EP1259224A2 - Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles - Google Patents

Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles

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Publication number
EP1259224A2
EP1259224A2 EP01903273A EP01903273A EP1259224A2 EP 1259224 A2 EP1259224 A2 EP 1259224A2 EP 01903273 A EP01903273 A EP 01903273A EP 01903273 A EP01903273 A EP 01903273A EP 1259224 A2 EP1259224 A2 EP 1259224A2
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EP
European Patent Office
Prior art keywords
biologically relevant
lipid
suspension
group
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01903273A
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German (de)
English (en)
Inventor
Leila Zarif
Tuo Jin
Ignacio Segarra
Raphael J. Mannino
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
Biodelivery Sciences Inc
Original Assignee
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
Biodelivery Sciences Inc
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Priority claimed from PCT/US2000/001684 external-priority patent/WO2000042989A2/fr
Application filed by University of Medicine and Dentistry of New Jersey, Rutgers State University of New Jersey, Biodelivery Sciences Inc filed Critical University of Medicine and Dentistry of New Jersey
Publication of EP1259224A2 publication Critical patent/EP1259224A2/fr
Withdrawn legal-status Critical Current

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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
    • A61K9/127—Liposomes
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases, cochleates; Sponge phases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals

Definitions

  • the present invention relates to a novel method for preparing a novel lipid-based cochleate delivery system, the preparations derived from the lipid-based cochleate delivery system, such as drugs, carbohydrates, vitamins, minerals, polynucleotides, polypeptides, lipids and the like, and the use of these preparations.
  • the ability of biologically relevant molecules to be administered via the oral route depends on several factors.
  • the biologically relevant molecule must be soluble in the gastrointestinal fluids in order for the biologically relevant molecule to be transported across biological membranes for an active transport mechanism, or have suitable small particle size that can be absorbed through the Peyer's Patches in the small intestine and through the lymphatic system. Particle size is an important parameter when oral delivery is to be achieved (see Couvreur et al, Adv. Drug Delivery Rev., 10 : 141 - 162 (1993)).
  • the primary issue in the ability to deliver drugs orally is the protection of the drug from proteolytic enzymes.
  • An ideal approach is to incorporate the drug in a hydrophobic material so that the aqueous fluids cannot penetrate the system.
  • Lipid-based cochleates are an ideal system that can achieve this purpose.
  • cochleates have a nonaqueous structure and therefore they: a) are more stable because of less oxidation of lipids; b) can be stored lyophilized, which provides the potential to be stored for long periods of time at room temperatures, making them advantageous for worldwide shipping and storage prior to administration; c) maintain their structure even after lyophilization, whereas liposome structures are destroyed by lyophilization; d) exhibit efficient incorporation of biologically relevant molecules into the lipid bilayer of the cochleate structure; e) have the potential for slow release of a biologically relevant molecule in vivo as cochleates dissociate; f) have a lipid bilayer which serves as a carrier and is composed of simple lipids which are found in animal and plant cell membranes, so that the lipids are non-toxic; g) are produced easily and safely; h) can be produced as defined formulations composed of predetermined amounts and ratios of drugs or antigens.
  • Cochleate structures have been prepared first by D. Papahadjopoulos as an intermediate in the preparation of large unilamellar vesicles (see U.S. Patent No. 4,078,052).
  • the use of cochleates to deliver protein or peptide molecules for vaccines has been disclosed in U.S. Patent Nos. 5,840,707 and 5,643,574.
  • the use of cochleates to orally deliver drugs, nutrients, and flavors have been described in U.S. Patent No. 5,994,318.
  • the method further comprises the steps required to encochleate at least one biologically relevant molecule in the hydrogel-isolated cochleates in an effective amount.
  • a "biologically relevant molecule” is one that has a role in the life processes of a living organism.
  • the molecule may be organic or inorganic, a monomer or a polymer, endogenous to a host organism or not, naturally occurring or synthesized in vitro, and the like.
  • examples include vitamins, minerals, flavors, amino acids, toxins, microbicides, microbistats, co-factors, enzymes, polypeptides, polypeptide aggregates, polynucleotides, lipids, carbohydrates, nucleotides, starches, pigments, fatty acids, hormones, cytokines, viruses, organelles, steroids and other multi-ring structures, saccharides, metals, metabolic poisons, drugs, and the like.
  • the biologically relevant molecule-cochleate comprises the following components: a) a biologically relevant molecule, b) a negatively charged lipid, and c) a cation component, wherein the particle size of the cochleate is less than one micron.
  • FIGURES Figure 1 is a schematic of the process by which the hydrogel-isolated cochleates of the present invention, with or without a biologically relevant molecule, are obtained.
  • Figures 2A and 2B illustrate a particle size distribution (weight analysis) of hydrogel-isolated cochleates either loaded with amphotericin B (AmB) (Fig. 2A) or empty (Fig. 2B) as measured by laser light scattering.
  • AmB amphotericin B
  • Figures 3A and 3B illustrate microscopic images of a mixture of liposomes in dextran dispersed into PEG gel solution.
  • the small black dots are dextran particles formed by dispersing the dextran phase in the PEG phase.
  • Fig. 3B Microscopic images of the sample shown in Fig. 3A after treatment with CaCl 2 solution. The black objects in circles, are cochleates formed by the addition of Ca ions.
  • Figures 4A-4F illustrate microscopic images of the sample shown in Figs. 3A and 3B after washing with a buffer containing 1 mM CaCl and 100 mM NaCl. Aggregates are formed by the cochleate particles (Fig. 4B).
  • AmB hydrogel-isolated cochleates precipitated with zinc according to the procedure described in Example 14 (Fig. 4D).
  • Cochleates displayed in Fig. 4C after treatment with EDTA (Fig. 4E).
  • Cochleates displayed in Fig. 4F are after treatment with EDTA.
  • Figure 5 illustrates micrographs of hydrogel-isolated cochleates after freeze fracture.
  • Figure 6 illustrates growth inhibition of Candida albicans by hydrogel-isolated cochleates loaded with AmB at 0.625 Eg AmB/ml. Comparison is made to AmB in DMSO and AmBisome R .
  • Figure 7 illustrates the effect of hydrogel-isolated cochleates on the viability of Candida albicans after 30 hours.
  • FIGS 8A and 8B illustrate the efficacy of Amphotericin B-cochleates on macrophage cultures.
  • Figure 9 illustrates Amphotericin B tissue levels after administration of Amphotericin B-cochleates.
  • Figure 10 illustrates the time profile tissue concentration of AmB after a single dose administration of hydrogel-isolated cochleates loaded with AmB.
  • Figure 11 illustrates AmB tissue level 24 hrs after single dose and 24 hrs after a multiple dose regime.
  • Figure 12 illustrates correlation between Amphotericin B tissue level and the level of Candida albicans after administration of Amphotericin B cochleates. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a solution to achieve effective oral delivery of drugs and other biologically relevant molecules by producing small-sized cochleates of less than one micron using new methods.
  • the new approach is based on the incompatibility between two polymer solutions, both of which are aqueous.
  • Aqueous two-phase systems of polymers are well used for protein purification due to a number of advantages such as freedom from the need for organic solvents, mild surface tension and the biocompatibility of aqueous polymers (see P.A. Albertsson, "Partition of cell particles and macromolecules", 3 rd edition, Wiley NY (1986); and "Separation using aqueous Phase System” D. Fisher Eds, Plenum NY (1989)).
  • the present invention there are provided methods for preparing small-sized, lipid-based cochleate particles and preparations derived therefrom, comprising a biologically relevant molecule incorporated into the particles.
  • the cochleate particles are formed of an alternating sequence of lipid bilayers/cation.
  • the biologically relevant molecule is incorporated either in the lipid bilayers or in the interspace between the lipid bilayers.
  • One of the methods for preparing the small-sized cochleates comprises: 1) preparing a suspension of small unilamellar liposomes or biologically relevant molecule-loaded liposomes, 2) mixing the liposome suspension with polymer A, 3) adding, preferably by injection, the liposome/Polymer A suspension into another polymer B in which polymer A is nonmiscible, leading to an aqueous two-phase system of polymers, 4) adding a solution of cation salt to the two-phase system of step 3, such that the cation diffuses into polymer B and then into the particles comprised of liposome/polymer A allowing the formation of small-sized cochleates, 5) washing the polymers out and resuspending the empty, drug or other biologically relevant molecule-loaded cochleates into a physiological buffer or any appropriate pharmaceutical vehicle.
  • a second method for preparing the small-sized cochleates comprises detergent and a biologically relevant molecule and cation.
  • the detergent is added to disrupt the liposomes. The method comprises the following steps:
  • a lyophilization procedure can be applied and the lyophilized biologically relevant molecule-cochleate complex can be filled into soft or hard gelatin capsules, tablets or other dosage form, for systemic, dermal or mucosal delivery.
  • the biologically relevant molecule partitions into either or both lipid bilayers and interspace, and the biologically relevant molecule is released from the cochleate particles by dissociation of the particles in vivo.
  • Alternative routes of administration may be systemic, such as intramuscular, subcutaneous or intravenous, or mucosal such as intranasal, intraocular, intravaginal, intraanal, or intrapulmonary. Appropriate dosages are determinable by, for example, dose-response experiments in laboratory animals or in clinical trials and taking into account body weight of the patient, absorption rate, half-life, disease severity and the like.
  • the number of doses, daily dosage and course of treatment may vary from individual to individual.
  • Other delivery routes can be dermal, transdermal or intradermal.
  • the first step of either method of the present invention which is the preparation of small liposomes, can be achieved by standard methods such as sonication or microfluidization or other related methods (see, for example,
  • the second step of either method comprises the addition, preferably by injection, of polymer A/liposome suspension into polymer B can be achieved mechanically by using a syringe pump at an appropriate controlled rate, for example a rate of 0.1 ml/min to 50 ml/min, and preferably at a rate of 1 to 10 ml/min.
  • hydrogel-isolated cochleates (with or without a biologically relevant molecule) is achieved in the third step by adding a positively charged molecule to the aqueous two-phase polymer solution containing liposomes.
  • the positively charged molecule can be a polyvalent cation and more specifically, any divalent cation that can induce the formation of a cochleate.
  • the divalent cations include Ca , Zn ++ , Ba ++ and Mg ++ or other elements capable of forming divalent ions or other structures having multiple positive charges capable of chelating and bridging negatively charged lipids, such as polycationic lipids. Addition of positively charged molecules to liposome-containing solutions is also used to precipitate cochleates from the aqueous solution.
  • cochleate precipitates are repeatedly washed in a fourth step with a buffer containing a positively charged molecule, and more preferably, a divalent cation. Addition of a positively charged molecule to the wash buffer ensures that the cochleate structures are maintained throughout the wash step, and that they remain as precipitates.
  • the medium in which the cochleates are suspended can contain salt such as calcium chloride, zinc chloride, cobalt chloride, sodium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, magnesium sulfate and sodium carbonate.
  • the medium can contain polymers, such as pluronics, and polyethylene glycols.
  • the biologically relevant molecule-cochleate is made by diluting into an appropriate biologically acceptable carrier (e.g., a divalent cation-containing buffer).
  • the lipids of the present invention are non-toxic lipids and include, but are not limited to simple lipids which are found in animal and plant cell membranes.
  • the lipid is a negatively charged lipid, more preferably a negatively charged phospholipid, and even more preferably a lipid from the group of phosphatidylserine, phosphatidylinositol, phosphatidic acid, and phosphatidyl glycerol.
  • the lipids may also include minor amounts of zwitterionic lipids, cationic lipids, polycationic lipids or neutral lipids capable of forming hydrogen bonds to a biologically relevant molecule such as PEGylated lipid.
  • the polymers A and B of the present invention can be of any biocompatible polymer classes that can produce an aqueous two-phase system.
  • polymer A can be, but is not limited to, dextran 200,000-500,000, Polyethylene glycol (PEG) 3,400-8,000
  • polymer B can be, but is not limited to, polyvinylpyrrolidone (PNP), polyvinylalcohol (PNA), Ficoll 30,000-50,000, polyvinyl methyl ether (PNMB) 60,000-160,000, PEG 3,400-8,000.
  • concentration of polymer A can range from between 2-20% w/w as the final concentration depending on the nature of the polymer. The same concentration range can be applied for polymer B.
  • Dextran PEG 5-20% w/w Dextran 200,000-500,000 in 4-10% w/w PEG 3,400-8,000
  • Dextran/PNP 10-20% w/w Dextran 200,000-500,000 in 10-20% w/w PNP 10,000-20,000
  • PEG/PNME 2-10% w/w PEG 3,500-35,000 in 6-15% w/w PNME 60,000-160,000.
  • the biologically relevant molecule is a molecule that has a role in the life processes of a living organism.
  • the molecule may be organic or inorganic, a monomer or a polymer, charged, either positively or negatively, hydrophilic, amphiphilic or hydrophobic in aqueous media, endogenous to a host organism or not, naturally occurring or synthesized in vitro and the like.
  • the biologically relevant molecule may be a drug, and the drug may be an antiviral, an anesthetic, an anti-infectious, an antifungal, an anticancer, an immunosuppressant, a steroidal anti-inflammatory, a non-steroidal anti- inflammatory, a tranquilizer or a vasodilatory agent.
  • Examples include Amphotericin B, acyclovir, adriamycin, carbamazepine, melphalan, nifedipine, indomethacin, naproxen, estrogens, testosterones, steroids, phenytoin, ergotamines, cannabinoids, rapamycin, propanidid, propofol, alphadione, echinomycine, miconazole nitrate, teniposide, taxol, taxotere, nystatin, rifampin, and vitamin A acid.
  • the biologically relevant molecule may be a polypeptide such as cyclosporin, angiotensin I, II and III, enkephalins and their analogs, ACTH, anti-inflammatory peptides I, II, III, bradykinin, calcitonin, beta-endorphin, dinorphin, leucokinin, leutinizing hormone releasing hormone (LHRH), insulin, neurokinins, somatostatin, substance P, thyroid releasing hormone (TRH) and vasopressin.
  • polypeptide such as cyclosporin, angiotensin I, II and III, enkephalins and their analogs, ACTH, anti-inflammatory peptides I, II, III, bradykinin, calcitonin, beta-endorphin, dinorphin, leucokinin, leutinizing hormone releasing hormone (LHRH), insulin, neurokinins, somatostatin, substance P, thyroid releasing hormone (TRH) and vasopress
  • the biologically relevant molecule may be an antigen, but the antigen is not limited to a protein antigen.
  • the antigen can also be a carbohydrate or a polynucleotide.
  • antigenic proteins include envelope glycoproteins from viruses, animal cell membrane proteins, plant cell membrane proteins, bacterial membrane proteins and parasitic membrane proteins.
  • a polynucleotide include a DNA or an RNA molecule.
  • the polynucleotide can also be in the form of a plasmid DNA.
  • the polynucleotide can be one that expresses a biologically active polypeptide, for example, an enzyme or a structural or housekeeping protein. Further, the polynucleotide need not be expressed, but may be an immunogen, a ribozyme or an antisense molecule.
  • the biologically relevant molecule may also be a nutrient such as vitamins, minerals, fatty acids, amino acids, and saccharides. Specific examples include vitamins A, D, E, or K; minerals such as calcium, magnesium, barium, iron or zinc; polyunsaturated fatty acids or essential oils; amino acids; and saccharides such as glucose and sucrose.
  • the biologically relevant molecule may also be a flavor substance. Examples include flavor substances generally associated with essential oils, such as cinnamon oil, and extracts obtained from botanical sources such as herbs, citrus, spices and seeds. Oils/extracts are sensitive to degradation by oxidation, and because the processing of the natural oils and extracts often involves multi- step operations, costs are generally considered to be higher.
  • Flavor-cochleates can also be incorporated into consumable food preparations as flavor enhancers.
  • the biologically relevant molecule is extracted from the source particle, cell, tissue, or organism by known methods. Biological activity of biologically relevant molecules need not be maintained. However, in some instances (e.g., where a protein has membrane fusion or ligand binding activity or a complex conformation which is recognized by the immune system), it is desirable to maintain the biological activity. In these instances, an extraction buffer containing a detergent which does not destroy the biological activity of the membrane protein is used.
  • Suitable detergents include ionic detergents such as cholate salts, deoxycholate salts and the like or heterogeneous polyoxyethylene detergents, such as Tween, BRIG or Triton. Utilization of this method allows reconstitution of antigens, more specifically proteins, into the liposomes with retention of biological activities, and eventually efficient association with the cochleates. This avoids organic solvents, sonication, or extreme pH, temperature, or pressure all of which may have an adverse effect upon efficient reconstitution of the antigen in a biologically active form.
  • Hydrogel-isolated cochleates may contain a combination of various biologically relevant molecules as appropriate.
  • the cochleate particles can be enteric.
  • the cochleate particles can be placed within gelatin capsules and the capsule can be enteric coated.
  • certain hydrophobic materials can be added to provide enhanced absorption properties for oral delivery of biologically relevant molecules. These materials are preferably selected from the group consisting of long chain carboxylic acids, long chain carboxylic acid esters, long chain carboxylic acid alcohols and mixtures thereof.
  • the hydrophobic materials can be added either initially to the lipid prior to the formation of liposomes or in a later step in the form of a fat vehicle such as an emulsion.
  • Step 1 Preparation of Small Unilamellar Vesicles From Dioleoylphosphatidylserine
  • a solution of dioleoyl phosphatidylserine (DOPS, Avanti Polar Lipids, Alabaster, AL, USA) in chloroform (10 mg/ml) was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at 35 ⁇ C.
  • the rotavapor was sterilized by flashing nitrogen gas through a 0.2 Iln filter.
  • the following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator (Laboratory Supplies Com., Inc.).
  • Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • Laser light scattering (weight analysis, Coulter N4 Plus) indicated that the mean diameter was 35.7 + 49.7 nm.
  • the liposome suspension obtained in step 1 was mixed with 40% w/w dextran-500,000 (Sigma) in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected with a syringe into 15% w/w PEG-8,000 (Sigma) (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM. Stirring was continued for one hour, and then a washing buffer containing 1 mM CaCl 2 and 150 mM NaCl was added to suspension B at the volumetric ratio of 1:1.
  • the suspension was vortexed and centrifuged at 3000 rpm, 2-4°C, for 30 min. After the supernatant was removed, additional washing buffer was added at the volumetric ratio of 0.5:1, followed by centrifugation under the same conditions.
  • a schematic of this new method of obtaining cochleates is detailed in Fig. 1.
  • the resultant pellet was reconstituted with the same buffer to the desired concentration.
  • Laser light scattering (weight analysis, Coulter N4 Plus) indicates that the mean diameter for the cochleate is 407.2 ⁇ 85 nm (Fig. 2B).
  • a solution of dioleoylphosphatidylserine (DOPS) and 1,2-distearoyl- sn-glycerol-3-phosphoethanolamine-n-(poly(ethylene glycol)-5000), (DSPE- PEG, Avanti Polar Lipids, Alabaster, AL, USA) in chloroform (ratio of DOPS:DSPS-PEG 100:1, w:w) was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at 35 ⁇ C. The rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter. The following steps were carried out in a sterile hood.
  • DOPS dioleoylphosphatidylserine
  • DSPE- PEG Avanti Polar Lipids, Alabaster, AL, USA
  • the dried lipid film was hydrated with de-ionized water to a concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator (Laboratory Supplies Com., Inc.). Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast optical microscope with a lOOOx magnification.
  • Step 2 Preparation of Hydrogel-isolated Cochleates
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar Vesicles
  • DOPS dioleoylphosphatidylserine
  • chloroform a solution of dioleoylphosphatidylserine (DOPS) in chloroform was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at 35 ⁇ C.
  • the rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter.
  • the following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with a solution of n-octyl-beta-D-gluco-pyranoside (OCG) at 1 mg/ml at a ratio of DOPS:OCG of 10: 1 w:w.
  • OCG n-octyl-beta-D-gluco-pyranoside
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated briefly in a
  • the suspension obtained in Step 1 was mixed with 40%) w/w dextran- 500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl 2 solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar AmB-Loaded, Vesicles from Dioleoylphosphatidylserine
  • the rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter. The following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator. Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear yellow (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • Step 2 Preparation of AmB-loaded, Hydrogel-isolated Cochleates.
  • the liposome suspension obtained in Step 1 was then mixed with
  • Step 1 Preparation of Small Unilamellar DXR-Loaded Vesicles from Dioleoylphosphatidylserine A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
  • DOPS dioleoylphosphatidylserine
  • step 1 Five milliliters of the liposome suspension obtained in step 1 was mixed with 40% w/w dextran-500,000 (Sigma) in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl 2 solution (100 mM) was added to the suspension to reach the final concentration of I mM.
  • Step 1 Preparation of Small Unilamellar CSPA-Loaded Vesicles from Dioleoylphosphatidylserine
  • a mixture of dioleoylphosphatidylserine (DOPS) in chloroform (10 mg/ml) and CSPA in methanol (0.5mg/ml) at a molar ratio of 10:1 was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at room temperature. The rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter. The following steps were carried out in a sterile hood. The dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml. The hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator. Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • DOPS dioleoylphosphatidy
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (Sigma) (PEG
  • Step 1 Preparation of Small Unilamellar NVIR-Loaded Vesicles from Dioleoylphosphatidylserine
  • a mixture of dioleoylphosphatidylserine (DOPS) in chloroform (10 mg/ml) and NVIR in methanol (0.5mg/ml) at a molar ratio of 10: 1 was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at RT. The rotavapor was sterilized by flashing nitrogen gas through a 0.2 LTn filter. The following steps were carried out in a sterile hood. The dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml. The hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator.
  • DOPS dioleoylphosphatidylserine
  • Step 2 Preparation of NVIR-Loaded, Hydrogel-isolated Cochleates
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 ⁇ m. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar RIF-Loaded Vesicles from Dioleoylphosphatidylserine A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
  • DOPS dioleoylphosphatidylserine
  • the liposome suspension obtained in step 1 was mixed with 40%) w/w dextran-500,000 (Sigma) in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15%) w/w PEG-8,000 (Sigma) (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Vitamin A acid (retinoic acid) is sensitive to air oxidation and is inactivated by UV light. Vitamin A is protected when embedded into lipid bilayers. The inco ⁇ oration is achieved as follows: A mixture of dioleoylphosphatidylserine (DOPS) in chloroform
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 in a ratio of suspension
  • A/PEG of Vi v/v (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B.
  • the rate of the stirring was 800-1,000 rpm.
  • a CaCl 2 solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • PFA's are biologically relevant molecules involved in the control of the level of cholesterol in blood and are the precursors of prostaglandins.
  • PFA's are sensitive to oxidation which limits their inco ⁇ oration into food. PFA's undergo, in the presence of oxygen, a series of reactions called autoxidation, leading to aldehydes and then ketones which have a fishy unpleasant odor and flavor. Embedding PFA in rigid, rolled-up, lipid bilayers helps prevent the autoxidation cascade.
  • PFA-cochleates is as follows: Step 1 : Preparation of Small Unilamellar PF A-Loaded Vesicles from Dioleoylphosphatidylserine
  • a mixture of dioleoylphosphatidylserine in chloroform (10 mg/ml) and PFA in methanol (0.5 mg/ml) at a molar ratio of 10: 1 was placed in a round- bottom flask and dried to a film using a rotary evaporator at RT.
  • the rotary evaporator was sterilized by flashing nitrogen gas through a 0.2 ITn filter.
  • the following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator. Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl 2 solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar Vitamin A-Loaded Vesicles from Dioleoylphosphatidylserine
  • a mixture of dioleoylphosphatidylserine (DOPS) in chloroform (10 mg/ml) and Vanillin in methanol (0.5mg/ml) at a molar ratio of lipid/vanillin of 10:1 was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at RT. The rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter. The following steps were carried out in a sterile hood. The dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml. The hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator. Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • DOPS dio
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 in a ratio of suspension A/PEG of Vi v/v (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 rpm. A CaCl 2 solution (100 mM) was added to the suspension to reach the final concentration of 1 mM. Stirring was continued for one hour, and then a washing buffer containing 1 mM CaCl 2 and 150 mM NaCl was added to suspension B at the volumetric ratio of 1:1.
  • the suspension was vortexed and centrifuged at 3000 ⁇ m, 2-4 ⁇ C, for 30 min. After the supernatant was removed, additional washing buffer was added at the volumetric ratio of 0.5: 1, followed by centrifugation under the same conditions (see Fig. 1). The resulting pellet was reconstituted with the same buffer to the desired concentration.
  • the amount of vanillin encapsulated in the cochleates was determined by UV abso ⁇ tion at 239 nm.
  • Step 1 Preparation of Small Unilamellar CinO-Loaded Vesicles from Dioleoylphosphatidylserine A mixture of dioleoylphosphatidyl serine (DOPS) in chloroform
  • DOPS dioleoylphosphatidyl serine
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15 % w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 ⁇ m. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar DNA-Loaded Vesicles from Dioleoylphosphatidylserine
  • a solution of dioleoylphosphatidylserine in chloroform (10 mg/ml) was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at RT.
  • the rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter. The following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with a solution of pCMV-beta-gal-DNA in TE buffer (at 1 mg/ml) to reach a concentration of DOPS:DNA of 10: 1 and a concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then vortexed for several minutes.
  • the DNA/liposome mixture was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 ⁇ m. A CaCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • Step 1 Preparation of Small Unilamellar Vesicles from Dioleoylphosphatidylserine
  • a solution of dioleoylphosphatidylserine (DOPS) in chloroform (10 mg/ml) was placed in a round-bottom flask and dried to a film using a Buchi rotavapor at 35 ⁇ C.
  • the rotavapor was sterilized by flashing nitrogen gas through a 0.2 ITn filter.
  • the following steps were carried out in a sterile hood.
  • the dried lipid film was hydrated with de-ionized water at the concentration of 10 mg lipid/ml.
  • the hydrated suspension was purged and sealed with nitrogen, then sonicated in a cooled bath sonicator. Sonication was continued (for several seconds to several minutes depending on lipid quantity and nature) until the suspension became clear (suspension A) and there were no liposomes apparently visible under a phase contrast microscope with a lOOOx magnification.
  • the liposome suspension obtained in step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension A)) under magnetic stirring to result in suspension B. The rate of the stirring was 800-1,000 ⁇ m. A ZnCl solution (100 mM) was added to the suspension to reach the final concentration of 1 mM.
  • the liposome suspension obtained in Step 1 was mixed with 40% w/w dextran-500,000 in a suspension of 2/1 v/v Dextran/liposome. This mixture was injected via a syringe into 15% w/w PEG-8,000 (PEG 8000/(suspension
  • Figs. 3 A, 3B and 4A-4F show the mo ⁇ hological changes at each preparation step of AmB loaded hydro gel- isolated cochleates precipitated with Ca ions.
  • the AmB/liposome- dextran mixture was dispersed into PEG solution, phase separation resulted as shown by Fig. 3A.
  • Partition of the liposomes favored the dispersed dextran phase as indicated by a yellow color of AmB. This partitioning ensures that liposomes are isolated in each dextran particle.
  • Addition of Calcium ions into the continued phase (PEG) resulted in formation of precipitates in the dispersed phase.
  • An in vitro yeast susceptibility assay was performed comparing the inhibitory and lethal effects of AmB-cochleates, AmBisomes (liposomal formulation of AmB) and AmB/DMSO.
  • AmB/cochleates AmB/DMSO and AmBisomes were added to 96 well plates to a final concentration of 0.078, 0.156, 0.3125, 0.625, 1.25 and 2.5 E ⁇ /ml of AmB.
  • the 96 well plates were incubated at 37 ⁇ C with gentle shaking and cell density was measured on a 96 well plate reader (Molecular Devices Spectramax 340) at 0, 2, 4, 6, 24 and 30 hours.
  • Fig. 6 shows that AmB-cochleates have a greater growth inhibitory effect than AmBisomes (liposomal formulation of AmB). Fungicidal Effect of Hydrogel-isolated Cochleates Loaded with Amphotericin B
  • Yeast cells treated with AmBisome, AmB/DMSO and AmB/cochleates were examined for the ratio of colony forming units to total cell number after 30 hours of incubation.
  • the results show that the AmB/cochleates had the greatest lethal effect on the yeast cells compared to the other antifungal agents tested.
  • the AmBisome was not as effective, resulting in 52% yeast viability (Fig. 7).
  • Particle scavenging cells such as macrophage
  • macrophage are the first line of defense against many microbial infections.
  • many microbes, which induce severe human clinical infections have been shown to infect macrophage and avoid destruction.
  • macrophage play an important role in the uptake of cochleates, via an endocytotic mechanism. Since macrophage also play an important role in the host defense and clearance of fungi and parasites, it is important to study the interaction between macrophage and cochleates.
  • cochleates are taken up by macrophage.
  • Large doses of AmB delivered to the macrophage were found to be non-toxic and remained within the macrophage in a biologically active form.
  • AmB cochleates provided protection for the macrophage against infection by Candida albicans when administered prior to or after fungal infection.
  • Prophylactic dose regime: J774A.1 macrophage (M) were subcultured into a 96-well plate at a concentration of lxlO 5 cells/ml in DMEM +10% FBS.
  • One-hundred IT AmB cochleates (AmBc 0.2, 0.6, 1.25, and 2.5 ⁇ g AmB/ml), Fungizone, or empty cochleates (EC at 2, 6, 12.5, and 25 ⁇ g lipid/ml) were added at the specified concentration. Plates were incubated overnight at 37°C and 5% CO . 24 hours later, the medium was replaced. This step was performed twice. Candida albicans (CA) was added to the plate at a concentration of 2.5x10 cells/ml, a ratio of 1 :200 with respect to the macrophage. Plates were incubated overnight under the conditions stated above.
  • CA Candida albicans
  • J774A.1 macrophage (M) were subcultured into a 96-well plate and then incubated overnight. Following incubation, the macrophage were infected with GA at a ratio of 200:1, then subsequently AmBc, Fungizone or EC was added at the specified concentrations. Twenty-four hours later, the cell cultures were observed and CFU's determined as described above.
  • Fungizone (AmB in deoxycholate), the most popular clinical form of AmB was extremely toxic and lethal to the macrophage in vitro.
  • AmB cochleates are not toxic to the macrophage even at the highest doses studied.
  • the AmB cochleates are accumulated at high levels resulting in large distended vacuoles. After washing of the macrophage and incubating again for 24 hours, most of the vacuoles had returned to the normal shape and size, yet a few were noticeably enlarged. A few macrophage were even noticed to be "moving" with the enlarged vacuoles. AmB cochleates are concentrated within the vacuoles and it is probable that AmB is released gradually over time.
  • AmB cochleates (0.05 ml/ 20 g) with a V cc U 100 insulin syringe with a 18 g Vi needle size.
  • animals were given anesthesia, their blood was collected via cardiac puncture, and then, the animals were euthanized and dissected.
  • Tissues of interest were removed (brain, lung, liver, spleen, kidneys, heart, fat, stomach, stomach contents, intestine and intestinal contents) and weighed.
  • extraction solvent (10% methanol, 35% water, 55% ethanol), homogenized, sonicated and centrifuged.
  • a 90 IT aliquot of supernatant was transferred into a micro vial, injected into the HPLC system in a Nova-Pak C- 18 column (3.9 x 150 mm, 4 ITn particle size), and kept at 40 ⁇ C.
  • Amphotericin B was eluted at a flow rate of 0.5 ml/min with 29% methanol, 30% acetonitrile and 41% 2.5 mM EDTA and then detected at 408 nm. The concentration of AmB was calculated with the help of an external standard curve.
  • Tissue and blood samples were processed as follows: tissues were diluted 1/20 or 1/10 by addition of extraction solvent (H 2 O 35%), methanol 10%, ethanol 55%) w/w/w nv/v/v) and homogenized with an Ultra-Turrex® device. A 0.5 ml aliquot was taken, sonicated for 1 min and centrifuged at 7260 rpm for 12 min at 4 ⁇ C. Supernatant was transferred to an HPLC micro-vial and 30 IT was injected on a C-18, 3.9 x 150 mm, 4 ITn particle sized analytical column with a flow rate of 0.5 ml, at 40 ⁇ C. Concentration of AmB detected at 408 nm was calculated with the help of an external calibration curve.
  • Fig. 10 shows the time profile of AmB in the tissues over a period of time of 24 hrs. Although only three time points are plotted, accumulation in key tissues (liver, lungs, spleen and kidneys) can be seen.
  • mice received a 10 mg/kg/day oral multiple dose regime for ten days and one group was sacrificed 24 hrs after the last dose and the other group 20 days after the last dose received. At the predetermined time points mice were anesthetized, sacrificed and dissected for tissue collection. Tissues were processed as in the single dose regime and the AmB level was determined by HPLC. Results from 24 hr after the 10 1 dose are depicted in Fig. 11 and show that hydrogel-isolated cochleates allow the delivery of AmB from the gastrointestinal tract at therapeutic levels. EXAMPLE 20
  • Fig. 12 shows the relationship between tissue levels of Amphotericin B (Efe/g tissue on left scale) and efficacy as decrease of Candida albicans infection (CFU/g on the right scale) after oral administration of AmB-cochleates.
  • orally administered AmB-cochleates were non-toxic even at the highest dose of 50 mg/kg (no lesions were found in kidneys, GI tract and other organs of mice given 10, 20 and 50 mg/kg of AmB-cochleates).
  • This high dose 50 mg/kg is equivalent to 100 times the lowest dose (0.5 mg/kg) that showed 100% ⁇ of survival in the Candida infected mouse model.

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Abstract

L'invention concerne un procédé de production d'une structure cochléaire de petite taille à base de lipide. Les structures cochléaires sont obtenues à partir de liposomes qui sont mis en suspension dans une solution aqueuse polymère à deux phases, ce qui permet de séparer de manière différentielle les structures à base de molécules polaires par séparation de phases. La solution polymère à deux phases contenant des liposomes, traitée à l'aide de molécules chargées positivement telles que Ca?2+ ou Zn2+¿, forme un précipité de structures cochléaires présentant des dimensions de particules inférieures à un micron. Le procédé peut être utilisé pour produire des structures cochléaires contenant des molécules biologiquement utiles.
EP01903273A 2000-01-24 2001-01-24 Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles Withdrawn EP1259224A2 (fr)

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WOPCT/US00/01684 2000-01-24
PCT/US2000/001684 WO2000042989A2 (fr) 1999-01-22 2000-01-24 Nouvelles formulations de structures cochleaires isolees dans des hydrogels, procede de preparation et utilisation de celles-ci pour administrer des molecules biologiquement utiles
US613840 2000-07-11
US09/613,840 US6592894B1 (en) 1999-01-22 2000-07-11 Hydrogel-isolated cochleate formulations, process of preparation and their use for the delivery of biologically relevant molecules
PCT/US2001/002299 WO2001052817A2 (fr) 2000-01-24 2001-01-24 Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles

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US20030219473A1 (en) * 2002-03-26 2003-11-27 Leila Zarif Cochleates made with purified soy phosphatidylserine
WO2004064805A1 (fr) * 2003-01-15 2004-08-05 Biodelivery Sciences International, Inc. Preparations cochleaires de nutriants fragiles
EP1631669A2 (fr) 2003-04-09 2006-03-08 Biodelivery Sciences International, Inc. Compositions contenant des structures cochleaires, dirigees contre l'expression de proteines
US20050013854A1 (en) 2003-04-09 2005-01-20 Mannino Raphael J. Novel encochleation methods, cochleates and methods of use
EP1674094A4 (fr) * 2003-10-15 2009-12-09 Nanoegg Res Lab Inc Composition contenant des nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent
KR101089534B1 (ko) * 2003-10-15 2011-12-05 가부시키가이샤 나노에그 다가 금속 무기염 피복 레티노인산 나노입자의 입경의조정방법 및 당해 조정방법에 의해 얻어진 나노입자
US20080220028A1 (en) 2006-12-15 2008-09-11 National Research Council Of Canada Archaeal polar lipid aggregates for administration to animals
US20140220108A1 (en) * 2011-05-05 2014-08-07 University Of Medicine And Dentistry Of New Jersey Cochleate compositions and methods of making and using same
US20180153807A1 (en) * 2015-04-22 2018-06-07 Matinas Biopharma Nanotechnologies, Inc. Compositions and methods for treating mycobacteria infections and lung disease
AU2016280276A1 (en) * 2015-06-18 2017-12-07 Matinas Biopharma Nanotechnologies, Inc. Compositions and methods for treating inflammatory disease or conditions
CN109689028A (zh) 2016-07-12 2019-04-26 马丁尼斯生物制药纳米技术公司 用于中枢神经系统递送和治疗隐球菌感染的脂质卷包封的抗真菌化合物
KR101989789B1 (ko) * 2017-11-30 2019-06-18 (주) 에이치엔에이파마켐 포스파티딜세린/음이온계면활성제/염화칼슘을 이용한 코킬레이트

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US5994318A (en) * 1993-10-04 1999-11-30 Albany Medical College Cochleate delivery vehicles
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US6153217A (en) * 1999-01-22 2000-11-28 Biodelivery Sciences, Inc. Nanocochleate formulations, process of preparation and method of delivery of pharmaceutical agents

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