WO2003022250A2

WO2003022250A2 - Unilamellar vesicles stabilized with short chain hydrophilic polymers

Info

Publication number: WO2003022250A2
Application number: PCT/CA2002/001365
Authority: WO
Inventors: Nancy Dos Santos; Marcel Bally; Lawrence Mayer
Original assignee: Celator Technologies Inc.
Priority date: 2001-09-10
Filing date: 2002-09-10
Publication date: 2003-03-20
Also published as: WO2003022250A3; AU2002325120A1

Abstract

Short chain hydrophilic polymers impart enhanced circulation longevity to unilamellar vesicles made without cholesterol, when the hydrophilic polymer is incorporated into the vesicles at low densities.

Description

VESICLES STABILIZED WITH SHORT CHAIN HYDROPHILIC POLYMERS

Field of the invention

This invention relates to unilamellar liposomes and in particular to unilamellar vesicles containing substantially no cholesterol.

Description of the prior art

Over the last decade significant progress has been made in the clinical development of liposomes for drug delivery of anti-cancer agents. Although chemotherapeutic agents are effective, there is significant toxicity to normal cells resulting in symptoms including nausea, alopecia, myelosuppression, cardio- and nephrotoxicity. Encapsulation of anti-cancer agents in drug delivery systems such as liposomes has proven to be beneficial because drug exposure to normal cells can be drastically reduced resulting in significantly lower toxic side effects.

Liposomes are microscopic particles that are made up of one or more lipid bilayers enclosing an internal compartment. Liposomes can be categorized into multilamellar vesicles, unilamellar vesicles and giant liposomes. Multilamellar liposomes (also known as multilamellar vesicles or "MLN") contain multiple concentric bilayers within each liposome particle, resembling the "layers of an onion".

Unilamellar liposomes enclose a single internal aqueous compartment although a small proportion of vesicles in a typical preparation of unilamellar vesicles may comprise two concentric bilayers and a few such vesicles may comprise more than two bilayers. Unilamellar liposomes include small unilamellar vesicles (SUN) and large unilamellar vesicles (LUN). LUNs and SUNs range in size from 50 to 500 and 20 to 50 nm, respectively.

The size of giant liposomes is typically in the thousands of nanometers, up to 50,000 nm and are used mainly for studying mechanochemical and interactive features of lipid bilayer vesicles in vitro (Νeedham et al., (2000) Colloids and Surfaces B:

Biointerfaces 18: 183). Giant liposomes are not used in vivo due to the poor circulation longevities exhibited by liposomes of greater than about 500 nm. Grafting a hydrophilic polymer, such as a polyalkylether, to the surface of liposomes has been utilized to stabilize liposomes by preventing surface-surface interactions and minimizing protein adsorption to liposomes. This results in enhanced blood stability, increased circulation time, and increased delivery to disease sites such as solid tumors (see: United States Patents 5013556 and 5593622; and Patel et al, (1992) Crit. Rev. Ther. Drug Carrier Syst. 9: 39). Typically, the polymer is conjugated to a lipid component of the liposome. A preferred hydrophilic polymer is polyethylene glycol (PEG). Such a polymer-conjugated lipid may be mixed with other lipids in preparation of liposomes or the conjugated lipid may be exchanged in the liposome from another source (such as from a vesicle or micelle containing the conjugated lipid). Alternatively, the polymer may be conjugated to a lipid component present on the exterior surface of a previously prepared liposome (see: United States Patent 6132763). Typically, PEG is conjugated to lipids having a head group that contains a primary amine but other PEG- lipid derivatives are known. As well, the literature describes various moieties that may be situated between a lipid and a hydrophilic polymer. A commonly used conjugate is PEG derivatized to distearylphosphatidylethanolamine (DSPE) with the resulting conjugate being termed PEG-DSPE.

Hydrophilic polymers grafted onto a liposome may exist in either the brush or the mushroom regime. The brush regime is defined with respect to the point at which lateral interactions occur between neighboring polymers, implying maximum surface coverage of the liposome surface. When the hydrophilic polymer is present in the mushroom regime, the polymer configuration is similar to that of a single chain in solution due to the absence of lateral interactions with neighbouring polymer chains. The concentration at which the brush to mushroom regime occurs depends on the percent incorporation and molecular weight of the hydrophilic polymer. Torchilin et al., have published means for calculating a theoretical transition from brush to mushroom regimes for PEGs of various molecular weights (Biochim. Biophys. Acta (1994) 1195: 11).

Literature dating back to the beginning of the use of hydrophilic polymers in liposomes suggests that the incorporation of hydrophilic polymers such as PEG of 1000 to 12,000 daltons imparts optimal circulation longevity to liposomes (Woodle et al, (1992) Biochimica et Biophysica Acta, 1105: 193). PEG1900 and 5000 have been reported to provide enhanced circulation longevity with about 15- 20 % of the initial injected dose remaining in the blood 24 hours after intravenous administration (Woodle et ah, (supra); Maryuama et al, (1992) Biochimica et Biophysica Acta 1128: 44) reported that PEG of 1000 or 2000 daltons provides adequate circulation longevity and Unezaki et al., have shown that PEG1000 also imparts adequate circulation lifetimes to liposomes (Pharmaceutical Research (1994) 11 (8): 1180).

Various groups have examined the ability of liposomes comprising lipid- hydrophilic polymer conjugates of molecular weights below 1000 daltons (referred to herein as short-chain hydrophilic polymers) to provide adequate circulation longevity. It has been reported that a key factor in determining the effectiveness of short chain hydrophilic polymers to provide adequate circulation longevity is the density of incorporation of the hydrophilic polymer in the liposome. Woodle et al, have shown that liposomes comprised of cholesterol and PHEPC (partially hydrogenated egg phosphatidylcholine) containing 10 mol % PEG of 750 daltons, exhibited severely compromised circulation lifetimes relative to PEG of 1900 daltons and PEG of 5000 daltons (Biochim. Biophys. Acta. (1992) 1113: 171). However, they suggested that incorporation of low molecular weight PEG at saturating densities to provide maximum surface coverage (hence the PEG will be in the "brush") might be adequate to extend circulation longevity. Kenworthy et al, suggested that 7.5 mol % PEG 750 in cholesterol-containing liposomes did not provide extended circulation longevity (as measured in Woodle et al, (1992) Biochimica et Biophysica Acta, 1105: 193) since at this density, PEG is in the "mushroom" regime as opposed to the "brush" regime (Biophysical Journal (1995) 68: 1921).

Most conventional liposome formulations contain greater than 30 mol % cholesterol to increase systemic circulation time and in vivo stability. Cholesterol decreases liposome elimination in the bloodstream by reducing interactions with serum proteins such as complement, lipoproteins and phospholipases. Plasma proteins associated with liposomes have been implicated as one of the dominant factors in determining the elimination rate and tissue distribution of liposomes in vivo. Binding of complement leads to rapid uptake by phagocytic cells and interaction with lipoproteins leads to phospholipid loss. Phospholipases cause hydrolysis of phospholipids and concomitant liposome degradation. While it may be advantageous to make use of cholesterol-free liposomes, one would expect that they would exhibit poor circulation longevity, particularly if they are not otherwise protected from in vivo interactions such as by incorporation of high molecular weight PEG.

Summary of the Invention

This invention is based on the finding that short chain hydrophilic polymers (below 1000 daltons) will impart enhanced circulation longevity to unilamellar vesicles even when the polymers are incorporated at low densities, such as densities where the hydrophilic polymer is present in the mushroom regime. These results are contrary to the previous wisdom concerning the ability of short chain hydrophilic polymers to impart enhanced circulation longevity to other forms of liposomes.

This invention is also based on the finding that short chain hydrophilic polymers incorporated at low densities in unilamellar vesicles impart enhanced circulation longevity to the vesicles, even when the vesicles contain substantially no cholesterol. Preferred embodiments of this invention are vesicles containing substantially no cholesterol.

The invention encompasses unilamellar vesicles comprising one or more hydrophilic polymer-conjugated lipids, wherein the hydrophilic polymer in the hydrophilic polymer-conjugated lipid is a short chain hydrophilic polymer and is incorporated into the vesicle at a density such that the hydrophilic polymer is substantially present in the mushroom regime. The molecular weight of the hydrophilic polymer in the hydrophilic polymer-conjugated lipid is preferably less than about 1000 daltons, and more preferably between about 200 to 900 daltons. The molecular weight of a hydrophilic polymer for use in this invention may be about 900, 800, 750, 600, 500, 400, 350 or 300 daltons. Depending upon the nature and size of the vesicle and the molecular weight of the polymer, unilamellar vesicles of this invention may contain from about 1 to about 6 mol % hydrophilic polymer-conjugated lipids, and up to about 99 mol % of one or more vesicle-forming lipids.

Vesicles of this invention may range in size from about 50 to about 500 nm, more preferably from about 80 to about 250, and even more preferably from about 80 to about 200 nm. Thus, preferred vesicles of this invention fall within the category of large unilamellar vesicles (LUVs). Preferred vesicle preparations of this invention will exhibit average particle sizes falling within the aforementioned ranges.

Vesicles of this invention may also comprise a therapeutic agent such as a drug or other biologically active agent suitable for liposomal delivery. In some embodiments, this invention provides large unilamellar vesicles comprising i) from about 1 to about 6 mol % of one or more hydrophilic polymer- conjugated lipids wherein the hydrophilic polymer in the hydrophilic polymer-conjugated lipid has a molecular weight of about 200 to 900 daltons; and ii) up to about 99 mol % of one or more vesicle- forming lipids, providing that the large unilamellar vesicle contains substantially no cholesterol. Such large unilamellar vesicles may also comprise a therapeutic agent.

As discussed herein, the invention provides unilamellar vesicles having circulation longevity suitable for administration to mammals. The invention thus also provides pharmaceutical formulations comprising the unilamellar vesicles of this invention. Also, encompassed are pharmaceutical compositions comprising unilamellar vesicles of this invention and a pharmaceutically acceptable carrier.

This invention also provides the use of the unilamellar vesicles of this invention in treatment, for preparation of pharmaceutical compositions for use in treatment, and for use as a carrier for therapeutic components or agents. This invention provides methods of administering a large unilamellar vesicle to a mammal, and methods of treating a mammal affected by or susceptible to or suspected of being affected by a disorder (e.g. cancer). In particular, the invention encompasses a method of administering a unilamellar vesicle of this invention to a subject, comprising administering a pharmaceutical composition comprising unilamellar vesicles of the invention. Methods of treatinent and/or administration may optionally further comprise a step of selecting or identifying a mammal, preferably a human, affected by or susceptible , to or suspected of being affected by a disorder. Methods of treatment or of administration will generally be understood to comprise administering the pharmaceutical composition at a dosage sufficient to ameliorate said disorder or symptoms thereof. Brief Description of the Drawings

Figure 1 : A graph showing the concentration injected dose remaining in the blood after intravenous injection of DSPC/DSPE-PEG750 (95:5 mol %; circles), DSPC/DSPE- PEG2000 (95:5 mol %; triangles) and DSPC/DSPE-PEG5000 (95:5 mol %; squares) liposomes into Balb/C mice as a function of time. Data points represent the mean values calculated from at least three mice. Error bars represent standard deviation.

Figure 2: A graph showing the percent injected dose remaining in the blood after intravenous injection of DSPC/DSPE-PEG2000 (95:5 mol %; filled circles),

DSPC/DSPE-PEG750 (95:5 mol %; open circles), DSPC/DSPE-PEG550 (95:5 mol %; filled inverted triangles) and DSPC/DSPE-PEG350 (95:5 mol %; open inverted triangles) liposomes into Balb/C mice as a function of time. Data points represent the mean values calculated from at least three mice. Error bars represent standard deviation.

Figure 3: A graph showing the liposomal lipid concentration (μmoles lipid/mL plasma) remaining in the blood after intravenous injection of DSPC/DSPE-PEG750 (95:5 mol %; closed circles), DSPC/DSPE-PEG550 (95:5 mol %; open circles), and DSPC/DSPE- PEG350 (95:5 mol %; inverted triangles) liposomes containing encapsulated idarubicin into Balb/C mice as a function of time. Data points represent the mean values calculated from at least three mice. Error bars represent standard deviation.

Figure 4: A histogram showing the percent injected dose remaining in the blood after intravenous injection of DSPC/DSPE-PEG750 (95:5 mol %), DSPC/DSPE-PEG750 (98:2 mol %) and DSPC/DSPE-PEG750 (99: 1 mol %) liposomes after 24 hrs. Error bars represent standard deviation.

Figure 5: A diagramatic representation of PEG grafted to vesicle surfaces in the mushroom and in the brush regimes. Relative extension of PEG from a vesicle surface for the two regimes is illustrated, as is the tendency for micelles to form at saturating densities of PEG. Detailed Description of the Invention

Throughout this specification, the following abbreviations have the indicated meaning:

PEG: polyethylene glycol; PEG-lipid: polyethylene glycol-lipid conjugate; PE: phosphatidylethanolamine; PE-PEG: polyethylene glycol-derivatized phosphatidylethanolamine; PEG preceded or followed by a number: the number is the molecular weight of PEG in Daltons; (for example: DSPE-PEG 2000 (or 2000 PEG- DSPE or PEG ooo-DSPE): l,2-distearoyl-577-glycero-3-phosphoethanolamine-N-

[polyethylene glycol 2000]; or DSPE-PEG 750 (or 750 PEG-DSPE or PEG₇₅₀-DSPE): 1 ,2-distearoyl-^/7-glycero-3-phosphoethanolamine-N-[polyethylene glycol 750]);

DSPC : 1 ,2-distearoyl-s^,«-glycero-3 -phosphocholine; DPPC : 1 ,2-dipalmaitoyl-5«- glycero-3-phosphocholine; DPPG: l,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(l- glycerol)]; MPPC: monopalmitoylphosphatidylcholine; PA: phosphatidic acid; PC: phosphatidylcholine; PI: phosphatidylinositol;

CH or Choi: cholesterol; HEPES: N-[2-hydroxylethyl]-piperazine-N-[2-ethanesulfonic acid]; HBS: HEPES Buffered Saline (20 mM HEPES, 150mM NaCl, pH 7.4).

The term "cholesterol-free" as used herein with reference to a liposome means that a liposome is prepared in the absence of cholesterol, or that the liposome contains substantially no cholesterol, or that the liposome contains essentially no cholesterol. The term "substantially no cholesterol" allows for the presence of an amount of cholesterol that is insufficient to significantly alter the phase transition characteristics of the liposome (typically less than 20 mol % cholesterol). 20 mol % or more of cholesterol broadens the range of temperatures at which phase transition occurs, with phase transition disappearing at higher cholesterol levels (e.g. greater than 30 mol %). Preferably, a liposome having substantially no cholesterol will have about 15 or less and more preferably about 10 or less mol % cholesterol. The term "essentially no cholesterol" means about 5 or less mol %, preferably about 2 or less mol % and even more preferably about 1 or less mol % cholesterol. Most preferably, no cholesterol will be present or added when preparing "cholesterol-free" liposomes.

The term "unilamellar vesicle" as used herein means single-bilayer vesicles or substantially single-bilayer vesicles encapsulating an aqueous phase wherein the vesicle is less than 500 nm. The unilamellar vesicle can be either a "large unilamellar vesicle

(LUV)" which is a unilamellar vesicle between 50 and 500 nm, preferably 80 to 200 nm or can be a "small unilamellar vesicle (SUV)" which is a unilamellar vesicle between 20 and 50 nm. Formation of the above vesicles requires the presence of "vesicle-forming lipids" which are amphipathic lipids capable of either forming or being incorporated into a bilayer structure. The latter term includes lipids that are capable of forming a bilayer by themselves or when in combination with another lipid or lipids. An amphipathic lipid is incoiporated into a lipid bilayer by having its hydrophobic moiety in contact with the interior, hydrophobic region of the membrane bilayer and its polar head moiety oriented toward an outer, polar surface of the membrane. Hydrophilicity arises from the presence of functional groups such as hydroxyl, phosphato, carboxyl, sulfato, amino or sulfhydryl groups. Hydrophobicity results from the presence of a long chain of aliphatic hydrocarbon groups.

Vesicles in accordance with this invention can be prepared by conventional techniques used to prepare vesicles. These techniques comprise the ether injection method (Deamer et al, (1978) Acad. Sci., 308: 250), the surfactant method (Brunner et al, (1976) Biochim. Biophys. Acta 455: 322), the freeze-thaw method (Pick et al, (1981) Arach. Biochim. Biophys., 212: 186), the reverse-phase evaporation method (Szoka et al, (1980) Biochim. Biophys. Acta, 601: 559-71), the ultrasonic treatment method (Huang et al, (1969) Biochemistry, 8: 344), the ethanol injection method (Kremer et al, (1977) Biochemistry, 16: 3932), the extrusion method (Hope et al, (1985) Biochimica et

Biophysica Acta 812: 55) and the french press method (Barenholz et al, (1979) FEBS Lett., 99: 210). All of the above processes are basic technologies for the formation of liposome vesicle and these processes can be used in combinations, respectively proved or modified. Preferably, LUVs are prepared by the ether injection method, the surfactant method, the freeze-thaw method, the reverse-phase evaporation method or the extrusion method. Preferably, SUNs are prepared by the ultrasonic treatment method, the ethanol injection method and the French press method. Vesicle-forming lipids that may be incorporated into liposomes or lipid carriers of this invention may be selected from a variety of amphipathic lipids, typically including phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylglycerol (PG); ceramides and, sphingolipids such as sphingomyelin. In this specification, the terms "bulk" or "structural" with reference to lipids means a vesicle-forming lipid which contributes to the structure of a lipid carrier or liposome.

The term "hydrophilic polymer-lipid conjugate" refers to a vesicle-forming lipid covalently joined at its polar head moiety to a hydrophilic polymer, and is typically made from a lipid that has a reactive functional group at the polar head moiety in order to attach the polymer. Suitable reactive functional groups are for example, amino, hydroxyl, carboxyl or formyl. The lipid may be any lipid described in the art for use in such conjugates such as phospholipids, sphingolipids and ceramides as mentioned above. Preferably, the lipid is a phospholipid such as PC, PE, PA or PI, having two acyl chains comprising between about 6 to about 24 carbon atoms in length with varying degrees of unsaturation. Most preferably, the lipid in the conjugate is a PE, preferably with an acyl chain that is miscible with the acyl chains comprising the bulk lipids. The polymer is a biocompatible polymer characterized by a solubility in water that permits polymer chains to effectively extend away from a liposome surface with sufficient flexibility that produces uniform surface coverage of a liposome. Preferably, the polymer is a polyalkylether, including polymethylene glycol, polyhydroxy propylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyacrylic acid and copolymers thereof, as well as those disclosed in United States Patents 5,013,556 and 5,395,619. A preferred polymer is polyethylene glycol (PEG). Mixtures of conjugates may be incorporated into liposomes for use in this invention.

The term "PEG-conjugated lipid" as used herein refers to the above-defined hydrophilic polymer-lipid conjugate in which the polymer is PEG.

The term "mushroom regime" as used herein refers to the conformation hydrophilic polymers assume at low polymer densities wherein the shape of an individual polymer chain is not affected by the presence of other polymer chains; the polymer configuration is similar to that of a single chain in solution (see Figure 5). This is in contrast to the "brush regime" where complete coverage of the surface with grafted hydrophilic polymer is attained to the point where the chains are roughly overlapping. In a mushroom regime, the hydrophilic polymer occupies a region next to the liposome membrane which can be determined by the Flory radius. Whether or not a hydrophilic polymer is present in the mushroom regime is dependent on the grafting density and the molecular weight of the conjugate. Methods for calculating the conformation of the polymers are set forth below in Example 5. Computer simulations available in the art and other literature may also be consulted to determine whether the hydrophilic polymer is present in the mushroom regime or the amount of polymer that must be grafted to enter the brush regime. The terms "substantially present in a mushroom regime" or "substantially in a mushroom regime" with reference to hydrophilic polymer density in this specification means that hydrophilic polymers grafted on the surface of a vesicle are substantially unaffected by the presence of adjacent polymer chains and the configuration of most of the polymers are such that the polymer chains are substantially limited to occupying a region next to the vesicle surface characteristic of the mushroom regime. The person of skill in the art may determine whether grafted polymers are substantially in the mushroom regime (for example by following the methodology in Example 5) to determine a theoretical polymer density for a vesicle beyond which polymers may tend to the brush configuration. Thus, vesicles of this invention having polymers substantially in the mushroom regime may exceed a theoretical transition density by about 50% or less of the theoretical transition density value. Preferably, the density will not exceed about 150%, more preferably about 140%, more preferably about 130%, more preferably about 125%, more preferably about 120%, more preferably about 115%, more preferably about 110%) and more preferably about 105% of a theoretical transition density calculated according to Example 5. Embodiments of this invention may comprise a polymer density at or below a theoretical transition density, such as that calculated according to the methods shown in Example 5.

A hydrophilic polymer-lipid conjugate may be prepared to include a releasable lipid-polymer linkage such as a peptide, ester or disulfide linkage which can be cleaved under selective physiological conditions so as to expose a LUV carrier surface once a desired biodistribution has been achieved, such as is disclosed in United States Patent No. 6,043,094; or Kirpotin, D., et al. (1996) FEBS Letters, 388: 115. Alternatively, the lipid in the conjugate, and in particular, its acyl chain length may be selected to provide for a desired rate of exchange of the polymers from a liposome to expose a reactive surface over time (Adlakha-Hutcheon, G., et al. (1999) Nature Biotechnology 17: 775).

The hydrophilic polymer-lipid conjugate may also be prepared to include a targeting ligand. The term "targeting ligand" as used herein, refers generally to all molecules capable of specifically binding to a particular target molecule and forming a bound complex. Thus, the targeting ligand and its corresponding target molecule form a specific binding pair. Examples include, but are not limited to antibodies, lymphokines, cytokines, receptor proteins, solubilized receptor proteins, hormones, growth factors, and the like which specifically bind desired target cells, and nucleic acids which bind corresponding nucleic acids through base pair complementarity. Vesicles in accordance with this invention can contain a ligand bound to the surface of the vesicles by attachment to surface lipid components. Generally, such a ligand is coupled to the polar head group of a vesicle-forming lipid and various methods have been described for attachment of ligands to lipids. The affinity moiety may be coupled to the lipid, by a coupling reaction to form a targeting ligand-lipid conjugate. This conjugate can be added to a solution of lipids during formation of the vesicles. A vesicle-forming lipid may be activated for covalent attachment of ligand to a lipid. The formed liposomes are exposed to the ligand to achieve attachment of the ligand to the activated lipids. A metal/metal-affinity tag association, such as a nickel/ histidine tag association, may be used to link targeting ligands modified with a metal-affinity tag to liposomes. Targeting ligands, conjugated with affinity tags such as repeating units of histidine, may be prepared by recombinant methods or by chemical crosslinking of the affinity tag to the targeting ligand. A metal ion (such as Nickel) is attached to the liposome by chelation to a head group of a lipid making up the liposome.

A hydrophilic polymer-lipid conjugate may also include a targeting ligand attached at the free end of the polymer to direct the liposome to specific cells. Derivatives of polyethyleneglycol that allow conjugation of a targeting ligand are for example, methoxy(hydrazido)polyethyleneglycol and bis(hydrazido)polyethyleneglycol. A wide variety of agents may be delivered by the liposomes of the present invention. "Therapeutic agent" and "drug" as used herein refer to chemical moieties used in therapy and for which liposome-based drug delivery is desirable. The term "anti- neoplastic agent" as used herein refers to chemical moieties having an effect on the growth, proliferation, invasiveness or survival of neoplastic cells or tumours. Anti- neoplastic therapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics and various plant alkaloids and their derivatives. Agents may be encapsulated inside liposomes of the present invention by passive or active loading techniques known in the art. A particularly suitable encapsulation or liposome loading technique is pH gradient loading. With this technique there are multiple means of using the pH gradient to actively load agents, non-limiting examples include citrate and ammonium sulfate loading. In citrate-based loading, liposomes are formed which encapsulate an aqueous phase of a selected pH and a buffer (e.g. citrate-phosphate) chosen to minimize changes in the selected pH caused by drug loading. Hydrated liposomes are placed in an aqueous environment of a different pH selected to increase the proportion of drug, or other agent to be encapsulated, in a neutral form. Once the drug moves inside the liposome, the pH of the interior results in a charged drug state, which prevents the drug from permeating the lipid bilayer, thereby entrapping the drug in the liposome. Similarly, loading with ammonium sulfate relies on the drug, in the neutral form, being able to readily cross the lipid bilayer and once inside the liposome being converted into a protonated or charged state that is incapable of permeating the bilayer. Alternatively, liposomes prepared with an ion may be combined with an ionophore that is capable of transporting the ion out of the liposome in exchange for protons which are transported inside and thus establishes a pH gradient.

Liposomes of the present invention may be prepared such that they are sensitive to elevations of the temperature in the surrounding environment. The temperature-sensitivity of such liposomes allows the release of compounds entrapped within the interior aqueous space of the liposome, and/or the release of compounds associated with the lipid bilayer, at a target site that is either heated (as in the clinical procedure of hyperthermia) or that is at an intrinsically higher temperature than the rest of the body (as in inflammation). Liposomes that allow release of compounds in a temperature dependent manner are termed "theπnosensitive liposomes" and contain low levels of cholesterol. Liposomes of the present invention comprise a lipid possessing a gel-to-liquid crystalline transition temperature in the hyperthermic range (e.g., the range of from approximately 38°C to approximately 45°C). Preferred are phospholipids with a phase-transition temperature of from about 38°C to about 45°C, and more preferred are phospholipids whose acyl groups are saturated. A particularly preferred phospholipid is dipalmitoylphosphatidylcholine (DPPC). DPPC is a common saturated chain (C16) phospholipid with a bilayer transition of 41.5°C (Blume (1983) Biochemistry 22: 5436; Albon and Sturtevant (1978) Proc. Natl. Acad. Sci. USA 75: 2258). Thermosensitive liposomes containing DPPC and other lipids that have a similar or higher transition temperature, and that mix ideally with DPPC (such as DPPG; Tc=41.5°C and DSPC; Tc=55.1°C) have been studied (Kastumi Iga et al, (1989) Intl. J. Pharmaceutics, 57: 241; Bassett et al, (1985) J. Urology, 135: 612; Gaber et al, (1995) Pharmacol. Res. 12: 1407). Thermosensitive liposomes of the present invention may incorporate a relatively- water soluble surface active agent, such as a lysolipid, into a bilayer composed primarily of a relatively water-insoluble molecule, such as a di-acyl phospholipid (e.g. DPPC). Incorporation of the surface active agent in the gel phase of the primary lipid component enhances the release of contents from the resulting liposome when heated to the gel-liquid crystalline phase transition temperature of the primary lipid. Preferred surface active agents are lysolipids, and a particularly preferred surface active agent is monopalmitoylphosphatidylcholine (MPPC). Suitable surface-active agents are those that are compatible with the primary lipid of the bilayer, and that desorb when the lipid melts to the liquid phase. Additional suitable surface-active agents for use in phospholipid bilayers include palmitoyl alcohols, stearoyl alcohols, palmitoyl, stearoyl, polyethylene glycol, glyceryl monopalmitate, glyceryl monooleate, and therapeutic lipids. Therapeutic lipids include, for example, C-18 ether linked lysophoshpatidylchohline.

Liposomes of this invention may also be prepared such that the liquid crystalline ' transition temperature is greater than 45°C. In this case, vesicle-forming lipids making up the liposome are phospholipids such as PC, PE, PA or PE. The preferred phospholipid is PC. When selecting lipids, precautions should be taken since phase separation may occur if acyl chain lengths of these lipids differ by four or more methylene groups. Preferably the lipid will have two saturated fatty acids, the acyl chains of which being independently selected from the group consisting of stearoyl (18:0), nonadecanoyl (19:0), arachidoyl (20:0), heniecosanoyl (21 :0), behenoyl (22:0), tricosanoyl (23:0), lingnoceroyl (24:0) and cerotoyl (26:0). Preferably, at least one (and more preferably both) of the acyl chains will be 19:0, or longer. The liposomes of the present invention may be administered to warm-blooded animals, including humans. These liposome and lipid carrier compositions may be used to treat a variety of diseases in warm-blooded animals. Examples of medical uses of the compositions of the present invention include but are not limited to treating cancer, treating cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis, treating bacterial, fungal or parasitic infections, treating and/or preventing diseases through the use of the compositions of the present inventions as vaccines, treating inflammation or treating autoimmune diseases. For treatment of human ailments, a qualified physician will determine how the compositions of the present invention should be utilized with respect to dose, schedule and route of administration using established protocols. Such applications may also utilize dose escalation should bioactive agents encapsulated in liposomes and lipid carriers of the present invention exhibit reduced toxicity to healthy tissues of the subject.

Pharmaceutical compositions comprising the liposomes of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Typically, these formulations will comprise a solution of liposomes suspended in the acceptable carrier, which is preferably an aqueous carrier. Particular formulations suitable for the purpose of this invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). Generally, normal saline will be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.9% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, or globulin. These compositions may be sterilized by conventional, well- known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid- peroxidative damages on storage. Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

The concentration of liposomes, in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5%> to as much as 10 to 30%) by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. Alternatively, liposomes composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. For diagnosis, the amount of liposomes administered will depend upon the particular label used, the disease state being diagnosed and the judgement of the clinician but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight.

Preferably, the pharmaceutical compositions are administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

Dosage for the liposome formulations will depend on the ratio of drug to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.

The methods of the present invention may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like.

Pharmaceutical preparations containing vesicles of this invention may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, "open" or "closed" procedures. By "topical", it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. "Open" procedures are those procedures include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. "Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.

Compositions of the present invention which further comprise a targeting antibody on the surface of the liposome may be particularly useful for the treatment of certain malignant diseases, as known in the art.

Examples

Example 1

Low density PEG 750 imparts enhanced circulation longevity to cholesterol-free LUVs

Lipids were prepared in chloroform and subsequently dried under a stream of nitrogen gas and placed in a vacuum pump overnight. The samples were then hydrated with 300 mM citrate buffer pH 4.0, frozen (liquid nitrogen) and thawed (65°C water bath) five times and subsequently passed through an extrusion apparatus (Lipex

Biomembranes, Vancouver, BC) 10 times with three 100 nm polycarbonate filters at 65°C to produce cholesterol-free large unilamellar vesicles (LUVs). Average vesicle size was determined by quasi-elastic light scattering using a NICOMP 370 submicron particle sizer at a wavelength of 632.8 nm. CD-I female mice or Balb/C mice were injected with 3.3 μmoles total lipid (165 μmoles/kg) and blood was collected by cardiac puncture at 1,4, and 24 hours post drug administration. Blood plasma was isolated by centrifugation (3000 rpm, 10 minutes) followed by determination of the lipid concentration by liquid scintillation counting.

Figure 1 shows that LUVs prepared with 5 mol % PEG750, PEG2000 and PEG5000 all demonstrate similar circulation longevities. These results are in contrast to previous teaching concerning the ability of liposomes comprising hydrophilic polymers with low molecular weights to exhibit extended circulation longevity.

Example 2

Multiple short-chain hydrophilic polymers impart enhanced circulation longevity to cholesterol-free LUVs

LUNs were prepared according to the methods of Example 1. Figure 2 shows that LUVs prepared with 5 mol % PEG750, PEG550 and PEG350 demonstrated similar circulation longevities to that of LUVs prepared with 5 mol %> PEG2000.

Example 3

Short-chain hydrophilic polymers impart enhanced circulation longevity to cholesterol-free LUVs containing encapsulated drug

Circulation of DSPC/DSPE-PEG LUVs was further analyzed with liposomes containing encapsulated idarubicin. LUVs were prepared as described in Example 1 except that trace amounts of ³H-CHE were added to determine plasma lipid concentration and after liposome size was determined a pH gradient was established across the liposomal membrane. To achieve this, the external liposomal buffer was exchanged by passage down a Sephadex G-50 column equilibrated in HBS (20 mM HEPES, 150mM ΝaCl; pH 7.4). Idarubicin was subsequently pH gradient loaded into the liposomes by incubating the drug and liposomes together at 37°C for approximately 90 minutes. Balb/C mice were administered 17.6 mg/kg idarubicin (33 μmoles/kg) and at the indicated time points (3 mice per time point), blood was collected by cardiac puncture and placed into EDTA coated microtainers. Blood plasma was isolated as above followed by determination of the lipid concentration by liquid scintillation counting. Idarubicin was isolated from plasma with an Idarubicin extraction assay and subsequently analyzed by fluorescence spectrometry.

Figure 3 shows that DSPC/DSPE-PEG liposomes encapsulating idarubicin and prepared with either 5 mol % PEG750, PEG550 or PEG350 have similar circulation longevities. The circulation times of these drug-loaded LUVs are also comparable to those of the empty liposomes seen in Figure 1, demonstrating that enhanced circulation longevity is observed regardless of whether or not the liposomes contain encapsulated drug.

Example 4

Short-chain hydrophilic polymers incorporated at 1, 2 and 5 mol % impart enhanced circulation longevity to cholesterol-free LUVs

LUVs were prepared according to the methods of Example 1. Figure 4 shows that LUVs prepared with 1, 2 and 5 mol% PEG750 exhibit circulation longevities of 7, 11 and 17 percent injected dose, respectively, remaining in the blood after 24 hrs. Error bars represent standard deviation. In contrast, previous studies showed that cholesterol containing liposomes prepared with 10 mol%> PEG750 exhibited undetectable plasma lipid levels after 24 hrs (Woodle et al, (1992) Biochim. Biophys. Acta., 1113: 171-199).

Example 5 Calculation of the phase transition of a hydrophilic polymer

Theoretical transition of a hydrophilic polymer from mushroom to brush regime for cholesterol containing liposomes has been calculated by a number of methods known in the art. Preferred calculations for cholesterol-free (or gel-phase) liposomes are described below. The exemplified values are based on 100 nm (1000 angstroms) LUVs consisting of DSPC/DSPE-PEG750 (95:5 mol %). These calculations are based on those previously established by Torchilin et al., (supra). If one assumes polymers are in the mushroom regime, the surface area protected by one PEG chain can be calculated from the Flory radius (R/) of the polymer. As defined by deGennes, in the mushroom regime the polymer "occupies a half-sphere with a radius comparable to the Flory radius for a coil in a good solvent" (deGennes 1980):

A_P = π(R_fY;whereR_f = aN% (1) where N is the number of "repeat" units in the chain (N= polymer molecular weight / molecular weight of monomer (44 g/mole) and a is the monomer length which is 0.35 nm for PEG (Kenworthy et al, (1995) Biophys. J. 68: 1921), giving a value of approximately 19 angstroms for the Flory radius of PEG 750. The projected area of the region corresponding to this Flory radius is A_p and gives the area of membrane surface covered by a single PEG750 "mushroom".

Using total surface area of the liposome (A_LIPOSOM_E, equation 3), the values of A_p (equation 1) may be used to calculate a theoretical total number of PEG molecules that could may incorporated while maintaining a mushroom configuration (N_PEGTOTA_L)- T- _ ^-LIPOSOME ^V PEG Total ^~ _A (2)

Surface area (S. A.) of the inner and outer bilayer (A_LIPOSOME) = S. A. of outer bilayer + S. A. of inner bilayer

±LIPOSOME = 4πr_v ² + 4π(r_v - b)² (3)

where r_v is the radius of the liposome, b is the bilayer thickness which is 40 A for a DSPC/DSPE liposome.

For these calculations one assumes that the polymer chains are in the mushroom regime and that the PEG lipid distributes itself evenly over the inner and outer leaflets of the lipid membrane. Once the total number of PEG molecules is calculated (equation 2), the mole fraction of PEG-lipid (%> PEG; equation 5) occupying the liposome in the mushroom regime can be calucated if the total number of lipid molecules in a liposome (NTOTAL LIPID) is determined.

_ 4πιA 4π(r_v -T)²

^TotalLlpid . ⁺ _j V where r_v is the radius of the liposome T is the bilayer thickness

A_L is the average weighted area of the lipid headgroup (A₁ + A₂) For example: Ai = 52 angstroms for a choline headgroup (incorporated at 95%>) in a gel-phase liposome A = 41 angstroms for an ethanolamine headgroup (incorporated at 5%)

% PEG = N_{PEG Tota}ι / (NPEG Total + N_TOTALLIPID) 100% (5)

Preferably, the percentage of PEG used to prepare vesicles of this invention should not exceed 150% of a value as derived by equation (5).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.

Claims

We claim:

1. A unilamellar vesicle comprising hydrophilic polymer-conjugated lipids incorporated in the vesicle at a density at which hydrophilic polymers of the polymer-conjugated lipids are substantially present in a mushroom regime, said hydrophilic polymers having a molecular weight of less than 1000 daltons, and wherein the vesicle contains substantially no cholesterol.

2. The vesicle of claim 1 , wherein the vesicle size is about 50 to about 500 nm.

3. The vesicle of claim 1 or 2, comprising from about 1 to about 6 mol % of the hydrophilic polymer-conjugated lipids.

4. The vesicle of any one of claims 1 -3, wherein the hydrophilic polymers have a molecular weight of about 200 to about 900 daltons.

5. The vesicle of any one of claims 1 -4, wherein the vesicle is a large unilamellar vesicle.

6. A large unilamellar vesicle comprising: i) from about 1 to about 6 mol %> of one or more hydrophilic polymer- conjugated lipids wherein hydrophilic polymers in the hydrophilic polymer-conjugated lipids have a molecular weight of about 200 to about 900 daltons; ii) up to about 94 mol % of one or more vesicle-forming lipids, providing that the large unilamellar vesicle contains substantially no cholesterol.

7. The vesicle of claim 5 or 6, wherein the vesicle size is about 80 to about 250 nm.

8. The vesicle of claim 5 or 6, wherein the vesicle size is about 80 to about 200 nm.

9. The vesicle of any one of claims 1-8, containing essentially no cholesterol.

10. The vesicle of any one of claims 1-8, containing no cholesterol.

11. The vesicle of any one of claims 1-10, wherein the hydrophilic polymer of the polymer-conjugated lipids is PEG.

12. The vesicle of claim 11 , wherein the PEG has a molecular weight from about 300 to 800 daltons.

13. The vesicle of claim 11 , wherein the PEG has a molecular weight from about 400 to 700 daltons.

14. The vesicle of any one of claims 1-13, further comprising a therapeutic agent.

15. Use of a vesicle according to any one of claims 1 - 13, as a carrier for a therapeutic agent in the bloodstream of a mammal.

16. Use of a vesicle according to any one of claims 1-13, for preparation of a pharmaceutical composition or medicament.