WO2009123595A1 - Système de délivrance en nanoémulsion lipide-huile-eau pour agents interagissant avec les microtubules - Google Patents

Système de délivrance en nanoémulsion lipide-huile-eau pour agents interagissant avec les microtubules Download PDF

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Publication number
WO2009123595A1
WO2009123595A1 PCT/US2008/004393 US2008004393W WO2009123595A1 WO 2009123595 A1 WO2009123595 A1 WO 2009123595A1 US 2008004393 W US2008004393 W US 2008004393W WO 2009123595 A1 WO2009123595 A1 WO 2009123595A1
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Prior art keywords
pharmaceutical composition
lipid
oil
paclitaxel
esters
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PCT/US2008/004393
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English (en)
Inventor
Robert Shorr
Robert Rodriguez
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Robert Shorr
Robert Rodriguez
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Application filed by Robert Shorr, Robert Rodriguez filed Critical Robert Shorr
Priority to PCT/US2008/004393 priority Critical patent/WO2009123595A1/fr
Priority to CA2720390A priority patent/CA2720390A1/fr
Priority to EP08742550.0A priority patent/EP2262369A4/fr
Priority to KR1020107024777A priority patent/KR20110009128A/ko
Priority to CN200880129433XA priority patent/CN102046011A/zh
Priority to AU2008354007A priority patent/AU2008354007A1/en
Priority to BRPI0821895-1A2A priority patent/BRPI0821895A2/pt
Priority to JP2011502906A priority patent/JP2011516472A/ja
Publication of WO2009123595A1 publication Critical patent/WO2009123595A1/fr
Priority to IL208386A priority patent/IL208386A0/en

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    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to therapeutic and diagnostic agents, and more particularly to the formulation of microtubule-interacting agents in a lipid-oil-water nanoemulsion suitable for introduction into a patient and capable of promoting active selective agent concentration into cells characterized by hyperproliferation, including tumor cells.
  • lipids have been found to play essential roles in membrane structure, growth and metastasis, signal transduction, and transport processes.
  • EPR enhanced permeability and retention
  • lipid-based nanoparticles or HDL- or LDL-based drug carriers that mimic the natural receptor ligand and increase targeting and drug uptake into cancer cells have been prepared with various therapeutic agents, however, optimized formulations for the promotion of active tumor cell drug uptake for the treatment of cancer have been lacking.
  • LCMs gas- or air-filled lipid coated microbubbles
  • the production process for LCMs is based on simple mechanical shaking of an aqueous suspension of nonionic lipids, such as saturated glycerides and cholesterol esters, of specific chain lengths and in a fixed ratio. In all cases, the majority of lipids added (99%) flocculate or precipitate with additional loss of material on filtration with yields less than 1%. Still, these artificial LCMs were found to be very long-lived, lasting over 6 months in vitro.
  • LCMs are sufficiently small and pliable enough to pass across the fenestrated capillary walls of tumor-tissue microcirculation.
  • Paclitaxel is a member of the class of drugs known as taxanes and has been isolated primarily from the bark of the Pacific yew tree, Taxus brevifolia, and related species. Although allergic reactions were observed in formulation excipients, taxanes have been found useful in the treatment of various cancers such as ovarian, breast, non-small cell lung, and head and neck carcinomas. A difficulty in administrating taxanes is that the drug is not typically water-soluble. Paclitaxel has consequently been formulated in a 1:1 mixture of Cremophor EL® (a polyethoxylated castor oil) and ethanol to create Taxol® (Bristol-Myers Squibb, Inc.).
  • Cremophor EL® a polyethoxylated castor oil
  • Taxol® has been shown to be neurotoxic, causing sensory and sometimes accompanying motor neuropathy in patients. Cremophor EL® is in part responsible for the neuropathy, as it is itself not free of significant side effects. Attempts to formulate paclitaxel in a stable lipid emulsion have similarly been unsuccessful. The drug is reported to be insoluble in lipid emulsions such as Intralipid®, which contains primarily soybean oil, or Liposyn®, which contains a mixture of soybean and safflower oils.
  • Administering therapeutic agents with an appropriate delivery vehicle that simultaneously promotes accumulation and cellular internalization in a tumor mass but limits accumulation in healthy tissues is highly desirable. With more efficient delivery, systemic and healthy tissue concentrations of therapeutic agents may be reduced while achieving the same or better therapeutic results with fewer or diminished side effects. Such delivery of agents with inherent degrees of tumor cell selectivity would offer additional advantages. Further, a delivery vehicle that would not be limited to a single tumor type but would allow for selective accumulation into a tumor mass and cellular internalization into diverse cancer cell types would be especially desirable and allow for safer more effective treatment of cancer. A delivery vehicle that would also allow for elevated loading capacity for the therapeutic agent would be a significant advance in the art.
  • microtubule-interacting agents including taxanes such as paclitaxel, which would promote uptake into tumor cells yet exhibit minimal side effects.
  • the present invention broadly provides a pharmaceutical composition useful for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals, including humans, wherein the pharmaceutical composition includes a lipid nanoemulsion comprised of lipid particles as hereinafter defined, uniformly dispersed in an aqueous phase capable of being selectively and preferentially internalized within a diseased cell, including a cancer cell; an effective amount of at least one therapeutic or diagnostic agent associated with the lipid nanoemulsion; and a pharmaceutically-acceptable carrier.
  • the lipid particles each comprise at least one non-bilayer-forming lipid.
  • Such suitable lipid particles have been found to enhance significantly the targeted delivery and concentration of the therapeutic or diagnostic agent into diseased cells characterized by hyperproliferation, including cancer cells, for improved treatment efficacy and bioavailability while reducing or at least maintaining those dosage amounts and frequencies of administration necessary to achieve the desired therapeutic benefits.
  • the nanoemulsion exhibits exceptional physical and chemical stability for an extended duration of time, thereby greatly facilitating prepackaging of the pharmaceutical composition in stable, ready-to-administer forms, and also thereby eliminating the problems and inconvenience associated with bedside dilution and formulation as currently practiced with compositions in the prior art containing similar active agents.
  • microtubule-interacting agents include, but are not limited to, taxanes, such as, for example, paclitaxel; epothilones; vinca alkaloids, such as, for example, vincristine; eleutherobins; discodermolide; dolastatins; colchicine; combrestatins; phomopsin A; halichondrin B; spongistatin 1; sarcodictyins; laulimalides; and derivatives, analogs, congeners, and combinations of each of the aforementioned agents thereof.
  • the microtubule-interacting agent is present in an amount sufficient to kill or at least suspend the growth of the hyperproliferated cells.
  • a method of preparing the pharmaceutical composition disclosed herein comprising the steps of: a) mixing the at least one non-bilayer-forming lipid with an effective amount of at least one diagnostic or therapeutic agent to yield a lipid portion; b) adding the lipid portion to an aqueous phase to yield a dispersion; and c) agitating the dispersion under high-shear conditions sufficient to disperse the aforementioned lipid therethrough to form a lipid nanoemulsion comprised of lipid particles.
  • FIGURE 1 illustrates particle size distribution and zeta potential for lipid particles loaded with 10% paclitaxel in 20% DMSO in accordance with the present invention.
  • FIGURE 2 shows the incorporation of paclitaxel into lipid particles in accordance with the present invention by a graph plotting the percent solubility of paclitaxel-loaded lipid particles in a sucrose density study.
  • FIGURES 3A and 3B depict graphs showing that cellular lipid particle uptake according to the present invention can be quantified by fluorescence-activated cell sorting.
  • FIGURES 4A-4D portray graphs showing cellular lipid particle uptake according to the present invention by tumor cell lines relative to HT-29 colon and SF-539 lung tumor cells.
  • FIGURE 5 is a graph showing that tumor cells take up a greater amount of paclitaxel formulated in one particular mixture of lipid particles according to the present invention than paclitaxel formulated in Cremophor EL®.
  • FIGURE 6 depicts a graph showing that cell uptake of paclitaxel reached a plateau after two hours of drug incubation for both LN and Cremophor EL®.
  • FIGURE 7 illustrates the results of an experiment to determine if paclitaxel amount is saturating for cell uptake in lipid particles according to the present invention.
  • FIGURES 8A and 8B depict graphs showing that cholesterol is a critical component in cellular uptake of lipid particles according to the present invention.
  • FIGURES 9A and 9B portray graphs showing that paclitaxel in lipid particles according to the present invention is internalized to a greater extent than paclitaxel in Cremophor EL®.
  • FIGURES 10A- 1OD depicts graphs showing the cytotoxicity of paclitaxel alone versus paclitaxel in lipid particles according to the present invention.
  • FIGURE 11 illustrates that paclitaxel is more cytotoxic when formulated in the lipid particles of the present invention than when formulated in Cremophor EL®.
  • FIGURES 12A and 12B illustrate graphs showing that paclitaxel has significantly greater anti-tumor activity when formulated in the lipid particles of the present invention than when formulated in Cremophor EL®.
  • FIGURE 13 shows representative images of A549 cells treated with EmPAC, Abraxane®, or Taxol® and stained with paclitaxel-specific antibodies.
  • the present invention is generally directed to pharmaceutical compositions for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof, in warm-blooded animals.
  • Such animals include those of the mammalian class, such as humans, horses, cattle, domestic animals including dogs and cats, and the like, subject to disease and other pathological conditions and syndromes characterized by cellular hyperproliferation, including cancer.
  • the pharmaceutical composition of the present invention comprises a nanoemulsion comprised of lipid particles, as defined below, operatively associated with at least one therapeutic or diagnostic microtubule-interacting agent, for which the lipid particles have an enhanced loading capacity, and a pharmaceutically-acceptable carrier or excipient therefor, thereby making the lipid particles particularly well-suited for the selective delivery to and effective concentration within such diseased cells and tissues as tumorous ones.
  • the lipid particles of the nanoemulsion are structured to facilitate both elevated passive accumulation and active internalization into diseased cells and tissues, including tumor cells and tissues.
  • the lipid particles are taken into these cells through active metabolic uptake as they passively accumulate in the vascular area of the diseased tissue.
  • the lipid particles of the present invention provide a delivery vehicle selectively and preferentially targeted for uptake and internalization by cells characterized by hyperproliferation, including tumor and cancer cells. "Internalization" as used herein means that the lipid particles are actively taken up by the cell.
  • Such elevated internalization levels coupled with a high loading capacity of the particles for the therapeutic or diagnostic agent, provides a potent vehicle for treatment or diagnosis of these targets by delivering an effective amount of the therapeutic or diagnostic agent to such targets, thereby inducing a therapeutically-beneficial effect, including stopping growth, inducing differentiation, or killing the cell.
  • the lipid particles of the present invention hence not only enhance delivery of the therapeutic agent to the diseased cells and tissues but also reduce the amount of the therapeutic agent needed to achieve the desired efficacy, especially as compared to delivery systems in the prior art.
  • the lipid particles of the present invention are exceptionally physically and chemically stable over an extended period of time and hence experience minimal loss of the therapeutic or diagnostic agent due to undesirable precipitation, aggregation, or insolubility that is typically exhibited in delivery systems in the prior art. Moreover, these lipid particles display other favorable characteristics including controlled release; enhanced drug stability; positive drug loading capacity; better compatibility with hydrophobic drugs; relatively low biotoxicity; and low organic solvent content.
  • the present lipid particles are also relatively simple and convenient to prepare and to administer.
  • the term "lipid particle" is meant to encompass any lipid-containing structures, typically nanosized, which are at least substantially-intact particles forming part of a nanoemulsion.
  • substantially-intact means that the particles maintain their shape in the absence of a membrane, as contrasted with a liposome.
  • the lipid particles are comprised of at least one non-bilayer-forming lipid.
  • a lipid bilayer structure or arrangement is typically formed by certain kinds of lipids having a hydrophilic end (polar head region) and a hydrophobic end (nonpolar tail region), including amphipathic molecules such as phospholipids, which exhibit the ability and/or tendency to self-organize into two opposing layers of lipid molecules in aqueous solution.
  • non-bilayer-forming lipid encompasses a lipid that lacks such ability and/or tendency to form a lipid bilayer structure or arrangement in an aqueous environment.
  • non-bilayer-forming lipids include lipids that are no more than weakly polar, preferably lipids that are substantially non- polar or neutral.
  • the more-preferred lipids in the present invention are neutral lipids.
  • the lipid particles of the present invention are distinguishable from the gas-containing microbubbles described in U.S. Patent Nos. 4684479 and 5215680, and are also structurally distinguishable from liposomes, such as those described, for example, in U.S. Patent Nos. 6565889 and 6596305, all herein incorporated by reference.
  • the lipid particles are formed by a mixture of non-bilayer-forming lipids that are physiologically acceptable and at least substantially free from the presence of charged or polar lipids, including, for example, phospholipids.
  • non-bilayer-forming lipids include those selected from glycerol monoesters of saturated and unsaturated carboxylic acids; glycerol monoesters of saturated aliphatic alcohols; sterol aromatic acid esters; sterols; terpenes; bile acids; alkali metal salts of bile acids; sterol esters of aliphatic acids; sterol esters of sugar acids; esters of sugar acids; esters of aliphatic alcohols; esters of sugars; esters of aliphatic acids; sugar acids; saponins; sapogenins; glycerol; glycerol di-esters of aliphatic acids; glycerol tri-esters of aliphatic acids; glycerol diesters of aliphatic alcohols; glycerol triesters of aliphatic alcohols; and combinations thereof.
  • the lipid particles are prepared by first forming a mixture of a select group of non-bilayer-forming lipids which provides the lipid particles with a size described hereinafter that facilitates high internalization levels when applied to targeted diseased tissues and cells.
  • the lipid mixture generally comprises: a) at least one first member selected from the group consisting of glycerol monoesters of carboxylic acids containing from about 9 to 18 carbon atoms and aliphatic alcohols containing from about 10 to 18 carbon atoms; b) at least one second member selected from the group consisting of sterol aromatic acid esters; c) at least one third member selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids; d) at least one optional fourth member selected from the group consisting of sterol esters of aliphatic acids containing from about 1 to 18 carbon atoms; sterol esters of sugar acids; esters of sugar acids and aliphatic alcohols containing from about 10 to 18 carbon atoms, esters of sugars and aliphatic acids containing from about 10 to 18 carbon atoms; sugar acids, saponins; and sapogenins; and e) at least one optional fifth member selected from the group
  • lipid mixture described above only includes the presence of members (a) through (c), it is more preferred to incorporate members (d) and/or (e) because the long-term stability and uniformity of size of the lipid particles are theoretically enhanced by the presence of these two optional members.
  • the five members (including the two optional members) making up the lipid mixture forming the lipid particles of the present invention are combined in a weight ratio of (a):(b):(c):(d):(e) of (l-5):(0.25-3):(0.25-3):(0- 3):(0-3), respectively.
  • glycerol monoesters of saturated carboxylic acids containing from about 10 to 18 carbon atoms it is contemplated that glycerol monoesters of mono- or polyunsaturated carboxylic acids containing from about 9 to 18 carbon atoms, such as but not limited to the 9-carbon oleic or elaidic acids, are also useful in the construction of the lipid mixture.
  • the proportions of the members of the lipid mixture may vary depending on several factors, including, but not limited to, the type of cells and/or tissues being targeted for delivery, the therapeutic or diagnostic agent being loaded, the desired dosage of the therapeutic or diagnostic agent, the pharmaceutically-acceptable carrier used, the mode of administration, the presence of other excipients or additives, and so forth.
  • factors that enable the lipid particles to be selectively internalized by targeted diseased tissues and cells include not only the composition of the lipid mixture and the structure of the resulting lipid particles but also the size and molecular weight of the particles as described hereinafter.
  • the lipid particles of the present invention maintain a desirable particle size distribution, preferably where a major portion of the particles have a mean average particle size ranging from about 0.02 to 0.2 ⁇ (micron), preferably 0.02 ⁇ to 0.1 ⁇ , with varying minor amounts of particles falling above or below the range and some lipid particles only ranging up to about 200 nm.
  • the particle size ranges attainable in the lipid particles of the present invention further lead to enhanced physical and chemical stability over an extended period of time, and substantial reduction in undesirable agglomeration and drug precipitation. Furthermore, this range is particularly suitable for the treatment of cancer; larger particles may be appropriate for other uses (e.g., targeting of other types of cells or tissues).
  • the range provided herein will be determined in part by the lipid mixture employed and the type and amount of the therapeutic or diagnostic agent added.
  • the therapeutic or diagnostic agents employed in the present invention may be uncharged or charged, nonpolar or polar, natural or synthetic, and so on.
  • therapeutic agent includes any substance including, but not limited to, drugs, hormones, vitamins, nutrients, substances, and the like, that affect microtubule production, structure, association, function, and destruction, and thus are useful in prevention and treatment of a disease, condition, syndrome, characterized by cellular hyperproliferation, or symptoms thereof, including cancer.
  • the therapeutic agents useful in the present invention include all types of drugs, lipophilic polypeptides, cytotoxins, oligonucleotides, cytotoxic antineoplastic agents, antimetabolites, hormones, and radioactive molecules, which affect microtubule production, structure, association, function, and destruction.
  • oligonucleotides includes both antisense oligonucleotides and sense oligonucleotides, (e.g., nucleic acids conventionally known as vectors). Oligonucleotides may be "natural” or "modified” with regard to subunits or bonds between subunits.
  • the therapeutic agent is a microtubule-interacting agent selected from a group consisting of taxanes, such as, for example, paclitaxel, docetaxel, cephalomannine baccatin-III, 10-deacetyl baccatin III, deacetylpaclitaxel, and deacetyl-7-epipaclitaxel; vinca alkaloids, such as, for example, vincristine, vinblastine, vinorelbine, vindesine, and analogs thereof; epothilones; eleutherobins; discodermolide; dolastatins; colchicine; combrestatins; phomopsin A; halichondrin B; spongistatin 1; sarcodictyins; laulimalides; derivatives, analogs, congeners, and combinations of each of the aforementioned agents thereof; and similar drugs or substances known to exhibit such microtubule-interacting activity.
  • taxanes such as, for example, pac
  • Taxanes such as paclitaxel may also be used in smaller time-release doses as an anti- inflammatory agent. This use is especially important in the field of biomedical devices to be placed surgically within patients, such as stents. While some accumulation of cells around and inside the stent is desirable as this accumulation forms a smooth cover and thereby incorporates the device into the artery itself, such cellular accumulation can also clog the interior channel and cause restenosis of the artery. As a consequence, Boston Scientific Corporation manufactures a paclitaxel-eluting coronary stent system coated with a proprietary polymer which binds paclitaxel onto the stent surface.
  • the paclitaxel-polymer complex allows precise control over the dosage and time-release characteristics for paclitaxel, permitting elution of a sufficient amount of the medication to inhibit cellular accumulation around the stent and significantly prevent restenosis and revascularization around the stent. It is contemplated that the pharmaceutical compositions of the present invention, especially where the therapeutic agent is a taxane or other microtubule-interacting agent, will be similarly useful to regulate cellular accumulation around surgically-implanted biomedical devices.
  • compositions of the present invention exhibit long-term physical and chemical stability, allowing such compositions to be conveniently pre-packaged into stable, ready-to-administer dosage forms and thereby eliminating the need for the bedside dilution and formulation prior to administration typically associated with similar compositions in the prior art.
  • the pharmaceutical compositions of the present invention exhibit desirable drug and emulsion stability over an extended time period (e.g., at least 14 days at about 30 0 C and at least 12 months at 4 0 C).
  • compositions of the present invention contain lipid particles in an amount of from about 0.1 ⁇ g/mL to 1000 ⁇ g/mL, preferably from about 10 ⁇ g/mL to 800 ⁇ g/mL, and most preferably from about 200 ⁇ g/mL to 600 ⁇ g/mL.
  • Typical concentrations of the therapeutic or diagnostic agent based on the total volume of the pharmaceutical composition may be at least 0.001% w/v, preferably 0.001% to 90% w/v, and more preferably from about 0.1% to 25% w/v.
  • the amount of the therapeutic or diagnostic agent present in the pharmaceutical composition may range from about 0.001 ⁇ g/mL to 1000 ⁇ g/mL, preferably from about 0.1 ⁇ g/mL to 800 ⁇ g/mL, and more preferably from about 60 ⁇ g/mL to 400 ⁇ g/mL.
  • the pharmaceutical composition of the present invention may further include emulsion-enhancing agents selected from a plant-based fat source, a solvent, a surfactant, or combinations thereof.
  • emulsion-enhancing agents have been found, individually or in combination, to enhance the stability and maintain the small particle size properties of the lipid particles theoretically by reducing or minimizing undesirable precipitation or aggregation of the lipid particles, thereby positively influencing and facilitating the active uptake of the lipid particles into the cancer cells.
  • the emulsion-enhancing agents should also improve the physical and chemical stability and drug-carrying capacity of the pharmaceutical compositions of the present invention.
  • the plant-based fat sources include vegetable-derived fatty acids generally in the form of vegetable oil, such as, for example, soybean oil, flaxseed oil, hemp oil, linseed oil, mustard oil, rapeseed oil, canola oil, safflower oil, sesame oil, sunflower oil, grape seed oil, almond oil, apricot oil, castor oil, corn oil, cottonseed oil, coconut oil, hazelnut oil, neem oil, olive oil, palm oil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil, walnut oil, and mixtures thereof.
  • the more preferred vegetable oil is soybean oil.
  • the vegetable oil is generally present in amounts sufficient to permit higher surface tension in the nanoemulsion which in turn increases the probability of hydrophobic interactions with the plasma membranes of the target cell, or receptors thereupon.
  • the plant- based fat source may be present in amounts of from about 0.001% v/v to 5.0% v/v, more preferably from about 0.005% v/v to 4.0% v/v, and most preferably from about 0.01% v/v to 2.5% v/v.
  • the surfactants are those selected from non-ionic surfactants.
  • non-ionic surfactants include sorbitan esters and mixtures thereof, such as fatty-acylated sorbitan esters and polyoxyethylene derivatives thereof, and mixtures thereof including, but not limited to, Poloxamer compounds (188, 182, 407 and 908), Tyloxapol, Polysorbate 20, 60 and 80, sodium glycolate, sodium dodecyl sulfate and the like, and combinations thereof.
  • More preferred non-ionic surfactants are detergent polysorbates, such as, for example, Tween®-80.
  • the surfactant is generally present in amounts sufficient to increase the kinetic stability of the nanoemulsion by stabilizing the interface between the hydrophobic and hydrophilic components of the nanoemulsion and keeping the hydrophobic components from coalescing, such that, once formed, the nanoemulsion does not significantly change in storage.
  • the surfactant may be present in amounts of from about 0.01% w/v to 4.0% w/v, more preferably from about 0.1% w/v to 3.0% w/v, and most preferably from about 0.2% w/v to 2.5% w/v.
  • the solvents include any pharmaceutically-acceptable water-miscible diluents or solvents such as, for example, polar protic and polar aprotic solvents.
  • solvents are preferably selected from 1,3-butanediol; dimethyl sulfoxide; alcohols such as methanol, butanol, benzyl alcohol, isopropanol, and ethanol; and the like.
  • a more preferred solvent is benzyl alcohol.
  • the solvent is generally present in amounts sufficient to control the extent of the aggregation of non-ionic surfactants in the nanoemulsion.
  • the solvent may be present in amounts of from about 0.001% v/v to 99.9% v/v, more preferably 0.005% v/v to 80% v/v, and most preferably from about 0.005% v/v to 70% v/v.
  • composition of the present invention does not modify or alter the underlying pharmacological activity or chemical properties of the therapeutic or diagnostic agent but simply enhances the agent's delivery to and internalization into the diseased cell or tissue, including cancerous cells or tissue, to impart therapeutic or diagnostic benefits.
  • Examples of teachings related to the use of taxanes as therapeutic agents in treating cancer are disclosed, for example, in U.S. Patent Nos. 6346543; 6384071; 6387946; 6395771; 6403634; and 6500858, each incorporated herein by reference.
  • the pharmaceutical compositions of the present invention are prepared by combining the lipid particles with the therapeutic or diagnostic agent and thoroughly mixing the same.
  • the lipid mixture may be mixed with a surfactant in combination with a plant- based fat source prior to mixing with the therapeutic or diagnostic agent, which themselves may be mixed with a water-miscible solvent for dissolution.
  • the lipid particle- therapeutic/diagnostic agent combination is then mixed with water, preferably purified water.
  • the resulting mixture is then subjected to high shear forces typically produced in standard conventional shear-intensive homogenizing mixers or homogenizers to produce a nanoemulsion comprising the lipid particles dispersed within the aqueous phase.
  • Sufficient high shear forces can be produced with a suitable shear-intensive homogenizing mixer or homogenizer such as Microfluidizer® Fluid Materials Processors marketed by Microfluidics of Newton, MA.
  • the resulting nanoemulsion may be further treated to yield a more purified form, which may be used for administration to warm-blooded animals, including humans.
  • the nanoemulsion may be processed through dialysis to remove the impurities, with the resulting dialysate retained for pharmaceutical use. Dialysis is a preferred method of removing any non-particulated lipid mixture components, drugs, and/or solvents and achieving any desired buffer exchange or concentration.
  • Dialysis membrane nominal molecular weight cutoffs of 5000 to 500000 can be used, with a molecular weight of 10000 to 300000 being preferred.
  • the lipid particles produced as described, when purified such as by dialysis to remove non-particulated drug, may be characterized to determine the extent to which the lipid particles may be internalized in targeted cells, such as, for example, C 6 glioma cells.
  • compositions of the present invention may further include a pharmaceutically- acceptable carrier or excipients.
  • pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof.
  • the amount of such additives depends on the properties desired, which can readily be determined by one skilled in the art.
  • compositions of the present invention may routinely contain salts, buffering agents, preservatives, and compatible carriers, optionally in combination with other therapeutic ingredients.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically- and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • the present invention additionally provides methods for treating or diagnosing a patient with therapeutic or diagnostic agents by delivering an effective amount of at least one therapeutic or diagnostic agent to cells for implementing the prevention, diagnosis, or treatment of a disease, condition, or syndrome characterized by cellular hyperproliferation, or symptoms thereof.
  • Improved treatments of cancer are especially contemplated, including treatment of primary tumors by the control of tumoral cell proliferation, angiogenesis, metastatic growth, apoptosis, and treatment of the development of micrometastasis after or concurrent with surgical removal; and radiological or other chemotherapeutic treatment of a primary tumor.
  • the pharmaceutical composition of the present invention is useful in such cancer types as primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.
  • the pharmaceutical composition can be administered directly to a patient when combined with a pharmaceutically-acceptable carrier. This method may be practiced by administering the therapeutic or diagnostic agent alone or in combination with an effective amount of another therapeutic or diagnostic agent, which may or may not be a second microtubule-interacting agent.
  • this second agent may be, but is not limited to, a cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a podophyllotoxin, an antitumor antibiotic, a chemotherapeutic agent, an apoptosis-inducing agent, and combinations thereof.
  • Such therapeutic agents may further include metabolic inhibition reagents. Many such therapeutic agents are known in the art.
  • the combination treatment method provides for simultaneous, sequential, or separate use in treating such conditions as needed to amplify or ensure patient response to the treatment method.
  • compositions of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • formulations specifically suited for parenteral administration are preferred, the compositions of the present invention can also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. If such a formulation is desired, other additives well-known in the art may be included to impart the desired consistency and other properties to the formulation.
  • the particular mode of administering the therapeutic or diagnostic agent depends on the particular agent selected; whether the administration is for treatment, diagnosis, or prevention of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being treated or diagnosed; and the dosage required for therapeutic efficacy.
  • a preferred mode of administering an anticancer agent for treatment of leukemia would involve intravenous administration, whereas preferred methods for treating skin cancer could involve topical or intradermal administration.
  • effective amount refers to the dosage or multiple dosages of the therapeutic or diagnostic agent at which the desired therapeutic or diagnostic effect is achieved.
  • an effective amount of the therapeutic or diagnostic agent may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated.
  • the precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired therapeutic effect or in the event of any complication.
  • the dosage amount of the therapeutic agent used should be sufficient to inhibit or kill tumor cells while leaving normal cells substantially unharmed.
  • the therapeutic or diagnostic agent included in the pharmaceutical compositions of the present invention can be prepared in any amount desired up to the maximum amount that can be solubilized by, suspended in, or operatively associated with the given lipid particles.
  • the amount of the diagnostic agent or therapeutic agent may range from 0.001 ⁇ g/mL to 1000 ⁇ g/mL, preferably from about 0.1 ⁇ g/mL to 800 ⁇ g/mL, and more preferably about 300 ⁇ g/mL.
  • the lipid particles will be delivered in a manner sufficient to administer an effective amount to the patient.
  • the dosage amount may range from about 0.1 mg/kg to 175 mg/kg, preferably from about 1 mg/kg to 80 mg/kg, and more preferably 5 mg/kg to 60 mg/kg.
  • the dosage amount may be administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day. In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance. Multiple doses per day are contemplated to achieve appropriate systemic or targeted levels of the therapeutic or diagnostic agent.
  • the microtubule-interacting agents may be used as diagnostic agents in vitro.
  • different microtubule-interacting agents may be more or less effective at inhibiting distinct tumor classes.
  • testing of a culture of tumor cells in vitro with microtubule-interacting agents known to target specific tumor cell types provides an alternative approach for identifying tumor types and effective treatments.
  • a method of preparing the pharmaceutical composition of the present invention there is provided a method of preparing the pharmaceutical composition of the present invention.
  • the lipid mixture is incorporated into the therapeutic or diagnostic agent in amounts such that, upon processing with an aqueous phase, the composition forms a lipid nanoemulsion comprising a dispersion of lipid particles wherein the dispersed phase of lipid particles are present in the form of macromolecules or clusters of small molecules on the nanoscale order of particle size.
  • the lipid mixture and the therapeutic or diagnostic agent are combined with an aqueous phase comprising water, preferably filtered water.
  • the resulting mixture is processed to form lipid particles having a mean average particle size range typically, but not always, in the range of up to 200 nm, a size particularly suited for the treatment of cancer, with larger particles appropriate for other uses.
  • the range obtained will in part be affected by the lipid mixture employed, the type and amount of the therapeutic or diagnostic agent added to the lipid mixture, and the technique used to produce the lipid particles.
  • the pharmaceutical compositions of the present invention can be made using conventional dispersion-producing techniques or processes known in the art. Such techniques include, but are not limited to, high-shear homogenization, ultrasonic agitation or sonication, high-pressure homogenization, solvent emulsification/evaporation, and the like.
  • the lipid particles may be prepared through conventional high-pressure homogenization techniques using a suitable high-pressure homogenizer. Homogenizers of suitable sizes are commercially available.
  • High-pressure homogenizers are generally designed to push a fluid through a narrow gap spanning about a few microns at high pressure, typically from about 100 to 2000 bar.
  • the pressurized fluid accelerates over a very short distance to a very high velocity of over 1000 km/hr.
  • Pressurized fluids containing the lipid mixture encounter very high-shear stress and cavitation forces, effectively disrupting and comminuting the lipid mixture into particles in the submicron range.
  • a major portion of the lipid particles should have a mean average particle size ranging from about 0.02 ⁇ to 0.2 ⁇ , preferably 0.02 ⁇ to 0.1 ⁇ , with varying minor amounts of particles falling above or below the range, especially with some lipid particles ranging up to about 200 nm.
  • the lipid mixture may be mixed with a plant-based fat source, such as a vegetable oil, and a surfactant, such as a non- ionic surfactant, to yield a lipid phase.
  • a surfactant such as a non- ionic surfactant
  • the therapeutic or diagnostic agent may be mixed with a solvent, such as a water-miscible solvent, to yield a therapeutic or diagnostic agent phase.
  • the lipid and therapeutic or diagnostic agent phases are thereafter mixed and blended together in the presence of an aqueous phase, preferably through sonication.
  • the resulting mixture is thereafter homogenized under high-shear forces to produce the corresponding nanoemulsion of the present invention.
  • the nanoemulsion may then be filtered through a 0.2 ⁇ membrane, sterilizing and/or removing impurities such as unused lipid materials, excess therapeutic or diagnostic agent, and so on, to yield a purified form suitable for delivery as a pharmaceutical composition to warm-blooded animals, including humans, in need of treatment or diagnosis.
  • Paclitaxel was selected as the first drug candidate to be tested for formulation via loading onto lipid nanoparticles (LN) and to be developed into a commercially viable product.
  • the paclitaxel/LN formulations were prepared directly in water and 4% ethanol followed by four cycles of high pressure homogenization at 18000 psi using a HOY Microfluidics Microfluidizer® high-pressure homogenizer (Model M-I lOY, Microfluidics, Inc., Newton, MA).
  • a HOY Microfluidics Microfluidizer® high-pressure homogenizer Model M-I lOY, Microfluidics, Inc., Newton, MA.
  • LN were first prepared in different alcohol contents (e.g., 50% v/v, 25% v/v, 12.5% v/v). It was observed that even particles without drug are not stable in ethanol content less than or equal to 25% v/v. Furthermore, only LN at 12.5% v/v alcohol remained stable for at least a day. Accordingly, 10% paclitaxel was loaded onto LN with 12.5 % v/v alcohol content. After three hours, gel formation was observed, strongly suggesting that a high level of alcohol does not stabilize paclitaxel formulations in LN.
  • alcohol contents e.g. 50% v/v, 25% v/v, 12.5% v/v
  • Ethyl alcohol was also generally used as a demulsifying agent. This could be the reason that even a high percentage of ethanol does not help in getting a stable drug-loaded formulation.
  • dimethyl sulfoxide (DMSO) is a solvent of choice, as paclitaxel known to have a very good solubility in DMSO.
  • paclitaxel-loaded samples were prepared in 10% DMSO at 15000 psi and three cycles on a second machine, Microfluidizer® high-pressure homogenizer (Model M-I lOEH, Microfluidics, Inc., Newton, MA) M-I lO EH.
  • polyether sulfone (PES) hydrophilic membrane was the most suitable membrane for filtration of the formulation.
  • PES polyether sulfone
  • a precipitated fraction was found at the bottom of the gradient, which consisted primarily of insoluble paclitaxel.
  • a band was found in the gradient which was established to be LN, and radioactive contents in both the precipitate as well as the LN fraction were analyzed, hi the presence of lipids comprising LN, very little of the radioactive label was found in the bottom precipitate. In the absence of the lipids, a greater amount of precipitate is found, and a much smaller fraction of the radiolabel is found in the precipitate fraction.
  • paclitaxel In the clinical setting, intravenous infusion of approximately 2.5L over a 3-24 hour period is generally considered to be safe and tolerable for most patients.
  • the dosage of paclitaxel currently in clinical use ranges from 135 to 175 mg/m 2 over a 3-24 hour period. Assuming an average-sized male of ⁇ 1.8 m 2 surface area, a concentration of ⁇ 97 ⁇ g/mL of paclitaxel is require to achieve such a dosage.
  • paclitaxel In order to deliver such a therapeutic dosage of paclitaxel within these time and volume parameters, paclitaxel must be at sufficiently high concentration. Consequently, it is desirable to formulate a paclitaxel-loaded LN sample achieving such a dosage as a benchmark.
  • the present invention is capable of selectively targeting tumor cells, the therapeutic dosage necessary for paclitaxel-incorporated LN may actually be much lower. Thus, a much greater fraction of drugs administered will end up in the tumor cells.
  • a second paclitaxel-LN formulation consisting of 60 ⁇ g/mL paclitaxel; 300 ⁇ g/mL lipid mixture; 0.5% butanol; 0.5% soybean oil; and 0.25% Tween®-80. Stability studies performed on this second formulation revealed that formulations using 400 ⁇ g/mL of lipids and a constant concentration of paclitaxel are stable for at least 24 hours. Results are summarized in Table 4.
  • Paclitaxel load (% Time after Description Mean particle ( ⁇ g/mL) w/w) preparation diameter (nm)
  • the second paclitaxel-LN formulation was stable at room temperature for at least seven days and at 2-8°C for at least thirty days. Table 5. Stability of second formulation
  • EXAMPLE 2 CELL UPTAKE OF EMULSIPHAN AND EmPAC The studies described herein were performed in order to determine if human tumor cells take up LN and if they display differential ability to take up LN. It was found not only that most tumor cell lines tested took up fluorescent LN readily but also that tumor cell lines from different tumor cell lineages displayed differential ability to take up LN.
  • the LN formulation used for these experiments were prepared as follows: the appropriate lipids were solubilized in 95% ethanol to 10mg/mL by sonication for 10 minutes. Next, 100 ⁇ L of 0.5mg/mL cholesteryl BODIPY-FL (Molecular Probes, Eugene, OR) in ethanol was added to 1 mL of the 10 mg/mL solubilized lipids. Lastly, the lipid and cholesteryl BODIPY-FL mixture was added to 50 mL of a solution of 1 mM sodium pyrophosphate in water and processed through a HOY Microfluidics Microfluidizer® high- pressure homogenizer (Model M-11OY, Microfluidics, Inc., Newton, MA).
  • FACS fluorescence- activated cell sorting
  • LN was prepared with 200 ⁇ g/mL lipids and 2.5 ⁇ g/mL DiO by microfluidization. Labelled LN was added to C6 cells and incubated at 37°C. After this, media was removed, the cells washed with phosphate buffered saline (PBS), trypsinized, and washed again before fixation in 4% formaldehyde. LN was added at 0, 12.5, 25, 50, and lOO ⁇ g/mL and incubated for 60 minutes. 50 ⁇ g/mL LN was added to cells and incubated for 0, 5, 10,15, 30, and 60 minutes before removal of media and processing for FACS analysis. As evident from the results seen in FIGURES 3A and 3B, which respectively show that fluorescence intensity per cell was directly proportional to increased concentrations and to increased incubation time, it is evident that LN uptake can be assessed by FACS.
  • PBS phosphate buffered saline
  • the NCI cell line panel consists of cell lines derived from a number of different human tumor lineages, with several different cell lines from each represented human tumor lineage.
  • the cell lines used included HS-578T, MDA-MB-231, and MX-I breast cancer; H23, H460, and H522 lung cancer; SF-539 liver; and HT29, SW- 620, and COLO205 colon tumor cell lines.
  • LN was prepared with 200 ⁇ g/mL lipids and 0.5% w/w cholesteryl-BODIPY-FL, by microfluidization. Labelled LN was added to cells and incubated at 37°C. After this, media was removed, the cells washed with PBS, trypsinized, and washed again before fixation in 4% formaldehyde. Samples were analyzed by FACS and average fluorescence intensity per cell was determined. Cells derived from colon, breast, central nervous system (CNS), and lung were compared to those of the HT-29 colon carcinoma cell line and SF-539 lung cancer cell line, which have been shown to take up low and high quantities of fluorescent LN, respectively. Fluorescent LN uptake by each LN-treated cell sample was obtained by subtracting the fluorescence intensity from the same cell line, which had been untreated.
  • LN uptake varies among cell types of different lineages. Although some variability was seen among cells from the same tumor lineage, relative uptake was fairly consistent for each tumor lineage. For example, breast and lung tumor cell lines generally displayed higher LN uptake than did colon tumor cell lines. The greatest LN uptake was found in cell lines from lung tumors and from the CNS. All results are summarized in Table 7.
  • EmPAC C-labelled paclitaxel
  • DMSO DMSO
  • SF539 glioma and A549 lung cancer cells were added to SF539 glioma and A549 lung cancer cells in 12- well replicates for 1 hour. Media was removed, and cell monolayers were solubilized after removal of drug and washing in PBS with identical volumes of scintillation cocktail containing toluene to determine the paclitaxel associated with each monolayer sample. Radiolabel counts for each sample was expressed as a fraction of the total radiolabel added initially to each sample. Results from this experiment suggested that SF539 internalized significantly greater paclitaxel formulated in LN (i.e., EmPAC) than Taxol®, as demonstrated in FIGURE 5.
  • EmPAC Emphosphonuclear paclitaxel
  • lipid and radiolabeled paclitaxel amounts held constant. As seen in FIGURE 7, it appears that paclitaxel amount is saturating for cell uptake, although this is not entirely clear from this particular experiment. Since labelled paclitaxel is constant, while total paclitaxel is varied, it may merely represent a smaller or larger fraction of total paclitaxel. Increased concentration of paclitaxel may also increase the total number of particles.
  • FIGURE 9A It is evident from FIGURE 9A that paclitaxel is internalized and is localized to microtubules. Since paclitaxel is known to bind to microtubules in vivo, this suggests that paclitaxel formulated in EmPAC, as well as in Cremophor EL®, is internalized. However, the intracellular fluorescence intensity of a number of fields of cells for each sample was also quantified by tracing the edges of each cell and quantifying the fluorescence intensity within the boundaries traced. It was found, as seen in FIGURE 9B, that EmPAC-treated cells had roughly twice the fluorescence intensity of that of cell treated with Taxol®, thereby confirming earlier findings.
  • EmPAC was compared either with paclitaxel dissolved in DMSO, as recommended by the manufacturer, or Taxol®.
  • paclitaxel dissolved in DMSO
  • Taxol® Taxol®.
  • MTS cell proliferation assay was used to assess relative cytotoxicity.
  • MTS is a tetrazolium compound, which is bioreduced by live cells into a soluble formazan product.
  • the absorbance of the formazan product at 490 run can be used to determine the relative number of living cells.
  • the MTS can be used to assess the relative cytotoxic potency of paclitaxel alone versus that of EmPAC.
  • the cell lines tested included the uterine sarcoma cell line MES-SA, and its drug-resistant sub-line MES-S A-DX5, to see if incorporation of paclitaxel into LN impacts paclitaxel's cytotoxicity in drug-resistant cells. Accordingly, MES-SA, MES-SA-DX5, A549, and MX-I cell lines were plated at subconfluent density.
  • EmPAC displayed roughly less cytotoxicity compared to paclitaxel in DMSO in MES-SA and A549 cells at lower paclitaxel concentrations but appeared to kill a greater fraction of cells at higher concentrations than paclitaxel alone, as also seen in FIGURE 1OC. This may have been due to the fact that paclitaxel alone is extremely insoluble in aqueous media; its hydrophobic properties cause paclitaxel to aggregate at higher concentrations, precluding its entry into cells. Incorporation of paclitaxel into LN may have allowed paclitaxel to remain stable in aqueous media, preventing aggregation and allowing higher concentrations of paclitaxel to enter the cells.
  • EmPAC also displayed significantly greater cytotoxicity than paclitaxel alone in MX-I breast cancer cells, as evident in FIGURE 10D, and moderately greater cytotoxicity than paclitaxel alone towards the MES-S A-DX5 cell line, as seen in FIGURE 1OB.
  • MES-SA DX-5 drug-resistant cell line
  • EmPAC does not get pumped out of cells as efficiently as does paclitaxel alone because it is buried in lipids and is, at least initially, treated as a component of LN. Lipid particles are taken up into cells by specific mechanisms, which may be unaffected by the cell machinery underlying drug resistance.
  • EmPAC Compared to Cremophor EL®.
  • the EmPAC formulation used in this experiment was prepared by solubilization of proprietary lipid mixture in the presence of Tween®-80 and soybean oil by sonication, followed by addition of this mixture to paclitaxel that had been solubilized by sonication in butanol.
  • the mixture was processed by microfluidization on a 11 OEH Microfluidics Microfluidizer® high-pressure homogenizer (Model M-I lOY, Microfluidics, Inc., Newton, MA).
  • paclitaxel formulations were compared after exposure to SF539 glioma cells for one hour before drug removal, as this short-term exposure to drugs more closely mimics in vivo tumor cell exposure to anti-tumor drugs than continuous exposure to drugs, since paclitaxel has a bioavailable half-life in vivo of less than one hour (Wiernik et ah, 1987; Rowinsky et al, 1990).
  • MTT assay utilizes mitochondrial dehydrogenase present in live cells to measure cell viability, as live cells are expected to have higher dehydrogenase activity than dying or dead cells and thus greater
  • EL® was assessed for each drug concentration by student's t-test. As seen in FIGURE 11, while EmPAC alone had no significant effect on cell killing and cell survival (data not shown), EmPAC was significantly more cytotoxic to the glioma cells than Taxol®.
  • mice were implanted subcutaneously with H23 lung tumor cells, which were allowed to grow for 25 days before drug was administered.
  • Tumor-bearing animals were injected IP with 2mL of 60 ⁇ g/mL drugs, each at days 25, 27, 29, 32, 34, 36, 39, 42. Tumor volumes were measured at each of these injection days. The last tumor volume measurement was at day 45 after tumor implantation. Compositions of the drug formulations injected are shown in Table 8.
  • FIGURE 12 A Over a period of 20 days after the first injection of drugs, as seen in FIGURE 12 A, tumors in animals treated with EmPAC decreased in volume over the entire time period while rumors in animals treated with Taxol® displayed regression in tumor volume for the first eleven days before resuming a course of steady growth for the rest of the experiment. Tumors from animals treated with LN controls or untreated animals displayed steady growth throughout the study. Thus, EmPAC was able to reduce tumor sizes for a longer time than did Taxol®. Data in FIGURE 12A were plotted to show mean tumor volume over the course of drug treatment. Turning to FIGURE 12B, percent tumor regression was determined by comparing tumor volume differences between the first and last days of drug treatment.
  • mice treated with Taxol® were treated with EmPAC (p ⁇ 0.0005). Each group represents the mean of 3 ⁇ SEM.
  • tumors from animals treated with EmPAC regressed by approximately 71% (71.4 ⁇ 2.4%), whereas tumors from animals treated with Taxol® regressed by approximately 19% (18.7 ⁇ 0.9%).
  • EmPAC has significantly greater antitumor activity than Taxol®.
  • EmPAC tumor growth inhibition by EmPAC with different concentrations of paclitaxel
  • nude mice implanted with H460 human lung tumor cells were injected with EmPAC formulated with twofold more paclitaxel than in the third formulation of EXAMPLE 1.
  • Taxol® was also administered at one to four times the dose given in previous TGI experiments. Results show that EmPAC is roughly as efficacious as fourfold higher Taxol®.
  • EmPAC formulations of 60 and 137 ⁇ g/mL paclitaxel and equivalent amounts of paclitaxel were used, hi addition, Taxol® at roughly four times the dose given at the original third EmPAC formulation (i.e., 60 ⁇ g/mL paclitaxel) was administered to H460 tumor-bearing mice.
  • mice tumors were allowed to grow to a larger size than in previous experiments. Animals were dosed with drug three times weekly, with two days off for 3 weeks, with tumors measured before injection of drug such that animals whose tumors reached above a cutoff size limit were automatically euthanized. Relative tumor volumes over time were calculated and plotted as a function of time. T-tests were performed to determine if there were significant differences in tumor growth inhibition between animals treated with different drugs. In addition, a survival curve was generated in which the terminal endpoint was defined as animal death by treatment-related causes or by euthanasia, from the tumors reaching a cutoff size limit.
  • EmPAC is roughly as efficacious as four times the concentration of Taxol®.
  • EmPAC PHARMACOKINETICS AND BIODISTRIBUTION To determine the difference in EmPAC pharmacokinetics for IP versus IV, and the biodistribution of EmPAC, nude mice implanted with A549 lung tumors were injected IP or IV with either EmPAC or paclitaxel in Taxol®, each containing radiolabeled paclitaxel.
  • EmPAC formulation containing 14 C-labelled paclitaxel was prepared as follows: A) PREPARATION OF BUFFER. 1) To IL of water add 466.1 mg of sodium pyrophosphate to make a ImM sodium pyrophosphate solution.
  • Blood was drawn by cardiac puncture, and tissues from selected organs (e.g., whole brain, kidney, lung, liver, stomach, spleen, pancreas, tumor, muscle from contralateral side from tumor, urine, feces, whole blood, and tumor muscle from under tumor) were harvested. All tissues and blood were processed for counting of radiolabeled drug. All tissues were weighed and homogenized. Since the tissues were too small to be accurately weighed by the scale used (scale reads to one decimal place), tissue homogenates, including buffers used, were weighed. Aliquots of the homogenates were weighed before scintillation counting in order to determine the fraction of total homogenate. Total 14 -C-labelled paclitaxel in whole organ tissues were determined.
  • organs e.g., whole brain, kidney, lung, liver, stomach, spleen, pancreas, tumor, muscle from contralateral side from tumor, urine, feces, whole blood, and tumor muscle from under tumor. All tissues were processed for counting of radiolabeled drug
  • Taxol® (paclitaxel in Cremophor EL®:ethanol 1 :1) has been in clinical use for a number of years to treat a variety of different cancer indications, including non small cell lung cancer and breast cancer.
  • Abraxane® a formulation of paclitaxel formulated with HSA, has been approved by the FDA for cancer treatment.
  • the objective of this study was to determine if EmPAC differs from Taxol® and from Abraxane® in influencing the cellular uptake of paclitaxel in vitro in short term exposure experiments using A549 human lung cancer and MDA MB 435 breast cancer cell lines exposed for one hour to each paclitaxel formulation. The cells were then fixed and stained with antibodies against paclitaxel, followed by fluorescently-labelled secondary antibodies.
  • Intracellular paclitaxel as detectable by intracellular fluorescence staining was visualized by epifluorescence microscopy. Digital images of fluorescently-labelled cells were captured with a cooled CCD camera. Fluorescence intensity of labelled cells was quantified using digital imaging software, and mean intracellular fluorescence intensity for cells in each experimental group was compared.
  • A549 human non small cell lung tumor cells purchased from American Type Culture Collection (ATCC), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) and subcultured at 1 :3 to 1 :8 ratio.
  • RPMI Roswell Park Memorial Institute
  • FCS fetal calf serum
  • MDA MB 435 human breast cancer cells purchased from ATCC, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS) and subcultured at 1 :3 to 1 :8 ratio.
  • Taxol® (Lot #729537) was obtained from Ben Venue Laboratories, Inc., Bedford, OH.
  • EmPAC (Batch #T343) was prepared by combining 90.42 mg paclitaxel dissolved by sonication in 0.3 mL benzyl alcohol; a sonicated mixture containing 5.6 g soybean oil, 6.61 g Tween®80. and 123.23 mg lipid mixture powder; and 300 mL HPLC-grade water, and emulsifying by high pressure homogenization at 1250 bar. 1O g of dextrose was then added to 200 mL of this solution, sonicated to dissolve, and sterile filtered through a 0.22 ⁇ M filter.
  • the resulting product contained 2.0% soybean oil, 2.2% Tween® 80, 400 ⁇ g/mL lipid mixture powder, 0.1% benzyl alcohol, and 300 ⁇ g/ml paclitaxel (lot # 28042/kl).
  • Abraxane® for Injectable Suspension (Lot #200495, expiration April 2007; Abraxis
  • BioScience, Inc. purchased from GlobalRx, Inc (Efland, NC), was supplied as a powder containing 100 mg of paclitaxel and 900 mg of HSA.
  • Abraxane® powder was then reconstituted by adding 20 mL of sterile PBS to create a suspension containing 5 mg/mL paclitaxel 45 mg/mL HSA. Reconstituted Abraxane® was utilized for experiments within 24 hours, according to package instructions, in order to avoid loss of stability.
  • test articles were placed into new tubes and were relabelled by a person not directly involved in the experiments; the identity of the test articles was revealed after results were calculated.
  • volume of cells (A x IO 4 cells/mL)(V tota ⁇ of cells needed in mL) ⁇ (2 df) (B cells/mL) where: df is dilution factor; A is the number of cells counted on the hemocytometer; and B is the concentration of cells/mL required for the experiment.
  • df dilution factor
  • A is the number of cells counted on the hemocytometer
  • B is the concentration of cells/mL required for the experiment.
  • A549 lung tumor and MDA MB 435 breast cancer cells were passaged into 8-well chamber slides, at 4 x 10 4 cells/cm 2 . Cells were allowed to incubate overnight for 24 hours before test articles were added.
  • Test articles each containing 2.5 ⁇ M paclitaxel, and control complete medium without test articles were added to duplicate chamber slide wells and incubated for one hour at standard conditions used to culture mammalian cells (5% CO 2 . 95% 02; 37°C).
  • cell samples were washed once with warm PBS by addition of 1 mL PBS, followed by aspiration of the warm PBS.
  • 3 mL of ice cold methanol were added to each tissue culture well, and cells were fixed in the methanol at -2O 0 C for 15 minutes. Slides were placed in PBS containing 0.02% sodium azide until staining.
  • Cell samples were blocked with 10% normal chicken serum (NCS) in TBS, pH 8.0 for one hour.
  • NCS normal chicken serum
  • Mean intracellular paclitaxel level as determined by mean fluorescence intensity per area for each treated sample was compared between cells treated with the different test articles.
  • Taxol®, or Abraxane® was determined. Mean fluorescence intensity per cell for each test article treated A549 cell sample is shown in Table 9. Cells treated with no test article showed no specific staining (data not shown). Therefore, fluorescence intensity of untreated cells was not determined. Also, fluorescence intensity of MDA-MB-435 cells was not determined since they became rounded in shape upon treatment with paclitaxel, which made the cytoplasm and cell periphery difficult to visualize.
  • results from this study are consistent with the results from EXAMPLE 2, in which we compared cellular uptake of radiolabeled paclitaxel formulated in EmPAC with that formulated in Taxol®.
  • the results of EXAMPLE 2 suggested significantly greater paclitaxel uptake by EmPAC-treated A549 cells relative to paclitaxel uptake of Taxol®- treated cells.
  • greater paclitaxel uptake of EmPAC-treated cells relative to Taxol®- treated cells was observed in this study, the difference was less than statistically significant. This difference between results may be due to the fact that the EmPAC formulation was different in the two studies.
  • the present invention is directed to a delivery system in the form of a composition for delivering therapeutic and diagnostic agents, including anticancer agents, for treating cancerous cells and tissues. Accordingly, all anticancer agents are within the scope of the present invention as well as all diseased tissues and cells exhibiting aberrant lipid metabolism and elevated uptake of lipids, including cancer cells, which may be treated by such therapeutic agents.
  • therapeutic and diagnostic agents including anticancer agents
  • all anticancer agents are within the scope of the present invention as well as all diseased tissues and cells exhibiting aberrant lipid metabolism and elevated uptake of lipids, including cancer cells, which may be treated by such therapeutic agents.

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Abstract

L'invention concerne une composition pharmaceutique, et des procédés pour l'utiliser et la préparer, composition qui est utile pour traiter, diagnostiquer et prévenir une maladie, un état, un syndrome ou des symptômes de ceux-ci, caractérisés par une hyperprolifération cellulaire, comme un cancer, chez des animaux à sang chaud, notamment des humains. La composition comprend une nanoémulsion lipidique, préparée par un traitement d'homogénéisation, constituée de particules lipidiques comprenant chacune au moins un lipide ne formant pas de bicouche susceptible d'être préférentiellement et sélectivement activement internalisée dans une cellule malade, une quantité efficace d'au moins un agent thérapeutique ou diagnostique interagissant avec les microtubules associé à la nanoémulsion, et un support pharmaceutiquement acceptable. Dans un mode de réalisation préféré, la composition peut également comprendre une molécule support protéique et/ou des agents favorisant l'émulsion comme un tensioactif, une source de graisse d'origine végétale, un solvant et des combinaisons de ceux-ci.
PCT/US2008/004393 2008-04-04 2008-04-04 Système de délivrance en nanoémulsion lipide-huile-eau pour agents interagissant avec les microtubules WO2009123595A1 (fr)

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PCT/US2008/004393 WO2009123595A1 (fr) 2008-04-04 2008-04-04 Système de délivrance en nanoémulsion lipide-huile-eau pour agents interagissant avec les microtubules
CA2720390A CA2720390A1 (fr) 2008-04-04 2008-04-04 Systeme de delivrance en nanoemulsion lipide-huile-eau pour agents interagissant avec les microtubules
EP08742550.0A EP2262369A4 (fr) 2008-04-04 2008-04-04 Système de délivrance en nanoémulsion lipide-huile-eau pour agents interagissant avec les microtubules
KR1020107024777A KR20110009128A (ko) 2008-04-04 2008-04-04 미세소관-상호작용 제제를 위한 지질-오일-물 나노에멀젼 전달 시스템
CN200880129433XA CN102046011A (zh) 2008-04-04 2008-04-04 微管相互作用剂的脂质-油-水纳米乳递送系统
AU2008354007A AU2008354007A1 (en) 2008-04-04 2008-04-04 Lipid-oil-water nanoemulsion delivery system for microtubule-interacting agents
BRPI0821895-1A2A BRPI0821895A2 (pt) 2008-04-04 2008-04-04 Composiçao farmacêutica, método de tratamento ou prevenção de uma doença, uma condição e sintomas das mesmas em animais de sangue quente, incluindo seres humanos, método de preparação da composição farmacêutica e método para diagnosticar uma doença, condição, síndrome.
JP2011502906A JP2011516472A (ja) 2008-04-04 2008-04-04 微小管相互作用薬の脂質−油−水型ナノエマルジョンデリバリシステム
IL208386A IL208386A0 (en) 2008-04-04 2010-10-03 Lipid-oil-water nanoemulsion delivery system for microtubule-interacting agents

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DE102009056871A1 (de) * 2009-12-03 2011-06-22 Novartis AG, 4056 Impfstoff-Adjuvantien und verbesserte Verfahren zur Herstellung derselben
JP2013512890A (ja) * 2009-12-03 2013-04-18 ノバルティス アーゲー ワクチンアジュバントの製造の間の親水性濾過
US8940786B2 (en) 2012-10-01 2015-01-27 Teikoku Pharma Usa, Inc. Non-aqueous taxane nanodispersion formulations and methods of using the same
USRE46906E1 (en) 2009-12-03 2018-06-26 Novartis Ag Methods for producing vaccine adjuvants
US10220095B2 (en) 2013-03-15 2019-03-05 Taiwan Liposome Company, Ltd Controlled drug release liposome compositions and methods thereof
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Cited By (16)

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WO2011062420A3 (fr) * 2009-11-20 2011-10-27 Yuhan Corporation Nanoparticules de ciblage de tumeurs et leurs procédés de préparation
WO2011062420A2 (fr) * 2009-11-20 2011-05-26 Yuhan Corporation Nanoparticules de ciblage de tumeurs et leurs procédés de préparation
US10463615B2 (en) 2009-12-03 2019-11-05 Novartis Ag Circulation of components during microfluidization and/or homogenization of emulsions
DE102009056871A1 (de) * 2009-12-03 2011-06-22 Novartis AG, 4056 Impfstoff-Adjuvantien und verbesserte Verfahren zur Herstellung derselben
JP2013512890A (ja) * 2009-12-03 2013-04-18 ノバルティス アーゲー ワクチンアジュバントの製造の間の親水性濾過
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US10799454B2 (en) 2009-12-03 2020-10-13 Novartis Ag Hydrophilic filtration during manufacture of vaccine adjuvants
USRE46906E1 (en) 2009-12-03 2018-06-26 Novartis Ag Methods for producing vaccine adjuvants
US10213383B2 (en) 2009-12-03 2019-02-26 Novartis Ag Hydrophilic filtration during manufacture of vaccine adjuvants
US10842770B2 (en) 2010-05-03 2020-11-24 Teikoku Pharma Usa, Inc. Non-aqueous taxane pro-emulsion formulations and methods of making and using the same
US9763880B2 (en) 2012-10-01 2017-09-19 Teikoku Pharma Usa, Inc. Non-aqueous taxane formulations and methods of using the same
US9308195B2 (en) 2012-10-01 2016-04-12 Teikoku Pharma Usa, Inc. Non-aqueous taxane formulations and methods of using the same
US8940786B2 (en) 2012-10-01 2015-01-27 Teikoku Pharma Usa, Inc. Non-aqueous taxane nanodispersion formulations and methods of using the same
US10220095B2 (en) 2013-03-15 2019-03-05 Taiwan Liposome Company, Ltd Controlled drug release liposome compositions and methods thereof
US11147881B2 (en) 2013-03-15 2021-10-19 Taiwan Liposome Company, Ltd. Controlled drug release liposome compositions and methods thereof
US11497715B2 (en) 2013-03-15 2022-11-15 Cureport, Inc. Methods and devices for preparation of lipid nanoparticles

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CA2720390A1 (fr) 2009-10-08
EP2262369A4 (fr) 2013-05-29
IL208386A0 (en) 2010-12-30
JP2011516472A (ja) 2011-05-26
CN102046011A (zh) 2011-05-04
KR20110009128A (ko) 2011-01-27
BRPI0821895A2 (pt) 2014-10-07
AU2008354007A1 (en) 2009-10-08
EP2262369A1 (fr) 2010-12-22

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