WO2007049279A2 - A liposomal combination and uses thereof - Google Patents

Abstract

The invention provides a liposomal combination comprising a composition comprising a first population of liposomes encapsulating an agent; a composition comprising a second population of liposomes free of the agent. The first population of liposomes is less permeable to the agent than the second population of liposomes. Also provided is a method for reducing an amount of non-encapsulated agent in a composition comprising a first population of liposomes encapsulating an agent, as well as a method for reducing toxicity following storage of a first composition comprising a first population of liposomes encapsulating an agent and an amount of free agent. Both methods comprise mixing the first population of liposomes with a second population of liposomes free of the agent, the first population of liposomes being less permeable to the agent than the second population of liposomes.

Description

A LIPOSOMAL COMBINATION AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to liposome technology and in particular to liposome based delivery systems.

LIST OF PRIOR ART 1. US Patent No. 5,316,771;

2. US Patent No. 5,192,549;

3. US Patent No. 5,939,096;

4. US Patent No. 6,162,462;

5. Barenholz Y., In: Medical Applications of Liposomes (Lasic, D.D. and Papahadjopoulos, D., eds.), Elsevier Science, Amsterdam, pp. 541-565 (1998).

6. Barenholz Y., Curr. Opin. Colloid Interface ScI 6:66-77 (2001).

7. International Patent Application Publication No. WO/2000/09089.

BACKGROUND OF THE INVENTION

Liposomes were first described nearly forty years ago and have been useful models for studying the physical chemistry of lipid bilayers and the biology of the cell membrane. It was also realized that liposomes might be used as vehicles for the delivery of drugs and other active agents as well as in the field of gene transfer.

Liposome technology faces at least two challenges. The first challenge is to achieve a high level of loading of an active agent in the liposome and to make that loading stable during handling and storage. The second is to be able to fit the release rate of the loaded/associated active agent to specific aims of the liposome formulation.

Loading of an agent into liposomes has proven to be a measure of their utility. A poor liposome loading leads to low drug/lipid ratio, and therefore the use of liposomes as a vehicle becomes inefficient at the target site. In addition, with poor loading, there is a great loss of the active agent, which makes such liposome-based drugs uneconomical.

So far, several methods have been developed for loading agents into liposomes. The simplest method of agent loading includes a passive entrapment of water soluble agents during a dry lipid film by hydration of the lipid components. The loading efficiency of this method is generally low as it depends on the entrapping volume of the liposome, on the concentration of the drug and its solubility in the hydration medium as well as on the amount of lipids used to prepare them.

Improved passive entrapment of an agent into liposomes has been achieved by using a dehydration-rehydration method according to which preformed liposomes are added to an aqueous solution containing the agent, followed by dehydration of the mixture, by lyophilization, evaporation or by a freeze-thaw processing method. This results in an increase in trapped aqueous volume and equilibrating of the solute throughout all the intraliposomal aqueous phase. Another method of encapsulating hydrophilic agents involves reverse evaporation from an organic solvent. According to this approach, a mixture of a hydrophilic agent and vesicle-forming lipids are emulsified in a water-in-oil emulsion, followed by solvent removal to form an unstable lipid-monolayer gel. When the gel is agitated, typically in the presence of added aqueous phase, the gel collapses to form oligolamellar liposomes with high efficiency of encapsulation of the agent. However, the limitation of agent solubility and trapped volume still applies.

In the case of amphiphatic weak acids or bases, loading can be achieved by forming a transmembrane pH gradient and/or ion gradient. Typically, the agent contains an ionizable amine group, and is loaded by adding it to a suspension of liposomes prepared to have an inside/outside pH gradient. The uncharged species of amphiphatic weak acid or base diffuse through the liposome membrane and due to the pH inside the intra-liposome aqueous phase being more acidic (for weak bases) or more alkaline (for weak amphiphatic acids), the species become charged. When using an ion gradient, the solubility and thus permeability through the membrane of the amphiphatic weak acid or amphiphatic weak base depends on the counter-ion thereof (cation or anion, respectively; see also WO 03/032947). For example, when using an ammonium sulfate gradient, the ammonium ion within the liposomes are in equilibrium with ammonia, which is highly permeable through the liposome membrane, while protons or sulfate anions have low permeability through the liposome membrane. Thus, accumulation of protons and sulfate occurs while ammonia is removed from the liposomes. This leads to the lower inside/outside pH gradient. The high ammonium ion concentration phase within the liposomes provides a reservoir of protons, to maintain the liposome pH gradient over time.

The release rate of the loaded agent from liposomes was shown to be dependent on: the anion of the ammonium salt (which inside the liposome will become the anion of the amphiphatic weak base once it is taken up by the liposome, see WO 03/032947); temperature; medium-related properties (medium composition, ionic strength, pH, etc.); liposome-related properties (membrane lipid composition, liposome type, number of lamellae, liposome size, physical state of phospholipid membrane, e.g. liquid-disordered (LD), liquid-ordered (LO), or solid-ordered (SO), etc.); and loaded-molecule-related properties (lipophilicity, hydrophilicity, size, etc.) [Haran G., et ah, Biochim Biophys. Acta 1151:201-215, (1993)].

A high inter-liposome/low intra-liposome transmembrane (ion) gradient may be achieved, for example with calcium acetate which permit remote loading of weak amphiphatic acids. 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)]. Other issues that must be resolved prior to the introduction of liposomal formulations for clinical use include the stability of the formulation during prolonged storage, batch-to-batch variability in physicochemical characteristics, and adaptability of the method for up-scaling for large batch sizes. It is well known that lipids undergo various thermotropic phase transitions and that this is highly relevant to liposome stability and integrity. Phase transition occurs between a more fluid state, referred to as liquid crystalline or liquid disordered (LD) phase, and a more rigid and ordered state, referred to - A -

as gel or solid ordered (SO) phase. The transition between the two is referred to as the "main transition" which can be characterized by a range of temperatures over which the main transition occurs and by temperature (the T_m) being the temperature at which the processes leading to the transition are maximal. It has been established that transition to the liquid disordered phase results in increased permeability of the liposomes and thereby leakage of active agents from the interior of liposomes to the surrounding. The LD to SO phase transition can be abolished by including cholesterol at above 33 mole-% in the liposome membrane, forming a new state referred to as the liquid ordered (LO) phase. However even in the presence of such high mole-% cholesterol, some molecules entrapped in the liposomes still leak at a rate that makes the drug loaded liposomes non-useful for drug delivery purposes. This leakage is overcome by the present invention.

SUMMARY OF THE INVENTION

The present invention is based on the novel understanding that it is possible to minimize the amount of a free agent (e.g., weak amphiphatic acid or weak amphiphatic base) within composition comprising liposomes into which the agent was a priori loaded. Specifically, it has been found that by mixing such a priori drug-loaded liposomes with other liposomes, the latter having a transmembrane pH and/or ion gradient and lacking any (remote-loaded) amphiphatic weak acid or base agent results in a reduced amount of free agent in the combined liposomal composition. The added liposomes (having no agent loaded therei) are referred to herein as "empty" liposomes. Without wishing to be bound by any theory, the empty liposomes may serve as a "sink- like" device for any (free) agent released from the agent-loaded liposomes. The effect is evident when the empty liposomes have a greater permeability than the permeability of the agent-loaded liposomes. Thus, in accordance with a first of its aspects, the present invention provides a liposomal combination comprising:

(a) a first composition comprising a first population of liposomes encapsulating an agent; and

(b) a second composition comprising a second population of liposomes being free of said agent; wherein said first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes.

The second liposome population (which are the empty liposomes) has a built-in driving force (pH and/or ion gradient) to pump/actively diffuse into them the agent released from the agent-loaded liposome population.

The liposomal combination may be provided with instructions for mixing the composition of said first population of liposomes encapsulating said agent and the composition comprising said second population of liposomes free of said agent to form a liposomal mixture prior to administration of the liposomal mixture to a subject in need. The liposomal combination may be provided in the form of a kit or package containing the first and second compositions, stored in two separate containers or in one container having two separate compartments, one containing the agent-loaded liposomes and the other the empty liposomes.

It is noted that the liposomes of the first population and the liposomes of the second population may have the same composition, i.e. formed from the same liposome-forming lipids, and may comprise their components at the same ratios. However, for facilitating the present invention, it is preferable that the two liposomes are different, such that the empty liposomes have a higher permeability as defined herein. In accordance with a further aspect, the invention provides a method for reducing an amount of non-encapsulated agent in a composition comprising a first population of liposomes encapsulating said agent, the method comprising mixing said first population of liposomes with a second population of liposomes being free of said agent (empty liposomes), wherein said first population of liposomes has a permeability to said agent which is lower than the permeability of said second population of liposomes to said agent.

In yet a further aspect of the invention there is provided a method for reducing toxicity following storage of a first composition comprising a first population of liposomes encapsulating an agent and an amount of free agent, the method comprising mixing said first composition with a second composition comprising a second population of liposomes free of said agent, wherein the first population of liposomes has a permeability to said agent which is lower than the permeability of said second population of liposomes to said agent.

In accordance with one preferred embodiment, the empty liposomes have a trans-membrane pH and/or ion gradient. Preferably the mixing of the first composition and the second composition should take place before administration to the subject, while allowing the mixture of the two compositions sufficient time to be incubated together so that the empty liposomes can "trap" the free (typically released during storage) agent.

The invention additionally provides a liposomal formulation comprising a first population of liposomes encapsulating an agent and a second population of liposomes encapsulating said agent, said first population of liposomes having a permeability to said agent which is lower than the permeability of said second population of liposomes to said agent.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a liposomal composition carrying an agent (e.g., a drug), in which a reduction of undesirable side effects related to the level of free agent released during liposome storage is achieved.

It has already been envisaged that during storage of compositions comprising agent-carrying liposomes the agent may leak (be released, diffuse) from the liposomes which then remain in the composition as a free agent. The existence of free agent in such compositions may affect the efficacy and toxicity of the composition upon administration to a subject in need thereof. Therefore, there is great interest in preventing or reducing the level of such free agent in the composition prior to administration. The solution provided by the present invention is based on the combination, prior to administration to a subject, of two populations of liposomes, one encapsulating an agent and the other being empty and having a higher permeability to the agent to be delivered to said subject. " Permeability" is generally defined by the amount of material that permeates or leaks per unit of area and unit of time. In the context of the present invention, "permeability" also denotes the capability of the lipid bilayer to transfer over time a substance, e.g. a drug or other agent, from one side of the liposome membrane (e.g. from the inter-liposomal aqueous medium, also referred to as extra-liposome medium) to the other side of the membrane (e.g., to the intra-liposomal surrounding). Permeability of the liposomes may be determined by methods known in the art to measure cell and liposome permeability. For example, leakage of an agent can be measured by separating the liposomes from any material which has leaked out, using methods such as gel permeation chromatography, dialysis, ultra-filtration or the like, and assaying in a known manner for any leaked material (see also in this connection sections relating to permeability in: Liposomes: A Practical Approach. V Weissig & V Torchilin (eds), 2^nd edition; New, R. C. C₅ Liposomes: A Practical Approach, Oxford 1^st edition). In accordance with the invention, the two populations of liposomes include: (i) a first population of liposomes encapsulating an agent, where, upon storage, some agent leaks from the liposomes, and thus this population of liposomes may be in the form of a mixture with free agent; and (ii) a second population of liposomes being free of the agent (herein also referred to by the term "empty liposomes"). An a priori requirement in accordance with the invention is that the first population of liposomes has permeability to the agent which is lower than that of said second population of liposomes.

Distinctively different permeabilities may be achieved by using different components within the bilayer of the two liposome populations. The liposomes may comprise one or more liposome-forming lipids as well as other substances as will be further discussed hereinbelow. In fact, lipid composition is the main factor in the determination of liposome permeability, and this correlates with a lipid's (when using one lipid to form the liposome) T_m. Permeability may be affected also by membrane active sterols (like cholesterol) as well as other membrane active substances (substances forming the membrane). Cholesterol will slow down leakage (i.e. reduce permeability) when all the lipids in the bilayer are in the LO phase (in many cases with PCs it is obtained with an amount of cholesterol being equal or above 33 mole-%). For liposoraes composed of a mixture of liposome-forming lipids the parameters may be complex, as appreciated and known by those versed in the art.

Temperature also affects permeability and thus the invention may make use of two liposome populations where at a specific temperature, one liposome population is in the LD phase and the other is in SO or LO phase. The permeability of liposomes in the LD phase will be higher than the permeability of liposome populations in the other phases. For example, a first liposome population may be composed of HSPC (T_m 52⁰C) and the other from DPPC (T_m of 41⁰C). At 45⁰C (above the T_m of DPPC), DPPC is in the LD phase, and thus liposomes formed therefrom are more permeable than the HSPC-based liposomes. At 55⁰C₅ when both liposome populations are in the LD phase, some difference in the permeabilities of the two liposome populations will still exist, as the HSPC is nevertheless more rigid as compared to the DPPC-based liposomes. The difference in permeability, however, will be smaller in the latter case.

Size of liposomes may have some effect as the membrane permeability, as large liposomes (e.g., 100 nm and above) have less curvature than the membranes of liposomes smaller than 100 nm. Another difference between large and small liposomes is in the surface area/volume ratio which, for small liposomes, is smaller than for large liposome; and therefore more agent will leak from the small liposomes as compared to larger liposomes. However, these differences are mild. Finally, as appreciated, for leakage or permeation through a membrane of multilamellar vesicles (MLV) it is required that the agent cross more than one bilayer to become free (or entrapped). MLV may therefore be regarded as less permeable than unilamellar liposomes. The same understanding should apply to multivesicular vesicles. However, also in this case, the differences in permeability between multi- and univesicular vesicles are mild.

It is worth noting that another important factor affecting permeability is the existence of a trans-membrane, proton, hydroxyl or other ion gradient with respect to the empty liposomes. Such a gradient is a driving force for the free agent (released from the agent-loaded liposomes). The different types of gradient are further discussed hereinbelow. - ci -

In accordance with one embodiment, the difference in the permeabilities of the two liposome populations is achieved by constructing liposomes of different gel to liquid crystalline phase transition temperatures (T_m).

A thermotropic phase transition from gel (i.e. solid) to liquid crystalline (i.e. fluid) or from liquid crystalline to gel phase undergone by lipids and liposomes is known to affect the free volume and degree of rigidity of the lipid bilayer of the liposome. When in LO phase, the lipids in both leaflets forming the bilayer are

"loosely" aligned according to their hydrophilic and lipophilic regions. This packing enables a large level of "free volume" which facilitates diffusion across the liposome membrane. Below the range of main transition (i.e. when in the SO phase), the lipid molecules are more closely packed, and have much less free volume, and therefore permeability is reduced to a large extent, if not eliminated entirely.

It is noted that at the temperature range in which LD and SO phases co-exist there are interface regions in which packing is disturbed and there are packing defects, as the two phases do not fit to each other. At this range permeability is usually the highest.

Permeability may also be designed by adding to the liposome composition membrane active sterols (as briefly discussed above). For example, in liposomes composed mainly of PCs and/or sphingomyelins (or any other liposome forming lipid, excluding those having polyunsaturated acyl chains) and having cholesterol in an amount equal to or above 33% (mole-%), the bilayer is in the LO phase (see above). As a result, permeability is reduced compared to liposomes in the LD phase. However there is no risk of going through the main transition as this is abolished by the high level of cholesterol (or other membrane active sterol). However, when comparing permeability of liposomes of different liposome-forming lipids with the same level of membrane active sterol (like cholesterol), permeability will be determined by each liposome- forming lipid's T_m. For example, permeability of HSPC/cholesterol (T_m of HSPC is 52⁰C) is lower than that of DPPC/Cholesterol (T_m of DPPC 42°C) or that of DMPC/Cholesterol (T_m of DMPC is 24°C). It is also worth noting that permeability of a membrane to an agent may also depend on the characteristics of the specific agent and in particular, the agent's octanol to aqueous phase partition coefficient (Kp). For example, doxorubicin has a low Kp and bupivacaine a much higher Kp. This explains the differences in their leakage rate from the same liposomes or from liposomes of similar composition. For this reason, bupisomes (bupivacaine-loaded liposomes) leak during storage at 4°C while Doxil (doxorubicin-loaded liposomes) do not [see also Haran et al. 1993, ibid.].

The difference in permeability may allow, under suitable conditions, free agent, which leaked from agent-carrying liposomes, to be trapped by the empty liposomes mixed therewith. To facilitate this entrapment by the empty liposomes, it is possible in an embodiment that the liposomes carrying the agent (the first population of liposomes) have a gel to liquid crystalline phase transition temperature range which is greater than that of agent-free liposomes (the second population of liposomes referred to as empty liposomes). In accordance with one embodiment, the Tm of the second population of liposomes is equal to or below 45° (and thus the Tm of the first population of liposomes is above this parameter). Lipids having a relatively high Tm may be referred to as "rigid" lipids, typically those having saturated, long acyl chains, while lipids with a relatively low Tm may be referred to as "fluid" lipids. Fluidity or rigidity of the liposome may be determined by selecting lipids with pre-determined fluidity/rigidity for use as the liposome-forming lipids.

A difference in permeability may also be achieved by combining liposome- forming lipids with other liposome constituents. Liposome-forming lipids are amphiphilic molecules essentially characterized by a packing parameter of about 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 times the mole fraction of each component) in the range between 0.74 and 1. In addition to liposome-forming lipids (like PCs and sphingomyelins), cholesterol and/or phosphatidylethanolamines can be included in the liposomal formulation in order to decrease a membrane's free volume and thereby permeability and leakage of agent encapsulated therein. In accordance with one embodiment, the liposomes, either the first population of liposomes, the second population of liposomes, or both, may comprise cholesterol. Independently, the lipid/cholesterol mole/mole ratio of the liposomes in the two liposome populations may be in the range of between about 80:20 and 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 phosphatidylglycerol, may also be incorporated into the liposome bilayer to decrease vesicle-vesicle fusion, and to increase interaction with cells. Buffers at a pH suitable to make the liposome surface's pH 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.

Variations in ratios between the 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.

Liposome-forming lipids in accordance with the invention are those 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 chain, 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 same, 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 a good alternative to glycerophospholipids.

Typically, a substituting chain, e.g. the acyl, alkyl 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 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 vesicle- forming lipids and naturally-occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), dimyristoyl phosphatidylglycerol (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), and the sphingophospholipids such as sphingomyelins (SM) having 12- to 24-carbon atom acyl or alkyl chains. The above-described lipids and phospholipids whose hydrocarbon chain (acyl/alkyl/alkenyl chains) have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids include in the liposomes are glyceroglycolipids and sphingoglycolipids and sterols (such as cholesterol or plant sterol).

Preferably, the liposome-forming lipids composing the first population of liposomes are selected from those having a Tm above 45°C, such as, without being limited thereto, phosphatidylcholine (PC) and derivatives thereof having an acyl chain with 16 or more carbon atoms. A preferred example of a PC derivative which forms the basis for the first population of liposomes is hydrogenated soy PC (HSPC) having a Tm of 52°C. The first population of liposomes may comprise sphingomyelins of various N acyl chains, such as N-stearoyl sphingomyelin. Further, preferably, the liposome-forming lipids of the second population of liposomes are selected from those having a T_m below 45⁰C, such as, without being limited thereto: Dimyristoylphosphatidylcholine (DMPC), having a T_m of 24⁰C; Dipalmitoylphosphatidylcholine (DPPC), having a T_m of 41.3°C; dipentadecanoyl PC, having a Tm of 33°C; and N-palmitoyl sphingomyelin having a T_m of 41.2°C. Those versed in the art will know how to select a lipid with a T_m either below or above 45°C (see also Barenholz, Y., Liposome application: problems and prospects. Curr. Opin. Colloid Interface Sci. 6, 66-77 (2001); Barenholz, Y. and Cevc, G., Structure and properties of membranes. In Physical Chemistry of Biological Surfaces (Baszkin, A. and Norde, W., eds.), Marcel Dekker, NY (2000) pp. 171-241) 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 where the lipid 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-trimethylammonium propane (DMTAP); l,2-dioleyloxy-3- (trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy)propyl]-N,N-dimeth- yl-N-hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N~ dimethyl-N-hydroxy ethyl ammonium bromide (DORIE); N-[l-(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-dimethy 1-2,3 -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.

Liposomes may be composed of liposome-forming lipids and lipids such as phosphatidylethanolamines (which are not liposome forming lipids) and derivatization of such lipids with hydrophilic polymers the latter forming lipopolymers which in most cases are not liposomes-forming lipids. Examples have been described in 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, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. The polymers may be employed as homopolymers 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 include 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 lipopolymer 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 is from about 750Da to about 20,000 Da. More preferably, the molecular weight is from about 750 Da to about 12,000 Da₅ 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 2000 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. Different types of liposomes may be employed in the context 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 liposomes of the first population may be of the same type as those forming the second population or may be of a different type. In accordance with one embodiment, the liposomes of the first population and second population are LMVV. LMVV may be prepared by methods known in the art. For example, LMVV may be prepared by: (a) vortexing a lipid film with an aqueous solution, such as a solution of ammonium sulfate; (b) homogenizing the resulting suspension to form a suspension of small unilamellar vesicles (SUV); and (c) repeatedly freeze-thawing said suspension of SUV in liquid nitrogen followed by water. Preferably, the freeze-thawing is repeated at least five times. The extraliposomal ammonium sulfate is then removed, e.g. by dialysis against normal saline. The therapeutic agent is encapsulated within the liposomes by incubating a suspension of the LMVV liposomes with a solution of the agent. This method is as also described in detail in International Patent Publication No. WO/20000/9089 (the LMVV referred to therein by the abbreviation GMV). The inventors have now developed a further approach for the preparation of liposomes which includes mixing dry liposome constituents, such as liposome-forming lipids, cholesterol, etc.; solvating or dissolving the mixture in a protic organic solvent which is miscible in water, such as ethanol; and adding to the dissolved/solvated mixture to a solution comprising a salt (pH forming aqueous solution, e.g. (NHU)₂SO₄) to obtain a liposome suspension having an amount of the organic solvent in the final volume (may even be up to 25% of the organic solvent from final volume).

By this method typically MLVs are formed. The MLVs may be converted to other structures, such as MLVV₅ by one or more cycles of freezing and thawing. Transmembrane H⁺ or ion gradients may then be formed by methods as described. This approach is described in detail in a co-pending International Patent Application publication No. claiming priority from US Provisional Application No.

60/730,045, and in the following examples.

An active agent in accordance with the invention is any substance having a utility in therapy or diagnostics. In accordance with one embodiment, the active substance is an amphiphatic weak acid or base. In accordance with yet another embodiment, the agent is an amphiphatic weak acid or base drug.

Amphiphatic weak base drugs include, among others, the following non-limiting list: tampamine (TMN), doxorubicin, epirubicin, daunorubicin, carcinomycin, N- acetyladriamycin, rubidazone, 5-imidodaunomycin, N-acetyldaunomycine, all anthracyline drugs, daunoryline, topotecn, irinotecan propranolol, pentamidine, dibucaine, bupivacaine, tetracaine, procaine, cblorpromazine, 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 prednisolone (hemi)succinate

(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 heeimisuccinate; 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; and 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 used interchangeably with the terms "encapsulation" or "carrying" or "loading".

The passive entrapment method is most suited for entrapping of lipophilic drugs in the liposome membrane and for entrapping 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 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 which 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.

More specifically, remote loading occurs due to a pH or ion, such as ammonium or ammonium-like (with norganic, organic or polymeric anions, e.g. alkylamine) ion gradient aggregation due to a high intraliposome concentration of the agent and the formation of agent-counter ion salt. 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^" cm/s). Typically, the pH of the intraliposome aqueous phase composed of an ammonium salt solution may be decreased by lowering the external concentration of ammonium and ammonia [Haran, et al., (1993) (ibid); Barenholz et al. 2001 (ibid.)]. The decrease of intraliposomal pH results from the release from the liposome of the unprotonated ammonia compound (NH₃) leaving within the liposome protons (H⁺) and the counter anion (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 accumulate 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. 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.

Liposomes having an H⁺ and/or ion gradient across the liposome bilayer for use in remote loading can be prepared by a variety of techniques. A typical procedure comprises dissolving a mixture of lipids at a ratio that forms stable liposomes in a suitable organic solvent and evaporated in a vessel to form a thin lipid film. The film is then covered with an aqueous medium containing the solute species that will form the aqueous phase in the liposome interior space. After liposome formation, the vesicles may be sized to achieve a size distribution of liposomes within a selected range, according to known methods. The liposomes utilized in the present invention are preferably uniformly sized to a selected size range between about 70-100 nm, inclusive, preferably about 80 nm.

After sizing, the external medium of the liposomes is treated to produce an ion gradient across the liposome membrane (typically with the same buffer used to form the liposomes), which is typically a higher inside/lower outside ion concentration gradient. This may be done in a variety of ways, e.g., by: (i) diluting the external medium; (ii) dialysis against the desired final medium; (iii) gel exclusion chromatography, e.g., using Sephadex G-50, equilibrated in the desired medium which is used for elution; or (iv) repeated high-speed centrifugation and resuspension of pelleted liposomes in the desired final medium. The selection of the external medium will depend on the type of gradient, on the mechanism of gradient formation, the external solute and pH desired, as will now be described.

In a simpler approach for generating an ion and/or H⁺ gradient, the lipids are hydrated and sized in a medium having a selected internal-medium pH. The suspension of the liposomes is titrated until the external liposome mixture reaches the desired final pH, or treated as above to exchange the external phase buffer with one having the desired external pH. For example, the original hydration medium may have a pH of 5.5, in a selected buffer, e.g., glutamate, citrate, succinate, fumarate buffer, and the final external medium may have a pH of 8.5 in the same or different buffer. The common characteristic of these buffers is that they are formed from acids which are essentially liposome impermeable. The internal and external media are preferably selected to contain about the same osmolarity, e.g., by suitable adjustment of the concentration of buffer, salt, or low molecular weight non-electrolyte solute, such as dextrose or sucrose.

In another general approach, the gradient is produced by including in the liposomes a selected ionophore. To illustrate, liposomes prepared to contain valinomycin in the liposome bilayer are prepared in a potassium buffer, sized, then the external medium 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 hydrate the lipid and to form multilamellar liposome in high concentration of magnesium sulfate. The magnesium sulfate gradient is created by dialysis against 20 raM 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 an inner liposome high- concentration/outer liposome lowconcentration proton gradient [Senske DB et al. (Biochim. Biophys. Acta 1414: 188-204 (1998)].

In another preferred approach, the proton gradient used for drug loading is produced by creating an ammonium ion gradient across the liposome membrane, as described, for example, in US Patent Nos. 5,192,549 and 5,316,771, incorporated herein by reference. The liposomes are prepared in an aqueous buffer containing an ammonium salt, such as ammonium sulfate, ammonium phosphate, ammonium citrate, etc., typically about 0.1 to about 0.3 M ammonium salt, at a suitable pH, e.g., about 5.5 to about 7.5. The gradient can also be produced by including in the hydration medium sulfated polymers, such as dextran sulfate ammonium salt, heparin sulfate ammonium salt or sucralfate. After liposome formation and sizing, the external medium is exchanged for one lacking ammonium ions. In this approach, during the loading the amphiphatic weak base is exchanged with the ammonium ion.

Yet, another approach is described in US 5,939,096, incorporated herein by reference. In brief, the 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 amphiphatic 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 (e.g. calcium ions)-promoted precipitation or low permeability across the liposome membrane, namely, the amphiphatic weak acid is exchanges with the acetic acid.

In the case of a weak base, the H⁺ or 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, and others inorganic or organic anions, or an anionic polymer such as dextran sulfate, dextran phosphate, dextran borate, carboxymethyl dextran and the like. 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. This means that 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 are those in which the high interliposomal/low intraliposomal trans-membrane gradient is formed by using calcium acetate.

The liposomal combination in accordance with the invention may also include instructions for use by the practitioner (physician, nurse, pharmacist etc.) or by the individual user (e.g. the subject in need of the agent). The instruction comprises mixing of the composition of the first population of liposome and the composition of the second population of liposomes prior to administration to the subject in need. The mixture of the two populations are then incubated together for a time sufficient to allow permeation and trapping of any free agent from the first composition by the second population of liposomes (e.g. as a result of leakage from the first population of liposomes during storage or a priori inefficient loading of the agent into the liposomes etc.). This incubation time will depend on the transition temperature of the second population of liposomes and the temperature at which the mixture is incubated. For example, incubation for about 30 min. at room temperature of a liposomal mixture having a first population of liposomes comprising HSPC and cholesterol (lipidxholesterol mole:mole ratio of about 60:40) and a second population of liposomes comprising DSPC or DPPC and cholesterol (lipid:cholesterol moleimole ratio of about 60:40) suffice to entrap a substantial portion of the free agent within the mixture.

It is noted that the mixing ratio between the first population of liposomes and the second population of liposomes will depend on various factors as known to those versed in the art. Such factors will include, inter alia, the specific liposomal compositions (i.e. the different constituents forming the liposomes' bilayers) and thus the permeability of the liposomal membrane, the specific characteristics of the active agent, the amount of free agent present in the composition, etc. The person skilled in the art may easily take these factors into consideration so as to determine the amount of agent free liposomes required for mixing and entrapping any free agent present in the agent carrying composition.

The invention also provides a method for reducing an amount of non- encapsulated agent in a composition comprising a first population of liposomes encapsulating said agent, the method comprising mixing said first population of liposomes with a second population of liposomes being free of said agent, said first population of liposomes have permeability to said agent which is lower than that of said second population of liposomes.

The invention also provides a method for reducing toxicity following storage of a first composition comprising a first population of liposomes encapsulating an agent and an amount of free agent, the method comprising mixing said first composition with a second composition comprising a second population of liposomes free of said agent, the first population of liposomes having permeability to said agent which is lower than that of said second population of liposomes.

Finally, the invention provides a liposomal formulation comprising a first population of liposomes encapsulating an agent and a second population of liposomes encapsulating the same agent, wherein said first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes. This liposomal formulation is obtainable either by the method described above or by any other method of combining two populations of liposomes.

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

It is noted that the forms V, "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 include the recited constituents, but does not exclude others which may be optional in the formation or composition of the liposome, such as antioxidants, cryo- protectants 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 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 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.

DESCRIPTION OF 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 , Switzeranld;

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. Liposomes preparation and characterization Preparation of liposomes for drus loading

Preparation of multilamellar vesicles (MLV)

MLV liposomes were prepared by weighting 450mg of dry HSPC and 154mg of dry cholesterol (a 60:40 mole: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 mM, 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 (-196⁰C) and thawing was performed using a water bath (37⁰C). 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 amphiphilic weak bases such as Bupivacaine (BUP). The presence of a transmembrane H⁺ or ion 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 6O⁰C 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, 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 (mole/mole) was calculated from the parameters obtained.

Characterization of Drug Loaded Liposome 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 an inter liposome low/intra liposome high transmembrane pH gradient being larger than 3.

Kinetics of Drug Loaded Liposome

Kinetics of drug leakage from liposomes was measured at 4°C during storage for one month (for BUP) or three months (for LID) after preparation. Tables 1 and 2 provide parameters indicative of leakage at the different time points. Table 1: Kinetics of BUP leakage from liposomes.

Time (days Peak area μg/ml mg/ml mM BUP % - free % from BUP BUP BUP liposomal preparation) BUP

1 20.63 54.56 5.46 18.94 100*

5 2.5 6.97 0.70 2.42 12.8 87.2

6 3.76 10.28 1.03 3.57 18.8 81.2

8 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

12 4.07 11.10 1.11 3.85 20.3 79.7

14 4.49 12.20 1.22 4.24 22.4 77.6

26 2.59 7.21 0.72 2.50 13.2 86.8

27 4.87 13.20 1.32 4.58 24.2 75.8

32 4.18 11.38 1.14 3.95 20.9 79.1

34 4.89 13.25 1.32 4.60 24.3 75.7

35 5.28 14.27 1.43 4.96 26.2 73.8

39 4.59 12.46 1.25 4.33 22.8 77.2

41 3.14 8.65 0.87 3.00 15.9 84.1

49 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 Lidocaine (LID) leakage from liposomes

* liposomal + residual free LID (after wash) Preparation of Empty LMW Formulation

Two types of LMVV with high interliposomal/low intraliposomal transmembrane ammonium sulfate gradient were prepared using the procedure described above with either a combination of dimyristoylphosphatidylcholine (DMPC) and cholesterol or a combination of dipalmitoylphosphatidylcholine (DPPC) Cholesterol. These two phospholipids were selected based on their gel to liquid crystalline phase transition temperatures (Tm): for DMPC, Tm of 24°C; and for DPPC Tm = 41.3⁰C being lower than that of HSPC (Tm = 52°C). Lower Tm liposomes were used in order to facilitate the mixture of the liposomes at room temperature without heating, enabling the penetration of the free drug through the membrane of the liposome. Due to the lower Tm of the liposome-forming lipid in the empty liposomes, the fluidity of the lipid bilayer is comparably greater than the fluidity of the lipids forming the drug-loaded liposome (HSPC), therefore aiding the capturing of the leaking drug by the empty liposomes.

Combination of empty LMW with drug-loaded liposomes

It has been found that during storage, the drug encapsulated into liposomes tends to leak from the liposome. The following approach was applied in order to reduce the amount of free drug obtained during storage of drug-loaded liposomal formulations. Mixing the Empty and Drug Loaded Liposomes

Empty DMPC/Chol or DPPC/Chol LMVV were mixed (without storage after preparation) at room temperature (RT) with a liposomal formulation comprising HSPC/Chol LMVV loaded with BUP and free BUP (see Tables 3A and 3B). It was postulated that the empty LMVV will capture the free BUP. The amount of drug entrapped in the empty LMVV may vary and will depend on the phospholipid ratio between BUP-loaded LMVV and empty LMVV as well as on the mixing time and a temperature of mixing.

The results are presented in Tables 3A and 3B showing the percentage of free, non-encapsulated, BUP in the composition relative to the total BUP prior to mixing with empty liposomes (the total BUP) and is provided in the column under the heading "% free from total"; the percentage of free, non-encapsulated, BUP in the composition relative to the total BUP after mixing with empty liposomes and is provided in the column under the heading "% fi-ee after mixing"; and the percentage of BUP reloaded into liposomes following mixing with empty liposomes (corresponding to the difference in free agent following mixing) is provided in the column under the heading "% free removed". This column shows the efficiency of the free drug removal in relative terms.

Table 3A: Percentage of entrapped BUP 1 hour after mixing at RT

Table 3B: Percentage of entrapped BUP 1 hour after mixing at RT

Specifically, Tables 3A and 3B show that there was no significant difference between the respective DMPC- and DPPC-based LMVV and that equilibrium between free and trapped BUP is achieved 1 hour following mixing of the drug-loaded and empty LMVV. It is noted that this equilibrium may be achieved also after a shorter incubation period if the temperature of incubation is increased.

Further, Tables 3 A and 3B show that the amount of free BUP in the liposomal formulation was reduced. Level of reduction was increased with the increasing of the amount of the DMPC- or DPPC-based liposomes (the liposomes used for the preparation of empty liposomes) and there was no significant difference between the type of phospholipid employed, DMPC/Chol- or DPPC/Chol-based LMVV on the level of free BUP re-entrapment by the empty LMVV.

Analεesic Effect of Mixed Liposomal Formulation In a further experiment the analgesic effect of a formulation comprising a combination of BUP-loaded LMVV and empty LMVV was examined. Specifically, BUP-loaded LMVV prepared as described above in the concentrations indicated above, and after 20 days of storage was mixed with empty LMVV, either DPCC/cholesterol based LMVV or DMPC/cholesterol based LMVV in a 60:40 lipid-to-cholesterol ratio (mole/mole), having an ammonium sulfate transmembrane gradient. As a control, drug- loaded LMVVs were provided to animals without prior incubation with empty liposomes and diluted respectively with an equal volume of normal saline. The concentration of drag given to animals was identical in both cases, as determined by HPLC analysis prior to administration (as described above). The results demonstrate that while the level of free drug in the liposomal formulation following storage was reduced (as a result from leakage), the addition of the empty liposomes to the liposomal formulation prior to administration did not affect the prolongation of the analgesic effect obtained by using liposomal BUP vs. free BUP.

The advantage of combining empty liposomes with BUP-loaded liposomes (from which some BUP leaked and is present in free form) is also clear from a further assay conducted by the inventors (data not shown). Specifically, 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 (LD₅₀) following intraperitoneal injection. 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 is consistent with slow release of drug from the liposomal depot. It also confirms that the use of LMVV will enable the safe administration of a much greater dose local anesthetic than is currently permitted.

Claims

CLAIMS:

1. A liposomal combination comprising:

(a) a first composition comprising a first population of liposomes encapsulating an agent;

(b) a second composition comprising a second population of liposomes and being free of said agent; wherein said first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes.

2. A liposomal combination according to Claim 1, wherein said first population of liposomes has a gel to liquid crystalline phase transition temperature (Tm) which is greater than that of said second population of liposomes.

3. The liposomal combination of Claim 2, wherein the Tm of the second population of liposomes is equal or below 45°.

4. The liposomal combination of any one of Claims 1 to 3, wherein said first population of liposomes comprise cholesterol.

5. The liposomal combination of any one of Claims 1 to 3 and 5, wherein said second population of liposomes comprise cholesterol.

6. The liposomal combination of Claim 4 or 5, comprising a lipid to cholesterol mole ratio of between 80:20 and 50:50.

7. The liposomal combination of any one of Claims 1 to 6, wherein said agent is encapsulated in the interior aqueous phase of said liposomes.

8. The liposomal combination of any one of Claims 1 to 7, wherein the first population of liposomes is selected from multilamellar vesicles (MLV)₅ small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), stericaly stabilized liposomes (SSL), multivesicular vesicles (MVV), large multivesicular vesicle (LMVV).

9. The liposomal combination of any one of Claims 1 to 8, wherein the second population of liposomes is selected from multilamellar vesicles (MLV), small unilamellar vesicles (SUV)₅ large unilamellar vesicles (LUV), stericaly stabilized liposomes (SSL), multivesicular vesicles (MVV), large multivesicular vesicle (LMVV).

10. The liposomal combination of any one of Claims 1 to 9, wherein said first population of liposomes comprise one or more phospholipids.

11. The liposomal combination of Claim 12, wherein said phospholipid is hydrogenated soy phosphatidylcholines (HSPC).

12. The liposomal combination of Claim 11, wherein said first population of liposomes comprise cholesterol.

13. The liposomal composition of any one of Claims 1 to 9, wherein said second population of liposomes comprise one or more phospholipids.

14. The liposomal combination of Claim 13, wherein said phospholipid is selected from Dimyristoylphosphatidylcholine (DMPC), Dipalmitoylphosphatidylcholine (DPPC) or combination of same.

15. The liposomal combination of Claim 14, wherein said first population of liposomes comprise cholesterol

16. The liposomal combination of any one of Claims 1 to 15, wherein said firs population of liposomes and second population of liposomes comprise a transmembrane pH gradient.

17. The liposomal combination of any one of Claims 1 to 16, wherein said second population of liposomes comprise a low inter liposomal/high intra liposomal transmembrane pH or ion gradient, and said agent is an amphiphatic weak base.

18. The liposomal combination of Claim 17, wherein said first population of liposomes comprise a low inter liposomal/high intra liposomal trans-membrane pH or ion gradient.

19. The liposomal combination of Claim 18, comprising a low inter liposomal/high intra liposomal trans-membrane ammonium salt gradient.

20. The liposomal combination of Claim 16, wherein said second population of liposomes comprise a high inter liposomal/low intra liposomal trans-membrane pH gradient, and said agent is an amphiphatic weak acid.

21. The liposomal combination of Claim 2O₅ wherein said first population of liposomes comprise a high inter liposomal/low intra liposomal trans-membrane pH or ion gradient.

22. The liposomal combination of Claim 21, wherein said first population of liposomes and second population of liposomes comprise a high inter liposomal/low intra liposomal trans-membrane acetate salt gradient.

23. The liposomal combination of any one of Claims 1 to 22, in combination with instructions for mixing the composition of said first population of liposomes encapsulating said agent and the composition comprising said second population of liposomes free of said agent to form a liposomal mixture prior to administration of the liposomal mixture to a subject in need.

24. The liposomal combination of Claim 23, wherein said instructions comprises mixing of said first population of liposomes and second population of liposomes at least 30 min. at room temperature prior to administration.

25. A method for reducing an amount of non-encapsulated agent in a composition comprising a first population of liposomes encapsulating an agent, the method comprises mixing said first population of liposomes with a second population of liposomes being free of said agent, said first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes.

26. A method for reducing toxicity following storage of a first composition comprising a first population of liposomes encapsulating an agent and an amount of free agent, the method comprises mixing said first composition with a second composition comprising a second population of liposomes free of said agent, the first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes.

27. The method of Claim 25 or 26, wherein said mixing is for a time sufficient to allow at least a portion of free agent in said first composition to permeate into the second population of liposomes.

28. The method of Claim 27, wherein said mixing is for at least 30 min at room temperature.

29. A liposomal formulation comprising a first population of liposomes encapsulating an agent and a second population of liposomes encapsulating said agent, said first population of liposomes has permeability to said agent which is lower than that of said second population of liposomes.

30. The liposomal formulation of Claim 29 obtainable by the method of any one of Claims 25 to 28.