationic polymer such as dextran spermine, dextran spermidine, aminoethyl dextran, trimethyl ammonium dextran, diethylaminoethyl dextran, polyethyleneimine dextran and the like. The counter-ion may be present in the form of a free small ion or attached to a polymer, or in both forms simultaneously. A specific embodiment for liposomes carrying weak amphiphatic acids is those in which the high inter-liposomal/low intra- liposomal trans-membrane gradient is formed by using an acetate salt, such as calcium acetate, sodium acetate or potassium acetate. Ca²⁺ acetate is a preferred acetate salt.

The release rate of the loaded agent from liposomes was shown to be dependent on a variety of factors, including, without being limited thereto, the counter ion which forms a salt with the active agent (see in this connection WO03/032947, "A method for preparing liposome formulations with a predefined release profile", incorporated herein its entirety by reference), temperature, medium-related properties (medium composition, ionic strength, pH), liposome-related properties (membrane lipid composition, liposome type, number of lamellae, liposome size, physical state of phospholipid membrane i.e., liquid- disordered (LD), liquid-ordered (LO), solid-ordered (SO)), and loaded-molecule-related properties (lipophilicity, hydrophilicity, size) [Haran G., et ah, Biochim Biophys. Acta 1151:201-215, (1993)].

The invention also provides pharmaceutical compositions comprising a physiologically acceptable carrier and an amount of the agent-carrying liposomes prepared in accordance with the invention, the amount being effective to treat or prevent a disease or disorder.

The pharmaceutical composition may be provided as a single dose, however it may be preferably administered to a subject in need of treatment over an extended period or time (e.g. to produce a cumulative effective amount), in a single daily dose for several days, in several doses a day, etc. The treatment regimen and the specific formulation to be administered will depend on the type of disease to be treated and may be determined by various considerations, known to those skilled in the art of medicine, e. g. physicians. The term "effective amount" or "amount effective" is used herein to denote the amount of the agent which, when loaded in the liposome, is sufficient in a given therapeutic regimen to achieve a desired therapeutic effect with respect to the treated disease or disorder. The amount is determined by such considerations as may be known in the art and depends on the type and severity of the condition to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors, including the mode of administration, type of vehicle carrying the amphipathic weak acid/base, the reactivity of the active agent (the weak amphiphatic acid or base), the liposome's distribution profile within the body, a variety of pharmacological parameters such as half-life in the body after being released from the liposome, undesired side effects, if any, factors such as age and gender of the treated subject, etc.

The term "administering" (or "administration") is used to denote the contacting or dispensing, delivering or applying of the liposomal formulation to a subject by any suitable route of delivery thereof to the desired location in the subject, including oral, parenteral (including subcutaneous, intramuscular and intravenous, intra-arterial, intraperitoneal, etc.) and intranasal administration, as well as intrathecal and infusion techniques. According to one embodiment, the composition used in accordance with the invention is in a form suitable for injection. The requirements for effective pharmaceutical vehicles for injectable formulations are well known to those of ordinary skill in the art [See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4^th ed., pages 622-630 (1986)]. Further, the invention concerns a method of treating a subject for a disease or disorder, the method comprising administering to said subject an amount of agent-carrying liposomes prepared by the method of the invention.

As used herein the term "treatment" (or "treating") denotes curing of an undesired pathological condition or prevention of a condition from developing. For the purpose of curing, the term "treatment" includes ameliorating undesired symptoms associated with the condition, slowing down progression of the condition, delaying the onset of a progressive stage of the condition, slowing down deterioration of such symptoms, enhancing onset of a remission period of the condition, if existing, delaying onset of a progressive stage, improving survival rate or more rapid recovery from the condition, lessening the severity of or curing the condition, etc. Treatment also includes prevention of a disease or disorder. The term "prevention" includes, without being limited thereto, administering an amount of the composition to prevent the condition from developing or to prevent irreversible damage caused by the condition, to prevent the manifestation of symptoms associated with the condition before they occur, to inhibit the progression of the condition etc.

It is noted that the forms "a", "an" and "the" as used in the specification include singular as well as plural references unless the context clearly dictates otherwise. For example, the term "a lipid" includes one or more, of the same or different lipids.

Similarly, reference to the plural includes the singular, unless the context clearly dictates otherwise.

Further, as used herein, the term "comprising" is intended to mean that the liposome includes the recited constituents, but does not exclude others which may be optional in the formation or composition of the liposome, such as antioxidants, cryoprotectants, etc. The term "consisting essentially of is used to define a substance, e.g. liposome, that includes the recited constituents but excludes other constituents that may have an essential significant effect on a parameter of the substance (e.g., in the case 006/001229

- 22 -

of liposomes, the stability, release or lack of release of the agent from the liposome as well as on other parameters characterizing the liposomes). "Consisting of^x shall thus mean excluding more than trace amounts of such other constituents. Embodiments defined by each of these transition terms are within the scope of this invention. Further, all numerical values, e.g. when referring the amounts or ranges of the elements constituting the composition or liposome components, are approximations which are varied (+) or (-) by up to 20%, at times by up to 10% from the stated values. It is to be understood, even if not always explicitly stated, that all numerical designations are preceded by the term "about". It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art, and it is explicitly intended that the invention include such alternatives, modifications and variations.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification.

DETAILED DESCRIPTION OF SOME NON-LIMITING EXEMPLARY EMBODIMENTS

Materials

Hydrogenated soy phosphatidylcholine (hereinafter referred to by the abbreviation HSPC) was obtained from Lipoid, Ludwigsahfen, Germany.

Cholesterol was obtained from Sigma.

Bupivacaine hydrochloride (hereinafter referred to by the abbreviation BUP) was obtained from Orgamol, Evionnar, Switzerland. Lidocaine hydrochloride (hereinafter referred to by the abbreviation LID) was obtained from Sigma.

Dimyristoylphosphatidylcholine (hereinafter referred to by the abbreviation DMPC) was obtained from Lipoid, Ludwigsahfen, Germany. Dipalmitoylphosphatidylcholine (hereinafter referred to by the abbreviation

DPPC) was obtained from Lipoid, Ludwigsahfen, Germany.

Liposome preparation and characterization

Preparation of multilamellar vesicles (MLV) MLV liposomes were prepared by weighing 450 mg of dry HSPC and 154 mg of dry cholesterol (a 60:40 mole ratio). The dry phospholipid/cholesterol mixture was then dissolved in 1 ml ethanol at 80°C and the dissolved mixture was added to an aqueous solution of (NHU)₂SO₄ (250 niM, prepared by adding 297 mg of ammonium sulfate to 9 ml of water), to obtain a preparation having a final phospholipid concentration of 60 mM. Ethanol volume was 10% of final volume. The thus obtained MLVs were heated at 65⁰C for 45 min.

Preparation of large multi-vesicular vesicles (LMW)

MLV prepared as above were freeze-thawed either once or more (up to a total of

10 freeze-thawing cycles). Freezing was performed using liquid nitrogen (-196°C) and thawing was performed using a water bath (37°C). Freezing time was proportional to the volume of liposome preparation such that for each milliliter of preparation, one minute freezing was executed (i.e. for 10 ml, 10 minute freezing took place).

To create a transmembrane ammonium sulfate gradient, the liposome preparation was centrifuged 4 times sequentially in normal saline (4⁰C, 1000 g, 5 min). This is effective to create an inside-to-outside ammonium ion gradient across the liposomal membrane. The ammonium ion concentration gradient provides the driving force for loading of amph philic weak bases such as Bupivacaine (BUP). The presence of a transmembrane pH gradient was verified by determining the distribution of amphiphatic weak base acridine orange (AO), as described in Haran, G. et al. Biochim. Biophys. Acta 1151:201-215 (1993) and Clerc S., Barenholz Y. Anal Biochem. 259(l):104-ll (1998).

Drug loading to LMW Liposome

The drug, BUP or LID was remote-loaded into the liposomes by incubating the liposome preparation with 4.5% of appropriate drug solution (50 mg/ml solution of drug) at 60°C for 45 min.

Non-entrapped drug was removed from LMVV suspension by centrifugation in normal saline (4°C, 1000 g, 5 min). The pH of the final medium was about 5.5. This pH was retained to ensure the drug's solubility and prevent precipitation. The amount of trapped and free drug after one day of storage and for at least one month after storage was measured using high performance liquid chromatography (HPLC) (Grant G. et al. Pharm. Res., 18 N3:336-343 (2001) and Grant G. et al. Anasthesiology, 101:133-137 (2004)); the amount of phospholipid in the liposomal formulation was determined using the Bartlett method (Shmeeda, H. et al. Methods Enzymol. 367:272-292 (2003)). The drug-to-lipid ratio was calculated from the parameters obtained.

Characterization of drug-loaded liposomes Drug-to-Lipid Ratio

The drug-to-lipid ratio obtained in the liposomal formulation prepared as described above was greater than 2 (mole drug/mole lipid > 2).

Liposome Size

The size of the liposomes was determined using laser Fraunhofer diffraction (LS 13320 Laser Diffraction Particle Sizer Analyzer, Beckman Coulter UK). The instrument's Software expresses particle size as the volume median diameter. The mean size of LMVV was ~ 8.5 ± 6.5 micron. The level of transmembrane pH gradient according to AO distribution was ~ 89% verifying a low inter-liposome /high intra- liposome transmembrane pH gradient, being larger than 3. Kinetics of Drug Loaded Liposome

Kinetics of drug leakage from liposomes was measured at 4⁰C during storage for one month (for BUP) or three months (for LID) after preparation. Tables 1 and 2 provide parameters indicating leakage at the different time points.

Table 1: Kinetics of BUP leakage from liposomes.

Days after Peak area μg/ml Mg/ml mM BUP % free % mixing BUP BUP BUP liposomal

BUP

1 20.63 54.56 5.46 18.94 - 100*

5 2.5 6.97 0.70 2.42 12.8 87.2

7 3.76 10.28 1.03 3.57 18.8 81.2

9 4.8 13.01 1.30 4.52 23.8 76.2

11 4.48 12.2 1.22 4.23 22.3 77.7

13 4.07 11.10 1.11 3.85 20.3 79.7

15 4.49 12.20 1.22 4.24 22.4 77.6

27 2.59 7.21 0.72 2.50 13.2 86.8

29 4.87 13.20 1.32 4.58 24.2 75.8

31 4.18 11.38 1.14 3.95 20.9 79.1

33 4.89 13.25 1.32 4.60 24.3 75.7

34 5.28 14.27 1.43 4.96 26.2 73.8

36 4.59 12.46 1.25 4.33 22.8 77.2

38 3.14 8.65 0.87 3.00 15.9 84.1

46 5.51 14.87 1.49 5.16 27.3 72.7

53 4.76 12.91 1.29 4.48 23.7 76.3

57 5.51 14.87 1.48 5.13 27.1 72.9

60 6.33 17.03 1.70 5.90 31.1 68.9

65 6.57 17.66 1.76 6.11 32.2 67.8

69 6.74 18.10 1.81 6.29 33.2 66.8

74 7.17 19.23 1.92 6.68 35.2 64.8

78 7.71 20.65 2.06 7.17 37.8 62.2

61 7.82 20.94 2.09 7.27 38.4 61.6

63 7.77 20.81 2.08 7.22 38.1 61.9

68 8.83 23.59 2.36 8.19 43.2 56.8

* liposomal + residual free BUP (after wash) Table 2: Kinetics of LID leakage from liposomes

Day after Peak area μg/ml LID mg/ml mM LID % free % mixing LID LID liposomal

LID

1 24.54 53.0 5.3 19.6 - 100*

3 1.15 1.8 0.2 0.7 3.5 97

7 1.73 3.1 0.3 1.2 5.9 94

14 1.51 2.6 0.3 1.0 5.0 95

24 1.59 2.8 0.3 1.0 5.3 95

36 1.97 3.6 0.4 1.3 6.9 93

* liposomal + residual free LID (after wash)

The effect of number offreeze-thaw cycles on drug/lipid ratio and drus release level

The effect of number of freezing-thawing cycles on LMVV properties was studied. Two independent experiments were performed. In the second experiment a batch of liposomes that were not freeze-thawed (MLV) were examined. Transmembrane pH gradient and BUP loading were prepared as described above. According to AO distribution measurements the transmembrane pH gradient was the same for all preparations, i.e., ~ 85% - 90%.

The results presented in Table 3 show the percentage of free, non-encapsulated BUP in the composition relative to the total BUP is provided after preparation (overnight) and following one week of storage.

Table 3: The effect of F&T on drug/Iipid ratio and drug leakage from liposomes mM PL mg/ml mM BUP drug/lipid % free % free Free BUP % free

BUP total total BUP from total after from total overnight overnight week after week mg/ml

MLV initial 38.13

1 F&T 8.84 3.2 11.22 1.27 0.39 11.93 0.56 17.38

3 F&T 9.01 3.1 10.63 1.18 0.30 9.93 0.45 14.85

5 F&T 7.97 3.5 12.21 1.53 0.43 12.21 0.46 13.22

7 F&T 7.75 4.1 14.34 1.85 0.28 6.80 0.50 12.12

9 F&T 8.27 4.9 17.16 2.08 0.44 8.89 0.58 11.78

MLV initial 43.8

MLV 11.2 3.3 11.32 1.01 0.67 20.60 0.65 19.82

F&T*

1 F&T 12.3 3.9 13.53 1.10 0.80 20.65 0.90 23.14

3 F&T 10.9 4.7 16.33 1.50 0.67 14.23 0.69 14.77

5 F&T 10.1 4.9 16.89 1.68 0.80 16.43 0.97 19.90

7 F&T 11.4 5.0 17.22 1.51 0.69 13.85 0.79 15.91

9 F&T 10.4 6.3 21.75 2.09 0.97 15.49 0.80 12.80

Table 3 shows that the number of F&T had no significant effect on the drug-lipid ratio. Further, the size distribution of the liposomes was measured as a function of the number of F&T.

The number of freezing-thawing cycles had no significant effect on liposome size distribution as determined based on the volume of vesicles using Universal Liquid

Model of Beckman Coulter (using a combination of light diffraction and PIDS which enable size range measurements of 0.040 to 2000 μm, optical model of

Fraunhofer.rf780d (for size distribution calculation) and with a precision size standard diameter of 1.27 μm, Cat No. 64035 of Polyscience, Inc) (data not shown). Specifically, there was no significant difference between drug/lipid ratio in cases of 1-5 freeze-thaw cycles in the first experiment and even 1-7 freeze-thaw cycles in the second one. At the same time the highest drug/lipid ratio was obtained after 9 freeze-thaw cycles in both experiments. The second preparation showed higher drug release levels.

The effect of the initial phospholipid concentration on drug/lipid ratio and drug release level

Stock solutions of different initial lipid concentrations were prepared and the effect of the number of freezing-thawing cycles (0, 1, and 9 F&T cycles) was evaluated. Two independent experiments were performed. In the first experiment BUP leakage from liposomes was measured two days after drug loading, and in the second experiment, BUP leakage was determined during two weeks after drug loading. Results are presented in Table 4 (first experiment) and Table 5 (second experiment).

Table 4: The effect of the initial phospholipid (PL) concentration on drug/lipid ratio and drug leakage

PL denotes phospholipids

^²' As measured two days after preparation

⁽³⁾ F&T denotes number of Freezing and Thawing cycles

Table 5 - The effect of the initial phospholipid (PL) concentration on drug/lipid ratio and drug releases mM PL Mg/ml mM BUP BUP/PL Free BUP Free BUP Free BUP Free BUP Free BUP Free

BUP total overnight /total after IW /total after 2W BUP/total

(mg/ml) overnight (mg/ml) after IW (mg/ml) after 2 W

MLV 48.2 initial

O F&T 12.9 2.3 7.92 0.62 0.58 25.65 0.62 27.27 0.69 30.24

1 F&T 11.4 4.9 16.89 1.48 0.72 14.81 0.73 14.91 0.77 15.91

9 F&T 11.2 6.9 24.06 2.14 0.85 12.31 1.06 15.26 1.14 16.51

MLV 53.5 W initial

O F&T 13.8 4.2 14.49 1.05 0.43 10.29 0.48 11.57 0.58 13.84

1 F&T 13.0 4.2 14.49 1.12 0.55 13.28 0.65 15.55 0.62 10.18

9 F&T 10.16 6.1 21.09 2.08 0.81 13.33 0.90 14.88 0.75 17.87

MLV 86.1 initial

O F&T 18.0 7.7 26.59 1.48 0.83 10.84 1.04 13.57 1.09 14.21

1 F&T 16.3 9.2 31.93 1.96 1.00 10.91 1.09 11.22 0.97 7.86

9 F&T 18.4 12.4 42.98 2.34 0.77 6.23 : 1.00 8.04 0.91 9.86

mM PL Mg/ml mM BUP BUP/PL Free BUP Free BUP Free BUP Free BUP Free BUP Free

BUP total overnight /total after IW /total after 2W BUP/total

(mg/ml) overnight (mg/ml) after IW (mg/ml) after 2W

MLV 93.5 initial

O F&T 23.8 7.6 26.27 1.10 1.11 14.65 1.18 15.56 1.19 15.70

1 F&T 28.5 11.6 40.36 1.42 0.76 6.57 0.85 7.34 0.81 6.94

9 F&T 27.7 10.7 37.30 1.35 0.93 8.70 1.02 9.48 1.46 19.34

According to the data presented in Tables 4 and 5 it is evident that increasing the initial phospholipid concentrations, as high as about 97 raM), affected the drug/lipid ratio in all preparations with MLV (no F&T cycles (0). In LMW obtained after 9 F&T cycles there was no significant effect of the different initial phospholipid concentration on the drug/lipid ratio. It is noted that the higher the initial lipid concentration, the lower the drug leakage (after two weeks). However, it is further noted that additional increase of the phospholipid initial concentration up to 118 mM resulted in a decreased drug/lipid ratio. Loading of BUP in liposomes has various advantages, amongst others, in reducing toxicity of the drug in a free form. It is known that BUP, which is an amide- linked local anesthetic of high potency, results in cardiovascular and central nervous system toxicity when high concentrations of the drug gain access to the circulation. Thus, the systemic toxicity of standard BUP and LMVV BUP was evaluated by determining the dose that was lethal in 50% of mice (LD₅o) following intraperitoneal injection (data not shown). To minimize the number of animals required, the up-and- down technique of Dixon and Massey was used. For all study solutions, 0.3 ml/10 g animal weight was injected, and mice were observed for 6 hours after injection for signs of toxicity. The results showed that the LD₅₀ for standard BUP was 71 mg/kg and the LD₅₀ for LMVV BUP was 565 mg/kg. This eight-fold increase in LD₅₀ in LMW BUP was consistent with a slow release of the drug from the liposomal depot. It also shows that the use of LMVV will enable the safer administration of a much greater dose of local anesthetic than currently permitted.

Claims

CLAIMS:

1. A method of preparing liposomes comprising:

(a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid; (b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid of step (a) with a protic organic solvent to form a solution or dispersion of said lipid; and

(c) adding the solution or dispersion of step (b) to ions containing aqueous solution to form a liposome suspension; wherein the liposomes are capable of encapsulating an agent at a ratio between the agent and the liposome-forming lipid of greater than 1.0.

2. A method of preparing liposomes consisting of:

(a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid; (b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid with a protic organic solvent to form a solution or dispersion of said lipid; and

(c) adding the solution or dispersion to an ions-containing aqueous solution to form a liposome suspension; wherein the liposomes are capable of encapsulating an agent at a ratio between the agent and the liposome-forming lipid of greater than 1.0.

3. A method of preparing liposomes comprising:

(a) providing a dry liposome-forming lipid or a dry mixture comprising a liposome-forming lipid; (b) dissolving the liposome-forming lipid or said mixture comprising a liposome-forming lipid with a protic organic solvent to form a solution or dispersion of said lipid;

(c) adding the solution or dispersion to ions containing aqueous solution to form a liposome suspension wherein the liposomes are capable of encapsulating an agent at a ratio between the agent and the liposome forming lipid of greater than 1.0; the method being characterized in that one or more of the following steps are excluded: drying the solution or dispersion to form a dry lipid film; down-sizing liposomes in said liposome suspension to form small unilamellar vesicles (SUV).

4. A method of preparing agent-carrying liposome, the method comprising: (i) providing a liposome suspension by the method of any one of Claims 1 to 3; and

(ii) incubating said liposome suspension with a solution comprising said agent to form agent-carrying liposomes, wherein said agent-carrying liposomes comprise a mole/mole ratio between said agent and said liposome-forming lipid of greater than 1.0.

5. The method of any one of Claims 1 to 4, wherein said liposome suspension comprises multilamellar vesicles (MLV).

6. The method of any one of Claims 1 to 5, wherein said liposome-forming lipid is a phospholipid.

7. The method of any one of Claims 1 to 6, wherein said dry mixture comprises cholesterol.

8. The method of any one of Claims 1 to 7, wherein said protic organic solvent is miscible in water.

9. The method of Claim 8, wherein said organic solvent is selected from ethanol, methanol or tertiary butanol.

10. The method of any one of Claims 1 to 9, wherein said solution or suspension of said liposome-forming lipid comprises a lipid concentration of at least 2OmM.

11. The method of Claim 10, wherein said lipid concentration is between about 30 mM and about 100 mM.

12. The method of any one of Claims 1 to 11, comprising at least one freezing and thawing (F&T) cycle of the liposome suspension to obtain large multivesicular vesicles

(LMVV).

13. The method of any one of Claims 2 to 12, wherein said agent-to-lipid ratio is equal to or above 1.8.

14. The method of any one of Claims 6 to 13, wherein said phospholipid is hydrogenated soy phosphatidylcholines (HSPC).

15. The method of any one of Claims 1 to 14, wherein said agent is a weak amphiphatic base or a weak amphiphatic acid.

16. The method of any one of Claims 2 to 15, wherein said incubation of the weak amphiphatic base is with liposomes having a low inter liposomal/high intra liposomal trans-membrane pH or ion gradient.

17. The method of Claim 16, wherein said liposomes have a low inter liposomal/high intra liposomal trans-membrane ammonium salt gradient.

18. The method of any one of Claims 2 to 15, wherein said incubation of the weak amphiphatic acid is with liposomes having a high inter liposomal/low intra liposomal trans-membrane pH or ion gradient.

19. The method of Claim 18, wherein said liposomes have a high inter liposomal/low intra liposomal trans-membrane acetate salt gradient.

20. A pharmaceutical composition comprising a physiologically acceptable carrier and agent carrying liposomes prepared by the method of any one of Claims 2 to 19.

21. A method of treating a subject comprising providing said subject with an amount of agent carrying liposomes prepared by the method of any one of Claims 2 to 20.