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WO1997030725A1 - Vecteurs d'apport de structures cochleaires - Google Patents

Vecteurs d'apport de structures cochleaires Download PDF

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Publication number
WO1997030725A1
WO1997030725A1 PCT/US1997/002632 US9702632W WO9730725A1 WO 1997030725 A1 WO1997030725 A1 WO 1997030725A1 US 9702632 W US9702632 W US 9702632W WO 9730725 A1 WO9730725 A1 WO 9730725A1
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Prior art keywords
cochleate
nutrient
formulation
drug
cochleates
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PCT/US1997/002632
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English (en)
Inventor
Raphael James Mannino
Susan Gould-Fogerite
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Raphael James Mannino
Gould Fogerite Susan
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Publication date
Priority claimed from PCT/US1996/001704 external-priority patent/WO1996025942A1/fr
Application filed by Raphael James Mannino, Gould Fogerite Susan filed Critical Raphael James Mannino
Priority to CA002246754A priority Critical patent/CA2246754C/fr
Publication of WO1997030725A1 publication Critical patent/WO1997030725A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18834—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the instant invention relates to cochleates and use thereof to stabilize biologic molecules, such as carbohydrates, vitamins, minerals, polynucleotides, polypeptides, lipids and the like.
  • Cochleates are insoluble stable lipid-divalent cation structures into which is incorporated the biologic molecule. Because cochleates can be biologically compatible, cochleates can be administered to hosts by conventional routes and can serve to deliver the biologic molecule to a targeted site in a host.
  • proteoliposomes resulting from polypeptide-cochleates have been shown to be effective i munogens when administered to animals by intraperitoneal and intramuscular routes of immunization (G. Goodman-Snitkoff, et al., J. Immunol.. Vol. 147, p.410 (1991); M.D. Miller, et al., J. EXP. Med.. Vol. 176, p. 1739 (1992)).
  • the proteoliposomes are effective delivery vehicles for encapsulated proteins and DNA to animals and to cells in culture (R.J. Mannino and S. Gould-Fogerite, Biotechniques.
  • the formulation can be used to deliver a biologic molecule to the gut for absorption or to a targeted organ, tissue or cell.
  • a suitable biologic molecule is a polynucleotide or a bioactive compound such as a lipophilic drug.
  • Suitable biologic molecules are polypeptides such as hormones and cytokines or nutrients such as vitamins, minerals, and fatty acids.
  • biologic molecules are essential oils which impart flavor.
  • a cochleate formulation comprising the following components: a) a biologically relevant molecule component to be stabilized or delivered, b) a negatively charged lipid component, and c) a divalent cation component.
  • the cochleate formulation is administered orally.
  • the instant invention further provides a cochleate formulation containing a polynucleotide wherein said polynucleotide-cochleate comprises the following components: a) a polynucleotide component, b) a negatively charged lipid component, and c) a divalent cation component.
  • the polynucleotide can be one which is expressed to yield a biologically active polypeptide or polynucleotide.
  • the polypeptide may serve as an immunogen or, for example, have enzymatic activity.
  • the polynucleotide may have catalytic activity, for example, be a ribozyme, or may serve as an inhibitor of transcription or translation, that is, be an antisense molecule. If expressed, the polynucleotide would include the necessary regulatory elements, such as a promoter, as known in the art.
  • the instant invention further provides a cochleate formulation containing a polypeptide, wherein said polypeptide-cochleate comprises the following components: a) a polypeptide component b) a negatively charged lipid component, and c) a divalent cation component.
  • a specific example is an insulin cochleate.
  • the instant invention also provides a cochleate formulation containing a lipophilic drug, wherein said drug-cochleate comprises the following components: a) at least one drug, b) at least one negatively charged lipid component, and c) at least one divalent cation component.
  • the drug may be an inhibitor of viral replication such as that used in the treatment of HERPES (acyclovir) , or one prescribed for it's antifungal effect on mycotic infections (miconazole nitrate) .
  • the drugs may also be those with specific targeted effects on different physiological systems such as anesthetics (propofol) which effect the nervous system, or immunosuppressants, such as cyclosporin A, which inhibit immune cell function.
  • Other lipophilic drugs may also be selected from the groups of anti-infectious, anti-cancer, steroidal anti-inflammatory, non-steroidal anti-inflammatory, tranquilizer, or vasodilatory agents.
  • the instant invention further provides a cochleate formulation containing a nutrient, wherein said nutrient-cochleate comprises the following components: a) at least one nutrient, b) at least one negatively charged lipid component, and c) at least one divalent cation component.
  • vitamin A- polyunsaturated fatty acids- and mineral- cochleates.
  • the instant invention further provides a cochleate formulation containing a flavor, wherein said flavor-cochleate comprises the following components: a) at least one essential oil or extract, b) at least one negatively charged lipid component, and c) at least one divalent cation component.
  • Examples include flavor substances generally associated with essential oils 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 multistep operations, costs are generally considered to be higher. The advantage of an oil/extract-cochleate would be in the stabilization of these otherwise volatile and expensive flavor substances. Flavor-cochleates can also be incorporated into consumable food preparations as flavor enhancers.
  • cochleates have a nonaqueous structure while not having an internal aqueous space, and therefore cochleates:
  • (b) can be stored lyophilized which provides the potential to be stored for long periods of time at room temperatures, which would be advantageous for worldwide shipping and storage prior to administration;
  • lipid bilayer matrix 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, non-immunogenic and non-inflammatory;
  • (g) contain high concentration of divalent cation, such as, calcium, an essential mineral;
  • cochleates are safe, the cochleates are non-living subunit formulations, and as a result the cochleates have none of the risks associated with use of live vaccines, or with vectors containing transforming sequences, such as life threatening infections in immunocompromised individuals or reversion to wild type infectivity which poses a danger to even healthy people;
  • (j) can be produced as defined formulations composed of predetermined amounts and ratios of biologically relevant molecules, including polypeptides, carbohydrates and polynucleotides, such as DNA, lipophilic drugs, and nutrients such as vitamins, minerals and fatty acids.
  • biologically relevant molecules including polypeptides, carbohydrates and polynucleotides, such as DNA, lipophilic drugs, and nutrients such as vitamins, minerals and fatty acids.
  • oral administration also are numerous.
  • An oral route has been chosen by the WHO Children's Vaccine Initiative because of ease of administration.
  • Oral vaccines are less expensive and much safer to administer than parenterally (intramuscular or subcutaneous) administered vaccines.
  • parenterally intramuscular or subcutaneous administered vaccines.
  • the use of needles adds to the cost, and also, unfortunately, in the field, needles are often reused.
  • Figure 1 is a schematic representation of a plain lipid cochleate.
  • Figure 2 shows the structure of polypeptide-lipid vesicles with integrated membrane proteins.
  • FIG. 3 summarizes the various alternative procedures for the preparation of cochleates.
  • Figures 4(A) and 4(B) show serum antibody titers in mice following oral administration of influenza polypeptide-cochleates.
  • Figure 5 is a graph showing the results of oral administration of polypeptide-cochleates when challenged with live virus.
  • Figure 6 is a graphic representation of serum antibody titers in mice following oral administration of Sendai-cochleates.
  • Figure 7 is a graph depicting the induction of antigen-specific cytotoxic splenocytes following oral administration of Sendai cochleates.
  • Figure 8 provides a series of bar graphs depicting serum glucose levels before and after oral insulin administration.
  • cochleates themselves be used as means for stabilizing and delivering biologic molecules.
  • the cochleates survive the harsh acid environment of the stomach, protecting the susceptible biologic molecules immersed therein, probably by virtue of their unique multilayered precipitate structure. It is likely that cochleates then are taken up by microfold cells (M cells) in the small intestine.
  • M cells microfold cells
  • the instant inventors have demonstrated that oral administration by drinking cochleates containing the glycoproteins and viral lipids from the surface of influenza or Sendai viruses plus phosphatidylserine and cholesterol, stimulate both mucosal and circulating antibody responses. In addition, strong helper cell (proliferative) and killer (cytotoxic) cell responses also are generated. Perhaps most impressively, oral administration of the influenza cochleates protects against intranasal challenge with live virus.
  • cochleates would survive the stomach and protect the polypeptides associated with them from the acid environment and degradative enzymes. It is known that without the presence of at least 3 mM calcium, the cochleates begin to unwind and form liposomes. It was possible, in fact likely, that the cochleates would not remain intact during the transit from the mouth, down the esophagus and through the stomach. If cochleates did come apart, they would be digested as food.
  • the cochleates would interact in an effective way with the mucosal and circulating immune systems was unknown and unexpected.
  • the cochleates deliver molecules which retain biologic activity at the delivery site within the host.
  • immune response means either antibody, cellular, proliferative or cytotoxic activities, or secretion of cytokines.
  • the term "antigen" is meant to indicate the polypeptide to which an immune response is directed or an expressible polynucleotide encoding that polypeptide.
  • Polynucleotide includes DNA or RNA, as well as antisense and enzymatically active molecules.
  • the biologically relevant molecule can be the polynucleotide itself, the transcript thereof or the translated polypeptide encoded thereby.
  • Polypeptide is any oligomer or polymer of amino acids.
  • the amino acids can be L-amino acids or D-amino acids.
  • 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, amino acids, toxins, icrobicides, microbistats, co-factors, enzymes, polypeptides, polypeptide aggregates, polynucleotides, lipids, carbohydrates, nucleotides, starches, pigments, fatty acids, fatty acids of polyunsaturated form, flavored essential oils or extracts, hormones, cytokines, viruses, organelles, steroids and other multi-ring structures, saccharides, metals, metabolic poisons, drugs and the like.
  • the instant invention also can be practiced using whole cells other subcellular replicative entities, such as viruses and viroids.
  • bacteria, yeasts, cell lines, viruses and the like can be mixed with the relevant lipid solution, caused to precipitate to yield structures wherein the cells and the like are fixed within the cochleate structure.
  • Polypeptides are suitable molecules to be incorporated with cochleates.
  • the procedure for preparing cochleates is set forth in greater detail hereinbelow.
  • the polypeptide is suspended in a suitable aqueous buffer.
  • the lipids are dried to form a thin film.
  • the aqueous buffer is added to the lipid film.
  • the vessel is vortexed and then the sample dialyzed against a cation-containing buffer.
  • cochleates carrying insulin can be obtained.
  • the insulin cochleates were made with a 1 mg/ml solution of insulin, but various other beginning concentrations of insulin can be used to obtain cochleates loaded with varying concentrations of insulin.
  • cochleates are stable structures which can withstand a variety of physiologic conditions
  • cochleates are suitable means for delivering biologic molecules, such as, polypeptides or polynucleotides, to a selected site in a host.
  • the polypeptide or polynucleotide is incorporated into and integral with the cochleate structure.
  • the polypeptide or polynucleotide which may need to be expressed, are protected from degrading proteases and nucleases.
  • the cochleates used in the instant invention can be prepared by known methods such as those described in U.S. Patent No. 4,663,161, filed 22 April 1985, U.S. Patent No. 4,871,488, filed 13 April 1987, S. Gould-Fogerite et al., Analytical Biochemistry. Vol. 148, pages 15-25 (1985); S. Gould-Fogerite et al. , Advances in Membrane Biochemistry and Bioenerqetics. edited by Kim, CH. , Tedeschi, T. , Diwan, J.J. , and Salerno, J.C., Plenum Press, New York, pages 569-586 (1988) ; S. Gould-Fogerite et al.. Gene.
  • the polynucleotide can be one which expresses a polypeptide, that is, pathogen membrane polypeptides, aberrant or atypical cell polypeptides, viral polypeptides and the like, which are known or which are suitable targets for host immune system recognition in the development of immunity thereto.
  • the polynucleotide may express a polypeptide which is biologically active, such as, an enzyme or structural or housekeeping protein.
  • the polynucleotide may be one which necessarily is not expressed as a polypeptide but nevertheless exerts a biologic effect.
  • examples are antisense molecules and RNA's with catalytic activity.
  • the expressed sequence may on transcription produce an RNA which is complementary to a message which, if inactivated, would negate an undesired phenotype, or produce an RNA which recognizes specific nucleic acid sequences and cleaves same at or about that site and again, the non-expression of which would negate an undesired phenotype.
  • the polynucleotide need not be expressed but may be used as is.
  • the polynucleotide may be an antisense molecule or a ribozyme.
  • the polynucleotide may be an immunogen.
  • the relevant coding sequence is subcloned downstream from a suitable promoter, other regulatory sequences can be incorporated as needed, in a vector which is expanded in an appropriate host, practicing methods and using materials known and available in the art.
  • pDOLHIVenv AIDS Research and Reference Reagent Program, Jan. 1991 catalog p. 113; Freed et al. J. Virol. 63: 4670 (1989)
  • pCMVHIVLenv Dr. Eric Freed, Laboratory of Molecular Immunology, NJAID, NIH
  • pDOLHIVenv AIDS Research and Reference Reagent Program, Jan. 1991 catalog p. 113; Freed et al. J. Virol. 63: 4670 (1989)
  • pCMVHIVLenv Dr. Eric Freed, Laboratory of Molecular Immunology, NJAID, NIH
  • the plasmids contain the open reading frames for the env, tat and rev coding regions of HIV-1 (LAV strain) .
  • pDOLHIVenv was constructed by introducing the Sall-Xhol fragment from the full length infectious molecular clone pNL4-3 into the Sail site of the retrovirus vector, pDOL (Korman et al. Proc. Natl. Acad. Sci. 84: 2150 (1987)). Expression is from the Moloney murine virus LTR.
  • pCMVHIVLenv was constructed by cloning the same Sall-Xhol fragment into the Xhol site of the cytomegalovirus (CMV)-based expression vector p763.
  • the polynucleotide can be configured to encode multiple epitopes or epitopes conjugated to a known immunogenic peptide to enhance immune system recognition, particularly if an epitope is only a few amino acids in size.
  • lipid present should be negatively charged.
  • One type of lipid can be used or a mixture of lipids can be used.
  • Phosphatidylserine or phosphatidylglycerol generally have been used.
  • Phosphatidylinositol also forms a precipitate which converts to liposomes on contact with EDTA.
  • a substantial proportion of the lipid can, however, be neutral or positively charged.
  • the instant inventors have included up to 40 mol% cholesterol based on total lipid present and routinely make polypeptide-lipid or polynucleotide-lipid cochleates which contain 10 mol% cholesterol and 2 0 % v i r a l m e m b r a n e l i p i d s .
  • Phosphatidylethanolamine, plain or cross-linked to polypeptides also can be incorporated into cochleates.
  • negatively charged lipid While negatively charged lipid can be used, a negatively charged phospholipid is preferred, and of those phosphatidylserine, phosphatidylinositol, phosphatidic acid and phosphatidylglycerol are most preferred.
  • One skilled in the art can determine readily how much lipid must be negatively charged by preparing a mixture with known concentrations of negative and non-negative lipids and by any of the procedures described herein, determining whether precipitates form.
  • a suitable procedure for making cochleates is one wherein a negatively charged lipid such as phosphatidylserine, phosphatidylinositol , phosphatidic acid or phosphatidylglycerol in the absence or presence of cholesterol (up to 3:1, preferably 9:1 w/w) are utilized to produce a suspension of multilamellar lipid vesicles containing or surrounded by a biologically relevant molecule (polypeptide, polysaccharide or polynucleotide, such as DNA) which are converted to small unilamellar protein lipid vesicles by sonication under nitrogen.
  • a biologically relevant molecule polypeptide, polysaccharide or polynucleotide, such as DNA
  • the biologically relevant molecule can be added to the solution following sonication.
  • the vesicles are dialyzed at room temperature against buffered divalent cation, e.g., calcium chloride, resulting in the formation of an insoluble precipitate which may be presented in a form referred to as a cochleate cylinder. After centrifugation, the resulting pellet can be taken up in buffer to yield the cochleate solution utilized in the instant invention.
  • buffered divalent cation e.g., calcium chloride
  • an amount of negatively charged lipid e.g., phosphatidylserine and optionally, cholesterol in the same proportions as above and equal to from about 1 to 10 times the weight, preferably equal to four times the weight of the viral or other additional lipids (including polyunsaturated fatty acids or essential oils) are utilized to prepare the cochleates.
  • a polypeptide, a mineral such as calcium, magnesium, barium, iron or zinc, a vitamin such as vitamins A, D, E or K, a lipophilic drug, a flavor, a carbohydrate or polynucleotide, such as DNA is added to the solution. That solution then is dialyzed against buffered divalent cation, e.g. , calcium chloride, to produce a precipitate which can be cabled a DC (for direct calcium dialysis) cochleate.
  • buffered divalent cation e.g., calcium chloride
  • LC method liposomes before cochleates
  • the initial steps involving addition of extracted polypeptide, polysaccharide,polynucleotide, such as DNA or combinations thereof, to dried down negatively charged lipid and cholesterol are the same as for the DC method.
  • the solution next is dialyzed against buffer (e.g., 2 mM TES, 2 mM L-histidine, 100 mM NaCI, pH 7.4) to form small liposomes containing the polypeptide, polynucleotide, such as DNA, and/or polysaccharide.
  • a divalent cation e.g., calcium, then is added either directly or by dialysis to form a precipitate which can consist of cochleates.
  • the divalent cation can be any divalent cation that can induce the formation of a cochleate or other insoluble lipid-antigen structures.
  • suitable divalent cations include Ca , Mg , Ba , and Zn + or
  • Fe +2 other elements capable of forming divalent ions or other structures having multiple positive charges capable of chelating and bridging negatively charged lipids.
  • Cochleates made with different cations have different structures and convert to liposomes at different rates. Because of those structural differences, the rate of release of the biologically relevant molecules contained therewith varies. Accordingly, by combining cochleates made with different cations, formulations which will release the biologically relevant molecule over a protracted period of time are obtainable.
  • the amount of biologically relevant molecule incorporated into the cochleates can vary. Because of the advantageous properties of cochleates generally, lesser amounts of biologically relevant molecule can be used to achieve the same end result as compared to using known delivery means.
  • the optimized ratio for any one use may range from a high ratio, for example, to minimize the use of a rare biologically relevant molecule, to a low ratio to obtain maximal amount of biologically relevant molecule in the cochleates.
  • Cochleates can be lyophilized and stored at room temperature indefinitely or can be stored in a divalent cation-containing buffer at 4°C for at least six months.
  • the cochleate formulations also can be prepared both with and without fusogenic molecules, such as Sendai virus envelope polypeptides.
  • fusogenic molecules such as Sendai virus envelope polypeptides.
  • Prior studies with proteoliposomes have demonstrated that cytoplasmic delivery of liposome contents requires a fusogenic liposome bilayer.
  • the exact role of Sendai virus envelope polypeptides in facilitating the immune response to polypeptide-cochleates as yet is not clear. It is preferred to use cochleates without fusogenic molecules over fusogenic molecule cochleates because of a more simple structure and ease of preparation favors eventual use in humans.
  • polynucleotides are hydrophilic molecules and cochleates are hydrophobic molecules that do not contain an internal aqueous space, it is surprising polynucleotides can be integrated into cochleates. The polynucleotides are not exposed on the surface of the cochleates because the polynucleotides are resistant to nucleases.
  • a suitable scheme for determining dosing is as follows.
  • the initial dose of polynucleotides in cochleates administered by injection to animals is selected to be about 50 ⁇ g, although it is know that as little as 2 ⁇ g of tested plasmids is effective. That dose is proposed to maximize the probability of observing a positive response following a single administration of a cochleate. Any formulations which do not elicit a response at that dose are to be considered ineffective but retained for further study.
  • Immune response or enzymatic activity are responses easily monitored when expression of the polynucleotide is required. Altered phenotype is another response for tracking efficacy of antisense or ribozyme type molecules.
  • T cell proliferation, CTL and antibody presence at specific body sites can be evaluated, using known methods, to assess the state of specific immune response.
  • Animals which fail to develop a detectable response on first exposure can be re-inoculated (boosted) to provide insights into the ability of the low dose formulations to prime the immune system for later stimulation.
  • compositions can be of solid form including tablets, capsules, pills, bulk or unit dose powders and granules or of liquid form including solutions, fluid emulsions, fluid suspensions, semisolids and the like.
  • the formulation would comprise suitable art-recognized diluents. carriers, fillers, binders, emulsifiers, surfactants, water-soluble vehicles, buffers, solubilizers and preservatives.
  • cochleates are stable of the composition.
  • cochleates can be administered orally or by instillation without concern, as well as by the more traditional routes, such as topical, subcutaneous, intradermal, intramuscular and the like.
  • Direct application to mucosal surfaces is an attractive delivery means made possible with cochleates.
  • the cochleates of the instant invention can be used as a means to transfect cells with an efficacy greater than using currently known delivery means, such as liposomes.
  • the polynucleotide cochleates of the instant invention provide a superior delivery means for the various avenue of gene therapy, Mulligan, Science 260: 926-931 (1993) .
  • Mulligan the many possibilities of treating disease by gene-based methods will be enhanced by improved methods of gene delivery.
  • the cochleates of the instant invention also serve as excellent means for delivering other biologically relevant molecules to a host.
  • biologically relevant molecules include nutrients, vitamins such as vitamins A, D, E or K, co-factors, enzymes, fatty acids such as polyunsaturated forms, minerals including divalent cations such as calcium, magnesium, zinc, iron or barium, flavors and the like.
  • the biologically relevant molecule is contained within the cochleate, in a non-aqueous environment, the biologically relevant molecule essentially is stabilized and preserved.
  • the biologically relevant molecule is added to the lipid solution and processed to form a precipitated structure comprising lipid and biologically relevant molecule.
  • hydrophilic molecules can be "cochleated", that is, can be made part of the cochleate structure, with little difficulty.
  • lipophilic biologically relevant molecules such as drugs and other therapeutic compounds
  • suitable lipophilic biologically relevant molecules are amenable to cochleation.
  • lipophilic drugs such as cyclosporin, ivermectin and amphotericin are readily cochleated.
  • lipophilic drugs which are amenable to incorporation into cochleates are acyclovir, propanidid, propofol, alphadione, echinomycine, miconazole nitrate, teniposide, didemnin B, hexamethylmelamine, taxol, taxotere, melphalan, adria ycin, 18-hydroxydeoxycorticosterone, rapamycine, prednisolone, dexamethazone, cortisone, hydrocortisone, pyroxicam, naproxen, diazepam, verapamil, nifedipine.
  • Bovine brain phosphatidylserine in chloroform was purchased from Avanti Polar Lipids, Birmingham, Alabama in glass ampules and stored under nitrogen at -20°C. Cholesterol (porcine liver) grade I, ⁇ -D-octyl-glucopyranoside (OCG) , fluorescein isothiocyanate (FITC)-dextran (average mol. wt. 67,000), metrizamide grade I, and chemicals for buffers and protein and phosphate determinations, were obtained from Sigma Chemical Company, St. Louis, Missouri. Organic solvents were purchased from Fisher Scientific Co. , Fairlawn, New Jersey. Reagents for polyacrylamide gel electrophoresis were from BioRad Laboratories, Richmond, California.
  • S1000 Sephacryl Superfine was obtained from Pharmacia, Piscataway, New Jersey. Thick walled polycarbonate centrifuge tubes (10 ml capacity) from Beckman Instruments, Palo Alto, California, were used for vesicle preparations, washes, and gradients. A bath type sonicator, Model G112SP1G, from Laboratory Supplies Company, Hicksville, New York was used for sonications.
  • Virus was grown and purified essentially as described by M.C. Hsu et al.. Virology. Vol. 95, page 476 (1979) .
  • Sendai (parainfluenza type I) and influenza (A/PR8/34) viruses were propagated in the allantoic sac of 10 or 11 day old embryonated chicken eggs.
  • Eggs were inoculated with 1-100 egg infectious doses (IO 3 to 10 5 viral particles as determined by HA titer) in 0.1 ml of phosphate buffered saline (0.2 gm/L KC1, 0.2 gm/L KH 2 P0 4 , 8.0 gm/L NaCI, 1.14 gm/L Na 2 H-P0 4 , 0.1 gm/L CaCl 2 , 0.1 gm/L MgCl 2 6H 2 0 (pH 7.2)). Eggs were incubated at 37°C for 48 to 72 hours, followed by incubation at 4°C for 24 to 48 hours.
  • phosphate buffered saline 0.2 gm/L KC1, 0.2 gm/L KH 2 P0 4 , 8.0 gm/L NaCI, 1.14 gm/L Na 2 H-P0 4 , 0.1 gm/L CaCl 2 , 0.1 gm/L MgC
  • Allantoic fluid was collected and clarified at 2,000 rpm for 20 minutes at 5°C in a Damon IEC/PR-J centrifuge. The supernatant was then centrifuged at 13,000 rpm for 60 minutes. This and all subsequent centrifugations were performed in a Sorvall RC2-B centrifuge at 5°C using a GG rotor. The pellets were resuspended in phosphate buffered saline (pH 7.2) by vortexing and sonicating, followed by centrifugation at 5,000 rpm for 20 minutes. The pellet was resuspended by vortexing and sonicating, diluting, and centrifuging again at 5,000 rpm for 20 minutes.
  • phosphate buffered saline pH 7.2
  • the two 5,000 rpm supernatants were combined and centrifuged at 13,000 rpm for 60 minutes.
  • the resulting pellets were resuspended in phosphate-buffered saline by vortexing and sonicating, aliquoted, and stored at -70°C.
  • Sterile technique and materials were used throughout viral inoculation, isolation, and purification.
  • Virus stored at -70°C was thawed, transferred to sterile thick-walled polycarbonate tubes and diluted with buffer A (2 mM TES, 2 mM L-histidine, 100 mM NaCI (pH 7.4)). Virus was pelleted at 30,000 rpm for 1 hour at 5 ⁇ C in a Beckman TY65 rotor. The supernatant was removed and the pellet resuspended to a concentration of 2 mg viral protein per ml of extraction buffer (EB) (2 M NaCI, 0.02 M sodium phosphate buffer (pH 7.4)) by vortexing and sonicating.
  • EB extraction buffer
  • the nonionic detergent ⁇ -D-octyl-glucopyranoside was then added to a concentration of 2% (w/v) .
  • the suspension was mixed, sonicated for 5 seconds and placed in a 37°C water bath for 45 minutes. At 15, 30 and 45 minute incubation times, the suspension was removed briefly for mixing and sonication. Nucleocapsids were pelleted by centrifugation at 30,000 rpm for 45 minutes in a TY65 rotor. The resulting clear supernatant was removed and used in the formation of viral glycoprotein-containing cochleates.
  • the final dialysis routinely used is 6 mM Ca * , although 3 mM Ca * is sufficient and other concentrations may be compatible with cochleate formation.
  • the ratio of dialyzate to buffer for each change was a minimum of 1:100.
  • the resulting white calcium-phospholipid precipitates have been termed DC cochleates.
  • the suspension When examined by light microscopy (x 1000, phase contrast, oil) , the suspension contains numerous particulate structures up to several microns in diameter, as well as needle-like structures.
  • influenza virus was grown, purified, and the glycoproteins and lipids extracted and isolated as described in Example 1. Protein-cochleates were made according to the "LC cochleate" procedure described above.
  • Cochleate vaccines containing the glycoproteins and lipids from the envelope of influenza virus and phosphatidylserine and cholesterol were given to mice by gradually dispensing 0.1 ml liquid into the mouth and allowing it to be comfortably swallowed.
  • Figures 4(A) (from Experiment A) and 4(B) (from Experiment B) show resulting total circulating antibody levels specific for influenza glycoproteins, as determined by ELISA.
  • Antibody titer is defined as the highest dilution that still gives the optimal density of the negative control.
  • the antibody response is long lived. Titers 13 weeks after the third immunization (Figure 4(A) , bleed 5) and 12 weeks after the second immunization ( Figure 4(B), bleed 4) remained the same or within one dilution higher or lower than seen at 3 weeks after the previous boost.
  • mice described in Experiment B of Example 2 were immunized with cochleates at 0, 3 and 15 weeks.
  • the immunized mice were challenged by intranasal appli ,cation of 2.5 x 109 parti.cles of influenza virus at 16 weeks.
  • Three days after viral challenge mice were sacrificed, and lungs and trachea were obtained.
  • the entire lung or trachea was triturated and sonicated, and aliquots were injected into embryonated chicken eggs to allow amplification of any virus present.
  • allantoic fluid was obtained from individual eggs and hemagglutination (HA) titers were performed.
  • HA hemagglutination
  • mice were also challenged with live influenza intranasally following oral cochleate administration in Experiment A of Example 2. Lungs were obtained three days later and cultured to detect presence of virus. The combined data for the two experiments is given in Table 1. The results also are shown graphically in Figure 5.
  • mice from Experiment B 1. Mice from Experiment B. 2. Mice from Experiment B. 3. Mice from Experiments A and B.
  • mice in Table 1 show that all five of the unvaccinated mice had sufficient virus in the trachea to infect the embryonated chicken eggs (greater than 10 particles per trachea or at least one egg infectious dose (EID) per 0.1 ml of suspension) .
  • the oral vaccine provided a high degree of protection from viral replication in the trachea.
  • All mice in groups 1, 3 and 5 of Experiment B were negative for virus.
  • Two mice in group 2, 1 in group 4, and 4 in group 6 (the lowest vaccine dose) of Experiment B had sufficient virus to test positive in this very sensitive assay used to detect presence of virus.
  • the oral protein cochleate vaccine also provided protection against viral replication in the lungs. All twenty mice which received the four highest doses of vaccine were negative for virus when lung suspensions were cultured in embryonated chicken eggs (Table 1) . All mice in the groups immunized with 6.25 ⁇ g and 3.1 ⁇ g glycoproteins and all mice in the unvaccinated control were positive for virus.
  • C57BL/6 mice were given cochleates containing Sendai virus glycoproteins orally at 0 and 3 weeks. They were bled at 0 (bleed 1), 3 (bleed 2), and 6 (bleed 3) weeks. Group 1 received approximately 50 ⁇ g protein, Group 2 about 25 ⁇ g. Group 3 about 12.5 ⁇ g. Group 4 about 6.25 ⁇ g, and Group 5 (negative control) received 0 ⁇ g protein.
  • the levels of Sendai specific antibodies in the serum pooled from 5 mice in each dose group were determined by ELISA. The results are shown in Figure 6. It can be seen that strong antibody responses were generated, that the magnitude of the response was directly related to the immunizing dose, and that the magnitude of the response increased (boosted) after a second immunization.
  • the response was extremely long-lived.
  • the response is predominantly IgG, indicative of the involvement in T cell help and establishment of long-term memory cells associated with a secondary immune response.
  • the lowest dose which initially had the lowest response now had the highest circulating antibody levels. This may be due to the immune system's down regulation of the very high responses originally but allowing the low response to slowly climb. This may also indicate a persistence and slow release of antigen. It is also interesting and consistent with the use of the oral route of immunization that significant IgA titers are generated and maintained.
  • a 50 ⁇ g protein dose of Sendai glycoprotein-containing cochleates was given orally. Two weeks later the animal (BALB/c mouse) was sacrificed and spleen cells obtained. Cytolytic activity of the spleen cells was measured by their ability to cause the release of chromium-51 from target cells presenting Sendai antigens. The non-immunized mouse did not kill Sendai virus (SV) pulsed cells with in culture restimulation (N/SV/SV) or non-Sendai presenting cells (N/N/N) . ( Figure 7) In contrast, Sendai cochleate immunized mice killed SV pulsed targets to a very high degree and non-pulsed targets to a lesser degree.
  • Sendai cochleate immunized mice killed SV pulsed targets to a very high degree and non-pulsed targets to a lesser degree.
  • Cytolytic activity is crucial to clearance of cells infected viruses, or intracellular parasites or to cancer cells. It is a highly desirable activity for a vaccine to induce, but classically has not been seen with most non-living vaccines. This is an important feature of protein-cochleate vaccines.
  • mice Eight week old BALB/c female mice were immunized IM twice with various polynucleotide-cochleate formulations, polynucleotide alone and controls and then splenocytes from the mice were tested for the ability to proliferate in response to a protein encoded by the polynucleotide.
  • Cochleates with and without fusogenic Sendai virus protein were prepared as described hereinabove.
  • the polynucleotide used was the pCMVHIVLenv plasmid.
  • the solution containing lipid and extracted Sendai virus envelop proteins as described hereinabove and polynucleotide were mixed at a 10:1 (w/w) ratio and 50:1 (w/w) ratio. That protocol yielded four groups, cochleate/DNA, 10:1; cochleate/DNA, 50:1; SV-cochleate/DNA, 10:1; and SV-cochleate/DNA, 50:1. Naked DNA was used at a rate of 10 ⁇ g/mouse and 50 ⁇ g/mouse.
  • the control was buffer alone. Mice were immunized twice, 15 days apart at 50 ⁇ l/mouse.
  • Splenocytes were obtained and tested in a T-cell proliferation assay using tritiated thymidine, as known in the art.
  • Control cultures contained no antigen or con A.
  • the antigen used was pl ⁇ peptide, at 1 mM, 3 mM and 6 mM.
  • Cells were harvested at days 2, 4 and 6 following preparation of the splenocyte cultures.
  • the DNA concentration range at the 10:1 ratio was about 120-170 ⁇ g/ml.
  • the DNA concentration was about 25-35 ⁇ g/ml.
  • the polynucleotide-cochleates were exposed to micrococcal nuclease and little or no nucleic acid degradation was observed.
  • the polynucleotide encapsulation efficiency was found to be about 50% based on quantification of free DNA from lipid, that is present in the supernatant following a precipitation reaction. After washing the precipitate and opening the structures by removing cation about 35% of the DNA was recovered.
  • splenocytes from animals immunized as described in Example 4 were tested for antigen specific cytotoxic activity using a chromium release assay using labelled H-2 compatible target cells known to express an HIV protein, such as gpl60.
  • the responder cells can be stimulated by brief exposure to purified HIV peptides.
  • mice were given insulin cochleate samples orally. Serum glucose levels were measured at 0 time, (prior to cochleate administration) , 30 min. and 60 min. post administration using standard methods. Cochleate formulations of Example 6 with a starting concentration of 1 mg insulin/ml solution were used. Each mouse was administered 100 ul or 200 ul of the designated preparations as indicated. For comparison, one mouse was given the standard commercial human insulin, Humulin R, by intraperitoneal administration. Sample Volume Given Serum Glucose mg/d ⁇ l
  • Oral administration of insulin affected serum glucose levels.
  • Insulin cochleates as produced in Example 6 were fed orally to three-month-old female BALB/c mice made diabetic through intraperitoneal injection of streptozotocin, practicing known methods. Two days after exposure to streptozotocin, the mice were allocated into groups of five and administered with oral insulin cochleates at 200 ⁇ l per mouse. Other mice were injected with 2 IU of Humulin R.
  • Serum samples were obtained at time 0, prior to insulin dosing, and two hours post insulin administration. Glucose levels were measured using a kit from Sigma (St. Louie) . Control animals were untreated, that is, received no streptozotocin or Figure 8. Orally administered insulin, simply by drinking, was effective in reducing blood glucose levels. No reduction in blood glucose was observed in control animals.
  • Vitamin A (retinol) is sensitive to air- oxidation and is inactivated by ultraviolet light. Stability of vitamin A is enhanced by its encapsulation into the intra-bilayers of cochleates. Incorporation of vitamin A into the intra-bilayer phospholipid region of a cochleate was achieved as follows: appropriate proportions of vitamin A, phosphatidylserine and cholesterol were dissolved in an organic solvent such as chloroform or a 1:1 methanol:chloroform mixture. The solvent was then removed under reduced pressure to yield a lipid-vitamin film. Buffer was added and the mixture was vortexed for several minutes.
  • Cinnamon oil in cochleates Flavors are volatile and sensitive to oxidation. Controlled release and enhanced physical and chemical stability can be achieved by the encapsulation of flavors into cochleates.
  • Incorporation of a flavor based on cinnamon oil into the intra-bilayer phospholipid region of a cochleate can be achieved as follows: phosphatidylserine and cholesterol were dissolved in an organic solvent such as chloroform or a 1:1 methanol:chloroform mixture, and an appropriate proportion of cinnamon oil dissolved in ethanol was added. The solvent was then removed under reduced pressure to yield a film. Buffer was added and the mixture was vortexed for several minutes. The resultant dispersion was then dialyzed at room temperature as in example 2.A against three changes of buffer A containing 3 mM CaCl 2 . Cinnamon oil- cochleates were obtained as a precipitate.
  • acyclovir/phosphatidylserine in an appropriate drug to lipid ratio was dissolved in an organic solvent such as chloroform or a 1:1 methanol:chloroform mixture. The solvent was then removed under reduced pressure to yield a homogenous film. Buffer was added and the mixture was vortexed for several minutes at a temperature above the transition temperature of the lipid. The excess drug, if any, was separated from the liposome containing acyclovir by repeated washing with PBS and centrifugation, the supernatant was discarded, and the pellet resuspended in PBS. The liposome suspension was then dialyzed at room temperature as in example 2.A against three changes of buffer A containing 3 mM CaCl 2 . Acyclovir- cochleates were obtained as a precipitate.

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Abstract

La présente invention se rapporte à des structures cochléaires comportant (a) un composant moléculaire biologiquement utile, (b) un composant lipidique négativement chargé et (c) un composant cationique divalent. Ladite structure cochléaire a une durée de conservation prolongée, même sous sa forme déshydratée. Elle peut être avantageusement ingérée. La molécule biologiquement utile peut être un polynucléotide ou un polypeptide.
PCT/US1997/002632 1996-02-22 1997-02-21 Vecteurs d'apport de structures cochleaires WO1997030725A1 (fr)

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Cited By (12)

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WO1999058158A1 (fr) * 1998-05-11 1999-11-18 Nycomed Imaging As Milieux de contraste
WO2000035421A2 (fr) * 1998-12-14 2000-06-22 University Of Maryland Formulations cochleaires d'integration de proteine-adn et techniques de transformation de cellules
WO2001052817A2 (fr) * 2000-01-24 2001-07-26 Biodelivery Sciences, Inc. Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles
US6592894B1 (en) 1999-01-22 2003-07-15 Biodelivery Sciences International, Inc. Hydrogel-isolated cochleate formulations, process of preparation and their use for the delivery of biologically relevant molecules
WO2004064805A1 (fr) * 2003-01-15 2004-08-05 Biodelivery Sciences International, Inc. Preparations cochleaires de nutriants fragiles
WO2004091572A2 (fr) * 2003-04-09 2004-10-28 Biodelivery Sciences International, Inc. Compositions contenant des structures cochleaires, dirigees contre l'expression de proteines
EP1674094A1 (fr) * 2003-10-15 2006-06-28 LTT Bio-Pharma Co., Ltd. Composition contenant des nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent
EP1674093A1 (fr) * 2003-10-15 2006-06-28 LTT Bio-Pharma Co., Ltd. Procede de controle de la taille particulaire de nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent et nanoparticules obtenues par ledit procede
AU2007200813B2 (en) * 1999-01-22 2010-06-03 Biodelivery Sicencies, Inc. Novel hydrogel isolated cochleate formulations, process of preparation and their use for the delivery of biologically relevant molecules
WO2011016043A2 (fr) 2009-08-06 2011-02-10 Technion Research & Development Foundation Ltd. Libération de médicament antibiotique et potentialisation
US8642073B2 (en) 2003-04-09 2014-02-04 Biodelivery Sciences International, Inc. Encochleation methods, cochleates and methods of use
US9566237B2 (en) 2002-11-01 2017-02-14 Rutgers, The State University Of New Jersey Geodate delivery vehicles

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US4906476A (en) * 1988-12-14 1990-03-06 Liposome Technology, Inc. Novel liposome composition for sustained release of steroidal drugs in lungs
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US4078052A (en) * 1976-06-30 1978-03-07 The United States Of America As Represented By The Secretary Of Health, Education And Welfare Large unilamellar vesicles (LUV) and method of preparing same
US4725442A (en) * 1983-06-17 1988-02-16 Haynes Duncan H Microdroplets of water-insoluble drugs and injectable formulations containing same
US4874795A (en) * 1985-04-02 1989-10-17 Yesair David W Composition for delivery of orally administered drugs and other substances
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US4871488A (en) * 1985-04-22 1989-10-03 Albany Medical College Of Union University Reconstituting viral glycoproteins into large phospholipid vesicles
US4906476A (en) * 1988-12-14 1990-03-06 Liposome Technology, Inc. Novel liposome composition for sustained release of steroidal drugs in lungs
US5571517A (en) * 1990-08-13 1996-11-05 Yesair; David W. Mixed lipid-bicarbonate colloidal particles for delivering drugs or calories

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058158A1 (fr) * 1998-05-11 1999-11-18 Nycomed Imaging As Milieux de contraste
WO2000035421A2 (fr) * 1998-12-14 2000-06-22 University Of Maryland Formulations cochleaires d'integration de proteine-adn et techniques de transformation de cellules
WO2000035421A3 (fr) * 1998-12-14 2000-11-09 Univ Maryland Formulations cochleaires d'integration de proteine-adn et techniques de transformation de cellules
US6340591B1 (en) 1998-12-14 2002-01-22 University Of Maryland Integrative protein-DNA cochleate formulations and methods for transforming cells
US6592894B1 (en) 1999-01-22 2003-07-15 Biodelivery Sciences International, Inc. Hydrogel-isolated cochleate formulations, process of preparation and their use for the delivery of biologically relevant molecules
US20120294901A1 (en) * 1999-01-22 2012-11-22 University Of Medicine And Dentistry Of New Jersey Novel cochleate formulations
US20140220109A1 (en) * 1999-01-22 2014-08-07 Rutgers, The State University Of New Jersey Novel cochleate formulations
AU2007200813B2 (en) * 1999-01-22 2010-06-03 Biodelivery Sicencies, Inc. Novel hydrogel isolated cochleate formulations, process of preparation and their use for the delivery of biologically relevant molecules
WO2001052817A2 (fr) * 2000-01-24 2001-07-26 Biodelivery Sciences, Inc. Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles
WO2001052817A3 (fr) * 2000-01-24 2002-02-21 Biodelivery Sciences Inc Nouvelles formulations cochleaires, procede de preparation et utilisation de celles-ci dans l'administration de molecules biologiquement utiles
US9566237B2 (en) 2002-11-01 2017-02-14 Rutgers, The State University Of New Jersey Geodate delivery vehicles
WO2004064805A1 (fr) * 2003-01-15 2004-08-05 Biodelivery Sciences International, Inc. Preparations cochleaires de nutriants fragiles
US9974745B2 (en) 2003-04-09 2018-05-22 Rutgers, The State University Of New Jersey Encochleation methods, cochleates and methods of use
US8642073B2 (en) 2003-04-09 2014-02-04 Biodelivery Sciences International, Inc. Encochleation methods, cochleates and methods of use
US9259392B2 (en) 2003-04-09 2016-02-16 Rutgers, The State University Of New Jersey Cochleate compositions directed against expression of proteins
WO2004091572A3 (fr) * 2003-04-09 2005-05-12 Biodelivery Sciences Internati Compositions contenant des structures cochleaires, dirigees contre l'expression de proteines
WO2004091572A2 (fr) * 2003-04-09 2004-10-28 Biodelivery Sciences International, Inc. Compositions contenant des structures cochleaires, dirigees contre l'expression de proteines
US8546555B2 (en) 2003-04-09 2013-10-01 Biodelivery Sciences International, Inc. Cochleate compositions directed against expression of proteins
EP1674094A1 (fr) * 2003-10-15 2006-06-28 LTT Bio-Pharma Co., Ltd. Composition contenant des nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent
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
EP1674093A4 (fr) * 2003-10-15 2009-12-09 Nanoegg Res Lab Inc Procede de controle de la taille particulaire de nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent et nanoparticules obtenues par ledit procede
EP1674093A1 (fr) * 2003-10-15 2006-06-28 LTT Bio-Pharma Co., Ltd. Procede de controle de la taille particulaire de nanoparticules d'acide retinoique revetues d'un sel inorganique metallique polyvalent et nanoparticules obtenues par ledit procede
WO2011016043A2 (fr) 2009-08-06 2011-02-10 Technion Research & Development Foundation Ltd. Libération de médicament antibiotique et potentialisation

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