CA2361914A1

CA2361914A1 - Improved cholesterol-free liposomes

Info

Publication number: CA2361914A1
Application number: CA002361914A
Authority: CA
Inventors: Lawrence Mayer; Ludger Ickenstein; Marcel Bally; Paul Tardi
Original assignee: CELATOR TECHNOLOGIES Inc
Current assignee: Individual
Priority date: 2001-11-13
Filing date: 2001-11-13
Publication date: 2003-05-13

Description

IMPROVED CHOLESTEROL-FREE LIPOSOMES
Technical Field This invention is directed toward improved thermosensitive liposomes having phase transition temperatures at mildly hyperthermic conditions which have improved drug retention and circulation longevity, and their uses in the treatment of disease.
Background of the Invention Liposomes and other lipid-based carrier systems have been extensively developed and analyzed for their ability to improve the therapeutic index of drugs by altering the pharmacokinetic and tissue distribution properties of drugs. This approach is aimed at reducing exposure of healthy tissues to therapeutic agents while increasing drug delivery to a diseased site.
I 5 Some drugs, and in particular, many anti-neoplastic drugs, are knownto have a short half life in the bloodstream such that their parenteral use is not optimized.
The use of lipid-based carriers such as liposomes for site-specific delivery of such drugs via the bloodstream presents possible means to improve the use of such drugs. However, the use of liposomes for site-specific delivery is limited by the rate of clearance of liposomes from the blood, for example by cells of the Mononuclear phagocytic system (MPS). Furthermore, drugs encapsulated into liposomes are often not retained in the liposome after intravenous administration. In order for therapeutic effectiveness of liposome encapsulated drugs to be realized, such drugs must be effectively retained within a liposome after intravenous administration and the liposomes must have a sufficient circulation lifetime to permit the desired drug delivery.
It has long been established that incorporation of membranerigidification agents such as cholesterol into a liposomal membrane enhances circulation lifetime of the liposome as well as retention of drugs within the liposome. Inclusion of cholesterol in liposomal membranes has been shown to reduce release of drug after intravenous administration (for example, see: United States Patents 4,756,910; 5,077,056; 5,225,212; and 5,843,473; Kirby, C., et al.
(1980) Biochem. J.
186:591-598; and, Ogihara-Umeda, I. and Kojima, S. (1989) Eur. J. Nucl. Med 15:617-7).
Generally, cholesterol increases bilayer thickness and fluidity while decreasing membrane permeability, protein interactions, and lipoprotein destabilization of theliposome. Conventional approaches to liposome formulation dictate inclusion of substantial amounts (e.g. 30-45mo1 %) cholesterol or equivalent membrane rigidification agents (such as other sterols) into liposomes.

More recently, means for providing targeted release of liposome contents via the use of "thermosensitive" drug carriers have been developed (for example, see United States Patent 6,200,598; and, Gaber, M., et al. (1996) Int. J. Radiation Oncology Biol.
Phys. 36:1177-1187).
Thermosensitive liposomes are designed to have a phase transition temperature slightly above body temperature so that the liposomes remain in a gel state while in circulation but exceed the phase transition temperature upon application of heat to a patient's body or specific tissues. When heated, the liposome releases an encapsulated drug because the liposome bilayer becomes much more permeable above the transition temperature. However, since cholesterol has the effect of broadening the phase transition temperature (inclusion of about 30 mol % or more cholesterol will usually eliminate phase transition entirely) thermosensitive liposomes are made without cholesterol. Further, to have a phase transition temperature su~ciently close to normal human body temperature (e.g. 40-45°C), the lipid composition of the liposome is carefully tailored. A
preferred lipid for use in thermal-sensitive liposomes is DPPC, which has an acyl chain length of 16 carbon atoms.
Incorporation of any substantial amount of lipids having longer acyl chain lengths will raise the phase transition temperature of the liposome beyond the point of usefulness in thermosensitive applications.
While circulation lifetime of a thermosensitive liposome may be enhanced by inclusion of PEG-conjugated lipids into the liposome just as in more conventional liposomes (see: United States Patent 5,843,473; Unezaki, S., et al. (1994) Pharm. Res. 11:1180-5; Maruyama, K., et al. (1993) Biochimica et Biophysica Acta 1149:209-206; Blume, G. and Cevc, G. ~l> & c2~ Biochimica et Biophysica Acta (1990)1029:91-97~'~ & (1993) 1146:157-168~2~), thermosensitive liposomes exhibit poor drug retention in vivo. It is apparent that liposomes with surface conjugated PEG
moieties still require cholesterol to exhibit optimal circulation behavior and that these liposomes would exhibit inferior characteristics for therapeutic applications in vivo.
Summary of Invention In the present invention, the inventors have provided liposomes which, prepared in the absence of cholesterol, can be made to behave comparably to cholesterol-containing liposomes through the incorporation of a hydrophilic polymer conjugated lipid. The present invention provides a method of preparing or selecting liposomes using a testing format based on the comparison of a cholesterol-free liposome having a phase transition temperatures mildly hyperthermic to a subject's body temperature to a cholesterol-containing liposome. By providing preferred liposome compositions and guidance on how to select liposomes, the inventors allow for increased liposome stability and drug retention properties.
3 5 The methods set forth below are based on the finding that liposomes having phase transition temperatures useful for thermosensitive applications display enhanced drug retention properties and circulation longevities if the temperature of recipient is maintained below the phase transition temperature of the liposomes. Results of this observation are set forth in Example 3.
Further, the inventors provide liposomes in which the hydrophilic polymer stabilization effects due to use of PEG-modified lipid incorporation are not substantially dependent on the concentration of the polymer or polymer molecular weight. The inventors provide that concentrations as low as 0.5 mol% PEG-2000 can cause a significant increase in Area-Under-the Curve (AUC) when compared to the same liposome prepared without the PEG-lipid and that PEG
350 at concentrations of S mol% can cause a significant improvement in AUC
when compared to the same liposome prepared without the PEG lipid, the increase in AUC being comparable to the improvement observed when using 5 mol% PEG 2000. The resulting liposomes provide much enhanced longevity of the liposomes while in blood circulation.
'This invention provides a liposome comprising a drug, the liposome identified or prepared by a process comprising:
(i) comparing drug retention or blood circulation longevity of (a) a liposome having a drug encapsulated therein, said liposome 1) having a phase transition temperature greater than that of the body of a subject to be treated but less than 45°C and/or 2) being substantially free of cholesterol, and (b) a substantially equivalent (e.g. containing substantially the same lipids and in the same proportions) cholesterol-containing liposome having a drug encapsulated therein; and (ii) identifying a liposome of step (a) demonstrating drug retention or blood circulation longevity comparable to or improved over that of the substantially equivalent cholesterol-containing liposome of step (b). The method optimally comprises the additional step of preparing liposomes so identified for use.
Preferably the liposome of the invention is both substantially cholesterol-free and has a phase transition temperature greater than that of the body of a subject to be treated but less than 45°C.
Additionally, this invention provides a method of designing or selecting a liposome for design, the liposome comprising an encapsulated drug, comprising the steps o~
(a) providing a liposome having a drug encapsulated therein, said liposome 1 ) having a phase transition temperature greater than that of the body of a subject to be treated but less than 45°C
and/or 2) being substantially free of cholesterol;
(b) providing a liposome having a drug encapsulated therein, said liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
(c) comparing drug retention or blood circulation longevity of the liposomes of steps (a) and (b);

(d) identifying a liposome of step (a), which liposome demonstrates comparable or improved drug retention or blood circulation longevity compared to a liposome containing substantially the same lipids and in the same proportions with at least 20 mol % cholesterol.
Preferably, drug retention time of a liposome compositions is compared, which preferably involves determining the drug:lipid ratio of the liposome after administration of said liposome to the bloodstream a non-human mammal. However, in other embodiments of the methods of the invention, circulation longevity of liposome compositions are compared instead of or in addition to comparing drug retention. Thus, optionally, in the methods described herein, the invention may encompass determining the proportion of liposome present in the bloodstream of an animal at least one fixed time point subsequent to administration instead of or in addition to determining the drug:lipid ratio.
In preferred embodiments, the invention provides a method for designing or selecting a liposome for an improved liposome composition, comprising the steps of (a) providing a liposome having a drug encapsulated therein, said liposome 1) having a phase transition temperature greater than that of the body of a subject to be treated but less than 45°C
and/or 2) being substantially free of cholesterol;
(b) providing a liposome having a drug encapsulated therein, said liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol cholesterol;
(d) administering the liposomes of (a) and (b) to the bloodstream of a non-human mammal;
(e) for the liposomes of each of (a) and (b), determining drug:lipid ratios in the blood of the mammals at least one fixed time point subsequent to administration; and (f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and (g) identifying a liposome of step (a) demonstrating a drug:lipid ratio at a fixed time point in a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said fixed time point of a liposome of step (b) containing substantially the same lipids and in the same proportions as the liposome in step (a) with at least 20 mol % cholesterol.
In further aspects, the invention provides a method for designing or selecting aliposome for an improved liposome composition, said method comprising the steps o~
(a) providing a liposome 1) containing substantially no cholesterol and/or 2) having a phase transition temperature greater than that of the body of a subject to be treated and less than 45°C;
(b) providing a liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
(c) encapsulating the drug into the liposomes of (a) and (b);

(d) administering the liposomes of (a) and (b) after encapsulation of the drug to the bloodstream of a non-human mammal;
(e) determining drug:lipid ratios in the blood of the mammals at least one fixai time point subsequent to administration; and (f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and (g) identifying a liposome of step (a) demonstrating a drug:lipid ratio at a fixed time point in a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said fixed time point of a liposome of step (b) containing substantially the same lipids and in the same proportions as the liposome in step (a) with at least 20 mol % cholesterol.
In yet further aspects, the invention provides a method for designing or selecting aliposome for an improved liposome composition, said method comprising the steps o~
a) providing a liposome having a drug encapsulated therein, said liposome 1 ) having a phase transition temperature greater than that of the body of a subject to be treated but less than 45°C and/or 2) being substantially free of cholesterol;
b) assessing the drug:lipid ratios of the liposome of step (a) after administration of said liposome to the bloodstream of a non-human mammal;
c) comparing the drug:lipid ratio of step (b) so assessed to the drug:lipid ratio of a liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
wherein the liposome of step (a) demonstrates a drug:lipid ratio at a fixed time point in a mammal which is comparable to or better than the drug:lipid ratio in a mammal at said fixed time point of a liposome of step (c).
It will be appreciated that the drug:lipid ratio obtained for the liposome of step (c) can be obtained by administering the liposome of step (c) to the bloodstream of a non-human mammal, or by consulting literature providing the drug:lipid ratio for said liposome composition at particular time points and under particular conditions.
As mentioned, in the preferred methods of the invention preferably, the drug:lipid ratio of the liposome after administration of said liposome to the bloodstream a non-human mammal is determined. However, in other embodiments of the methods of the invention, circulation longevity of liposome compositions are compared instead of or in addition to comparing drug:lipid ratio. Thus, it will be appreciated that in the methods of the invention, 'drug:lipid ratio' may be substituted for 'drug retention property' if desired.
The methods according to the invention of designing or selecting aliposome can thus further comprise additional steps to improve the liposome complex, or can involve repeating any or all of the aforementioned steps. It will also be appreciated that in the steps relating to 'providing' a liposome, the term providing may be substituted with the term 'preparing'. Liposomes having desired lipid composition and proportion can be prepared according to known methods, several examples of which are provided herein, or can be obtained from commercial suppliers.
Phase transition temperature are preferably between about the temperature the body of a subject to be treated and 45° C, between about 38° C and 45° C, between 38° C and 43° C, and yet more preferably between 39°C and 41°C. Most preferably, the subject to be treated is a human.
Optionally, the subject to be treated is a non-human mammal. In general, an optimal phase transition temperature is preferably that at which mild hyperthermic conditions can cause release of liposome contents without causing damage or other effects adverse to the intended treatment, to the vasculature of a patient.
It will be appreciated that any suitable method for deterrnning the circulation longevity and/or drug retention of a liposome can be used. In this specification, the term "retention" with respect to a drug or other agent encapsulated in a liposome refers to retention of the drug in a liposome while the liposome is present in the bloodstream of a mammal. This term does not refer to a measure of drug that may be loaded or incorporated into a liposome or the ability of a liposome to retain the drug in ex vivo conditions. Most preferably, the methods of the invention for assessing drug retention comprise determining the drug:lipid ratio at least one time point upon administration to a non-human mammal. Circulation longevity is preferably expressed in terms of portion (percent) or lipid dose remaining in the blood of a mammal at a given time point. As used herein, a drug:lipid ratio or retention time which is deemed 'comparable' will depend on the circumstances, but is preferably at least 5%, 10%, 20%, 40%, 50%, 70%, 80%, 90%, or more preferably 95% of the drug retention time or drug:lipid ratio of a reference (e.g. cholesterol-containing) liposome. As further discussed herein, cholesterol-containing reference liposome will contain the same lipids and in the same proportions as substantially cholesterol-free liposomes of the invention, but will contain at least 20 mol % cholesterol. These cholesterol-containing reference liposomes may contain a hydrophilic polymer-conjugated lipid such as PEG, or may be free of hydrophilic polymer-conjugated lipid andlor free of PEG. The 'time point' is generally a number as measured in hours, minutes, etc.
A particularly suitable non-human mammal for use in the aforementioned method for comparing drug:lipid ratios is the mouse. The liposome compositions to be compared, that is the liposomes of steps (a) and (b) in the aforementioned methods, will each typically be administered to a separate (that is, individual) non-human mammal for determination of drug:lipid ratios. However, it can also be envisioned to compare drug lipid ratios for both compositions of steps (a) and (b) in the same non-human mammal if means (e.g. detectable labels) are used to distinguish each of the liposomes and drugs from one another. The drug encapsulated in the liposomes is preferably the same drug for both liposomes to compared, but may also be different drugs so long as the drugs have similar retention properties in a liposome.

Preferably, the amount of cholesterol in the liposome at (b) will be about 30 to about 50 mol %. Preferably, the drug:lipid ratios will be determined in step (e) at a series of intervals subsequent to administration with the comparison at (f) being of the ratios determined over the series of intervals.
'This invention also provides methods for determining whether retention of a particular drug is enhanced by elimination of cholesterol from a liposome. This method allows conditions to be standardized during thermosensitive liposome design such that improvement can be evaluated more accurately.
This invention further provides a method for determining whether retention of a drug in a liposome may be improved, as well as a method for designing or selecting an improved liposome composition. Said methods comprising the steps of:
(a) preparing a liposome 1) having a phase transition ~mperature greater than that of the body of a subject to be treated but less than 45°C and/or 2) being substantially free of cholesterol;
(b) preparing a liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
I 5 (c) encapsulating the drug into the liposomes of (a) and (b);
(d) administering the liposomes of (a) and (b) after encapsulation of the drug to the bloodstream of separate non-human mammals;
(e) determining drug:lipid ratios in the blood of the mammals at least one fixed time point subsequent to administration; and (f) comparing the ratios so determined for each mammal, wherein an increase in drug:lipid ratio in a mammal in which liposomes of (a) were administered as compared to drug:lipid ratio in a mammal in which liposomes of (b) were administered, is indicative of improvement in drug retention.
In principle, any suitable liposome composition may be used, as long as the liposome has the required phase transition temperatures. However, due to the well known effects of cholesterol on phase transition, liposomes of the invention will generally contain little or no cholesterol.
Preferably, liposome of the invention, including liposomes for use in step (a) of the preceding methods, will comprise at least 60, 70, 80, 85, 90 or 95 mol % of a phospholipid having two saturated fatty acids, wherein at least one of the acyl chains has 16 carbon atoms. A preferred phospholipid with acyl chains of 16 carbon atoms is dipalmitoylphosphatidylcholine (DPPC).
More preferably, liposomes for use in step (a) in the method above will have at least about 80, at least about 85, and even more preferably, at least 90 mol % of such a phospholipid. Preferably, DPPC is the predominant phospholipid. The remainder of the liposome may comprise one or more amphipathic lipids suitable for use in liposomes, but substantially no cholesterol.
Preferably, such other lipids will include a hydrophilic polymer-conjugated lipid. Preferably, the amount of such polymer-conjugated lipids present in the liposome will be from about 1 to about 15 mol %. Liposomes of the invention and liposomes for use in step (a) comprise a hydrophilic polymer-conjugated lipid. Preferably, the hydrophilic polymer-conjugated lipid is a PEG-lipid, preferably having a molecular weight from about 100 to about 5000 daltons, or from about 1000 to 5000 daltons. Preferably the liposome comprises 2 to about 15 mol %, or 5 to about 10 mol hydrophilic polymer-conjugated lipid.
Liposomes for use in the above method may be prepared using known and conventional techniques. Determination of phase transition temperatures, encapsulation of drug into liposomes (liposome loading), administration of liposomes, and determining drug:lipid ratios from blood may be carried out according to known and conventional techniques.
The above-described method may be used to select a liposome formulation to achieve optimal drug retention. Accordingly, this invention also provides a combination of a liposome and a drug wherein the liposome is a liposome as described above with respect to step (a) and the drug is an anti-neoplastic agent which exhibits greater retention in such a liposome, when the above-described method is performed. By "combination", it is meant that the drug is encapsulated in the liposome or is segregated but associated with the liposome (such as in a commercial package or kit comprising the liposome and the drug). Preferably, the liposome is one having the preferred characteristics of liposomes of step (a) as described above.
This invention also provides improved drug retention in liposomes for specific drugs which previously exhibited poor retention in conventional cholesterol-containing liposomes. Also provided are novel cholesterol-free liposome formulations that are particularly suited for use in this invention.
This invention also provides novel liposomes which are particularly suitable for use in this invention. The invention provides in preferred aspects a liposome comprising:
(a) at least 60 mol % of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, at least one of said acyl chains having 16 carbon atoms;
(b) from about 2 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mot % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; wherein the liposome displays a comparable or greater circulation longevity, or when encapsulating a drug displays a comparable or greater circulation longevity or drug:lipid ratio, at a fixed time point upon administration to a mammal than a liposome containing substantially the same lipids and in the same proportions, but with at least 20 mol % cholesterol.
In other embodiments, the liposome will comprise at least 70, 80, 85, 90 or 95 mol % of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, at least one of said acyl chains having 16 carbon atoms, preferably wherein the phospholipid is DPPC.

Preferably, the liposome will contain substantially no cholesterol.
Preferably, the liposome will have a phase transition temperature preferably between about the temperature the body of a subject to be treated and 45° C, between about 38° C and 45° C, between 38° C and 43° C, and yet more preferably between 39° C and 41° C.
This invention also provides the novel liposomes of this invention in combination with a drug and the use of such liposomes as a carrier for a drug encapsulated in the liposome. Such drugs include most preferably anti-neoplastic, anti-inflammatory or anti-infective agents.
Brief Description of the Drawings Figure 1: A graph showing lipid dose remaining in the blood of mice after intravenous injection of radiolabelled (a) a 90: 4 molar ratio of DPPC: DSPE-PEG2000 liposomes (80-100 mmoles total lipid) with and without thermal control (squares and triangles respectively), (b) a 55:45:4 mol ratio of DSPC: cholesterol: DSPE-PEG2000 liposomes (triangles) into female Balb/c mice as a function of time.
Figure 2: A graph showing doxorubicin: lipid remaining in the blood of mice after intravenous injection of radiolabelled (a) a 90: 4 molar ratio of DPPC: DSPE-PEG2000 liposomes (80-100 mmoles total lipid) with and without thermal control (squares and triangles respectively) into female Balb/c mice as a function of time.
Detailed Description of the Invention As mentioned above, the inventors have provided cholesterol-free liposomes, more particularly cholesterol-free liposomes suitable for thermosensitive applications in animals which can be made to be at least as stable in circulation and having drug retention characteristics comparable or better than their cholesterol-containing counterparts. In addition to providing such liposome compositions, the inventors have provided a method of preparing and selecting such cholesterol-free liposomes having advantageous properties.
The inventors have provided a means for the design wherein a liposome during development is tested by comparison with a similar liposome composition containing cholesterol. As an additional advantage, the inventors provide a means for assessing drug retention and/or in vivo serum stability based on the known properties of cholesterol containing liposomes, use of such a liposome as a reference liposome allows testing conditions to be carefully assessed.
As mentioned, the inventors provide liposomes in which the hydrophilic polymer stabilization effects due to use of PEG-modified lipid incorporation are not substantially dependent on the concentration of the polymer or polymer molecular weight. The inventors provide that concentrations as low as 0.5 mol% PEG-2000 can cause a significant (preferably greater than S, 10, or 15-fold increase in Area-Under-the Curve (AUC) when compared to the same liposome prepared without the PEG-lipid. Provided also is that for example PEG 350 at concentrations of 5 mol% can cause a significant (preferably greater than 10, 15 or 25 fold) improvement in AUC when compared to the same liposome prepared without the PEG lipid, and this increase in AUC
is comparable to a significant (preferably greater than 10, 15, 25 or 38-fold) improvement obtained when using 5 mol%
PEG 2000). The resulting liposomes provide much enhanced longevity of the liposomes while in blood circulation.
Throughout this specification, the following abbreviations have the indicated meaning.
PEG: polyethylene glycol; PEG preceded or followed by a number: the number is the molecular weight of PEG in Daltons; PEG-lipid: polyethylene glycol-lipid conjugate; PE-PEG: polyethylene glycol-derivatized phosphatidylethanolamine; PA: phosphatidic acid; PE:
phosphatidylethanolamine; PC: phosphatidylcholine; PI: phosphatidylinositol;
DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG 2000 (or 2000 PEG-DSPE or PEGZ~-DSPE):
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol 2000];
DSPE-PEG 750 (or 750PEG-DSPE or PEG~SO-DSPE): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol 750]; DPPE-PEG2000: 1,2-dipalmaitoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol 2000); DAPC: 1,2-arachidoyhrn-glycero-3-phosphocholine;
DBPC: 1,2-dibehenoyl-sn-glycero-3-phosphocholine; CH or Chol: cholesterol; DPPC: 1,2-dipalmaitoyl~sn-glycero-3-phosphocholine; HEPES: N-[2-hydroxylethyl]-piperazine-N-[2-ethanesulfonic acid].
As used in this specification and the appended claims, the singular forms "a,"
"an" and "the"
include plural references unless the context clearly dictates otherwise.
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. 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. Cholesterol free and liposomes having substantially no cholesterol are described incopending international patent application PCT/CA01/00655, which is incorporated herein by reference.

The term "liposome" as used herein means vesicles comprised of one or more concentrically ordered lipid bilayers encapsulating an aqueous phase. Formation of such 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 incorporated 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, phosphate, carboxyl, sulphate, amino or sulflrydryl groups.
Hydrophobicity results from the presence of a long chain of aliphatic hydrocarbon groups.
The term "hydrophilic polymer-lipid conjugate" refers to a vesicle-forming lipil 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 other than cholesterol.
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 of the distearoyl form. 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. Conventional liposomes suffer from a relatively short half life in the blood circulation due to their rapid uptake by macrophages of the liver and spleen (organs of the reticuloendothelial system or RES), and therefore do not accumulate in leaky tumor tissue. Liposome preparations have been devised which avoid rapid RES uptake and which have increased circulation times. See, e.g., Allen, UCLA
Symposium on Molecular and Cellular Biology, 89:405 (1989); Allen et al., Biochim. Biophys.
Acta 1066:29 (1991); Klibanov et al., FEBS Letters 268:235 (1990); Needham et al., Biochim.
Biophys. Acta 1108:40 (1992); Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA 88:11460 (1991); Wu et al., Cancer Research 53:3765 (1993); Klibanov and Huang, J. Liposome Research 2:321 (1992); Lasic and Martin, Stealth Liposomes, In: Pharmacology and Toxicology, CRC Press, Boca Raton, Fla.
(1995). See also U.S. Pat. No. 5,225,212 to Martin et al.; U.S. Pat. No.
5,395,619 to Zalipsky et al.
regarding liposomes containing polymer grafted lipids in the vesicle membrane.
The presence of polymers on the exterior liposome surface decreases the uptake of liposomes by the organs of the RES. A preferred polymer is polyethylene glycol (PEG). Preferably the polymer has a molecular weight between about 1000 and 5000 daltons. The conjugate may be prepared to include a releasable lipid-polymer linkage such as a peptide, ester, or disulfide linkage. The conjugate may also include a targeting ligand. 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 "phase transition temperature" is the temperature or range of temperatures at which a liposome changes from a gel state to a liquid crystalline state. A
convenient method for measuring phase transition temperature is to monitor energy absorption while heating a preparation of liposomes and noting the temperature or range in temperatures at which there is an energy absorbance.
The predominant vesicle-forming lipid in liposomes of this invention are responsible for achieving phase transition temperatures of betweefi the body temperature of a subject to be treated (e.g. human or non-human mammal) and 45°C. Preferably, the lipid is a phospholipid such as PC, PE, PA or PI. 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 ofcaproyl (6:0), octanoyl (8:0), capryl (10:0), lauroyl (12:0), mirystoyl (14:0) and palmitoyl (16:0).
As mentioned, liposomes used according to the invention comprise a lipid possessing a gel-to-liquid crystalline phase transition temperature in the hyperthermic range, and preferred are phospholipids whose acyl groups are saturated. A particularly preferred phospholipid is dipalmitoylphosphatidylcholine (DPPC). DPPC is a common saturated chain (C 16) phospholipid with a bilayer transition of 41.5° C. (Blume, Biochemistry 22:5436 (1983); Albon and Sturtevant, Proc. Natl. Acad. Sci. USA 75:2258 (1978)). Thermosensitive liposomes containing DPPC and other lipids that have a similar or higher transition temperature, and that can be mixed with DPPC (such as 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG) (Tc=41.5° C.) and 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) (Tc=55.1° C.)) have been studied.
Kastumi Iga et al, Intl. J.
Pharmaceutics, 57:241 (1989); Bassett et al, J. Urology, 135:612 (1985); Gaber et al, Pharmacol. Res.
12:1407 ( 1995).
As demonstrated in the Examples, a preferred example of a liposome formulation of the invention was prepared having a 90: 4 molar ratio of DPPC: DSPE-PEG2000.
Generally, preferred liposomes of the invention comprise at least 60 mole % of a phospholipid.
Preferably, DPPC is the predominant lipid. Most preferably the liposomes comprise at least 30, 40, 50, 60, 70, 80, 85, 90 or 95 mole/% DPPC. It will be appreciated, however, that any other suitable lipid composition may be used according to the invention and that the liposomes of the invention need not be limited to liposomes comprising DPPC. Moreover, it is often practice to prepare liposomes comprising several different lipids (e.g. to achieve optimal stability and drug retention characteristics, or as a surface active agent). Thus, liposomes of the invention may comprise lipids which by themselves would not have the desired transition temperatures so long as~the lipid (for example a hydrophilic polymer lipid conjugate) does not destabilize the membrane at processing temperatures where the bilayer is in the liquid phase, nor at physiological temperatures where the bilayer is in the gel phase. For example, other phase compatible components such as DSPE, DSPE-PEG or DSPC can optionally be included a liposome. Preferably, however, DSPC is not the predominant lipid (e.g. the main lipid component of liposome bilayer material) and more preferably DSPC is present at less than 40 mole/%, less than 20, 10 or 5 mole/%, or the liposome is essentially free of DSPC.
Preferably, the liposomes which are to be designed according to the methods of this invention, and the liposome compositions of the invention may comprise amphipathic lipids in addition to those described above, but no substantial amount of cholesterol.
Such lipids include sphingomyelins, glycolipids, ceramides and phospholipids. Such lipids may include lipids having therapeutic agents, targeting agents, ligands, antibodies or other such components which are used in liposomes, either covalently or non-covalently bound to lipid components.
Methods of preparation The liposomes that are the subject of the methods of the invention can be obtained from commercial sourcesor can be prepared according to known methods, as described herein or otherwise known.
Liposomes of the present invention or for use in the present invention may be generated by a variety of techniques including lipid film/hydration, reverse phase evaporation, detergent dialysis, freeze/thaw, homogenation, solvent dilution and extrusion procedures. Various known techniques are provided for example in U.S. Pat. No. 4,235,871; Published PCT
applications WO 96/14057;
New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic D D, Liposomes from physics to applications, Elsevier Science Publishers, Amsterdam, 1993; Liposomes, Marcel Dekker, Inc., New York (1983).
As shown in Example l, liposome comprising having a 90: 4 molar ratio of DPPC:
DSPE-PEG2000 were prepared. The liposomes were administrated to mice as detailed, and circulation longevity was assessed as shown in Example 3.
It will be appreciated that any suitable method for producing the liposomes of the invention can be used. A non-limiting example is provided for illustration as follows.
Liposomes having a desired molar ratio of lipids, comprising [at least one phospholipid] and 3 S at least one polymer-conjugated lipid, are prepared. A physiologically acceptable buffer is used for formation of the liposome, for example citrate having an acid pH of typically about pH 2 to about pH
6, about pH 3 to pH 5, and most preferably at about pH 4.
Once the liposomes are prepared with the entrapped acidic buffer, the liposomes can be sized to a desired size range. Liposomes of this invention or for use in this invention are typically greater than SOnm in diameter, more preferably between about SOnm and about lpm in diameter. However, preferred liposomes of this invention will be less than about 200 nm, preferably less than about 160 nm, and more preferably less than about 140 nm in diameter. 100-140 nm liposomes (cholesterol-free liposomes tend to be slightly larger than cholesterol containing ones) are employed in the Examples below. Liposomes are typically sized by extrusion through a filter (e.g. a polycarbonate filter) having pores or passages of the desired diameter. A liposome suspension may also be sonicated either by bath or probe down to small vesicles of less than about 0.05 microns in size.
Homogenization may also be used to fragment large liposomes into smaller ones.
In both methods the particle size distribution can be monitored by conventional laser-beam particle size discrimination or the like.
Therapeutic agents may be loaded into liposomes using passive and active loading methods described herein.
Passive methods of encapsulating therapeutic agents Therapeutic agents may be encapsulated using passive methods of encapsulation.
Passive methods of encapsulating therapeutic agents in liposomes involve encapsulating the agent during the synthesis of the liposomes. In this method, the drug may be membrane associated or encapsulated within an entrapped aqueous space. This includes a passive entrapment method described by Bangham et al., (J. Mol. Biol. 12, (1965), 238) where the aqueous phase containing the agent of interest is put into contact with a film of dried vesicle-forming lipids deposited on the walls of a reaction vessel. Upon agitation by mechanical means, swelling of the lipids will occur and multilamellar vesicles (MLV) will form. Using extrusion, the MLV's can be converted to large unilamellar vesicles (LUV) or small unilamellar vesicles (SUV) following sonication.
Another method of passive loading that may be used includes that described by Deamer et al (Biochim. Biophys. Acta 443, (1976), 629). This method involves dissolving vesicle-forming lipids in ether and, instead of first evaporating the ether to form a thin film on a surface, this film being thereafter put into contact with an aqueous phase to be encapsulated, the ether solution is directly injected into said aqueous phase and the ether is evaporated afterwards, whereby liposomes with encapsulated agents are obtained. A further method that may be employed is the Reverse Phase Evaporation (REV) method described by Szoka & Papahadjopoulos (P.N.A.S.
(1978) 75: 4194) in which a solution of lipids in a water insoluble organic solvent is emulsified in an aqueous carrier phase and the organic solvent is subsequently removed under reduced pressure.

Other methods of passive entrapment that may be used subjecting liposomes to successive dehydration and rehydration treatment, or freezing and thawing; dehydration was carried out by evaporation or freeze-drying. This technique is disclosed by Kirby et al (Biotechnology, November 1984, 979-984). Also, Shew et al (Biochim. Et Biophys. Acta 816 (1985), 1-8) describe a method wherein liposomes prepared by sonication are mixed in aqueous solution with the solute to be encapsulated, and the mixture is dried under nitrogen in a rotating flask. Upon rehydration, large liposomes are produced in which a significant fraction of the solute has been encapsulated.
Active methods of encapsulating therapeutic agents Therapeutic agents in accordance with this invention may be encapsulated using active methods of encapsulation. Active loading involves the use of transmembrane gradients across the liposome membrane to induce uptake of a therapeutic agent after the liposome has been formed. This can involve a gradient of one or more ions including Na+, K+, H+, and/or a protonated nitrogen moiety. Active loading techniques that may be used in accordance with this invention include pH gradient loading, charge attraction, and drug shuttling by an agent that can bind to the drug.
Liposomes may be loaded according to the pH gradient loading technique.
According to this technique, liposomes are formed which encapsulate an aqueous phase of a selected pH.
Hydrated liposomes are placed in an aqueous environment of a different pH
selected to remove or minimize a charge on the drug or other agent to be encapsulated. 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.
To create a pH gradient, the original external medium is replaced by a new external medium having a different concentration of protons. The replacement of the external medium can be accomplished by various techniques, such as, by passing the lipid vesicle preparation through a gel filtration column, e.g., a Sephadex column, which has been equilibrated with the new medium (as set forth in the examples below), or by centrifugation, dialysis, or related techniques. The internal medium may be either acidic or basic with respect to the external medium.
After establishment of a pH gradient, a pH gradient loadable agent is added to the mixture and encapsulation of the agent in the liposome occurs as described above.
PH gradient loading may be carried out according to methods described in US
patent nos.
5,616,341; 5,736,155 and 5,785,987 the disclosures of which are incorporated herein by reference.
Therapeutic agents that may be loaded using pH gradient loading comprise one or more ionizable moieties such that the neutral form of the ionizable moiety allows the drug to cross the liposome membrane and conversion of the moiety to a charged form causes the drug to remain encapsulated within the liposome. Ionizable moieties may comprise, but are not limited to comprising, amine, carboxylic acid and hydroxyl groups. PH gradient loadable agents that load in response to an acidic interior may comprise ionizable moieties that are charged in response to an acidic environment whereas drugs that load in response to a basic interior comprise moieties that are charged in response to a basic environment. In the case of a basic interior, ionizable moieties including but not limited to carboxylic acid or hydroxyl groups may be utilized. In the case of an acidic interior, ionizable moieties including but not limited to primary, secondary and tertiary amine groups may be used.
Preferably, the pH gradient loadable agent is a drug and most preferably an anti-neoplastic agent. Examples of some of the antineoplastic agents which can be loaded into liposomes by this method and therefore may be used in this invention include but are not limited to anthracyclines such as doxorubicin, daunorubicin, mitoxanthrone, idarubicin, epirubicin and aclarubicin; antineoplastic antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine and vinorelbine; alkylating agents such as cyclophosphamide and mechlorethamine hydrochloride; campthothecins such as topotecan, irinotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin; purine and pyrimidine derivatives such as 5-fluorouracil; cytarabines such as cytosine arabinoside.
This invention is not to be limited to those drugs currently available, but extends to others not yet developed or commercially available, and which can be loaded using the transmembrane pH
gradients.
Various methods may be employed to establish and maintain a pH gradient across a liposome all of which are incorporated herein by reference. This may involve the use of ionophores that can insert into the liposome membrane and transport ions across membranes in exchange for protons (see for example US patent no. 5,837,282). Buffers encapsulated in the interior of the liposome that are able to shuttle protons across the liposomal membrane and thus set up a pH gradient (see for example US patent no 5,837,282) may also be utilized. These buffers comprise an ionizable moiety that is neutral when deprotonated and charged when protonated. The neutral deprotonated form of the buffer (which is in equilibrium with the protonated form) is able to cross the liposome membrane and thus leave a proton behind in the interior of the liposome and thereby cause a decrease in the pH of the interior. Examples of such buffers include methylammonium chloride, methylammonium sulfate, ethylenediammonium sulfate (see US patent no. 5,785,987) and ammonium sulfate. Internal loading buffers that are able to establish a basic internal pH, can also be utilized. In this case, the neutral form of the buffer is protonated such that protons are shuttled out of the liposome interior to establish a basic interior. An example of such a buffer is calcium acetate (see LJS patent no.
5,939,096).

In other aspects, charge attraction methods may be utilized to actively load therapeutic agents. Charge amaction mechanisms for drug loading involves creating a transmembrane potential across the membrane by creating a concentration gradient for one or more charged species. Thus, for a drug that is negatively charged when ionized, a transmembrane potential is created across the membrane that has an inside potential which is positive relative to the outside potential. For a drug that is positively charged, the opposite transmembrane potential would be used.
Following a separation step as may be necessary to remove free drug from the medium containing the liposome, the liposome suspension is brought to a desired concentration in a pharmaceutically acceptable carrier for administration to the patient or host cells. Many pharmaceutically acceptable carriers may be employed in the compositions and methods of the present invention. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Generally, normal buffered saline (135-150 mM
NaCI) will be employed as the pharmaceutically acceptable carrier, but other suitable carriers will suffice. These compositions may be sterilized by conventional liposomal sterilization techniques, such as filtration. 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. These compositions may be sterilized techniques referred to above or produced under sterile conditions. 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 concentration of liposomes in the carrier may vary. Generally, the concentration will be about 20-200 mg/ml, usually about 50-150 mg/ml, and most usually about 75-125 mg/ml, e.g., about 100 mg/ml. Persons of skill may vary these concentrations to optimize treatment with different liposome components or for particular patients. For example, the concentration may be increased to lower the fluid load associated with treatment.
Thus, in the present method, one step comprises providing a liposome of the invention having a phase transition temperature greater than that of the body of a subject to be treated. The liposome is generally stable at body temperature but is capable of releasing an encapsulated drug at mildly hyperthermic conditions, which are generally understood to be between about 37°C and 45°C, or more preferably between about 37°C and 43°C.
Another step comprises providing a 'reference' liposome containing substantially the same lipids and in the same proportions as the liposome of the invention, but containing at least 20 mol cholesterol. The cholesterol-containing liposome may contain a hydrophilic polymer-conjugated lipid such as PEG, or may be free of hydrophilic polymer-conjugated lipid and/or free of PEG. The 'time point' is generally a number as measured in hours, minutes, etc.
The liposome compositions to be compared will generally have encapsulated therein a therapeutic agent. The therapeutic agent contained in each of the liposome compositions to be compared will preferably be the same agent; however, it will be appreciated that agents that are not identical but have structural similarities or having similar or identical liposome loading, stability or retention properties can also be used. The liposomes may be provided, when obtained from commercial sources for example, with a drug already encapsulated therein.
Typically, however, the method will involve preparing the liposomes to be compared, and encapsulating a drug into each of the liposomes compositions to be compared.
In an alternative, albeit less preferable embodiment, an agent which is not itself a therapeutic molecule may be used in the methods of the invention. Generally, to be useful in the present method, the marker will have physical properties allowing it to be indicative of drug behavior in the liposome.
That is, the step of encapsulating a drug into one or both of liposomes (e.g.
the thermosensitive and/or substantially cholesterol-free liposomes of the invention, and the cholesterol-containing reference liposomes) can be substituted with the step of encapsulating a marker molecule, and the steps of determining and comparing drug:lipid ratios can be substituted with determining and comparing marker: lipid ratios. Alternatively, said comparing step may comprise comparing a marker:lipid ratio to a drug:lipid ratio where the drug and marker have similar or substantially identical physical properties or retention properties in a liposome.
The terms "drug" and "therapeutic agent" as used herein generally refer to moieties used in therapy and for which liposome-based delivery is desirable. Active agents (including drugs, therapeutic agents or other agents) suitable for use in the present invention include therapeutic agents and pharmacologically active agents, nutritional molecules, cosmetic agents, diagnostic agents and contrast agents for imaging. Included are small molecule therapeutics as well as nucleic acids, polynucleotides, polypeptides or any other suitable agents. As used herein, activeagents include pharmacologically acceptable salts of active agents. Suitable therapeutic agents include, for example, antineoplastics, antitumor agents, antibiotics, antifungals, anti-inflammatory agents, immunosuppressive agents, anti-infective agents, antivirals, anthelminthic, and antiparasitic compounds. 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. In treating tumors or neoplastic growths, suitable compounds may include alkylating agents, antimetabolities, anthracycline antibiotics (such as doxorubicin, daunorubicin, carinomycin, N-acetyladriamycin, rubidazone, 5-imidodaunomycin, N30 acetyldaunomycin, and epirubicin) and plant alkaloids (such as vincristine, vinblastine, vinorelbine, etoposide, ellipticine and camptothecin). Other suitable agents include paclitaxel (Taxol); docetaxol (taxotere); mitotane, cisplatin, and phenesterine. Anti-inflammatory therapeutic agents suitable for use in the present invention include steroids and non-steroidal anti-inflammatory compounds, such as prednisone, methyl-prednisolone, paramethazone, 11-fludrocortisol, triamciniolone, betamethasone and dexamethasone, ibuprofen, piroxicam, beclomethasone; methotrexate, azaribine, etretinate, anthralin, psoralins;
salicylates such as aspirin;
and immunosuppresant agents such as cyclosporine. Antiinflammatory corticosteroids and the antiinflammatory and immunosuppressive agent cyclosporine are both highly lipophilic and are suited for use in the present invention. Other examples of agents that can be used according to the invention are shown in Table 1.

Table 1 CLASS TYPE OFAGENT NONPROPRIETARY DISEASE (Neoplastic) NAMES

(OTHER NAMES) Alkylating Agents NitrogenMechlorethamine Hodgkin's disease, Mustards (HN2) non-Hodgkin's lymphomas Cyclophosphamide Acute and chronic lymphocytic Ifosfamide leukemias, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma, brest, ovary, lung, Wihns' tumor, cervix, testis, soft-tissue sarcomas Melphalan (L-sarcolysin)Multimple myeloma, breast, ovary Chlorambucil Chronic lymphocytic leukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin's lymphomas Ethylenimenes and HexamethylmelamineOvary Thiotepa Bladder, breast, Methyhnelamines ovary Alkyl Sulfonates Busulfan Chronic granulocytic leukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease, non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma, malignant melanoma Lomustine (CCNU) Hodgkin's disease, non-Hodgkin's lymphomas, primary brain tumors, small-cell lung Semustine (methyl-CCNU)Primary brain tumors, stomach, colon Streptozocin (streptozotocin)Malignant pancreatic insulinoma, malignant carcinoid Triazines Dacarbazine (DTIC;Malignant melanoma, Hodgkin's dimethyltriazenoimidazole-disease, soft-tissue sarcomas Antimetabolites Folic Methotrexate (amethopterin)Acute lymphocytic Acid Analogs leukemia, choriocarcinoma, mycosis fungoides, breast, head and neck, lung, osteogenic sarcoma Pyrimidine Analogs Fluouracil (5-fluorouracil;Breast, colon, stomach, 5- pancreas, FU) ovary, head and neck, urinary bladder Floxuridine (fluorode-premalignant skin lesions (topical) oxyuridine; FudR) Cytarabine (cytosineAcute granulocytic and acute arabinoside) lymphocytic leukemias Purine Analogs and Mercaptopurine(6- Acute lymphocytic, acute Related Inhibitors mercaptopurine; granulocytic and chronic 6-MP) granulocytic leukemias Thioguanine(6-thioguanine;Acute granulocytic, acute TG) lymphocytic and chronic granulocytic leukemias Pentostatin(2- Hairy cell leukemia, mycosis deoxycoformycin) fungoides, chronic lymphocytic leukemia Natural Products Vinca Vinblastine (VLB) Hodgkin's disease, Alkaloids non-Hodgkin's lymphomas, breast, testis Vincristine Acute lymphocytic leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas, small-cell lung Epipodophyllotoxins Etoposide Testis, small-cell lung and other Tertiposide lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's sarcoma Antibiotics Dactinomycin (actinomycinChoriocarcinoma, Wilms' tumor, D) rhabdomyosarcoma, testis, Kaposi's sarcoma Daunorubicin (daunomycin;Acute granulocytic and acute;

rubidomycin) lymphocytic leukemias Doxorubicin Soft-tissue, osteogenic and other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, breast, genitourinary, thyroid, lung, stomach, neuroblastoma Bleomycin Testis, head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin's disease, non-Hodgkin's lymphomas Plicamycin (mithramycin)Testis, malignant hypercalcemia Mitomycin (mitomycinStomach, cervix, colon, C) breast, pancreas, bladder, head and neck Enzymes L-Asparaginase Acute lymphocytic leukemia Biological Response Interferon alfa Hairy cell leukemia, Kaposi's Modifiers sarcoma, melanoma, carcinoid, renal cell, ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma, chronic granulocytic leukemia Miscellaneous Platinum Cisplatin (cis-DDP)Testis, ovary, bladder, Coordination , head and Agents Complexes Carboplatin neck, lung, thyroid, cervix, endometrium, neuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acute granulocytic leukemia, breast Substituted Urea Hydroxyurea Chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis, malignant melanoma Methyl Hydrazine Procarbazine(N- Hodgkin's disease Derivative methylhydrazine, MIH) Adrenocortical Mitotane (o,p'-DDD)Adrenal cortex Suppressant AminoglutethimideBreast Hormones and AdrenocorticosteroidsPrednisone (severalAcute and chronic other lymphocytic Antagonists equivalent preparationsleukemias, non-Hodgkin's available) lymphomas, Hodgkin's disease, breast Progestins HydroxyprogesteroneEndometrium, breast caproate Medroxyprogesterone acetate Megestrol acetate Estrogens DiethylstilbestrolBreast, prostate Ethinyl estradiol (other preparations available) Antiestrogen Tamoxifen Breast Androgens Testosterone propionateBreast Fluoxymesterone (other preparations available) Antiandrogen Flutamide Prostate Gonadotropin-releasing Leuprolide Prostate hormone analog The methods of the invention comprise providing and comparing a first liposome having a phase transition temperature at temperatures mildly hyperthermic to the body of a subject to be treated, and a second liposome comprising substantially the same lipids and in the same proportions as the first liposome, but comprising cholesterol (e.g. preferably at least 20 mole/%).
Liposomes of this invention may be formulated for parenteral administration in a suitable carrier such as a sterile aqueous solution. The carrier may comprise excipients known to be tolerated by warm-blooded animals. When performing the assay method of this invention, a mammal such as a mouse will be injected with a liposome formulation and blood is removed from the mouse at fixed time intervals such as 1, 2, 3, 4, 8, 12, 18 or 24 hours post-administration.
A convenient means for obtaining blood at a fixed time interval is by cardiac puncture. Following removal of whole blood, the plasma is isolated and subjected to suitable techniques known in the art for measuring the amount of lipid and drug present. For example, the lipid component may be radioactively labeled and the plasma subjected to liquid scintillation counting. The amount of drug can be determined for example by a spectraphotometric assay.
It will be appreciated that the drug:lipid ratios of the liposomes can be determined according to any suitable method. One convenient method for determining the lipid component is liquid scintillation counting. For example, liposomes are labeled with ~H]-CHE as a non-exchangeable, non-metabolizeable lipid marker. The liposome are injected to a mouse via the lateral tail vein with a lipid dose of 50 mg/kg and an injection volume of 200 pL into ~ 22 g female CD-1 mice. At various times, three mice from each group are terminated by COz asphyxiation. Blood is collected by cardiac puncture, and placed into EDTA-coated or heparin-coated microtainer collection tubes (Becton-Dickinson). After centrifuging the blood samples at 4°C for 15 minutes at 1000 x g, plasma is isolated. Aliquots of the plasma obtained are counted directly in 5.0 mL
scintillation fluid. ~H]- and ['4C]-CHE labels are available from NEN/Dupont. It will be appreciated however that any other suitable method of determining the drug retention time of a liposome can be used. In this specification, the term "retention" with respect to a drug or other agent encapsulated in a liposome refers to retention of the drug in a liposome while the liposome is present in the bloodstream of a mammal. This term does not refer to a measure of drug that may be loaded or incorporated into a liposome or the ability of a liposome to retain the drug inex vivo conditions.
The liposomes according to the invention result in enhanced longevity (circulation time) while the liposome or lipid carrier of this invention is present in the bloodstream of a warm blooded animal. Preferably, a liposome or lipid carrier of this invention will be made such that the amount that would remain in the bloodstream of an animal at 4, 6, 12, 18, 24, 36 or 48 hours after intravenous administration is at least about 10%, 20%, 40%, 50%, 60%, 70%, 80% or 90% of the amount administered. Example 3 demonstrates the circulation longevity of exemplary liposomes of the invention at specified time points after administration. These DPPC-DSPE-PEG2000-liposomes demonstrated substantial stability in the bloodstream as shown in Figure 1. In another aspect, liposomes in accordance with the invention may display a circulation longevity, preferably the proportion of injected liposome remaining in the bloodstream at a fixed time point after administration to a mammal, is which is comparable to or better than the circulation longevity in a mammal of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol. For liposome circulation longevity, the data obtained from a model animal system can be reasonably extrapolated to humans and veterinary animals of interest.
Liposome uptake by liver and spleen has been found to occur at similar rates in several mammalian species, including mouse, rat, monkey, and human (Gregoriadis, G., and Neerunjun, D. (1974) Eur. J.
Biochem. 47, 179-185; Jonah, M. M., et al. (1975) Biochem. Biophys. Acta 401, 336-348;
Kimelberg, H. K., et al. (1976) Cancer Res. 36,2949-2957; Juliano, R. L., and Stamp, D. (1975) Biochem. Biophys. Res. Commun. 63. 651-658; Richardson, V.J., et al. (1979) Br. J. Cancer 40, 3543; Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378). 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.
Optionally, any or all of the steps of the method of designing or preparing liposomes of the invention can be repeated. For example, the steps of providing liposomes and comparing retention properties of cholesterol-free or thermosensitive liposomes of the invention and cholesterol-containing liposomes can be repeated. With these iterations, adjustments to the respective liposome compositions can be made. In this way, liposomes can be optimized and the resulting compositions can be assessed for drug retention and/or circulation longevity properties.
It will be appreciated that a panel of liposomes can be prepared, liposomes of said panel having a phase transition temperatures greater than that of the body of a subject to be treated, preferably above 37°C but less than 45°C, having differences in lipids and in lipid proportions, so long as at least one of the liposomes of said panel is compared against a liposome comprising substantially the same lipids and in the same proportions, but comprising cholesterol (e.g. preferably at least 20 mole %). A panel of liposomes can comprise for example at least 2, 3, 4, 5, 8, 10, 20, 50, 100 different liposome compositions.
A liposome having the aforementioned phase transition properties, preferably a liposome that is substantially cholesterol-free can thus be identified, which liposome has drug retention characteristics comparable or better than a similar cholesterol-containing liposome. In the context of identifying said liposome, the terms 'identifying' and 'selecting' can be used interchangeably. This liposome with a drug incorporated therein can be used as such as a medicament for treatment of a subject, or can be the subject of further optimization or design steps to modify the composition as desired. In particular, steps of adjusting the liposome composition can be carried out, and the steps of the method of the present invention can be repeated to compare drug:lipid ratios with a comparable cholesterol-containing liposome. In other aspects, the improved liposome obtained using the method of the invention can also be used with different drugs. For example, it will be appreciated that different drugs having similar retention properties to the drug or marker used in the drug:lipid ratio comparison of the present method can be incorporated the liposome in place of the drug used in the drug:lipid comparison. Furthermore, if desired, tests can be carried out to compare drug:lipid ratios of the two drugs in the thermosensitive liposomes of the invention.
Administration and therapeutic use The liposomes of the invention allows the,use of thermosensitive liposomes in combination with hyperthermia at the desired target site, permitting improved targeting at 'mildly hyperthermic' temperatures which otherwise do not cause damage to the patient. Applications of such liposomes have been reported for example in Magin and Weinstein In: Liposome Technology, Vol. 3, (Gregoriadis, G., ed.) p. 137, CRC Press, Boca Raton, Fla. (1993); Gaber et al., Intl. J. Radiation Oncology, Biol. Physics, 36(5):1177 (1996).
Liposome of the present invention may be administered to warm-blooded animals to be treated, including humans. Liposomes of the present invention may be administered using methods that are known to those skilled in the art, including but not limited to delivery into the bloodstream (e.g. administered intravenously) of a subject or subcutaneous administration of liposomes. These liposomes may be used to treat a variety of diseases in warm-blooded animals, the application of which depending on the particular bioactive agent incorporated in the liposome. Examples of medical uses of the compositions of the present invention include but are not limited to treating cancer, treating cancer, inflammation, treating bacterial, fungal or parasitic infections. 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 of the present invention, exhibit reduced toxicity to healthy tissues of the subject.
3 5 For medical applications, formulations of the liposomes of the present invention for parenteral administration are preferably in a sterile aqueous solution optimally comprised of excipients known to be tolerated by warm-blooded animals. For oral or topical applications, the liposome and lipid carrier compositions of the present invention may be incorporated in vehicles commonly used for the respective applications such as but not limited to creams, salves, ointments and slow release patches for topical medical applications and tablets, capsules, powders, suspensions, solutions and elixirs for oral applications.
The liposomes and lipid carrier compositions of the present invention may also be used for diagnostic purposes where the controlled release of liposome contents can provide improved delivery of distribution of a diagnostic agent. Liposomes of the present invention can be administered as a parenteral agent to warm-blooded animals to detect the presence of specific disease sites or markers of disease. Such compositions may contain imaging agents including, but not limited to, radionuclides, magnetic resonance contrast agents and heavy atom contrast agents.
EXAMPLES
Example 1 Preparation of liposomes Solutions of DPPC, DSPE-PEG2000 and cholesterol in chloroform were combined to give a 90: 4 molar ratio of DPPC: DSPE-PEG2000 (80-100 pmoles total lipid), and a 55:45:4 mol ratio of DSPC: cholesterol: DSPE-PEG2000 with 50,000 dpm/mg lipid of3H-cholesteryl hexadecyl ether (3CHE) as a radiolabelled marker. The resulting mixture was dried under a stream of nitrogen gas and placed in a vacuum pump overnight. The samples were then hydrated with 300 mM citrate pH

4.0 and subsequently passed through an extrusion apparatus (Lipex Biomembranes, Vancouver, BC) 10 times with 1 X 80 nm and 1 X 100 nm polycarbonate filters at 55 °C.
Average liposome size was determined by quasi-elastic light scattering using a NICOMP 370 submicron particle sizer at a wavelength of 632.8 nm.
Example 2 Encapsulation of drug For each of the radiolabelled solutions of Example 1, a 90: 4 molar ratio of DPPC: DSPE-PEG2000 (80-100 pmoles total lipid), and a 50:45:5 mol ratio of DSPC:
cholesterol: DSPE-PEG2000, the solution was run down a Sephadex G50 column equilibrated with HBS
(20 mM
HEPES, 150 mM NaCI, pH 7.45) in order to create a transmembrane pH gradient by exchange of the exterior buffer. Resulting pH gradient liposomes were combined with doxorubicin to give a final concentration of 5 mM lipid and 1 mM doxorubicin (0.2:1 drug:lipid ratio) in a final volume of 1 mL
adjusted with HBS. The resulting mixture was incubated at 37°C prior to assaying the amount of encapsulated doxorubicin. At various time points, samples were fractionated on a 1 mL mini-Sephadex G-50 spin column to remove unencapsulated doxorubicin. The voided fraction was assayed for liposomal lipid by scintillation counting. To measure levels of doxorubicin, a defined volume of the eluant was adjusted to 100 pL followed by addition of 900 pL, of 1% Triton X-100 to dissolve the liposomal membrane. The sample was heated until cloudy in appearance and the Abs480 was measured after equilibration at room temperature. Concentrations of doxorubicin were calculated by preparing a standard curve.
Example 3 Administration of thermosensitive liposomes and assessment of circulation longevity and drug:
lipid ratio DPPC: DSPE-PEG2000 (90:4 mol %) liposomes were prepared and loaded with doxorubicin as outlined in the methods of Example 1 and 2 respectively.
Non-thermally controlled mice were treated with cholesterol-free liposomes as follows:
Adult female Rag-2 mice were injected with DPPC: DSPE-PEGz~o (90:4 mol%) liposomes via the tail vein. Mice were killed and blood was collected by cardiac puncture into EDTA-coated microtainers at 10 min, 1h, 2h, and 4h after treatment.
Non-thermally controlled mice were treated with cholesterol-containing liposomes as follows:
Adult female Rag-2 mice were injected with DSPC: Cholesterol:DSPE-PEGz~
(55:45:4 mol%) via the tail vein. Mice were killed and blood was collected by cardiac puncture into EDTA-coated microtainers 1h and 4h after treatment.
Thermally controlled mice were treated with cholesterol-free liposomes as follows:
Late time points (2 and 4 hours post injection):
Twelve mice were anaesthetized for at least 1h with ketamine/xylazine (160/10 mg/kg). Mice were placed in groups of four mice per cage in a temperature controlled cage incubator that was pre-heated at 37°C. After being fully anaesthetized, mice were removed from the cage incubator and injected with DPPC:DSPE-PEGZOOO (90:4 mol%) liposomes via the tail vein. Mice were subsequently placed back in the cage incubator with the heat turned off. After 2h, the mice had fully recovered from the anesthesia and their body temperature was therefore not controlled thereafter.
Mice were terminated at 2h and 4h post injection and blood was collected by cardiac puncture into EDTA-coated microtainers.
Early time points (10 minutes and 1 hour post injection):
The remaining 12 mice were anaesthetized as described above and placed individually in a custom-made mouse-incubator that was preheated at 37°C. Mice were injected with DPPC: DSPE-PEC~ooo (90:4 mol%) liposomes via the tail vein. Mice were terminated after lOmin and 1h. Blood was collected by cardiac puncture into EDTA-coated microtainers.
Lipid and plasma doxorubicin concentrations were determined as follows:
Plasma was separated by centrifugation at 750g for lOmin and the lipid concentration in plasma was determined by liquid scintillation counting. Doxorubicin was extracted and quantified as follows:
A defined volume of plasma was adjusted to 200 mL with distilled water and the following reagents were added: 600 mL of distilled water, 100 mL of a 10% sodium dodecyl sulfate solution, and 100 mL of 10 mM HZS04. To the resulting mixture, 2mL of isopropanol/chloroform (1:1 vol/vol) was added and mixed vigorously. Samples were frozen at -20°C overnight or at 80°C for 1 hour to promote protein aggregation, brought to room temperature, mixed again and centrifuged at 3000 rpm for 10 minutes. The bottom organic layer was removed and assayed by fluorescence spectroscopy (~,eX: 470 nm, 7<,em: 550 nm).
Doxorubicin-containing DPPC:DSPE-PEGZOOO liposomes exhibited extended circulation longevity (Figure 1) and enhanced drug retention (Figure 2) in temperature controlled mice similar to the cholesterol-containing formulation and in contrast to doxorubicin-containing DPPC:DSPE-PEGzooo liposomes administered to mice without thermal control. At 4h after injection, the body temperature of thermally controlled mice was likely increased to temperature at which liposomes start releasing the drug (39°C) since the anesthetic wore off starting at 1.5h - 2h after injection. The body temperature in mice can increase to values up to 40.5°C as a stress-response in non anesthetized mice, which may explain why lipid and drug levels were decreased 4h after administration (due to lack of thermal control).
Results depicted in Figures 1 and 2 are contrary to observations set forth in the state of the art (see review article: Kong et al. (1999) Int. J. Hyperthermia 15(5): 345-370).
Most likely this is due to the absence of thermal control in previous studies described in the art.
Example 4 Delayed release of cholesterol-free, thermosensitive liposomes DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes are prepared and loaded with doxorubicin as outlined in the materials and methods of Example 1 and 2 respectively.
The resulting doxorubicin loaded liposomes are administered to a mouse(however, the body temperatures of the mice cannot be controlled according to the methods of Example 3 due to time points in the study beyond 1 hr) in a final volume of 200 pL immediately after preparation (within 1-2 hrs). Subsequent to administration, local hyperthermia (42°C) at the tumor site using a radiofrequency oscillator or a water bath (with specially designed holders that allow the tumor to be placed in a water bath) is started at 4, 6, 12,18,24,36 and 48 hours after administration and continued for a set period of time (typically not exceeding 2 hrs). Just prior to and after thehyperthermia treatment, blood is collected and tumors excised. Lipid levels are measured by liquid scintillation counting. To determine drug levels, tumors are frozen at-70 °C and extracted with chloroform and silver nitrate to determine doxorubicin concentrations (Cummings et al. (1986) Br. J. Cancer 53: 835-838). Samples may also be extracted with only chloroform for comparison to determine the amount of doxorubicin bound to DNA or RNA (thereby giving a measure of released drug). Concentrations of doxorubicin in tumour samples are quantified using high performance liquid chromatography.
In order to determine drug and lipid levels, in the blood afterhyperthermia, blood samples are quantitated for levels of doxorubicin and lipid as in Example 3.
Example S
Assessing survival time upon administration of liposomal doxorubicin DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes are prepared and loaded with doxorubicin as outlined in the materials and methods of Example 4 (except temperature control is not possible at time points greater than 1 hour according to the methods of Example 3).
P388/wt cells are maintained by passage in vivo (in the peritoneum) of BDF-1 female mice. Cells are only used for experiment between the 3~d and 20'h passage.
Cells are harvested 7 days post inoculation, diluted in Hepes Buffered Saline (HBS) to 2 x 106 cells/mL, and 0.5 mL is injected intraperitoneally into BDF-1 mice. Two days after tumor cell inoculation, BDF1 female mice are administered by intravenous administration one of the following: HBS;
doxorubicin (1 mg/kg); DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes (1 mg/kg) loaded with doxorubicin.
Percent survival is calculated based on 4 mice per group.
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 incorporated herein by reference.

Claims

1. A method for designing an improved liposome composition containing a drug, said method comprising the steps of (a) preparing a liposome having a phase transition temperature greater than that of the body of a subject to be treated, and less than 45°C;
(b) preparing a liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
(c) encapsulating the drug into the liposomes of (a) and (b);
(d) administering the liposomes of (a) and (b) after encapsulation of the drug to the bloodstream of a separate non-human mammal;
(e) determining drug:lipid ratios in the blood of the mammals at least one fixed time subsequent to administration; and (f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and (g) identifying a liposome of step (a) having a drug:lipid ratio at a fixed time point in a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said fixed time point of a liposome of step (b) containing substantially the same lipids and in the same proportions as the liposome in step (a) with at least 20 mol % cholesterol.

2. The method of claim 1, wherein the liposome of step (a) has a phase transition temperature of between 39°C and 41°C.

3. The method of claim 1 or 2, wherein the liposome of step (a) containing substantially no cholesterol.

4. The method of claim 1, 2 or 3, wherein the liposome of step (a) comprises (a) at least 60 mol % of a phospholipid;
(b) from about 1 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mol % of one or more vesicle-forming lipids.

5. The method of anyone of claims 1-4, wherein the liposome of step (a) comprises a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

6. The method of anyone of claims 1-5, wherein the liposome of step (a) comprises at least 60 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms.

7. The method of claim 6, wherein the liposome of step (a) comprises at least 80 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms.

8. The method of claim 6, wherein the liposome of step (a) comprises at least 90 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms.

9. The method of one of claims 4-8, wherein the phospholipid is DPPC.

10. The method of anyone claims 1-9, wherein the drug is an antineoplastic drug.

11. The method of any of claims 1-10 wherein the liposome of step (a) contains essentially no cholesterol.

12 The method of any of claims 1-10 wherein the liposome of step (a) contains about 1 or less mol % cholesterol.

13. The method of any of claims 1-12 wherein the liposome of step (a) comprises a hydrophilic polymer-conjugated lipid.

14. The method of claim 13 wherein the hydrophilic polymer-conjugated lipid is a PEG-lipid

15. The method of claim 14, wherein PEG in the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

16. The method of claim 14, wherein PEG in the PEG-lipid has a molecular weight from about 1000 to about 5000 daltons.

17. The method of anyone of claims 1-16, wherein the liposome of step (a) comprises from about 5 to about 10 mol % PEG-lipid.

18. The method of any of claims 1-17 wherein the liposome of step (a) further comprises one or more phospholipids selected from the goup consisting of: PC, PE, PA and PI.

19. A liposome designed according to the method of any of claims 1 to 18.

20. A liposome designed according to the method of any of claims 1 to 18, wherein the liposome comprises:
(a) at least 60 mol % of a phospholipid;
(b) from about 1 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mol % of one or more vesicle-forming lipids.

21. A method for designing an improved liposome composition, said method comprising the steps of:
(a) preparing a liposome containing substantially no cholesterol;
(b) preparing a liposome containing substantially the same lipids and in the same proportions as the liposome in (a) with at least 20 mol % cholesterol;
(c) encapsulating the drug into the liposomes of (a) and (b);
(d) administering the liposomes of (a) and (b) after encapsulation of the drug to the bloodstream of a separate non-human mammal;
(e) determining drug:lipid ratios in the blood of the mammals at least one fixed time subsequent to administration; and (f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and (g) identifying a liposome of step (a) having a drug:lipid ratio at a fixed time point in a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said fixed time point of a liposome of step (b) containing substantially the same lipids and in the same proportions as the liposome in step (a) with at least 20 mol % cholesterol.

22. The method of claim 21, wherein the liposome of step (a) has a phase transition temperature Beater than that of the body of a subject to be treated, and less than 45°C.

23. The method of claim 21, wherein the liposome of step (a) has a phase transition temperature of between 39°C and 41°C.

24. The method of claim 21, 22 or 23, wherein the liposome of step (a) comprises (a) at least 60 mol % of a phospholipid;
(b) from about 1 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mol % of one or more vesicle-forming lipids.

25. The method of anyone of claims 21-24, wherein the liposome of step (a) comprises a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

26. The method of anyone of claims 21-25, wherein the liposome of step (a) comprises at least 60 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

27. The method of claim 26, wherein the liposome of step (a) comprises at least 80 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different;
wherein at least one of said acyl chains has 16 carbon atoms

28. The method of claim 26, wherein the liposome of step (a) comprises at least 90 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

29. The method of any of claims 24-28, wherein the phospholipid is DPPC.

30. The method of anyone of claims 21-29, wherein the drug is an antineoplastic drug.

31. The method of anyone of claims 21-30, wherein the liposome of step (a) contains essentially no cholesterol.

32. The method of anyone of claims 21-30, wherein the liposome of step (a) contains about 1 or less mol % cholesterol.

33. The method of anyone of claims 21-32, wherein the liposome of step (a) comprises a hydrophilic polymer-conjugated lipid.

34. The method of claim 33, wherein the hydrophilic polymer-conjugated lipid is a PEG-lipid

35. The method of claim 34, wherein PEG in the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

36. The method of claim 34, wherein PEG in the PEG-lipid has a molecular weight from about 1000 to about 5000 daltons.

37. The method of anyone of claims 34-36, wherein the liposome of step (a) comprises from about 5 to about 10 mol % PEG-lipid.

38. The method of anyone of claims 21-37, wherein the liposome of step (a) further comprises one or more phospholipids selected from the group consisting of PC, PE, PA and PI.

39. A liposome designed according to the method of any one of claims 21-38.

40. A liposome designed according to the method of any one of claims 21-38, wherein the liposome comprises:
(a) at least 60 mol % of a phospholipid;
(b) from about 1 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mol % of one or more vesicle-forming lipids.

41. A liposome comprising:
(a) at least 60 mol % of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms;
(b) from about 1 to about 15 mol % hydrophilic polymer-conjugated lipid; and (c) up to about 38 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol;
wherein the liposome, when encapsulating a drug, displays a comparable or greater drug:lipid ratio at a fixed time point upon administration to a mammal than a liposome containing substantially the same lipids and in the same proportions, but with at least 20 mol % cholesterol.

42. The liposome of claim 41, wherein the liposome has a phase transition temperature greater than that of the body of a subject to be treated, and less than 45°C.

43. The liposome of claim 41, wherein the liposome has a phase transition temperature of between 39°C and 41°C.

44. The liposome of claim 41, 42 or 43, wherein the liposome comprises a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

45. The liposome of claim 41, 42 or 43, wherein the liposome comprises at least 60 mol% of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

46. The liposome of claim 45, wherein the liposome comprises at least 80 mol%
of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

47. The liposome of claim 45, wherein the liposome comprises at least 90 mol%
of a phospholipid comprising two saturated fatty acids, the acyl chain of each being the same or different, wherein at least one of said acyl chains has 16 carbon atoms

48. The liposome of anyone of claims 44-47, wherein the phospholipid is DPPC.

49. The liposome of anyone of claims 44-48, further comprising a drug encapsulated therein.

50. The liposome of claim 49, wherein the drug is an antineoplastic drug.

51. The liposome of any of claims 41 to 50, wherein said liposome contains essentially no cholesterol.

52. The liposome of any of claims 41 to 50, wherein said liposome contains about 1 or less mol % cholesterol.

53. The liposome of any of claims 41 to 52, wherein said liposome comprises a hydrophilic polymer-conjugated lipid.

54. The liposome of claim 53 wherein the hydrophilic polymer-conjugated lipid is a PEG-lipid

55. The liposome of claim 54, wherein PEG in the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

56. The liposome of claim 54, wherein PEG in the PEG-lipid has a molecular weight from about 1000 to about 5000 daltons.

57. The liposome of anyone of claims 54-56, comprising from about 5 to about 10 mol %
PEG-lipid.

58. The liposome of any of claims 41 to 57, wherein said liposome comprises one or more phospholipids selected from the group consisting of: PC, PE, PA and PI.