Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/02—Halogenated hydrocarbons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
  • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
  • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
  • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
  • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0026—Blood substitute; Oxygen transporting formulations; Plasma extender
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L27/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Compositions of derivatives of such polymers
  • C08L27/02—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Compositions of derivatives of such polymers not modified by chemical after-treatment
  • C08L27/12—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Compositions of derivatives of such polymers not modified by chemical after-treatment containing fluorine atoms

Definitions

• This invention generally relates to compositions of fluorocarbon nanoemulsions and methods of their preparation and use. More particularly, the invention relates to unique fluorocarbon nanoemulsions stabilized with one or more of perfluoro-n-hexyl-oligoethyleneoxy-alcohols and phospholipids, and methods of preparation and application thereof.
• Emulsified fluorocarbon (FC)-based oxygen carriers have been reported, for example, utilizing higher molecular weight fluorocarbons (e.g., perfluorodecalin and perfluorooctylbromide). These compositions, however, have largely failed to meet the rigorous clinical requirements and many challenges remain to address the urgent needs for safe, reliable and effective oxygen carriers.
• FC fluorocarbon
• the present invention is based in part of the unexpected discovery that fluorocarbon nanoemulsions uniquely formulated with surfactants or stabilizing agents selected from perfluoro-n- hexyl-oligoethyleneoxy-alcohols, phospholipids or combinations thereof, resolve the critical issues of bioaccumulation of metabolites and degradation products, which form from fluorosurfactants with perfluoroalkyl groups of more than 6 fully fluorinated carbons.
• Fluorocarbon nanoemulsions of the invention derive a significant benefit from employing a shorter perfluorocarbon chain of the surfactant because excretion and metabolism products do not bioaccumulate as do those with larger perfluoroalkyl moieties.
• the invention generally relates to a composition of a fluorocarbon nanoemulsion that includes a fluorocarbon ranging from about 4 to about 8 carbons; and one or more surfactants selected from perfluoro-n-hexyl-oligoethyleneoxy-alcohols, phospholipids or
• the one or more surfactants comprise a perfluoro-n- hexyl-oligoethyleneoxy-alcohol and/or a mixture of three phospholipids.
• the invention generally relates to a method for forming a
• the method includes: preparing an aqueous first mixture comprising PEG Telomer B and a fluorocarbon; transferring via a homogeni/er comprising a bypass valve and a pneumatic unit the first mixture between a first container and a second container and back to the first container, wherein the bypass valve is open; initiating the pneumatic unit using a closed bypass valve to form a homogenized pri mary nanoemulsion; disposing the homogenized primary nanoemulsion into an aqueous solution of sucrose or another viscogen and optionally one or more of pharmaceutically acceptable buffer salts and microbiocidal agents, disposed in a first pressure vessel to form a second mixture; attaching the first pressure vessel to an input end of the homogeni/er; attaching a second pressure vessel to an output end of the homogeni/er; operatin the pneumatic unit with the bypass valve closed to form nanoemulsion until all of the second mixture is transferred to the second pressure vessel; and pressurizing the second pressure vessel to transfer and ster
• the invention generally relates to a method for forming a
• the method includes: preparing an aqueous first mixture comprisin one or more perfiuoro-n-hexyl-oligoethyleneoxy-alcohols where the oligoethyleneoxy moieties are from 1 to 16 units in length, and a fluorocarbon; transferrin via a homogeni/er comprisin a bypass valve and a pneumatic unit the first mixture between a first container and a second container and back to the first container, wherein the bypass valve is open: initiating the pneumatic unit with a closed bypass valve to form a homogenized primary emulsion; disposing the homogenized primary emulsion into a sucrose solution comprising optionally one or more of pharmaceutically acceptable buffer salts, viscogens and microbiocidal agents disposed in a first pressure vessel to form a second mixture; attaching the first pressure vessel to an input end of the homogenizer; attaching a second pressure vessel to an output end of the homogenizer; operating the pneumatic unit with the bypass valve closed to form an emul
• the invention generally relates to a method for forming a
• the method includes: preparing an aqueous first mixture comprising a peril uoro-n- hexyl-oligoethyleneoxy-alcohol. sucrose and optionally one or more of pharmaceutically acceptable buffer salts, viscogens and approved biocidal sterilants; disposing the first mixture into a vial using a syringe and a needle attached to the syringe; adding a fiuorocarbon into the vial, stoppering and crimp capping the vial, followed by ⁇ ortexing and sonicating the vial.
• the invention generally relates to a method for forming a
• the method includes: forming a mixture comprising one or more phospholipids, water, glycerol, monobasic sodium phosphate and dibasic sodium phosphate: transferring the mixture via a 0.2 micron filter into a sterile vessel; disposing the mixture into a vial; adding a fiuorocarbon to the vial and immediately stoppering and crimp capping the vial, followed by vortexing and sonicating the vial.
• the invention generally relates to a nanoemulsion formed by a method disclosed herein.
• FIG. 1 is a flowchart summarizing an exemplary embodiment of the method according to the invention to generate PEG Telomer B-stabilized perfluorocarbon nanoemulsions by high-pressure homogenization.
• FIG. 2 is a flowchart summarizing additional steps in an exemplary embodiment of the method according to the invention of FIG. 1.
• FIG. 3 graphically recites an exemplary embodiment of the method according to the invention stability data of TDFH-PTB nanoparticle emulsions stored at 4°C and the same emulsions stored at 23°C.
• FIG. 4 graphically recites an exemplary embodiment of the method according to the invention stability data of TDFH-PTB nanoparticle emulsions stored at 4°C and TDFH-Compound 20a (DuPont Capstone FS-3100) nanoparticle emulsions stored at 4°C.
• FIG. 5 is a flowchart summarizing an exemplary embodiment of the method according to the invention to generate phospholipids-stabilized-perfluorocarbon nanoemulsions.
• FIG. 6 is a flow chart summarizing an exemplary embodiment of the method according to the invention to form stabilized perfluorocarbon nanoemulsions comprising mixed components at their concentrations in the final product by vortexing and sonication.
• FIG. 7 is a diagram of an exemplary apparatus that measures uptake of dissolved oxygen from a solution before and after addition of a test solution of a nanoemulsion of perfluorocarbon and surfactant.
• FIG. 8 is a graph of dissolved oxygen content of an aqueous solution prior to and after addition of the perfluorocarbon nanoemulsion of Example 5.
• FIG. 9 is a graph showing decrease in dissolved oxygen level in aqueous solution post addition of the formulation of Example 7.
• FIG. 10 is a graph showing decrease in dissolved oxygen level in aqueous solution post addition of the formulation of Example 8.
• the invention provides fluorocarbon nanoemulsions uniquely formulated with surfactants or stabilizing agents selected from perfluoro-n-hexyl-oligoethyleneoxy-alcohols and phospholipids. These fluorocarbon nanoemulsions resolve key issues faced by conventional fluorocarbons-based artificial oxygen carriers, including bioaccumulation of metabolites and degradation products. These undesirable impurities form from fluorosurfactants with perfluoroalkyl groups of more than 6 fully fluorinated carbons and have led to continued setbacks in light of the stringent regulatory standards. Fluorocarbon nanoemulsions of the invention derive a significant benefit from using a shorter perfluorocarbon chain of the surfactant because excretion and metabolism products do not bioaccumulate as do those with larger perfluoroalkyl moieties.
• the invention generally relates to a composition of a tluorocarbon nanoemulsion that includes a fluorocarbon rangin from about 4 to about 8 carbons; and one or more surfactants selected from perfluoro-n-hexyl-oligoethyleneoxy-alcohols and phospholipids.
• the fluorocarbon comprises perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, or a mixture of two of more thereof.
• the fluorocarbon comprises perfluoropentane.
• the one or more surfactants comprise a perfluoro-n-hexyl- oligoethyleneoxy-alcohol and/or a mixture of three phospholipids.
• the perfluoro-n-hexyl-oligoethyleneoxy-alcohol comprises one or more of:
• n 5 and q is an integer from 1 to about 50.
• the perfluoro-n-hexyl-oligoethyleneoxy-alcohol is N-(2-aminoethyl)-2-aminoethyl-oligoethyleneoxy-alcohol
• n is 5, and q is an integer from 1 to about 16 (e.g., from about 3 to about 16, , from about 6 to about 16, from about 10 to about 16, from about 3 to about 10).
• Fluorocarbon may account for any suitable weight percentage in the nanoemulsion, for example, from about 1% to about 50% (e.g., from about 1% to about 40%, from about 1% to about 30%, from about 1% to about 20%, from about 1% to about 10%, from about 1% to about 5%).
• Perfluoro-n-hexyl-oligoethyleneoxy-alcohol may account for any suitable weight percentage in the nanoemulsion, for example, from about 0.10% to about 7.5% (e.g., from about 0.10% to about 5%, from about 0.10% to about 4%, from about 0.10% to about 3%, from about 0.10% to about 1.5%).
• the phospholipids have any suitable carbon chain length, for example, ranging from about 12 carbons to about 18 carbons (e.g., 12, 13, 14, 15, 16, 17, 18) in length.
• the phospholipids may account for any suitable weight percentage in the nanoemulsion, for example, from about 0.10% to about 7.5% (e.g., from about 0.10% to about 5%, from about 0.10% to about 4%, from about 0.10% to about 3%, from about 0.10% to about 1.5%).
• exemplary phospholipids and relative amounts there of may be, for example, from about 75 to about 87 mole % phosphatidylcholine, about 5 to about 15 mole % phosphatidylethanolamine and about 3 to about 20 mole % phosphatidylethanolamine-MPEG, wherein "MPEG” refers to a PEG group having a terminus methoxy group.
• the MPEG herein may have a molecular weight from about 350 to about 5,000 (e.g., from about 350 to about 4,000, from about 350 to about 3,000, from about 350 to about 2,000, from about 500 to about 5,000, from about 1,000 to about 5,000, from about 1,500 to about 5,000, from about 2,000 to about 5,000, from about 3,000 to about 5,000, from about 4,000 to about 5,000).
• Phosphatidylethanolamine-PEG where the oligoethyleneoxy portion of the molecule is terminated with a hydroxyl group as opposed to the methoxy terminus present in MPEG phospholipids can be substituted for the phosphatidylethanolamine-MPEG in the formulation.
• Combinations of phosphatidylethanolamine-MPEG and phosphatidylethanolamine-PEG may also be employed in any relative ratio, as the oligoethyleneoxy-bearing phospholipid component of these formulations.
• exemplary phospholipids and relative amounts there of may be, for example, from about 80 to about 85 mole % phosphatidylcholine, about 8 to about 13 mole % phosphatidylethanolamine and about 6 to about 11 mole % phosphatidylethanolamine-MPEG (or phosphatidylethanolamine-PEG).
• the phosphatidylethanolamine includes a PEG group with a molecular weight from about 350 to about 5,000 (e.g., from about 350 to about 4,000, from about 350 to about 3,000, from about 350 to about 2,000, from about 500 to about 5,000, from about 1,000 to about 5,000, from about 1,500 to about 5,000, from about 2,000 to about 5,000, from about 3,000 to about 5,000, from about 4,000 to about 5,000).
• compositions of the invention include PEG Telomer B (PTB) a custom purified medical grade of DuPont Zonyl FS-100 or DuPont FSO.
• compositions of the invention include perfluoro-n-hexyl-oligoethyleneoxy-alcohol.
• a particular form of perfluoro-n-hexyloligoethyleneoxy-alcohol is a fluorosurfactant product known as DuPont Capstone FS-3100 and, in certain embodiments, compositions of the invention include comprises that material or a custom refined version of that material.
• compositions of the invention include tetradecafluoro-n-hexane (TDFH).
• compositions of the invention include tetradecafluorohexane that may consist of a mixture of 2 or more of its possible structural isomers present in any proportions.
• compositions of the invention include dodecafluoro-n-pentane (DDFP).
• DDFP dodecafluoro-n-pentane
• compositions of the invention include dodecafluoropentane that may consist of a mixture of 2 or more of its possible structural isomers present in any proportions.
• compositions of the invention include one or more of
• dodecafluoro-n-pentane l,2-dipalmitoyl-OT-glycero-3-phosphatidylcholine, 1 ,2-palmitoyl-OT- glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-5000] salts such as the sodium salt, and l,2-dipalmitoyl-OT-glycero-3-phosphoethanolamine.
• compositions of the invention include one or more of 1 ,2- dimyristoyl-OT-glycero-3-phosphatidylcholine, l,2-dimyristoyl-OT-glycero-3-phosphoethanolamine- N-[methoxy(poly ethylene glycol)-2000] salts such as the sodium salt, and 1,2-dimyristoyl-OT- glycero-3-phosphoethanolamine.
• compositions of the invention include one or more of 1,2- didodecanoyl-sft-glycero-3-phosphatidylcholine, 1,2- didodecanoyl -OT-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] sodium salt, and 1,2- didodecanoyl- sft-glycero-3-phosphoethanolamine.
• step (110) the method prepares about a 6% wt/volume mixture of a fluorosurfactant in water for injection (WFI) using a magnetic stirrer in a vessel, disposed in a 2-5°C bath for a period of between 15 and 60 minutes, while the solution temperature is monitored and maintained at a temperature of 2-5°C.
• WFI water for injection
• the fluorocarbon of step (110) comprises tetradecafluoro-n- hexane. In certain embodiments, the fluorocarbon of step (110) comprises tetradecafluorohexane which may be a mixture of 2 or more structural isomers. In certain embodiments, the fluorocarbon of step (110) comprises dodecafluoro-n-pentane. In certain embodiments, the fluorocarbon of step (110) comprises dodecafluoropentane which may be a mixture of 2 or more structural isomers.
• the fluorocarbon of step (110) consists of tetradecafluoro-n- hexane. In certain embodiments, the fluorocarbon of step (110) consists of tetradecafluorohexane. In certain embodiments, the fluorocarbon of step (110) consists of dodecafluoro-n-pentane. In certain embodiments, the fluorocarbon of step (110) consists of dodecafluoropentane.
• step (110) method of the invention then quickly adds an aliquot of cold (0-4°C) perfluorocarbon to the vessel, closes a vessel cap, and then stirs the resulting first mixture for about 1 hour.
• the fluorocarbon surfactant of step (110) comprises Peg Telomer B (PTB), compound 21,
• the fluorocarbon surfactant of step (110) comprises peril uoro-n- hexyl-oligoethyleneoxy-alcohol.
• the fluorocarbon surfactant of step (1 10) consists of PEG Telomer B that is the mixture of compounds denoted as 21 above.
• the fluorocarbon surfactant of step (110) consists of perfluoro-n-hexyl- oligoethyleneoxy-alcohol.
• the fluorosurfactant of step (110) is perfluoro-n-hexyl- oligoethyleneoxy-alcohol surfactant 17, which has an invariant perfluoro-n- hexyl moiety 18, in combination with a variable ethylene oxide moiety 19.
• the fluorosurfactant of step (110) comprises a perfluoro-n-alkyl- oligoethyleneoxy-alcohol 20 wherein y is greater than 6. In certain embodiments, the fluorosurfactant of step (110) comprises compound 20 wherein y is 7, and wherein n is greater than or equal to 1 and less than or equal to 16.
• the fluorosurfactant of step (110) comprises a perfluoro-n-alkyl- oligoethyleneoxy-alcohol 20a wherein y is 6, and wherein n is greater than or equal to 1 and less than or equal to 16 (e.g., DuPont Capstone FS-3100 fluorosurfactant).
• compound 20a can be used as received from the manufacturer.
• compound 20a can be subjected to a custom refinement procedure or purification procedure before use.
• the fluorosurfactant of step (110) comprises compound 20b wherein y is 5, and wherein n is greater than or equal to 1 and less than or equal to 16.
• the fluorosurfactant of step (110) comprises compound 1. In certain embodiments, the fluorosurfactant of step (110) comprises compound 2. In certain embodiments, the fluorosurfactant of step (110) comprises compound 3. In certain embodiments, the fluorosurfactant of step (110) comprises compound 4. In certain embodiments, the fluorosurfactant of step (110) comprises compound 5. In certain embodiments, the fluorosurfactant of step (110) comprises compound 6. In certain embodiments, the fluorosurfactant of step (110) comprises compound 7. In certain embodiments, the fluorosurfactant of step 110 comprises compound 8. In certain embodiments, the fluorosurfactant of step (110) comprises compound 9. In certain embodiments, the fluorosurfactant of step (110) comprises compound 10. In certain embodiments, the fluorosurfactant of step (110) comprises compound 11. In certain embodiments, the fluorosurfactant of step (110) comprises compound 2. In certain embodiments, the fluorosurfactant of step (110) comprises compound 3. In certain embodiments, the fluorosurfactant of
• fluorosurfactant of step (110) comprises compound 12. In certain embodiments, the fluorosurfactant of step (110) comprises compound 13. In certain embodiments, the fluorosurfactant of step (110) comprises compound 14. In certain embodiments, the fluorosurfactant of step (110) comprises compound 15. In certain embodiments, the fluorosurfactant of step (110) comprises compound 16.
• the fluorosurfactant consists of mixtures of compounds 1 to 16 which may contain any combination of those compounds and may omit any of those compounds singly or selected members of the set of compounds from 1 to 16.
• fluorocarbon surfactants comprising perfluoro-moiety 18 in combination with different foaming and wetting properties are used to form perfluorocarbon nanoemulsions of the invention.
• other anionic surfactants based on sulfonic acids DuPont FS-10) or phosphoric acid esters (DuPont FS-61, FS63, FS-64); zwitterionic and amphoteric surfactants (DuPont FS-50 and DuPont FS-51 respectively); nonionic surfactants, based on perfluoroalkylated polyethyleneoxy alcohols, such as DuPont FS-30, DuPont FS-31, DuPont FS- 3100, DuPont FS34 and DuPont FS-35; and etc. can be utilized.
• fluorocarbon-compound comprising moiety 18 in combination with differing length ethylene oxide moieties 19 are utilized to form perfluorocarbon nanoemulsions of the invention.
• n is between about 3 and about 6. In other embodiments, n is greater than or equal to 1 and less than or equal to about 16 to form stable nanoemulsions with low molecular weight perfluorocarbons, such as, dodecafluoropentane (DDFP), tetradecafluorohexane (TDFH), hexadecafluoroheptane (HDFH), and octadecafluorooctane (ODFO).
• DDFP dodecafluoropentane
• TDFH tetradecafluorohexane
• HDFH hexadecafluoroheptane
• ODFO octadecafluorooctane
• pegylated perfluoroalkyloligoethyleneoxy alcohol surfactants of the form shown as compound 22 are provided.
• m is an integer from 1 to about 50 (e.g., 1 to about 25, 1 to about 12, 1 to about 6, 6 to about 50, 12 to about 50, 25 to about 50)
• n is an integer from 1 to about 50 (e.g., 1 to about 25, 1 to about 12, 1 to about 6, 6 to about 50, 12 to about 50, 25 to about 50)
• q is an integer from 1 to about 50 (e.g., 1 to about 25, 1 to about 12, 1 to about 6, 6 to about 50, 12 to about 50, 25 to about 50) are utilized to form perfluorocarbon nanoemulsions disclosed herein.
• a class of compounds that can be employed is selected from a subset of compound 22 shown below:
• a class of compounds that can be employed is selected from a subset of compound 22 shown below:
• individual members of the group of compounds or any combination of the individual members of the group of compounds represented as compound 22 are utilized as the fluorocarbon surfactant employed to form perfluorocarbon nanoemulsions disclosed herein.
• the mixture of compounds represented as structure 22 can be subjected to a custom refinement or purification protocol before being utilized to form
• any individual molecular species or combinations of individual members of the group of compounds represented by structure 22 can be subjected to a custom refinement or purification protocol before being utilized to form perfluorocarbon nanoemulsions disclosed herein.
• an Ultra Turrax or similar dispersing apparatus can replace the magnetic stirring system to generate an initial coarse emulsion.
• the Ultra Turrax has a high-speed stirring motor (1,000-25,000 rpm), to which a dispersing element is attached to via a shaft.
• the coarse emulsion is homogenized into a fine emulsion comprising mean particle diameters of less than or equal to 400 nm and a 99% cumulative distribution of which is less than or equal to 900 nm.
• a homogenizer (Avestin model C5) is used. In another embodiment, a homogenizer (Avestin model C50) is used. In another embodiment a Kirkland Products hand-held homogenizer with or without a pneumatic assist device is used. In certain embodiments, the homogenization pressure ranges from 1,000 psi to 14,000 psi. Further, in other embodiments, the homogenizer is used in continuous homogenization mode or in discrete homogenization mode. The number of discrete passes or the time for continuous homogenization can be adjusted to ensure achieving the fine emulsion.
• step (180) after the homogenization of the coarse emulsion to form a fine emulsion is completed, the resulting fine emulsion is transferred to another vessel containing a cooled (0-10°C) stirred continuous phase consisting of WFI.
• the continuous phase optionally contains a buffering agent, a viscogen, excipients, and preservatives (microbiocidal agents) to inhibit growth of any adventitiously introduced microbial species.
• the transfer may be conducted by passing the emulsion from the first vessel through the homogenizer and into the second vessel with the homogenizer optionally pressurized.
• the volume of the dispersed phase is between 1 and 100-fold of the emulsion transferred.
• the fine emulsion is stirred for 10-30 minutes under nitrogen pressure (2-10 psi) to insure that the fine emulsion is homogeneously distributed throughout the continuous phase.
• step (180) the method injects the fine emulsion into a sucrose solution disposed in a first pressure vessel (PV1) to form a second mixture.
• step (210) the method pressurizes the first pressure vessel (PV1) while stirring the second mixture of step (210).
• steps (220) and (230) the method transfers the second mixture into a second pressure vessel (PV2) using the homogenizer.
• the method connects the second pressure vessel (PV2) to the third pressure vessel (PV3) via a 0.8/0/2 micron filter and then pressurizes the second pressure vessel to filter and sterilize the second mixture, and to transfer that second mixture to a third pressure vessel.
• the filtration step utilizes a syringe membrane filter, cartridge membrane filter, or a capsule membrane filter, or any other membranes that are made of materials compatible with the medium and are capable of removing particulate matter, including microbial entities, such as bacteria, mold, mold spores, and fungus contaminants, as small as 0.2 micron or even 0.1 micron.
• the membrane material can be Supor® (polyethersulfone) membrane from Pall Sciences, the Pall EKV® filter series membrane, or Pall Sciences GHP Polypro® membrane.
• the method stirs the resulting sterilized second mixture under a light pressure of nitrogen to insure homogeneous dispersion of the filtered solution.
• the method further transfers the filtered second mixture into capped and crimped vials using a peristaltic pump, a metering pump, a gear pump, or other suitable fluid transfer apparatus. Precautions to avoid microbial contamination during all of these operations are taken and such precautions for aseptic filling operations are known to the person having ordinary skill in the art.
• the nanoemulsions comprising a fluorocarbon surfactant 20a or 21 for example can be prepared by sonication of the combined components.
• DDFP as the neat liquid
• phosphate-buffered aqueous sucrose as a solution
• compound 20a surfactant as the neat liquid or premixed with the aqueous phosphate buffered sucrose solution
• step (610) the method prepares an aqueous solution of phosphate-buffered sucrose and fluorosurfactant compound 21 (Peg Telomer B).
• step (620) the method charges a plurality of vials with the solution of step (610) and places stoppers over the mouth of each vial.
• step (630) the method places the vials in a chilled environment that brings the vials to a temperature such that when the perfluorocarbon is added to the solution of the vial there is expected no significant amount of evaporation of the perfluorocarbon.
• step (640) the fluorocarbon is added to each vial followed by immediate crimp capping of each vial.
• step (650) the method subjects each vial to vortexing followed by sonication to generate perfluorocarbon nanoemulsions of the invention.
• step (650) the components are initially mixed in the stoppered crimp capped vial by shaking or vortexing to create a coarse emulsion whose particle size distribution may range from about 200 nanometers to as high as 15 microns.
• the particle dimension sizes are illustrative and the person having ordinary skill in the art are aware that the dimensions in actual practice could fall below or above the given range.
• Method of the invention then sonicates the vial, for example, in an ultrasonic cleaning bath such as a VWR Aquasonic 75HT unit, which provides a sonication frequency of about 40 KHz and a total power output of about 75-80 watts for a period of time between 1 second and 1 hour to form a desired particle-size distribution of the nanoparticle emulsion.
• an ultrasonic cleaning bath such as a VWR Aquasonic 75HT unit, which provides a sonication frequency of about 40 KHz and a total power output of about 75-80 watts for a period of time between 1 second and 1 hour to form a desired particle-size distribution of the nanoparticle emulsion.
• one or more phospholipids are employed as an emulsifying surfactant system.
• the method prepares a first mixture of water and glycerol containing monobasic and dibasic phosphate buffer which is stirred at a temperature between ambient and 100°C.
• phospholipids are added to the first mixture as solid material or as a solution in a suitable solvent at a temperature between ambient and 100°C compatible with the process to form a second mixture.
• the second mixture is filtered through a 0.2 micron filter into a second vessel.
• the filtered solution is allowed to cool to ambient temperature if the temperature of its preparation exceeded ambient.
• step (550) the method transfers the solution of step (540) into a plurality of vials and affixes stoppers to the vials.
• step (560) the chosen fluorocarbon to be emulsified is added to the vials and the vials are immediately crimp capped.
• step (570) the method vortexes and then sonicates said vials of step (560) for a time between 1 second and 1 hour in order to provide the fluorocarbon emulsion.
• the sonication time may be adjusted to provide particle size distributions of desired sizes which may be optimal for specific applications.
• the one or more phospholipids are saturated, partially saturated, or fully unsaturated.
• a first nanoemulsion is formed using: DPPC (16:0), DPPE-MPEG-2000, or DPPE-MPEG5000, and optionally DPPE (16:0).
• a second nanoemulsion is formed using Egg yolk phospholipids singly or in combination with other added phospholipids or derivatized phospholipids.
• a third nanoemulsion is formed using 16:0-18: 1 PC, 16:0-18: 1 PE and 16:0-18: 1 PE-MPEG2000, or 16:0-18: 1 PE-MPEG5000.
• a fourth nanoemulsion is formed using 16:0-18: 1 PC, 16:0-18: 1 PE and 16:0-18: 1 PE-MPEG2000, or 16:0-18: 1 PE-MPEG5000.
• nanoemulsion is formed using DPPC (16: 1), DPPE-MPEG-2000 or DPPE-MPEG-5000, and optionally DPPE (16: 1).
• a fifth nanoemulsion is formed using DMPC, DMPE and DMPE- MPEG2000, or DMPE-MPEG5000.
• a sixth nanoemulsion is formed using 14: 1 (A9-Cis) PC, 14: 1 (A9-Cis) PE, 14: 1 (A9-Cis) MPEG2000, or 14: 1 (A9-Cis) MPEG5000.
• a sixth nanoemulsion is formed using; 14: 1 (A9-trans) PC, 14: 1 (A9-trans) PE, 14: 1 (A9-trans) MPEG2000, or 14: 1 ( ⁇ 9- trans) MPEG5000.
• a seventh nanoemulsion is formed using DLPC (12:0), DLPE-MPEG-2000 or DLPE-MPEG-5000, DLPE(12:0).
• the relative proportions of the phospholipids components described herein can be varied to optimize the formulation with respect to solubility, emulsion stability, and oxygen uptake and release kinetics.
• the first nanoemulsion can be formulated in a mole ratio of 82 DPPC (16:0), 8 DPPE (16:0)-MPEG-2000, and 10 DPPE (16:0) in a solution with water-propylene glycol-glycerol 85/10/5 v/v/v and a total lipids concentration of 0.75 mg/mL to as high as 50 mg/ml.
• the relative amounts of the propylene glycol and glycerol can be increased relative to water.
• the phospholipid components that can be employed are not limited to those described in the examples cited above. Certain formulations may require phospholipids mixtures consisting of a given fraction fully saturated phospholipids combined with a given fraction of their unsaturated congeners. The adjustments and tuning of properties, such as the gel to liquid crystal phase transition temperature, are well understood by the person having ordinary skill in the art.
• the addition of phospholipids with head groups that are homologous to those mentioned in the set of examples may be required.
• the relative proportions of the components in systems that employ MPEG2000 can be different by needing a larger proportion of MPEG phospholipids compared to those with a choline head group. This also applies wherein PEGylated phospholipids or
• choline type head groups may contain larger or smaller alkyl groups, or less than three alkyl groups.
• phospholipids with cationic moieties in their head groups such as diacyl phosphatidylethanolamine phospholipids
• small molecule substances can be employed as viscogen excipients, which can inhibit settling (inverse creaming) of the nanoparticulate fluorocarbon emulsion and adjust the overall density, viscosity, and tonicity of the solution to approximate that of blood into which the product solution is injected.
• substances such as propylene glycol, glycerin, and sugar alcohols, such as sorbitol, xylitol, mannitol, and erythritol can be used as viscogens.
• other polyhydroxy compounds such as mono-, di- or trisaccharides having appropriate solubility in the medium can serve as viscogens as well.
• Examples are fructose, glucose, xylose, sucrose, trehalose, raffinose, stachyose, alginates, cyclodextrins, substituted cyclodextrins, and dextrans.
• straight chain or multiarmed polyethylene glycols such as PEG300, PEG400, PEG600 and higher molecular weight PEGs up to MW 10,000 , can also be employed as viscogens.
• those skilled in the medical arts need to control the injection rate of the solution of the nanoemulsion to minimize the effects of tonicity mismatch between the solution of nanoemulsion and blood.
• the injected nanoemulsion needs not be isotonic.
• the said injected solution of nanoemulsion can be hypotonic or hypertonic so long as the degree of deviation from isotonicity does not result in discomfort to the patient or injury to tissue beyond transient effects.
• the said nanoemulsion can be injected into a larger volume of a diluent such as 0.9% saline optionally containing other components such as a phosphate buffer as employed for preparation of phosphate buffered saline.
• a diluent such as 0.9% saline
• other components such as a phosphate buffer as employed for preparation of phosphate buffered saline.
• buffers other than sodium phosphate buffering systems may be employed to maintain the pH of the said nanoemulsions.
• Said buffers can be salts or combinations of the free acid form and salt form of, for example, acetic acid, arginine, aspartic acid, benzoic acid, carbonic acid, citric acid, gluconic acid, gluconic lactone, glycine, histidine, lysine, meglumine, phosphoric acid, or tromethamine; wherein acid salts are part of the buffering system.
• the counterions are generally sodium, meglumine, or other cations that biochemically compatible and allowed for use in parenterals.
• a chelating agent such as disodium EDTA can be used to sequester amounts of oxidizing metal ions such as Fe 3+ in order to protect nanoemulsions containing unsaturated phospholipids.
• other antioxidant excipients such as acetone sodium bisulfate, argon 100% in the headspace, ascorbyl palmitate, ascorbate (sodium/acid), bisulfite sodium, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), cysteine/cysteinate HC1, dithionite sodium (Nahydrosulfite, Nasulfoxylate), gentisic acid, gentisic acid ethanolamine, glutathione, formaldehyde sulfoxylate, sodium metabisulfite, potassium metabisulfite, methionine, nitrogenl00% (in the headspace), propylgallate, sulfite sodium, tocopherol alpha
• antimicrobials such as benzalkonium chloride, benzyl alcohol, benzoic acid, chlorobutanol, m-cresol, myristyl gammapicolinium chloride, paraben methyl, paraben propyl, or Thimerosal in amounts that may range from 0.005% to 5% w/v depending on which of these is employed, can be added to the formulations.
• PVs stainless steel pressure vessels
• All process flow paths between PVs were staged with 1/4 inch I.D. flexible nylon tubing.
• the ends of the tubing lines were fitted with mini-1/2 inch sanitary triclamp fittings that were chosen to smoothly and aseptically connect flow paths between the homogenizer and the PVs.
• the process temperature was controlled at 4 - 6° C and the pressure in the vessel headspace was controlled at 5-7 psi using compressed nitrogen.
• the emulsion was recirculated (continuous homogenization) through the homogenizing valve for 6 passes (effectively) at 14,000 psi and then immediately filtered through a 0.2 ⁇ sterile filtration capsule.
• a Unispence ® (Wheaton, Millville, NJ) filling machine, the resulting product was filled into 5 mL vials that were promptly stoppered by hand and crimped with a pneumatic Power Crimper (Kebby Industries).
• the particle size distribution of the emulsion was assessed in triplicate at 0, 1, 2, 3 weeks and at 1, 2, 4, 6 and 11 months. At each time-point, 3 vials were selected at random and analyzed by dynamic light scattering using a PSS Nicomp 380 DLS submicron particle sizer (Particle Sizing Systems, Port Richie, FL). In order to minimize the effect of gradual temperature increase during size determinations, the sample temperature was controlled at 19° C.
• the average particle size which is given as the intensity weighted mean diameter (IWMD), did not exceed 260 nm. Furthermore, 99% of the particles were measured to have diameter less than or equal to 400 nm throughout the study. Less than 0.8% of the total volume of particles consisted of particles between 0.5 ⁇ and 50 ⁇ .
• IWMD intensity weighted mean diameter
• a 6% solution of custom purified medical grade compound 21 (J. Tech Sales, Boca Raton, FL) was prepared by combining a 1.2 g aliquot of the compound 21, wherein y is 5 and wherein n is 1 through 16, diluting to the mark of a premeasured 20 mL volume in a glass scintillation vial with water for injection (HyPure ® from Hyclone), and stirring at 5°C for 0.5 hr using a small cross-shaped magnetic stirrer. The solution was stored in the refrigerator at 4°C.
• the syringe cooling tank and the PV1-PV3 cooling tank were charged with ice-water.
• the pressure vessels containing floating magnetic stirrers were fitted with their caps, each of which bears a SwagelokTM bottom-entry two- position instrument ball valve attached to a 0.25" OD 316SS dip tube (for introduction and withdrawal of product from the vessels) and a three-way three-position stop cock which serves as a nitrogen inlet and a venting system.
• the PV1 with cap and large cross-shaped stirrer was charged with the solution of phosphate-buffered sucrose (216.8 mL) and placed into the PV1-PV3 cooling tank and stirring was initiated.
• PV2 was placed in the tank and by appropriate placement in the proximity of PV1 so that both stirrers were in motion.
• PV3 was placed in an ice bath for cooling prior to receipt of product from PV2.
• a 0.8/0.2 micron Pall AcrodiscTM (Supor membrane) was readied for attachment to the instrument valve inlet of PV3.
• a Kirkland products hand-held homogenizer (“homogenizer”) was purged of resident WFI and then rinsed with cold WFI (25 mL). Cold-jacketed 25 mL glass Per Stammum MicroMateTM syringes with glass plungers were attached to the input and output Luer lock fittings of the homogenizer. The receiving syringe was fitted with a two-way stopcock with Luer fittings. The input gas line (80 psi) was connected to the pneumatic piston unit of the homogenizer. At completion of the stirring period for the primary emulsion, the output syringe was removed from the homogenizer and fitted with a 17-gauge needle.
• the primary emulsion was withdrawn into the syringe, then the needle was removed and the syringe was rapidly affixed to the input side of the homogenizer.
• a BioRad Econopump peristaltic pump was employed to pump ice water through the syringes in series from the syringe bath cooling tank.
• the peristaltic pump was fitted with two lengths of 1/8" diameter Pharmed ® tubing which were joined at the input and output sides using Y connectors in order to access a total flow rate of 38 mL/min.
• the coolant lines from the peristaltic pump were attached in series to the output syringe and the input syringe and the circulation of the coolant was initiated.
• the bypass valve of the homogenizer was opened and the primary emulsion was transferred several times between the output syringe and the input syringe by alternate depression of the syringe plungers; the final position of the primary emulsion was in the input syringe.
• the bypass valve was closed and the pneumatic unit operation was initiated at a pumping rate of -30 mL/min.
• the bypass valve was rapidly opened, the plunger of the output syringe was depressed to quickly transfer the homogenized primary emulsion back to the input syringe, the bypass valve was closed and the second pass was begun. In this manner the primary emulsion was subjected to 20 discrete passes through the homogenizer. At the end of this procedure the primary emulsion was the output syringe.
• the output syringe was disconnected from the homogenizer outlet and the needle put about 2 inches below the surface of the stirred solution in PVl.
• the primary emulsion was injected from the syringe into PVl.
• PVl was pressurized with nitrogen (-8-10 psi) and the mixture was stirred for about 5 minutes during which time 1/16" tubing (braided silicone-platinum cured) was connected from the dip tube stopcock of PVl to the input side of the homogenizer and from the PV2 dip tube stopcock to the output side of the homogenizer.
• the nitrogen pressure was maintained in PV1 and PV2 was vented to atmosphere.
• the dip tube ball valve of PV2 was opened followed by that of PV1 with simultaneous starting of the homogenizer.
• the filtered solution in PV3 was transferred to a crystallization dish containing ice water, stabilized with a clamp, pressurized to about 8 psi with nitrogen and the solution within was stirred for -15 min. After this time two Wheaton vial trays with 50 nominal 2 mL serum vials (total capacity 3 mL) with gray butyl rubber stoppers were removed from the refrigerator. A 1/16" i.d. braided silicone tube fitted with a 12 gauge SS tube on its output side was connected to the output of the instrument valve on PV3. The pressure in PV3 was adjusted to about 5 psi and the dip tube stopcock was opened to the tubing. The vials were filled (in groups of 10) by removing the notched stopper from the vial, opening and closing the stopcock at the distal end of the tubing attached to PV3, and replacement of the notched stopper.
• Submicron particle sizing data using a PSS Nicomp 380 DLS Submicron Particle Sizer (Particle Sizing Systems, Port Richie, Fl) obtained immediately post preparation is displayed in Table 1.
• the average particle size of the TDFH-compound 21 nanoemulsion measured in IWMD does not vary and remains well within the specification for release. Furthermore, 99% of the particles were measured to have diameter less than or equal to 400 nm throughout the study.
• Particle size data in the region 0.5 to 50 microns was obtained using a PSS 780 SIS light obscuration instrument (Particle Sizing Systems, Port Richie, FL) and is displayed in Table 2. Less than 0.8% of the total volume of particles consisted of particles between 0.5 ⁇ and 50 ⁇ and major volume of particles consisted of particles smaller than threshold of 0.5 micron.
• TDFH Tetradecafluoro-n-hexane
• a 6% solution of compound 20a (J. Tech Sales, Boca Raton, FL) (20 mL) was prepared by adding a 1.2 g aliquot of the surfactant, diluting to the mark of a premeasured 20 mL volume in a glass scintillation vial with water for injection (HyPure from Hy clone), and stirring at 5 °C for 0.5 hr using a small cross-shaped magnetic stirrer. The solution was clear at 5 °C and was stored in the refrigerator (3 °C). This solution can be stored and employed for different experiments.
• PV1 was charged withlO mM phosphate-buffered (pH 7) sucrose (32% wt/vol sucrose, 104 mL) and placed into the PV1-PV3 cooling tank and stirring was initiated. PV2 and PV3 were placed in the tank as well and stirring was initiated.
• a homogenizer fitted to a pneumatic pumping unit was purged of resident WFI and then rinsed with cold WFI (25 mL).
• Cold-jacketed 10 mL glass Popper and Sons syringes with glass plungers were attached to the input and output Luer lock fittings of the homogenizer.
• the bypass valve of the homogenizer was opened and the primary emulsion was transferred several times between the output syringe and the input syringe by alternate depression of the syringe plungers, wherein the final position of the primary emulsion was in the input syringe.
• the bypass valve was closed and the pneumatic unit operation was initiated at an approximate pumping rate of 30 mL/min.
• the bypass valve was rapidly opened, then the plunger of the output syringe was depressed to quickly transfer the homogenized primary emulsion back to the input syringe, then the bypass valve was closed, and the second pass was begun. In this manner the primary emulsion was subjected to 20 discrete passes through the homogenizer. The primary emulsion was in the output syringe at the end of this operation.
• the output syringe was disconnected from the homogenizer outlet, attached to PVl and the primary emulsion was injected from the syringe into PVl .
• PVl was pressurized with nitrogen (-8-10 psi) and the mixture was stirred for about 5 minutes. The nitrogen pressure was maintained in PVl and PV2 was vented to atmosphere. The transfer of the stirred solution of the primary emulsion mixed with the 32% sucrose solution in PVl to PV2 was accomplished in a single pass through the homogenizer. Then a Pall Sciences 32 mm 0.8/0.2 micron Acrodisc syringe filter was attached between PV2 and PV3.
• PV2 was pressurized with nitrogen at -12 psi and PV3 was vented to the atmosphere to initiate filtration/transfer of the solution from PV2 to PV3. After -5 min the pressure was increased to 15 psi and after about 10 min up to 25 psi. The filtration required about 30-40 min to complete and the transfer was nearly quantitative.
• Particle size analysis of the product immediately post preparation was carried out using a Nicomp 380 DLS submicron particle sizer (Particle Sizing Systems, Port Richie, FL) on 5 vials of material selected from across the entire range of filled vials.
• the average particle size of the TDFH-compound 20a nanoemulsion, measured in IWMD, is about 216 nm and does not vary much among different samples. Further, the average 99% cumulative distribution of the TDFH-compound 20a nanoemulsion is less than 318 nm.
• Nanoparticle emulsion of DDFP and Compound 20a prepared by high-pressure homogenization
• Nanoparticle emulsion of DDFP and Compound 20a prepared by vortexing followed by sonication
• the three-way stopcock was positioned to allow aspiration of solution from the vessel into the syringe until a volume of 7.8 mL was loaded into the syringe. Then the position of the stopcock was set to allow delivery of solution from the syringe through the 18 gauge needle.
• a tray of 25 nominal 5 mL capacity (9 mL total capacity) serum vials (purged with dry nitrogen gas, fitted with halobutyl stoppers, and kept in a 4 °C refrigerator for 1 h) was charged with 7.8 mL aliquots of the solution by lifting the stopper slightly, positioning the needle tip in the mouth of the vial, and depressing the syringe plunger followed by rapid replacement of the stopper.
• each vial was quickly charged with cold ( ⁇ 0°C) DDFP (0.156 g, 0.089 mL) using a Becton and Dickinson 0.5 mL Lo Dose U-100 insulin syringe and immediately stoppered and crimp was capped.
• a vial was vortexed at 4500 rpm for 1 min upright, 1 min inverted, and 1 min upright. Then the vial was positioned up to the neck in the center of a VWR Aquasonic 75HT ultrasonic cleaning bath and sonicated for 5 minutes. A second vial was processed in the same manner and the particle size and distribution were analyzed using the PSS Nicomp 380 DLS submicron particle sizer (Particle Sizing Systems, Port Richie, FL). Table 11. Submicron Particle Sizing of DDFP - Compound 20a Nanoparticle Nanoemulsion Prepared by Vortexing and Sonication Using the Nicomp 380 DLS Submicron Particle Sizer
• the formulation is designed to imbibe oxygen into the peril uorocarbon in oxygen rich regions and to release oxygen upon transport to tissues where there is an oxygen deficit resulting in low oxygen tension (hypoxic tissue).
• hypoxic tissue or tissues wherein the dissolved oxygen concentration is lower than that in the nanoparticles, oxygen is released from the
• the propylene glycol solution of phospholipids was added dropwise over a period of about 2 min to a stirred mixture of water / glycerol 95/5 v/v (200.7 mL) containing monobasic sodium phosphate » H 2 0 (173.9 mg) and anhydrous dibasic sodium phosphate (137.87 mg) at 55°C.
• the solution was stirred for 5 min after the addition of the glycerol solution of the phospholipids.
• the solution was immediately push-filtered through a 32 mm Pall Sciences GH Polypro ® 0.2 micron filter into a 250 mL bottle which is then immediately purged with dry ultrapure nitrogen. The solution was allowed to cool to ambient temperature.
• VWMD Volume weighted mean diameter
• NWMD Number weighted mean diameter
• the propylene glycol solution of phospholipids was added dropwise over a period of about 2 min into a stirred mixture of water / glycerol 95/5 v/v (200.7 mL) containing monobasic sodium phosphate » H 2 0 (173.9 mg) and anhydrous dibasic sodium phosphate (137.87 mg) at 55 °C.
• the solution was stirred for 5 min after the addition of the glycerol solution of the phospholipids.
• the solution was immediately push- filtered through a 32 mm Pall Sciences GH Polypro ® 0.2 micron filter into a 250 mL bottle which was then immediately purged with nitrogen. The solution was allowed to cool to ambient temperature.
• the formulation was evaluated for its ability to imbibe oxygen from an oxygen rich environment as described for the evaluation of the formulation of Example 5.
• a clear decrease in oxygen level over the measurement period is shown in graphical form in FIG. 9.
• the corresponding graph using a saline control displayed no decrease in dissolved oxygen level over the same period.
• a clear decrease in oxygen level over the measurement period is shown in graphical form in FIG. 10.
• the corresponding graph using a saline control displayed no decrease in dissolved oxygen level over the same period.
• a 22 mL aliquot of propylene glycol in a 50 mL beaker is heated to 55 °C with stirring.
• l,2-Dimyristoyl-OT-glycero-3-phosphatidylcholine (677.9 mg)
• l,2-dimyristoyl-OT-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] sodium salt 174 mg
• 1,2- dimyristoyl-OT-glycero-3-phosphoethanolamine 50.95 mg
• the solution is allowed to cool to ambient temperature followed by addition of water for injection (20 mL) cooling to 10 °C and addition of cold DDFP (4.51 g, 2.61 mL) and stirring rapidly for 30 min at 10 °C.
• the resulting material is subjected to discrete homogenization using a Kirkland products hand held homogenizer at 5 °C for a total of 20 passes.
• This material is then added to a solution of glycerol in water for injection.
• the nanoemulsion is added to a stirred solution of 205.4 mL of 30% sucrose and stirred for 10 min under a nitrogen atmosphere (5 psi).
• the solution is transferred via the homogenizer to a second vessel kept at 2-5 °C after which it is stirred for 15 minutes and filtered through a Pall Sciences 0.8/0.2 micron Acropak 200 filter into a third vessel at 2-5 °C and stirred for 15 minutes at that temperature after transfer.
• the material is then filled into nominal 5 mL Wheaton vials, stoppered with gray halobutyl rubber stoppers and crimp capped and stored.
• a 22 mL aliquot of propylene glycol in a 50 mL beaker is heated to 55 °C with stirring.
• the phospholipids of Example 1 are added with stirring and the mixture is stirred until the phospholipids dissolve (-15 min).
• the propylene glycol solution of phospholipids is added dropwise over a period of about 2 min to a stirred mixture of water / glycerol 95/5 v/v (200.7 mL) containing monobasic sodium phosphate » H 2 0 (173.9 mg) and anhydrous dibasic sodium phosphate (137.87 mg) at 55 °C.
• the solution is stirred for 5 min after the addition of the glycerol solution of the phospholipids.
• the solution is immediately push-filtered through a 32 mm Pall Sciences GH Polypro ® 0.2 micron filter into a 250 mL bottle which is then immediately purged with nitrogen.
• the solution is allowed to cool to ambient temperature.
• nominal 5 mL serum vials (actual capacity 9 mL) are charged with a 7.56 mL aliquot of the phospholipid suspension followed by purging the headspace with dry ultrapure nitrogen and stoppering with halobutyl rubber stoppers.
• the stoppered vials (in trays) are placed in a refrigerator at 4 °C until the vial contents have equilibrated to that temperature.
• the trays are removed and the vials are charged with TDFH (Sigma Aldrich Co. St. Louis, MO) and crimp capped as described in Example 5.
• the nanoemulsion is prepared by vortexing the vial and sonication as described in Example 5.
• Example 6 The methods of Example 6 are followed except that the surfactant system comprises 30 mole percent compound 20a in addition to the C14-phospholipid-based system.
• the nano-emulsion is then prepared similarly. Similar procedures and techniques from Examples 1-6 and prophetic Examples 1 and 2 can be employed for preparation of nanoemulsions based on lauroyl (C12)-based phospholipid surfactant systems.
• the solution is allowed to cool to ambient temperature followed by addition of water for injection (20 mL) cooling to 10 °C and addition of cold DDFP (4.51 g, 2.61 mL) and stirring rapidly for 30 min at 10°C.
• the resulting material is subjected to homogenization using a Kirkland products hand held homogenizer at 5°C for a total of 20 passes.
• This material is then added to a solution of glycerol in water for injection.
• the nanoemulsion is added to a stirred solution of 205.4 mL of 30% sucrose and stirred for 10 min under a nitrogen atmosphere (5 psi).
• the solution is transferred via the homogenizer to a second vessel kept at 2-5 °C after which it is stirred for 15 minutes and filtered through a Pall Sciences 0.8/0.2 micron Acropak 200 filter into a third vessel at 2-5 °C and stirred for 15 minutes at that temperature after transfer.
• the material is then filled into nominal 5 mL Wheaton vials, stoppered with gray halobutyl rubber stoppers and crimp capped and stored.
• FIGs. 1, 2, 3, 4, and 5 The schematic flow chart diagrams included are generally set forth as logical flow-chart diagrams (e.g., FIGs. 1, 2, 3, 4, and 5). As such, the depicted order and labeled steps are indicative of one embodiment of the presented method. Other steps and methods may be conceived that are equivalent in function, logic, or effect to one or more steps, or portions thereof, of the illustrated method. Additionally, the format and symbols employed are provided to explain the logical steps of the method and are understood not to limit the scope of the method. Although various arrow types and line types may be employed in the flow-chart diagrams, they are understood not to limit the scope of the corresponding method (e.g., FIGs. 1, 2, 3, 4, and 5).
• arrows or other connectors may be used to indicate only the logical flow of the method. For instance, an arrow may indicate a waiting or monitoring period of unspecified duration between enumerated steps of the depicted method. Additionally, the order in which a particular method occurs may or may not strictly adhere to the order of the corresponding steps shown.

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