WO2010009075A1

WO2010009075A1 - Methods and compositions comprising crystalline nanoparticles of hydrophobic compounds

Info

Publication number: WO2010009075A1
Application number: PCT/US2009/050463
Authority: WO
Inventors: Leaf Huang; Feng Liu
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2008-07-14
Filing date: 2009-07-14
Publication date: 2010-01-21

Abstract

Provided herein are methods for producing crystalline nanoparticles of hydrophobic drugs, having adsorbed on the surface thereof surface stabilizers and stable dispersions comprising the same. Also provided herein are crystalline nanoparticles comprising crystalline hydrophobic compounds having adsorbed on the surface thereof surface stabilizers and stable dispersions comprising the same. The crystalline nanoparticles have a relatively low percentage of surface stabilizers by weight. Further, methods for treating a disease or unwanted condition, such as a cancer, in a subject by administering the crystalline nanoparticles of hydrophobic drugs or dispersions thereof are provided.

Description

METHODS AND COMPOSITIONS COMPRISING CRYSTALLINE NANOPARTICLES OF HYDROPHOBIC COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to the generation of nanoparticle formulations of hydrophobic compounds.

BACKGROUND OF THE INVENTION

In general, drug discovery entails high risk and the development of novel drugs into commercial products is costly. For example, out of every 6,200 designed and synthesized compounds, merely seven are tested in humans and only three reach phase III studies. Out of the three, only one will successfully make it to the market. The entire process can cost as much as one billion dollars (Leitner and Lindner (2006) Proteomics 6:5418-5434). Most of the failed compounds are due to toxicity, solubility, and bioavailability problems (Cachau et al. (2007) Curr Top Med Chem 7:1537-1540). Recently, the increased number of poorly soluble compounds in the drug discovery and development pipeline has become an industry-wide concern (Sugano et al. (2007) Drug Metab Pharmacokinet 22:225-254). A long-term solution to these bottleneck hurdles may come from the new and developing field of nanoparticle-based drug delivery systems (Mustelin (2006) Nanomed 1 :383-385).

Recent advances in nanotechnology have made it possible to design drug carriers with nanometer-scale features (e.g. nanoparticles) that have the potential to deliver drugs to specific tissues. Nanoparticles represent a broad range of materials, including liposomes, micelles, dendrimers, nanocrystals, metal colloids and fullerenes (Hall et al. (2007) Nanomed 2:789-803), many of which have been successfully used as platforms or carriers for insoluble or poorly soluble drugs to improve their pharmacokinetic and disposition profile. Currently, a few nanoparticle products are on the market and many more novel nanoparticles are being designed and characterized by different laboratories. Several variables, however, determine whether the product obtained in the laboratory can be translated into a commercially viable product (Morrow, Bawa, and Wei (2007) Med Clin North Am 91 :805-843). Even when well-established methodologies are applied, nanoparticle therapeutics pose some challenges (Bawa (2007) Nanomed 2:351-374). One challenge is the high production cost. Considering the drug industry is currently facing increased pressure to reduce healthcare costs, high production costs must be avoided in designing and developing new nanoparticle formulations (Bawa (2007) Nanomed 2:351-374). Another challenge is to improve the efficiency of drug loading, since nano-drug carriers are often characterized by a low drug to carrier ratio. An ideal nanoparticle should possess a high ratio of the drug to excipients, particularly for targeted nanoparticles. Upon reaching target cells, nanoparticles with a high drug load will unload an increased amount of the drug to the cells, resulting in an enhanced therapeutic effect. Further, a high drug to excipient ratio reduces the amount of excipient that is co-administered to the patient and thus, minimizes any adverse reactions to such excipients.

BRIEF SUMMARY OF THE INVENTION

The presently disclosed subject matter provides methods for preparing crystalline nanoparticles of hydrophobic compounds. Methods for producing stable dispersions of crystalline nanoparticles of hydrophobic compounds comprise dissolving a provided hydrophobic compound and a surface stabilizer in a nonpolar solvent, evaporating the solvent, dispersing the resultant precipitate with a liquid dispersion medium to generate a dispersion of a hydrophobic compound/surface stabilizer aggregate, and homogenizing the hydrophobic compound/surface stabilizer aggregate. The hydrophobic compound and surface stabilizer can be added to the nonpolar solvent at various hydrophobic compound: surface stabilizer weight/weight ratios, including but not limited to a weight/weight ratio of hydrophobic compound to surface stabilizer of between about 1 :5 and about 1 :1. The presently disclosed methods allow for about 90% to about 100% of the hydrophobic compound added to the nonpolar solvent to be incorporated into the crystalline nanoparticles, producing a formulation comprising a relatively high percentage by weight of the hydrophobic compound. In those instances wherein the hydrophobic compound comprises a drug, administration of the crystalline nanoparticles having a relatively high percentage by weight (e.g., up to about 50%) of the drug to a subject in need thereof results in enhanced delivery of the drug and reduced toxicity due to the reduced amount of surface stabilizer present in the formulation. The presently disclosed subject matter also provides crystalline nanoparticles comprising crystalline hydrophobic compounds having adsorbed on the surface thereof surface stabilizers, wherein the particles comprise a high percentage of the hydrophobic compound by weight (e.g., up to about 50%), and stable dispersions thereof. In some embodiments, the hydrophobic compound comprises at least one of paclitaxel, camptothecin, and C6-ceramide. Methods for treating a disease or unwanted condition in a subject, such as a cancer, are also provided herein, wherein the methods comprise administering the crystalline nanoparticles or dispersions comprising the same to the subject, wherein the hydrophobic compound comprises a hydrophobic drug (e.g., paclitaxel, camptothecin, C6-ceramide).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 presents transmission electron microscope (TEM) images of crystalline nanoparticles of paclitaxel (PTX) and camptothecin (CPT) generated using the presently disclosed methods; Figure IA presents a TEM image of crystalline nanoparticles of PTX; Figure IB shows a TEM image of the lyophilized and reconstituted nanoparticles of PTX; and Figure 1C shows the crystalline nanoparticles of CPT. Figure 2 presents a graph measuring the absorbance at OD₆Oo of a dispersion of

PTX and CPT crystals in the presence or absence of Pluronic® F 127.

Figure 3 provides a TEM image of the crystalline PTX nanoparticles following a four hour incubation with serum at 37°C.

Figure 4 presents a graph depicting the hemolytic activity of the PTX/F127 crystalline nanoparticles and Cremophor-EL^®-PTX. The hemolytic activities were detected in erythrocyte suspensions obtained from mouse blood (n = 3, mean + S. D.). ** p < 0.005.

Figures 5A-5C show the in vivo antitumor activity of crystalline PTX nanoparticles against H460 human lung (Figure 5A) and 4Tl murine breast cancers (Figures 5 B and 5C). PTX in the crystalline nanoparticles or Cremophor-EL^®-PTX were intravenously injected into the nude (Figure 5A) or BALB/c (Figure 5B) tumor- bearing mice 7 days after the tumor inoculation (drug administrations indicated by arrows). The lyophilized and reconstituted crystalline nanoparticles were injected at either a 20 mg/kg or 60 mg/kg dosage. Figure 5 C presents the results of experiments wherein BALB/c tumor-bearing mice were orally administered the lyophilized and reconstituted crystalline nanoparticles or PTX suspension that was freshly prepared by sonication of PTX in 20% sucrose. The mice were first administered two doses of 80 mg/kg by oral gavage every other day and then the dose was decreased to 60 mg/kg every other day thereafter (indicated by arrows).

Figure 6 provides images of amorphous precipitates of PTX. The left panel (a, c, and e) is of PTX/F127 and the right panel (b, d, and f) is of PTX alone.

Figure 7 illustrates the proposed mechanism of formation of the crystalline nanoparticles. Figure 8 A and Figure 8B present graphs depicting the viability of KB cells in the presence of crystalline nanoparticles of PTX:F127 or PTX-F 127-folate. The targeted crystalline nanoparticles contained 10% F 127-folate with different concentrations of PTX (Figure 8A) or varied amounts of F 127-folate with 2 μM PTX (Figure 8B). Untreated cells were set at 100% viability and data were presented as the mean ± SD (n=8). ** p < 0.01.

Figure 9A and Figure 9B provide TEM images of PTX/vitamin E tocopheryl polyethylene glycol succinate (TPGS) (1/1) crystalline nanoparticles (Figure 9A) and the combined drug crystalline nanoparticles (PTX:C6-CER:TPGS, 1 :1 :5, w/w/w) (Figure 9B). Figure 1OA and Figure 1OB provide graphs depicting the physical stability of the F 127 and TPGS-based crystalline nanoparticles, which was evaluated based on the particle size after storage of the crystalline nanoparticles at room temperature (Figure 10A) and 37 ⁰C (Figure 10B).

Figure 11 presents a graph depicting the release of PTX from the F 127 and TPGS-based crystalline nanoparticles at 37 ⁰C. ** p < 0.05.

Figures 12A-12C provides graphs depicting the cell viability of cells in the presence of various PTX formulations in NCI/ADR-RES (Figures 12A- 12C), or KB and H460 cells (Figure 12A), as determined by MTT assays. KB and H460 cells (Figure 12A) were treated with 2 μM PTX. NCI/ADR-RES cell in Figure 12A and Figure 12C were treated with 10 μM PTX.

Figures 13A-13C provide a flow cytometric analysis of apoptosis occurring in NCI/ARD-RES cells that were untreated (Figure 13A), treated with the PTX/TPGS crystalline nanoparticles (Figure 13B), or TPGS (Figure 13C). Apoptosis was analyzed 12 hours after the treatment. Figure 14A and Figure 14B provide an evaluation of the antitumor activity of various PTX formulations or TPGS alone in tumor-bearing mice (5 per group) by measuring tumor size (Figure 14A) and weight (Figure 14B). The tumor weight was measured when the experiment was terminated. ** p < 0.01. Figure 15 presents a graph depicting the hemolytic activity of the PTX/TPGS crystalline nanoparticles and PTX/Cremophor-EL^®.

DETAILED DESCRIPTION OF THE INVENTION

Orally administered poorly water soluble drugs are eliminated from the gastrointestinal tract before being absorbed into the circulation. In addition, intravenous administration of these drugs tends to be unsafe. One method for increasing the rate of dissolution of a particulate drug and thus, enhancing its bioavailability and pharmacokinetic profile, is to increase the surface area of the drug particles, i.e., decrease particle size. The presently disclosed subject matter provides methods for preparing crystalline nanoparticles and stable dispersions thereof that allow for the intravenous or oral administration of drugs that are otherwise poorly soluble in aqueous solutions.

Further, the presently disclosed methods can produce formulations of drugs that are otherwise poorly water soluble using a single surface stabilizer, wherein the formulations comprise a high percentage of the drug by weight and decreased amounts of the surface stabilizer, reducing any toxicities associated with the use of high levels of surface stabilizers and thus enhancing the therapeutic efficacy of these drugs.

Methods for preparing a stable dispersion of crystalline nanoparticles of hydrophobic compounds comprise dissolving a hydrophobic compound and a surface stabilizer in a nonpolar solvent, thereby producing a hydrophobic compound/surface stabilizer solution; evaporating the nonpolar solvent, thereby producing a hydrophobic compound/surface stabilizer precipitate; dispersing the hydrophobic compound/surface stabilizer precipitate with a liquid dispersion medium, thereby producing a dispersion of a hydrophobic compound/surface stabilizer aggregate; and homogenizing the hydrophobic compound/surface stabilizer aggregate, thereby producing the stable dispersion of crystalline nanoparticles of the hydrophobic drug.

Using the presently disclosed methods, hydrophobic drugs can be formulated into crystalline nanoparticles having adsorbed on the surface thereof a surface stabilizer. As used herein, the term "nanoparticle" refers to particles of any shape having at least one dimension that is less than about 1000 nm. In some embodiments, nanoparticles have at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, and 1000). In certain embodiments, the nanoparticles have at least one dimension that is less than about 100 nm. Particle size can be determined using any method known in the art, including, but not limited to, sedimentation field flow fractionation, photon correlation spectroscopy, disk centrifugation, and dynamic light scattering (using, for example, a submicron particle sizer such as the NICOMP particle sizing system from AutodilutePAT Model 370; Santa Barbara, CA).

"Crystalline nanoparticles" refer to those nanoparticles that have a substantially uniform, repeating three-dimensional structure. Crystalline nanoparticles can be of any shape, including, but not limited to, cubes, rods, and hexagons.

The hydrophobic compound can be any type of hydrophobic compound for which the production of a nanoparticle formulation with a high compound to surface stabilizer would be useful. In some embodiments, the hydrophobic compound comprises a hydrophobic drug. Crystalline nanoparticles of drugs can be useful by reducing the amount and number of excipients required to efficiently deliver the drug to the intended cell/tissue/organ of a subject with limited toxicity. As used herein, the term "drug" refers to any bioactive compound that can exhibit a therapeutic effect when administered to a living cell, tissue, or organism. Representative, non-limiting drugs include antimicrobials, antibiotics, antimycobacterial, antifungals, antivirals, chemotherapeutic agents, agents affecting the immune response, blood calcium regulators, agents useful in glucose regulation, anticoagulants, antithrombotics, antihyperlipidemic agents, cardiac drugs, thyromimetic and antithyroid drugs, adrenergics, antihypertensive agents, cholinergics, anticholinergics, antispasmodics, antiulcer agents, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of the allergic response, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotic agents, anticonvulsants, antipsychotics, anti-anxiety agents, antidepressant agents, anorexigenics, non-steroidal antiinflammatory agents, steroidal anti-inflammatory agents, antioxidants, vaso-active agents, bone-active agents, antiarthritics, and diagnostic agents.

The presently disclosed methods produce crystalline nanoparticles and dispersions thereof comprising hydrophobic drugs. As used herein, the term "hydrophobic" is a physical property of a molecule that is repelled from a mass of water. Hydrophobic compounds can be solubilized in nonpolar solvents, including but not limited to, organic solvents. Hydrophobicity can be conferred by the inclusion of apolar or nonpolar chemical groups that include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cyclo aliphatic or heterocyclic group(s).

Conversely, "hydrophilic" molecules are capable of hydrogen bonding with a water (H₂O) molecule and are therefore soluble in water and other polar solvents. The terms "hydrophilic" and "polar" can be used interchangeably. Hydrophilic characteristics derive from the presence of polar or charged groups, such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.

Hydrophobic molecules are poorly water soluble, i.e., having a solubility of less than about 10 mg/ml. In some embodiments, the hydrophobic compound of the crystalline nanoparticle can have a solubility of less than about 1 mg/ml in water. In other embodiments, the hydrophobic compound has a solubility in water of less than about 10 μg/ml, and in particular embodiments, about 1 μg/ml or 2.5 μg/ml. In other embodiments, the hydrophobic compound can have a solubility of about 0.001 μg/ml to about 10 mg/ml, including but not limited to 0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, 50 μg/ml, 100 μg/ml, 500 μg/ml, 1 mg/ml, 5 mg/ml, and 10 mg/ml, and any other concentration between 0.001 μg/ml and 10 mg/ml. Representative, non-limiting examples of drugs that can be formulated using the presently disclosed methods include paclitaxel (PTX) and camptothecin (CPT). Both PTX and CPT are very poorly soluble in water, with PTX having a solubility in water of less than about 1 μg/ml, and CPT having a water solubility of about 2.5 μg/ml.

In some embodiments, the crystalline nanoparticles can comprise more than one hydrophobic compound. In such embodiments, the multiple hydrophobic compounds are added, along with the surface stabilizer, to the nonpolar solvent. In some non- limiting embodiments, paclitaxel and C6-ceramide or paclitaxel and camptothecin are co-formulated into crystalline nanoparticles.

In other embodiments, crystalline nanoparticles can be generated that comprise a crystalline hydrophobic compound and a hydrophilic compound (e.g,. 5-fluorouracil), wherein the hydrophilic compound is conjugated to the surface stabilizer that is adsorbed to the surface of the crystalline hydrophobic compound. In some of these embodiments, the hydrophobic compound comprises paclitaxel or camptothecin and the hydrophilic compound comprises 5-fluorouracil (5-FU).

The crystalline nanoparticles of hydrophobic drugs disclosed herein are stabilized in dispersions through the adsorption of surface stabilizers on the surface of the nanoparticles. As used herein, the term "surface stabilizer" refers to a molecule that has the ability, at a sufficient concentration, to stabilize the size of a particle to which it is adsorbed to the surface thereof. Surface stabilizers are known in the art and many are commercially available. Any surface stabilizer can be used with the presently disclosed methods and compositions, including, but not limited to, those described in the Handbook of Pharmaceutical Excipients, 5 ^th edition, published jointly by the

American Pharmaceutical Association, 2005, which is hereby incorporated by reference in its entirety.

Suitable surface stabilizers include, but are not limited to, surfactants, which are molecules that can reduce the surface tension of a liquid. Surfactants have both hydrophilic and hydrophobic properties, and thus, can be solubilized to some extent in either water or nonpolar solvents. Surfactants are classified into four primary groups: cationic, anionic, non-ionic, and zwitterionic. In some embodiments of the presently disclosed methods and compositions, the surface stabilizer comprises a non-ionic surfactant. Non-ionic surfactants are those surfactants that have no charge when dissolved or dispersed in aqueous solutions. Thus, the hydrophilic moieties of non- ionic surfactants are uncharged, polar groups.

Representative non- limiting examples of non-ionic surfactants suitable for use for the presently disclosed methods and compositions include polysorbates, including but not limited to, polyethoxylated sorbitan fatty acid esters (e.g., Tween® compounds) and sorbitan derivatives (e,g., Span® compounds); ethylene oxide/propylene oxide copolymers (e.g., Pluronic® compounds, which are also known as poloxamers); polyoxyethylene ether compounds, such as those of the Brij® family, including but not limited to polyoxyethylene stearyl ether (also known as polyoxyethylene (100) stearyl ether and by the trade name Brij® 700); and ethers of fatty alcohols.

Polyethoxylated sorbitan fatty acid esters (polysorbates) are commercially available from multiple suppliers (e.g., Sigma-Aldrich, St Louis, MO) under the trade name Tween®, and include, but are not limited to, polyoxyethylene (POE) sorbitan monooleate (Tween® 80), POE sorbitan monostearate (T ween® 60), POE sorbitan monolaurate (Tween® 20), and POE sorbitan monopalmitate (Tween® 40).

Ethylene oxide/propylene oxide copolymers include the block copolymers known as poloxamers, which are also known by the trade name Pluronic® and can be purchased from BASF Corporation (Florham Park, New Jersey). Poloxamers are composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) and are represented by the following chemical structure: HO(C₂H₄OX(CsHeO)_I3(C₂H₄OXH; wherein the C2H4O subunits are ethylene oxide monomers and the C3H6O subunits are propylene oxide monomers, and wherein a and b can be any integer ranging from 20 to 150.

Table 1 presents a table of representative poloxamer compounds suitable for use with the presently disclosed methods and compositions, their corresponding trademarked names, and the lengths of their polyoxyethylene chains (represented as a) and polyoxypropylene chains (represented as b).

Table 1. Representative poloxamers.

a: number of ethylene oxide units in each polyoxyethylene chain; b: number of propylene oxide units in each polyoxypropylene chain

It is to be understood that the molecular weight and the lengths of the polyoxypropylene and polyoxyethylene chains will differ slightly within any preparation of these copolymers. Thus, the numbers provided in Table 1 for a and b are average lengths of the polyoxyethylene and polyoxypropylene chains, respectively, within many preparations of these copolymers.

In some embodiments, the average length of the polyoxyethylene chains of the poloxamer is greater than about 50 ethylene oxide subunits, and in particular embodiments, the average length of the polyoxyethylene chain is greater than about 80 ethylene oxide subunits. In certain embodiments, the non-ionic surfactant adsorbed to the surface of the crystalline nanoparticles is a poloxamer with polyoxyethylene chains composed of an average of about 101 ethylene oxide subunits. In other embodiments, the non-ionic surfactant adsorbed to the surface of the crystalline nanoparticles is a poloxamer with polyoxyethylene chains composed of an average of about 100 ethylene oxide subunits.

In certain embodiments, the average length of the polyoxypropylene chain is greater than about 20 propylene oxide subunits and in particular embodiments, the polyoxypropylene chain has an average length greater than about 30 propylene oxide subunits. In yet other embodiments, the polyoxypropylene chain of the poloxamer has an average length of greater than about 50. In still other embodiments, the non-ionic surfactant adsorbed to the surface of the crystalline nanoparticles is a poloxamer with polyoxypropylene chains composed of about 56 propylene oxide subunits, on average. In yet other embodiments, the polyoxypropylene chain of the poloxamer has an average length of about 65. In still other embodiments, the surface stabilizer is a poloxamer, wherein the average length of the polyoxyethylene chain is about 56, and the average length of the polyoxypropylene chain is about 101. In other particular embodiments, the surface stabilizer is a poloxamer, wherein the average length of the polyoxyethylene chain is about 65, and the average length of the polyoxypropylene chain is about 100. In some embodiments wherein the surface stabilizer is a surfactant, the surfactant has a relatively high hydrophilic/lipophilic balance (HLB). The term "hydrophilic/lipohilic balance" abbreviated as HLB, refers to a numerically calculated number used to classify surfactants that is based on the surfactant's molecular structure. The HLB provides a description of the water solubility of surfactant compounds, wherein surfactants with higher HLBs exhibit greater solubility in water. The HLB is an arbitrary number on a scale ranging from 0 to 40. Thus, in some embodiments of the presently disclosed methods and compositions, the non-ionic surfactant will have a HLB between about 10 and about 40, including but not limited to, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40.

Other non-limiting examples of surface stabilizers useful for the presently disclosed methods and compositions include cationic molecules, such as cationic lipids. As used herein, "cationic lipid" encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. Such lipids include, but are not limited to the cationic lipids of formula (I) disclosed in the International Application No. PCT/US2009/042476, entitled "Methods and Compositions Comprising Novel Cationic Lipids," which was filed May 1, 2009, and is herein incorporated by reference in its entirety. Non-limiting examples of cationic lipids of formula (I) include N,N-di- myristoyl-N-methyl-N-2[N'-(N⁶-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N-2[N²-guanidino-L-lysinyl] aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2[N'-(N2, N6-di-guanidino-L- lysinyl)] aminoethyl ammonium chloride, N-methyl-N-(2-(arginoylamino) ethyl)-N, N- Di octadecyl-aminium chloride or di stearoyl arginyl ammonium chloride (DSAA), N,N-di-stearoyl-N-methyl-N-2 [N ' -(N6-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DSGLA). Non-limiting examples of other cationic lipids include N ,N- dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleoyloxy) propyl)- N,N,N-trimethylammonium chloride ("DOTAP"); N-(2,3-dioleyloxy) propyl)-N,N,N- trimethylammonium chloride ("DOTMA") or other N-(N₅N- 1 -dialkoxy)-alkyl-N,N,N- trisubstituted ammonium surfactants; N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl) cholesterol ("DC-Choi") and N-(1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"); l,3-dioleoyl-3-trimethylammonium-propane, N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy- 1 ammonium trifiuoro-acetate (DOSPA); GAP-DLRIE; DMDHP; S-β^N-^N^N-diguanidino spermidine)-carbamoyl] cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)- carbamoyl] cholesterol (BGTC); N₅N ,N ,N Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N'-tetradecyl-S-tetradecyl-aminopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); l,3-dioleoyloxy-2-(6- carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-l-methyl- 1 H-imidazole (DPIM) N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3 dioleoyloxy-l,4-butanediammonium iodide) (Tfx-50); 1,2 dioleoyl-3-(4'- trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4'trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DORI (DL-1, 2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-l,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammoniu- m) (DORIE) or analogs thereof as disclosed in WO 93/03709; l,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES) or the cationic lipids disclosed in U.S. Pat. No. 5,283,185; cholesteryl-Sβ-carboxyl-amido-ethylenetrimethylammonium iodide; 1- dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide; cholesteryl-3-β-carboxyamidoethyleneamine; cholesteryl-3-β-oxysuccinamido- ethylenetrimethylammonium iodide; 1 -dimethylamino-S-trimethylammonio-DL^- propyl-cholesteryl-3-β-oxysuccinate iodide; 2-(2-trimethylammonio)- ethylmethylamino ethyl-cholesteryl-3-β-oxysuccinate iodide; and 3-β-N- (polyethyleneimine)-carbamoylcholesterol.

In general, surface stabilizers suitable for the presently disclosed methods and compositions are relatively non-toxic and are generally regarded as safe. Pharmaceutically acceptable surface stabilizers are described in the Handbook of Pharmaceutical Excipients, 5^th edition, published jointly by the American Pharmaceutical Association, 2005, which was cited elsewhere herein and has been incorporated by reference in its entirety.

In particular embodiments, the non-ionic surfactant that is adsorbed to the surface of the crystalline nanoparticles of the invention is selected from the group consisting of Pluronic® F 127 (poloxamer 407), Tween® 80 (polyoxyethylene sorbitan monooleate), and Brij® 700 (polyoxyethylene stearyl ether).

In other embodiments, the surface stabilizer comprises a vitamin E tocopheryl polyethylene glycol succinate (TPGS). TPGS inhibits the ATP-dependent transporter P-glycoprotein (P-gp), which is utilized by some tumor cells to increase the efflux of cytotoxic drugs (e.g., paclitaxel, doxorubicin, vincristine, vinblastine) out of the cell, thereby decreasing the intracellular levels of the drugs, leading to a multidrug-resistant (MDR) phenotype (Leonessa and Clarke (2003) Endocr Relat Cancer 10(l):43-73; Filipits et al. (1996) Clin Cancer Res 2(7): 1231-7; Dumontet and Sikic (1999) J Clin Oncol 17(3):1061-70; Ramachandra et al. (1998) Biochemistry 37(14):5010-9; and Schinkel et al. (1997) Proc Natl Acad Sci USA 94(8):4028-33). When drugs bind to P-gp, one of the ATP-binding domains is activated and hydrolysis of ATP causes a conformational change in P-gp, causing drugs to be released into the extracellular space (Ramachandra et al. (1998) Biochemistry 37(14):5010-9). P-gp can bind to a wide variety of drugs, including PTX, doxorubicin, vincristine, and vinblastine (Schinkel et al. (1997) Proc Natl Acad Sci USA 94(8):4028-33). While P-gp has been found abnormally expressed on the plasma membrane, it is also often detected on the nuclear envelope and the membrane of cytoplasmic organelles, which in turn reduces the amount of cytotoxic drug reaching the cytoplasmic organelles and nucleus (Abbaszadegan et al. (1996) Cancer Res 56(23):5435-42; Hipfner et al. (1996) Cancer Res 56(14):3307-14; Molinari et al. (2002) Curr Protein Pept Sci 3(6):653-70; and Gruol, King, and Kuehne (2002) MoI Pharmacol 62(5): 1238-48). Therefore, the crystalline nanoparticles comprising TPGS as the surface stabilizer can be useful in inhibiting the P-gp on both the plasma membrane and the membranes of cytoplasmic organelles. Further, as demonstrated elsewhere herein (see Experimental Example 7), the TPGS-based crystalline nanoparticles exhibit an enhanced thermal and physical stability. While the TPGS-based crystalline nanoparticles described herein (see Experimental Example 7) comprised vitamin E tocopheryl polyethylene glycol 1000 succinate (TPGS 1000), TPGS having varying chain lengths of polyethylene glycol (PEG) may also be used in the presently disclosed methods and compositions. TPGS with varying chain lengths of PEG (e.g., 2000, 3000, 5000) can be synthesized using methods known in the art and described elsewhere herein (Collnot et al. (2006) J Control Release 11 l(l-2):35-40, which is herein incorporated by reference in its entirety).

In some embodiments, the crystalline nanoparticle comprises more than one type of surface stabilizer. The presently disclosed methods, however, allow for the generation of crystalline nanoparticles of hydrophobic drugs and stable dispersions comprising the same using a single surface stabilizer as the excipient. As used herein, the term "excipient" refers to an inert substance with no therapeutic activity that is used to formulate and deliver an active drug. In these embodiments, only one type of surface stabilizer is adsorbed to the surface of the crystalline nanoparticles and is present in dispersions comprising the same. A crystalline nanoparticle is comprised of a single type of a surface stabilizer when the crystalline nanoparticle is essentially free of other types of surfaces stabilizers. By "essentially free" is intended that the crystalline nanoparticle comprises less than 10%, less than 5%, less than 1%, less than 0.1%, or less of other types of surface stabilizers by weight. In other words, greater than 90%, greater than 95%, greater than 99%, greater than 99.9% or more of the surface stabilizers of a crystalline nanoparticle are of a single type of surface stabilizers. The term "type" when referring to a surface stabilizer (e.g., surfactant) refers to a particular chemical species. Thus, a composition comprising one type or a single type of surface stabilizers comprises one or more molecules of one chemical species of surface stabilizers, in contrast to a composition that comprises a mixture of more than one chemical species of surface stabilizers. When referring to a polymeric molecule, including but not limited to a copolymer (e.g., poloxamer), one type of a polymeric molecule can refer to a group of chemically similar species that have the same chemical makeup, but differ only by the number of monomeric subunits, wherein the number of monomeric subunits between each species does not substantially differ from one another. Thus, one type of a polymeric surface stabilizer can include a group of polymers, wherein the number of monomeric subunits does not differ between any of the molecules by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, or more than 50%.

In certain embodiments, the surface stabilizers are conjugated to a targeting ligand. Surface stabilizer-targeting ligand conjugates that are adsorbed to the surface of crystalline hydrophobic compounds target the crystalline nanoparticles (which are referred to herein as targeted crystalline nanoparticles) to particular cell types, tissue, organs, or intracellular regions.

By "targeting ligand" is intended a molecule that targets associated compounds or particles to a targeted cell, tissue, organ, or intracellular region. Targeting ligands can include, but are not limited to, small molecules, peptides, lipids, sugars, oligonucleotides, hormones, vitamins, antigens, antibodies or fragments thereof, specific membrane-receptor ligands, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. Non-limiting examples of targeting ligands include asialo glycoprotein, insulin, low density lipoprotein (LDL), folate, benzamide derivatives, peptides comprising the arginine-glycine-aspartate (RGD) sequence and monoclonal and polyclonal antibodies directed against cell surface molecules.

Peptide ligands containing the Arginine-Glycine- Aspartate (RGD) triad have been developed to target the tumor-associated cells expressing the αvβ3 integrin receptors. Integrin αvβ3 is a glycoprotein membrane receptor that recognizes extracellular matrix proteins expressing an RGD peptide sequence (Pickles et al. (1998) J Virol 72(7):6014-23; Wang et al. (2005) Nat Med 11(5):515-21). The receptor is highly expressed on activated tumor vasculature endothelial cells, but not resting endothelial cells and normal organ systems, thus making αvβ3 an appropriate target for anti-angiogenic therapeutics (Haubner (2006) Eur J Nucl Med MoI Imaging 33 Suppl 1 :54-63). Therapeutic studies demonstrated that doxorubicin (DOX) coupled with RGD effectively targeted (DOX) to the tumor neovasculature and enhanced efficacy in human breast cancer xenografts in mice (Arap, Pasqualini, and Ruoslahti (1998) Science 279(5349):377-80). In another study, nanoparticles containing the RGD motif were capable of delivering this cytotoxic drug to the αvβ3 -positive tumor (human renal carcinoma) cell vasculature (Murphy et al. (2008) Proc Natl Acad Sci USA 105(27):9343-8). More recently, it has been reported that, by conjugating RGD to the 2'-OH-group of PTX through an aliphatic ester, the proliferation of human umbilical vein endothelial cells was significantly inhibited compared to free PTX (Ryppa et al.

(2009) IntJ Pharm 368(l-2):89-97). Thus, in some embodiments, the surface stabilizer is conjugated to a peptide comprising a RGD sequence.

Another strategy for tumor targeting is to directly deliver the drugs to the tumor by targeting the receptors on the tumor surface, for example folate receptor (FR) and sigma receptors (SR). Human folate receptor (FR) is a 38-40 kDa //-glycosylated protein. By immunohistochemical staining, elevated FR expression has been observed in ovarian, endometrial, colorectal, breast, lung, and renal cell carcinomas, as well as in brain metastases (Wu, Gunning, and Ratnam (1999) Cancer Epidemiol Biomarkers Prev 8(9):775-82; Zhao and Lee (2004) Adv Drug Deliv Rev 56(8): 1193-204). FR- targeting has been evaluated using a target ligand, folic acid (FA), for enhancing tumor cell selective delivery of a wide variety of therapeutic agents. Sigma receptors (SR), on the other hands, are membrane-bound protein receptors expressed in normal tissues such as the liver, endocrine glands, kidneys, lungs, central nervous system and ovaries at basal levels (Wolfe, CuIp, and De Souza (1989) Endocrinology 124(3): 1160-72; Hellewell et al. (1994) Eur J Pharmacol 268(1):9-18). The physiological roles of the sigma receptor in these normal tissues are not yet clear, nor has their over-expression in the tumor been elucidated (Garg, John, and Zalutsky (1995) Nucl Med Biol 22(4):467- 73; John et al. (1999) Nucl Med Biol 26(4):377-82; John et al. (1998) Nucl Med Biol 25(3): 189-94; John et al. (1996) Nucl Med Biol 23(6):761-6; Aydar, Palmer, and Djamgoz (2004) Cancer Res 64(15):5029-35; Bowen (2000) Pharm Acta HeIv 74(2- 3):211-8; Collier, Waterhouse, and Kassiou (2007) Curr Pharm Des 13(1):51-72). SR is overexpressed in many types of human tumors such as melanoma, breast cancer, small lung carcinoma and prostate cancer (Vilner, John, and Bowen (1995) Cancer Res 55(2):408-13; Al-Nabulsi et al. (1999) Br J Cancer 81(6):925-33; Li, Chono, and Huang (2008) MoI Ther 16(5):942-6; John et al. (1999) Cancer Res 59(18):4578-83). The high SR density has been exploited for tumor imaging (John et al. (1999) Cancer Res 59(18):4578-83; John et al (1998) J Med Chem 41(14):2445-50; John et al. (1995) Life Sci 56(26):2385-92; Kashiwagi et al. (2007) MoI Cancer 6:48; Pham et al. (2007) JNuclMed 48(8): 1348-56) and targeted delivery of anti-cancer drugs (Ostenfeld et al. (2008) Autophagy 4(4) :487-99; van Waarde et al. (2006) JNucl Med 47(9): 1538-45; Banerjee et al. (2004) Int J Cancer 112(4):693-700). Previous studies have demonstrated that tumor targeted delivery can be achieved via SR by conjugation with a ligand, anisamide (AA), on the surface of liposomes (Banerjee et al. (2004) Int J Cancer 112(4):693-700). Binding of anisamide-targeted liposomes is followed by rapid endocytosis and intracellular delivery of the entrapped drug (Banerjee et al. (2004) Int J Cancer 112(4):693-700). Therefore, in particular embodiments, the targeting ligand of the surface stabilizer/targeting ligand conjugate comprises folate (or folic acid) or a benzamide derivative, such as anisamide. By "targeted cell" is intended the cell to which a targeting ligand recruits an associated compound or particle. The targeting ligand can interact with one or more constituents of a target cell. The targeted cell can be any cell type or at any developmental stage, exhibiting various phenotypes, and can be in various pathological states (i.e., abnormal and normal states). For example, the targeting ligand can associate with normal, abnormal, and/or unique constituents on a microbe (i.e., a prokaryotic cell (bacteria), viruses, fungi, protozoa or parasites) or on a eukaryotic cell (e.g., epithelial cells, muscle cells, nerve cells, sensory cells, cancerous cells, secretory cells, malignant cells, erythroid and lymphoid cells, stem cells). Thus, the targeting ligand can associate with a constitutient on a target cell which is a disease-associated antigen including, for example, tumor-associated antigens and autoimmune disease- associated antigens. Such disease-associated antigens include, for example, growth factor receptors, cell cycle regulators, angiogenic factors, and signaling factors.

In some embodiments, the targeting ligand interacts with a cell surface protein on the targeted cell. In some of these embodiments, the expression level of the cell surface protein that is capable of binding to the targeting ligand is higher in the targeted cell relative to other cells. For example, cancer cells overexpress certain cell surface molecules, such as the HER2 receptor (breast cancer) or the sigma receptor. In certain embodiments wherein the targeting ligand comprises a benzamide derivative, such as anisamide, the targeting ligand targets the associated particle to sigma-receptor overexpressing cells, which can include, but is not limited to, cancer cells such as small- and non-small-cell lung carcinoma, renal carcinoma, colon carcinoma, sarcoma, breast cancer, melanoma, glioblastoma, neuroblastoma, and prostate cancer (Aydar, Palmer, and Djamgoz (2004) Cancer Res. 64:5029-5035). Thus, in some embodiments, the targeted cell comprises a cancer cell. The terms "cancer" or "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, "cancer cells" or "tumor cells" refer to the cells that are characterized by this unregulated cell growth. The term "cancer" encompasses all types of cancers, including, but not limited to, all forms of carcinomas, melanomas, sarcomas, lymphomas and leukemias, including without limitation, bladder carcinoma, brain tumors, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, endometrial cancer, hepatocellular carcinoma, laryngeal cancer, lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and thyroid cancer. In some embodiments, the targeted cancer cell comprises a lung cancer cell. The term "lung cancer" refers to all types of lung cancers, including but not limited to, small cell lung cancer (SCLC), non-small-cell lung cancer (NSCLC, which includes large-cell lung cancer, squamous cell lung cancer, and adenocarcinoma of the lung), and mixed small-cell/large-cell lung cancer. The targeting ligand is conjugated to the surface stabilizer, meaning there is a covalent bond between the targeting ligand and the surface stabilizer, wherein at least one pair of electrons is shared between two atoms of the two molecules. A "conjugate" refers to the complex of molecules that are covalently bound to one another, such as a surface stabilizer/targeting ligand conjugate. A targeting ligand can be conjugated to a surface stabilizer using any reaction chemistry known in the art.

Surface stabilizers are adsorbed to the surface of the presently disclosed crystalline nanoparticles of hydrophobic drugs. By "adsorbed" is intended the surface stabilizer is associated with the drug particles through non-covalent interactions. Non- covalent interactions do not involve the sharing of pairs of electrons (as with covalent interactions or bonds), but rather involve more dispersed variations of electromagnetic interactions, and can include hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bonds. Without being bound by any theory or mechanism of action, it is believed that the hydrophobic moieties of the non-ionic surfactants adsorb to the surface of the nanoparticles of hydrophobic drugs through hydrophobic interactions and the hydrophilic moieties serve to form ionic interactions with surrounding water molecules, dispersing the particles, and therefore, prevent aggregation and growth of the crystalline nanop articles. Thus, non-ionic surfactants with longer hydrophobic chains may adsorb more strongly to the surface of the crystalline nanoparticles and surfactants with longer hydrophilic chains may function as stronger dispersants.

The hydrophobic compound and surface stabilizer are provided at a weight ratio sufficient to produce crystalline nanoparticles that can be stabilized in a liquid dispersion medium (e.g., water). In some embodiments, the weight ratio of the hydrophobic compound to surface stabilizer present in the nonpolar solvent is between about 1 :10 and about 2:1, including but not limited to about 1 :10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1, and about 2:1. In particular embodiments, the weight ratio of hydrophobic compound to surface stabilizer is between about 1 :5 and about 1 :1.

The presently disclosed methods involve dissolving a provided hydrophobic compound and a surface stabilizer in a nonpolar solvent to produce a hydrophobic compound/surface stabilizer solution. As used herein, the term "dissolve" refers to the solubilization of a solid into a solvent as it passes into solution. A "solvent" is the substance in a solution that is present in the greater amount. The term "solution" refers to a substantially homogeneous mixture of a solute (e.g., solid) in a solvent (e.g., a liquid).

Nonpolar solvents include, but are not limited to, organic solvents. Non- limiting examples of a nonpolar solvent include chloroform, methanol, ether, acetyl acetate, n-hexane, and dichloromethane. In some embodiments, the organic solvent is one that is volatile. As used herein, the term "volatile" refers to a property of a solvent that can be readily evaporated at ambient temperature and pressure.

The hydrophobic compound and surface stabilizer can be dissolved in mixtures of nonpolar solvents, including, but not limited to mixtures of chloroform and methanol. In some embodiments, the nonpolar solvent comprises chloroform and methanol at a volume/volume ratio of about 1 : 10 to about 10:1, including but not limited to, 1 :10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1.

In some embodiments, the hydrophobic compound/surface stabilizer solution is essentially free of polar solvents (e.g., water). A nonpolar solvent or solution that is essentially free of polar solvents comprises a nonpolar solvent or a solution wherein less than 10%, less than 5%, less than 1%, less than 0.1% or less of the volume of the solvent or solution is a polar solvent (e.g., water).

Once the hydrophobic compound and surface stabilizer are dissolved in one or more nonpolar solvents, the one or more nonpolar solvents are evaporated using any means known in the art while allowing formation of a hydrophobic compound/surface stabilizer precipitate, including but not limited to, rotary evaporation, and evaporation facilitated by a steady stream of nitrogen gas. A "precipitate" refers to the solid formed in a solution during a chemical reaction, which has been subsequently substantially separated from the solvent or a solid that remains from a solution after the solvent has been removed. In some embodiments, the precipitate is amorphous. As used herein, an object that is "amorphous" has no definite, consistent form (e.g., crystal). Thus, an essentially amorphous hydrophobic compound/surface stabilizer precipitate following evaporation of the nonpolar solvent is essentially free of crystals of the hydrophobic compound, meaning that less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, or less of the precipitate comprises crystalline structures. While not being bound by any theory or mechanism of action, it is believed that the formation of an essentially amorphous precipitate versus one that is crystalline is important for the formation of the crystalline nanoparticles.

While not being bound by any theory or mechanism of action, it is believed that a relatively slow evaporative process is important for the formation of the hydrophobic compound/surface stabilizer precipitate, particularly one that is essentially amorphous in nature. Thus, in some embodiments, evaporation of the nonpolar solvent does not occur via spray drying, which is a relatively rapid evaporative process. In particular embodiments, the nonpolar solvent is evaporated through the provision of a steady stream of nitrogen gas over the drug solution. Traces of remaining solvent can be removed through such methods as exposing the crystals to a vacuum in the presence or absence of dessicants.

It should also be noted that the addition of a surface stabilizer after the evaporation of the nonpolar solvent having dissolved therein a hydrophobic compound does not result in the formation of crystalline nanoparticles upon dispersion and homogenization. For example, crystalline nanoparticles could only be prepared when both paclitaxel and F 127 were in physical contact at the beginning of the solidification process as the nonpolar solvent evaporated. If paclitaxel alone was precipitated, then hydrated with a F 127 solution and sonicated, large particles (> 1 μM) were formed and the particles precipitated. Therefore, the hydrophobic compound and surface stabilizer must be present together in the nonpolar solvent prior to precipitation.

The resultant hydrophobic compound/surface stabilizer precipitate can then be dispersed by adding a liquid dispersion medium as described elsewhere herein, thereby producing a dispersion of a hydrophobic compound/surface stabilizer aggregate. As used herein, the term "dispersion" refers to a continuous medium (e.g. liquid dispersion medium) in which another phase (e.g., hydrophobic compound) is dispersed (e.g., not dissolved). Due to the polar nature of the liquid dispersion medium, the hydrophobic compounds associate through hydrophobic interactions, forming a hydrophobic compound/surface stabilizer aggregate. In some embodiments, the aggregate is essentially amorphous and therefore, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, or less of the aggregate comprises crystalline structures. Without being bound by any theory or mechanism of action, the generation of a hydrophobic compound/surface stabilizer aggregate, particularly one that is essentially amorphous and essentially free of crystals, is important for the production of crystalline nanoparticles.

In some embodiments, the liquid dispersion medium comprises a polar solvent. In other embodiments, the liquid dispersion medium comprises an aqueous solution (e.g., water) and the dispersion is thus, referred to as an aqueous dispersion. In some of these embodiments, the liquid dispersion medium further comprises a sugar (e.g., dextrose, sucrose), which in some embodiments is present in the liquid dispersion medium or aqueous solution at a concentration of between about 0.5% and about 30%, including but not limited to, about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%. In particular embodiments, the concentration is between about 0.5% and 20%. In some of these embodiments, the sugar comprises dextrose or sucrose. In certain embodiments, the liquid dispersion medium comprises an aqueous solution comprising about 5% dextrose or about 20% sucrose.

The hydrophobic compound/surface stabilizer precipitate is incubated in the liquid dispersion medium for a period of time sufficient to generate the hydrophobic compound/surface stabilizer aggregate, but not long enough that crystalline particles of the hydrophobic compound form. In some embodiments, the amount of time which the precipitate is incubated in the liquid dispersion medium ranges from about 1 minute to about 5 hours, including but not limited to, about 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours. In particular embodiments, the period of time for incubation is between about 2 minutes and about 4 hours. In some of these embodiments, the incubation time is between about 30 minutes and about 1 hour. During the incubation, intermittent or continuous mixing of the particle dispersion can be performed.

In those embodiments wherein the hydrophobic compound comprises a hydrophobic drug, the volume of the liquid dispersion medium added to the precipitate can be sufficient to bring the final concentration of the hydrophobic drug to a concentration necessary for a therapeutic effect. In some embodiments, the final concentration of hydrophobic drug within the dispersion ranges from about 1 mg/ml to about 50 mg/ml, including but not limited to, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/ml.

According to the presently disclosed methods, the hydrophobic compound/surface stabilizer aggregate is then homogenized. As used herein, the term "homogenize" when referring to a particular dispersion or solids (e.g., aggregates) within a dispersion is intended the particles within the dispersion are reduced in size through a mechanical force and are dispersed. Any method known in the art can be used to homogenize the hydrophobic compound/surface stabilizer aggregate into nanoparticles, including but not limited to, sonication (e.g., with a bath-type sonicator), emulsification (e.g., with an emulsifying machine), and vortexing. In some embodiments, the term homogenization excludes milling (e.g., wet milling). In those embodiments wherein the dispersion of the hydrophobic compound/surface stabilizer aggregate is sonicated, a bath-type sonicator may be used and the dispersion will be sonicated for a sufficient period of time and at a sufficient frequency to produce the crystalline nanoparticle dispersion. In some embodiments, the dispersion is sonicated for about 5 to about 30 minutes, including but not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, and about 30 minutes. In some of these embodiments, an output of about 80 kilocycles (kc) and about 80 watts is required to form the crystalline nanoparticles of the desired size. In certain embodiments, it may be necessary to cool the sonicator or the dispersion intermittently or throughout the sonication step to avoid excessively heating the dispersion, which may denature the drug and reduce its activity.

In some embodiments, such as those wherein the dispersion of crystalline nanoparticles will be administered to a subject in vivo, the presently disclosed methods can comprise an additional step, wherein the dispersion is sterilized to remove microbial contaminants. The dispersion may be sterilized using any method known in the art while retaining the activity of the drug, including, but not limited to, filter sterilization. Alternatively, the liquid dispersion medium is sterilized prior to its addition to the crystallized particles.

Dispersions comprising crystalline nanoparticles of hydrophobic compounds are stabilized due to the adsorption of the surface stabilizers to the surface of the nanoparticles. When referring to a dispersion, the terms "stable" or "stabilized" refer to those dispersions wherein the solid phase (e.g., the crystalline nanoparticles) that is dispersed within the liquid dispersion medium remain of a substantially uniform size, and do not aggregate or grow in size over time. In certain embodiments, the crystalline nanoparticles within the stable dispersion remain of a substantially uniform size and do not aggregate for at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 2 weeks, at least about 5 weeks, at least about 10 weeks, at least about 25 weeks, at least about 1 year, or greater. Methods known in the art and described elsewhere herein can be used to measure the size of the particles in the dispersion. The turbidity of the dispersion can also provide information concerning the stability and size distribution of the crystalline nanoparticles within the dispersion. Turbidity of a dispersion can be measured by any method known in the art, including, but not limited to, absorbance of the dispersion at OD₆oo-

The presently disclosed methods advantageously allow for the production of stable dispersions of hydrophobic compounds at ambient temperatures and pressures. Thus, in some embodiments, the steps of the presently disclosed methods are performed at a temperature ranging from about 20⁰C to about 30⁰C, including, but not limited to 20⁰C, 2FC, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30⁰C.

In some embodiments, the presently disclosed methods further comprise a step of recovering the crystalline nanoparticles from the dispersion. In one embodiment, recovering the particles comprises removing the liquid dispersion medium (e.g., water) from the particles. Removing the liquid dispersion medium can be performed using any technique known to those skilled in the art, including spray drying, spray freeze drying, gellation, defined as gelling the particles within a polymeric matrix, lyophilization, drying with cold air, and filtration. In particular embodiments, the removal of the liquid dispersion medium does not comprise spray drying. In certain embodiments, the recovered crystalline nanoparticles can be reconstituted in a liquid dispersion medium prior to administration to a subject in need thereof. The ability to recover the crystalline nanoparticles from the dispersion permits storage of the drug for long periods of time under a more stable environment. The recovered crystalline nanoparticles of the hydrophobic drug can then be reconstituted by the addition of a liquid dispersion medium (e.g., water) to again form a stable dispersion of the crystalline nanoparticles. Advantageously, upon reconstitution, the recovered crystalline nanoparticles are dispersed and remain active.

The presently disclosed methods allow for the incorporation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the hydrophobic compound present within the nonpolar solvent into crystalline nanoparticles. The efficiency of incorporation using the presently disclosed methods for any given hydrophobic compound can be determined using any method known in the art, including, but not limited to, filtering out aggregates of the hydrophobic compound that have not been incorporated and quantifying the amount of incorporated hydrophobic drug using, for example, high performance liquid chromatography (HPLC). In some embodiments, dispersions can comprise crystalline nanoparticles of more than one hydrophobic compound (e.g., drug), wherein the dispersion is stabilized through the adsorption of surface stabilizers to the surfaces of the crystalline nanoparticles. Thus, crystalline nanoparticles of drug A can be mixed with crystalline nanoparticles of drug B in a liquid dispersion medium to produce a formulation wherein drugs A and B can be co-administered to subjects in need thereof. In other embodiments, crystalline nanoparticles of hydrophobic compound A can be added to a liquid dispersion medium that comprises compound B (hydrophobic or hydrophilic, as described elsewhere herein).

In other embodiments, the crystalline nanoparticles can comprise more than one type of compound. The hydrophobic compound present as a crystalline hydrophobic compound in the crystalline nanoparticles can further comprise a second type of hydrophobic compound. Alternatively, the crystalline nanoparticles comprising a crystalline hydrophobic compound having surface stabilizers adsorbed to the surface thereto can further comprise a hydrophilic compound conjugated to the surface stabilizer. A non- limiting example of crystalline nanoparticles that comprise more than one hydrophobic drug is described elsewhere herein (see Experimental Example 7), wherein crystalline nanoparticles are generated comprising crystalline paclitaxel and C6-ceramide coated with TPGS. The weight ratio of paclitaxel:C6-ceramide:TPGS added to the organic solvent in this non- limiting embodiment was 1 :1 :5.

As discussed elsewhere herein, in some embodiments, the stable dispersion can comprise additional additives, including but not limited to, sugars, salts, pectin, and citric acid. Representative non- limiting examples of sugars that can be added to the dispersions comprising the crystalline nanoparticles include dextrose, sucrose, and galactose. In some of these embodiments, the concentration of dextrose within the aqueous dispersion is from about 0.5% to about 30% (weight/volume; w/v), including but not limited to 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%. In some embodiments, stable aqueous dispersions of crystalline nanoparticles of hydrophobic drugs comprise dextrose or another sugar at a concentration of between about 0.5% and about 20%. In particular embodiments, stable aqueous dispersions of crystalline nanoparticles of hydrophobic drugs comprise dextrose at a concentration of 5% (w/v). In other embodiments, the stable aqueous dispersions comprise 20% sucrose (w/v). The dispersion additives can be present in the liquid dispersion medium when it is added to the hydrophobic compound/surface stabilizer precipitate or to the hydrophobic compound/surface stabilizer aggregate prior to homogenization or the additives can be added to the dispersion following homogenization, or can be present in the liquid dispersion medium used to reconstitute nanoparticles that have been recovered from a dispersion. The presently disclosed methods can be used to produce crystalline nanoparticles of hydrophobic compounds and dispersions comprising the same. Thus, the present invention provides crystalline nanoparticles comprising crystalline hydrophobic compounds, having a surface stabilizer adsorbed to the surface thereof, wherein a relatively high percentage of the nanoparticle by weight is the hydrophobic compound. In some embodiments, the crystalline nanoparticles comprise at least about 10% to about 50% by weight, including but not limited to, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, and about 50% of the hydrophobic compound by weight. Further provided are stable dispersions comprising these nanoparticles and a liquid dispersion medium. In some embodiments, these nanoparticles and dispersions thereof are produced according to the presently disclosed methods. In certain embodiments, crystalline nanoparticles comprising at least one of paclitaxel, camptothecin, C6-ceramide, and 5-FU are provided.

The presently disclosed crystalline nanoparticles of hydrophobic compounds and dispersions thereof, wherein the hydrophobic compound comprises a hydrophobic drug or the nanoparticle comprises a hydrophilic drug, are useful in mammalian tissue culture systems, in animal studies, and for therapeutic purposes. The nanoparticles or dispersions themselves can be administered for therapeutic purposes or pharmaceutical compositions comprising the nanoparticles or dispersions along with additional pharmaceutical carriers can be formulated for delivery, i.e., administering to the subject, by any available route including, but not limited, to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. In some embodiments, the route of delivery is intravenous, parenteral, transmucosal, nasal, bronchial, vaginal, and oral. As used herein the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds also can be incorporated into the compositions. As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions or dispersions such as those described elsewhere herein and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. In some embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, or sodium chloride are included in the formulation. Prolonged absorption of the injectable formulation can be brought about by including in the formulation an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by filter sterilization as described elsewhere herein. In certain embodiments, solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In those embodiments in which sterile powders are used for the preparation of sterile injectable solutions, the solutions can be prepared by vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The oral compositions can include a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the presently disclosed compositions can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used.

Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Guidance regarding dosing is provided elsewhere herein.

The present invention also includes an article of manufacture providing the presently disclosed crystalline nanoparticles of a hydrophobic drug or dispersions comprising the same.

The article of manufacture can include a vial or other container that contains the presently disclosed crystalline nanoparticles or dispersions together with any carrier, either dried or in liquid form. The article of manufacture further includes instructions in the form of a label on the container and/or in the form of an insert included in a box in which the container is packaged, for administering the composition to a subject. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow the subject or a worker in the field to administer the pharmaceutical composition. It is anticipated that a worker in the field encompasses any doctor, nurse, technician, spouse, or other caregiver that might administer the composition. The pharmaceutical composition can also be self-administered by the subject.

The crystalline nanoparticles of hydrophobic drugs and dispersions comprising the same can be administered to subjects in need thereof. Thus, the presently disclosed subject matter provides methods for the treatment of a disease or unwanted condition in a subject comprising administering to the subject the presently disclosed crystallized nanoparticles of hydrophobic drugs, stable dispersions comprising the same, or pharmaceutical compositions comprising the crystallized nanoparticles and a pharmaceutically acceptable carrier. By "therapeutic activity" when referring to a molecule is intended that the molecule is able to elicit a desired pharmacological or physiological effect when administered to a subject in need thereof.

As used herein, the terms "treatment" or "prevention" refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular infection or disease or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure of an infection or disease and/or adverse effect attributable to the infection or the disease. Accordingly, the method "prevents" (i.e., delays or inhibits) and/or "reduces" (i.e., decreases, slows, or ameliorates) the detrimental effects of a disease or disorder in the subject receiving the compositions of the invention.

The subject may be any animal, including a mammal, such as a human, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The subject that is administered the crystalline nanoparticle is afflicted with a disease or unwanted condition, which can encompass any type of condition or disease that can be treated therapeutically. In some embodiments, the disease or unwanted condition that is to be treated is a cancer, and the hydrophobic drug is a chemotherapeutic drug.

Methods to detect the inhibition of cancer growth or progression are known in the art and include, but are not limited to, measuring the size of the primary tumor to detect a reduction in its size, delayed appearance of secondary tumors, slowed development of secondary tumors, decreased occurrence of secondary tumors, and slowed or decreased severity of the secondary effects of disease. A "chemotherapeutic drug" is one that has therapeutic activity against a cancer. In general, chemotherapeutic drugs take advantage of the rapid growth associated with cancerous cells and inhibit growth or proliferation or induce the death of the cancerous cells. Chemotherapeutic drugs are well known in the art (see e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)) and include, but are not limited to, alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), toxins, and synthetics. In particular embodiments, the chemotherapeutic drug administered to a subject having a cancer is paclitaxel, camptothecin, C6-ceramide, 5-fluorouracil, or a combination thereof.

It will be understood by one of skill in the art that the presently disclosed compositions can be used alone or can be used in conjunction with other therapeutic modalities, including, but not limited to, surgical therapy, radiotherapy, or treatment with any type of therapeutic agent, such as a drug. In those embodiments in which the subject is afflicted with cancer, the presently disclosed compositions can be delivered in combination with any chemotherapeutic agent well known in the art, including, but not limited to, the chemotherapeutic agents described elsewhere herein, or any other cytotoxic drug, as described elsewhere herein or immunotherapy.

Paclitaxel is also useful for the prevention of restenosis. Restenosis refers to the closing of an artery that was previously opened during a surgical procedure, such as angioplasty.

In some embodiments, the use of the presently disclosed crystalline nanoparticles of hydrophobic drugs or dispersions comprising the same allows the effective dose of the hydrophobic drug that is administered to a subject to be increased. Delivery of a therapeutically effective amount of a hydrophobic drug that has been formulated into crystalline nanoparticles or dispersions comprising the same can be obtained via administration of the presently disclosed crystalline nanoparticles or dispersions thereof alone or in combination with a pharmaceutically acceptable carrier comprising a therapeutically effective dose of the hydrophobic drug. By "therapeutically effective amount" or "dose" is meant the concentration of a hydrophobic drug that is sufficient to elicit the desired therapeutic effect.

As used herein, "effective amount" is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. The effective amount of the hydrophobic drug will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound or complex, and, if desired, the adjuvant therapeutic agent being administered along with the hydrophobic drug formulation. Methods to determine efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996) Harrison 's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical methods in cell cultures or experimental animals, e.g., for determining the LD_5O (the dose lethal to 50% of the population) and the ED_5O (the dose therapeutically effective in 50% of the population).

The dose ratio between toxic (e.g., immunotoxic) and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. In some embodiments, compositions which exhibit high therapeutic indices are administered to subjects in need thereof. As a non- limiting example, the paclitaxel/Pluronic® F 127 (PTX/ 127) crystalline nanoparticle formulations disclosed herein has a much lower level of associated cytotoxicity (e.g., hemolytic activity) than the most commonly used Cremophor EL^®-paclitaxel formulation at equivalent doses (see Experimental Examples 3 and 4). Further, the PTX/127 crystalline nanoparticle formulation exhibited a higher therapeutically effective dose than the Cremophor EL^®-PTX formulation.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the presently disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. A therapeutically effective amount of a hydrophobic drug typically ranges from about 0.001 to 100 mg/kg body weight; in some embodiments, about 0.01 to 80 mg/kg body weight; in other embodiments, about 0.1 to 60 mg/kg body weight, and in still other embodiments, about 1 to 20 mg/kg body weight. The pharmaceutical composition can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments.

In another embodiment of the invention, a therapeutically effective dose of the hydrophobic drug formulation is administered intermittently. By "intermittent administration" is intended administration of a therapeutically effective dose of the hydrophobic drug, followed by a time period of discontinuance, which is then followed by another administration of a therapeutically effective dose, and so forth.

One of ordinary skill in the art upon review of the presently disclosed subject matter would appreciate that the presently disclosed compositions, including pharmaceutical compositions thereof, can be administered directly to a cell, a cell culture, a cell culture medium, a tissue, a tissue culture, a tissue culture medium, and the like. When referring to the crystalline nanoparticles and dispersion comprising the same, the term "administering," and derivations thereof, comprises any method that allows for the hydrophobic drug to contact a cell, tissue, or physiological site. The presently disclosed compounds or pharmaceutical compositions thereof, can be administered to (or contacted with) a cell or a tissue in vitro or ex vivo. The presently disclosed compounds, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, also can be administered to (or contacted with) a cell or a tissue in vivo by administration to an individual subject, e.g., a patient, for example, by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration), oral administration, or topical application, as described elsewhere herein.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nanoparticle" is understood to represent one or more nanoparticles. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. Throughout this specification and the claims, the words "comprise," "comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.

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

EXPERIMENTAL

Materials and Methods for Examples 1-4 Animals and materials

Female BALB/c and nude BALB/c mice (5 weeks of age) were purchased from the National Cancer Institute (NCI). The human lung cancer cell line, NCI-H460, was obtained from American Type Culture Collection. The murine breast cancer cell line, 4Tl, was kindly provided by Dr. Mark Dewhirst (Department of Radiation Oncology, Duke University Medical Center). All work performed on animals was in accordance with and permitted by the University of North Carolina Institutional Animal Care and Use committee. Paclitaxel (PTX) was purchased from Lc laboratories (Woburn, MA) and Pluronic® F-127 (Fl 27), Pluronic® F-68 (F68), Cremophor-EL^® and camptothecin (CPT) were purchased from Sigma. Preparation of the crystalline nanoparticles

Paclitaxel (PTX) and F 127 were dissolved in chloroform (in a glass tube) at a ratio of 3:1 (PTX/F127, mol/mol), which corresponds to an approximate PTX/F127 weight/weight ratio of about 1 :5, and then crystallized by evaporating the chloroform with a steady stream of nitrogen gas. Traces of chloroform were evaporated under a vacuum in the presence of desiccators for 2 to 4 hours. The crystals were hydrated in 5% dextrose for one hour and vortexed. Suspensions of the crystals (4 mg/mL PTX was prepared for the dose of 60 mg/kg) were sonicated for 10 to 15 min by a bath-type sonicator (output 80 kc, 80 Watts) to form the crystalline nanoparticles. Similar methods were utilized to prepare crystalline nanoparticles of camptothecin, except camptothecin (CPT) and F 127 were dissolved in a chloroform/methanol suspension (1 :1, volume/volume) at a 7:1 molar ratio of CPT to F 127, which corresponds to an approximate CPT:F127 weight ratio of 1 :5.

Characterization of the crystalline nanoparticles

Transmission electron microscopy (TEM) images of the resulting crystalline nanoparticles were acquired using a Phillips CM12 (FEI, Hillsboro, OR). Freshly prepared nanoparticles (5 μL) samples were dropped onto a 300 mesh carbon-coated copper grid (Ted Pella, Inc., Redding, CA), followed by a short incubation (5 min) at room temperature. Grids were then stained with 1% uranyl acetate (40 μL) and wicked dry. All images were acquired at an accelerating voltage of 100 kV. Gatan DigitalMicro graph software was used to analyze the images.

To assess the efficiency of the incorporation of PTX into the stable crystalline nanoparticles, the nanoparticles were incubated at room temperature for one hour, and were then centrifuged at 16,000 x g for 20 min in a 0.45 μm Centrifugal Filter Device (Millipore Co., Bedford, MA). Using the presently disclosed methods, in the absence of a surface stabilizer, free PTX does not form particles less than 0.45 μm in size; thus, the filter device removes free PTX particles, which are larger than 0.45 μm. The PTX incorporated into crystalline nanoparticles was quantified by HPLC. The distribution of particle size in the samples was measured using the submicron particle sizer

(NICOMP particle sizing systems, AutodilutePAT Model 370, Santa Barbara, CA) in the NICOMP mode. The zeta potential of the samples diluted in 1 mM KCl was determined by the Zeta Plus zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY).

Evaluation of hemolytic activity of the crystalline nanoparticles Blood was drawn from the nude BALB/c mice and centrifuged at 4⁰C. The centrifuged blood was washed twice with PBS buffer (pH 7.4) to isolate erythrocytes (RBC). RBC (2 x 10¹⁰) were re-suspended in 100 μl of PBS containing either the crystalline nanoparticles or Cremophor-EL^® with different concentrations of PTX. The above procedures were performed at about 0⁰C. After the samples were incubated at 37⁰C for 1 h and then centrifuged for 10 min at 16,000 x g, 70 μl of the supernatant was diluted with 100 μl PBS and measured at an optical density (OD) of 570 nm. The percentage of hemolysis was calculated as a function of OD according to the equation: % hemolysis = OD (sample)/OD (total hemolysis) x 100. The OD (total hemolysis) was measured from samples that were sonicated for 5 min before the measurement.

Animal model and treatments

The NCI-H460 cells (5 x 10⁶) were subcutaneous Iy injected into the right flank of the nude mice. The 4Tl cells (10⁵) were subcutaneously injected into the hair- trimmed belly of BALB/c mice. Mice were intravenously administered PTX in different formulations, with five mice in each treatment group. Tumor volume of the xenograft models was monitored every other day, which was calculated as {a² x b)/2, where a and b were the long and short dimensions of the tumor, respectively (Poirson- Bichat et al. (1997) Life Sci 60:919-931). Mice were sacrificed when the long dimension of tumor reached 2 cm. In the tumor studies, wherein the PTX formulations were orally administered, the lyophilized crystalline nanoparticles were examined and the control was freshly prepared PTX sonicated in 10% sucrose.

Statistical analysis

All statistical analyses were performed by a two-tailed student t-test. Data were considered statistically significant when the P value was less than 0.05. Example 1. Development of stabilized crystalline nanoparticles.

Currently, PTX is one of the most effective anticancer drugs in the clinic. Paclitaxel (PTX) promotes tubulin polymerization by forming a hyperstabilized structure, disrupting the normal tubule dynamics essential in cellular division, thereby inducing cell death (Spratlin and Sawyer (2007) Crit Rev Oncol Hematol 61 (3):222-9; Gligorov and Lotz (2004) Oncologist 9 Suppl 2:3-8). PTX has a significant antineoplastic activity in solid tumors and it is currently approved by both the FDA and the EMEA for the treatment of non- small cell lung cancer, breast cancer, ovarian cancer and AIDS-related Kaposi's sarcoma. It is also used either as a single agent or in combination with other antineoplastic agents in the treatment of thyroid, bladder, head and neck cancers (Ain, Egorin, and DeSimone (2000) Thyroid 10(7):587-94; Chougule et al. (2008) Head Neck 30(3):289-96; Crown, O'Leary, and Ooi (2004) Oncologist 9 Suppl 2:24-32; Galsky (2005) Oncologist 10(10):792-8; Haddad et al. (2005) Am J Clin Oncol 28(1): 104; and Voigt et al. (2005) J Cancer Res Clin Oncol 131(9):585-90). However, PTX has been shown to be a high affinity substrate for the multi-drug resistance 1 (MDRl) expressed protein, a P-glycoprotein (P-gp), which hinders its successful therapy in cancers (Yusuf et al. (2003) Curr Cancer Drug Targets 3(1): 1-19; Galletti et al. (2007) Chem Med Chem 2(7):920-42; and Geney et al. (2002) Clin Chem Lab Med 40(9):918-25). Moreover, the clinical application of PTX is limited by its low solubility in water (<1 μg/ml) (Fonseca, Simoes, and Gaspar (2002) J Control Release 83(2):273-286). Accordingly, the standard formulation of PTX (Taxol^®) requires the use of solvents, such as Cremophor^® EL (a 50/50 (v/v) mixture of polyoxyethylated castor oil and dehydrated alcohol). The ratio of PTX to the excipient (Cremophor-EL) is 1/90 (w/w). This formulation contributes to most of the toxicity associated with PTX- based therapy, including hypersensitivity, nephrotoxicity and neurotoxicity, which commonly occur during infusion, affecting 25-30% of patients (Xie, J. and CH. Wang, Self-assembled biodegradable nanoparticles developed by direct dialysis for the delivery of paclitaxel. Pharm Res, 2005. 22(12):2079-90; Szebeni, Muggia, and Alving (1998) J Natl Cancer Inst 90(4):300-6). The object of this study is to develop a Cremophor-EL^®-free nanoparticle formulation with decreased toxicity. These studies were initiated to determine if a surfactant can stabilize crystalline nanoparticles of PTX suspended in an aqueous solution. Initial experiments tested the stabilizing effects of two different excipients, the poloxamers Pluronic® F68 (F68) and Pluronic® F 127 (F 127) that have different physical properties (see Table 2; Croy and Kwon (2004) J Control Release 95:161-171).

Table 2. Physical Properties of Selected Poloxamers

MW: molecular weight; CMC: critical micelle concentration; a: number of ethylene oxide units in each polyoxyethylene chain; b: number of propylene oxide units in each polyoxypropylene chain

Transmission electron microscopy demonstrated that rod-shaped crystalline nanoparticles with an average long dimension of 139 ± 44 nm and a short dimension of 25 ± 6 nm could be formulated by the presently disclosed methods using F 127 as the surfactant at a 3:1 molar ratio of PTX:F127 (Figure IA), which corresponds to an approximate 1 :5 weight/weight ratio of PTX:F127. Crystalline nanoparticles of PTX and F 127 were also generated at a weight ratio of 1 :4 PTX:F127, but when the amount of F127 was reduced further (e.g., 1 :3 w/w of PTX:F127), no crystalline nanoparticles were formed. The crystalline nanoparticles could be lyophilized to powder and reconstituted to regain the rod-shaped crystal morphology (Figure IB). The ability to lyophilize and reconstitute the formulations while retaining activity will allow for long- term storage of the drug formulations. In contrast to F 127, using F68 as a surfactant resulted in unstable aggregates (data not shown). It is possible, however, that at a slightly lower drug: surfactant ratio, Pluronic® F68 would also be an effective stabilizer of PTX nanoparticles. PTX crystalline nanonp articles have been successfully generated using the presently disclosed methods with Tween® 80 and Brij® 700 as surfactants, wherein the drug: surfactant ratio was 1 :1. To test whether the methodology described above can be used to formulate other hydrophobic drugs in a crystalline nanop article form, camptothecin (CPT) was selected and prepared using similar methods, Transmission electron microscopic analysis of the resulting nanoparticles (Figure 1C) demonstrated that CPT could be formulated into cubic crystalline nanoparticles [102 (± 51) x 82 (± 35 nm)] with a ratio of 7:1 (CPT: F 127, mol/mol), which corresponds to an approximate weight ratio of CPT:F127 of about 1 :5. Additional crystalline nanoparticle formulations were successfully generated with a weight/weight ratio of CPT to F127 of 1 :4. It is important to note that both PTX and CPT alone without any surfactant also assumed the same crystalline morphology as in the presence of F 127 when prepared using similar methods. In other words, pure PTX formed rod-shaped crystals and pure CPT formed cubic crystals (data not shown). However, these pure crystals aggregated and precipitated from the suspension shortly after preparation (see Figure 2). Thus, it appears that the inclusion of F 127 in a drug formulation did not change the drug's crystalline structure; it only stabilizes the crystals in their nano-sized physical state. It also suggests that surfactant does not co-crystallize with the drug.

Example 2. Characterization of the crystalline nanoparticles.

Additional evidence that the F 127 can stabilize the PTX and CPT crystalline nanoparticles came from turbidity studies. When F 127 was incorporated into either PTX or CPT, as shown in Figure 2, both nanoparticles were well dispersed and no change in turbidity was observed for these nanoparticle formulations. In contrast, PTX and CPT alone (without F 127) showed a high absorption value (OD 600 nm) at time zero due to of aggregation. With time, the aggregates precipitated and the turbidity of the suspension decreased. In addition, we examined the zeta potential, the size distribution of the nanoparticles, and the incorporation efficiency of PTX into the nanoparticles. Large crystals composed of PTX alone, even after mixing with F 127 and sonication, could not pass through either a 0.22 μm or a 0.44 μm filter. In contrast, 100% and 96.5% of PTX in the crystalline nanoparticles were recovered in the 0.44 μm and 0.22 μm filtrate, respectively, under the same experimental conditions. Neither preparation could pass through a 3,000 MWCO Microcon filter. Therefore, the crystalline nanoparticles incorporated about 100% of PTX that was added to the nonpolar solvent. The crystalline nanoparticles exhibited a zeta potential close to neutral (0.8 mV), with a size distribution of 122 ± 35 nm (measured by a submicron particle sizer). These parameters remained the same for at least 12 h in storage at room temperature, indicating the nanoparticles were stable. Also, the nanoparticles exhibited in vitro cytotoxicity similar to Cremophor-EL^®-PTX in H460 and 4Tl tumor cell lines (data not shown). The crystalline nanoparticles containing 0.72 mg PTX were incubated in a 250 μl solution of 45% mouse serum at 37⁰C for 4 h and were then examined by TEM. As shown in Figure 3, the rod-shaped nanoparticles still existed after a 4 h incubation in the serum solution, demonstrating that the nanoparticles were stable in the presence of biological fluid.

Table 3. Characterization of PTX crystalline nanoparticles.

* The samples passed through 0.22 or 0.44 μm Ultrafree-MC (Millipore Co., Bedford,

MA).

** The samples passed through a 3,000 MWCO Microcon (Millipore Co., Bedford,

MA).

N/A: the formulation was a precipitate such that no reproducible measurements could be made.

Example 3. Evaluation of the toxicity of the crystalline nanoparticles.

The low toxicity and biocompatibility of Pluronic® F 127 has made it an attractive candidate in designing pharmaceutical vehicles to deliver drugs through different routes of administration (Escobar-Chavez et al. (2006) J Pharm Pharm Sci 9:339-358). The PTX nanoparticles developed and described herein consisted of a very small amount of F 127, and are, therefore, expected to exhibit lower toxicity than Cremophor-EL^®-PTX. The toxicity of the nanoparticle formulations was first evaluated in mice by determining the maximum tolerated dose. When female nude mice (18-2Og) were intravenously injected with PTX in Cremophor-EL^®, six out of six injected mice died as the dose reached 30 mg/kg; however, all six mice that were treated with the PTX crystalline nanoparticles survived at doses as high as 60 mg/kg.

High levels of surfactants are known to cause hemolysis of the red blood cells. The hemolytic property of PTX administered in the commercial Cremophor-EL^® vehicle has been reported due to the high levels of the surfactant, Cremophor-EL^® (polyoxyethylated castor oil) in the formulation (Bitton, Figg, and Reed (1995) Drug Saf \2:\ 96-208). In order to determine that the PTX crystalline nanoparticles are safe for i.v. administration, the hemolytic potential of the formulations were evaluated. In vitro drug-induced hemolysis has been considered to be a simple and reliable estimation of the membrane damage caused by drugs in vivo (Nornoo, Osborne, and Chow (2008) IntJPharm 349:108-116). The in vitro hemolytic activity of the crystalline PTX nanoparticles and of the Cremophor-EL^® PTX formulations with molar concentrations ranging from 0 to 800 μM was assessed in a murine erythrocyte suspension, with results provided in Figure 4. It can be observed that the nanoparticles were significantly less hemolytic than the Cremophor-EL^®-PTX at a PTX concentration of 200 μM (p < 0.005). If a mouse (20 g) is intravenously injected with 20 mg/kg PTX, the blood concentration of PTX is approximately 320 μM (the blood volume is estimated to be 7.3% of the body weight for mice (Wu et al. (1981) Biochim Biophys Acta 674: 19-29)). As shown in Figure 4, Cremophor-EL^® with a PTX concentration of approximately 320 μM PTX induced about 40% hemolysis, with no observed hemolysis associated with the crystalline nanoparticle formulation. As the concentration increases to 800 μM, no hemolysis was detected with the nanoparticles but 100% hemolysis was observed for Cremophor-EL^®-PTX. The results of these assays measuring the hemolytic activity and maximum tolerated dose indicate that the nanoparticles are much safer than Cremophor-EL^®-PTX.

Example 4. Inhibition of tumor growth by the stabilized crystalline nanoparticles.

PTX is highly efficacious in the treatment of nonsmall-cell lung cancer, breast cancer, ovarian carcinoma and head and neck cancers (Mekhail and Markman (2002) Expert Opin Pharmacother 3:755-766). In this study, the effect of the PTX nanoparticles on tumor growth was evaluated in two tumor models, H460 human lung cancer in a xenograft model and murine 4Tl breast cancer in a syngeneic model. For the H460 tumor model shown in Figure 5 A, the untreated tumors grew rapidly, reaching 1676 ± 238 mm³ in volume 17 days after the tumor cell inoculation. In contrast, tumors in the group of mice intravenously injected with the crystalline PTX nanoparticles (20 mg/kg PTX) had only reached 144 ± 35 mm in volume at the same time point, which was significantly smaller than that of the untreated group (p<0.001). Similar tumor-growth inhibition was observed in the group treated with 20 mg/kg PTX in Cremophor-EL^® (p > 0.5 compared to the crystalline nanoparticles group). The low toxicity of the PTX crystalline nanoparticles allowed the effects on tumor growth to be examined at a higher dose of this formulation, i.e., 60 mg/kg. As shown in Figure 5A, the higher dose could further inhibit tumor growth and the tumor size was as small as 100.2 ± 46 mm³29 days after the tumor implantation, but the tumor reached 1,689 ± 538 mm³ and 1,599 ± 413 mm³ in the groups treated with the lower dose (20 mg/kg) of Cremophor-EL^® and the nanoparticles, respectively.

Next, tumor inhibition was studied in the murine breast cancer model using PTX crystalline nanoparticles that had been lyophilized and reconstituted (Figure 5B). The lyophilized and reconstituted PTX/F127 nanoparticles demonstrated a similar therapeutic pattern in the 4Tl breast tumor model as seen in the H460 lung cancer model, i.e., the nanoparticles were as efficient as the Cremophor-EL^® formulation in tumor inhibition at a lower dose (20 mg/kg) and exhibited significantly enhanced efficacy at the higher dose of 60 mg/kg.

The stability of the nanoparticle formulations prompted studies examining tumor inhibition through an oral administration route. Currently, PTX is only marketed as an intravenous (i.v.) formulation. Oral administration of PTX is attractive because it may enable the development of chronic treatment schedules, resulting in plasma concentrations at a pharmacologically relevant level for a prolonged period of time

(Veltkamp et al. (2007) Cancer Chemother Pharmacol 60:635-642). In addition, oral administration is more convenient and patients are more compliant with orally administered medications than those administered intravenously. However, PTX is a high affinity substrate for the drug efflux transporter P-glycoprotein (P-gp) in the membrane of gastrointestinal tract (Sparreboom et al. (1997) Proc Natl Acad Sci USA 94:2031-2035). PTX stabilized by F127 may prevent PTX from being recognized by P-gp allowing the drug to be transported into the blood circulation. This was tested in the 4Tl tumor-bearing mice that were orally administered either a PTX suspension or the lyophilized and reconstituted PTX crystalline nanoparticles. Indeed, no antitumor activity was observed in the mice treated with the PTX suspension; there was no significant difference in tumor growth between the untreated and treated (PTX suspension) groups (p>0.5). Oral administration of the lyophilized and reconstituted nanoparticles, however, significantly inhibited tumor growth (p<0.01). The tumor volume in the mice treated with the crystalline nanoparticles was three times smaller than that of mice treated with the PTX suspension.

Since the crystalline nanoparticles were formulated with a single excipient of F 127 with a high ratio of drug to F 127, the crystalline nanoparticles exhibit the capability to attenuate toxicities, enhance delivery of hydrophobic drugs to the desired biological site (such as the gastroentero epithelium), and improve the therapeutic efficacy.

It is well known that intravenously administered colloidal particles are normally removed efficiently by the reticuloendothelial cells of the liver and spleen. By coating the model microspheres with F 127, the F 127 on the surface of the microparticles redirected the colloidal particles from the liver and the spleen to the bone marrow (Ilium and Davis (1987) Life Sci 40:1553-1560; Porter et al. (1992) FEBS Lett 305:62- 66). Therefore, the crystalline nanoparticles described herein may also be used to treat metastases in organs including the bone marrow. In summary, the results of this study strengthen the validity of the novel crystalline nanoparticles as a drug delivery vehicle for cancer therapy. This formulation has great potential for marketability due to the following advantages. First, the crystalline nanoparticles achieved very high efficiency of drug loading, with a ratio of 3 to 1 (PTX/F127, mol/mol), which indicates that every molecule of the excipient can carry three molecules of the drug. Second, the methodology for the preparation of the crystalline nanoparticles is simple and without any chemical modifications for the drug and the excipient. The process can be easily scaled up for manufacturing. Third, it is cost-effective to prepare the nanoparticles because the commonly used F 127 is the only excipient in this formulation and the price of F 127 is comparable to Cremophor- EL^®. Lastly, this study is the first to show that a nanoparticle based formulation can achieve antitumor activity through both i.v. and oral administrations.

Example 5. Examination of the mechanism of crystalline nanoparticle formation.

To elucidate the possible mechanism by which the crystalline nanoparticles were generated, kinetic morphology changes of PTX/F127 and PTX alone were examined. Chloroform containing PTX with or without F 127 was placed onto a glass slide. After drying and dessicating, a drop of water was added to each sample, the slides were placed into a humidified chamber at room temperature for various hydration times and pictures were taken. Without being bound by any theory or mechanism of action, based on the imaging results (Fig. 6), it is believed that the mechanism by which the crystalline nanoparticles form involves three phases: phase 1, an amorphous precipitate; phase 2, a hydrated amorphous aggregate; and phase 3, stabilized nanocrystal (F127 coated nanocrystal; see Figure 7). The amorphous solid in phase 1 was formed when PTX and F 127 co-precipitated as the chloroform was evaporated. PTX without F 127 also formed the amorphous solid. The amorphous structure still existed after 2 min of hydration for both PTX/F127 and PTX alone (Fig. 6a and 6b). When the amorphous precipitate was hydrated for a longer period of time (i.e., 30 min) in phase 2, the PTX/F127 formulation formed hydrated amorphous aggregates (Fig. 6c, circled). For the PTX sample without F127, however, the hydration resulted in the formation of crystals (Fig. 6d, circled). If the hydration occurred for a longer period of time (i.e., 4 h) partial amorphous aggregates (PTX/F127) started to form small crystals (Fig. 6e, circled) and the crystals formed from PTX without F 127 grew even larger (Fig. 6f, circled). Upon nanonization (sonication for 10 min) of the hydrated amorphous aggregate, the crystalline nanoparticles (F 127 coated nanocrystals) were formed (Fig. 1). Sonication performed before the formation of the hydrated amorphous aggregate (a short period of hydration) required an increase in sonication time (> 20 min) in order to form the crystals, which were heterogeneous in size. Heterogeneous crystals were also observed when the sonication was performed after the formation of the crystalline (4h hydration, Fig. 6E), which may be due to the fact that the nanonization cannot break down the crystalline PTX into nanocrystals, suggesting that the formation of crystalline nanoparticles may require the formation of the hydrated amorphous aggregates of phase 2 or at least the absence of crystalline PTX prior to sonication.

Example 6. Folate receptor-targeted crystalline nanoparticles.

The folate receptor (FR), over-expressed in a broad spectrum of malignant tumors, is an attractive target for selective delivery of anticancer drugs to tumor cells (Zhao, Li, and Lee (2008) Expert Opin DrugDeliv 5(3):309-19). Folic acid, therefore, was conjugated to pluronic F 127 and folate receptor-targeted crystalline nanoparticles were prepared using a protocol identical to that of the non-targeted formulation. Cytotoxicity of FR-targeted and non-targeted crystalline nanoparticles was determined in FR-positive KB cells (human oral carcinoma cells) using the MTT assay. The results in Fig. 8 A showed that the targeted crystalline nanoparticles containing 10% (F127-folate/F127, w/w) of F127-folate resulted in significantly lower viability (p < 0.001) compared with the non-targeted crystalline nanoparticles as the concentration of PTX varied from 0.08 to 10 μM. As shown in Fig. 8B, the cytotoxicity decreased when the F127-folate decreased from 10% to 5% (p < 0.05). No further enhancement of the cytotoxicity was observed at higher concentrations of F127-folate (> 10%). The differential cytotoxicity was abolished in the presence of ImM free folate as a competitor (Fig. 8B), indicating the FR-specifϊc cytotoxicity of the FR-targeted crystalline nanoparticles.

Example 7. Vitamin E tocopherol polyethylene glycol succinate (TPGS)-based crystalline nanoparticles.

The encouraging results of anticancer therapy from the F127-based crystalline nanoparticles prompted the development of crystalline nanoparticles for the treatment of drug-resistant cancers. For these studies, the crystalline nanoparticles were prepared using TPGS as the surface stabilizer, which serves dual functions, stabilizing the crystalline nanoparticles and inhibiting the drug efflux transporter P-glycoprotein (P- gp)-

The TPGS crystalline nanoparticles were also prepared using the methods described herein. Replacing F 127 with TPGS did not affect the morphology or particle size of the crystalline nanoparticles (Fig. 9A). The TPGS crystalline nanoparticles still kept exhibited a rod shape with an average long dimension of 148 nm and a short dimension of 24 nm, which did not change when the weight ratio of PTX to TPGS varied from 1/1 to 1/5.

When the ratio was lower than 1/1 (further reduction in the amount of TPGS), the crystalline nanoparticles could not be formed. Thus, the maximum drug loading ratio of the TPGS crystalline nanoparticles was found to be as high as 50%. The TPGS crystalline nanoparticles could also be lyophilized to a powder and reconstituted to regain the rod-shaped crystals (data not shown).

To assess the possibility of formulating the crystalline nanoparticles comprising more than one type of hydrophobic drug, C6-Ceramide (C6-CER) was selected as the additional hydrophobic drug that was combined with PTX (at a weight ratio of PTX:C6-CER:TPGS of 1 : 1 :5) during the formation of crystalline nanoparticles. As shown in Fig. 9B, the combined drug crystalline nanoparticles were also rod-shaped, with a slightly larger size and width than that of PTX crystalline nanoparticles. It should be noted, however, that the conditions for this preparation were not optimized.

Next, the particle size of the crystalline nanoparticles was examined over a period of two weeks at both room temperature (RT) and 37°C to evaluate the physical stability of the PTX/TPGS crystalline nanoparticles, The particle size of the PTX:F127 crystalline nanoparticles slowly increased after storage at RT (Fig. 10A) and increased more rapidly when stored at 37°C (Fig. 10B). In contrast, as shown in Fig. 1OA and 1OB, the physical stability of the crystalline nanoparticles, particularly the thermal stability, was greatly increased by TPGS. TPGS crystalline nanoparticles, with either a 1/1 or 1/5 weight ratio, showed no growth in particle size for at least two weeks, under the tested storage conditions at RT or 37°C.

The amount of PTX released from the crystalline nanoparticles with a weight ratio of 1/5 (PTX/F127 or TPGS) at 37°C was determined in an effort to assess whether PTX would be released from crystalline nanoparticles prior to cellular uptake. The results shown in Fig. 11 revealed a slow and sustained release of PTX from both F 127 and TPGS crystalline nanoparticles. An initial burst of release of PTX was not observed for either crystalline nanoparticle formulation, which indicated that PTX was likely not present at or near the surface of the crystalline nanoparticles, but instead the PTX was encapsulated by the nonionic surfactants coated on the surface of the crystalline PTX. In addition, the crystalline nanoparticles coated and stabilized with TPGS exhibited a significantly lower amount of PTX release when compared to that of F 127 crystalline nanoparticles. The cumulative release of PTX after 72 h was about 70% from F 127 crystalline nanoparticles and about 50% from TPGS crystalline nanoparticles. The attenuated drug release of the PTX:TPGS formulations could result in a more favorable pharmacokinetic profile in vivo (Stevens, Sekido, and Lee (2004) Pharm Res 2\(\2):2\53-7).

Previous studies have shown that the NCI/ ADR-RES cell line, which was derived from human ovarian carcinoma cells overexpresses P-gp and is highly resistant to anticancer drugs such as PTX and doxorubicin (Liscovitch and Ravid (2007) Cancer Lett 245 ( 1 -2) : 350-2) . This cell line, therefore, was treated with PTX in different formulations and the cell viability was assessed with an MTT assay 48 h after the treatment. As shown in Fig. 12A, a similar cell-killing efficacy was observed for PTX alone, PTX/Cremophor-EL^®, F 127- and TPGS-based crystalline nanoparticles (with a 1/5 weight ratio of PTX/F127 or TPGS) in two cell lines (KB and H460), irrespective of whether PTX was free or encapsulated in the crystalline nanoparticles. No cell- killing was detected in NCI/ADR-RES cells for PTX alone or F 127 crystalline nanoparticles (p > 0.05, compared to PBS treatment). Treatment with the TPGS crystalline nanoparticles, however, resulted in very significant cell killing, with 35% cell viability (p < 0.001). PTX/Cremophor-EL^® was able to induce cell killing, with 74% cell viability (p < 0.01), which is not surprising considering Cremophor can act as a P-gp inhibitor (Bogman et al. (2003) J Pharm Sci 92(6): 1250-61). The TPGS crystalline nanoparticles had an IC50 (the half maximal inhibitory concentration) value that was four times lower than that of PTX/Cremophor-EL^® (Fig. 12B), although the amount of Cremophor was about 18-fold higher than that of TPGS (PTX/Cremophor = 1/90, PTX/TPGS = 1/5, w/w). The cell-killing efficacy of the TPGS crystalline nanoparticles depended on the ratio of PTX to TPGS; the higher the ratio, the more cell killing was observed (Fig. 12C). For example, the crystalline nanoparticles with a weight ratio of 1/1 achieved about 70% cell viability, which decreased to approximately 32% as the ratio increased to 1/5 (PTX/TPGS).

Apoptotic cell death of NCI/ADR-RES cells was detected using flow cytometry (Fig. 13). The crystalline nanoparticles (10 μM) induced a higher percentage of apoptotic cell death (51.8%) compared to the untreated cells (3.4%) and cells treated with TPGS alone (9.2%). All in vitro data presented herein, including the cytotoxicity and apoptotic studies, indicate that the TPGS-based PTX crystalline nanoparticles can efficiently overcome MDR.

The crystalline nanoparticles with a weight ratio of 1/5 (PTX/TPGS) were used in studies to assess the antitumor efficacy of the formulations in MDR cell-bearing mice because of their high level of cytotoxicity (Fig. 12C). Female nude mice, 7 days after the tumor cell inoculation (6 x 10⁶ NCI/ADR-RES cells per mouse), were injected (every three days for a total of five times) with TPGS alone or PTX (10 mg/kg) carried by the TPGS crystalline nanoparticles or Cremophor-EL^®. The results of tumor volume changes as a function of time after the injections are shown in Fig. 14 A. In this xenograft model, only PTX formulated in the TPGS crystalline nanoparticles were able to overcome the MDR, which significantly inhibited NCI/SDR-RES cell-growth. For example, on day 38, when the experiment was terminated, the tumor volume in the mice treated with the crystalline nanoparticles was 216 mm³ with a weight of 0.16 g (Fig. 14B), but 487 mm³ (0.49 g), 512 mm³ (0.51 g), and 458 mm³ (0.46 g) in untreated mice, mice treated with TPGS alone or PTX/Cremophor-EL^®, respectively. There was no significant difference between the untreated group and that treated with TPGS alone. It is important to note that while PTX/Cremophor-EL^® showed some cytotoxicity in this cell line in vitro (Fig. 12A and 12B), this commercially available formulation did not show therapeutic effect in vivo. This may be due to the very short half- life (approximately one hour) of PTX (Taxol) (Eiseman et al. (1994) Cancer Chemother Pharmacol 34(6):465-71) or its in vitro cytotoxicity against this MDR cell is too low to be reflected in this in vivo model.

The toxicity of the crystalline nanoparticles was evaluated by the hemolysis assay. The mouse (20 g) was intravenously injected with 10 mg/kg PTX. The blood concentration of PTX is approximately 150 μM (the blood volume is estimated to be 7.3% of the body weight for mice (Wu et al. (1981) Biochim Biophys Acta 674(1): 19- 29)). Hemolytic activity, therefore, was evaluated for the crystalline nanoparticles with 150 and 300 μM of PTX. As shown in Fig. 15, the crystalline nanoparticles with 150 μM of PTX did not induce hemolysis, and hemolysis was not observed as the PTX concentration was doubled (300 μM). In contrast, hemolysis was significantly higher for PTX/Cremophor-EL^®, with 31% and 48% hemolysis at 150 and 300 μM of PTX, respectively.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

THAT WHICH IS CLAIMED:

1. A method of preparing a stable dispersion of crystalline nanoparticles of a hydrophobic compound, said method comprising: a) dissolving a hydrophobic compound and a surface stabilizer in a nonpolar solvent, thereby forming a hydrophobic compound/surface stabilizer solution; b) evaporating said nonpolar solvent from said hydrophobic compound/surface stabilizer solution, thereby producing a hydrophobic compound/surface stabilizer precipitate, wherein said evaporating does not comprise spray drying; c) dispersing said precipitate with a liquid dispersion medium, thereby producing a dispersion of a hydrophobic compound/surface stabilizer aggregate; and d) homogenizing said hydrophobic compound/surface stabilizer aggregate, thereby producing said stable dispersion of said crystalline nanoparticles of said hydrophobic compound.

2. A method of preparing a stable dispersion of crystalline nanoparticles of a hydrophobic compound, said method comprising: a) dissolving a hydrophobic compound and a surface stabilizer in a nonpolar solvent, thereby forming a hydrophobic compound/surface stabilizer solution; b) evaporating said nonpolar solvent from said hydrophobic compound/surface stabilizer solution, thereby producing an essentially amorphous hydrophobic compound/surface stabilizer precipitate; c) dispersing said precipitate with a liquid dispersion medium, thereby producing a dispersion of an essentially amorphous hydrophobic compound/surface stabilizer aggregate; and d) homogenizing said hydrophobic compound/surface stabilizer aggregate, thereby producing said stable dispersion of said crystalline nanoparticles of said hydrophobic compound.

3. The method of claim 2, wherein said evaporating does not comprise spray drying.

4. The method of any one of claims 1-3, wherein said evaporating comprises providing a steady stream of nitrogen gas over said hydrophobic compound/surface stabilizer solution.

5. The method of any one of claims 1 -4, wherein said nonpolar solvent comprises at least one of chloroform and methanol.

6. The method of any one of claims 1-5, wherein said hydrophobic compound/surface stabilizer solution is essentially free of polar solvents.

7. The method of any one of claims 1 -6, wherein said hydrophobic compound/surface stabilizer solution comprises a weight/weight ratio of said hydrophobic compound to said surface stabilizer of between about 1 :5 and about 1 :1.

8. The method of claim 7, wherein said weight/weight ratio of said hydrophobic compound to said surface stabilizer is about 1 :5.

9. The method of claim 7, wherein said weight/weight ratio of said hydrophobic compound to said surface stabilizer is about 1 :1.

10. The method of any one of claims 1 -9, wherein at least about 90% of said hydrophobic compound in said nonpolar solvent is incorporated into said crystalline nanoparticles.

11. The method of claim 10, wherein at least about 95% of said hydrophobic compound in said nonpolar solvent is incorporated into said crystalline nanoparticles.

12. The method of claim 11, wherein about 100% of said hydrophobic compound in said nonpolar solvent is incorporated into said crystalline nanoparticles.

13. The method of any one of claims 1-12, wherein said hydrophobic compound has a solubility in water of less than about 1 mg/ml.

14. The method of claim 13, wherein said hydrophobic compound has a solubility in water of less than about 10 μg/ml.

15. The method of any one of claims 1-14, wherein said surface stabilizer comprises a non-ionic surfactant, a cationic surfactant, an anionic surfactant, or a zwitterionic surfactant.

16. The method of claim 15, wherein said non-ionic surfactant is selected from the group consisting of a poloxamer, a polysorbate, a polyoxyethylene ether, and a vitamin E tocopheryl polyethylene glycol succinate (TPGS).

17. The method of claim 16, wherein said TPGS comprises a vitamin E tocopheryl polyethylene glycol 1000 succinate.

18. The method of claim 15 , wherein said non-ionic surfactant is selected from the group consisting of Pluronic® F127, Tween® 80, and Brij® 700.

19. The method of any one of claims 1-18, wherein said surface stabilizer is a single type of surface stabilizer.

20. The method of any one of claims 1-19, wherein said liquid dispersion medium comprises an aqueous solution.

21. The method of claim 20, wherein said aqueous solution further comprises a sugar.

22. The method of claim 21 , wherein said concentration of said sugar in said aqueous solution is from about 0.5% to about 20% weight per volume.

23. The method of claim 21 or claim 22, wherein said sugar is dextrose or sucrose.

24. The method of claim 23, wherein said aqueous solution comprises dextrose at a concentration of about 5%.

25. The method of claim 23, wherein said aqueous solution comprises sucrose at a concentration of about 20%.

26. The method of any one of claims 1-25, wherein said hydrophobic compound/surface stabilizer precipitate is incubated in said liquid dispersion medium for a period of time of between about 2 minutes and about 4 hours.

27. The method of claim 26, wherein said period of time is between about 30 minutes and about 1 hour.

28. The method of any one of claims 1-27, wherein said homogenizing comprises sonicating.

29. The method of any one of claims 1-28, wherein at least one dimension of said crystalline nanoparticles is less than 100 nm.

30. The method of any one of claims 1-29, wherein said hydrophobic compound comprises at least one of paclitaxel, camptothecin, and C6-ceramide.

31. The method of any one of claims 1 -30, further comprising sterilizing said stable dispersion.

32. The method of claim 31 , wherein said sterilizing comprises filter sterilizing said stable dispersion.

33. The method of any one of claims 1 -32, further comprising a recovery step, wherein said nanoparticles are recovered from said stable dispersion thereof, wherein said recovery step comprises removing said liquid dispersion medium from said stable dispersion, thereby producing a plurality of nanoparticles of said hydrophobic compound.

34. The method of claim 33, wherein said removing said liquid dispersion medium comprises lyophilizing.

35. A stable dispersion of nanoparticles of a hydrophobic drug produced by the method of any one of claims 1-32.

36. A plurality of nanoparticles of said hydrophobic drug produced by the method of claim 33 or claim 34.

37. A crystalline nanoparticle comprising a crystalline hydrophobic compound having adsorbed on the surface thereof a surface stabilizer, wherein said crystalline nanoparticle comprises between about 20% and about 50% by weight of said hydrophobic compound.

38. The crystalline nanoparticle of claim 37, wherein about 25% of said nanoparticle by weight is said hydrophobic compound.

39. The crystalline nanoparticle of claim 38, wherein about 50% of said nanoparticle by weight is said hydrophobic compound.

40. The crystalline nanoparticle of any one of claims 37-39, wherein at least one dimension of said crystalline hydrophobic compound is less than 100 nm.

41. The crystalline nanoparticle of any one of claims 37-40, wherein said surface stabilizer comprises a non-ionic surfactant, a cationic surfactant, an anionic surfactant, or a zwitterionic surfactant.

42. The crystalline nanoparticle of claim 41 , wherein said non-ionic surfactant is selected from the group consisting of a poloxamer, a polysorbate, a polyoxyethylene ether, and a vitamin E tocopheryl polyethylene glycol succinate (TPGS).

43. The crystalline nanoparticle of claim 42, wherein said TPGS comprises a vitamin E tocopheryl polyethylene glycol 1000 succinate.

44. The crystalline nanoparticle of claim 41 , wherein said non-ionic surfactant is selected from the group consisting of Pluronic® F 127, Tween® 80, and Brij® 700.

45. The crystalline nanoparticle of any one of claims 37-44, wherein said crystalline hydrophobic compound has adsorbed on its surface only one type of surface stabilizer.

46. The crystalline nanoparticle of any one of claims 37-45, wherein said hydrophobic compound has a solubility in water of less than about 1 mg/ml.

47. The crystalline nanoparticle of claim 46, wherein said hydrophobic compound has a solubility in water of less than about 10 μg/ml.

48. The crystalline nanoparticle of any one of claims 37-47, wherein said hydrophobic compound comprises a hydrophobic drug.

49. The crystalline nanoparticle of claim 48, wherein said hydrophobic drug comprises at least one of paclitaxel, camptothecin, and C6-ceramide.

50. A stable dispersion comprising a plurality of said crystalline nanoparticles of any one of claims 37-49 and a liquid dispersion medium.

51. The stable dispersion of claim 50, wherein said liquid dispersion medium comprises an aqueous solution.

52. The stable dispersion of claim 51, wherein said aqueous solution further comprises a sugar.

53. The stable dispersion of claim 52, wherein said concentration of said sugar in said aqueous solution is from about 0.5% to about 20% weight per volume.

54. The stable dispersion of claim 52 or claim 53, wherein said sugar is dextrose or sucrose.

55. The stable dispersion of claim 54, wherein said aqueous solution comprises dextrose at a concentration of about 5%.

56. The method of claim 54, wherein said aqueous solution comprises sucrose at a concentration of about 20%.

57. A pharmaceutical composition comprising one or more of the nanoparticles of claim 48 or claim 49 and a pharmaceutical carrier.

58. A method for treating a disease or an unwanted condition in a subject, said method comprising administering the pharmaceutical composition of claim 57 to said subject, wherein said hydrophobic drug has therapeutic activity against said disease or said unwanted condition.

59. A method for treating a disease or an unwanted condition in a subject, said method comprising administering the stable dispersion of any one of claims 50-56 to said subject, wherein said hydrophobic compound is a hydrophobic drug having therapeutic activity against said disease or said unwanted condition.

60. The method of claim 58 or claim 59, wherein said administering comprises orally administering or intravenously administering.