WO2010009075A1 - Procédés et compositions comprenant des nanoparticules cristallines de composés hydrophobes - Google Patents

Procédés et compositions comprenant des nanoparticules cristallines de composés hydrophobes Download PDF

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Publication number
WO2010009075A1
WO2010009075A1 PCT/US2009/050463 US2009050463W WO2010009075A1 WO 2010009075 A1 WO2010009075 A1 WO 2010009075A1 US 2009050463 W US2009050463 W US 2009050463W WO 2010009075 A1 WO2010009075 A1 WO 2010009075A1
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Prior art keywords
hydrophobic compound
crystalline
nanoparticles
surface stabilizer
hydrophobic
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PCT/US2009/050463
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English (en)
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Leaf Huang
Feng Liu
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The University Of North Carolina At Chapel Hill
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention relates to the generation of nanoparticle formulations of hydrophobic compounds.
  • 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.
  • 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.
  • nanoparticles with a high drug load 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.
  • 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.
  • 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.
  • 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).
  • a hydrophobic drug e.g., paclitaxel, camptothecin, C6-ceramide
  • 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;
  • Figure 1C shows the crystalline nanoparticles of CPT.
  • Figure 2 presents a graph measuring the absorbance at OD 6 Oo of a dispersion of
  • Figure 3 provides a TEM image of the crystalline PTX nanoparticles following a four hour incubation with serum at 37°C.
  • 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 ⁇ M PTX ( Figure 8B).
  • 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 0 C ( Figure 10B).
  • Figure 11 presents a graph depicting the release of PTX from the F 127 and TPGS-based crystalline nanoparticles at 37 0 C. ** 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 ⁇ M PTX. NCI/ADR-RES cell in Figure 12A and Figure 12C were treated with 10 ⁇ M 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 ® .
  • Orally administered poorly water soluble drugs are eliminated from the gastrointestinal tract before being absorbed into the circulation.
  • 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.
  • 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.
  • nanoparticle refers to particles of any shape having at least one dimension that is less than about 1000 nm.
  • 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).
  • 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).
  • 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.
  • 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.
  • drug refers to any bioactive compound that can exhibit a therapeutic effect when administered to a living cell, tissue, or organism.
  • 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
  • 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).
  • hydrophilic molecules are capable of hydrogen bonding with a water (H 2 O) 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.
  • the hydrophobic compound of the crystalline nanoparticle can have a solubility of less than about 1 mg/ml in water.
  • the hydrophobic compound has a solubility in water of less than about 10 ⁇ g/ml, and in particular embodiments, about 1 ⁇ g/ml or 2.5 ⁇ g/ml.
  • the hydrophobic compound can have a solubility of about 0.001 ⁇ g/ml to about 10 mg/ml, including but not limited to 0.001 ⁇ g/ml, 0.01 ⁇ g/ml, 0.1 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, 500 ⁇ g/ml, 1 mg/ml, 5 mg/ml, and 10 mg/ml, and any other concentration between 0.001 ⁇ g/ml and 10 mg/ml.
  • PTX paclitaxel
  • CPT camptothecin
  • the crystalline nanoparticles can comprise more than one hydrophobic compound.
  • the multiple hydrophobic compounds are added, along with the surface stabilizer, to the nonpolar solvent.
  • paclitaxel and C6-ceramide or paclitaxel and camptothecin are co-formulated into crystalline nanoparticles.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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®
  • Polyethoxylated sorbitan fatty acid esters 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).
  • POE polyoxyethylene
  • Tween® 80 polyoxyethylene
  • POE sorbitan monostearate T ween® 60
  • POE sorbitan monolaurate Tween® 20
  • 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(C 2 H 4 OX(CsHeO) I3 (C 2 H 4 OXH; 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).
  • a number of ethylene oxide units in each polyoxyethylene chain
  • b number of propylene oxide units in each polyoxypropylene chain
  • 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.
  • 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.
  • 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.
  • 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).
  • HLB hydrophilic/lipophilic balance
  • HLB hydrophilic/lipohilic balance
  • 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.
  • 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.
  • cationic molecules such as cationic lipids.
  • 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'-(N 6 -guanidino-L-lysinyl)] aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N-2[N 2 -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-
  • 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-(N 5 N- 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-hydroxyeth
  • 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.
  • 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).
  • 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.
  • cytotoxic drugs e.g., paclitaxel, doxorubicin, vincristine, vinblastine
  • 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.
  • 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.
  • TPGS-based crystalline nanoparticles described herein 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
  • TPGS with varying chain lengths of PEG 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).
  • the crystalline nanoparticle comprises more than one type of surface stabilizer.
  • the presently disclosed methods allow for the generation of crystalline nanoparticles of hydrophobic drugs and stable dispersions comprising the same using a single surface stabilizer as the excipient.
  • the term "excipient" refers to an inert substance with no therapeutic activity that is used to formulate and deliver an active drug.
  • 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.
  • 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.
  • type when referring to a surface stabilizer (e.g., surfactant) refers to a particular chemical species.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • LDL low density lipoprotein
  • RGD arginine-glycine-aspartate
  • Integrin ⁇ v ⁇ 3 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 ⁇ v ⁇ 3 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).
  • nanoparticles containing the RGD motif were capable of delivering this cytotoxic drug to the ⁇ v ⁇ 3 -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.
  • the surface stabilizer is conjugated to a peptide comprising a RGD sequence.
  • FR folate receptor
  • SR sigma receptors
  • Human folate receptor (FR) is a 38-40 kDa //-glycosylated protein.
  • 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.
  • FFA folic acid
  • Sigma receptors (SR) 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).
  • 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.
  • the targeting ligand of the surface stabilizer/targeting ligand conjugate comprises folate (or folic acid) or a benzamide derivative, such as anisamide.
  • target 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).
  • 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).
  • a target cell which is a disease-associated antigen including, for example, tumor-associated antigens and autoimmune disease- associated antigens.
  • diseases-associated antigens include, for example, growth factor receptors, cell cycle regulators, angiogenic factors, and signaling factors.
  • the targeting ligand interacts with a cell surface protein on the targeted cell.
  • 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.
  • cancer cells overexpress certain cell surface molecules, such as the HER2 receptor (breast cancer) or the sigma receptor.
  • 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).
  • 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).
  • the targeted cell comprises a cancer cell.
  • cancer or “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer cells refer to the cells that are characterized by this unregulated cell growth.
  • 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.
  • the targeted cancer cell comprises a lung cancer cell.
  • 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.
  • SCLC small cell lung cancer
  • NSCLC non-small-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.
  • 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.
  • 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).
  • a liquid dispersion medium e.g., water
  • 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.
  • 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.
  • solvent 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.
  • 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.
  • the organic solvent is one that is volatile.
  • 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.
  • 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.
  • 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).
  • 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.
  • the precipitate is amorphous.
  • an object that is "amorphous" has no definite, consistent form (e.g., crystal).
  • 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.
  • evaporation of the nonpolar solvent does not occur via spray drying, which is a relatively rapid evaporative process.
  • 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.
  • 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.
  • 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 ⁇ M) 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.
  • a liquid dispersion medium e.g. liquid dispersion medium
  • another phase e.g., hydrophobic compound
  • the hydrophobic compounds associate through hydrophobic interactions, forming a hydrophobic compound/surface stabilizer aggregate.
  • 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.
  • the generation of a hydrophobic compound/surface stabilizer aggregate is important for the production of crystalline nanoparticles.
  • the liquid dispersion medium comprises a polar solvent.
  • the liquid dispersion medium comprises an aqueous solution (e.g., water) and the dispersion is thus, referred to as an aqueous dispersion.
  • 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%.
  • the concentration is between about 0.5% and 20%.
  • the sugar comprises dextrose or sucrose.
  • 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.
  • 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.
  • 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.
  • intermittent or continuous mixing of the particle dispersion can be performed.
  • 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.
  • 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.
  • the hydrophobic compound/surface stabilizer aggregate is then homogenized.
  • 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.
  • the term homogenization excludes milling (e.g., wet milling).
  • 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.
  • 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.
  • an output of about 80 kilocycles (kc) and about 80 watts is required to form the crystalline nanoparticles of the desired size.
  • 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.
  • 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.
  • 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.
  • 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 OD 6 oo-
  • the presently disclosed methods advantageously allow for the production of stable dispersions of hydrophobic compounds at ambient temperatures and pressures.
  • the steps of the presently disclosed methods are performed at a temperature ranging from about 20 0 C to about 30 0 C, including, but not limited to 20 0 C, 2FC, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30 0 C.
  • the presently disclosed methods further comprise a step of recovering the crystalline nanoparticles from the dispersion.
  • 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.
  • the removal of the liquid dispersion medium does not comprise spray drying.
  • 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.
  • a liquid dispersion medium e.g., water
  • 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).
  • HPLC high performance liquid chromatography
  • 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.
  • hydrophobic compound 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.
  • 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).
  • 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.
  • 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.
  • the stable dispersion can comprise additional additives, including but not limited to, sugars, salts, pectin, and citric acid.
  • additional additives including but not limited to, sugars, salts, pectin, and citric acid.
  • sugars that can be added to the dispersions comprising the crystalline nanoparticles include dextrose, sucrose, and galactose.
  • 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%.
  • 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%.
  • 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 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.
  • 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.
  • stable dispersions comprising these nanoparticles and a liquid dispersion medium.
  • these nanoparticles and dispersions thereof are produced according to the presently disclosed methods.
  • 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.
  • parenteral e.g., intravenous
  • intradermal subcutaneous
  • opthalmic transdermal (topical)
  • transmucosal rectal, and vaginal routes.
  • the route of delivery is intravenous, parenteral, transmucosal, nasal, bronchial, vaginal, and oral.
  • 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 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.
  • 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.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition should be sterile and should be fluid to the extent that easy syringability exists.
  • 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.
  • 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.
  • polyol for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like
  • 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.
  • 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.
  • solutions for injection are free of endotoxin.
  • 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.
  • 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.
  • compositions can include a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring.
  • a sweetening agent such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • 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.
  • 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.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • 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.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Dosage unit form 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disease or unwanted condition which can encompass any type of condition or disease that can be treated therapeutically.
  • the disease or unwanted condition that is to be treated is a cancer
  • the hydrophobic drug is a chemotherapeutic drug.
  • chemotherapeutic drug is one that has therapeutic activity against a cancer.
  • 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.
  • the chemotherapeutic drug administered to a subject having a cancer is paclitaxel, camptothecin, C6-ceramide, 5-fluorouracil, or a combination thereof.
  • 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.
  • 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.
  • 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.
  • therapeutically effective amount or “dose” is meant the concentration of a hydrophobic drug that is sufficient to elicit the desired therapeutic effect.
  • an 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).
  • compositions which exhibit high therapeutic indices are administered to subjects in need thereof.
  • 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.
  • 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 IC 50 (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.
  • 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.
  • treatment of a subject can include a single treatment or, in many cases, can include a series of treatments.
  • a therapeutically effective dose of the hydrophobic drug formulation is administered intermittently.
  • 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.
  • compositions 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.
  • 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.
  • systemic administration e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration
  • oral administration e.g., topical application, as described elsewhere herein.
  • a or “an” entity refers to one or more of that entity; for example, “a nanoparticle” is understood to represent one or more nanoparticles.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise.
  • 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.
  • 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.
  • mice Female BALB/c and nude BALB/c mice (5 weeks of age) were purchased from the National Cancer Institute (NCI).
  • 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.
  • CPT camptothecin
  • 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.
  • TEM images of the resulting crystalline nanoparticles were acquired using a Phillips CM12 (FEI, Hillsboro, OR). Freshly prepared nanoparticles (5 ⁇ L) 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 ⁇ L) and wicked dry. All images were acquired at an accelerating voltage of 100 kV. Gatan DigitalMicro graph software was used to analyze the images.
  • 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 ⁇ m Centrifugal Filter Device (Millipore Co., Bedford, MA).
  • a 0.45 ⁇ m Centrifugal Filter Device Millipore Co., Bedford, MA.
  • free PTX does not form particles less than 0.45 ⁇ m in size; thus, the filter device removes free PTX particles, which are larger than 0.45 ⁇ m.
  • 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
  • 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).
  • % 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.
  • the NCI-H460 cells (5 x 10 6 ) were subcutaneous Iy injected into the right flank of the nude mice.
  • the 4Tl cells (10 5 ) 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 ⁇ a 2 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.
  • the lyophilized crystalline nanoparticles were examined and the control was freshly prepared PTX sonicated in 10% sucrose.
  • 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.
  • 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 ⁇ g/ml) (Fonseca, Simoes, and Gaspar (2002) J Control Release 83(2):273-286).
  • MDRl multi-drug resistance 1
  • P-gp P-glycoprotein
  • PTX Texol ®
  • solvents such as Cremophor ® EL (a 50/50 (v/v) mixture of polyoxyethylated castor oil and dehydrated alcohol).
  • Cremophor-EL a 50/50 (v/v) mixture of polyoxyethylated castor oil and dehydrated alcohol.
  • the ratio of PTX to the excipient 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.
  • 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
  • the crystalline nanoparticles could be lyophilized to powder and reconstituted to regain the rod-shaped crystal morphology ( Figure IB).
  • Figure IB The ability to lyophilize and reconstitute the formulations while retaining activity will allow for long- term storage of the drug formulations.
  • F 127 using F68 as a surfactant resulted in unstable aggregates (data not shown).
  • 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.
  • camptothecin 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.
  • Example 2 Characterization of the crystalline nanoparticles.
  • 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 ⁇ l solution of 45% mouse serum at 37 0 C 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.
  • 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.
  • Pluronic® F 127 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.
  • mice 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.
  • 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).
  • 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.
  • H460 tumor model shown in Figure 5 A the untreated tumors grew rapidly, reaching 1676 ⁇ 238 mm 3 in volume 17 days after the tumor cell inoculation.
  • 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.
  • the higher dose could further inhibit tumor growth and the tumor size was as small as 100.2 ⁇ 46 mm 3 29 days after the tumor implantation, but the tumor reached 1,689 ⁇ 538 mm 3 and 1,599 ⁇ 413 mm 3 in the groups treated with the lower dose (20 mg/kg) of Cremophor-EL ® and the nanoparticles, respectively.
  • 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
  • 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).
  • P-gp drug efflux transporter P-glycoprotein
  • F127 PTX stabilized by F127 may prevent PTX from being recognized by P-gp allowing the drug to be transported into the blood circulation.
  • 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.
  • 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.
  • 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.
  • 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 ® .
  • 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.
  • phase 1 an amorphous precipitate
  • phase 2 a hydrated amorphous aggregate
  • phase 3 stabilized nanocrystal
  • 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).
  • the crystalline nanoparticles F 127 coated nanocrystals
  • Sonication performed before the formation of the hydrated amorphous aggregate 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.
  • Example 6 Folate receptor-targeted crystalline nanoparticles.
  • FR folate receptor
  • FR-positive KB cells human oral carcinoma cells
  • Example 7 Vitamin E tocopherol polyethylene glycol succinate (TPGS)-based crystalline nanoparticles.
  • TPGS polyethylene glycol succinate
  • 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.
  • the crystalline nanoparticles could not be formed.
  • 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).
  • 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.
  • PTX at a weight ratio of PTX:C6-CER:TPGS of 1 : 1 :5
  • 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.
  • 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).
  • the physical stability of the crystalline nanoparticles particularly the thermal stability, was greatly increased by TPGS.
  • 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.
  • 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).
  • 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.
  • 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 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).
  • 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).
  • the crystalline nanoparticles with a weight ratio of 1/5 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).
  • the results of tumor volume changes as a function of time after the injections are shown in Fig. 14 A.
  • the tumor volume in the mice treated with the crystalline nanoparticles was 216 mm 3 with a weight of 0.16 g (Fig. 14B), but 487 mm 3 (0.49 g), 512 mm 3 (0.51 g), and 458 mm 3 (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.
  • 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 ⁇ M (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 ⁇ M of PTX.
  • the crystalline nanoparticles with 150 ⁇ M of PTX did not induce hemolysis, and hemolysis was not observed as the PTX concentration was doubled (300 ⁇ M).
  • hemolysis was significantly higher for PTX/Cremophor-EL ® , with 31% and 48% hemolysis at 150 and 300 ⁇ M of PTX, respectively.

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Abstract

L'invention porte sur des procédés de production de nanoparticules cristallines de médicaments hydrophobes à la surface desquelles sont adsorbés des stabilisateurs de surface, et sur des dispersions stables les comprenant. L'invention porte également sur des nanoparticules cristallines comprenant des composés hydrophobes cristallins à la surface desquelles sont adsorbés des stabilisateurs de surface, et des dispersions stables les comprenant. Les nanoparticules cristallines comportent un pourcentage en poids relativement faible de stabilisateurs de surface. En outre, l'invention porte sur des méthodes de traitement d'une maladie ou d'un état indésirable, tel qu'un cancer, chez un sujet, par l’administration des nanoparticules cristallines de médicaments hydrophobes ou de dispersions de celles-ci.
PCT/US2009/050463 2008-07-14 2009-07-14 Procédés et compositions comprenant des nanoparticules cristallines de composés hydrophobes WO2010009075A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010045292A2 (fr) * 2008-10-15 2010-04-22 The University Of North Carolina At Chapel Hill Compositions de nanoparticules comportant des noyaux d'huiles liquides
EP2635260A2 (fr) * 2010-11-02 2013-09-11 Board Of Regents Of The University Of Nebraska Compositions et procédés pour l'administration d'agents thérapeutiques
WO2015138819A1 (fr) * 2014-03-14 2015-09-17 Livepet, Llc. Améliorations cellulaires par l'intermédiaire de combinaisons de groupes lipofullerènes et peptidiques
US9808428B2 (en) 2014-01-14 2017-11-07 Board Of Regents Of The University Of Nebraska Compositions and methods for the delivery of therapeutics
CN112426535A (zh) * 2019-08-26 2021-03-02 复旦大学 一种肿瘤靶向药物纳米晶递送系统
US11311545B2 (en) 2014-10-09 2022-04-26 Board Of Regents Of The University Of Nebraska Compositions and methods for the delivery of therapeutics
US11458136B2 (en) 2018-04-09 2022-10-04 Board Of Regents Of The University Of Nebraska Antiviral prodrugs and formulations thereof
US11839623B2 (en) 2018-01-12 2023-12-12 Board Of Regents Of The University Of Nebraska Antiviral prodrugs and formulations thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0577215A1 (fr) * 1992-07-01 1994-01-05 NanoSystems L.L.C. Nanoparticules d'un agent anticancereux avec une surface modifiée
WO1998030205A1 (fr) * 1997-01-07 1998-07-16 Sonus Pharmaceuticals, Inc. Excipient emulsionne pour medicaments faiblement solubles
US20030054045A1 (en) * 1994-12-30 2003-03-20 Elan Pharma International Limited Formulations for therapeutic agents absorbed through mucous membranes
US20040247624A1 (en) * 2003-06-05 2004-12-09 Unger Evan Charles Methods of making pharmaceutical formulations for the delivery of drugs having low aqueous solubility
US20060073199A1 (en) * 2000-12-22 2006-04-06 Mahesh Chaubal Surfactant systems for delivery of organic compounds

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0577215A1 (fr) * 1992-07-01 1994-01-05 NanoSystems L.L.C. Nanoparticules d'un agent anticancereux avec une surface modifiée
US20030054045A1 (en) * 1994-12-30 2003-03-20 Elan Pharma International Limited Formulations for therapeutic agents absorbed through mucous membranes
WO1998030205A1 (fr) * 1997-01-07 1998-07-16 Sonus Pharmaceuticals, Inc. Excipient emulsionne pour medicaments faiblement solubles
US20060073199A1 (en) * 2000-12-22 2006-04-06 Mahesh Chaubal Surfactant systems for delivery of organic compounds
US20040247624A1 (en) * 2003-06-05 2004-12-09 Unger Evan Charles Methods of making pharmaceutical formulations for the delivery of drugs having low aqueous solubility

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010045292A2 (fr) * 2008-10-15 2010-04-22 The University Of North Carolina At Chapel Hill Compositions de nanoparticules comportant des noyaux d'huiles liquides
WO2010045292A3 (fr) * 2008-10-15 2010-07-22 The University Of North Carolina At Chapel Hill Compositions de nanoparticules comportant des noyaux d'huiles liquides
AU2011323458B2 (en) * 2010-11-02 2017-02-23 Board Of Regents Of The University Of Nebraska Compositions and methods for the delivery of therapeutics
JP2013542945A (ja) * 2010-11-02 2013-11-28 ボード オブ リージェンツ オブ ザ ユニバーシティ オブ ネブラスカ 治療を送達するための組成物および方法
EP2635260A4 (fr) * 2010-11-02 2014-07-09 Univ Nebraska Compositions et procédés pour l'administration d'agents thérapeutiques
EP2635260A2 (fr) * 2010-11-02 2013-09-11 Board Of Regents Of The University Of Nebraska Compositions et procédés pour l'administration d'agents thérapeutiques
RU2632445C2 (ru) * 2010-11-02 2017-10-04 Боард Оф Реджентс Оф Де Юниверсити Оф Небраска Композиции и способы доставки терапевтических средств
US9808428B2 (en) 2014-01-14 2017-11-07 Board Of Regents Of The University Of Nebraska Compositions and methods for the delivery of therapeutics
WO2015138819A1 (fr) * 2014-03-14 2015-09-17 Livepet, Llc. Améliorations cellulaires par l'intermédiaire de combinaisons de groupes lipofullerènes et peptidiques
US11311545B2 (en) 2014-10-09 2022-04-26 Board Of Regents Of The University Of Nebraska Compositions and methods for the delivery of therapeutics
US11839623B2 (en) 2018-01-12 2023-12-12 Board Of Regents Of The University Of Nebraska Antiviral prodrugs and formulations thereof
US11458136B2 (en) 2018-04-09 2022-10-04 Board Of Regents Of The University Of Nebraska Antiviral prodrugs and formulations thereof
CN112426535A (zh) * 2019-08-26 2021-03-02 复旦大学 一种肿瘤靶向药物纳米晶递送系统
CN112426535B (zh) * 2019-08-26 2024-04-30 复旦大学 一种肿瘤靶向药物纳米晶递送系统

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