CA2361946A1

CA2361946A1 - Delayed drug release using thermosensitive liposome

Info

Publication number: CA2361946A1
Application number: CA 2361946
Authority: CA
Inventors: Paul Tardi; Lawrence Mayer; Marcel Bally; Ludger Ickenstein
Original assignee: CELATOR TECHNOLOGIES Inc
Current assignee: Individual
Priority date: 2001-11-13
Filing date: 2001-11-13
Publication date: 2003-05-13

Description

DELAYED DRUG RELEASE USING THERMOSENSITIVE LIPOSOME
TPChnical FiPlri S This invention is directed toward methods of delivering a therapeutic agent to a target tissue in a mammal using long-circulating thermosensitive liposomes combined with a mild hyperthermic treatment.
Background of the Invention The successful treatment of breast tumors, head and neck tumors, prostate tumors and other deep seated tumors (malignant or benign) within the human body is a difficult task. The main objective of the treatment is to reduce in size or completely remove the tumor mass by one or more modalities available at the treatment facility. The most common modalities are surgery, radiation therapy and chemotherapy. Surgical treatment of breast cancer often involves substantial disfigurement, and surgery for other deep seated cancers often creates complications for surrounding vital organs and healthy tissue. Radiation therapy of deep seated tumors also puts surrounding healthy tissues at risk.
It has long been established that incorporation of membrane rigidification 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 the liposome. Conventional approaches to liposome formulation dictate inclusion of substantial amounts (e.g. 30-45 mol %) 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 Yatvin et al., Science 202:1290 (1978); 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, the efficacy of liposomes targeted to diseased sites by hyperthermia depends on the stability of the liposome in the blood stream (when liposomes are administered to the circulatory system), and on the amount of active agent released by the liposome at the target site. For example, liposomes described by Yatvin et al, Science 202:1290 ( 1978) released only a portion of the drug carried by the liposome at hyperthermic temperatures.
While the use of thermosensitive liposomes in hyperthermia therapy is promising, the tissue targeting effect depends upon how stably the liposomes administered circulate through the circulatory system at the normal body temperature and how much the liposomes release the drug at the site of tumor at the temperature of hyperthermia. The thermosensitive liposomes reported so far have problems with respect to the stability and the release by heating, and may not be expected to show their full effect.
For example, the liposomes described in Science, 202, 1290 (1978) release only a small amount of the drug at the temperature of hyperthermia, and the liposomes described in J. Urol., 135, 162 (1986) release a certain amount of the drug already at a temperature too low for effective treatment conditions. Thus the liposomes prepared by the conventional methods have problems to be solved with respect to release by heating and stability.
Other liposomes, particularly liposomes containing a substantial amount of cholesterol, require an elevated temperature (generally above 45° C) to release the drug, which presents disadvantages in that collateral tissue damage may occur. 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 sufficiently 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. (1) & (2) Biochimica et Biophysica Acta (1990)1029:91-97(1) & (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. As a result, currently available liposomes either suffer from low stability and low drug retention rates, or are not able to release their contents at the 'mildly' hyperthermic temperatures needed to be useful in human therapy.
Summary of Invention The present inventors provide a method which allows improved delivery of an active agent to a tissue of interest in a mammal. Provided is a method of delivering an agent to a site of interest in a subject by administering thermosensitive liposomes containing an active agent to a subject, allowing an extended time period for liposome localization to the site of interest, and subsequently administering a hyperthermic treatment at the site of interest to cause the release of the liposome contents. The period between the administration of liposomes and the administration of a hyperthermic treatment is preferably at least about 4 hours, but may be at least about 8, 12, 24 or 48 hours.
The 'delaying' of the hyperthermia treatment which allows improved tissue localization is enabled by the discovery of means for preparing thermosensitive liposomes having phase transition temperatures at 'mildly' hyperthermic temperatures, extended circulation longevity and improved drug retention. The inventors provide liposomes prepared. in the absence of cholesterol that can be made to behave comparably to cholesterol-containing liposomes through the incorporation of a hydrophilic polymer conjugated lipid. These results are contrary to the previous wisdom concerning liposomes prepared without cholesterol. The methods set forth below are based on the finding that liposomes having phase transition temperatures useful for thermosensitive applications display drug retention and circulation longevities that are comparable or better than cholesterol containing liposomes in mouse models of disease if the temperatures of the mice are maintained below the phase transition temperature of the liposomes.
Results of this observation are set forth in Example 3.
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.
Further, it has been discovered that the hydrophilic polymer stabilization effects due to use of PEG-modified lipid incorporation are, surprisingly, not substantially dependent on the concentration of the polymer (concentrations as low as 0.5 mol% PEG-2000 can cause a greater than 15-fold increase in Area-Under-the Curve (AUC) when compared to the same liposome prepared without the PEG-lipid) or polymer molecular weight (PEG 350 at concentrations of 5 mol% can cause a greater than 25 fold improvement in AUC when compared to the same liposome prepared without the PEG lipid and this increase in AUC is comparable to the greater than 38-fold improvement observed when using 5 mol% PEG 2000). The resulting liposomes exhibit much enhanced longevity of the liposomes while in blood circulation.
These results are contrary to the previous wisdom concerning the incorporation of PEG-lipids into a liposome.
The methods disclosed herein allow the controlled release of an active agent from a liposome upon administration of hyperthermia. This controlled release has heretofore not been possible with liposomes having long circulation times and/or sufficient drug retention. The treatment method allows both higher levels of drug to be administered, due to reduced drug toxicity in liposomes, and greater drug efficacy, due to selective liposome localization in a target tissue, preferably in the intercellular fluid of a tumor. The methods will allow comparable or better toxicity and tissue localization than methods using liposomes containing substantially the same lipids and in the same ratio but containing at least 20mo1% cholesterol, which do not have the thermosensitivity characteristics of the present liposomes.
The invention therefore discloses a method of administering an agent to a mammalian subject, comprising:
(a) administering to the bloodstream of a subject thermosensitive liposomes comprising a therapeutic agent, said liposomes having a phase transition temperature greater than that of the body of the subject to be treated, and less than 45°C;
(b) administering a hyperthermic treatment to a localized site on the body of the subject a mild hyperthermic treatment at a time point at least 4 hours after administration of the liposomes of step (a); and (c) said hyperthermic treatment delivered in an amount and for a time sufficient to cause the release of enapsulated therapeutic agent from the administered liposomes.
The invention provides improved treatment methods by allowing the administered liposomes to circulate in the bloodstream of the subject until a desired biodistribution of the liposomes is achieved. An agent of interest such as a therapeutic or diagnostic agent can thereby be exposed to a target surface or target tissue in the subject mammal, Also provided is a method of preparing an agent for localization in a target tissue by extravasation of liposomes containing the agent into the target tissue, when the agent is administered by intravenous injection, comprising:
entrapping the agent in liposomes which are characterized by:
(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 mol % of one or more vesicle-forming lipids;
wherein the liposome displays a drug:lipid ratio in the bloodstream at a time point at least 4 hours after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.
In another aspect, the invention discloses a method of localizing a therapeutic agent in a target tissue in a subject by extravasation of liposomes containing the therapeutic agent into the target tissue comprising:
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids;
(d) having a therapeutic agent encapsulated therein; and injecting the composition intravenously in the subject in an amount effective to localize a therapeutically effective quantity of the therapeutic agent in the target tissue. This method may achieve localization of the liposomes in the target tissue, 4 hours after intravenous administration, that is comparable or better than that of liposomes containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.
In a further aspect, the invention provides a method of administering a therapeutic agent to a target tissue in a subject, comprising:
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids; and (d) having a therapeutic agent encapsulated therein injecting the composition intravenously to the subject in an amount effective to localize a therapeutically effective quantity of the agent in the target tissue; and administering hyperthermia to the target tissue at least 4 hours after injection the composition intravenously to the subject in a dose sufficient to cause release of therapeutic agent contained in liposomes.
Another aspect of the invention relates to a method of administering a therapeutic agent to a target tissue in a subject, comprising:
selecting a subject suffering from a neoplastic disease;
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids; and (d) having a therapeutic agent encapsulated therein injecting the composition intravenously to the subject in an amount effective to localize a therapeutically effective quantity of the agent at a neoplastic lesion; and administering hyperthermia to a neoplastic lesion at least 4 hours after injection the composition intravenously to the subject in a dose sufficient to cause release of therapeutic agent contained in liposomes.
In any of the methods of the invention, the liposome administered to the subject will most preferably contain substantially no cholesterol, or will contain less than 1 mol % cholesterol, or more preferably essentially no cholesterol. As discussed herein, the absence of cholesterol will generally provide optimal phase transition at temperatures which are 'mildly thermosensitive' resulting in optimal release of encapsulated agent from liposomes.
Furthermore, the liposome for use in the methods of the invention preferably comprise at least 60, 70, 80 or 90 mol % of a phospholipid comprising two saturated fatty acids. Most preferably, the liposomes comprise : (a) at least 60, 70, 80 or 90 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 mol % of one or more vesicle-forming lipids. A

particularly useful phospholipid is dipalmitoylphosphatidylcholine (DPPC). The hydrophilic polymer-conjugated lipid is preferably a PEG lipid.
As further described herein, the agent encapsulated in the liposome may be any suitable agent, including preferably a therapeutic agent (drug) or a diagnostic agent.
According to the methods of the invention, the period of time between liposome administration and administration of the hyperthermic treatment will generally be sufficient such that increased accumulation of therapeutic agent in a target tissue (for example a tumor) is achieved when compared with free therapeutic agent (e.g. delivered in a non-liposomal formulation). The hyperthermic treatment is preferably administered at least 4, 8, 12, 24 or 48 hours after administration of the liposomes of step (a). The hyperthermic treatment is preferably delivered locally at a tumor site, at a site of inflammation or at a site of infection. The hyperthermic treatment preferably comprises administering to a tissue of interest a temperature greater than that of the body of a subject to be treated but less than 45°C, and more preferably a temperature is between 39° C and 42° C.
According to preferred methods of the invention, the liposomes for use in accordance with the methods of the invention display: (i) a circulation longevity at a fixed time point after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said fixed time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol; and/or (ii) when encapsulating a drug, a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol. The fixed time point is preferably at least 4, 8 ,12, 18, 24, 36 or 48 hours after liposome administration.
As discussed, while any suitable liposome composition in accordance with the methods of the invention can be used, the inventors provide particularly preferred liposome compositions.
The invention thus encompasses a composition 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 mol % 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 and/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.
Preferably, in accordance with the invention, the fixed time point is a time point at least about 4, 8, 12, 18, 24, 36 or 48 hours after administration of the liposomes to the subject. It will be appreciated that said time point may also be a time point greater than about 4, 8, 12, 18, 24, 36 or 48 hours after administration of the liposomes to the subject.
The liposome will preferably contain substantially no cholesterol, or may optionally contain less than 1 mol % cholesterol or may contain essentially 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 42° C.
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.
It will be appreciated that any suitable method for determining 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. A
drug: lipid ratio or retention time which is deemed 'comparable' will depend on the circumstances, but is preferably at least 60 %, 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 and/or free of PEG. The 'time point' is generally a number as measured in hours, minutes, etc.
Liposomes for use in the present methods may be prepared using known and conventional techniques. Selection or design of liposomes having the desired circulation longevity and drug retention characteristics can be obtained according to methods described herein. 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 as well as the techniques presented in Examples 1 to 5. 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.
1 S 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 5, 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 DSPE): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol 2000]; DSPE-PEG 750 (or 750PEG-DSPE or PEG750-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]; 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.
As used herein, the term "hyperthermia" refers to the elevation of the temperature of a subject's body, or a part of a subject's body, compared to the normal temperature of the subject.
Such elevation may be the result of a natural process (such as inflammation) or artificially induced for therapeutic or diagnostic purposes.
In mammals, a normal body temperature is ordinarily maintained due to the thermoregulatory center in the anterior hypothalamus, which acts to balance heat production by body tissues with heat loss. "Hyperthermia" refers to the elevation of body temperature above the hypothalamic set point due to insufficient heat dissipation. In contrast to hyperthermia, "fever"
refers to an elevation of body temperature due to a change in the thermoregulatory center. The overall mean oral temperature for a healthy human aged 18-40 years is 36.8.+-Ø4°C. (98.2.+-Ø7°F.). See, e.g., Harrison's Principles of Internal Medicine (Fauci et al., Eds.) 14th Edition, McGraw-Hill, New York, p. 84 (1998).
As used herein, "hyperthermic administration" of an active agent refers to its administration in conjunction with the use of clinical hyperthermia in the subject at a preselected target site, to deliver a larger amount of active agent to the target site compared to that which would result from the administration of the active agent in the absence of hyperthermia.
As used herein, "solid tumors" are those growing in an anatomical site other than the bloodstream (in contrast to blood-borne tumors such as leukemias). Solid tumors require the formation of small blood vessels and capillaries to nourish the growing tumor tissue.
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 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 liposomes are described in copending international patent application 10 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, sulfate, amino or sulfhydryl 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 lipid covalently joined at its polar head moiety to a hydrophilic polymer, and is typically made from a lipid that has a reactive functional group at the polar head moiety in order to attach the polymer.
Suitable reactive functional groups are for example, amino, hydroxyl, carboxyl or formyl. The lipid may be any lipid described in the art for use in such conjugates 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, polylactie acid, polyglycolic acid, polyacrylic acid and copolymers thereof, as well as those disclosed in United States Patents 5,013,556 and 5,395,619, the disclosures of which are incorporated herein by reference. 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. Regarding liposomes containing polymer grafted lipids in the vesicle membrane, 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, the disclosures of all of the references being incorporated herein by reference in their entireties. 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 between 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 of caproyl (6:0), octanoyl (8:0), capryl (10:0), lauroyl (12:0), myristoyl (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 (C16) 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), the disclosures of which are incorporated herein by reference.
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.
The liposomes may comprise amphipathic lipids in addition to those described above, but preferably 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 sources or 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), the disclosures of which are incorporated herein by reference.
As shown in Example 1, 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.
Advantageous methods of designing and preparing thermosensitive and/or substantially cholesterol free liposomes having improved circulation longevity and/or drug retention characteristics are described in the copending Application No. [] titled "Improved cholesterol-free liposomes" filed on the same day as the present application and the disclosure of which is incorporated herein by reference.
In summary, this method comprises:
(i) comparing a drug retention property 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 comparable to or improved over that of the substantially equivalent cholesterol-containing liposome of step (b).

In preferred aspects, said method comprises 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.
The liposome of the steps (a) in the method of designing liposomes will typically comprise at least 60, 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, wherein at least one of said acyl chains has 16 carbon atoms. Most preferably, the liposome comprises DPPC, most preferably at least 30, 50, 60, 80 or 95 mol% DPPC. Said liposome may further comprises one or more phospholipids selected from the group consisting of: I, PA and PE. As in the liposomes of the invention, the hydrophilic polymer-conjugated lipid is preferably a PEG-lipid, preferably having a molecular weight from about 100 to about 5000 daltons, or from about 1000 to about 5000 daltons. The PEG lipid will be present in the liposome of step (a) from about 2 to about 10 mol % PEG-lipid.
Moreover, preferably the liposome of step (a) will contain substantially no cholesterol and/or 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, or more preferably between 39° C and 41 ° C.
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 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 180 nm 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. 80-140 nm liposomes 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 SOnm 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.
Active agents may be loaded into liposomes using passive and active loading methods described herein.
Passive methods of encapsulating active agents Therapeutic agents may be encapsulated using passive methods of encapsulation.
Passive methods of encapsulating active agent in liposomes involve encapsulating the agent during the synthesis of the liposomes. In this method, the active agent 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 agent 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 an 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 active agent.
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 therapeutic agent or other agent to be encapsulated.
Once the agent 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 active agent 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 active agent to cross the liposome membrane and conversion of the moiety to a charged form causes the active agent 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 active agents 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 US patent no.
5,939,096).
In other aspects, charge attraction methods may be utilized to actively load therapeutic agents. Charge attraction 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.
The liposomes will have 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 a mildly hyperthermic conditions, which are generally understood to be between that of the body of a subject to be treated and about 45°C, or more preferably between that of the body of a subject to be treated and about 43°C, or more preferably between that of the body of a subject to be treated and about 42°C.
The terms "drug", "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, active agent includes 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.
The anti-neoplastic agent which may be used is any compound, including the ones listed herein, which can be stably entrapped in liposomes at a suitable loading factor and administered at a therapeutically effective dose (indicated below in parentheses after each compound). These include amphipathic anti-tumor compounds such as the plant alkaloids vincristine (1.4 mg/m2), vinblastine (4-18 mg/m2) and etoposide (35-100 mg/m2), and the anthracycline antibiotics including doxorubicin (60-75 mg/m2), epirubicin (60-120 mg/m2) and daunorubicin (25-45 mg/m2). The water-soluble anti-metabolites such as methotrexate (3 mg/m2), cytosine arabinoside (100 mg/m2), and fluorouracil (10-15 mg/kg), the antibiotics such as bleomycin (10-units/m2), mitomycin (20 mg/m2), plicamycin (25-30 µg/2) and dactinomycin (15 µg/m2), and the alkylating agents including cyclophosphamide (3-25 mg/kg), thiotepa (0.3-0.4 mg/Kg) and BCNU (150-200 mg/m2) are also useful in this context. Preferred examples, the 15 plant alkaloids exemplified by vincristine and the anthracycline antibiotics including doxorubicin, daunorubicin and epirubicin are preferably actively loaded into liposomes, to achieve drug/lipid ratios which are several times greater than can be achieved with passive loading techniques. Also the liposomes may contain encapsulated tumor-therapeutic peptides and protein drugs, such as IL-2, and/or TNF, and/or immunomodulators, such as GM-CSF, which are present alone or in 20 combination with anti-neoplastic agents, such as an anthracycline antibiotic drug.
Other examples of agents that can be used according to the invention are shown in Table 1.

Table 1 CLASS TYPE OF AGENT 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, Wilms' 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, Methylmelamines 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 Malignant pancreatic insulinoma, (streptozotocin) malignant carcinoid Triazines Dacarbazine (DTIC;Malignant melanoma, Hodgkin's dimethyltriazenoimidazole-disease, soft-tissue sarcomas Antimetabolites Folic Methotrexate Acute lymphocytic Acid Analogs leukemia, (amethopterin) choriocarcinoma, mycosis fungoides, breast, head and neck, lung, osteogenic sarcoma Pyrimidine Analogs Fluouracil (5-fluorouracil;Breast, colon, stomach, pancreas, 5-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, Wilins' 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, C) colon, 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 Diethylstilbestrol Breast, prostate Ethinyl estradiol (other preparations available) Antiestrogen Tamoxifen Breast Androgens Testosterone propionate Breast Fluoxymesterone (other preparations available) Antiandrogen Flutamide Prostate Gonadotropin- Leuprolide Prostate releasing hormone analog 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.
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 of a subject (intravenous or "IV") or subcutaneous administration of liposomes. Where liposomes according to the present invention are used in conjunction with hyperthermia, the liposomes may be administered by any suitable means that results in delivery of the liposomes to the treatment site.
For example, liposomes may be administered intravenously and thereby brought to the site of a tumor by the normal blood flow; heating of this site results in the liposomal membranes being heated to the phase transition temperature so that the liposomal contents are preferentially released at the site of the tumor.
In accordance with the present invention, the anti-tumor or anti-neoplastic agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers. The resulting liposomal formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration. Tumors characterized by an acute increase in permeability of the vasculature in the region of tumor growth are particularly suited for treatment by the present methods. Administration of liposomes is followed by heating of the treatment site to a temperature that results in release of the liposomal contents.
Where site-specific treatment of inflammation is desired, effective liposome delivery of an active agent requires that the liposome have a long blood halflife, and be capable of penetrating the continuous endothelial cell layer and underlying basement membrane surrounding blood vessels adjacent to the site of inflammation. Liposomes of smaller sizes have been found to be more effective at extravasation through the endothelial cell barrier and into associated inflamed regions. See, e.g., U.S. Pat. No. 5,356,633 to Woodle et al. In accordance with the present invention, the anti-inflammatory agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers. The resulting liposomal formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration. Inflamed regions characterized by an acute increase in permeability of the vasculature in the region of inflammation, and by a localized increase in temperature, are particularly suited for treatment by the present methods.
It will further be appreciated that the liposomes of the present invention may be utilized to deliver of anti-infective agents to sites of infection, via the bloodstream.
The use of liposomes containing a vesicle-forming lipid derivatized with a hydrophilic polymer, and having sizes ranging between 0.07 and 0.2 microns, to deliver therapeutic agents to sites of infection is described in published PCT patent application WO 93/19738. In accordance with the present invention, the anti-infective agent of choice is entrapped within a liposome having a membrane according to the present invention, and the resulting liposomal formulation is administered parenterally to a subject, preferably by intravenous administration. If desired, localized hyperthermia may be induced at the site of infection to cause the preferential release of liposomal contents at that site.
Upon administration, the liposomes are allowed time to reach the site of disease.
Preferably, where liposome are administered to the bloodstream time is allowed for liposomes to extravasate into target tissues, e.g. tumors, inflamed tissues, infected tissues. The thermosensitive liposomes of the invention allow extended stability such that extended circulation times and drug retention are obtained. The invention thus comprises allowing an extended period of time before administering hyperthermic treatment to a tissue, which has heretofore not been possible.
The invention thus comprises administering a mild hyperthermic treatment to a subject at least about 4, 6, 8, 12, or 18 hours, or more preferably at least about 24, 36, or 48 hours following the administration of the liposomes to the subject. The hyperthermic treatment is preferably administered locally, such as to a disease site, so as to provide a localized release of active agent from liposomes.
Hyperthermia can be administered by any suitable method. Several methods and apparati are known and available. Preferably a minimally invasive RF, microwave, or ultrasound based hyperthermia delivery system is used to administer the hyperthermic treatment.
The hyperthermic treatment is thus preferably minimally invasive and targeted, capable for example of targeting large tumor masses or large-volume infected or arthritic tissue or other diseased tissue deep within the body. The hyperthermia delivery system thus produces heat which activates thermosensitive liposomes and releases drugs in targeted tissue in accordance with the invention. Adaptive phased array systems are available, further described in U.S. Patent No.
5,510,888, Fenn, et al, Int J Hyperthermia 15 (1): 45-61 (1999); and Gavrilov et al, Int J
Hyperthermia 15(6): 495-507 ( 1999), the disclosures of which are incorporated herein by reference, and are available from Celsion Corp (Maryland). Other hyperthermia delivery devices include a surface temperature controlled microwave ring radiator embedded in an contoured epoxy plaque base, which allowed circulation of water inside the plaque to cool the tissue surface and provide deeper heating field penetration for tumor treatment, described in Huang et al (Cancer Res. 54: 5135-5143 (1994) the disclosure of which is incorporated herein by reference).
Although minimally invasive systems which allow specific localization of heat at tissues deep within the body are preferred, other less sophisticated or surface based heating devices can also be used. Such systems are particularly useful for animal studies. For example, a Positive Temperature Coefficient Thermistor (PTC) heater, (Tokyo Denki Kagaku, Tokyo) can be used on the tumor surface (Katsumi et al, J. Pharm. Sciences 80(6): 522-525 (1991), incorporated herein by reference). Alternatively, for testing, an animal can be treated in a water bath such as used to heat implanted leg or flank tumors. In another example, a gas can be used as a hyperthermic potentiator by incorporating it in a liposome for use with ultrasound imaging devices. Devices specially designed for administering ultrasonic hyperthermia are described U.S. Pat. Nos.
4,620,546, 4,658,828, and 4,586,512, the disclosures of which are incorporated herein by reference.
Hyperthermia delivery apparati can thus be used to heat a target site where release of liposome contents is to be stimulated. For the treatment of solid tumors, for example, an apparatus is used to administer hyperthermia to the solid tumor, thereby causing the release of drug at or near the tumor site. Similarly, for the treatment of inflammatory and infectious disease, hyperthermia will be focused on one or more sites of inflammation or infection. Hyperthermia dosage and intensity required to release liposome contents can be readily determined by the skilled person. Commercially available apparati permit for example the targeting of tumors in the liver, breast, rectum, cervix, pancreas, lung, and other areas deep within the torso. Hyperthermia can also be applied to the target site prior to administration of the therapeutic agent, followed by a second application of heat after at least 4 hours post administration.
Assessment of hyperthermia treatment The treatment methods of the invention can readily be compared to conventional methods. Commonly used studies examine tumor drug uptake and tumor growth delay. For each of tumor drug uptake and tumor growth delay, two ratios can be calculated:
1 ) (HT + Lip)/(Lip), comparing the endpoint reached when treated with thermosensitive liposomes combined with hyperthermia treatment (HT + Lip) to liposomes without hyperthermia (Lip); and 2) (HT + Lip)/(HT + drug), comparing the endpoint reached when treated with hyperthermia and thermosensitive liposomes to that of hyperthermia and free drug.
For drug uptake studies, the endpoint used to calculate the therapeutic ratios are the tumor drug levels achieved at the end of a study. The endpoint used to calculate the therapeutic ratios for the tumor growth delay studies can be either the number of days to reach a predetermined tumor volume or the tumor volumes reached at the end of the study. For tumor growth delay studies, liposomes can be administered to a mouse via tail vein, and flank or leg tumors are heated in a water bath. Tumor volumes can be followed over time until a certain tumor size is attained or time point is reached (Maekawa et al, Cancer Treatment Reports 71:1053-1059 (1987); and Nishimura et al, Radiation Res. 122:161-167 (1990), the disclosures of which are incorporated herein by reference).
Tumor models are well known in the art, and any suitable tumor type of test animal can be used. Preferably mice are used. Commonly used tumors include C-26 colon carcinoma, J-6456 lymphoma, B16F10 melanoma, Meth A fibrosarcoma, RIF-1, Walker carcinosarcoma 256 in liver and rous sarcoma virus induced glioma.
Assessment of circulation longevity The liposomes according to the invention result in enhanced longevity (circulation time).
Preferably, a liposome or lipid carrier of this invention will be made such that the lipid dose remaining in the bloodstream of an animal at one or more selected time points at least 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 and retention of drug in the bloodstream as shown in Figure 1.
This observation is contrary to conventional wisdom concerning the ability of thermosensitive liposomes to display extended circulation longevity and drug retention (see the review article:
Dewirst et al. (1999) Int. J. Hyperthermia 15(S): 345-370). This increased stability was due to the maintenance of the body temperature of the mice below the Tm of the liposomes.
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, which is comparable to or better than the circulation longevity in a mammal of a 'reference' liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol. These cholesterol-containing 'reference' 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.
For liposome circulation longevity, the data obtained from a model animal system can be reasonably extrapolated to humans and veterinary animals of interest.
Liposomes 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.
Assessment of drug retention: drug: lipid ratio The ability of the liposomes to retain an amphipathic anti-tumor drug while circulating in the bloodstream over the extended periods after administration are also an important factor for liposomes which are to reach and enter a site such as a distant solid tumor.
An extended period can be for example at least about 4, 6, 12, 18, 24, 36 or 48 hour period, that is the time allowed in the present method between liposome administration and stimulated release of a therapeutic agent. For example, a therapeutic agent can be encapsulated into DPPC-PEG
liposomes of the invention, and the drug:lipid ratio can be determined at one or more specified time points after administration of the liposomes to a mammal, preferably a mouse. Blood is removed from the mammal at fixed time intervals such as 1, 2, 3, 4, 8, 12, 18, 24 or 48 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 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.
A liposome in accordance with the invention preferably displays a drug:lipid ratio at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.
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 [3H]-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 C02 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. [3H]- and [14C]-CHE labels are available from NEN/Dupont.
It will be appreciated however that any 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 in ex vivo conditions. As used in the context of determining a drug:lipid ratio, the term drug refers generally to the active agent encapsulated in the liposome administered.
Assessment of extravasatioh into Tumors As mentioned, the high stability/high drug retention liposomes of the invention allow methods of treatment which maximize benefit from liposome extravasation into a site of disease, e.g. tumor, infectious disease or inflammation. Liposomes can extravasate through the endothelial cell barrier and underlying basement membrane separating a capillary from for example the tumor cells supplied by the capillary. This feature is optimized in liposomes with sizes between about 50nm and 200nm in diameter, although liposomes with smaller size are also expected to extravasate. However, in the examples provided herein, liposomes have sizes of about 80 nm to 140nm are expected to allow a su~ciently large drug-carrying capacity.
Previous studies with cholesterol-containing non-thermosensitive liposomes have indicated that delivery to the tumor offers advantages in drug targeting (see for example U.S.
Patent No. 5,510,888). A study which can be used to demonstrate the ability of preferred liposomes to reach a target site involves inoculating mice subcutaneously with the J-6456 lymphoma, which forms a solid tumor mass of about 1 cm3 after one to two weeks. The animals are then injected either with free drug or drug loaded into the liposomes of the invention, preferably substantially cholesterol-free PEG-liposomes. The tissue distribution (heart, muscle, and tumor) of the drug can then assayed at 4, 8, 12, 18, 24, 36 and/or 48 hours after drug administration, as shown in Example 4 or according to conventional techniques known in the art.
Drug levels accumulated in a target tissue such as a tumor can thereby be compared to drug levels in other tissues, and can serve to demonstrate that the liposomes of the invention result in drug accumulating at higher levels in the tumor site as compared to heart, muscle, liver, etc. than free drug or drug encapsulated in liposomes containing substantially the same lipids and in the same proportions as the thermosensitive liposomes of the invention but comprising at least 20mo1%
cholesterol.
Preferably the liposomes of the invention show increased drug accumulation into the target tissue (e.g. tumor, site of inflammation or site of infection).
Preferably, the target tissue contains at least 2, 4, 8, 10, 20, 50 or 100 times more drug compared with healthy muscle and at least 2, 4, 6, 8, 10, 20 or 100 times the amount in heart following administration of the liposome of the invention (for example, at 12, 18, 24 or 48 hours post administration).
To demonstrate that increase in drug accumulation are due to the entry of intact liposomes into the extravascular region of a tumor, tumor tissue can be separated into cellular and supernatant (intercellular fluid) fractions, and the presence of liposome-associated and free drug in both fractions can be determined. To assay liposome-associated drug, the supernatant is passed through a gel filtration column to remove free drug (Gabizon, A et al, (1989) J. Natl. Cancer Inst.
81, 1484-1488), and the drug remaining in the supernatant is assayed. Further demonstration of liposome extravasation into tumor cells can be obtained by direct microscopic observation of liposome distribution in normal liver tissue and in solid tumors, at a time point, e.g. 12, 18, 24, 48 hours, after IV injection of liposomes of the invention.
Assessment of tumor Localization and treatment e~cacy The liposomes of the invention provide means for achieving improved localization of an anti-tumor, anti-inflammatory or anti-infective agents specifically in a target tissue or region by virtue of the extended lifetime of the liposomes in the bloodstream and a liposome size which allows both extravasation into tumors, a relatively high drug carrying capacity and minimal leakage of the entrapped drug during the time required for the liposomes to distribute to and enter the tumor (the first 24-48 hours following injection). In a preferred embodiment, the liposomes thus provide an effective method for localizing a compound selectively to a solid tumor, by entrapping the compound in such liposomes and injecting the liposomes IV into a subject. In this case, for an IV injected liposome (and its entrapped anti-tumor drug) to reach the tumor site it must leave the bloodstream and enter the tumor. In one embodiment, the method is used for tumor treatment by localizing an anti-tumor drug selectively in the tumor. The anti-tumor drug which may be used is any compound, including the ones listed herein, which can be stably entrapped in liposomes at a suitable loading factor and administered at a therapeutically effective dose (indicated below in parentheses after each compound).
Studies to compare treatment efficacy of delayed release methods using the liposomes of the invention to conventional methods can be carried out in animal models as described above.
Alternatively, studies may also measure endpoints such as tumor growth or survival. For example, in one comparison animals can be treated with free drug or drug entrapped in liposomes of the invention. In another comparison, animals can be treated with drug entrapped in liposomes of the invention with the delivery of hyperthermia immediately after liposome administration (e.g.
5, 10, 30 minutes post administration), or at extended times as in the "delayed-release" methods of the invention (e.g. at least 4, 8, 12, 18, 24, 36 or 48 hours post administration). The study may assess for example percent survivors over a period of a certain number of days (for example a 100-day period) or tumor growth delay following tumor implantation.
Since reduced toxicity has been observed in model animal systems and in a clinical setting in tumor treatment by doxorubicin entrapped in conventional liposomes (as reported, for example, in U.S. Pat. No. 4,797,285), studies can be carried out to determined the degree of toxicity protection provided in the treatment method of the present invention.
In one example, animals are injected intravenously with increasing doses of a drug in free form or entrapped in conventional or thermosensitive liposomes of the invention, and the maximum tolerated dose (MTD) for the various drug formulations is determined. Preferably, entrapment in liposomes of the invention provides an MTD for the drug which is comparable or better than that seen in liposomes containing substantially the same lipids and in the same proportions but containing at least 20mo1% cholesterol. Preferably, entrapment in liposomes of the invention provides an MTD
for the drug which is at least 2, 4, 10, 20 or 100 times better than with free drug.
It will be appreciated that the ability to localize a compound selectively in a tumor, by liposome extravasation, can also be exploited for improved targeting of an imaging agent to a tumor, such as for tumor diagnosis. Here the imaging agent, typically a radioisotope in chelated form, or a paramagnetic molecule, is entrapped in liposomes, which are then administered IV to the subject being examined. After a selected period, typically 12 to 24 or 24 to 48 hours, the subject is then monitored, for example by gamma scintillation radiography in the case of the radioisotope, or by nuclear magnetic resonance (NMR) in the case of the paramagnetic agent, to detect regions of local uptake of the imaging agent.
Also, as noted above, it is anticipated that long circulating thermosensitive liposomes would be useful for delivery of anti-infective drugs to regions of infections.
Sites of infection, like tumors, often exhibit compromised leaky endothelial barriers--as evidenced by the fact that edema (fluid uptake from the bloodstream) is quite often found at these sites.
It is expected that liposomes containing antibiotics (such as aminoglycosides, cephalosporins, and beta lactams) would improve drug localization at sites of infection, thereby improving the therapeutic index of such agents--particularly ones which exhibit dose-related toxicities, such as the aminoglycosides.

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 of 3H-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 moles 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-PEG2000 (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-PEGZ000 (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 1 h 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-PEG2000 (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-PEG2000 (90:4 mol%) liposomes via the tail vein. Mice were terminated after l Omin 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 H2S04. 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, ~,em: 550 nm).
Doxorubicin-containing DPPC:DSPE-PEG2000 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-PEG2000 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 the hyperthermia 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 after hyperthermia, blood samples are quantitated for levels of doxorubicin and lipid as in Example 3.
Example 5 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 3rd and 20th 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 of administering a therapeutic agent to a mammalian subject, comprising:
(a) administering to the bloodstream of a subject thermosensitive liposomes comprising a therapeutic agent, said liposomes having a phase transition temperature greater than that of the body of the subject to be treated, and less than 45°C;
(b) administering a hyperthermic treatment to a localized site on the body of the subject a hyperthermic treatment at a time point at least 4 hours after administration of the liposomes of step (a); and (c) said hyperthermic treatment delivered in an amount and for a time sufficient to cause the release of enapsulated therapeutic agent from the administered liposomes.

2. The method of claim 1, wherein said hyperthermic treatment comprises administering to a site on the body of the mammalian subject a temperature greater than that of the body of a subject to be treated but less than 45°C.

3. The method of claim 2, wherein the temperature is between 39° C and 41 ° C.

4. The method of claim 1, wherein the liposome administered to the subject contains substantially no cholesterol.

5. The method of claim 1, wherein the liposome administered to the subject comprises at least 60 mol % of a phospholipid.

6. The method of claim 1, wherein the liposome administered to the subject comprises:
(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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol.

7. The method of claim 6, wherein the liposome administered to the subject comprises at least 70 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.

8. The method of claim 6, wherein the liposome administered to the subject comprises at least 80 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.

9. The method of claims 6, 7 or 8, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

10. The method of claim 6, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

11. The method of claim 6, wherein the liposome contains less than 1 mol %
cholesterol.

12. The method of claim 10, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

13. The method of any of claims 1 to 8, wherein the hyperthermic treatment is administered at least 8 hours after administration of the liposomes of step (a).

14. The method of any of claims 1 to 8, wherein the hyperthermic treatment is administered at least 12 hours after administration of the liposomes of step (a).

15. The method of any of claims 1 to 8, wherein the hyperthermic treatment is administered at least 24 hours after administration of the liposomes of step (a).

16. The method of any of claims 1 to 8, wherein the hyperthermic treatment is delivered locally at a tumor site.

17. The method of claim 1, wherein the liposome, when encapsulating a drug, displays a circulation longevity at a fixed time point after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said fixed time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

18. The method of claim 1 or 17, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

19. A liposome composition for use in localizing a compound in a target tissue via the bloodstream, by liposome extravasation into the target tissue, 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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and wherein the liposome, when encapsulating a drug, displays a circulation longevity at a time point at least 4 hours after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.

20. The liposome of claim 19, wherein the liposome, when encapsulating a drug, displays a circulation longevity at a time point at least 8 hours after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

21. The liposome of claim 19, wherein the liposome, when encapsulating a drug, displays a circulation longevity at a time point at least 12 hours after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

22. The liposome of claim 19, wherein the liposome, when encapsulating a drug, displays a circulation longevity at a time point at least 24 hours after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

23. The liposome of any of claims 19 to 22, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

24. The liposome of claim 19, wherein said liposome has a phase transition temperature greater than that of the body of the subject to be treated, and less than 45°C.

25. The liposome of claim 19, wherein said liposome has a phase transition temperature between 39° C and 41 ° C..

26. The liposome of claim 19, wherein the liposome administered to the subject comprises at least 70 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.

27. The liposome of claim 19, wherein the liposome administered to the subject comprises at least 80 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.

28. The liposome of claim 19, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

29. The liposome of any of claims 19 and 24 to 27, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

30. The liposome of any of claims 19 and 24 to 27, wherein the liposome contains less than 1 mol % cholesterol.

31. The liposome of claim 29, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

32. A liposome composition for use in localizing a compound in a target tissue via the bloodstream by liposome extravasation into the target tissue, 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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a time point at least 4 hours after administration to a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol cholesterol.

33. The liposome of claim 32, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a time point at least 8 hours after administration to a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

34. The liposome of claim 32, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a time point at least 12 hours after administration to a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

35. The liposome of claim 32, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a time point at least 24 hours after administration to a mammal is which is comparable to or better than the drug:lipid ratio in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

36. The method of claim 32, wherein said liposome has a phase transition temperature greater than that of the body of the subject to be treated, and less than 45°C.

37. The method of claim 36, wherein said liposome has a phase transition temperature between 39° C and 41 ° C.

38. The method of claim 32, wherein the liposome administered to the subject comprises at least 70 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.

39. The method of claim 32, wherein the liposome administered to the subject comprises at least 80 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.

40. The method of claim 32, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

41. The method of any of claims 32 to 40, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

42. The method of claim 32 to 40, wherein the liposome contains less than 1 mol cholesterol.

43. The method of claim 41, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons..

44. A method of preparing an agent for localization in a target tissue by extravasation of liposomes containing the agent into the target tissue, when the agent is administered by intravenous injection, comprising:

entrapping the agent in liposomes which are characterized by:

(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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and wherein the liposome displays a circulation longevity at a time point at least 4 hours after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

45. The method of claim 44, wherein the agent is a therapeutic agent.

46. The method of claim 44, wherein the agent is a diagnostic agent.

47. The method of claim 44, wherein the liposome administered to the subject comprises at least 70 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.

48. The method of claim 44, wherein the liposome administered to the subject comprises at least 80 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.

49. The method of claim 44, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

50. The method of any of claims 44 to 49, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

51. The method of any of claims 44 to 49, wherein the liposome contains less than 1 mol %
cholesterol.

52. The method of any of claims 50, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons..

53. A method of localizing a therapeutic agent in a target tissue in a subject by extravasation of liposomes containing the therapeutic agent into the target tissue comprising:

providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and (d) having a therapeutic agent encapsulated therein;

injecting the composition intravenously in the subject in an amount effective to localize a therapeutically effective quantity of the therapeutic agent in the target tissue, and by said injecting, achieving a localization of the liposomes in the target tissue, 4 hours after intravenous administration, that is comparable or better than that of liposomes containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.

54. The method of claim 53, wherein the liposome displays a circulation longevity at a fixed time point after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said fixed time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.

55. The method of claim 53 or 54, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

56. The method of claim 53, wherein the liposome administered to the subject comprises at least 70 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.

57. The method of claim 53, wherein the liposome administered to the subject comprises at least 80 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.

58. The method of claim 53, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

59. The method of any of claims 53 and 56 to 58, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

60. The method of any of claims 53 and 56 to 58, wherein the liposome contains less than 1 mol % cholesterol.

61. The method of claim 59, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

62. A method of administering a therapeutic agent to a target tissue in a subject, comprising:
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and (d) having a therapeutic agent encapsulated therein injecting the composition intravenously to the subject in an amount effective to localize a therapeutically effective quantity of the agent in the target tissue; and administering hyperthermia to the target tissue at least 4 hours after injection the composition intravenously to the subject in a dose sufficient to cause release of therapeutic agent contained in liposomes.

63. The method of claim 62, wherein the liposome displays a circulation longevity at a fixed time point after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said fixed time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.

64. The method of claim 62 or 63, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

65. The method of claim 62, wherein the liposome administered to the subject comprises at least 70 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.

66. The method of claim 62, wherein the liposome administered to the subject comprises at least 80 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.

67. The method of claim 62, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

68. The method of any of claims 62 and 65 to 67, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

69. The method of any of claims 62 and 65 to 67, wherein the liposome contains less than 1 mol % cholesterol.

70. The method of claim 68, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.

71. A method of administering a therapeutic agent to a target tissue in a subject, comprising:
selecting a subject suffering from a neoplastic disease;
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids, providing that the liposome contains substantially no cholesterol; and (d) having a therapeutic agent encapsulated therein injecting the composition intravenously to the subject in an amount effective to localize a therapeutically effective quantity of the agent at a neoplastic lesion; and administering hyperthermia to a neoplastic lesion at least 4 hours after injection the composition intravenously to the subject in a dose sufficient to cause release of therapeutic agent contained in liposomes.

72. The method of claim 71, wherein the liposome displays a circulation longevity at a fixed time point after administration to a mammal is which is comparable to or better than the circulation longevity in a mammal at said fixed time point of a liposome containing substantially the same lipids and in the same proportions but with at least 20 mol %
cholesterol.

73. The method of claim 71 or 72, wherein the liposome, when encapsulating a drug, displays a drug:lipid ratio in the bloodstream at a fixed time point after administration to 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 containing substantially the same lipids and in the same proportions but with at least 20 mol % cholesterol.

74. The method of claim 71, wherein the liposome administered to the subject comprises at least 70 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.

75. The method of claim 71, wherein the liposome administered to the subject comprises at least 80 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.

76. The method of claim 71, wherein said phospholipid is dipalmitoylphosphatidylcholine (DPPC).

77. The method of any of claims 71 and 74 to 76, wherein said hydrophilic polymer-conjugated lipid is a PEG lipid.

78. The method of any of claims 71 and 74 to 76, wherein the liposome contains less than 1 mol % cholesterol.

79. The method of claim 77, wherein the PEG-lipid has a molecular weight from about 100 to about 5000 daltons.