EP2262369A1 - Lipid-oil-water nanoemulsion delivery system for microtubule-interacting agents - Google Patents

Abstract

A pharmaceutical composition, and methods of use and preparation thereof, beneficial in treating, diagnosing, and preventing a disease, condition, syndrome, or symptoms thereof, characterized by cellular hyperproliferation such as cancer, in warm-blooded animals, including humans, incorporates a lipid nanoemulsion, prepared by processing through homogenization, comprised of lipid particles each comprising at least one non-bilayer- forming lipid capable of being preferentially and selectively actively internalized within a diseased cell; an effective amount of at least one therapeutic or diagnostic microtubule- interacting agent associated with the nanoemulsion; and a pharmaceutically-acceptable carrier. In a preferred embodiment, the composition may also be comprised of a protein carrier molecule and/or emulsion-enhancing agents such as a surfactant, a plant-based fat source, a solvent, and combinations thereof.

Description

Lipid-Oil-Water Nanoemulsion Delivery System for Microtubule-Interacting Agents

Field of the Invention This invention relates to therapeutic and diagnostic agents, and more particularly to the formulation of microtubule-interacting agents in a lipid-oil-water nanoemulsion suitable for introduction into a patient and capable of promoting active selective agent concentration into cells characterized by hyperproliferation, including tumor cells.

Background of the Invention

The role of drug distribution within tumors and their microenvironment has not been well studied. Currently, to see benefit, high doses of even the most potent drugs must be given to drive diffusion and tissue distribution. However, increasing drug concentration in a tumor mass does not always lead to increased cell drug concentration or the ability to reach an intracellular target. Indeed, while some drugs may be increased concentration in a rumor mass by conjugation to polymers, liposome encapsulation, or solid lipid nanoparticle formulation, these may actually inhibit cellular uptake and distribution. Additionally, high dosing to drive tumor penetration by diffusion typically can result in those side effects associated with the high morbidity of cancer treatment. It is therefore desirable to devise a drug delivery technology that would improve drug penetration through tumor tissue and increase its concentration within tumor cells while simultaneously presenting an increased safety and efficacy profile. While diverse approaches have been explored for the passive accumulation of drugs into tumors, however, few technologies are available for the promotion of active tumor cell uptake. Lipid-soluble drugs readily penetrate cell membranes and may be transported through cells. Cells characterized by hyperproliferation, such as cancer cells, generally exhibit an aberrant lipid metabolism, marked by their greatly-increased preferential uptake of lipids and fatty acids. Thus, with respect to cancer, lipids have been found to play essential roles in membrane structure, growth and metastasis, signal transduction, and transport processes. Caused by the characteristic leaky features of tumor vasculature, the phenomenon known as the enhanced permeability and retention (EPR) effect for lipid particles, liposomes, synthetic nanoparticles, and other macromolecular agents is universal in solid tumors and has been explored for more selective targeting and tumor mass accumulation of, for example, polymer-conjugated anticancer drugs. The use of encapsulating agents such as polymeric vesicles, liposomes, and solid polymeric or lipid nanoparticles, each prepared in a variety of compositions, formulations, and structures and administered under a variety of physiological conditions, has been reported in the prior art. However, these agents permit only passive accumulation in tumors. Moreover, these agents are often rapidly cleared from the circulation by extensive co-accumulation in the reticuloendothelial system, and often the use of these agents is associated with cytotoxicity and heightened immunoresponses.

Studies have shown that multiple receptor types and subtypes, transport proteins, and lipid raft phenomena are likely to be involved in lipid uptake into cancer cells, even among patients with the same disease but altered genetics. While lipid-based nanoparticles or HDL- or LDL-based drug carriers that mimic the natural receptor ligand and increase targeting and drug uptake into cancer cells have been prepared with various therapeutic agents, however, optimized formulations for the promotion of active tumor cell drug uptake for the treatment of cancer have been lacking.

US Patents 4684479 and 5215680 to D'Arrigo, herein incorporated by reference, describe the formation of gas- or air-filled lipid coated microbubbles (LCMs), and methods of production and use thereof, to be used as imaging agents for ultrasound methods and for drug delivery. The production process for LCMs is based on simple mechanical shaking of an aqueous suspension of nonionic lipids, such as saturated glycerides and cholesterol esters, of specific chain lengths and in a fixed ratio. In all cases, the majority of lipids added (99%) flocculate or precipitate with additional loss of material on filtration with yields less than 1%. Still, these artificial LCMs were found to be very long-lived, lasting over 6 months in vitro. Also, despite their low filtration yields, LCMs are sufficiently small and pliable enough to pass across the fenestrated capillary walls of tumor-tissue microcirculation. Of particular note is the highly-selective, temperature-dependent, apparently saturable uptake of LCMs into rodent brain tumor cells as well as into spontaneous tumors in dogs, likely mimicking cancerous cells' natural uptake of certain lipids. To date, however, there has not been an efficacious formulation of a pharmaceutical compound utilizing LCM technology which presents minimal to nonexistent levels of adverse side effects upon patient administration.

Paclitaxel is a member of the class of drugs known as taxanes and has been isolated primarily from the bark of the Pacific yew tree, Taxus brevifolia, and related species. Although allergic reactions were observed in formulation excipients, taxanes have been found useful in the treatment of various cancers such as ovarian, breast, non-small cell lung, and head and neck carcinomas. A difficulty in administrating taxanes is that the drug is not typically water-soluble. Paclitaxel has consequently been formulated in a 1:1 mixture of Cremophor EL® (a polyethoxylated castor oil) and ethanol to create Taxol® (Bristol-Myers Squibb, Inc.). Reconstitution in a suitable carrier stable for approximately twelve hours is required, along with in-line filtration to remove any drug which may crystallize out. Taxol® has been shown to be neurotoxic, causing sensory and sometimes accompanying motor neuropathy in patients. Cremophor EL® is in part responsible for the neuropathy, as it is itself not free of significant side effects. Attempts to formulate paclitaxel in a stable lipid emulsion have similarly been unsuccessful. The drug is reported to be insoluble in lipid emulsions such as Intralipid®, which contains primarily soybean oil, or Liposyn®, which contains a mixture of soybean and safflower oils. Heating paclitaxel in either soybean or safflower oil, or even upon sonication, does not result in the dissolution of appreciable amounts of the drug, and addition of paclitaxel to a lipid emulsion during a homogenization step meets with equally disappointing results. Emulsions incorporating paclitaxel up to 15 mg/mL have been formulated with triacetin, L-alpha-lecithin, Polysorbate 80, Pluronic F-68, ethyloleate, and glycerol. However, these emulsions are highly toxic and unstable. Paclitaxel has also been combined with Cremophor EL® and ethanol in a LCM formulation. However, while the tumor uptake selectivity of LCM is intriguing from a drug delivery perspective, the manufacturing yield and drug payload capacity according to the prior art are too low to be practical. The use of Cremophor EL® and ethanol with paclitaxel also likely contributes to this formulation's toxicity. Additionally, production methods are complex and small changes in conditions can result in significant differences in particle size and performance features. Typical approaches include high-shear homogenization and ultrasound, high-pressure homogenization, hot homogenization, cold homogenization, and solvent emulsification evaporation. Sterilization by aseptic filtration or radiation may also be complex. Thus, attempts to increase drug payload and solubility of lipids in LCMs with addition of a drug in ethanol and aqueous solution followed by high-shear homogenization result in the formation of poorly-stable non- gas-containing particles that contain little if any drug.

Administering therapeutic agents with an appropriate delivery vehicle that simultaneously promotes accumulation and cellular internalization in a tumor mass but limits accumulation in healthy tissues is highly desirable. With more efficient delivery, systemic and healthy tissue concentrations of therapeutic agents may be reduced while achieving the same or better therapeutic results with fewer or diminished side effects. Such delivery of agents with inherent degrees of tumor cell selectivity would offer additional advantages. Further, a delivery vehicle that would not be limited to a single tumor type but would allow for selective accumulation into a tumor mass and cellular internalization into diverse cancer cell types would be especially desirable and allow for safer more effective treatment of cancer. A delivery vehicle that would also allow for elevated loading capacity for the therapeutic agent would be a significant advance in the art.

There is therefore a clear need for a stable, easily-prepared, biocompatible, efficacious formulation of microtubule-interacting agents, including taxanes such as paclitaxel, which would promote uptake into tumor cells yet exhibit minimal side effects.

Objects of the Invention and Industrial Applicability

Consequently, it is an object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of diseases, conditions, and syndromes characterized by cellular hyperproliferation, such as cancer, which exhibits rapid and increased selective and preferential uptake into tumor cells.

It is a further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of diseases, conditions, and syndromes characterized by cellular hyperproliferation, such as cancer, which causes minimal side effects upon administration.

It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of diseases, conditions, and syndromes characterized by cellular hyperproliferation, such as cancer, which is easily manufactured at the least possible cost and is capable of being stored for the longest possible period. Summary of the Invention

To achieve these objects, the present invention broadly provides a pharmaceutical composition useful for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals, including humans, wherein the pharmaceutical composition includes a lipid nanoemulsion comprised of lipid particles as hereinafter defined, uniformly dispersed in an aqueous phase capable of being selectively and preferentially internalized within a diseased cell, including a cancer cell; an effective amount of at least one therapeutic or diagnostic agent associated with the lipid nanoemulsion; and a pharmaceutically-acceptable carrier. The lipid particles each comprise at least one non-bilayer-forming lipid. Such suitable lipid particles have been found to enhance significantly the targeted delivery and concentration of the therapeutic or diagnostic agent into diseased cells characterized by hyperproliferation, including cancer cells, for improved treatment efficacy and bioavailability while reducing or at least maintaining those dosage amounts and frequencies of administration necessary to achieve the desired therapeutic benefits. Moreover, the nanoemulsion exhibits exceptional physical and chemical stability for an extended duration of time, thereby greatly facilitating prepackaging of the pharmaceutical composition in stable, ready-to-administer forms, and also thereby eliminating the problems and inconvenience associated with bedside dilution and formulation as currently practiced with compositions in the prior art containing similar active agents.

The lipid particles have associated therewith a pharmaceutically-active therapeutic microtubule-interacting agent. Suitable microtubule-interacting agents include, but are not limited to, taxanes, such as, for example, paclitaxel; epothilones; vinca alkaloids, such as, for example, vincristine; eleutherobins; discodermolide; dolastatins; colchicine; combrestatins; phomopsin A; halichondrin B; spongistatin 1; sarcodictyins; laulimalides; and derivatives, analogs, congeners, and combinations of each of the aforementioned agents thereof. The microtubule-interacting agent is present in an amount sufficient to kill or at least suspend the growth of the hyperproliferated cells.

In a further aspect of the present invention, there is provided a method of diagnosing, treating, or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, including cancer, in warm-blooded animals, including humans, wherein the method comprises administering to a warm-blooded animal an effective amount of the pharmaceutical composition disclosed herein.

In a still further aspect of the present invention, there is provided a method of preparing the pharmaceutical composition disclosed herein, comprising the steps of: a) mixing the at least one non-bilayer-forming lipid with an effective amount of at least one diagnostic or therapeutic agent to yield a lipid portion; b) adding the lipid portion to an aqueous phase to yield a dispersion; and c) agitating the dispersion under high-shear conditions sufficient to disperse the aforementioned lipid therethrough to form a lipid nanoemulsion comprised of lipid particles.

Brief Description of the Drawings

The following drawings are illustrative of embodiments of the invention and are not intended to limit the scope of the application as encompassed by the entire specification and claims.

FIGURE 1 illustrates particle size distribution and zeta potential for lipid particles loaded with 10% paclitaxel in 20% DMSO in accordance with the present invention.

FIGURE 2 shows the incorporation of paclitaxel into lipid particles in accordance with the present invention by a graph plotting the percent solubility of paclitaxel-loaded lipid particles in a sucrose density study. FIGURES 3A and 3B depict graphs showing that cellular lipid particle uptake according to the present invention can be quantified by fluorescence-activated cell sorting.

FIGURES 4A-4D portray graphs showing cellular lipid particle uptake according to the present invention by tumor cell lines relative to HT-29 colon and SF-539 lung tumor cells.

FIGURE 5 is a graph showing that tumor cells take up a greater amount of paclitaxel formulated in one particular mixture of lipid particles according to the present invention than paclitaxel formulated in Cremophor EL®.

FIGURE 6 depicts a graph showing that cell uptake of paclitaxel reached a plateau after two hours of drug incubation for both LN and Cremophor EL®.

FIGURE 7 illustrates the results of an experiment to determine if paclitaxel amount is saturating for cell uptake in lipid particles according to the present invention.

FIGURES 8A and 8B depict graphs showing that cholesterol is a critical component in cellular uptake of lipid particles according to the present invention. FIGURES 9A and 9B portray graphs showing that paclitaxel in lipid particles according to the present invention is internalized to a greater extent than paclitaxel in Cremophor EL®.

FIGURES 10A- 1OD depicts graphs showing the cytotoxicity of paclitaxel alone versus paclitaxel in lipid particles according to the present invention. FIGURE 11 illustrates that paclitaxel is more cytotoxic when formulated in the lipid particles of the present invention than when formulated in Cremophor EL®.

FIGURES 12A and 12B illustrate graphs showing that paclitaxel has significantly greater anti-tumor activity when formulated in the lipid particles of the present invention than when formulated in Cremophor EL®. FIGURE 13 shows representative images of A549 cells treated with EmPAC, Abraxane®, or Taxol® and stained with paclitaxel-specific antibodies.

Detailed Description of the Invention

The present invention is generally directed to pharmaceutical compositions for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals. Such animals include those of the mammalian class, such as humans, horses, cattle, domestic animals including dogs and cats, and the like, subject to disease and other pathological conditions and syndromes characterized by cellular hyperproliferation, including cancer. The pharmaceutical composition of the present invention comprises a nanoemulsion comprised of lipid particles, as defined below, operatively associated with at least one therapeutic or diagnostic microtubule-interacting agent, for which the lipid particles have an enhanced loading capacity, and a pharmaceutically-acceptable carrier or excipient therefor, thereby making the lipid particles particularly well-suited for the selective delivery to and effective concentration within such diseased cells and tissues as tumorous ones.

The lipid particles of the nanoemulsion are structured to facilitate both elevated passive accumulation and active internalization into diseased cells and tissues, including tumor cells and tissues. The lipid particles are taken into these cells through active metabolic uptake as they passively accumulate in the vascular area of the diseased tissue. Thus, the lipid particles of the present invention provide a delivery vehicle selectively and preferentially targeted for uptake and internalization by cells characterized by hyperproliferation, including tumor and cancer cells. "Internalization" as used herein means that the lipid particles are actively taken up by the cell. Such elevated internalization levels, coupled with a high loading capacity of the particles for the therapeutic or diagnostic agent, provides a potent vehicle for treatment or diagnosis of these targets by delivering an effective amount of the therapeutic or diagnostic agent to such targets, thereby inducing a therapeutically-beneficial effect, including stopping growth, inducing differentiation, or killing the cell. The lipid particles of the present invention hence not only enhance delivery of the therapeutic agent to the diseased cells and tissues but also reduce the amount of the therapeutic agent needed to achieve the desired efficacy, especially as compared to delivery systems in the prior art.

The lipid particles of the present invention are exceptionally physically and chemically stable over an extended period of time and hence experience minimal loss of the therapeutic or diagnostic agent due to undesirable precipitation, aggregation, or insolubility that is typically exhibited in delivery systems in the prior art. Moreover, these lipid particles display other favorable characteristics including controlled release; enhanced drug stability; positive drug loading capacity; better compatibility with hydrophobic drugs; relatively low biotoxicity; and low organic solvent content. The present lipid particles are also relatively simple and convenient to prepare and to administer. As used herein, the term "lipid particle" is meant to encompass any lipid-containing structures, typically nanosized, which are at least substantially-intact particles forming part of a nanoemulsion. The term "substantially-intact" means that the particles maintain their shape in the absence of a membrane, as contrasted with a liposome. The lipid particles are comprised of at least one non-bilayer-forming lipid. A lipid bilayer structure or arrangement is typically formed by certain kinds of lipids having a hydrophilic end (polar head region) and a hydrophobic end (nonpolar tail region), including amphipathic molecules such as phospholipids, which exhibit the ability and/or tendency to self-organize into two opposing layers of lipid molecules in aqueous solution. The two opposing layers of lipid molecules are arranged so that their hydrophobic ends face one another to form an oily core, while their hydrophilic ends face the aqueous solutions on either side of the bilayer structure. In the present invention, the term "non-bilayer-forming lipid" encompasses a lipid that lacks such ability and/or tendency to form a lipid bilayer structure or arrangement in an aqueous environment. Examples of non-bilayer-forming lipids include lipids that are no more than weakly polar, preferably lipids that are substantially non- polar or neutral. The more-preferred lipids in the present invention are neutral lipids.

The lipid particles of the present invention are distinguishable from the gas-containing microbubbles described in U.S. Patent Nos. 4684479 and 5215680, and are also structurally distinguishable from liposomes, such as those described, for example, in U.S. Patent Nos. 6565889 and 6596305, all herein incorporated by reference. In particular, the lipid particles are formed by a mixture of non-bilayer-forming lipids that are physiologically acceptable and at least substantially free from the presence of charged or polar lipids, including, for example, phospholipids. Suitable examples of non-bilayer-forming lipids include those selected from glycerol monoesters of saturated and unsaturated carboxylic acids; glycerol monoesters of saturated aliphatic alcohols; sterol aromatic acid esters; sterols; terpenes; bile acids; alkali metal salts of bile acids; sterol esters of aliphatic acids; sterol esters of sugar acids; esters of sugar acids; esters of aliphatic alcohols; esters of sugars; esters of aliphatic acids; sugar acids; saponins; sapogenins; glycerol; glycerol di-esters of aliphatic acids; glycerol tri-esters of aliphatic acids; glycerol diesters of aliphatic alcohols; glycerol triesters of aliphatic alcohols; and combinations thereof. In an embodiment of the present invention, the lipid particles are prepared by first forming a mixture of a select group of non-bilayer-forming lipids which provides the lipid particles with a size described hereinafter that facilitates high internalization levels when applied to targeted diseased tissues and cells. The lipid mixture generally comprises: a) at least one first member selected from the group consisting of glycerol monoesters of carboxylic acids containing from about 9 to 18 carbon atoms and aliphatic alcohols containing from about 10 to 18 carbon atoms; b) at least one second member selected from the group consisting of sterol aromatic acid esters; c) at least one third member selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids; d) at least one optional fourth member selected from the group consisting of sterol esters of aliphatic acids containing from about 1 to 18 carbon atoms; sterol esters of sugar acids; esters of sugar acids and aliphatic alcohols containing from about 10 to 18 carbon atoms, esters of sugars and aliphatic acids containing from about 10 to 18 carbon atoms; sugar acids, saponins; and sapogenins; and e) at least one optional fifth member selected from the group consisting of glycerol, glycerol di- or tri-esters of aliphatic acids containing from about 10 to 18 carbon atoms and aliphatic alcohols containing from about 10 to 18 carbon atoms.

While the lipid mixture described above only includes the presence of members (a) through (c), it is more preferred to incorporate members (d) and/or (e) because the long-term stability and uniformity of size of the lipid particles are theoretically enhanced by the presence of these two optional members.

In a preferred embodiment of the present invention, the five members (including the two optional members) making up the lipid mixture forming the lipid particles of the present invention are combined in a weight ratio of (a):(b):(c):(d):(e) of (l-5):(0.25-3):(0.25-3):(0- 3):(0-3), respectively.

While the first member of the lipid mixture has been described as including glycerol monoesters of saturated carboxylic acids containing from about 10 to 18 carbon atoms, it is contemplated that glycerol monoesters of mono- or polyunsaturated carboxylic acids containing from about 9 to 18 carbon atoms, such as but not limited to the 9-carbon oleic or elaidic acids, are also useful in the construction of the lipid mixture.

It will be understood that the proportions of the members of the lipid mixture may vary depending on several factors, including, but not limited to, the type of cells and/or tissues being targeted for delivery, the therapeutic or diagnostic agent being loaded, the desired dosage of the therapeutic or diagnostic agent, the pharmaceutically-acceptable carrier used, the mode of administration, the presence of other excipients or additives, and so forth. Furthermore, factors that enable the lipid particles to be selectively internalized by targeted diseased tissues and cells include not only the composition of the lipid mixture and the structure of the resulting lipid particles but also the size and molecular weight of the particles as described hereinafter.

The lipid particles of the present invention maintain a desirable particle size distribution, preferably where a major portion of the particles have a mean average particle size ranging from about 0.02 to 0.2 μ (micron), preferably 0.02 μ to 0.1 μ, with varying minor amounts of particles falling above or below the range and some lipid particles only ranging up to about 200 nm. The particle size ranges attainable in the lipid particles of the present invention further lead to enhanced physical and chemical stability over an extended period of time, and substantial reduction in undesirable agglomeration and drug precipitation. Furthermore, this range is particularly suitable for the treatment of cancer; larger particles may be appropriate for other uses (e.g., targeting of other types of cells or tissues). The range provided herein will be determined in part by the lipid mixture employed and the type and amount of the therapeutic or diagnostic agent added.

The therapeutic or diagnostic agents employed in the present invention may be uncharged or charged, nonpolar or polar, natural or synthetic, and so on. The term "therapeutic agent" as used herein includes any substance including, but not limited to, drugs, hormones, vitamins, nutrients, substances, and the like, that affect microtubule production, structure, association, function, and destruction, and thus are useful in prevention and treatment of a disease, condition, syndrome, characterized by cellular hyperproliferation, or symptoms thereof, including cancer. Thus, the therapeutic agents useful in the present invention include all types of drugs, lipophilic polypeptides, cytotoxins, oligonucleotides, cytotoxic antineoplastic agents, antimetabolites, hormones, and radioactive molecules, which affect microtubule production, structure, association, function, and destruction. The term "oligonucleotides" includes both antisense oligonucleotides and sense oligonucleotides, (e.g., nucleic acids conventionally known as vectors). Oligonucleotides may be "natural" or "modified" with regard to subunits or bonds between subunits. However, in a particular aspect of the present invention the therapeutic agent is a microtubule-interacting agent selected from a group consisting of taxanes, such as, for example, paclitaxel, docetaxel, cephalomannine baccatin-III, 10-deacetyl baccatin III, deacetylpaclitaxel, and deacetyl-7-epipaclitaxel; vinca alkaloids, such as, for example, vincristine, vinblastine, vinorelbine, vindesine, and analogs thereof; epothilones; eleutherobins; discodermolide; dolastatins; colchicine; combrestatins; phomopsin A; halichondrin B; spongistatin 1; sarcodictyins; laulimalides; derivatives, analogs, congeners, and combinations of each of the aforementioned agents thereof; and similar drugs or substances known to exhibit such microtubule-interacting activity.

Taxanes such as paclitaxel may also be used in smaller time-release doses as an anti- inflammatory agent. This use is especially important in the field of biomedical devices to be placed surgically within patients, such as stents. While some accumulation of cells around and inside the stent is desirable as this accumulation forms a smooth cover and thereby incorporates the device into the artery itself, such cellular accumulation can also clog the interior channel and cause restenosis of the artery. As a consequence, Boston Scientific Corporation manufactures a paclitaxel-eluting coronary stent system coated with a proprietary polymer which binds paclitaxel onto the stent surface. The paclitaxel-polymer complex allows precise control over the dosage and time-release characteristics for paclitaxel, permitting elution of a sufficient amount of the medication to inhibit cellular accumulation around the stent and significantly prevent restenosis and revascularization around the stent. It is contemplated that the pharmaceutical compositions of the present invention, especially where the therapeutic agent is a taxane or other microtubule-interacting agent, will be similarly useful to regulate cellular accumulation around surgically-implanted biomedical devices.

The pharmaceutical compositions of the present invention exhibit long-term physical and chemical stability, allowing such compositions to be conveniently pre-packaged into stable, ready-to-administer dosage forms and thereby eliminating the need for the bedside dilution and formulation prior to administration typically associated with similar compositions in the prior art. The pharmaceutical compositions of the present invention exhibit desirable drug and emulsion stability over an extended time period (e.g., at least 14 days at about 30⁰C and at least 12 months at 4⁰C). The pharmaceutical compositions of the present invention contain lipid particles in an amount of from about 0.1 μg/mL to 1000 μg/mL, preferably from about 10 μg/mL to 800 μg/mL, and most preferably from about 200 μg/mL to 600 μg/mL. Typical concentrations of the therapeutic or diagnostic agent based on the total volume of the pharmaceutical composition may be at least 0.001% w/v, preferably 0.001% to 90% w/v, and more preferably from about 0.1% to 25% w/v. The amount of the therapeutic or diagnostic agent present in the pharmaceutical composition may range from about 0.001 μg/mL to 1000 μg/mL, preferably from about 0.1 μg/mL to 800 μg/mL, and more preferably from about 60 μg/mL to 400 μg/mL.

The pharmaceutical composition of the present invention may further include emulsion-enhancing agents selected from a plant-based fat source, a solvent, a surfactant, or combinations thereof. The emulsion-enhancing agents have been found, individually or in combination, to enhance the stability and maintain the small particle size properties of the lipid particles theoretically by reducing or minimizing undesirable precipitation or aggregation of the lipid particles, thereby positively influencing and facilitating the active uptake of the lipid particles into the cancer cells. The emulsion-enhancing agents should also improve the physical and chemical stability and drug-carrying capacity of the pharmaceutical compositions of the present invention.

In a preferred embodiment of the present invention, the plant-based fat sources include vegetable-derived fatty acids generally in the form of vegetable oil, such as, for example, soybean oil, flaxseed oil, hemp oil, linseed oil, mustard oil, rapeseed oil, canola oil, safflower oil, sesame oil, sunflower oil, grape seed oil, almond oil, apricot oil, castor oil, corn oil, cottonseed oil, coconut oil, hazelnut oil, neem oil, olive oil, palm oil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil, walnut oil, and mixtures thereof. The more preferred vegetable oil is soybean oil. The vegetable oil is generally present in amounts sufficient to permit higher surface tension in the nanoemulsion which in turn increases the probability of hydrophobic interactions with the plasma membranes of the target cell, or receptors thereupon. The plant- based fat source may be present in amounts of from about 0.001% v/v to 5.0% v/v, more preferably from about 0.005% v/v to 4.0% v/v, and most preferably from about 0.01% v/v to 2.5% v/v.

In another preferred embodiment of the present invention, the surfactants are those selected from non-ionic surfactants. Examples of non-ionic surfactants include sorbitan esters and mixtures thereof, such as fatty-acylated sorbitan esters and polyoxyethylene derivatives thereof, and mixtures thereof including, but not limited to, Poloxamer compounds (188, 182, 407 and 908), Tyloxapol, Polysorbate 20, 60 and 80, sodium glycolate, sodium dodecyl sulfate and the like, and combinations thereof. More preferred non-ionic surfactants are detergent polysorbates, such as, for example, Tween®-80.

The surfactant is generally present in amounts sufficient to increase the kinetic stability of the nanoemulsion by stabilizing the interface between the hydrophobic and hydrophilic components of the nanoemulsion and keeping the hydrophobic components from coalescing, such that, once formed, the nanoemulsion does not significantly change in storage. The surfactant may be present in amounts of from about 0.01% w/v to 4.0% w/v, more preferably from about 0.1% w/v to 3.0% w/v, and most preferably from about 0.2% w/v to 2.5% w/v. In yet another preferred embodiment of the present invention, the solvents include any pharmaceutically-acceptable water-miscible diluents or solvents such as, for example, polar protic and polar aprotic solvents. Such solvents are preferably selected from 1,3-butanediol; dimethyl sulfoxide; alcohols such as methanol, butanol, benzyl alcohol, isopropanol, and ethanol; and the like. A more preferred solvent is benzyl alcohol. The solvent is generally present in amounts sufficient to control the extent of the aggregation of non-ionic surfactants in the nanoemulsion. The solvent may be present in amounts of from about 0.001% v/v to 99.9% v/v, more preferably 0.005% v/v to 80% v/v, and most preferably from about 0.005% v/v to 70% v/v.

The composition of the present invention does not modify or alter the underlying pharmacological activity or chemical properties of the therapeutic or diagnostic agent but simply enhances the agent's delivery to and internalization into the diseased cell or tissue, including cancerous cells or tissue, to impart therapeutic or diagnostic benefits. Examples of teachings related to the use of taxanes as therapeutic agents in treating cancer are disclosed, for example, in U.S. Patent Nos. 6346543; 6384071; 6387946; 6395771; 6403634; and 6500858, each incorporated herein by reference. Generally, the pharmaceutical compositions of the present invention are prepared by combining the lipid particles with the therapeutic or diagnostic agent and thoroughly mixing the same. The lipid mixture may be mixed with a surfactant in combination with a plant- based fat source prior to mixing with the therapeutic or diagnostic agent, which themselves may be mixed with a water-miscible solvent for dissolution. The lipid particle- therapeutic/diagnostic agent combination is then mixed with water, preferably purified water. The resulting mixture is then subjected to high shear forces typically produced in standard conventional shear-intensive homogenizing mixers or homogenizers to produce a nanoemulsion comprising the lipid particles dispersed within the aqueous phase. Sufficient high shear forces can be produced with a suitable shear-intensive homogenizing mixer or homogenizer such as Microfluidizer® Fluid Materials Processors marketed by Microfluidics of Newton, MA. The resulting nanoemulsion may be further treated to yield a more purified form, which may be used for administration to warm-blooded animals, including humans.

In some instances, it may be desirable to remove unduly large particles from the mixing process, so as to maintain the particle size distribution within a desired range. Suitable filtration systems, such as those from Millipore Corporation of Waltham, Massachusetts, are available for this purpose. The selection of a suitable filter system therefore may be a factor in controlling the particle size distribution of the lipid particles within a desirable range and is within the routine skill of the skilled artisan. Alternatively, the nanoemulsion may be processed through dialysis to remove the impurities, with the resulting dialysate retained for pharmaceutical use. Dialysis is a preferred method of removing any non-particulated lipid mixture components, drugs, and/or solvents and achieving any desired buffer exchange or concentration. Dialysis membrane nominal molecular weight cutoffs of 5000 to 500000 can be used, with a molecular weight of 10000 to 300000 being preferred. The lipid particles produced as described, when purified such as by dialysis to remove non-particulated drug, may be characterized to determine the extent to which the lipid particles may be internalized in targeted cells, such as, for example, C₆ glioma cells.

The compositions of the present invention may further include a pharmaceutically- acceptable carrier or excipients. Examples of pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof. The amount of such additives depends on the properties desired, which can readily be determined by one skilled in the art.

The pharmaceutical compositions of the present invention may routinely contain salts, buffering agents, preservatives, and compatible carriers, optionally in combination with other therapeutic ingredients. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically- and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

The present invention additionally provides methods for treating or diagnosing a patient with therapeutic or diagnostic agents by delivering an effective amount of at least one therapeutic or diagnostic agent to cells for implementing the prevention, diagnosis, or treatment of a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof. Improved treatments of cancer are especially contemplated, including treatment of primary tumors by the control of tumoral cell proliferation, angiogenesis, metastatic growth, apoptosis, and treatment of the development of micrometastasis after or concurrent with surgical removal; and radiological or other chemotherapeutic treatment of a primary tumor. The pharmaceutical composition of the present invention is useful in such cancer types as primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer. For therapeutic and diagnostic applications, the pharmaceutical composition can be administered directly to a patient when combined with a pharmaceutically-acceptable carrier. This method may be practiced by administering the therapeutic or diagnostic agent alone or in combination with an effective amount of another therapeutic or diagnostic agent, which may or may not be a second microtubule-interacting agent. When not a microtubule- interacting agent, this second agent may be, but is not limited to, a cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a podophyllotoxin, an antitumor antibiotic, a chemotherapeutic agent, an apoptosis-inducing agent, and combinations thereof. Such therapeutic agents may further include metabolic inhibition reagents. Many such therapeutic agents are known in the art. The combination treatment method provides for simultaneous, sequential, or separate use in treating such conditions as needed to amplify or ensure patient response to the treatment method.

The methods of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Although formulations specifically suited for parenteral administration are preferred, the compositions of the present invention can also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. If such a formulation is desired, other additives well-known in the art may be included to impart the desired consistency and other properties to the formulation.

Those skilled in the art will recognize that the particular mode of administering the therapeutic or diagnostic agent depends on the particular agent selected; whether the administration is for treatment, diagnosis, or prevention of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being treated or diagnosed; and the dosage required for therapeutic efficacy. For example, a preferred mode of administering an anticancer agent for treatment of leukemia would involve intravenous administration, whereas preferred methods for treating skin cancer could involve topical or intradermal administration. As used herein, "effective amount" refers to the dosage or multiple dosages of the therapeutic or diagnostic agent at which the desired therapeutic or diagnostic effect is achieved. Generally, an effective amount of the therapeutic or diagnostic agent may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired therapeutic effect or in the event of any complication. Importantly, when used to treat cancer, the dosage amount of the therapeutic agent used should be sufficient to inhibit or kill tumor cells while leaving normal cells substantially unharmed.

The therapeutic or diagnostic agent included in the pharmaceutical compositions of the present invention can be prepared in any amount desired up to the maximum amount that can be solubilized by, suspended in, or operatively associated with the given lipid particles. The amount of the diagnostic agent or therapeutic agent may range from 0.001 μg/mL to 1000 μg/mL, preferably from about 0.1 μg/mL to 800 μg/mL, and more preferably about 300 μg/mL.

Generally, the lipid particles will be delivered in a manner sufficient to administer an effective amount to the patient. The dosage amount may range from about 0.1 mg/kg to 175 mg/kg, preferably from about 1 mg/kg to 80 mg/kg, and more preferably 5 mg/kg to 60 mg/kg. The dosage amount may be administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day. In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance. Multiple doses per day are contemplated to achieve appropriate systemic or targeted levels of the therapeutic or diagnostic agent.

In yet another embodiment of the present invention, the microtubule-interacting agents may be used as diagnostic agents in vitro. As stated earlier, depending on the specific tumor cell or cell type in question, different microtubule-interacting agents may be more or less effective at inhibiting distinct tumor classes. Thus, for example, in cases where diagnosis or selection of an appropriate chemotherapeutic strategy may be difficult, testing of a culture of tumor cells in vitro with microtubule-interacting agents known to target specific tumor cell types provides an alternative approach for identifying tumor types and effective treatments. In another aspect of the present invention, there is provided a method of preparing the pharmaceutical composition of the present invention. The lipid mixture is incorporated into the therapeutic or diagnostic agent in amounts such that, upon processing with an aqueous phase, the composition forms a lipid nanoemulsion comprising a dispersion of lipid particles wherein the dispersed phase of lipid particles are present in the form of macromolecules or clusters of small molecules on the nanoscale order of particle size. In preparing the compositions of the present invention, the lipid mixture and the therapeutic or diagnostic agent are combined with an aqueous phase comprising water, preferably filtered water. The resulting mixture is processed to form lipid particles having a mean average particle size range typically, but not always, in the range of up to 200 nm, a size particularly suited for the treatment of cancer, with larger particles appropriate for other uses. The range obtained will in part be affected by the lipid mixture employed, the type and amount of the therapeutic or diagnostic agent added to the lipid mixture, and the technique used to produce the lipid particles. The pharmaceutical compositions of the present invention can be made using conventional dispersion-producing techniques or processes known in the art. Such techniques include, but are not limited to, high-shear homogenization, ultrasonic agitation or sonication, high-pressure homogenization, solvent emulsification/evaporation, and the like. In one embodiment of the present invention, the lipid particles may be prepared through conventional high-pressure homogenization techniques using a suitable high-pressure homogenizer. Homogenizers of suitable sizes are commercially available. High-pressure homogenizers are generally designed to push a fluid through a narrow gap spanning about a few microns at high pressure, typically from about 100 to 2000 bar. The pressurized fluid accelerates over a very short distance to a very high velocity of over 1000 km/hr. Pressurized fluids containing the lipid mixture encounter very high-shear stress and cavitation forces, effectively disrupting and comminuting the lipid mixture into particles in the submicron range. As previously discussed, a major portion of the lipid particles should have a mean average particle size ranging from about 0.02 μ to 0.2 μ, preferably 0.02 μ to 0.1 μ, with varying minor amounts of particles falling above or below the range, especially with some lipid particles ranging up to about 200 nm.

In another particular method of preparing the lipid particles, the lipid mixture may be mixed with a plant-based fat source, such as a vegetable oil, and a surfactant, such as a non- ionic surfactant, to yield a lipid phase. The therapeutic or diagnostic agent may be mixed with a solvent, such as a water-miscible solvent, to yield a therapeutic or diagnostic agent phase. The lipid and therapeutic or diagnostic agent phases are thereafter mixed and blended together in the presence of an aqueous phase, preferably through sonication. The resulting mixture is thereafter homogenized under high-shear forces to produce the corresponding nanoemulsion of the present invention. The nanoemulsion may then be filtered through a 0.2 μ membrane, sterilizing and/or removing impurities such as unused lipid materials, excess therapeutic or diagnostic agent, and so on, to yield a purified form suitable for delivery as a pharmaceutical composition to warm-blooded animals, including humans, in need of treatment or diagnosis.

The following examples are provided to facilitate understanding of the pharmaceutical compositions of the present invention.

EXAMPLE 1

FORMULATION OF EmPAC

Paclitaxel was selected as the first drug candidate to be tested for formulation via loading onto lipid nanoparticles (LN) and to be developed into a commercially viable product. The paclitaxel/LN formulations were prepared directly in water and 4% ethanol followed by four cycles of high pressure homogenization at 18000 psi using a HOY Microfluidics Microfluidizer® high-pressure homogenizer (Model M-I lOY, Microfluidics, Inc., Newton, MA). Although earlier studies had shown that paclitaxel could be incorporated into LN up to -25% w/w lipid concentration, it was found that the formulations were stable for only a few hours, although such issues seemed more related to stability than to drug incorporation. After the finding that LN prepared in ImM sodium pyrophosphate (pH 9.5) remained stable for at least two months, ImM sodium pyrophosphate (pH 9.5) was selected as the first trial medium for the drug incorporation study.

It was found that if paclitaxel was incorporated into LN with 4% ethanol content in 1 mM sodium pyrophosphate at 15000 psi at 3 passes, only the formulation with 5% drug loading is stable, and even this last formulation was not stable for a significant time period. On the second day after incorporation, slight turbidity was observed in the 5% loaded sample, which became complete precipitation on the third day. While immediate cloudiness observed in samples at 33%, and 16% drug loading. These findings are summarized in Table 1. The possible reasons for such decreased stability could be poor solubility of paclitaxel in water, low alcohol content (4%), high pH of the final solution (pH 11.5), and/or incompatible lipid concentrations. Table 1. Paclitaxel loading into LN prepared in 4% ethanol

Lipid cone. % Drug Particle size Zeta potential (mV) Description (μg/mL) loading (μm)

200 0.0 0.149 -39.0 clear

200 5.0 0.101 -32.30 clear

200 16.2 — — precipitation

200 33.2 — — precipitation

It is quite possible that varying the organic content, which in this case is ethyl alcohol, could improve stability of drug incorporated particles. Hence, LN were first prepared in different alcohol contents (e.g., 50% v/v, 25% v/v, 12.5% v/v). It was observed that even particles without drug are not stable in ethanol content less than or equal to 25% v/v. Furthermore, only LN at 12.5% v/v alcohol remained stable for at least a day. Accordingly, 10% paclitaxel was loaded onto LN with 12.5 % v/v alcohol content. After three hours, gel formation was observed, strongly suggesting that a high level of alcohol does not stabilize paclitaxel formulations in LN. Ethyl alcohol was also generally used as a demulsifying agent. This could be the reason that even a high percentage of ethanol does not help in getting a stable drug-loaded formulation. Among other solvents, dimethyl sulfoxide (DMSO) is a solvent of choice, as paclitaxel known to have a very good solubility in DMSO. Thus, paclitaxel-loaded samples were prepared in 10% DMSO at 15000 psi and three cycles on a second machine, Microfluidizer® high-pressure homogenizer (Model M-I lOEH, Microfluidics, Inc., Newton, MA) M-I lO EH.

Two controls were used, one with no paclitaxel and another with no lipid components. From the second control, it is clear that lipid components do play a role in the stability of drug-incorporated LN, as this sample precipitated in the absence of lipid components. However, it was observed that the sample with 10% paclitaxel precipitated on the second day, while the sample with 5% paclitaxel remained clear. On high performance liquid chromatography (HPLC) assay of these samples, it was observed that paclitaxel degraded in these samples.

Since paclitaxel is highly soluble in DMSO, the stability of LN prepared with paclitaxel was tested in different concentrations of DMSO. It was seen that a concentration of 20% DMSO resulted in the greatest stability for 200μg/mL LN with 10% w/w paclitaxel. Representative particle size distribution and zeta potential values are shown in FIGURE 1. Data for stability of this modified formulation is shown in Table 2.

Table 2. Stability of paclitaxel-loaded LN in modified DMSO formulation Lipid cone. % Drug Particle size Zeta potential (mV) Description* (μg/mL) loading (μm)

200 0 0.089 -46.36 clear

200 5 0.106 -38.71 clear

200 10 0.093 -46.46 clear

0 10 — — precipitation

^¥Samples were analyzed after 24 hours at room temperature.

It was hypothesized that the aforementioned degradation of paclitaxel, almost 100% within 24 hours, may have been due to high pH (11.0) in the formulation. Addition of 1 mM sodium pyrophosphate decreased pH to 9.5. Thus, the effect on the stability of lipid components as well as of paclitaxel by adjusting the pH via ImM sodium pyrophosphate was studied.

It was found that that at pH equal to or exceeding 6.0, the formulation remained clear, with no precipitation observed. However, there was considerable degradation of paclitaxel at pH exceeding 6.0. Thus, the optimal solution was to utilize 1 mM sodium pyrophosphate with pH adjusted to 6.0 with dilute phosphoric acid in order to keep both lipid components and paclitaxel chemically and physically stable in the formulation. All results are summarized in Table 3.

Table 3. Effect of pH on stability of lipid nanoparticles^¥

As it was thus discovered that DMSO and control of pH can lead to long-term stability of the formulation, the next avenue of study was if filtration affects the lipid components and the drug potency of paclitaxel-loaded LN samples, as filtration may be utilized to sterilize product during manufacturing.

After initial screening for different types of membranes such as Nylon, PVDF, and PES, it was concluded that polyether sulfone (PES) hydrophilic membrane was the most suitable membrane for filtration of the formulation. When 10% paclitaxel-loaded LN prepared in 20% DMSO were filtered through a 0.22 μm PES membrane filter, at least 98% of the paclitaxel passed through the membrane. When lipid components from these the same formulations were analyzed by ELSD, on average, 93% of all the components passed through the membrane. It was thus demonstrated that filtration should enhance the stability as well as the quality of the formulation. To determine if paclitaxel was incorporated into LN in the presence of 20% DMSO or whether DMSO prevents incorporation of paclitaxel into LN by partitioning out paclitaxel into the aqueous phase, separation of ¹⁴C-labelled paclitaxel from LN was performed using a sucrose density gradient. Measurement of the distribution of the radioactive counts provides data as to which fraction contained paclitaxel. Hence, 200 μg/mL of LN was prepared in the presence of 10% w/w paclitaxel, including some ¹⁴C-labelled paclitaxel, and the preparation was fractionated on a sucrose density gradient. Fractions were collected for analysis of radioactive counts in order to determine which fraction contained paclitaxel. A precipitated fraction was found at the bottom of the gradient, which consisted primarily of insoluble paclitaxel. A band was found in the gradient which was established to be LN, and radioactive contents in both the precipitate as well as the LN fraction were analyzed, hi the presence of lipids comprising LN, very little of the radioactive label was found in the bottom precipitate. In the absence of the lipids, a greater amount of precipitate is found, and a much smaller fraction of the radiolabel is found in the precipitate fraction.

As seen in FIGURE 2, then, approximately 80% of the paclitaxel precipitated out in the absence of the lipids, whereas only 1.8% of the paclitaxel precipitated in the presence of lipids. This supports the notion that most of the paclitaxel is incorporated into the lipids in LN.

In the clinical setting, intravenous infusion of approximately 2.5L over a 3-24 hour period is generally considered to be safe and tolerable for most patients. The dosage of paclitaxel currently in clinical use ranges from 135 to 175 mg/m² over a 3-24 hour period. Assuming an average-sized male of ~ 1.8 m² surface area, a concentration of ~97 μg/mL of paclitaxel is require to achieve such a dosage. In order to deliver such a therapeutic dosage of paclitaxel within these time and volume parameters, paclitaxel must be at sufficiently high concentration. Consequently, it is desirable to formulate a paclitaxel-loaded LN sample achieving such a dosage as a benchmark. However, since the present invention is capable of selectively targeting tumor cells, the therapeutic dosage necessary for paclitaxel-incorporated LN may actually be much lower. Thus, a much greater fraction of drugs administered will end up in the tumor cells.

As evident later in Table 4, it is possible to prepare up to 400 μg/mL of LN, with 10% paclitaxel loading, that is stable for at least 5 hours without signs of aggregation. This preparation has a paclitaxel concentration of 40 μg/mL and was stable over the time necessary for intravenous administration.

After filtration through a 0.22 μm PES membrane, a sample of 10% paclitaxel in 20% DMSO in 1 mM sodium pyrophosphate (pH-6.0) loaded onto LN remained stable for at least three days. After changing the sodium pyrophosphate concentration to 0.5 mM, the sample remained stable for 7 days. Initially, all samples were analyzed in filtered and unfiltered form for paclitaxel content and particle size distribution. These data thus suggested that a paclitaxel formulation of at concentrations as those currently utilized clinically can be prepared in LN.

Accordingly, a second paclitaxel-LN formulation was prepared, consisting of 60 μg/mL paclitaxel; 300 μg/mL lipid mixture; 0.5% butanol; 0.5% soybean oil; and 0.25% Tween®-80. Stability studies performed on this second formulation revealed that formulations using 400 μg/mL of lipids and a constant concentration of paclitaxel are stable for at least 24 hours. Results are summarized in Table 4.

Table 4. Effect of lipid concentration on nanoparticle stability

Lipid Cone. Paclitaxel load (% Time after Description Mean particle (μg/mL) w/w) preparation diameter (nm)

200 20 0 hour ^δ clear 114

5 hours ^δ clear 113

24 hours * clear 126

400 10 0 hour * clear 153

5 hours ^δ clear 145

24 hours * clear 180

600 5 0 hour ^δ clear 162

5 hours ^δ clear 170

24 hours * turbid 286

⁸ Samples were kept at room temperature ¥ Samples were kept frozen at -80°C.

Specifically, it was found that, as seen in Table 5, the second paclitaxel-LN formulation was stable at room temperature for at least seven days and at 2-8°C for at least thirty days. Table 5. Stability of second formulation

However, as butanol was found to be toxic in mice, causing lethargy upon injection (data not shown), the butanol was replaced by benzyl alcohol. The resulting third formulation consists of 60 μg/mL paclitaxel, 400 μg/mL lipid mixture, 0.06% benzyl alcohol, 0.5% soybean oil, and 0.25% Tween®-80. This third formulation was found to be stable for at least three weeks at room temperature and for over eighty days at 2-8°C, as summarized in Table 6.

Table 6. Stability of third formulation

EXAMPLE 2 CELL UPTAKE OF EMULSIPHAN AND EmPAC The studies described herein were performed in order to determine if human tumor cells take up LN and if they display differential ability to take up LN. It was found not only that most tumor cell lines tested took up fluorescent LN readily but also that tumor cell lines from different tumor cell lineages displayed differential ability to take up LN.

The LN formulation used for these experiments were prepared as follows: the appropriate lipids were solubilized in 95% ethanol to 10mg/mL by sonication for 10 minutes. Next, 100 μL of 0.5mg/mL cholesteryl BODIPY-FL (Molecular Probes, Eugene, OR) in ethanol was added to 1 mL of the 10 mg/mL solubilized lipids. Lastly, the lipid and cholesteryl BODIPY-FL mixture was added to 50 mL of a solution of 1 mM sodium pyrophosphate in water and processed through a HOY Microfluidics Microfluidizer® high- pressure homogenizer (Model M-11OY, Microfluidics, Inc., Newton, MA). In order to first establish that extent of cell uptake of fluorescent-labelled nanoparticles is directly proportional to cell fluorescence intensity through fluorescence- activated cell sorting (FACS), it was determined that average fluorescence intensity per cell is directly proportional to concentration of LN, as seen in FIGURE 3A, and also to time of incubation in the presence of LN, as evident from FIGURE 3B. Indeed, it was visually confirmed that fluorescence intensity was proportional to extent of fluorescent LN uptake with identically treated cells fixed on glass coverslips (data not shown). The LN were labelled with the fluorescent lipophilic dye DiO (Molecular Probes, Eugene, OR), which can be detected using filter sets for fluorescein. LN was prepared with 200μg/mL lipids and 2.5μg/mL DiO by microfluidization. Labelled LN was added to C6 cells and incubated at 37°C. After this, media was removed, the cells washed with phosphate buffered saline (PBS), trypsinized, and washed again before fixation in 4% formaldehyde. LN was added at 0, 12.5, 25, 50, and lOOμg/mL and incubated for 60 minutes. 50μg/mL LN was added to cells and incubated for 0, 5, 10,15, 30, and 60 minutes before removal of media and processing for FACS analysis. As evident from the results seen in FIGURES 3A and 3B, which respectively show that fluorescence intensity per cell was directly proportional to increased concentrations and to increased incubation time, it is evident that LN uptake can be assessed by FACS.

In order to determine if cells from different cell lineages display differential ability to take up LN and also to assess relative abilities of cells from different tumor types to take up LN, LN uptake was tested in a diverse panel of tumor cell lines. The cell lines chosen were selected from those used by the Developmental Therapeutics Program In Vitro Screening of the National Cancer Institute (NCI). The NCI cell line panel consists of cell lines derived from a number of different human tumor lineages, with several different cell lines from each represented human tumor lineage. The cell lines used included HS-578T, MDA-MB-231, and MX-I breast cancer; H23, H460, and H522 lung cancer; SF-539 liver; and HT29, SW- 620, and COLO205 colon tumor cell lines.

LN was prepared with 200μg/mL lipids and 0.5% w/w cholesteryl-BODIPY-FL, by microfluidization. Labelled LN was added to cells and incubated at 37°C. After this, media was removed, the cells washed with PBS, trypsinized, and washed again before fixation in 4% formaldehyde. Samples were analyzed by FACS and average fluorescence intensity per cell was determined. Cells derived from colon, breast, central nervous system (CNS), and lung were compared to those of the HT-29 colon carcinoma cell line and SF-539 lung cancer cell line, which have been shown to take up low and high quantities of fluorescent LN, respectively. Fluorescent LN uptake by each LN-treated cell sample was obtained by subtracting the fluorescence intensity from the same cell line, which had been untreated.

As seen in FIGURES 4A through 4D, comparison of LN uptake by cell lines from the NCI panel reveals that LN uptake varies among cell types of different lineages. Although some variability was seen among cells from the same tumor lineage, relative uptake was fairly consistent for each tumor lineage. For example, breast and lung tumor cell lines generally displayed higher LN uptake than did colon tumor cell lines. The greatest LN uptake was found in cell lines from lung tumors and from the CNS. All results are summarized in Table 7.

Table 7. Uptake of lipid nanoparticles by tumor cell lines

⁸ Average fluorescence intensity per cell was obtained for each fluorescent LN-treated sample, by comparing and subtracting fluorescence from the same respective untreated cell line.

^£ ND- Not determined. Particle diameter not determinable because of high level of precipitation.

Next, the uptake of paclitaxel formulated in LN (EmPAC) was compared with paclitaxel formulated in the traditional vehicle, Cremophor/ethanol 1 :1 (Cremophor EL®), known as Taxol®, in a series of experiments analyzing cultured tumor cell uptake of radiolabeled paclitaxel.

In the first experiment, ^l4C-labelled paclitaxel was incorporated into LN (thereby forming EmPAC) and in Taxol®. EmPAC formulation used in this experiment was the first formulation containing DMSO (see Example 1). These formulations, consisting of the same concentration of paclitaxel, were added to SF539 glioma and A549 lung cancer cells in 12- well replicates for 1 hour. Media was removed, and cell monolayers were solubilized after removal of drug and washing in PBS with identical volumes of scintillation cocktail containing toluene to determine the paclitaxel associated with each monolayer sample. Radiolabel counts for each sample was expressed as a fraction of the total radiolabel added initially to each sample. Results from this experiment suggested that SF539 internalized significantly greater paclitaxel formulated in LN (i.e., EmPAC) than Taxol®, as demonstrated in FIGURE 5.

This experiment was repeated essentially as described previously, except that cells were treated with the second formulation of EmPAC and were harvested at various times after incubation in drug. Essentially the same amount of radiolabeled drug for each formulation was added to each cell sample. Shown are raw counts of radiolabeled drug associated with cell monolayers. Additionally, paclitaxel uptake was analyzed as a function of time of drug incubation, in order to assess kinetics of cell uptake. Previous studies with SF539 glioma cells demonstrated that uptake increased over a period of two hours before reaching a plateau. This was also observed for both EmPAC and for Taxol®, suggesting the difference was not due to relative insolubility of paclitaxel in the Cremophor EL® formulation, as seen in FIGURE 6. However, there was a significantly greater amount of paclitaxel in EmPAC associated with these cells than for Taxol®. To determine whether paclitaxel or the lipid mixture in EmPAC can saturate cell uptake, an experiment was conducted in which total unlabelled paclitaxel was varied but lipid and radiolabeled paclitaxel amounts held constant. As seen in FIGURE 7, it appears that paclitaxel amount is saturating for cell uptake, although this is not entirely clear from this particular experiment. Since labelled paclitaxel is constant, while total paclitaxel is varied, it may merely represent a smaller or larger fraction of total paclitaxel. Increased concentration of paclitaxel may also increase the total number of particles. Therefore, even if the same number of drug-loaded particles is taken up, a smaller amount of labelled paclitaxel may be taken up. Subsequently, a series of experiments was performed to ascertain the contributions of each lipid component of LN on cell uptake. LN samples labelled by trace substitution of ¹⁴C- labelled cholesterol were prepared with systematic absence of one component. The LN sample was prepared using 300 μg/mL lipids and 20% DMSO. All other components were increased proportionately to compensate for the missing component. This sample was added to SF539 human glioma cells for two hours. The same experiment was also performed using single components mixed 1 : 1 with cholesterol to make LN particles. FIGURES 8 A and 8B together confirm that cholesterol plays the most important role of all the lipid components found in LN in promoting cell uptake.

Although it was hypothesized that the radiolabeled paclitaxel associated with cell monolayers were a result of internalization of paclitaxel, a formal possibility existed that the paclitaxel was located on the outside of the cells, rather than being internalized. In order to determine if paclitaxel is internalized, A549 human lung tumor cells were treated with paclitaxel formulated in EmPAC or in Cremophor EL® for 2 hours. Cells were subsequently fixed with ice-cold methanol, stained with an anti-taxane antibody (Hawaii Biotech, Honolulu, HI), followed by a secondary antibody conjugated to the fluorescent Alexa Fluor 488 molecule (Molecular Probes, Eugene, OR). It is evident from FIGURE 9A that paclitaxel is internalized and is localized to microtubules. Since paclitaxel is known to bind to microtubules in vivo, this suggests that paclitaxel formulated in EmPAC, as well as in Cremophor EL®, is internalized. However, the intracellular fluorescence intensity of a number of fields of cells for each sample was also quantified by tracing the edges of each cell and quantifying the fluorescence intensity within the boundaries traced. It was found, as seen in FIGURE 9B, that EmPAC-treated cells had roughly twice the fluorescence intensity of that of cell treated with Taxol®, thereby confirming earlier findings.

EXAMPLE 3

CYTOTOXICITY OF EmPAC

The objective of this set of experiments was to determine the relative tumor growth inhibition and cytotoxicity of EmPAC. EmPAC was compared either with paclitaxel dissolved in DMSO, as recommended by the manufacturer, or Taxol®. To determine if paclitaxel incorporated into LN retains the ability to kill cells, the cytotoxic effects of paclitaxel alone, dissolved into a DMSO stock solution, were compared with equimolar amounts of EmPAC in a number of different cell lines. The MTS cell proliferation assay was used to assess relative cytotoxicity. MTS is a tetrazolium compound, which is bioreduced by live cells into a soluble formazan product. The absorbance of the formazan product at 490 run can be used to determine the relative number of living cells. Thus, the MTS can be used to assess the relative cytotoxic potency of paclitaxel alone versus that of EmPAC. The cell lines tested included the uterine sarcoma cell line MES-SA, and its drug-resistant sub-line MES-S A-DX5, to see if incorporation of paclitaxel into LN impacts paclitaxel's cytotoxicity in drug-resistant cells. Accordingly, MES-SA, MES-SA-DX5, A549, and MX-I cell lines were plated at subconfluent density. Equimolar concentrations of paclitaxel alone and EmPAC, diluted in tissue culture media, were added to cells the day after cells were plated and allowed to incubate with the cells for 72 hours. Media was replaced, and MTS assay was performed according to instructions from the manufacturer (Promega Corp, Madison, WI). Percent survival was calculated based on normalization to untreated cells from each respective cell line. Each data point in FIGURES HA through HD represents the mean of 6 samples ± SEM.

As seen in FIGURE 1OA, EmPAC displayed roughly less cytotoxicity compared to paclitaxel in DMSO in MES-SA and A549 cells at lower paclitaxel concentrations but appeared to kill a greater fraction of cells at higher concentrations than paclitaxel alone, as also seen in FIGURE 1OC. This may have been due to the fact that paclitaxel alone is extremely insoluble in aqueous media; its hydrophobic properties cause paclitaxel to aggregate at higher concentrations, precluding its entry into cells. Incorporation of paclitaxel into LN may have allowed paclitaxel to remain stable in aqueous media, preventing aggregation and allowing higher concentrations of paclitaxel to enter the cells. EmPAC also displayed significantly greater cytotoxicity than paclitaxel alone in MX-I breast cancer cells, as evident in FIGURE 10D, and moderately greater cytotoxicity than paclitaxel alone towards the MES-S A-DX5 cell line, as seen in FIGURE 1OB. Although the mechanism underlying these observations is unknown, one hypothesis is that, in the case of the drug-resistant cell line MES-SA DX-5, EmPAC does not get pumped out of cells as efficiently as does paclitaxel alone because it is buried in lipids and is, at least initially, treated as a component of LN. Lipid particles are taken up into cells by specific mechanisms, which may be unaffected by the cell machinery underlying drug resistance.

The aforementioned experiment was modified to compare EmPAC versus Cremophor EL®. The EmPAC formulation used in this experiment was prepared by solubilization of proprietary lipid mixture in the presence of Tween®-80 and soybean oil by sonication, followed by addition of this mixture to paclitaxel that had been solubilized by sonication in butanol. The mixture was processed by microfluidization on a 11 OEH Microfluidics Microfluidizer® high-pressure homogenizer (Model M-I lOY, Microfluidics, Inc., Newton, MA). These paclitaxel formulations were compared after exposure to SF539 glioma cells for one hour before drug removal, as this short-term exposure to drugs more closely mimics in vivo tumor cell exposure to anti-tumor drugs than continuous exposure to drugs, since paclitaxel has a bioavailable half-life in vivo of less than one hour (Wiernik et ah, 1987; Rowinsky et al, 1990).

After removal of drugs, cells were subsequently allowed to proliferate for another 72 hours before survival and proliferation was assayed by MTT. The MTT assay utilizes mitochondrial dehydrogenase present in live cells to measure cell viability, as live cells are expected to have higher dehydrogenase activity than dying or dead cells and thus greater

MTT activity level. Survival of each cell sample was calculated as fraction relative to the respective control cell line, which was exposed to no drug. Percent survival was plotted as a function of drug concentration, each curve was fitted on a four-parameter logistic curve, and the EC₅₀ value was calculated. Statistical significance between EmPAC versus Cremophor

EL® was assessed for each drug concentration by student's t-test. As seen in FIGURE 11, while EmPAC alone had no significant effect on cell killing and cell survival (data not shown), EmPAC was significantly more cytotoxic to the glioma cells than Taxol®.

EXAMPLE 5 TUMOR GROWTH INHIBITION OF EmPAC IN MOUSE TUMOR MODELS

When nude mice subcutaneously implanted with H23 human lung tumor were treated with EmPAC or with equivalent doses of Taxol®, it was observed that there was a significantly greater tumor growth inhibition by EmPAC compared to equivalent doses of paclitaxel in Taxol®.

Nude mice were implanted subcutaneously with H23 lung tumor cells, which were allowed to grow for 25 days before drug was administered. Tumor-bearing animals were injected IP with 2mL of 60μg/mL drugs, each at days 25, 27, 29, 32, 34, 36, 39, 42. Tumor volumes were measured at each of these injection days. The last tumor volume measurement was at day 45 after tumor implantation. Compositions of the drug formulations injected are shown in Table 8.

Table 8. Animal experimental groups for H23 TGI

Over a period of 20 days after the first injection of drugs, as seen in FIGURE 12 A, tumors in animals treated with EmPAC decreased in volume over the entire time period while rumors in animals treated with Taxol® displayed regression in tumor volume for the first eleven days before resuming a course of steady growth for the rest of the experiment. Tumors from animals treated with LN controls or untreated animals displayed steady growth throughout the study. Thus, EmPAC was able to reduce tumor sizes for a longer time than did Taxol®. Data in FIGURE 12A were plotted to show mean tumor volume over the course of drug treatment. Turning to FIGURE 12B, percent tumor regression was determined by comparing tumor volume differences between the first and last days of drug treatment. A statistically significant difference in tumor regression was shown between mice treated with Taxol® and mice treated with EmPAC (p< 0.0005). Each group represents the mean of 3 ± SEM. By the end of the study, tumors from animals treated with EmPAC regressed by approximately 71% (71.4 ± 2.4%), whereas tumors from animals treated with Taxol® regressed by approximately 19% (18.7 ± 0.9%). The data therefore suggests that EmPAC has significantly greater antitumor activity than Taxol®.

To compare amounts of tumor growth inhibition by EmPAC with different concentrations of paclitaxel, nude mice implanted with H460 human lung tumor cells were injected with EmPAC formulated with twofold more paclitaxel than in the third formulation of EXAMPLE 1. Taxol® was also administered at one to four times the dose given in previous TGI experiments. Results show that EmPAC is roughly as efficacious as fourfold higher Taxol®. In this experiment, EmPAC formulations of 60 and 137 μg/mL paclitaxel and equivalent amounts of paclitaxel were used, hi addition, Taxol® at roughly four times the dose given at the original third EmPAC formulation (i.e., 60 μg/mL paclitaxel) was administered to H460 tumor-bearing mice. Mice tumors were allowed to grow to a larger size than in previous experiments. Animals were dosed with drug three times weekly, with two days off for 3 weeks, with tumors measured before injection of drug such that animals whose tumors reached above a cutoff size limit were automatically euthanized. Relative tumor volumes over time were calculated and plotted as a function of time. T-tests were performed to determine if there were significant differences in tumor growth inhibition between animals treated with different drugs. In addition, a survival curve was generated in which the terminal endpoint was defined as animal death by treatment-related causes or by euthanasia, from the tumors reaching a cutoff size limit.

Unfortunately, the animal groups were not randomized, so that control group animals started out with the smallest tumors while those treated with the higher dose drugs started out with larger tumors before treatment. Nevertheless, the results suggest that EmPAC is roughly as efficacious as four times the concentration of Taxol®.

EXAMPLE 6 EmPAC PHARMACOKINETICS AND BIODISTRIBUTION To determine the difference in EmPAC pharmacokinetics for IP versus IV, and the biodistribution of EmPAC, nude mice implanted with A549 lung tumors were injected IP or IV with either EmPAC or paclitaxel in Taxol®, each containing radiolabeled paclitaxel. EmPAC formulation containing ¹⁴C-labelled paclitaxel was prepared as follows: A) PREPARATION OF BUFFER. 1) To IL of water add 466.1 mg of sodium pyrophosphate to make a ImM sodium pyrophosphate solution.

2) Adjust pH to 4.0 by adding dilute phosphoric acid.

3) Filter through 0.22 μm PES filter.

4) Add 10 μL of 10% Tween®-80 to 10 mL of this buffer. B) LIPIDS STOCK SOLUTION.

1) Measure lOmg of lipid mixture powder and dissolve in 10 mL of DMSO to make a lmg/mL lipid/DMSO solution.

2) Sonicate until a clear solution is obtained. C) HOT PACLITAXEL: 1) Take 50 μL of hot paclitaxel. 2) Evaporate the ethyl acetate completely in 8O⁰C water bath. D) FORMULATION: Add 800 μL of lipid/DMSO solution to the hot paclitaxel solution. Mix completely and vortex. Take this solution and then add to 3.2 mL of aqueous buffer. Animals were euthanized 0 (immediately after injection), 0.5, 1, 6, or 12 hours after injection. Blood was drawn by cardiac puncture, and tissues from selected organs (e.g., whole brain, kidney, lung, liver, stomach, spleen, pancreas, tumor, muscle from contralateral side from tumor, urine, feces, whole blood, and tumor muscle from under tumor) were harvested. All tissues and blood were processed for counting of radiolabeled drug. All tissues were weighed and homogenized. Since the tissues were too small to be accurately weighed by the scale used (scale reads to one decimal place), tissue homogenates, including buffers used, were weighed. Aliquots of the homogenates were weighed before scintillation counting in order to determine the fraction of total homogenate. Total ¹⁴-C-labelled paclitaxel in whole organ tissues were determined. Results suggest that there is no significant difference in pharmacokinetics between drugs that are injected IP versus IV. These results also suggest similar pharmacokinetics between paclitaxel in EmPAC versus paclitaxel in Taxol®. Similar experiments where the tissues were harvested at 0.25, 0.75, 3, 6, or 12 hours after injection suggest that EmPAC and Taxol® has essentially the same pharmacokinetic profile as well, with tissue paclitaxel levels peaking at approximately three hours after injection. Furthermore, upon changing the EmPAC formulation to the third formulation described in EXAMPLE 1, results suggest again that EmPAC formulation #3 has the same pharmacokinetic profile as that of Taxol®. EXAMPLE 7

CELLULAR UPTAKE OF PACLIT AXEL FORMULATED AS EmPAC COMPARED WITH PACLITAXEL FORMULATED AS TAXOL® AND AS ABRAXANE®

Taxol® (paclitaxel in Cremophor EL®:ethanol 1 :1) has been in clinical use for a number of years to treat a variety of different cancer indications, including non small cell lung cancer and breast cancer. Abraxane®, a formulation of paclitaxel formulated with HSA, has been approved by the FDA for cancer treatment. The objective of this study was to determine if EmPAC differs from Taxol® and from Abraxane® in influencing the cellular uptake of paclitaxel in vitro in short term exposure experiments using A549 human lung cancer and MDA MB 435 breast cancer cell lines exposed for one hour to each paclitaxel formulation. The cells were then fixed and stained with antibodies against paclitaxel, followed by fluorescently-labelled secondary antibodies. Intracellular paclitaxel, as detectable by intracellular fluorescence staining was visualized by epifluorescence microscopy. Digital images of fluorescently-labelled cells were captured with a cooled CCD camera. Fluorescence intensity of labelled cells was quantified using digital imaging software, and mean intracellular fluorescence intensity for cells in each experimental group was compared.

MATERIALS AND METHODS Cell lines

A549 human non small cell lung tumor cells, purchased from American Type Culture Collection (ATCC), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) and subcultured at 1 :3 to 1 :8 ratio. MDA MB 435 human breast cancer cells, purchased from ATCC, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) and subcultured at 1 :3 to 1 :8 ratio.

Test articles

Taxol® (Lot #729537) was obtained from Ben Venue Laboratories, Inc., Bedford, OH.

EmPAC (Batch #T343) was prepared by combining 90.42 mg paclitaxel dissolved by sonication in 0.3 mL benzyl alcohol; a sonicated mixture containing 5.6 g soybean oil, 6.61 g Tween®80. and 123.23 mg lipid mixture powder; and 300 mL HPLC-grade water, and emulsifying by high pressure homogenization at 1250 bar. 1O g of dextrose was then added to 200 mL of this solution, sonicated to dissolve, and sterile filtered through a 0.22 μM filter. The resulting product contained 2.0% soybean oil, 2.2% Tween® 80, 400 μg/mL lipid mixture powder, 0.1% benzyl alcohol, and 300 μg/ml paclitaxel (lot # 28042/kl). Abraxane® for Injectable Suspension (Lot #200495, expiration April 2007; Abraxis

BioScience, Inc.), purchased from GlobalRx, Inc (Efland, NC), was supplied as a powder containing 100 mg of paclitaxel and 900 mg of HSA. Abraxane® powder was then reconstituted by adding 20 mL of sterile PBS to create a suspension containing 5 mg/mL paclitaxel 45 mg/mL HSA. Reconstituted Abraxane® was utilized for experiments within 24 hours, according to package instructions, in order to avoid loss of stability.

Reagents

1) Milli Q water

2) Benzyl alcohol, Sigma, Lot# 02748PC 3) Soybean oil, Sigma, Iot#074k0169 4) Tween® 80, Sigma Ultra, lot#073K00641

5) Paclitaxel (R&D grade), Indena, lot#28042/kl

6) Dextrose (anhydrous powder), Fisher, lot#024291

7) Emulsiphan lipids mixture #003/05

Antibodies

1) Mouse anti-paclitaxel IgG. Cat. No. TA 12; Hawaii Biotechnology, Inc., Aiea, Hawaii.

2) AlexaFluor 488-conjugated chicken anti-mouse IgG. Molecular Probes, Inc., Eugene, OR.

Additional antibody staining materials

1) Nunc Lab Tek Cell chamber slide system. Nalge Nunc International, Inc., Rochester, NY. 2) Tris Buffered Saline (TBS). Boston Bioproducts. Worcester, MA.

STUDY DESIGN AND PROCEDURES

The experiment was designed such that the investigator was blinded to the identity of each test article. The test articles were placed into new tubes and were relabelled by a person not directly involved in the experiments; the identity of the test articles was revealed after results were calculated.

To cells grown to >50% confluency, medium was removed and the cell monolayers were washed briefly by addition of 5 mL of PBS followed by aspiration. 2 mL of Trypsin ethylenediaminetetraacetic acid (EDTA) was added to each flask, and the flask was placed in the tissue culture incubator for five minutes. 10 mL of complete medium was added to halt the enzymatic reactions, and cells were disaggregated by repeated resuspension with serological pipet. 10 μL of cells were added to 10 μL of 0.4% Trypan Blue solution, mixed, and -10-20 μL of this cell solution was placed in a chamber of a hemocytometer. The number of viable cells was determined by counting the number of cells that excluded Trypan Blue in the 4 corner squares of the hemocytometer chamber at IOOX total magnification.

The volume of cells needed was determined by the following formula: Volume of cells = (A x IO⁴ cells/mL)(V_totaι of cells needed in mL) ÷ (2 df) (B cells/mL) where: df is dilution factor; A is the number of cells counted on the hemocytometer; and B is the concentration of cells/mL required for the experiment. A549 lung tumor and MDA MB 435 breast cancer cells were passaged into 8-well chamber slides, at 4 x 10⁴ cells/cm². Cells were allowed to incubate overnight for 24 hours before test articles were added.

Test articles, each containing 2.5 μM paclitaxel, and control complete medium without test articles were added to duplicate chamber slide wells and incubated for one hour at standard conditions used to culture mammalian cells (5% CO₂. 95% 02; 37°C). At the end of the incubation period, cell samples were washed once with warm PBS by addition of 1 mL PBS, followed by aspiration of the warm PBS. 3 mL of ice cold methanol were added to each tissue culture well, and cells were fixed in the methanol at -2O⁰C for 15 minutes. Slides were placed in PBS containing 0.02% sodium azide until staining. Cell samples were blocked with 10% normal chicken serum (NCS) in TBS, pH 8.0 for one hour. After blocking solution was removed, cells were incubated with TBS containing 2% NCS and mouse anti-paclitaxel at 1 :100 dilution for one hour and at room temperature. At the end of the primary antibody incubation, cell samples were briefly rinsed with TBS in a wash bottle, and the entire chamber slide was immersed in a Coplin staining jar containing TBS. Washing of chamber slides were accomplished by agitation of TBS with a stir bar on a stir plate for 10 minutes. Chamber slides were washed in three changes of TBS. Cells were stained with TBS containing 2% NCS and chicken anti-mouse IgG conjugated to Alexa Fluor 488, at 1:100 dilution for 30 minutes, at room temperature. At the end of the secondary antibody incubation, cells were briefly rinsed with TBS in a wash bottle and washed as described above. Stained cells were mounted cell with Fluoromount G mounting medium (Southern Biotechnology, Inc.) containing 14.3 μM 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) and covered with a coverslip.

Slides and cells were viewed with epifluorescence microscopy using excitation at 480 nm and emission at 520 nm in order to visualize the Alexa Fluor secondary staining of anti- paclitaxel. DAPI chromosomal staining was visualized using filters of excitation 358 nm and emission at 461 nm. Images were obtained using a Zeiss Axiocam HR digital camera and Axiovision image acquisition and analysis software. Images of paclitaxel staining and DAPI staining were simultaneously captured for each cell field with the Zeiss Axiocam HR digital camera. Intracellular fluorescence intensity per cell area of paclitaxel staining was determined by using the Axiovision software. Images of 8 to 11 non-overlapping fields of cells, each containing 4 to 19 cells, were taken randomly from both of the duplicate wells in each sample. In all, a total of 123-147 cells were analyzed for each cell sample. Cell perimeters were manually defined for each cell by digitally tracing the cell edges with a cursor mouse. Calculations and statistical analyses

Data from fluorescence intensity and cell area were copied into Excel spreadsheets, and mean fluorescence intensity per area for individual cells were calculated in Excel.

To calculate mean cell fluorescence intensity, was used the following formula was used: f(Ii/Aι)+(l2/A₂)+(l₃/Aj)+...(I^A' _n)J/n = Mean fluorescence intensity per cell area where: I is the intensity of each cell area as defined by manual tracing of cell perimeter; A is the area of each defined cell area; and n is the number of cells sampled.

Mean intracellular paclitaxel level, as determined by mean fluorescence intensity per area for each treated sample was compared between cells treated with the different test articles.

Statistical significant differences in mean fluorescence intensity values among EmPAC-, Taxol®-, and Abraxane®-treated cells were compared using Student's t-test.

RESULTS Mean fluorescence intensity of 108-147 A549 cells, each treated with EmPAC,

Taxol®, or Abraxane® was determined. Mean fluorescence intensity per cell for each test article treated A549 cell sample is shown in Table 9. Cells treated with no test article showed no specific staining (data not shown). Therefore, fluorescence intensity of untreated cells was not determined. Also, fluorescence intensity of MDA-MB-435 cells was not determined since they became rounded in shape upon treatment with paclitaxel, which made the cytoplasm and cell periphery difficult to visualize.

Table 9. Intracellular fluorescent paclitaxel in A549 cells exposed for 1 hour to various paclitaxel formulations

EmPAC Abraxane® Taxol®

Relative intracellular 116.73 ± 2.03 86.32 ± 1.54 111.97 ± 1.78 fluorescent paclitaxel (mean N=I 06 N=I 08 N=147 density/area ± SEM)

§Measured from than 109-147 cells in 10-12 fields, for each group.

Table 10. Results of Student t-tests comparing the intracellular fluorescent paclitaxel intensity per cell areas of paclitaxel formulations in A549 tumor cells

As seen in FIGURE 13, cells treated with EmPAC, Taxol®, or Abraxane® all displayed fluorescence staining that had a perinuclear fibrous pattern typical of microtubules This suggests that the anti-paclitaxel staining was specific, since paclitaxel binds avidly to microtubules and is localized primarily on microtubules in cells.

Student's t-test indicated that cells treated with either EmPAC or Taxol® had a significantly higher mean intracellular paclitaxel levels relative to that of Abraxane®, as indicated by mean fluorescence intensity per cell area values (Tables 9 and 10; P<0.0001 for comparison between EmPAC and Abraxane®, and between Taxol®and Abraxane®). It was further found that EmPAC had moderately higher intracellular paclitaxel level than Taxol® (Table 9, 116.7 ± 2.0 and 112 ± 1.8, respectively) although the difference was less than statistically significant (P=0.08).

DISCUSSION/CONCLUSION

The results from this study suggest that both EmPAC and Taxol® formulations permit significantly greater cell uptake of paclitaxel than did Abraxane®. These results appear to be consistent with results of previous studies of cell growth inhibition by these paclitaxel formulations, which suggested that EmPAC and Taxol® possessed greater cell growth inhibition activities relative to Abraxane® when exposed briefly to cells for one hour. Since these three formulations have the same active ingredient, one possible explanation for the differences among these three formulations may be accounted for by their vehicles. In this case, it is possible that the vehicle of Abraxane® inhibits cellular uptake of paclitaxel, relative to the vehicles of EmPAC and Taxol®.

Results from this study are consistent with the results from EXAMPLE 2, in which we compared cellular uptake of radiolabeled paclitaxel formulated in EmPAC with that formulated in Taxol®. However, the results of EXAMPLE 2 suggested significantly greater paclitaxel uptake by EmPAC-treated A549 cells relative to paclitaxel uptake of Taxol®- treated cells. Although greater paclitaxel uptake of EmPAC-treated cells relative to Taxol®- treated cells was observed in this study, the difference was less than statistically significant. This difference between results may be due to the fact that the EmPAC formulation was different in the two studies.

REFERENCES

Ibrahim NK, Desai N, Legha S, Soon-Shiong P, Theriault RL, Rivera E, Esmaeli B, Ring SE, Bedikian A, Hortobagyi GN, and Ellerhorst JA. 2002. Phase I and pharmacokinetics study of ABI-007, a Cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel. Clin. Cancer Res. 8:1038 1044.

Singla AK, Garg A, and Aggarwal D. 2002. Paclitaxel and its formulations. Int. J. Pharm. 235:179-192.

It will be understood that the present invention is directed to a delivery system in the form of a composition for delivering therapeutic and diagnostic agents, including anticancer agents, for treating cancerous cells and tissues. Accordingly, all anticancer agents are within the scope of the present invention as well as all diseased tissues and cells exhibiting aberrant lipid metabolism and elevated uptake of lipids, including cancer cells, which may be treated by such therapeutic agents. The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. Furthermore, while exemplary embodiments have been expressed herein, others practiced in the art may be aware of other designs or uses of the present invention. Thus, while the present invention has been described in connection with exemplary embodiments thereof, it will be understood that many modifications in both design and use will be apparent to those of ordinary skill in the art, and this application is intended to cover any adaptations or variations thereof. It is therefore manifestly intended that this invention be limited only by the claims and the equivalents thereof.

Claims

The invention to be claimed is:

1. A pharmaceutical composition useful for treating or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals, including humans, said pharmaceutical composition comprising: a lipid nanoemulsion comprised of lipid particles each comprising at least one non- bilayer-forming lipid capable of being preferentially and selectively internalized within a diseased cell; an effective amount of at least one therapeutic agent associated with the lipid nanoemulsion; and a pharmaceutically-acceptable carrier, wherein said pharmaceutical composition is produced by processing through homogenization.

2. The pharmaceutical composition of claim 1, wherein the therapeutic agent is a microtubule-interacting agent.

3. The pharmaceutical composition of claim 2, wherein the microtubule- interacting agent is selected from the group consisting of a taxane, a vinca alkaloid, an epothilone, an eleutherobin, a dolastatin, a combrestatin, a sarcodictyin, a laulimalide, discodermolide, colchicine, phomopsin A, halichondrin B, spongistatin 1, and derivatives, analogs, congeners, and combinations thereof.

4. The pharmaceutical composition of claim 3 wherein the taxane is selected from the group consisting of paclitaxel, cephalomannine, docetaxel, baccatin-III, 10-deacetyl baccatin III, deacetylpaclitaxel, deacetyl-7-epipaclitaxel, and derivatives, congeners, analogs, and combinations thereof.

5. The pharmaceutical composition of claim 3, wherein the vinca alkaloid is selected from the group consisting of vincistine, vinblastine, vinorelbine, vindesine, and derivatives, congeners, analogs, and combinations thereof.

6. The pharmaceutical composition of claim 1, wherein the amount of the therapeutic agent is at least 0.001% by weight based on the total volume of the pharmaceutical composition.

7. The pharmaceutical composition of claim 6, wherein the amount of the therapeutic agent is present from about 0.1% to 90% by weight based on the total volume of the pharmaceutical composition.

8. The pharmaceutical composition of claim 1, wherein the non-bilayer-forming lipid is no more than weakly polar.

9. The pharmaceutical composition of claim 8, wherein the non-bilayer-forming lipid is substantially non-polar or neutral.

10. The pharmaceutical composition of claim 8, wherein the non-bilayer-forming lipid is neutral.

11. The pharmaceutical composition of claim 1 , wherein the lipid particles are comprised of a mixture of non-bilayer forming-lipids.

12. The pharmaceutical composition of claim 1, wherein the lipid particles are stably dispersed in an aqueous phase for at least seven days.

13. The pharmaceutical composition of claim 12, wherein the lipid particles are stably dispersed in an aqueous phase for at least fourteen days.

14. The pharmaceutical composition of claim 13, wherein the lipid particles are stably dispersed in an aqueous phase for at least one month.

15. The pharmaceutical composition of claim 14, wherein the lipid particles are stably dispersed in an aqueous phase for at least six months.

16. The pharmaceutical composition of claim 15, wherein the lipid particles are stably dispersed in an aqueous phase for at least one year.

17. The pharmaceutical composition of claim 1, further comprising emulsion- enhancing agents.

18. The pharmaceutical composition of claim 17, wherein the emulsion-enhancing agents are selected from the group consisting of a surfactant, a plant-based fat source, a solvent, and combinations thereof.

19. The pharmaceutical composition of claim 18, wherein the surfactant is a non- ionic surfactant.

20. The pharmaceutical composition of claim 19, wherein the non-ionic surfactant is a detergent polysorbate.

21. The pharmaceutical composition of claim 20, wherein the detergent polysorbate is Tween®-80.

22. The pharmaceutical composition of claim 18, wherein the non-ionic surfactant is a sorbitan ester.

23 The pharmaceutical composition of claim 22, wherein the sorbitan ester is selected from the group consisting of fatty-acylated sorbitan esters and polyoxyethylene derivatives thereof, and mixtures thereof.

24. The pharmaceutical composition of claim 18, wherein the surfactant is present in an amount sufficient to prevent the nanoemulsion from deteriorating in storage.

25. The pharmaceutical composition of claim 24, wherein the surfactant is present in amounts of from about 0.01% w/v to 4.0% w/v.

26. The pharmaceutical composition of claim 25, wherein the surfactant is present in amounts of from about 0.1% w/v to 3.0% w/v.

27. The pharmaceutical composition of claim 18, wherein the solvent is a water- miscible solvent.

28. The pharmaceutical composition of claim 27, wherein the water-miscible solvent is selected from the group consisting of a polar solvent and combinations thereof.

29. The pharmaceutical composition of claim 28, wherein the solvent is selected from the group consisting of a polar aprotic solvent, a polar protic solvent, and combinations thereof.

30. The pharmaceutical composition of claim 29, wherein the polar aprotic solvent is dimethyl sulfoxide.

31. The pharmaceutical composition of claim 29, wherein the polar protic solvent is an alcohol.

32. The pharmaceutical composition of claim 31, wherein the alcohol is selected from the group consisting of butanol, propanol, ethanol, methanol, benzyl alcohol, and combinations thereof.

33. The pharmaceutical composition of claim 18, wherein the solvent is present in an amount sufficient to control the extent of aggregation of the surfactant.

34. The pharmaceutical composition of claim 33, wherein the solvent is present in amounts of from about 0.001% v/v to 99.9% v/v.

35. The pharmaceutical composition of claim 34, wherein the solvent is present in amounts of from about 0.005% v/v to 80% v/v.

36. The pharmaceutical composition of claim 18, wherein the plant-based fat source is vegetable oil.

37. The pharmaceutical composition of claim 36, wherein the vegetable oil is selected from the group consisting of soybean oil, flaxseed oil, hemp oil, linseed oil, mustard oil, rapeseed oil, canola oil, safflower oil, sesame oil, sunflower oil, grape seed oil, almond oil, apricot oil, castor oil, corn oil, cottonseed oil, coconut oil, hazelnut oil, neem oil, olive oil, palm oil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil, walnut oil, and combinations thereof.

38. The pharmaceutical composition of claim 37, wherein the vegetable oil is soybean oil.

39. The pharmaceutical composition of claim 18, wherein the plant-based fat source is present in an amount sufficient to permit higher surface tension in the nanoemulsion, thereby increasing the probability of hydrophobic interactions with the plasma membrane of a target cell.

40. The pharmaceutical composition of claim 39, wherein the plant-based fat source is present in amounts of from about 0.001% v/v to 5.0% v/v.

41. The pharmaceutical composition of claim 40, wherein the plant-based fat source is present in amounts of from about 0.005% v/v to 4.0% v/v.

42. The pharmaceutical composition of claim 1, wherein the at least one non- bilayer-forming lipid is selected from the group consisting of glycerol monoesters of carboxylic acids, aliphatic alcohols, sterol aromatic acid esters, sterols, terpenes, bile acids, alkali metal salts of bile acid, sterol esters of aliphatic acids, sterol esters of sugar acids, esters of sugar acids, esters of aliphatic alcohols, esters of sugars, esters of aliphatic acids, sugar acids, saponins, sapogenins, glycerol, glycerol di-esters of aliphatic acids, glycerol tri- esters of aliphatic acids, glycerol di-esters of aliphatic alcohols, and glycerol tri-esters of aliphatic alcohols, and combinations thereof.

43. The pharmaceutical composition of claim 1, wherein the at least one non- bilayer-forming lipid is in the form of a lipid mixture comprising: a) at least one first member selected from the group consisting of glycerol monoesters of carboxylic acids containing from about 9 to 18 carbon atoms and aliphatic alcohols containing from about 10 to 18 carbon atoms; b) at least one second member selected from the group consisting of sterol aromatic acid esters; and c) at least one third member selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids.

44. The pharmaceutical composition of claim 43, wherein the glycerol monoesters of carboxylic acids are saturated.

45 The pharmaceutical composition of claim 43, wherein the glycerol monoesters of carboxylic acids are unsaturated.

46. The pharmaceutical composition of claim 43, wherein the lipid mixture has a weight ratio of (a):(b):(c) of (l-5):(0.25-3):(0.25-3) based on the total weight of the lipid particles.

47. The pharmaceutical composition of claim 43 wherein the lipid mixture further comprises: d) at least one fourth member selected from the group consisting of sterol esters of aliphatic acids containing from about 1 to 18 carbon atoms; sterol esters of sugar acids; esters of sugar acids and aliphatic alcohols containing from about 10 to 18 carbon atoms; esters of sugars and aliphatic acids containing from about 10 to 18 carbon atoms; sugar acids, saponins; and sapogenins; and e) at least one fifth member selected from the group consisting of glycerol; glycerol di- and tri-esters of aliphatic acids containing from about 10 to 18 carbon atoms; and aliphatic alcohols containing from about 10 to 18 carbon atoms.

48. The pharmaceutical composition of claim 47 wherein the lipid mixture has a weight ratio of (a):(b):(c):(d):(e) of (l-5):(0.25-3):(0.25-3):(0.25-3):(0.25-3) based on the total weight of the lipid particles.

49. The pharmaceutical composition of claim 1, wherein the lipid particles are present in an amount of from about 0.1 μg/mL to 1000 μg/mL.

50. The pharmaceutical composition of claim 49, wherein the lipid particles are present in an amount of from about 10 μg/mL to 800 μg/mL.

51. The pharmaceutical composition of claim 50, wherein the lipid particles are present in an amount of from about 200 μg/mL to 600 μg/mL.

52. The pharmaceutical composition of claim 1, wherein the mean average particle size of the lipid particles is up to 0.2 μ (micron).

53. The pharmaceutical composition of claim 52, wherein the mean average particle size of the lipid particles is from about 0.02 μ to 0.2 μ.

54. A pharmaceutical composition useful for diagnosing a disease, condition, syndrome, or symptoms thereof in warm-blooded animals, including humans, said pharmaceutical composition comprising: a lipid nanoemulsion comprised of lipid particles each comprising at least one non- bilayer-forming lipid capable of being preferentially and selectively internalized within a diseased cell, including a cancer cell; an effective amount of at least one diagnostic agent associated with the lipid nanoemulsion; and a pharmaceutically acceptable carrier, wherein said pharmaceutical composition is produced by processing through homogenization.

55. A method of treating or preventing a disease, condition and symptoms thereof in warm-blooded animals, including humans, comprising administering to the animal an effective amount of the pharmaceutical composition of claim 1.

56. The method of claim 55, wherein the the pharmaceutical composition is imbued upon the surface of a biomedical device surgically inserted into the body of the animal.

57. The method of claim 56, wherein the biomedical device is a stent.

58. A method of preparing the pharmaceutical composition of claim 1, comprising the steps of: a) mixing the at least one non-bilayer-forming lipid with an effective amount of at least one therapeutic agent to yield a lipid portion; b) adding the lipid portion to an aqueous phase to yield a dispersion; and c) agitating the dispersion under high-shear conditions to sufficiently disperse the at least one non-bilayer-forming lipid therethrough to form a lipid nanoemulsion comprised of lipid particles.

59. The method of claim 58, further comprising the step of adding an emulsion- enhancing agent to the pharmaceutical composition.

60. The method of claim 59, wherein the emulsion-enhancing agent is selected from the group consisting of a surfactant, a solvent, a plant-based fat source, and combinations thereof.

61. The method of claim 60, further comprising the step of mixing the surfactant with the at least one non-bilayer-forming lipid prior to the step of mixing with the therapeutic agent.

62. The method of claim 60, further comprising the step of mixing the plant-based fat source with the at least one non-bilayer-forming lipid prior to the step of mixing with the therapeutic agent.

63. The method of claim 60 further comprising the step of mixing the solvent with the therapeutic agent prior to the step of mixing with the at least one non-bilayer forming lipid.

64. The method of claim 58, wherein the agitating step comprises processing the lipid mixture by a fluid homogenization process.

65. The method of claim 64, wherein the fluid homogenization process is selected from the group consisting of high-shear homogenization, ultrasound homogenization, high- pressure homogenization, and combinations thereof.

66. The method of claim 58, further comprising sterilizing the nanoemulsion.

67. The method of claim 58, further comprising processing the nanoemulsion by aseptic filtration.

68. The method of claim 67, wherein the processing step comprises passing the nanoemulsion through a 0.2 μ membrane.

69. A method of diagnosing a disease, condition, syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals, including humans, comprising administering to the warm-blooded animal an effective amount of the pharmaceutical composition of claim 54.