WO2011102905A1 - Système d'administration diagnostique de nano-émulsion lipide-huile-eau imageable - Google Patents

Système d'administration diagnostique de nano-émulsion lipide-huile-eau imageable Download PDF

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WO2011102905A1
WO2011102905A1 PCT/US2011/000313 US2011000313W WO2011102905A1 WO 2011102905 A1 WO2011102905 A1 WO 2011102905A1 US 2011000313 W US2011000313 W US 2011000313W WO 2011102905 A1 WO2011102905 A1 WO 2011102905A1
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nanoemulsion
lipid
oil
esters
imaging
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PCT/US2011/000313
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English (en)
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Robert Shorr
Robert Rodriguez
Frank Gibson
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Robert Shorr
Robert Rodriguez
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0078—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion microemulsion, nanoemulsion

Definitions

  • This invention relates to therapeutic and diagnostic agents, and more particularly to a lipid-oil-water nanoemulsion suitable for introduction into a patient and capable of promoting both active and selective imaging and diagnostic agent concentration into cells characterized by aberrant lipid metabolism, including but not limited to tumor cells, and imaging and treatment of a primary or metastatic tumor formed by such cells.
  • RECIST Solid Tumors
  • Measurement and response is determined by comparative measurements of the most recent imaging results to measurements taken at time of diagnosis or at baseline prior to entering a clinical trial. Staging of disease may also taking into account alternative imaging techniques such as positron emission tomography (PET) using radiolabelled glucose, ultrasound, or imaging using radiolabelled antibodies which target l tumor specific antigens.
  • PET positron emission tomography
  • a complete response is disappearance of all target lesions, while a partial response is a 30% decrease in the sum of the longest diameter of target lesions.
  • Progressive disease is a 20% increase in the sum of the longest diameter of target lesions and/or the appearance of new tumors. Stable disease is considered to be small changes that do not meet the above criteria.
  • anatomical imaging has been fundamental to the evaluation of patients receiving drugs that are cytotoxic and expected to shrink tumors. It has been assumed not only that early determination of tumor shrinkage may potentially spare patients from experiencing those debilitating side effects accompanying chemotherapy but also that tumor shrinkage would correlate well with longer term survival. What has emerged in retrospective studies, however, is not only that tumor shrinkage may not always correlate well with longer term survival but also that metastatic disease as well as local relapse can contribute to disease-related death. Additionally, not all patients may respond to a drug at the same rate or in the same way. Furthermore, the emergence of new drugs with novel mechanisms of action has made the use of anatomical imaging questionable as an indicator for tumor response to drug.
  • a drug has been found to be cytostatic
  • patients who would have been characterized under RECIST as having stable disease or a slowed trajectory of disease progression have gone on to achieve longer term survival than those patients receiving cytotoxic agents who would be considered responders under RECIST.
  • some drugs are able to help cancer be "managed" so that patients bearing tumors may nevertheless enjoy longer life expectancy and better life quality.
  • An additional confounding consideration for RECIST is that with some targeted therapies, the drug-related mechanism of tumor cell death can influence what appears in an anatomical image and vary with time of treatment. For example, the dissolution and shrinkage of tumors is part of the final steps in a complicated cascade of cellular and subcellular local and immune system changes as a response to therapy.
  • tumor swelling, edema, and inflammation can occur, phenomena which may suggest tumor enlargement or growth on an anatomical image.
  • Such effects make the timing of taking the image, the kinetics of these effects, and the resolution of these effects critical.
  • contrast agents and additional imaging methods may help better elucidate both what is occurring within the tumor and the kinetics thereof, in many situations an imaging center may lack the expertise, equipment, or resources to accurately reach the proper interpretation of the tumor image presented. Cancer specific necrosis, apoptosis, or autophagy, or a combination of these and the precise impact of the mechanism of cell death on the nature of an image obtained by the various imaging modalities is unknown.
  • Targeted molecular imaging of cancer in combination with an anatomical imaging modality permits a physician to characterize a cancer mass' biological properties accurately and ultimately to select the treatment most likely to be effective based on the tumor's characteristics.
  • Conventional imaging modalities which often use additional contrast agents administered to the patient to enhance the quality of the images generated; these contrast agents often differ by the imaging modality used, and each contrast agent demonstrates different inherent strengths and limitations, such as toxicity.
  • radiolabelled agents include 18-fluorodeoxyglucose (FDG) to measure metabolic activity of cancer cells versus normal cells, or radiolabeled nucleotides to measure cellular proliferation.
  • FDG 18-fluorodeoxyglucose
  • PET imaging has its shortfalls: the technique requires exposure to radiation, and some drugs may alter the ability for the radiolabeled agent to be taken up intracellularly without killing the tumor cells at a rate that is consistent with what appears on image. Additionally, false positives can occur on inflammation and invasion of tumors with infiltrating lymphocytes, or even on healing and fibrosis. Given these shortfalls, the ability to utilize molecular targeted ligands that are specific for cancer cells is therefore extremely important so as to enable distinguishing true disease response or progression from artifact.
  • molecularly-targeted isotopes can be delivered to specific targets within a cancer cell, concentrating cell-killing doses of radiation directly to a tumor mass, thereby decreasing radiation exposure to healthy tissue.
  • Lipid-soluble drugs readily penetrate cell membranes and may be transported through cells.
  • Cells characterized by hyperproliferation, such as cancer cells generally exhibit an aberrant lipid metabolism, marked by their greatly-increased preferential uptake of lipids and fatty acids. Indeed, it is not uncommon for cancer patients to present with low or depleted levels of serum lipids, including cholesterol.
  • Upregulation of lipid receptors as well as changes in plasma membrane lipid raft compositions have been suggested to be associated with diverse solid tumor cell types and increased lipid uptake.
  • lipids have been found to play essential roles in membrane structure, growth and metastasis, signal transduction, and transport processes.
  • Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-terminal kinase (JNK).
  • CML chronic myelogenous leukemia
  • JNK c-JUN N-terminal kinase
  • Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK. Cell Cycle 7:3362-70, herein incorporated by reference.) Additionally, in US Patent 5773431 to Javitt, herein incorporated by reference, 26-aminocholesterol and the related compound 27-hydroxycholesterol were demonstrated to have selective and potent cell-killing effects on LI 210 mouse leukemia cells, -12 rat colon adenocarcinoma cells, and HCT-8 human colon carcinoma cells.
  • 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, especially agents for the imaging of cancer, have been lacking.
  • fluorescent dyes have relatively only recently been explored for their capability to act as targeted molecular imaging agents for cancer. While many classes of highly fluorescent organic compounds are known, over the past two decades dyes from the difluoro-boraindacene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) family (BODIPY) have been recognized as a photostable substitute for fluorescein.
  • BODIPY difluoro-boraindacene
  • BODIPY inherent photophysical properties, including excellent thermal and photochemical stability relative to fluorescein; high fluorescence quantum yield; negligible triplet-state formation and nanosecond excited-state lifetimes; intense absorption profile and large absorption coefficients; narrow emission profiles; good solubility; relative insensitivity to changes in polarity and pH; and large range of colors have all added to the general attractiveness of these fluorophore materials.
  • BODIPY-based dyes have thus been designed for and used as biological labelling reagents, with most current labelling studies involving the use of commercial materials bearing an anchor with which to attach the dye to the biological host.
  • DiO 3,3'-dioctadecyloxacarbocyanine perchlorate
  • Eosin Y an acidic fluorescent red dye
  • Eosin Y is a tetrabromo derivate of fluorescein used to stain basic parts of the cell, such as cytoplasm, collagen, red blood cells, and muscle fibers, for examination under the microscope.
  • Eosin is most often used as a counterstain to Hematoxylin in H&E (Hematoxylin and Eosin) staining.
  • H&E Hematoxylin and Eosin
  • Eosin Y is typically used in concentrations of 1 to 5 percent weight by volume, dissolved in water or ethanol; a small concentration (0.5 percent) of acetic acid usually gives a deeper red stain to the tissue.
  • Erythrosine B the disodium salt of 2,4,5, 7-tetraiodofluorescein, is used as a cherry-pink biological stain and a radiopaque medium with a maximum absorbance at 530 nm in an aqueous solution. Because Erythrosine B is identical in structure to Eosin Y except that iodine is substituted for the bromines at each position on Eosin Y, Erythrosine B is often used as a substitute for Eosin Y staining.
  • the stain Rose Bengal (4,5,6,7-tetrachloro-2 , ,4 , ,5',7'-tetraiodofluorescein) is a nontoxic dye whose sodium salt is commonly used in eye drops to stain damaged conjunctival and corneal cells and thereby identify damage to the eye.
  • This fluorescent stain has also been studied for its separate property as having a therapeutic effect on metastatic melanoma.
  • 26 target lesions in eleven metastatic melanoma patients were directly injected with 10% w/v Rose Bengal in saline at a dose of 0.5 ml/cc lesion volume, with twenty-eight additional untreated lesions observed for potential bystander effect.
  • labelled cholesterol-derived sterols have been formulated in the past to be used to study the metabolism and intracellular localization of cholesterol, including a variety of photoreactive, spin-labelled, and fluorescent cholesterol analogues.
  • photoreactive, spin-labelled, and fluorescent cholesterol analogues See, e.g., Gimpl G and Gehrig-Burger K (2007). Cholesterol reporter molecules. Biosci. Rep. 27:335-358, herein incorporated by reference.
  • these analogues possess all of the properties of the parental cholesterol molecule, these molecules have been reported to localize in cholesterol-enriched microdomains.
  • Photoreactive cholesterol probes such as cholesteryl diazoacetate; 3a-azido-5-cholestene; 3a- (4-azido-3-iodosalicylic)-cholest-5-ene; p-azidophenacyl 3a-hydroxy-5P-cholan-24-ate; 25- azidonorcholesterol; sterols with diazoacetate, aryldiazirines or fluorodiazirine attached at C- 22 or C-24; [ 3 H]22-(p-benzoylphenoxy)-23,24-bisnorcholan-5-en-3P-ol; and [ 3 H]6-azi-5a- cholestanol (i.e., photocholesterol) and [ 3 H]7-azi-5a-cholestanol have been used to visualize the nicotinic acetylcholine receptor and oxytocin receptors and may be used to identify and analyze other putative cholesterol binding proteins at the molecular level
  • dehydroergosterol and cholestatrienol include those in which a fluorophore or photoreactive group is attached (e.g., NBD-cholesterol, BODIPY-cholesterol, and dansyl-cholestanol).
  • Such technology has also been used to analyze the potential efficacy of cholesterol- utilizing agents in the treatment of cancer.
  • VIP vasoactive intestinal peptide
  • SSL sterically stabilized liposomes
  • researchers covalently inserted VIP into BODIPY-cholesterol-labelled SSL and then incubated rat breast cancer tissue sections in vitro with the liposomes .
  • 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.
  • there has not been an efficacious formulation of a targeted molecular imaging agent utilizing LCM technology which presents minimal to nonexistent levels of adverse side effects upon patient administration.
  • Typical approaches include high-shear homogenization and ultrasound, high-pressure homogenization, hot homogenization, cold homogenization, and solvent emulsification evaporation. Sterilization by aseptic filtration or radiation may also be complex.
  • attempts to increase drug payload and solubility of lipids in LCMs with addition of a drug in ethanol and aqueous solution followed by high-shear homogenization result in the formation of poorly-stable non- gas-containing particles that contain little if any drug.
  • Lipid-oil-water nanoemulsions have also been described and prepared in the art. (See, e.g. , US Patent 7387790 to Shorr et al. ; Constantinides PP, Chaubal MV, and Shorr R (2008). Advances in lipid nanodispersions for parenteral drug delivery and targeting. Adv Drug Deliv Rev 60:757-67; and Barbarese E, Ho SY, D'Arrigo JS, and Simon RH (1995). Internalization of microbubbles by tumor cells in vivo and in vitro. J Neurooncol 26:25-34, all incorporated by reference.) Stability was achieved by modification of conditions of high shear homogenization. To date, however, there has not been an efficacious formulation of a targeted molecular imaging agent using lipid-oil-water nanoemulsion technology which presents minimal to nonexistent levels of adverse side effects upon patient administration.
  • Administering imaging agents with an appropriate delivery vehicle that simultaneously promotes accumulation and cellular internalization in a cancer mass but limits accumulation in healthy tissues to promote accurate imaging of the cancer mass is highly desirable. With more efficient delivery, systemic and healthy tissue concentrations of imaging agents may be reduced while achieving the same or better imaging results with fewer or diminished side effects. Further, a delivery vehicle that would not be limited to a single type of cancer but would allow for selective accumulation into a cancer 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 imaging agent would be a significant advance in the art.
  • labelled nanoparticles may act as biomarkers that are specific for cancer cells and hence capable of facilitating the diagnosis of the cancer.
  • one team of researchers performed cell viability experiments and confocal fluorescence microscopy using 3-mercaptopropionic acid-capped gold nanoparticles to determine whether these nanoparticles could enhance the delivery of daunorubicin to drug- resistant leukemia 562/ADM cells.
  • Both fluorescence microscopy and electrochemical studies demonstrated that visualization of the cells was much greater when the nanoparticles were present than when either daunorubicin or the bare nanoparticles were administered alone.
  • the study concluded that the use of functionalized nanoparticles merited further exploration as a sensitive method for the early diagnosis of certain cancers.
  • a targeted molecular imaging agent to be used for the imaging of diseases, conditions, and syndromes characterized by aberrant lipid metabolism, such as cancer, which exhibits rapid and increased selective and preferential uptake into tumor cells.
  • a targeted molecular imaging agent to be used for the imaging of diseases, conditions, and syndromes characterized by aberrant lipid metabolism, such as cancer, which acts as a biomarker for diseased, injured, or mutated tissue, cells, and/or at least one organelle thereof, that are amenable to delivery of diagnostic and imaging agents by the nanoemulsion.
  • the present invention broadly provides a stable lipid-oil- water nanoemulsion that demonstrates dramatically increased and selective uptake and concentration into the cytosol of diseased, injured, or mutated cells of warm-blooded animals, including humans, compared to healthy cells.
  • This nanoemulsion is useful for selectively and preferentially imaging a diseased, injured, or injuriously mutated tissue, cell, or at least one organelle thereof, and treating the disease, injury, or injurious mutation.
  • the disease, injury, or mutation is characterized by aberrant lipid metabolism, and in a more preferred embodiment of the present invention, the disease is cancer.
  • the lipid-oil-water nanoemulsion is comprised of lipid particles as hereinafter defined, uniformly dispersed through homogenization 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 pharmaceutically-acceptable imaging agent associated with the lipid nanoemulsion; an effective amount of at least one pharmaceutically- acceptable diagnostic agent associated with the lipid nanoemulsion; and a pharmaceutically- acceptable carrier or excipient therefor.
  • the lipid particles each comprise at least one non- bilayer-forming lipid, among which the imaging agent is physically or chemically integrated.
  • Such suitable lipid particles have been found to enhance significantly the targeted delivery and concentration of an associated payload into diseased cells characterized by aberrant lipid metabolism, including cancer cells.
  • the nanoemulsion exhibits exceptional physical and chemical stability for an extended duration of time, thereby greatly facilitating prepackaging of the nanoemulsion in stable, ready-to-administer forms.
  • the imaging agent may be a fluorescent dye, such as, but not limited to, BODIPY, DiO, Rose Bengal, Eosin Y, and Erythrosine B.
  • the imaging agent may be a labelled sterol, such as, but not limited to, BODIPY-cholesterol, NBD-cholesterol, and dansyl-cholesterol, and their associated cholesteryl esters.
  • the imaging agent is radiolabeled, such as, but not limited to, l4 C-cholesterol.
  • the demonstration of increased uptake into cancer cells associated with a particular tumor type upon use of an imaging modality such as x-ray, CT, PET, or MRI, without limitation, or lack thereof, is expected to be a useful indicator of the potential benefits to be achieved by delivering a chemotherapeutic agent known to be efficacious in treating that tumor type.
  • an imaging modality such as x-ray, CT, PET, or MRI
  • chemotherapeutic agent known to be efficacious in treating that tumor type.
  • the naked specificity of the nanoemulsion for both tumor mass and tumor cell uptake allows the nanoemulsion to act as a biomarker for the treatment of those targeted diseased, injured, or mutated cells that form the cancerous mass.
  • any component of the nanoemulsion of the present invention may act as an antigen, or conversely as an antibody, which may be detected through immunologic-based imaging modalities, such as immunofluorescence.
  • the at least one pharmaceutically-acceptable diagnostic agent is to be present in an amount sufficient to produce an observable effect that assists in diagnostic interpretation of the presented disease, injury, or injurious mutation.
  • the diagnostic agent is, without limitation, a drug, prodrug, protein, lipid, nucleic acid, carbohydrate, hormone, vitamin, nutrient, or other substance shown to produce such an observable effect. These substances can be radioactive where necessary.
  • the method of diagnosing the disease, injury, or injurious mutation comprises administering to the patient an effective amount of the nanoemulsion of the present invention, obtaining an ex vivo sample from the patient, and performing a diagnostic procedure thereupon.
  • ex vivo samples may constitute a tissue biopsy, cerebrospinal fluid, urine, saliva, hair, blood, or DNA obtained through any collection method.
  • the result of the diagnostic procedure is a colorimetric (e.g., fluorometric), chemilumenscent, or radioisotopic readout.
  • FIGURE 1 depicts C6 glioma cells treated with a labelled nanoemulsion according to the present invention.
  • FIGURE 2 shows murine B16 melanoma cells treated with a labelled nanoemulsion according to the present invention.
  • the present invention is generally directed to a stable lipid-oil-water nanoemulsion that demonstrates dramatically increased and selective uptake and concentration into the cytosol of diseased, injured, or mutated cells in warm-blooded animals compared to healthy cells.
  • warm-blooded animals include those of the mammalian class, such as humans, horses, cattle, domestic animals including dogs and cats, and the like, subject to disease, injury, mutation, and other pathological conditions and syndromes.
  • This nanoemulsion is useful for selectively and preferentially imaging a diseased, injured, or injuriously mutated tissue, cell, or at least one organelle thereof, and treating the disease, injury, or injurious mutation.
  • the disease, injury, or mutation is characterized by aberrant lipid metabolism, and in a more preferred embodiment of the present invention, the disease is cancer.
  • the nanoemulsion is comprised of lipid particles as hereinafter defined, uniformly dispersed through homogenization in an aqueous phase capable of being selectively and preferentially internalized within a cell characterized by aberrant lipid metabolism, including a cancer cell; an effective amount of at least one pharmaceutically-acceptable imaging agent associated with the lipid nanoemulsion; an effective amount of at least one pharmaceutical ly-acceptable dianostic agent associated with the lipid nanoemulsion; 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 aberrant lipid metabolism, including tumor and cancer cells.
  • Internalization as used herein means that the lipid particles are actively taken up by the cell.
  • the lipid particles of the nanoemulsion are exceptionally physically and chemically stable over an extended period of time and hence experience minimal loss of imaging or diagnostic capability 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 lipid particles are also relatively simple and convenient to prepare and to administer.
  • 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 used in the nanoemulsion 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 to be used to form the nanoemulsion 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:
  • 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;
  • At least one third member selected from the group consisting of sterols, terpenes, bile acids and alkali metal salts of bile acids;
  • the lipid mixture described above only includes the presence of members (a) through (c), it is more preferred to incorporate members (d) and/or (e) because the long-term stability and uniformity of size of the lipid particles are theoretically enhanced by the presence of these two optional members.
  • 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 structure and desired dosage of any payload to be encapsulated within or otherwise associated with the nanoemulsion, 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 ⁇ , 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 imaging and diagnostic agents added.
  • the imaging agent associated with the nanoemulsion of the present invention may be a fluorescent dye, such as, but not limited to, BODIPY, DiO, Eosin Y, and Erythrosine B.
  • the imaging agent may be a labelled sterol, such as, but not limited to, BODIPY-cholesterol, NBD-cholesterol, and dansyl-cholesterol, and their associated cholesteryl esters.
  • the imaging agent is radiolabelled, such as but not limited to l4 C-cholesterol.
  • the demonstration of increased uptake into cancer cells associated with a particular tumor type upon use of an imaging modality such as x-ray, CT, PET, or MRI, without limitation, or lack thereof, is expected to be a useful indicator of the potential benefits to be achieved by delivering a chemotherapeutic agent known to be efficacious in treating that tumor type.
  • an imaging modality such as x-ray, CT, PET, or MRI
  • chemotherapeutic agent known to be efficacious in treating that tumor type.
  • the naked specificity of the nanoemulsion for both tumor mass and tumor cell uptake allows the nanoemulsion to act as a biomarker for the treatment of those targeted diseased, injured, or mutated cells that form the cancerous mass.
  • the at least one pharmaceutically-acceptable diagnostic agent for which the lipid particles have an enhanced loading capacity, should be present in a pharmaceutically- sufficient amount to produce an observable effect that assists in diagnostic interpretation of the presented disease, injury, or injurious mutation.
  • the capacity of the nanoemulsion for elevated internalization levels within a target cell coupled with a high loading capacity of the particles for the diagnostic agent, provides a potent vehicle for diagnosis of a disease, condition, syndrome, or symptom thereof, initiating from these target diseased, injured, or injuriously-mutated cells, as well as imaging through detection of the imaging agent, by delivering an effective amount of the diagnostic agent to such targets, thereby inducing an observable effect, including stopping growth, inducing differentiation, or killing the cell.
  • the lipid particles of the present invention hence not only enhance delivery of the diagnostic agent to the diseased cells and tissues but also reduce the amount of the diagnostic agent needed to achieve the observed effect, especially as compared to delivery systems in the prior art.
  • the diagnostic agents employed in the present invention may be uncharged or charged, nonpolar or polar, natural or synthetic, and so on.
  • the term "diagnostic agent” as used herein includes any substance including, but not limited to, drugs, prodrugs, proteins, lipids, nucleic acids, carbohydrates, hormones, vitamins, nutrients, substances, and the like, shown to produce an observable effect that assists in diagnostic interpretation of the presented disease, condition, syndrome, characterized by cellular hyperproliferation as a subset of aberrant lipid metabolism, or symptoms thereof, including cancer.
  • the present invention further discloses a method of diagnosing the disease, injury, or injurious mutation which comprises administering to the patient an effective amount of the nanoemulsion of the present invention, obtaining an ex vivo sample from the patient, and performing a diagnostic procedure thereupon.
  • ex vivo samples may constitute a tissue biopsy, cerebrospinal fluid, urine, saliva, hair, blood, or DNA obtained through any collection method.
  • the result of the diagnostic procedure is a colorimetric (e.g., fluorometric), chemilumenscent, or radioisotopic readout.
  • the nanoemulsion of the present invention exhibits 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 nanoemulsion of the present invention exhibits desirable drug and emulsion stability over an extended time period (e.g. , at least 14 days at about 30°C and at least 12 months at 4°C).
  • the nanoemulsion of the present invention contains lipid particles in an amount of from about 0.1 ⁇ .
  • Typical concentrations of the diagnostic agent based on the total volume of the nanoemulsion 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 diagnostic agent potentially present in the nanoemulsion 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 nanoemulsion 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 nanoemulsion of the present invention.
  • the plant-based fat sources may be produced, derived, or modified from natural, chemical, or recombinant means.
  • 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 imaging agent and diagnostic agent but simply enhances their delivery to and internalization into the diseased cell or tissue, including cancerous cells or tissue, to impart imaging and/or diagnostic benefits.
  • the nanoemulsion of the present invention is prepared by combining the lipid particles with the imaging and diagnostic agents 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 imaging agent, which themselves may be mixed with a water- miscible solvent for dissolution.
  • the lipid particle-imaging 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.
  • 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.
  • the nanoemulsion 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 nanoemulsion 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 nanoemulsion can be administered directly to a patient when combined with a pharmaceutically-acceptable carrier.
  • the methods of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • 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 imaging and 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 imaged or diagnosed; and the dosage required for efficacy.
  • an effective amount refers to the dosage or multiple dosages of the imaging agent and/or diagnostic agent at which the desired imaging and/or diagnostic effect is achieved.
  • an effective amount of the imaging agent and/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 imaging and/or diagnostic effect or in the event of any complication.
  • the imaging agent and diagnostic agent included in the nanoemulsion 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 imaging agent or diagnostic 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 imaging agent and/or diagnostic agent.
  • a method of preparing the nanoemulsion of the present invention comprising 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 imaging agent and/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 nra, 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 diagnostic and imaging agent added to the lipid mixture, and the technique used to produce the lipid particles.
  • the nanoemulsion 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 plant-based fat source such as a vegetable oil
  • a surfactant such as a non- ionic surfactant
  • the imaging agent and/or diagnostic agent may be mixed with a solvent, such as a water-miscible solvent, to yield an imaging/diagnostic phase.
  • the lipid and imaging/diagnostic 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 diagnostic or imaging agent, and so on, to yield a purified form suitable for delivery as a nanoemulsion to warm-blooded animals, including humans, in need of imaging and/or treatment.
  • the objective of this experiment is to determine whether there is a significant difference observed in the uptake of labelled nanoemulsion particles in cancer cells versus that observed in non-cancer cells.
  • Nanoemulsion particles prepared according to the specification were provided with 0.01% w/w either of lipid cholesteryl BODIPY-FL C12 (Molecular Probes, Eugene, Oregon) or of DiO before processing through high-shear homogenization.
  • Samples (5 mL) were dialyzed (30 minutes) to remove any non-incorporated dye against distilled water (1.2 L) with two changes.
  • C6 glioma, murine normal-derived NIH 3T3, ras-transformed NIH 3T3, and murine B16 melanoma cells cultured in 96-well plates at a seeding density of 104 cells/well were seeded one day prior to each experiment and grown in complete medium consisting of DMEM and 10% fetal bovine serum.
  • the samples were diluted in complete media to 50 ⁇ g/mL and added to the cells.
  • the cells were incubated with diluted samples at 37°C for 30 minutes. After appropriate incubation time, lipid nanoparticle samples were aspirated from each well. Wells were then washed once by addition of 100 ⁇ , or PBS followed by aspiration and replacement of the same volume of PBS.
  • the fluorescence intensity of the cells was quantified using a microplate fluorimeter (FLUOstar Optima, BMG Labtechnologies, Inc., Durham, NC).
  • the present invention is directed to a delivery system in the form of a composition for delivering imaging and diagnostic agents, including anticancer agents, for treating cancerous cells and tissues. Accordingly, all amenable agents are within the scope of the present invention, as well as all diseased tissues and cells exhibiting cellular hyperproliferation and aberrant lipid metabolism and elevated uptake of lipids, including cancer cells, which may be diagnosed by such agents.

Abstract

La présente invention a pour objet une nano-émulsion lipide-huile-eau stable associée à un agent d'imagerie et à un agent diagnostique utile à la fois pour l'imagerie de cellules caractérisées par un métabolisme lipidique aberrant et/ou une hyperprolifération cellulaire, telles que des cellules cancéreuses, et pour le diagnostic du cancer d'une manière extrêmement spécifique et sélective par rapport à des cellules non transformées.
PCT/US2011/000313 2010-02-19 2011-02-22 Système d'administration diagnostique de nano-émulsion lipide-huile-eau imageable WO2011102905A1 (fr)

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