US20130230457A1 - Thermosensitive Nanoparticle Formulations and Method of Making The Same - Google Patents

Thermosensitive Nanoparticle Formulations and Method of Making The Same Download PDF

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US20130230457A1
US20130230457A1 US13/768,840 US201313768840A US2013230457A1 US 20130230457 A1 US20130230457 A1 US 20130230457A1 US 201313768840 A US201313768840 A US 201313768840A US 2013230457 A1 US2013230457 A1 US 2013230457A1
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liposomes
less
doxorubicin
concentration
phospholipids
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Robert A. Reed
Daishui SU
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Imunon Inc
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Celsion Corp
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Priority to US15/364,003 priority patent/US10251901B2/en
Assigned to CELSION CORPORATION reassignment CELSION CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: REED, ROBERT A., SU, Daishui
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K9/127—Liposomes
    • 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
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a formulation of thermosensitive liposomes, and more specifically to a formulation of liposomes comprising phospholipids and a surface active agent, wherein the liposomes support long term storage at temperatures less than or equal to about 8° C., control degradate formation to maximize product potency and release its contents at mild hyperthermic temperatures. Methods of making formulations are also described.
  • Liposomes are composed of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.25 ⁇ m in diameter; large unilamellar vesicles (LUVs) are typically larger than 0.25 ⁇ m. Oligolamellar large vesicles and multilamellar large vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.25 ⁇ m. Liposomes with several nonconcentric membranes, i.e., several small vesicles contained within a larger vesicle are termed multivesicular vesicles.
  • SUVs Small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • Oligolamellar large vesicles and multilamellar large vesicles have multiple
  • Liposomes may be formulated to carry therapeutic agents, drugs or other active agents either contained within the aqueous interior space (water soluble active agents) or partitioned into the lipid bilayer (water-insoluble active agents). Liposomes may also be conjugated to, an antibody or targeting molecule that permits the delivery of active agent to a specific target site. Encapsulation of a drag in a liposome (1) reduces toxicity of the drug, (2) avoids the body's defenses that normally recognize foreign particles and target them for removal by the reticuloenclothelial system (RES) of the liver and spleen, and (3) allows targeting of the drag carrier to the therapeutic site of action, and once there, to release the drug rapidly so that it can act on the target tissue.
  • RES reticuloenclothelial system
  • RES reticuloendothelial system
  • Liposomes can be designed to be not leaky but will become so if a pore occurs in the liposome membrane, or if the membrane becomes fluid (e.g. undergoes a phase transition from a solid or gel phase to a liquid phase), or if the membrane degrades or dissolves. Such a breakdown in permeability can be induced by the application of electric fields (electroporation), or exposure of the liposome to enzymes or surfactants. Another met wolves raising the temperature of the membrane to temperatures in the vicinity of its gel to liquid phase transition temperature, where it appears that porous defects at phase boundary regions in the partially liquid and partially solid membrane allow for increased transport of water, ions and small molecules across the membrane. The clinical elevation of temperature in the body is called hyperthermia.
  • Hyperthermia causes multiple biologic changes. For a review refer to Issels R D. Hyperthermia adds to Chemotherapy, European J of Cancer (2008) 44:2546-2554. Temperatures in the mild hyperthermia range (39-44° C.) mediate localized physiological changes such as increases in blood flow, vasculature permeability and tissue oxygenation. The vasculature supporting solid tumors is chaotic in structure and the endothelial cells lining the micro-vasculature do not seal together normally resulting in a porous quality.
  • Hyperthermia causes an increase in the pore size in the abnormal tumor microvasculature and therefore enhances the extravasation of nanoscale molecules, such as liposomes of about 100 nm diameter, into the tumor interstitium (Bates D A, Mackillop W J Hyperthermia, adriamycin transport, and cytotoxicity in drug-sensitive and -resistant Chinese hamster ovary cells, Cancer Res (1986) 46:5477-5481, Nagaoka S, Kawasaki Sasaki K, Nakanishi T. Intracellular uptake, retention and cytotoxic effect of adriamycin combined with hyperthermia in vitro. Jpn J Cancer Res (1986) 77:205-211). For these reasons mild hyperthermia is selectively lethal to tumor cells, with the antitumor effect increasing as the temperature increases.
  • Heat sensitive liposomes carry a high concentration of chemotherapeutic agent to solid tumors and the supporting vasculature and release drug locally when heated.
  • Hyperthermia selectively increases liposomal uptake, liposomal permeability, stimulates localized drug release, increases the influx of drug into tumor cells, and increases drug binding to tumor cell DNA (the latter being essential to the mechanism of action of a number of chemotherapeutic agents).
  • liposome formulations capable of delivering therapeutic amounts of active agents in response to mild hyperthermic conditions, i.e., for clinically attainable temperatures in the range 39-45° C.
  • U.S. Pat. No. 6,726,925 describes liposomes that are sensitive to alterations in the temperature of the surrounding environment.
  • the liposomes are loaded with, inter alia, doxorubicin, an approved and frequently used oncology drug for the treatment of a wide range of cancers.
  • the doxorubicin containing liposomal formulation is administered intravenously and in combination with hyperthermia can provide local tumor control and improve quality of life. Localized mild hyperthermia (39.5-45 degrees Celsius) releases the entrapped doxorubicin from the liposome.
  • This delivery technology enables high concentrations of doxorubicin to be deposited preferentially in a targeted tumor.
  • U.S. Pat. No. 7,901,709 describes a method for heat-activated liposomal encapsulation of doxorubicin.
  • WO 2007/024826 describes a method of storing a liposome or nanoparticle formulation including freezing such a formulation.
  • the formulation describes a method of storing liposomes having enhanced stability and storage characteristics.
  • a hyperthermically activated liposomal-drug formulation Key design principles that are required for a hyperthermically activated liposomal-drug formulation to be effective are: 1) near complete encapsulation of active agent to allow the drug to be associated with the liposome in the systemic circulation, 2) a membrane that is engineered to retain drug at normal body temperatures (37° C.) and release drug at mild hyperthermia temperatures 41-43° C.), 3) a membrane composition and particle size that allows the liposome to remain in the systemic circulation long enough to allow the application of a heating modality to trigger the release of the drag to its target, and 4) liposome size that permits its extravasation from the blood stream across leaky tumor micro-vasculature permitting targeting of chemotherapeutic drugs to a tumor site.
  • the present invention solves a persistent problem with drug degradation in doxorubicin liposomal formulation that results in a citrate complex (co-crystal or salt). It has been found that the citrate complex plays a significant role in doxorubicin instability and formation of degradates. More specifically, the formation of 8-desacetyl-8-carboxy daunorubicin and impurity A can be significantly reduced by changing the formation of a citrate complex to a sulfate complex (co-crystal or salt). In addition, the present invention maintains the key design principles listed above for an efficacious hyperthermically activated liposomal formulation containing an active agent.
  • the invention provides a pharmaceutical composition, comprising a suspension of liposomes having, a gel-phase lipid bilayer and doxorubicin entrapped inside the liposomes; said lipid bilayer comprising:
  • the invention provides a method for loading doxorubicin into temperature sensitive liposomes, comprising:
  • the invention comprises a liposomal preparation made by the method set forth above.
  • the invention comprises a liposomal preparation comprising doxorubicin and an imaging agent. In yet another aspect, the invention comprises a liposomal preparation comp doxorubicin and another drug.
  • FIG. 1 schematically represents a liposome having a bilayer membrane containing dipalmitoylphosphatidylcholine (DPPC) as a phospholipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000 (DSPE-MPEG), and monosteroyl-phosphatidylcholine (MSPC) as a lysolipid.
  • DPPC dipalmitoylphosphatidylcholine
  • DSPE-MPEG 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000
  • MSPC monosteroyl-phosphatidylcholine
  • FIG. 2 is a schematic representation of the ammonium loaded doxorubicin liposomes manufacturing process.
  • FIG. 3 is a particle size distribution of NH 4 + -loaded doxorubicin liposomes formed by the extrusion process.
  • FIG. 4 depicts a differential scanning calorimetry graph (scan rate of 2° C. per minute) of pH loaded liposomes prepared using known methods in the art.
  • FIG. 5 depicts a differential scanning calorimetry graph (scan rate of 2° C. per minute) of NH 4 + -loaded liposomes of the present invention.
  • FIG. 6 depicts tunneling electron micrograph of pH loaded liposomes prepared a according to known methods in the art.
  • FIG. 7 depicts a tunneling electron micrograph of NH 4 + -loaded liposomes prepared according to the present invention.
  • FIG. 8 shows a comparison of doxorubicin release profiles as a function of solution temperature for the pH-loaded and NH 4 + -loaded liposomes
  • FIG. 9 a shows a comparison of the levels of 8-desacetyl-8-carboxy daunorubicin in the pH-loaded and NH 4 + -loaded doxorubicin liposomes.
  • FIG. 9 b shows a comparison of the levels of Impurity A in the pH-loaded or NH 4 + -loaded doxorubicin liposomes.
  • the “A” and “B” bars denote impurity levels for a formulation prepared using excipients sourced from different suppliers.
  • FIG. 9 a and FIG. 9 b also show the levels of impurities 8-desacetyl-8-carboxy daunorubicin and impurity A for three replicate runs of NH 4 + -loaded doxorubicin liposomes prepared according to the present invention.
  • FIG. 10 shows the levels of doxorubicin in pH-loaded and NH 4 + -loaded doxorubicin liposomes upon storage for prolonged periods of time at 2-8° C.
  • FIG. 11 shows the levels of degradate growth in pH-loaded and NH 4 + -loaded doxorubicin liposomes upon storage for prolonged periods of time at 2-8° C.
  • FIG. 12 shows the levels of doxorubicin in pH-loaded and NH 4 + -loaded doxorubicin liposomes upon storage for prolonged periods of time at ⁇ 20° C.
  • FIG. 13 shows the levels of degradate growth in pH-loaded and NH 4 + -loaded doxorubicin liposomes upon storage for prolonged periods of time at ⁇ 20° C.
  • the invention provides a liposomal preparation, comprising a suspension of liposomes having a gel-phase lipid bilayer and an active agent entrapped inside the liposomes; said lipid bilayer comprising:
  • the active agent is doxorubicin
  • the relative concentration of impurity A after 6 months of storage at less than or equal to 8° C. is less than 0.5%, wherein impurity A is a peak with a relative retention time approximately 1.4 in a high performance liquid chromatography (HPLC) with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.
  • HPLC high performance liquid chromatography
  • the relative concentration of impurity A after 6 months of storage at less than or equal to 8° C. is less than about 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%.
  • the relative concentration of impurity A after about 1 year of storage at less than or equal to 8° C. is less than about 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%.
  • the relative concentration of impurity A after about 2 years of storage at less than or equal to 8° C. is less than about 1%, 0.75%, 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%.
  • the relative concentration of 8-desacetyl-8-carboxy daunorubicin after 6 months of storage at less than or equal to 8° C. is less than about 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%.
  • the relative concentration of 8-desacetyl-8-carboxy daunorubicin after about 1 year of storage at less than or equal to 8° C. is less than about 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%.
  • the relative concentration of 8-desacetyl-8-carboxy daunorubicin after about 2 years of storage at less than or equal to 8° C. is less than about 2.0%, less than 1.6%, less than 1.5%, less than 1.0%, less than 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%.
  • the concentration of doxorubicin after 150 days of storage at a temperature of about less than or equal to 8° C. is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%, of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.
  • the concentration of doxorubicin after about one year of storage at a temperature of about less than or equal to 8° C. is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%, of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.
  • the concentration of doxorubicin after about one year of storage at a temperature of about less than or equal to 8° C. is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%, of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.
  • the concentration of doxorubicin after about two years of storage at a temperature of about less than or equal to 8° C. is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%, of the initial doxorubicin concentration, as determined by HPLC with a C18 reverse phase column with an acetic acid/methanol solvent gradient elution conditions.
  • the invention is a pharmaceutical composition, wherein the formation of total degradation products after 150 days of storage at a temperature of about less than or equal to 8° C. is less than 1%, or less than 0.5%.
  • the invention is a pharmaceutical composition, wherein the formation of total degradation products after about six months of storage at a temperature of about less than or equal to 8° C. is less than 1%, or less than 0.5%.
  • the invention is a pharmaceutical composition, wherein the formation of total degradation products after about one year of storage at a temperature of about less than or equal to 8° C. is less than 1%, or less than 0.5%.
  • the invention is a pharmaceutical composition, wherein the formation of total degradation products after about two years of storage at a temperature of about less than or equal to 8° C. is less than 2.5%, less than 1% or less than 0.5%.
  • the liposomes are suspended in a buffer comprising a saccharide.
  • the saccharide may be a monosaccharide, such as lactose, or a disaccharide such as sucrose.
  • the buffer further comprises histidine.
  • the invention provides a method for loading an active agent into temperature sensitive liposomes, comprising:
  • the active agent is doxorubicin. In one embodiment, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the doxorubicin present in the solution is taken up into the liposomes.
  • the concentration of doxorubicin taken up into the liposomes is about 1 mM to about 200 mM, preferably about 10 to about 65 mM, and most preferably about 45 mM to about 55 mM. In a further embodiment the concentration of doxorubicin taken up into the liposomes is about 50 mM. In another embodiment the concentration of doxorubicin taken up into the liposomes is about 75 mM.
  • Liposomes of the present invention are composed of phospholipids selected from the group consisting of phosphatidyl cholines, phosphatidyl glycerols, phosphatidyl inositols, and phosphatidyl ethanolamines.
  • the phospholipids preferably possess a solid or gel form to liquid transition temperature in the lower end of the hyperthermic range (e.g., the range of from approximately 38° C. to approximately 45° C.). More preferred are phospholipids whose acyl groups are saturated.
  • the one or more phospholipids have two same or different C 14 -C 20 acyl groups, such as, for example dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidyl glycerol (DSPG), or a combination thereof.
  • DPPC dipalmitoylphosphatidylcholine
  • DSPG distearoylphosphatidyl glycerol
  • the liposomes of the present invention are composed of one or more lysolipids.
  • the lysolipid is monopalmitoylphosphatidylcLoline (MPPC), monolaurylphosphatidylcholine (MLPC), monornyristoylphosphatidylcholine (MMPC), monostearoylphosphatidylcholine (MSPC), or a mixture thereof.
  • the total concentration of lipids in the final liposomal formulation is about 10-50 mg/ml, about 20-50 mg/ml, about 30-40 mg/ml, about 20 mg/ml, about 30 mg/ml, or 40 mg/ml.
  • the concentration of doxorubicin in the liposomal formulation is about 0.2-40 mg/ml, about 0.5-30 mg/ml, about 1-20 mg/ml, about 2-10 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml or about 5 mg/ml.
  • the doxorubicin to lipid ratio is 0.02-10, about 0.05, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10.
  • Liposomes of the present invention include polymer-derivatized lipids to decrease liposome uptake by the RES and thus increase the circulation time of the liposomes.
  • Suitable polymers include hydrophilic polymers such as polyethylene glycol, polyvinylpymlidine, olylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyvinyl alcohols, polyvinylpyrrolidone, dextrans, oligosaccharides, along with mixtures of the above.
  • the one or more phospholipids derivatized with a hydrophilic polymer is a polyethylene glycol derivatized (PEGylatal) lipid.
  • the PEGylated lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol) 2000].
  • the invention provides a method for loading a liposome with an active agent which is bleomycin, dacarbazine, daunorubicin, dactinomycin, fludarabine, gemcitabine, idarubicin, methotrexate, mitomycin, mitoxantrone, vinblastine, vinorelbine, or vincristine.
  • an active agent which is bleomycin, dacarbazine, daunorubicin, dactinomycin, fludarabine, gemcitabine, idarubicin, methotrexate, mitomycin, mitoxantrone, vinblastine, vinorelbine, or vincristine.
  • the said preparing comprises preparing the liposomes in the presence of an ammonium salt, provided as an ammonium sulfate solution.
  • an ammonium salt provided as an ammonium sulfate solution.
  • the concentration of ammonium sulfate in the solution is about 100 mM to about 300 mM, preferably about 200 mM.
  • the ammonium salt is provided as a salt of adipic acid.
  • the ammonium salt in the solution is about 100 mM to about 300 mM, preferably about 200 mM.
  • the ammonium ions outside the liposomes are replaced with a monosaccharide or disaccharide solution.
  • concentration of the monosaccharide or disaccharide solution is about 5-15%, preferably about 10%. This replacement or exchange can be earned out by techniques such as dialysis or diafiltration.
  • ammonium ions outside the liposomes are replaced with a monosaccharide solution, such as for example, a lactose solution.
  • ammonium ions outside the liposomes are replaced with a disaccharide solution, such as for example, a sucrose solution.
  • a histidine buffer is added to the liposomal preparation after step (b).
  • the concentration of the histidine buffer is about 5 mM to about 15 mM, preferably about 10 mM.
  • a method of preparing a liposomal formulation according to the present invention comprises mixing the bilayer components in the appropriate proportions in a suitable organic solvent.
  • Useful solvents include chloroform, acetone, methanol or methylene chloride.
  • the solvent is then evaporated to form a dried lipid film.
  • the film is rehydrated (at temperatures above the phase transition temperature of the lipid mixture) using an aqueous solution containing an equilibrating amount of the lysolipid and a desired active agent, e.g., doxorubicin.
  • the liposomes formed after rehydration are extruded to form liposomes of a desired size.
  • aqueous solution used to rehydrate the lipid film comprises an equilibrating amount of lysolipid monomers (e.g., a concentration equal to the Critical Micelle Concentration of MSPC, about 1 micromolar).
  • the manufacturing process for large scale batches of the ammonium loaded formulation is described below.
  • the process can be employed to produce various size batches of formulation, for example, a 2-2000 L scale batch.
  • a proposed manufacturing process is illustrated schematically in FIG. 2 .
  • an ammonium sulfate buffer by dissolving appropriate quantities of ammonium sulfate in water for injection (WFI) followed by a bioburden reduction filtration.
  • the molarity of the buffer may be, for example, 200 mM.
  • Extrude the hydrated lipid mixture through filter membranes having a certain pore size at an elevated temperature, in order to obtain liposomes of desired size. For example, the hydrated lipid mixture is extruded through 80 nm polycarbonate filter membranes at 65° C.
  • the invention is a liposomal preparation made by a method for loading doxorubicin into temperature sensitive liposomes, comprising:
  • Liposomes of between 0.05 to 0.3 microns in diameter have been reported as suitable for tumor administration (U.S. Pat. No. 5,527,528 to Allen et al.). Sizing of liposomes according to the present invention may be carried out according to methods known in the art, and taking into account the active agent contained therein and the effects desired (see, e.g., U.S. Pat. No. 5,225,212 to Martin et al; U.S. Pat. No. 5,527,528 to Allen et al., the disclosures of which are incorporated herein by reference in their entirety).
  • liposomes are from about 0.05 microns or about 0.1 microns in diameter, to about 0.3 microns or about 0.4 microns in diameter.
  • Liposome preparations may contain liposomes of different sizes.
  • these liposomes comprise, lipid mixtures set forth herein and are therefore temperature-sensitive, with an ability to release contained drug, as described.
  • the liposomes are prepared to have substantially homogeneous sizes in a selected size range.
  • One effective sizing method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond roughly with the average sizes of liposomes produced by extrusion through that membrane. See e.g., U.S. Pat. No. 4,737,323.
  • liposomes are from about 50 nm, 100 nm, 120 nm, 130 nm, 140 nm or 150 nm, up to about 175 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm or 500 nm in diameter.
  • the liposomal preparation of the present invention is stored at a temperature of less than or equal to 8° C., from about 2° C. to about 8° C., from about ⁇ 80° C. to about ⁇ 15° C., from about ⁇ 30° C. to about ⁇ 15° C., or from about ⁇ 15° C. to about 2° C.
  • the liposomal preparation comprises doxorubicin and an imaging or diagnostic agent.
  • the ability to encapsulate an imaging agent in a liposome or an imaging agent in combination with a therapeutic is desirable for a number of reasons.
  • the therapeutic efficacy of the active agent will be increased with the ability to visualize release of the imaging agent and thus infer the release of drug. This would provide the tools to determine the drug's tissue penetration and concentration.
  • combining a drug with an imaging agent in a liposome will permit monitoring and quantitation of drug release over time, tissue distribution, and drug clearance.
  • a liposome carrying and releasing imaging agent will allow for the opportunity to pre-screen patients.
  • a select patient population may be identified as likely to benefit from the therapeutic liposome based on the “leakiness” of tumor vasculature.
  • This leakiness is an indicator of ability of the active agent to extravasate across the microvasculature and any fibrotic tissue to access and treat the tumor.
  • imaging or diagnostic agents include, but are not limited to, agents for X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging or nuclear medicine imaging.
  • the liposomal preparation comprises doxorubicin and an X-ray contrast agent.
  • X-ray contrast agents are generally based on heavy elements, and include barium salts such as barium sulphate, which may be used to enhance visualization of the gastrointestinal system and iodinated contrast agents, which may be used in visualization of the gastrointestinal system and in parenteral studies.
  • Iodinated X-ray contrast agents include, but are not limited to, iohexyl, iopentol, iopamidol, iodixanol, iopromide, iotrolan, metrizamide, metrizoic acid, diatriazoic acid, iothalamic acid, ioxaglic acid and salts of these acids.
  • the liposomal preparation comprises doxorubicin and an MRI contrast agent.
  • MRI contrast agents include paramagnetic chelates, for example based on manganese (2+), gadolinium (3+) or iron (3+).
  • Hydrophilic chelates such as GdDTPA, GdDOTA, GdHPDO3A and GdDTPA-BMA are distributed extracellularly and eliminated renally. Such compounds are useful in, for example, visualizing lesions in the central nervous system.
  • Other more organ- or tissue-specific agents include MnDPDP, GdBOPA, GdEOB-DTPA, paramagnetic porphyrins, macromolecular compounds, particles and liposomes.
  • the liposomal preparation comprises doxorubicin and an ultrasonic imaging, agent.
  • Ultrasonic imaging is based on penetration of ultrasound waves, e.g. in the frequency range 1-10 MHz, into a human or animal subject via a transducer, the ultrasound waves interacting with interfaces of body tissues and fluids. Contrast in an ultrasound image derives from differential reflection/absorption of the sound waves at such interfaces; results may be enhanced by the use of Doppler techniques, including the use of color Doppler to evaluate blood flow.
  • ultrasound contrast agents examples include Echovist®, based on gas-containing galactose microcrystals; Levovist®, comprising gas-containing galactose microcrystals coated with fatty acid; and Infoson®, which comprises gas bubbles encapsulated by partially denatured human serum albumin.
  • imaging or diagnostic agents include, but are not limited to, fluorescent agents such as 6-carboxyfluorescem, radioactive agents (such as radioisotopes or compounds containing radioisotopes, including iodo-octanes, halocarbons, and renografin), and the like.
  • fluorescent agents such as 6-carboxyfluorescem
  • radioactive agents such as radioisotopes or compounds containing radioisotopes, including iodo-octanes, halocarbons, and renografin
  • the liposomal preparation further comprises an additional active agent, for e.g., another chemotherapeutic drug.
  • Liposomes containing 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline which comprises 86% (mole %) of the liposome membrane; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol 2000 (DSPE-mPEG), at approximately 4% (mole %); and 1-stearoyl-2-hydroxy-sn-glycero phosphatidyl choline (MSPC) at approximately 10% (mole %) are prepared by the following technique: The appropriate lipid composition is first hydrated in 200 mM ammonium sulfate buffer, forming multi-lamellar liposomes. Small uni-lamellar liposomes are then formed by extrusion through 80 nm filters to form approximately 100 nm spheres in 200 mM ammonium sulfate buffer.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphatid
  • the liposomes prepared in the previous step were then subjected to a dialysis or diafiltration step exchanging the ammonium sulfate that is external to the liposome with a 10% sucrose solution, forming an ammonium concentration gradient across the liposome membrane (i.e. 200 mM inside, less than 1 mM outside).
  • a 500 mM sodium carbonate solution is then added to the liposomes prepared in the previous step, increasing the external solution to a pH of ⁇ 7.5. It is known (see for example, Mayer L B, Bally M B, Cullis P R., Uptake of adriamyacin into large unilamellar liposomes in response to a pH gradient, Biochimica et Biophysiea Acta 857 (1986) 123-126) that the pH gradient formed across the membrane can effectively, and near quantitatively, promote the loading of an added doxorubicin solution to the internal volume of the liposome at elevated temperatures. Doxorubicin was entrapped within the inner aqueous volumes of liposomes by incubation at 35-39° C.
  • Table 1 displays a comparison between formulations according to Example 1, and a conventional pH loaded liposome, according to Example 2. As seen from Table 1, both formulations contain 2.0 mg/mL of doxorubicin.
  • the formulation according to the present invention compares well to a more conventional liposomal doxorubicin formulation. All raw materials used were of pharmaceutical grade.
  • the final product is characterized for total doxorubicin content, doxorubicin degradation products, pH, osmolality, particle size distribution, MSPC content, DPPC content, DSPE-mPEG content, % encapsulated doxorubicin, drug release at 37° C., and drug release at 41° C. to effectively complete assessment of the product.
  • the target total doxorubicin content is between about 1.8 to about 2.2 mg/mL.
  • the drug encapsulation was typically greater than 90%, and showed limited release, e.g. ⁇ 10%, at normal body temperature (i.e. 37° C.), and exhibited enhanced release, typically >80%, at 41.0° C.
  • the volume averaged particle size of the liposomes as measured by dynamic light scattering is between about 50 to about 150 nm.
  • the physicochemical properties of the liposomes formed in the above Example 1 are comparable to a liposomal preparation formed using a conventional buffer. As shown in FIG. 3 , the particle size distribution of ammonium sulfate hydrated liposome is essentially identical to a citrate buffer hydrated liposome.
  • the lipid composition of the liposomal preparation of the present invention is identical to the lipid composition of the liposomal, preparation known in the art.
  • the functionality of the lipid membrane composition is also confirmed by testing the differential drug release at both 37° C. and 41.0° C.
  • the present invention provides a liposomal product designed to utilize a remote loading, procedure (see for example, Haran G, Cohen R, Bar L K and Barenholz Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases, Biochimica et Biophysica Acta, 1151 (1993) 201-215 201), to encapsulate greater than 90% of the doxorubicin in the internal aqueous core.
  • the % of doxorubicin encapsulated is calculated by measuring unencapsulated doxorubicin (free Dox), separated by ultrafiltration, and the total doxorubicin in the product. Current studies have shown that greater than 95% encapsulation can b achieved for the ammonium loaded formulation.
  • thermal release properties of each batch % release at 37° C. % release at 41° C., have been very reproducible from batch to batch, and are comparable, as shown in FIG. 8 .
  • the overall size and morphology of the two formulations were also compared using the high resolution technique of tunneling electron microscopy (TEM). Again, the comparison between pH-loaded product produced in a GMP manufacturing facility at the current manufacturing scale ( FIG. 6 ), which is currently being used in Phase III clinical studies, to product made using the NH 4 + -loaded formulation at the laboratory scale at Celsion ( FIG. 7 ) was performed.
  • the liposomes for the two formulations show similar vesicle diameters, predominately unilamellar membranes, and exhibit a classical single crystal inside each liposome, which is attributed to the doxorubicin drug complex formation inside the liposome during the loading step. Overall, the TEMs show that the liposomes generated using either pH or NH 4 + -loading system are quite similar.
  • the temperature release profiles measuring the amount of doxorubicin released as a function of temperature from 35 to 45° C. was determined by incubating each sample at the specified temperature for 10 minutes. The results of the tests are shown in FIG. 8 .
  • the comparison was made between pH-loaded product produced in a GMP manufacturing facility at the current manufacturing scale, which is currently being used in Phase III clinical studies, to product made using the NH 4 + -loaded formulation at the laboratory scale at Celsion ( FIG. 8 ).
  • the release curves are very similar for the two formulations, both showing minimal release at temperatures below 39° C., and near 90% release at 41.0° C. and above.
  • both formulations support the design target of limiting doxorubicin release at normal body temperature, i.e. 37° C., with the majority of the drug being released with mild hyperthermia, or temperatures in the 41-45° C. range.
  • the temperature release data is also the best measure of the microscopic uniformity of the lipid membrane composition.
  • the majority of the liposomes i.e. the 100 nm vesicles
  • the appropriate lipid composition to demonstrate the thermal triggered release for the bulk product.
  • incorrect levels of DSPE-MPEG or MSPC will adversely affect the extent and rate of release for doxorubicin from these liposomes.
  • the transition temperatures are nearly identical, in conjunction with the comparative DSC scans ( FIGS. 4 and 5 ), leads to the conclusion that the change in the buffer sys em has negligible impact on liposome membrane and, therefore should have negligible impact on its drug release properties.
  • the combined levels of 8-desacetyl-8-carboxy daunorubicin and Impurity A for the NH 4 + -loaded formulations were less than 0.2%, even with four hour incubation times at 35° C.
  • the levels of degradate formation are shown as the initial time point in the stability data shown in FIG. 11 , and correlate well with the doxorubicin values shown in FIG. 10 .
  • Comparative stability data were generated for the pH-loaded and NH 4 + -loaded formulations. While the pH-loaded formulation requires storage at ⁇ 15° C. to ⁇ 30° C., the stability comparison as generated both at ⁇ 20° C. and under accelerated stability condition, i.e., at +5° C. storage.
  • the results of the doxorubicin assay after 739 days showed a loss of ⁇ 4% doxorubicin for the ammonium-loaded formulation. In contrast, the loss of doxorubicin after the same time period was ⁇ 60% for the pH loaded formulation.
  • the loss of doxorubicin assay data is summarized in FIG. 10 and Table 2. The total degradate growth supports the same trend, i.e. significant increase in degradates are observed for the pH-loaded formulation, with very low levels of degradate, growth for the NH 4 + -loaded formulation ( FIG. 11 and Table 2
  • FIG. 12 and FIG. 13 show the loss of doxorubicin assay data at ⁇ 20° C.
  • the data demonstrate that the NH 4 + -loaded formulation exhibits very low levels of degradate growth and increased doxorubicin stability compared to the pH-loaded formulation.
  • the identity of the degradation products formed from the pH-loaded and NH 4 + -loaded formulations are the same confirmed by LC/MS, although formation occurs to a lesser extent for the NH 4 + -loaded formulation.
  • the NH 4 + -loaded formulation exhibits improved doxorubicin HCl stability, in addition to lower levels of degradation product growth, through at least two years of storage.
  • the solution pH, liposome particle size, % encapsulation, and % release of doxorubicin at 41.0° C. for the NH 4 + -loaded formulation remain through at least two years storage at temperatures of less than or equal to 8° C.

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