Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
• • 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
  • A61K31/28—Compounds containing heavy metals
• • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy

Definitions

• the present invention demonstrates a novel phospholipids composition and its application in delivering various therapeutic agents to which tissue and/or membranes are impermeable.
• This composition comprises negatively charged lipid bilayers which interact with organic multi- cations to rolled up forming a cylindrical multi-layer structure.
• Cochleates are spiral rolls formed of negatively charged phospholipid bilayers which are rolled up through the interaction with multivalent counter ions (Ca 2+ or Zn 2+ ) as the bridging agents between the bilayers [9].
• cochleates possess unique properties that they offer superior mechanical stability and better protection for drugs encapsulated than liposomes due to their solid matrix, yet they remain the phospholipid bilayer structure. These solid particles are so flexible that they can readily convert to liposomes by extracting the bridging counter ions out of the inter bilayer spaces.
• Such unique properties have made cochleates an ideal system for delivering insoluble ingredients which can be loaded in the matrix of a phospholipid bilayer but avoid the instability problem of liposomes [10].
• nanometer-sized cochleates (nanocochleates) [11].
• These size-reduced cochleates showed capability for oral delivery of Amphoterecin B (AmB), a hydrophobic drug currently administrated through IV injection in the form of liposomes or micelles.
• AmB Amphoterecin B
• Oral availability of AmB achieved by nanocochleates encourage the inventor to design a new cochleate system by which highly charged and membrane-impermeable therapeutics may be encapsulated and delivered orally.
• hydrophobic drug molecules are incorporated in the matrix of phospholipid bilayer prior to cochleation (formation of cochleates by addition of metal cations). Drug loading capacity is limited by how much drug can be "dissolved” in the lipid matrix without destroying its bilayer structure. This structure limits application of cochleates to delivery of hydrophobic molecules.
• the present invention demonstrates a new type of cochleates and nano-cochleates that allow charged, soluble but tissue-impermeable molecules, including relatively small therapeutic peptides, be encapsulated in the inter-bilayer space and delivered cross tissue-membrane.
• Cochleates and nano-cochleates are phospholipid-calcium (or zinc) precipitates that are formed by calcium (or zinc) induced fusion of unilamellar liposomes into large lipid bilayer sheets which then fold spirally into cylinders.
• the new cochleates and nano-cochleates differ from conventional systems in that i) the fusion of unilamellar liposomes is no longer induced by Ca 2+ , Zn 2+ or other metal ions but by the molecules to be encapsulated (See Figure 1); ii) charged, hydrophilic and tissue impermeable drugs can be encapsulated in the structure with improved loading capacity. Since no additional metal cations (such as Ca 2+ or Zn 2+ ) are existing during the new cochelation process, there is no such possibility that the molecules to be encapsulated are precipitated outside of the cochleate structure as in the conventional cochelation.
• the new cochleates and nano-cochleates showed some similarities in physical chemical properties and drug delivery functions as the conventional systems.
• the cylindrical structure could open-up and converted to liposomes upon adding a cation carrier, such as EDTA.
• the new system showed the ability to deliver encapsulated ingredients across cell membranes by fusion with the membrane (See Figure 2).
• This invention offers a simplified method to prepare nano-cochleates.
• poly-cations such as polypeptides with net charge over 5
• nano-cochleates were easily prepared by adding the polycations directly into the lipsomal suspension, without using complicated hydrogel-isolation technique.
• Figure 1 Schematic description of compalxatiqn of phospholipids bilayers with Ca 2+ , and with organic cations.
• FIG. 1 Schematic description of fusion of cochleates formed by interaction with drug molecules which function as the bridging agent between phospholipids bilayers.
• the loaded drug molecues are delivered across cell membranes due to fusion of cochleates with the cell membrane.
• FIG. 1 Microscopic image of cochelates formed by complexation with 2,3,5,6- tetraaminopyrimidine as the bridging agent.
• Figure 4 Microscopic image of nano-cochelates formed by complexation with 2,3,5,6- tetraaminopyrimidine as the bridging agent. A: before treatment with EDTA; B: after treatment with EDTA. Figure 5. Microscopic image of cochelates formed by complexation with tobramycin as the bridging agent. A: before treatment with EDTA; B: after treatment with EDTA.
• FIG. 1 Microscopic image of nano-cochelates formed by complexation with tobramycin as the bridging agent. A; before treatment with EDTA; B: after treatment with EDTA.
• Figure 7 Distribution of dynamic sizes of nano-cochleates formed by complexation with tobramycin.
• Figure 8 Microscopic image of cochelates formed by complexation with polylysine as the bridging agent. A: before treatment with EDTA; B: after treatment with EDTA.
• Figure 9 Antibiotic activity of tobramycin formulated in solution, cochleates and nanocochleates. The drug of various doses were added to E.Coli prior to incubation at 37 °C, followed by counting the colonies.
• This invention provides a new cochleate system and a nano-cochleate system for which the agents that bridge lipid bilayers together to form a multi-layer structure are organic multi-valent cations.
• the new cochleates systems is defined as a spiral phospholipids bilayer that rolled up by complexation with organic multi-valent cations which bring two surfaces of charged lipid bilayers together through ionic bonds (See figure 1).
• the multi- bilayer systems formed by interaction with the organic cations may or may not form a cylindrical shape.
• the new systems can allow charged and hydrophilic therapeutics, such as peptides, be microencapsulated into the cochleate structure while conventional cochleates cannot.
• the new systems showed the similar properties observed from conventional cochleates such as conversion back to liposomes when treated with cation carriers, and the ability to deliver drugs across tissue membranes. These properties (loading hydrophilic drugs and delivery across membrane) make the new systems promising to deliver peptides orally.
• cochleate and nano-cochleate systems disclosed herein can be used for microencapsulation and delivery of therapeutics wherein the therapeutics agents are loaded in the cochleate structure as the bridging agents between lipid bilayers.
• the therapeutics includes but is not limited to peptides, poly-amino acids, nucleotides and hydrophilic chemical drugs which possess two or more net charges.
• cochleate systems are used for oral delivery of peptides, polyamino acids, nucleotides and hydrophilic chemical drugs which possess more than two net positive charges.
• Other drugs may be used.
• An ordinary skilled artisan may use the drugs exemplified herein or the guidelines provided in other drugs.
• the delivery of therapeutics is through inhalation.
• This invention also provides a method of preparing the new cochleate systems comprising direct cochleation [9], hydrogel-isolated cochleation [11], and size-controlled cochleation using poly-cations (See Example 5).
• Organic cations can be added to a suspension of unilamellar liposomes directly with stirring or vortex, or added to an polymer aqueous two-phase system for which liposomes are partitioned in the dispersed phases and isolated within each droplet
• nano-cochleates can be prepared without using the complicated hydrogel-isolation technique [11].
• the sizes of cochleates formed can be controlled by the charge ratio of the polycations over liposomes.
• nano-cochleates can be formed by adding the polycations directly to the liposomal suspension (Example 5). Due to their multiple net charges and long chain, polycations may be partially associated with the lipids of opposite charge, leaving some charged species dangling at the cochleate surfaces.
• This invention offers a significantly simplified method to prepare nano-cochleates.
• DOPS dioleoyl phosphatidyl serine
• the TAS solution was added to the liposome suspension drop-wise under magnetic stirring until precipitation occurred.
• the precipitates were examined using a microscope, and the microscopic image showed that the lipids formed needle shape structures (See Figure 3A).
• Other organic cationic molecules such as antibiotics and polypeptides, can also be used to form cochleate structure.
• Example 2 Preparation of nano-cochleates with 2,3,5,6-tetraaminopyrimidine sulfate.
• Nano-sized cochleates can be prepared with 2,3,5,6-tetraaminopyrimidine sulfate using hydrogel- isolated methods that the inventor has patented previously [11].
• a liposome suspension prepared as in Example 1 was added into a dextran solution (5-25%) with lipid content of 0.2-2%. This suspension was then dispersed into a polyethylene glycol (PEG) solution (5-25%) and well stirred.
• PEG polyethylene glycol
• the solutions of dextran and PEG were immiscible and formed an aqueous two-phase system.
• the TAS solution prepared as in Example 1 was added drop-wise to the aqueous two-phase system under stirring with the charge of the organic cations was more than that of the lipids.
• FIG 4A show a microscopic image of recovered cochleates. Needle structure was not detectable by optical microscope due to the particle size. A laser scattering measurement showed that the cochleate sizes were in sub- micron.
• Figure 4B shows the microscopic image of nano-cohcleates after treatment with EDTA. Liposomes formed from nano-cochleates are much smaller than those from the cochleates formed without hydrogel-isolation (compare Figure 3B with Figure 4B).
• Cohcleates and nano-cochleates were prepared by repeating the experimental procedure in example 1 and 2 uisng a drug, tobramycin chloride, as the bridging agent inseatd of TAS.
• Tobramycin is an antibiotic, soluble in water in salt form and administrated by injection.
• the molecule has molecular weight of 467 and 5 amino groups.
• tobramycin was prepared by dissolving 100 mg tobramycin with 100 ml water. Prior to cochleation, the solution was divided to several parts with pH adjusted to 1.2, 2.5, 3.5 and 5, respectively. These drug solutions were added dropwse to liposome solutions prepared as in Example 1, respectively. Visible precipitates were formed for the samples treated with tobramycin solution with pH of 1.2 and 2.5, suggesting that sufficient ionization of the amino groups of tobramycin is required.
• the formed cochleates and their response to EDTA were examined using an optical microscope. The images were shown in Figure 5A and 5B, respectively. For the precipitates showed needle shapes (Figure 5A) before treatment with EDTA, and converted to giant liposomes when EDTA was added ( Figure 5B).
• Cochelates can also be prepared by adding a solution of peptide into the lipsomal suspension as in Example 1.
• DOPS polylysine
• the final ratio of DOPS and polylysine was 1 :1.2.
• a microscopic image showed that the precipitated particles possess a needle shape ( Figure 8A).
• neddle shape particles readily opened up and converted to giant liposomes ( Figure 8B) as those prepared with other bridgling agents (Ca 2+ [1 1], Zn 2+ [11], TAS, and tobramycin).
• Nanometer sized cochleates can be prepared with peptides as the bridging agent in a way without using the hydrogel-isolation [11].
• the liposomal suspention prepared as in Example 1 was added into the polylisine solution as in Example 4 under stirring with the final lipid to polylysine ratio of 1 :4.
• the clear liquids (polylysine solution and liposomal suspension) readiliy turned to cloudy. No visible particles was observed under optical microscope.
• a particle size measurement was carried out using a Nicomp Submicron particle sizer, suggesting the mean dynamic size of the particles was about 60 - 100 nm.
• the mechanism of the size reduction due to increased polylysine to liposome ratio may be similar to that in the complexation between DNA and cationic polymers [14].
• Example 6 Loading capacity of cochelates for molecules as bridging agents. Loading capacities of the new cochelates and nano-cochleates for TAS and tobramycin were determined.
• a solution of 2,4-dinitrofluourobene (10mg/ml in alcohol) was prepared within 5 dyas prior to use and refrigerated.
• a water solution of tris (hydroxymethyl) aminomethane (15 mg/ml) was also prepared as a stock solution. Within 4 hours of analysis, this stock solution, 40 ml, was diluted with dimethyl suifoxide (DMSO) to 200ml.
• DMSO dimethyl suifoxide
• the supernatant (the sample to be measured) 1ml of 5.5mg/ml of tobramycin solution was added into a liposome suspension with lipid to drug ratio of 1:5. After precipitation, the supernatant was collected and diluted to 50ml with water.
• Example 7 Antibiotic activity of tobramycin loaded in cochleates and nano-cochleates. Tobramycin was selected as a model drug to examine the capability of cochelates and nanocochleates to delivery hydrophilic drugs across cell membranes for that the antibiotic function of tobramycin relies on its binding to ribosomes inside of cells. In another word, for tobramycin, its antibiotic activity reflects internization of the drug into the cells.
• tobramycin loaded cochleates and nano-cochleates prepared as in Example 2 were incubated with E.Coli at various dose. As a control, a tobramycin solution was also incubated with E.Coli under identical conditions.
• E.Coli cell line DH5a
• 5 ul of the DH5a culture solution was diluted to 2 ml and added with tobramycin in the forms of cochleates, nano-cochleates at the final concentration of 0, 0.5, 1.0, 2.5 and 5.0 ug/ml, respectively.
• the tobramycin-added cell cultures were incubated at 37 °C, with shacking at 200 rpm, for 24 hrs.
• nano-cochleates became the most active dosage form.
• the cell counts for nano-cochleates treated culture was ten times lower than that by tobramycin solution and 100 times lower than that of large cochleates.
• cell counts became zero for all the three formulations. It is clearly that nano-cochleates significantly enhanced antibiotic activity of the drug.
• nano-cochleates can facilitate cross-membrane diffusion for charged and impermeable molecules have a wide application in drug delivery.
• Many therapeutic agents, such as peptides are soluble but impermeable to tisse membranes.
• Cross-membrane permeation is especially important for those agents for which the binding sites are inside of cells rather than cell surface receptors.
• the system may also facilitate oral absorption for peptide drugs that possess net positive charge.
• the nano-cochleates demonstrated in the inventor's previous invention [11] showed significant in vivo bioavailability and therapeutic efficacy for oral delivery of amphotericin B, a hydrophobic anti-fungus agent normally administrated through IV route.
• the new nano- cochleates although differing from the previous one by using organic cations as the bridging agents, possess similar physical chemical properties including the ability of fusing with cell membranes (Example 7). Therefore, the new system is expected to have the ability to deliver impermeable therapeutics orally.

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• 2003
  • 2003-08-06 WO PCT/CN2003/000634 patent/WO2004012709A1/en not_active Ceased
  • 2003-08-06 AU AU2003254598A patent/AU2003254598A1/en not_active Abandoned
  • 2003-08-06 CN CNA038185695A patent/CN1674869A/zh active Pending
• 2008
  • 2008-09-29 US US12/239,847 patent/US20090036417A1/en not_active Abandoned

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