WO1999058111A1 - Methods of using lipid based drug delivery vehicles - Google Patents

Abstract

Methods and compositions for administering agents with lipid based carriers to a host are provided. In the subject methods, both a lipid composition including the agent and a lipid composition free of the agent are administered to the host, usually at different times. In many embodiments, the lipid composition is a liposome composition. The subject methods provide for one or more advantages in agent delivery, such as reduced agent dosage, reduced toxicity, more specific targeting and the like.

Description

METHODS OF USING LIPID BASED DRUG DELIVERY VEHICLES

CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 60/084,772 filed May 8, 1998, the disclosure of which is herein incorporated by reference.

INTRODUCTION Technical Field The field of this invention is pharmacology, particularly drug delivery and more particularly liposome mediated drug delivery. Background of the Invention

Liposomes are made of molecules with hydrophilic and hydrophobic ends that form spherical lipid bilayers, either unilamellar or multilamellar, enclosing a polar, usually aqueous medium. As such, hydrophilic agents can be entrapped in the interior of the medium, and hydrophobic agents can be entrapped in the lipid bilayer component. The preparation of liposomes with entrapped solutes was first demonstrated by A. D. Bangham in 1965. Bangham, J. Mol. Biol. (1965) 13: 238-252.

Liposomes have been extensively investigated as drug delivery vehicles for a variety of agents, including: small molecule drugs, proteins and nucleic acids. Liposomes are of interest as delivery vehicles for drugs in that they can provide for a number of advantages, including modulation of the half-life of a drug, modulation of the biodistribution of a drug, targeting of a drug to particular cell or tissue types, and the like. As such, much effort has been expended in both academic and industrial settings in the development of liposome based drug delivery technology.

However, despite some early successes in liposome technology, there is a great need

1 for the identification of new liposomal based therapeutic approaches. Relevant Literature

Patents of interest include U.S. Patent No. 5,023,087. The use of liposomes as drug delivery vehicles is reviewed in: Allen, Drugs (Nov. 1998) 56:747-756; Allen, Drugs (1997) 54 Suppl 4: 8-14; Allen, Trends Pharmacol Sci. (July 1994) 15: 215-220; Lasic & Martin, Stealth Liposomes (CRC Press)(1995); Mouritsen & Jorgensen, Pharm. Res. (Oct. 1998) 15: 1507-1519; Desormeaux & Bergeron, J. Drug Target. (1998) 6: 1-15; Sallovitz et al.. Vet. Res. (Sep.-Oct. 1998) 29:409-430; Langer, Nature (April 30, 1998) 392 (6670 Suppl):5-10; Wisner et al., J. Med. Chem. (1997) 40:3992-3996.

SUMMARY OF THE INVENTION

Methods and compositions for administering an agent to a host are provided. In the subject methods, at least a first lipid composition that includes the active agent and a second lipid composition that lacks the active agent are administered to the host. In many preferred embodiments, the first and second lipid compositions are administered at different times, e.g. the lipid composition including the agent is administered to the host before or after the lipid composition free of the agent. The lipid composition is typically, though not necessarily, a liposome composition or a micelle composition. Also provided are kits for use in the subject methods, where such kits include a lipid composition comprising the agent and a lipid composition free of the agent.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is graph of normalized mean signal intensity of tumor periphery and tumor center measured at various time intervals before and after injection of 110 nm diameter liposomes containing Gd-DTPA-BMA. Error bars indicate one standard deviation. Peak contrast enhancement, i.e. maximum uptake of liposomes, is at 24 hours when there is no co- injection of empty liposomes.

DEFINITIONS The term "Lipid Composition" as used herein means single layer or bilayer lipid structures arranged in roughly spherical or elliptical shapes, although other shapes including irregular shapes are also possible, where the lipids are generally surface active lipids that may

2 either be ionic or nonionic. Within the lipid structures themselves, the individual lipid molecules are typically not covalently bonded to one another, but instead are generally free to move relative to one another in a fluid manner analogous to that observed in naturally occurring lipid membranes, e.g. cell membranes. Lipid compositions include liposomes, micelles, lipid coated microbubbles, lipid-nucleic acid particles, and the like.

The term "Lipid/Agent Composition" means a lipid composition as defined above that includes an active agent, e.g. a therapeutic agent, a diagnostic agent, etc.

The term "Agent Free Lipid Composition" means a lipid composition that lacks an active agent, e.g. "empty" liposomes. The term "Liposome" as used herein refers to any lipid structure that is a unilamellar or multilamellar vesicle, by which is meant that the structure comprises one or more concentrically ordered assemblies of lipid bilayers.

The term "Prolonged Circulation Liposome" refers to liposomes with prolonged circulation times, where the chemical composition of such liposomes has been modified to prolong circulation times. Liposomes with prolonged circulation times have, for example, surfaces that have been modified with surfactant or polymer molecules. Surface modification can, for example, be achieved with polymeric substances such as polyethyleneglycol, poly(2- methyl-2-oxazolidine)(PMOZ), poly(2-ethyl-2-oxazolidine)(PEOZ), or proteins, or glycolipids, or ganglioside gml . Liposomes composed of sphingomyelin and cholesterol and having an acidic intraliposomal pH also have prolonged circulation time. Amphiphilic polyacrylamide and polyvinyl(pyrrolidone) containing liposomes also have prolonged circulation times. Prolonged circulation liposomes also include small, uncharged liposomes. Liposomes composed from phospholipids with saturated hydrocarbon chains, most notably stearic, and preferably containing high amounts of cholesterol, or liposomes doped with hydrogenated plant phosphatidylinositol can also have prolonged circulation time.

The term "Conventional Liposomes" as used herein refers to liposomes whose surface has not been modified and generally any liposome formulation that does not have prolonged circulation time. Conventional liposomes are taken up preferentially by the liver, spleen, and other reticuloendothelial tissues. Because uptake occurs predominantly in liver and spleen and because circulation time is shorter, the liposome dose delivered to a lesion, e.g. a tumor, is typically lower with conventional liposomes than with liposomes with prolonged circulation time.

The term "Therapeutic Agent" means any agent that exhibits a pharmacologic activity, e.g. a drug, particularly a pharmacologic activity of which results in a desirable therapeutic outcome, e.g. an amelioration of a disease symptom, cure of a disease condition, etc.

The term "Diagnostic Agent" means any agent that is useful in the diagnosis of a physical condition, where diagnostic agents include: imaging or visualization agents that allow one to locate the agent with a device, e.g. contrast agents; etc.

The term "Dosing Event" means the administration of at least one agent/lipid composition and an at least one agent free lipid composition.

The term "Tissue Saturation Dose" means the liposome concentration at which further dose increases do not results in additional liposome accumulation in the target tissue

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods and compositions for administering an agent to a host are provided. In the subject methods, a first, lipid/agent composition and a second, agent free lipid composition are administered to the host. In many preferred embodiments, the first and second lipid compositions are administered at different times. Thus, the lipid composition that includes the active agent may be administered before or after the lipid composition that is free of the active agent. In many embodiments of the subject invention, the lipid composition is a liposome composition. Also provided are kits for practicing the subject methods, where such kits at least include a lipid/agent composition and an agent free lipid composition. In further describing the subject invention, the subject methods will be described first in greater detail, followed by a discussion of the lipid compositions that find use in the subject methods as well as a more detailed description of kits for use in carrying out the subject methods.

Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms "a," "an" and "the' include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

As summarized above, the subject invention provides a method of delivering one or more active agents to a host using lipid compositions as delivery vehicles. A critical feature of the invention is that during administration of the agent(s) to the host, a first lipid composition that includes the active agent and a second lipid composition that lacks or is free of the active agent are administered to the host. By free of the agent is meant that the second lipid composition does not have the active agent incorporated into it, e.g. either sequestered inside of it, interspersed among a plurality of lipids, present on a surface of a lipid structure, and the like. As such, in the subject methods both a lipid composition comprising the agent (i.e. a lipid/agent composition) and a lipid composition free of the agent (i.e. an agent free lipid composition) are administered to the host.

As mentioned above, the first and second lipid compositions are administered over the course of a dosing event, where a dosing event is defined as the administration of the agent/lipid composition and the agent free lipid composition(s). For example, a dosing event might actually include the administration of four distinct lipid compositions over a period of time, but only one of the lipid compositions will be a lipid/agent composition. Thus, where a given treatment protocol requires four different administrations of agent over a given period of time, the treatment protocol includes four different dosing events. Within any given dosing event, the number of lipid compositions (i.e. agent/lipid compositions and agent free lipid compositions) will vary, but will generally range from about 2 to 30, usually from about 2 to 25 and more usually from about 2 to 20.

During any given dosing event, the first and second lipid compositions may be administered at the same time or at different times. As such, the lipid compositions may be administered substantially simultaneously or sequentially. Where the lipid compositions are administered sequentially, i.e. at different times, the order in which the compositions are administered may vary. Thus, the lipid composition that includes the agent may be administered to the host before the lipid composition that is free of the agent. Conversely, the lipid composition that includes the active agent may be administered after the lipid composition that is free of the agent. In yet other embodiments, two or more agent free lipid compositions are administered to the host in a given dosing event, with at least one agent free lipid composition administered before and after the lipid/agent composition, such that the lipid/agent composition is administered between administrations of the agent free lipid composition. While the first and second lipid compositions may be administered at substantially the same time, including simultaneously, during a given dosing event (as mentioned above), in many preferred embodiments, the first and second lipid compositions are administered at different times.

Where the lipid compositions are administered at different times, the period of time that separates administration of the agent/lipid composition and the agent free lipid composition will vary depending on a number of parameters, including the nature of the agent being administered, the nature of the host, the disease condition being treated, the lipid composition being used, etc. In many embodiments, the period of time between administration of the two compositions will be at least about 30 min, usually at least about 1 hr and more usually at least about 6 hr, where the period of time may be 1 day or longer, but will generally not exceed about 2 weeks and usually will not exceed about 1 week.

In one embodiment, the agent free lipid composition is administered to the host at least once prior to administration of the lipid/agent composition, where the number of times the agent free composition is administered may range from about 1 to 20, usually from about 1 to 5 times prior to administration of the lipid/agent composition (i.e. the lipid composition carrying the therapeutic agent). In this first embodiment, the agent free lipid composition is administered at least once, usually at least twice and more usually at least three times prior to the lipid/agent composition, where the period of time between administrations may be as great as 1 to 6 hrs, but will usually not exceed about 7 days. In this first embodiment, the time period between administration of the agent free lipid composition and the agent/lipid composition may vary, but typically will range from about 3 hours to 7 days, usually from about 24 to 72 hours and more usually from about 24 to 48 hours.

In another embodiment, the agent free lipid composition is administered after the lipid/agent composition. In this embodiment, the agent free lipid composition is administered at least once, usually at least twice and more usually at least five to ten or more times after the lipid/agent composition. The agent free lipid composition is preferably first administered to the host at a time when maximum accumulation of agent/lipid composition in the target tissue(s) has been achieved. Although this time may vary depending on the nature of the host, the disease condition being treated and the lipid/agent composition, the first administration of agent free lipid composition in this embodiment is typically from about 3 hours to 7 days, usually from about 24 hours to 72 hours and more usually from about 24 hours to 48 hours following administration of the lipid/agent composition. Where a plurality of agent free lipid compositions are administered to the host in a given dosing event, the period of time between administrations of agent free lipid compositions may be as great as 1 to 2 days, but will usually not exceed about 1 week. In this second embodiment, the number of times that the agent free lipid composition is administered to the host in a given dosing event will be at least one, where the agent free lipid composition may be administered to the host a plurality of times, where by plurality is meant 2 to 30 times, usually 2 to 10 times.

The order in which the two compositions are administered, i.e. which embodiment is employed, depends on the particular nature of the condition being treated and the results desired, as the order of administration, as well as the compositional and physical characteristics of the lipid compositions, can be selected to obtain specific desired results, as described in greater detail infra.

To further modulate the distribution of the active agent in the host, administration of the lipid compositions may be accompanied by one or more additional treatment modalities. For example, administration of the lipid compositions may be accompanied by an additional treatment that alters, e.g. increases or decreases, the vascular permeability of the target tissue(s) for the lipid compositions. Additional treatment modalities to which the host may be subjected include hypothermia treatment, ultrasound shock wave treatment, radiation treatment, hyperthermia or heat treatment and the like.

Where hypothermia is included in the treatment regimen, the temperature of the host will be lowered by an amount sufficient to modulate the host's circulation system, i.e. to decrease blood flow to the exterior of the host. In these embodiments, the temperature of the host will be decreased by at least about 3 °C, usually at least about 10°C and more usually at least about 15°C, but will not be decreased by more than about 40°C and usually will not be decreased by more than about 20°C. Any convenient means of accomplishing the desired decrease in temperature may be employed, where such means include: immersion in cold water; wrapping with a cold blanket; and the like. The hypothermic treatment may be

7 administered during the entire dosing event or a portion thereof, depending on the particular treatment regimen being performed.

In yet other embodiments, the overall treatment protocol may include a heat treatment step. For example, in embodiments where the agent free lipid composition is administered to the host first, the host may undergo one or more preliminary heat treatments, such as local mild heating to the external surface, eg. by warm bath or heating suit, to increase distribution of the agent free lipid composition into the skin prior to administration of the lipid/agent composition. This type of therapeutic approach reduces agent accumulation in non-target tissue, thereby reducing the occurrence and/or severity of adverse or toxic reactions to the agent, e.g. foot hand syndrome, etc. In these embodiments, the temperature of the host during heat treatment will typically be raised by a value of at least about 2°C, usually at least about 5°C and more usually at least about 10°C, where the value may be as large as 30°C or larger, but will generally not exceed about 15°C.

Another treatment which may be incorporated into the overall treatment region is ultrasound treatment. Where ultrasound treatment is employed, the ultrasound will typically be localized to one or more target tissues, e.g. organs, in which lipid composition accumulation is desired. Means of applying ultrasound to a host are known in the art, and include those described in U.S. Patent Nos. 5,409,002; 5,143,073; 5,111,822; 5,080,102; 5,080,101; 5,065,761; 5,065,741; and 5,060,650; the disclosures of which are herein incorporated by reference.

When the host is subjected to one or more additional treatment protocols as described above, such treatment protocols may occur prior to, during or after the administration of one or both of the two different lipid compositions, as determined with respect to the nature of the condition being treated, the nature of the additional treatment protocol, the lipid compositions, the active agent, the target tissue, and the like.

Turning now to the lipid compositions that may be employed in the subject methods, a variety of different lipid structures may be employed as the lipid component of the lipid compositions. The lipid structures are generally single layer or bilayer lipid structures arranged in roughly spherical or elliptical shapes, although other shapes including irregular shapes are also possible, where the lipids are generally surface active lipids that may either be ionic or nonionic. Within the lipid structures themselves, the individual lipid molecules are

8 typically not covalently bonded to one another, but instead are generally free to move relative to one another in a fluid manner analogous to that observed in naturally occurring lipid membranes, e.g. cell membranes. The diameter of the lipid structures or particles may vary from nanometers to micrometers, where the diameter of the lipid structures will generally be at least about 10 nm, usually at least about 50 nm and more usually at least about 100 nm, and may be as great as 300 nm or greater, but will usually not exceed about 1000 nm.

One type of lipid structure of interest are lipid-coated microbubbles: roughly spherical structures of a monolayer of non-ionic surface active lipids, such as saturated glycerides and cholesterol esters, surrounding a non-polar or gaseous medium. Lipid-coated microbubbles and methods for their preparation are described in U.S. Patent Nos. 4,684,479 and 5,215,680, the disclosures of which are herein incorporated by reference.

Another type of lipid composition of interest are spherical structures prepared from one or more amphipathic lipid molecules having both hydrophilic and hydrophobic moieties, such as phospholipids, e.g. phosphatidylcholine, phosphatidylserine, synthetic diplamitoyl- DL-alpha-phosphatidylcholine, phosphatidylinositol, glycolipids, etc. The lipid molecules may form a monolayer or bilayer spherical structure surrounding an aqueous core, where the bilayer structure may be unilamellar or multilamellar. Lipid structures of this type include both micelles and liposomes.

In many preferred embodiments of the subject invention, the lipid component will be liposomal, i.e. made up of liposomes. The liposome structure is a unilamellar or multilamellar vesicle, by which is meant that the structure comprises one or more concentrically ordered assemblies of lipid bilayers. The liposomes that find use in the subject invention can be cationic or anionic, depending on the particular use. As desired, the properties of the liposomes may be modulated by doping the lipid bilayer with one or more modulation agents, e.g. cholesterol, charged lipids, e.g. stearylamine or phophatidic acid, and the like. The liposomes can be modified to modulate their expected in vivo half-life, e.g. the half-life may be extended by attaching biologically inert polymers to the liposome surface, such as polyethylene glycol (PEG), and the like, to produce prolonged circulation liposomes. Alternatively, or in addition, the surface may be modified to present one or more targeting moieties that serves to direct the liposome to its target under in vivo conditions, where such targeting moieties include antibodies, receptor ligands and the like. A variety of liposome compositions have been prepared and used in agent delivery.

Representative liposomes that have been prepared and may find use in the present invention. as well as methods for their preparation, are described in: 5,744,158; 5,741,516; 5,739,271 ;

5,736,156; 5,736,155; 5,733,572; 5,723,147; 5,720,976; 5,715,824; 5,71 1,964; 5,705,187; 5,705,385; 5,693.769; 5,686,101 ; 5,665,379; 5,660,855; 5,656.211 ; 5,654,006; 5,653.998;

5,637,564; 5,616,341; 5,595,756; 5,593,688; 5,580,575; 5,571,497; 5,556,948; 5,552,157;

5,552,156; 5,552,155; 5,545,395; 5,542,935; 5,527,528; 5,512,299; 5,492,696; 5,456,901 ;

5,422,120; 5,409,704; 5,407.660; 5,401,51 1 ; 5,399,331; 5,393,530; 5,387,410; 5.277,913;

5,223,263; 5,104.736; 5,077,056; 5,064,655; 5,049,389; 5,043.165; 5,043,107; 5,023.087; 5,019,369; 5,013.556; 4,999,199; 4,985,233; 4,963,367; 4,963,362; 4,948,590; 4.946.683;

4,937,078; 4,921,757; 4,920,016; 4,917,951 ; 4,906,476; 4,873,088; 4,855,090; 4,853,228;

4,844,904; 4,839,175; 4,822,777; 4,812,312; 4,804,539; 4,801,459; 4,687,661; 4,610.868;

4,551,288; 4,356,167; and 3,932,657, the disclosures of which are herein incorporated by reference. The agent component of the lipid/agent compositions employed in the subject invention may be a number of different compounds. The agent component is one that provides for a desired activity during a given treatment regimen. The agent may be hydrophobic or hydrophilic. As such, the agent may be a therapeutic agent, a diagnostic agent, etc. Diagnostic agents of interest are those agents that make detection of the lipid composition following administration possible, where such agents include contrast agents, radioactive agents, fluorescent agents and the like. Therapeutic agents are agents that have a desirable pharmacologic activity and are therefore useful in a particularly therapeutic treatment regimen, where therapeutic agents of interest include anti-inflammatory agents, antibacterial agents, antimicrobial agents, chemotherapeutic agents (antineoplastic agents), antiviral agents, antifungal agents, immunotherapeutic agents, gene therapy agents (e.g. nucleic acids encoding a product of therapeutic value) and the like, where the agent may be a nucleic acid, e.g. DNA, RNA, antisense, peptide nucleic acid, ribozymes, etc, a proteins, a peptides, an organic small molecule, and the like.

Depending on the specific nature of the agent, the agent may be present in the interior of the lipid structure, on the exterior of the lipid structure and/or incorporated into the lipid layer of the lipid structure. Two or more agents may be used simultaneously. These two or

10 more agents can be carried by different lipid/agent compositions or the same lipid/agent composition. If the same lipid/agent composition is used, both agents may be present in the interior of the lipid structure or both agents may be present on the exterior of the lipid structure or both agents may be incorporated into the lipid layer of the lipid structure. Alternatively, one agent may be present in the interior of the lipid structure and one agent may be present on the exterior of the lipid structure or one agent may be present in the interior of the lipid structure and one agent is incorporated into the lipid layer of the lipid structure or one agent is present on the exterior of the lipid structure and one agent is incorporated into the lipid layer of the lipid structure. As described above, first and second lipid compositions (a lipid composition including agent and a lipid composition free of agent) are administered to the host, usually at different times. In lipid compositions that include agent, the agent or agents will be incorporated into the structure of lipid component, e.g. sequestered in the interior, present on the surface, or present in the bilayer of a liposome. In the lipid compositions that are free of the agent, these compositions will not comprise the agent which is present in the lipid/agent compositions used in the invention, i.e. they will not comprise the same biologically active agent. In preferred embodiments, the agent free lipid compositions are empty, i.e. these lipid compositions comprise nothing but structural lipid, e.g. an empty liposome, or have one or more biologically inactive or inert compounds associated with it, e.g. sequestered within in it or present on the surface, where these biologically inactive or inert compounds play no role in the treatment regimen being performed.

Although the first and second lipid compositions employed in the subject methods are different in that one has active agent associated with it and the other lacks agent, the lipid components of the lipid compositions are generally the same type of structure, e.g. they are both liposomal, micellar, LCM etc. While the lipid components of the lipid compositions are the same type of structure, they may differ from each other compositionally and/or physically. For example, the lipid component of each of the compositions may be liposomal, but the liposomes of the first composition may have a different diameter than the liposomes of the second composition. Likewise, the liposomes of the first composition may be non- surface modified liposomes, where the liposomes of the second composition may be surface modified to change plasma half-life, tissue half-life, tissue uptake, extra- and intracellular

1 1 uptake, biodistribution, therapeutic efficacy, and the like, e.g. PEGylated, labeled with antibody, etc, to achieve differential stability profiles, tissue targeting, etc.

Desired results in the subject methods can be achieved by selecting lipid compositions with specific compositional and/or physical characteristics, as well as the proper order of administration. In other words, by tailoring the physical or compositional characteristics of the lipid compositions and selecting the proper order of administration, desired results can be obtained. For example, empty liposomes can be injected prior to therapeutic agent comprising liposomes to decrease tissue accumulation of therapeutic agent loaded liposomes in non- target tissue and at least diminish accumulation of therapeutic agent in these tissues. To further modulation biodistribution of the lipid compositions, additional treatments such as temperature modulation, e.g. hypothermia, heat treatment, etc. may be employed. To further enhance selective uptake of therapeutic agent loaded liposomes by the target tissue, the target tissue can be treated after administration of the empty liposomes to readily uptake the subsequently administered drug loaded liposomes. Treatments of interest include: hypothermia treatment, ultrasound shock wave treatment, radiation treatment and the like, where such protocols are known to those of skill in the art and have been described in greater detail supra. Another means of improving the selectivity of agent uptake by the target tissue and to decrease systemic agent toxicity is to administer agent free liposomes comprising antibodies to normal tissue on their surface first, followed by administration of agent loaded liposomes modified with target tissue antibodies. Selective uptake of therapeutic agent by tissues/cells other than those associated with the liver, spleen and reticuloendothelial system can be accomplished by administering conventional, e.g. non-surface modified, empty liposomes prior to, or at the same time as, administration of prolonged circulation drug loaded liposomes. In practicing the subject method, the lipid compositions are administered to the host using any convenient technique. Administration may be accomplished by a variety of different routes, depending on the specific nature of the lipid composition, the host to be treated, the disease condition to be treated, and the like. Representative routes of administration include: intravenous injection, intracavitary instillation, intraperitoneal injection, intraventricular injection, pleural injection, intraarticular injection, lymphatic injection, intraarterial injection, intratumoral injection, inhalation, etc. In many preferred

12 embodiments, the lipid compositions are systemically administered. A particularly preferred means of administration are means for introducing the lipid compositions directly into the circulatory system of the host, e.g. intraarterial or intravenous injection means. A particularly preferred means of administration is intravenous, especially in those embodiments in which the two different lipid compositions are administered at substantially the same time or simultaneously.

The dosage of the lipid composition that comprises the active agent will necessarily depend on the specific nature of the agent, the condition to be treated, the target tissue and the nature of the lipid component of the composition. Nevertheless, the dosage of the agent/lipid composition will be one that is effective to achieve a desired result, e.g. treatment of the condition, cessation or amelioration of a symptom, and the like. For any given specific agent/lipid composition and condition to be treated, the dosage can be determined empirically. As far as the dosage of agent free lipid composition is concerned, depending on the desired outcome, this composition may be administered at tissue saturating dose (i.e. the liposome concentration at which further dose increases do not results in additional liposome accumulation in the target tissue), below tissue saturation dose or above tissue saturation dose.

Administration of an agent in accordance with the subject methods results in improved results as compared to a control situation. The specific nature of the improved results necessarily depends on the type of agent being administered, but may include one or more of: enhanced target specificity; a longer retention of agent at a desired target site; a reduced dosage requirement; longer intervals between dosages; reduced toxicity; and the like. By control situation is meant administration of the agent by a method other than the subject invention, such as by drug loaded liposome alone, free agent alone, etc. Of particular interest are those embodiments of the subject invention that result in improved results as compared to administration of agent/lipid composition by itself. Improved results are obtained by the subject invention in many embodiments as administration of an agent according to the subject results in an altered biodistribution of the agent as compared to a control situation, where the altered biodistribution yields improved results, such as enhanced distribution of the agent to the target tissue(s), where specific target tissues to which delivery of the agent is enhanced by the subject methods include: neoplasms, breast cancer tissue; pediatric tumors, e.g.

13 osteogenic sarcoma, Ewingsarcoma, neuroblastoma; lymphoma; leukemia; infected tissue; inflamed tissue; tissue characterized by cells that lack a gene, cells that contain a dysfunctional gene, cells that contain an abnormal gene; tissues with abnormal capillary permeability; tissues altered by a collagen vascular disease; and the like. The term "host" as used herein generally refers to "mammals" or "mammalian species," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many- embodiments, the host will be human. Kits for use in the subject methods are also provided. The kits according to the subject invention at least comprise a lipid composition that includes agent and a lipid composition free of agent, where the agent may be a diagnostic agent, a therapeutic agent, etc.. as described above, where the two compositions can be in one container or in separate containers, but in many preferred embodiments will be in separate containers. The kit may also comprise one or more additional components necessary and/or desirable for carrying out the subject method, including administration means, e.g. needles, etc. The kit may further comprise instructional material describing how to practice the subject methods, where such instructional material may be associated with the labeling, a package insert and/or packaging of the kit.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL A. Four rats bearing osteogenic sarcoma were studied: two animals received only a single injection of 110 nm liposomal Gd-DTPA-BMA (dose of 0.05 mmol/kg body weight Gd- DPTA-BMA)(control group) via the rats' tail vein. Two animals received injection of 110 nm liposome encapsulated Gadolinium (dose of 0.05 mmol/kg body weight Gd-DTPA-BMA) immediately followed by injection of liposome encapsulated doxorubicin (Doxil, Sequus Pharmaceuticals, Menlo Park, CA)(dose of 4 mg/kg body weight)(treatment Group). The two animals in the treatment group received repeat injections of liposome encapsulated docorubicin 2 and 4 days after the initial injection using the same dose. Liposomes with

14 encapsulated GD-DTPA-BMA were not reinjected in either the treatment or control groups. All animals underwent follow-up MR imaging (Bruker Omega, 2.0T) using Tl -weighted (spin-echo, TR=400 msec, TE=12 msec, 4NEX) and intermediate-weighted imaging sequences (spin-echo, TR=600 msec, TE=50 msec, 2NEX) on days 2, 3, 4, 5, and 6 after the initial injection. Tumor volumes were estimated based on intermediate- weighted MR images (TR=600 msec, TE=50 msec) for each follow-up interval using the formula for elliptical masses.

In the control group, average increase in tumor volume was 3069%(standard deviation [SD] 909%) over the 6 day observation period. In the treatment group, average increase in tumor volume was only 81.6% (SD 39.5%) after 6 days.

In the control group, MR images obtained 24 hours after injection demonstrated prominent intratumoral enhancement (peak enhancement); enhancement decreased progressively at 2 days after injection. Four days post-injection, no contrast enhancement of the tumor was visible in the control group.

In the treatment group, both animals demonstrated prominent enhancement of the tumor 24 hours after injection comparable to that seen in the control group. However, contrast enhancement was seen to persist for the entire observation period: the tumors were still brightly enhanced at 3 days, 4 days, 5 days and 6 days after injection.

B. The following groups are prepared:

Day O Day 2 Day 4 Day 6 Day 8

Group 1 : liposomes with Gd-DTPA- ~ — ~ — BMA & liposomes with doxorubicin

Group 2 liposomes with Gd-DTPA- empty empty empty empty BMA & liposomes with liposomes liposomes liposomes liposomes doxorubicin

Each group consists of six tumor bearing animals. Both groups receive the same initial injection of liposomes with encapsulated Gd-DTPA-BMA at a dose of 0.05 mmol/kg body weight followed by liposomes with encapsulated doxorubicin at a dose of 2 mg/kg body weight. However, group 2 is injected with empty liposomes on days 2, 4, 6 and 8 after the initial injection. The retention of liposomes with encapsulated agent by the tumor is then observed in the two groups and compared. It is expected that at least one of the following occurs: (a) a persistent enhancement of the tumor in Group 2 beyond 24 hours that is greater

15 than that observed in Group 1 ; and (b) no or decreased tumor growth in Group 2 that is lower than that observed in Group 1.

C. The following groups are prepared:

Day O Day 2 Day 4 Day 6 Day 8

Group L liposomes with prolonged liposomes liposomes liposomes with liposomes with circulation time loaded with Gd- with with prolonged prolonged DTPA-BMA & liposomes with prolonged prolonged circulation circulation prolonged circulation time loaded circulation circulation time loaded time loaded with doxorubicin time loaded time loaded with with with with doxorubicin doxorubicin doxorubicin doxorubicin

Group 2 a conventional empty liposomes liposomes liposomes liposomes with liposomes with b 6 hours later liposomes with with with prolonged prolonged prolonged circulation time loaded prolonged prolonged circulation circulation with Gd-DTPA-BMA &lιposomes circulation circulation time loaded time loaded with prolonged circulation time time loaded time loaded with with loaded with doxorubicin with with doxorubicin doxorubicin doxorubicin doxorubicin

Group 3 a conventional empty liposomes conventional conventional conventional conventional b rats placed in 40 °C water tor empty emptv empty empty 30 min liposomes and liposomes and liposomes and liposomes and c 6 hours later liposomes with liposomes liposomes liposomes with liposomes with prolonged circulation time loaded with with prolonged prolonged with Gd-DTPA-BMA &lιposomes prolonged prolonged circulation circulation with doxorubicin circulation circulation time loaded time loaded time loaded time loaded with with with with doxorubicin doxorubicin

doxorubicin doxorubicin

Each group consists of 6 tumor bearing rats. Each group gets the same dosage schedule of doxorubicin liposomes with prolonged circulation time and liposomes with prolonged circulation time loaded with Gd-DTPA-BMA. Groups 2 and 3 additionally receive empty conventional liposomes designed to load normal tissues with empty liposomes. These liposomes have a brief circulation time and are not appreciably taken up by tumor. Using this approach, accumulation of drug liposomes is inhibited in normal liver, spleen and reticuloendothelial tissue thereby reducing adverse reactions. Accumulation/retention of drug liposomes in liver, skin, and tumor is determined on MRI images. In addition, production of skin rash or other signs of hand-foot syndrome is monitored for each group. Groups 2 and 3 exhibit less skin rash, lower quantities of drug liposome in liver, but similar quantities delivered to the tumor, based on MRI evaluation.

D. NEW TREATMENT APPROACH TO IMPROVE EFFICACY OF LIPOSOMAL DRUG THERAPY

16 Sixty patients with osteogenic sarcoma are selected for a clinical trial. A chemotherapeutic regimen, i.e. a combination of chemotherapeutic drugs used for treatment of the tumor, is chosen that includes the use of doxorubicin. Patients are subdivided into three groups, each consisting of 20 patients:

Group 1 receives the chemotherapeutic regimen with free, unencapsulated doxorubicin at the standard dose.

Group 2 receives the chemotherapeutic regimen with liposomes with prolonged circulation time with encapsulated doxorubicin at the same doxorubicin dose used with the free drug; however, no empty liposomes are injected subsequently.

Group 3 receives the chemotherapeutic regimen with liposomes with prolonged circulation time with encapsulated doxorubicin at the same doxorubicin dose used for the free drug; however, additionally, empty liposomes with prolonged circulation time are injected every 24 hours for 5 days and starting 24 hours after each therapeutic agent/liposome injection using the same lipid dose that is employed for liposomes with encapsulated doxorubicin.

Treatment response is monitored using diffusion weighted MR imaging (see also

Lang et al. Radiology 1998; (1) 206: 227-235).

Patients in Group 3 demonstrate enhanced efficacy of chemotherapeutic treatment, i.e. more extensive central tumor necrosis and more pronounced arrest of tumor growth, than patients in Groups 1 and 2.

E. NEW TREATMENT APPROACH TO DECREASE THE INCIDENCE AND SEVERITY OF HAND-FOOT SYNDROME

The same design of the clinical trial is used as in Example D is employed. However, patients in Group 3 are pre-treated with empty liposomes for three days prior to injection of therapeutic agent/liposomes: Empty liposomes with prolonged circulation time are injected every 24 hours using the same lipid dose that is employed for liposomes with encapsulated doxorubicin. Prior to injection of liposomes with prolonged circulation time with

17 encapsulated doxorubicin, tumors are treated locally with hyperthermia as a means of increasing capillary permeability and leakiness and as a means of increasing tissue extravasation of therapeutic agent liposomes.

Identical to Example D, Group 3 then receives the chemotherapeutic regimen with liposomes with prolonged circulation time with encapsulated doxorubicin at the same doxorubicin dose used for the free drug; additionally, empty liposomes with prolonged circulation time are injected every 24 hours for 5 days and starting 24 hours after each therapeutic agent/liposome injection using the same lipid dose that is employed for liposomes with encapsulated doxorubicin. Patients in Group 3 demonstrate (a) a better therapeutic efficacy, i.e. more extensive central tumor necrosis and more pronounced arrest of tumor growth, than patients in Groups 1 and 2. However, additionally, patients in Group 3 demonstrate a lower incidence of hand foot syndrome than patients in Group 2.

It is evident from the above results and discussion that the subject invention provides for a number of advantages over conventional liposome based delivery protocols in which only agent loaded liposomes are administered without re-injection of empty liposomes. Such advantages include one or more of reduced dosage requirements, longer time intervals between dosages and reduced host toxicity.

The following publications have been referenced above: A. D. Bangham in 1965. Bangham, J. Mol. Biol. (1965) 13: 238-252; Allen, Drugs (Nov. 1998) 56:747-756; Allen, Drugs (1997) 54 Suppl 4: 8-14; Allen, Trends Pharmacol Sci. (July 1994) 15: 215-220; Lasic & Martin, Stealth Liposomes (CRC Press)(1995); Mouritsen & Jorgensen, Pharm. Res. (Oct. 1998) 15: 1507-1519; Desormeaux & Bergeron, J. Drug Target. (1998) 6: 1-15; Sallovitz et al., Vet. Res. (Sep.-Oct. 1998) 29:409-430; Langer, Nature (April 30, 1998) 392 (6670 Suppl):5-10; Wisner et al., J. Med. Chem. (1997) 40:3992-3996; and Lang et al. Radiology 1998; (1) 206: 227-235. The following patents have been referenced above: 5,023,087; 5,409,002; 5,143,073; 5,111,822; 5,080,102; 5,080,101 ; 5,065,761 ; 5,065,741; 5,060,650; 4,684,479; 5,215,680; 5,744,158; 5,741,516; 5,739,271 ; 5,736,156; 5,736,155; 5,733,572; 5,723,147; 5,720,976; 5,715,824; 5,711,964; 5,705,187; 5,705,385; 5,693,769; 5,686,101; 5,665,379; 5,660,855; 5,656,211; 5,654,006; 5,653,998; 5,637,564; 5,616,341 ; 5,595,756; 5,593,688; 5,580,575; 5,571,497; 5,556,948; 5,552,157; 5,552,156; 5,552,155; 5,545,395;

18 5,542,935; 5,527,528; 5,512,299 5,492,696; 5,456,901; 5,422,120; 5,409,704; 5,407,660; 5,401,511 ; 5,399,331 ; 5,393,530 5,387,410; 5,277,913; 5,223,263; 5,104,736 5,077,056 5,064,655; 5,049,389; 5,043,165 5,043,107; 5,023,087; 5,019,369; 5,013,556 4,999,199 4,985,233; 4,963,367; 4,963,362 4,948,590; 4,946,683; 4,937,078; 4,921,757 4,920,016 4,917,951; 4,906,476; 4,873,088 4,855,090; 4,853,228; 4,844,904; 4,839,175 4,822,777

4,812,312; 4,804,539; 4,801,459; 4,687,661 ; 4,610,868; 4,551,288; 4,356,167 and

3,932,657.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A method of administering an agent to a host, said method comprising: administering to said host: (a) a lipid composition comprising said agent; and (b) a lipid composition free of said agent, wherein said administering is by a methods selected from the group consisting of: intravenous injection, intracavitary instillation, intraperitoneal injection, intraventricular injection, pleural injection, intraarticular injection, lymphatic injection, intraarterial injection, intratumoral injection, and inhalation.

2. The method according to Claim 1, wherein said lipid compositions are administered at substantially the same time.

3. The method according to Claim 1, wherein said lipid compositions are administered at different times.

4. The method according to Claim 3, wherein said lipid composition comprising said agent is administered prior to said lipid composition free of said agent.

5. The method according to Claim 3, wherein said lipid composition comprising said agent is administered after said lipid composition free of said agent.

6. The method according to Claim 1 , wherein said lipid composition is a liposome composition.

7. The method according to Claim 1 , wherein said lipid composition is a micelle composition.

8. The method according to Claim 1 , wherein said host is mammalian.

9. A method of administering an agent to a mammalian host, said method comprising: systemically administering a liposome composition comprising said agent and a liposome composition free of said agent to said mammalian host; wherein at least one of said liposome compositions comprises prolonged circulation liposomes.

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10. The method according to Claim 9, wherein said agent is selected from the group consisting of diagnostic agents and therapeutic agents.

11. The method according to Claim 10, wherein said therapeutic agent is selected from the group consisting of: chemotherapeutic agents, anti-inflammatory agents, antifungal agents, immunotherapeutic agents, antiviral agents and gene therapy agents.

12. The method according to Claim 9, wherein said liposome composition free of said agent comprises empty liposomes.

13. The method according to Claim 9, wherein said mammalian host is human.

14. The method according to Claim 9, wherein said liposome composition comprising said active agent and said liposome composition free of said agent are administered to said host at different times.

15. The method according to Claim 9, wherein said liposome composition free of said agent comprises prolonged circulation liposomes.

16. The method according to Claim 15, wherein said liposome composition free of said agent is administered prior to said liposome composition comprising said agent.

17. The method according to Claim 14, wherein said liposome composition comprising said agent comprises prolonged circulation liposomes.

18. The method according to Claim 17, wherein said liposome composition free of said agent is administered prior to said liposome composition comprising said agent.

19. The method according to Claim 9, wherein at least one of said liposome compositions comprises a targeting moiety.

20. The method according to Claim 19, wherein said targeting moiety is an antibody.

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21. The method according to Claim 20, wherein said targeting moiety is a receptor ligand.

22. The method according to Claim 9, wherein said method further comprises treating said host with at least one additional treatment modality.

23. The method according to Claim 22, wherein said additional treatment modality modulates vascular permeability for at least one of said lipid compositions.

24. A method of administering an agent to a human, said method comprising: administering a liposome composition comprising said agent and a liposome composition free of said agent to the circulatory system of said human.

25. The method according to Claim 24, wherein said agent is selected from the group consisting of diagnostic agents and therapeutic agents.

26. The method according to Claim 25, wherein said therapeutic agent is selected from the group consisting of: chemotherapeutic agents, anti-inflammatory agents, anti-infectious agents, anti-bacterial agents, antifungal agents, immunotherapeutic agents, antiviral agents and gene therapy agents.

27. The method according to Claim 24, wherein said liposome composition free of said agent comprises empty liposomes.

28. In a method of drug delivery in which liposomes are employed to deliver an active agent, the improvement comprising: administering a liposome composition free of said active agent following delivery of said active agent.

29. A kit for use in the administration of an agent to a host, said kit comprising: a lipid composition comprising said agent; and a lipid composition free of said agent.

30. The kit according to Claim 29, wherein said agent is a diagnostic agent.

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31. The kit according to Claim 29, wherein said agent is a therapeutic agent.

32. The kit according to Claim 30, wherein said therapeutic agent is selected from the group consisting of: chemotherapeutic agents, anti-inflammatory agents, anti-infectious agents, anti-bacterial agents, antifungal agents, immunotherapeutic agents, antiviral agents and gene therapy agents.

33. The kit according to Claim 29, wherein said lipid compositions are liposome compositions.

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