Liposomes with enhanced circulation time

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1. FIELD OF THE INVENTION

The present invention relates to liposome therapeutic compositions, and, more particularly, to liposome compositions which have enhanced circulation time when administered intravenously.

2. REFERENCES

Allen, T. M., (1981) Biochem. Biophys. Acta 640. 385397.

Allen, T. M., and Everest, J. (1983) J. Pharmacol. Exp. Therap. 226. 539-544.

Altura, B. M. (1980) Adv. Microcirc. 9, 252-294.

Alving, C. R. (1984) Biochem. Soc. Trans. 12. 342344.

Ashwell, G., and Morell, A. G. (1974) Adv. Enzymology 41, 99-128.

Czop, J. K. (1978) Proc. Natl. Acad. Sci. USA 75:3831.

Durocher, J. P., et al. (1975) Blood 45:11.

Ellens, H., et al. (1981) Biochim. Biophys. Acta 674. 10-18.

Gabizon, A., et al., J. Liposome Research 1:123 (1988).

Gregoriadis, G., and Ryman, B. E. (1972) Eur. J. Biochem. 24, 485-491.

Gregoriadis, G., and Neerunjun, D. (1974) Eur. J. Biochem. 47, 179-185.

Gregoriadis, G., and Senior, J. (1980) FEBS Lett. 119, 43-46.

Greenberg, J. P., et al (1979) Blood 53:916.

Hakomori, S. (1981) Ann. Rev. Biochem. 50, 733-764.

Hwang, K. J., et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030.

Jonah, M. M., et al. (1975) Biochem. Biophys. Acta 401, 336-348.

Juliano, R. L., and Stamp, D. (1975) Biochem. Biophys. Res. Commun. 63. 651-658.

Karlsson, K. A. (1982) In: Biological Membranes, Vol. 4, D. Chapman (ed.) Academic Press, N.Y., pp. 1-74.

Kimelberg, H. K., et al. (1976) Cancer Res. 36, 2949-2957.

Lee, K. C., et al., J. Immunology 125:86 (1980).

Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378.

Okada, N. (1982) Nature 299:261.

Poznansky, M. J., and Juliano, R. L. (1984) Pharmacol. Rev. 36. 277-336.

Richardson, V. J., et al. (1979) Br. J. Cancer 40, 3543.

Saba, T. M. (1970) Arch. Intern. Med. 126. 1031-1052.

Schaver, R. (1982) Adv. Carbohydrate Chem. Biochem. 40:131.

Scherphof, T., et al. (1978) Biochim. Biophys. Acta 542, 296-307.

Senior, J., and Gregoriadis, G. (1982) FEBS Lett. 145, 109-114.

Senior, J., et al. (1985) Biochim. Biophys. Acta 839, 1-8.

Szoka, F., Jr., et al. (1978) Proc. Natl. Acad. Sci. USA 75:4194.

Szoka, F., Jr., et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467.

Woodruff, J. J., et al. (1969) J. Exp. Med. 129:551.

3. BACKGROUND OF THE INVENTION

Liposome delivery systems have been proposed for a variety of drugs. For use in drug delivery via the bloodstream, liposomes have the potential of providing a controlled "depot" release of a liposome-entrapped drug over an extended time period, and of reducing toxic side effects of the drug, by limiting the concentration of free drug in the bloodstream. Liposome/drug compositions can also increase the convenience of therapy by allowing higher drug dosage and less frequent drug administration. Liposome drug delivery systems are reviewed generally in Poznansky et al.

One limitation of intravenous liposome drug delivery which has been recognized for many years is the rapid uptake of blood-circulating liposomes by the mononuclear phagocytic system (MPS), also referred to as the reticuloendothelial system (RES). This system, which consists of the circulating macrophages and the fixed macrophages of the liver (Kupffer cells), spleen, lungs, and bone marrow, removes foreign particulate matter, including liposomes, from blood circulation with a half life on the order of minutes (Saba). Liposomes, one of the most extensively investigated particulate drug carriers, are removed from circulation primarily by Kupffer cells of the liver and to a lesser extent by other macrophage populations.

A variety of studies on factors which effect liposome uptake by the RES have been reported. Early experiments, using heterogeneous preparations of multilamellar liposomes (MLV) containing phosphatidylcholine (PC) and cholesterol (CH) as their principal lipid constituents, demonstrated that these liposomes are rapidly removed from circulation by uptake into liver and spleen in a biphasic process with an initial rapid uptake followed by a slow phase of uptake (Gregoriadis, 1974; Jonah; Gregoriadis, 1972; Juliano). Half-time for removal of MLV from circulation was on the order of 5-15 min. following intravenous (IV) injection. Negatively charged liposomes are removed more rapidly from circulation than neutral or positively charged liposomes. Small unilamellar liposomes (SUV) are cleared with half-lives approximately three-to four-fold slower than MLV (Juliano; Allen, 1983). Uptake of liposomes by liver and spleen occurs at similar rates in several species, including mouse, rat, monkey, and human (Gregoriadis, 1974; Jonah; Kimelberg, 1976; Juliano; Richardson; Lopez-Berestein).

Liposomes which are capable of evading the RES would have two important benefits. One is the increased liposome circulation time in the blood, which would both increase the pharmacokinetic benefits of slow drug release in the bloodstream, and also provide greater opportunity for tissue targeting where the liver, spleen, and lungs are not involved. The second benefit is decreased liposome loading of the RES. In addition to the role of the RES in removing foreign particles, the RES is involved in several other functions, including host defense against pathogenic microorganisms, parasites, and tumor cells, host responses to endotoxins and hemorrhagic shock, drug response, and responses to circulating immune complexes (Saba, Altura). It is important, therefore, in liposome administration via the bloodstream, to avoid compromising the RES seriously, by massive short-term or accumulated liposome uptake.

One approach which has been proposed is to increase liposome circulation time by increasing liposome stability in serum. This approach is based on studies which have shown that factors which decrease leakage of liposome contents in plasma also decrease the rate of uptake of liposomes by the RES (Allen, 1983; Gregoriadis, 1980; Allen, 1981; Senior, 1982). One factor contributing to this effect appears to be bilayer rigidity, which renders the liposomes more resistant to the destabilizing effects of serum components, in particular high density lipoproteins (Allen, 1981; Scherphof). Thus, inclusion of cholesterol in the liposomal bilayer can reduce the rate of uptake by the RES (Gregoriadis, 1980; Hwang; Senior, 1985), and solid liposomes such as those composed of distearoylphosphatidylcholine (DSPC) or containing large amounts of sphingomyelin (SM) show decreased rate and extent of uptake into liver (Allen, 1983; Ellens; Senior, 1982; Hwang). However, this approach appears to have a limited potential for increasing liposome circulation times in the bloodstream.

Efforts designed to enhance liposome circulation time, by modifying the liposome outer surface to mimic that of the red blood cell, have also been reported. The role of cell surface carbohydrates in cellular recognition phenomena is widely appreciated (Ashwell, Hakomori, Karlsson). The chemistry, metabolism, and biological functions of sialic acid have been reviewed (Schauer). Surface sialic acid, which is carried by gangliosides, and glycoproteins such as glycophorin, plays an important role in the survival of erythrocytes, thrombocytes, and lymphocytes in circulation. Enzymatic removal of sialic acid, which exposes terminal galactose residues, results in rapid removal of erythrocytes from circulation, and uptake into Kupffer cells of the liver (Durocher). Desialylation of thrombocytes (Greenberg) and lymphocytes (Woodruff) also results in their rapid removal by the liver.

Although desialylated erythrocytes will bind to Kupffer cells or peritoneal macrophages in vitro in the absence of serum, serum must be added in order for significant phagocytosis to occur. The nature of the serum components mediating endocytosis is speculative, but immunoglobin[globulin?] and complement (C3b) are thought to be involved. Czop et al. (Czop) have shown that sheep erythrocytes, which are not normally phagocytosed by human monocytes, will bind C3b and be phagocytosed upon desialylation. Okada et al. (Okada) have demonstrated that sialyglycolipids on liposome membranes restrict activation of the alternative complement pathway and that removal of the terminal sialic acid from the glycolipids abolishes this restricting capacity and results in activation of the alternative complement pathway. Sialic acid, therefore, may be functioning as a nonrecognition molecule on cell membranes partly through its ability to prevent binding of C3b, thus preventing phagocytosis via the alternative complement pathway. Other immune factors may also be involved in liposome phagocytosis. Alving has reported that 50% of the test sera from individual humans contain naturally occurring "anti-liposome" antibodies which mediated complement-dependent immune damage to liposomes.

The observations reported above suggest that surface sialic acid, and/or other red-cell surface agents, incorporated into liposomes, for example, in the form of ganglioside or glycophorin, may lead to increased circulation half-lives of liposomes. This approach is proposed, for example, in U.S. Pat. No. 4,501,728 for "Masking of Liposomes from RES Recognition."

Co-owned U.S. Pat. No. 4,837,028 discloses a liposome composition which shows significantly enhanced circulation half-life, as measured by blood/RES ratios 2 hours after intravenous administration. Two factors were required for achieving high blood/RES ratios. The first was the presence of the specific ganglioside GM.sub.1, which produced blood/RES ratios significantly greater than those seen with a variety of other glycosides and/or negatively charged lipids which were examined. Secondly, high blood/RES ratios were only observed in the liposomes composed predominantly of membrane-rigidifying lipids, such as sphingomyelin or phospholipids with saturated acyl chains.

The results reported in the above-cited patent suggest that a combination of specific surface molecules, such as GM.sub.1, and rigid membrane lipid components, may be required for achieving effective liposome evasion of the RES. However, it is not known from these studies whether other surface molecules would be effective in enhancing liposome blood circulation times, and, if so, whether such molecules would be practical for use in liposomes designed for intravenous injection in humans. Further, the requirement for membrane-rigidifying membrane components may present limitations in liposome formulation methods, and also limit the ability to control the rate of drug release from circulating liposomes, by varying the "fluidity" of the lipids making up the liposomes.

4. SUMMARY OF THE INVENTION

One general object of the invention is to provide a liposome composition characterized by enhanced circulation time in the bloodstream.

Another object of the invention is to provide such a composition in liposomes composed of either fluid or rigid vesicle-forming lipids.

Still another object of the invention is to provide a method which utilizes the liposome composition to deliver drugs to a target tissue accessible via the bloodstream.

The invention includes, in one aspect, a liposome composition for administering a drug via the bloodstream. The composition includes liposomes containing the drug in liposome-entrapped form, and between 1-20 mole percent of an amphipathic lipid derivatized with a polyalkylether. One preferred amphipathic lipid is a phospholipid, such as phosphatidylethanolamine, derivatized with polyethyleneglycol. The liposomes in the composition preferably have a selected average size in the size range between about 0.05 and 0.5 microns.

The vesicle-forming lipids making up the liposomes may be predominantly rigid lipid components, such as sphingomyelin, or fluid lipids, such as phospholipids with predominantly unsaturated acyl chains.

In one embodiment, the polyalkylether is derivatized to the amphipathic lipid through an esterase- or peptidase-sensitive linkage. The rate of clearance of the liposomes from the bloodstream can then be modulated according to the rate of release of polyalkylesther groups from the liposome surface.

In another embodiment, the liposomes are formulated to contain surface-bound ligand molecules which are effective to bind specifically and with high affinity to ligand-binding molecules carried on the surface of specific target tissue or cells.

In another aspect, the invention includes a method of enhancing the blood-circulation time of liposomes administered intravenously. The enhanced circulation time is achieved by adding to the liposomes, in an amount between about 1-20 mole percent, an amphipathic lipid derivatized with a polyalkylether. The method is preferably effective to enhance the blood/RES ratio, 24 hours after intravenous administration, by up to tenfold or more over that observed with the same liposomes in the absence of the derivatized amphipathic lipid.

In still another aspect, the invention includes a method of administering a drug intravenously, for delayed drug release into the bloodstream. A suspension of liposomes containing the drug in liposome-entrapped form, and between about 1-20 mole percent of an amphipathic lipid derivatized with a polyalkylether, is administered parenterally, in an amount of the suspension containing a pharmacologically acceptable amount of the drug.

Also included in the invention is a method for delivering a drug selectively to a target tissue or cell type characterized by surface-bound tissue-specific ligand-binding molecules. A suspension of liposomes containing a surface-bound ligand effective to bind specifically and with high affinity to said ligand-binding molecule, and between 1-20 mole percent of an amphipathic lipid derivatized with a polyalkylether is administered parenterally. The target tissue may be a disease-related tissue, such as a solid tumor, or a circulating cell type, such as a virus-infected blood cell having virus-specific surface antigens.

A related aspect of the invention embraces a method of enhancing the uptake, by the reticuloendothelial system, of cells carrying surface-specific ligand-binding molecules which are characteristic of a disease state. The method is based on ligand-specific binding of liposomes containing between 1-20 mole percent of an amphipathic lipid derivatized with a polyalkylether, and a surface-bound ligand to the ligand-binding molecules on the cells.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general reaction scheme for derivatizing an amphipathic lipid amine with a polyalkylether;

FIG. 2 is a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol via a cyanuric chloride linking agent;

FIG. 3 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol by means of a carbonyl diimidazole activating reagent;

FIG. 4 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol by means of a trifluoromethane sulfonate reagent;

FIG. 5 illustrates an amphipathic lipid derivatized with polyethyleneglycol through a peptide (A), ester (B), and disulfide (C) linkage;

FIG. 6 is a plot of liposome retention time on the blood, expressed in terms of percent injected dose as a function of hours after IV injection, for PEG-PE liposomes containing different amounts of phosphatidyl glycerol; and

FIG. 7 is a plot similar to that of FIG. 6, showing retention time in liposomes composed of predominantly unsaturated phospholipid components.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As used herein, the term:

"Polyalkylether" refers to polyethyleneglycol and related homopolymers, such as polymethylethyleneglycol, polyhydroxypropyleneglycol, polypropyleneglycol, polymethylpropyleneglycol, and polyhydroxypropyleneoxide, and to heteropolymers of small alkoxy monomers, such as a polyethetylene/polypropyleneglycol, such polymers having a molecular weight of at least about 120 daltons, and up to about 20,000 daltons.

"Amphipathic lipid" refers to any lipid having an amphipathic hydrophobic group, typically including two acyl hydrocarbon chains or a steroid group by which the lipid can be anchored in the outer lipid layer of a lipid bilayer, and a polar group which contains a reactive chamical group, such as an amine, acid, ester, aldehyde, or alcohol group by which the lipid can be derivatized to a polyalkylether.

"Amphipathic lipid derivatized with a polyalkylether" and "polyalkylether lipids" refers to an amphipathic lipid which is covalently joined, at its polar group, to a polyalkylether.

I. PREPARATION OF POLYALKYLETHER LIPIDS

FIG. 1 shows a general reaction scheme for forming polyalkyl ethers. The polyalkyether which is employed, such as the polyethyleneglycol (PEG) molecule shown, is preferably capped by a methoxy, ethoxy or other unreactive group at one end. The polymer is activated at its other end by reaction with a suitable activating agent, such as cyanuric acid, carbonyl diimadozle, anhydride reagent, or the like, as described below. The activated compound is then reacted with a suitable amphipathic lipid, such as the phosphatidylethanolamine (PE) shown, to produce the derivatized lipid. Alternatively, the lipid group may be activated for reaction with the polyalkylether, or the two groups may be joined in a concerted coupling reaction, according to known coupling methods.

The polyalkylether, such as polyethyleneglycol or polypropyleneglycol, or the methoxy- or ethoxy-capped analogs, can be obtained commercially in a variety of polymer sizes, e.g., 120-20,000 dalton molecular weights. Alternatively, the homo- or heteropolymer can be formed by known polymer sysnthesis methods to achieve a desired monomeric composition and size. One preferred polyalkylether is PEG having a molecular weight between about 1,000 and 5,000 daltons.

The amphipathic lipid is preferably a vesicle-forming lipid having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), PE, phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. Also included in this class are the glycolipids, such as cerebroside and gangliosides.

Another amphipathic lipid which may be employed is cholesterol and related sterols. In general, cholesterol may be less tightly anchored to a lipid bilayer membrane, particularly when derivatized with a high molecular weight polyalkylether, and therefore be less effective in promoting liposome evasion of the RES in the bloodstream. Similarly, single-chain lipids, such as long-chain fatty acids, may be derivatized with a polyalkylether, but provide less effective anchoring to the bilayer membrane than a lipid having two or more hydrocarbon chains.

According to one important feature of the invention, the amphipathic lipid may be a relatively fluid lipid, typically meaning that the lipid phase has a relatively low lipid melting temperature, e.g., at or below room temperature, or relatively rigid lipid, meaning that the lipid has a relatively high melting temperature, e.g., up to 50.degree. C. As a rule, the more rigid, i.e., saturated lipids, contribute to greater membrane rigidity in a lipid bilayer structure and also contribute to greater bilayer stability in serum. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity and stability in lipid bilayer structures. Phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to known methods.

FIG. 2 shows a reaction scheme for producing a PE-PEG lipid in which the PEG is derivatized to PE through a cyanuric chloride group. Details of the reaction are provided in Example 1. Briefly, methoxy-capped PEG is activated with cyanuric chloride in the presence in sodium carbonate under conditions which produced the activated PEG compound shown at the top in the figure. This material is purified to remove unreacted cyanuric acid. The activated PEG compound is reacted with PE in the presence of triethyl amine to produce the desired PE-PEG compound shown at the bottom in the figure. The yield is about 8-10% with respect to initial quantities of PEG.

The method just described may be applied to a variety of lipid amines, including PE, cholesteryl amine, and glycolipids with sugar-amine groups.

A second method of coupling a polyalkylether, such as capped PEG to a lipid amine is illustrated in FIG. 3. Here the capped PEG is activated with a carbonyl diimidazole coupling reagent, to form the activated imidazole compound shown at the center in FIG. 3. Reaction with a lipid amine, such as PE, leads to PEG coupling to the lipid through a carbamate linkage, as illustrated in the PEG-PE compound shown at the bottom in the figure. Details of the reaction are given in Example 2.

A third reaction method for coupling a capped polyalkylether to a lipid amine is shown in FIG. 4. Here a polyalkylether, as exemplified by PEG is first protected at its free OH. The end-protection reaction is shown at the top in the figure, and involves the reaction of trimethylsilylchloride with PEG in the presence of triethylamine. The protected PEG is then reacted with the anhydride of trifluoromethyl sulfonate to form PEG activated with trifluoromethyl sulfonate. Reaction of the activated compound with a lipid amine, such as PE, in the presence of triethylamine, gives the desired derivatized lipid product, such as the PEG-PE compound, in which the lipid amine group is coupled to the polyether through the terminal methylene carbon in the polyether polymer. The trimethylsilyl protective group can be released by acid treatment, as indicated at the lower left in the figure, or by reaction with a quaternary amine fluoride salt, such as the fluoride salt of tetrabutylamine.

It will be appreciated that a variety of known coupling reactions, in addition to those just described, are suitable for preparing polyalkyletheramine lipid derivatives for use in the liposome composition of the invention. For example, the sulfonate anhydride coupling reagent illustrated in FIG. 4 can be used to join an activated polyalkylether to the hydroxyl group of an amphipathic lipid, such as the 5'OH of cholesterol. Other reactive lipid groups, such as an acid or ester lipid group may also be used for coupling, according to known coupling methods. For example, the acid group of phosphatidic acid can be activated to form an active lipid anhydride, by reaction with a suitable anhydride, such as acetic anhydride, and the reactive lipid can then be joined to a protected polyalkylamine, e.g., by reaction in the presence of an isothiocyanate reagent.

In another embodiment, the derivatized lipid components are prepared to include a labile lipid-polymer linkage, such as a peptide, ester, or disulfide linkage, which can be cleaved under selective physiological conditions, such as in the presence of peptidase or esterase enzyme present in the bloodstream. FIG. 5 shows exemplary lipids which are linked through (A) peptide, (B), ester, and (C), disulfide containing linkages. The peptide-linked compound can be prepared, for example, by first coupling a polyalkylether with the N-terminal amine of the tripeptide shown, e.g., via the reaction shown in FIG. 3. The peptide carboxyl group can then be coupled to a lipid amine group through a carbodiimide coupling reagent conventionally. The ester linked compound can be prepared, for example, by coupling a lipid acid, such as phosphatidic acid, to the terminal alcohol group of a polyalkylether, using alcohol via an anhydride coupling agent. Alternatively, a short linkage fragment containing an internal ester bond and suitable end groups, such as primary amine groups, can be used to couple the polyalkylether to the amphipathic lipid through amide or carbamate linkages. Similarly, the linkage fragment may contain an internal disulfide linkage, for use in forming the compound shown at C in FIG. 5.

II. PREPARATION OF LIPOSOME COMPOSITION

The liposome composition of the invention is designed for use in delivering a drug via the bloodstream, i.e., via a parenteral route in which the liposomes are accessible to clearance mechanisms involving the reticuloendothelial system (RES). Section IIA below describes the general procedure employed for determining liposome clearance times from the bloodstream, and Section IIB, lipid component parameters which effect blood retention times, in accordance with the invention, and procedures for producing the liposome composition of the invention.

A. Measuring liposome uptake by the RES in vivo

One method used for evaluating liposome circulation time in vivo measures the distribution of intravenously injected liposomes in the bloodstream and the primary organs of the RES at selected times after injection. In the standardized model which is used, RES uptake is measured by the ratio of total liposomes in the bloodstream to total liposomes in the liver and spleen, the principal organs of the RES. In practice, age and sex matched mice are injected intravenously (IV) through the tail vein with a radiolabeled liposome composition, and each time point is determined by measuring total blood and combined liver and spleen radiolabel counts. Experimental methods are detailed in Example 5.

Since the liver and spleen account for nearly 100% of the initial uptake of liposomes by the RES, the blood/RES ratio just described provides a good approximation of the extent of uptake from the blood to the RES in vivo. For example, a ratio of about 1 or greater indicates a predominance of injected liposomes remaining in the bloodstream, and a ratio below about 1, a predominance of liposomes in the RES. For most of the lipid compositions of interest, blood/RES ratios were determined at selected intervals intervals of between 15 minutes and 24 hours. A related method which is also used herein measures blood circulation lifetime, as determined from the decrease in percent dose in the bloodstream over time.

The data obtained with the model animal system can be reasonably extrapolated to humans and veterinary animals of interest. This is because, as mentioned above, uptake of liposomes by liver and spleen has been found to occur at similar rates in several mammalian species, including mouse, rat monkey, and human (Gregoriadis, 1974; Jonah; Kimelberg, 1976; Juliano; Richardson; Lopez-Berestein). This result likely reflects the fact that the biochemical factors which appear to be most important in liposome uptake by the RES--including opsinization by serum lipoproteins, size-dependent uptake effects, and cell shielding by surface moieties--are common features of all mammalian species which have been examined.

B. Lipid Components

The lipid components used in forming the liposomes of the invention may be selected from a variety of vesicle-forming lipids, typically including phospholipids and sterols. According to one important aspect of the invention, it has been discovered that the lipids making up the bulk of the vesicle-forming lipids in the liposomes may be either fluidic lipids, e.g., phospholipids whose acyl chains are relatively unsaturated, or more rigidifying membrane lipids, such as highly saturated phospholipids. This feature of the invention is seen in Example 6, which examines blood/RES ratios in liposomes formed with PEG-PE, cholesterol, and PC having varying degrees of saturation. As seen from the data in Table 4 in Example 6, high blood/RES ratios were achieved in substantially all of the liposome formulations, independent of the extent of lipid unsaturation in the bulk PC phospholipid, and no systematic trend, as a function of degree of lipid saturation, was observed.

Accordingly, the vesicle-forming lipids may be selected to achieve a selected degree of fluidity or rigidity, to control the stability of the liposomes in serum and the rate of release of entrapped drug from the liposomes in the bloodstream. The vesicle-forming lipids may also be selected, in lipid saturation characteristics, to achieve desired liposome preparation properties. It is generally the case, for example, that more fluidic lipids are easier to formulate and size by extrusion than more rigid lipid components, and can be readily formulated in sizes down to 0.05 microns.

Similarly, it has been found that the percentage of cholesterol in the liposomes may be varied over a wide range without significant effect on observed blood circulation lifetime. The studies presented in Example 7A show virtually no change in blood circulation lifetime in the range of cholesterol between 0-30 mole percent.

It has also been found, in accordance with the invention that blood circulation lifetime is also relatively unaffected by the percentage of charged lipid components, such as phosphatidylglycerol (PG). This can be seen from FIG. 6, which plots percent loss of encapsulated marker for PEG-PE liposomes containing either 4.7 mole percent PG (triangles) or 14 mole percent PG (circles). Virtually no difference in liposome retention in the bloodstream over a 24 hour period was observed. It is noted here that the PEG-PE lipid is itself negatively charged and thus the PG represents additional negative charge on the liposome surface.

Thus, according to one feature of the invention, total liposome charge may be varied to modulate liposome stability, to achieve desired interactions with or binding to drugs. The concentration of charged lipid may be about percent or higher.

As an example, in preparing liposomes containing entrapped doxorubicin or epirubicin, additional charged lipid components may be added to increase the amount of entrapped drug, in a lipid-film hydration method of forming liposomes.

The polyalkylether lipid employed in the liposome composition is present in an amount preferably between about 1-20 mole percent, on the basis of moles of derivatized lipid as a percentage of toal total moles of vesicle-forming lipids. As noted above, the polyalkylether moiety of the lipid preferably has a molecular weight between about 120-20,000 daltons, and more preferably between about 1,000-5,000 daltons. Example 7B, which examines the effect of very short ethoxy ether moieties (120 daltons) on blood circulation lifetime ratios indicates that polyether moieties of at least about 5 carbon atoms are required to achieve significant enhancement of blood/RES ratios.

C. Preparing the Liposome Composition

The liposomes may be prepared by a variety of techniques, such as those detailed in Szoka et al, 1980. One preferred method for preparing drug-containing liposomes is the reverse phase evaporation method described by Szoka et al and in U.S. Pat. No. 4,235,871. In this method, a solution of liposome-forming lipids is mixed with a smaller volume of an aqueous medium, and the mixture is dispersed to form a water-in-oil emulsion, preferably using pyrogen-free components. The drug or other pharmaceutical agent to be delivered is added either to the lipid solution, in the case of a lipophilic drug, or to the aqueous medium, in the case of a water-soluble drug.

After removing the lipid solvent by evaporation, the resulting gel is converted to liposomes, with an encapsulation efficiency, for a water-soluble drug, of up to 50%. The reverse phase evaporation vesicles (REVs) have typical average sizes between about 2-4 microns and are predominantly oligolamellar, that is, contain one or a few lipid bilayer shells. The REVs may be readily sized, as discussed below, by extrusion to give oligolamellar vesicles having a maximum selected size preferably between about 0.05 to 0.5 microns.

To form MLV's, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns. These vesicles, when unsized, show relatively poor blood/RES ratios, as seen in Table 9, for the unextruded MLV composition. Typically, MLVs are sized down to a desired size range of 0.5 or less, and preferably between about 0.05 and 0.2 microns by extrusion.

One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a polycarbonate membrane having a selected uniform pore size, typically 0.05, 0.08, 0.1, 0.2, or 0.4 microns (Szoka). The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times