AU753008B2 - Cochleate delivery vehicles - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT(S): Albany Medical College and University of Medicine and Dentistry of New Jersey ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: COCHLEATE DELIVERY VEHICLES The following statement is a full description of this invention, including the best method of performing it known to us: Q:\OPER\TDO\2292669.DIV 8/5/0 1A COCHLEATE DELIVERY VEHICLES FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION Plain lipid cochleates (Figure 1) have been described previously. Protein-cochleates or peptide-cochleates have been described heretofore and patented by the instant inventors, as 20 intermediate structures which can be converted to protein-lipid vesicles (proteoliposomes) (Figure 2) by the addition of calcium chelating agents (see U.S. Pat. No. 4,663,161 and U.S. Pat. No.

4,871,488, the disclosures of which expressly are incorporated herein by reference). Freeze-fracture electron micrographs of protein-cochleates containing Sendai glycoproteins made by the DC method show the rolled up lipid bilayer structures with a "bumpy" surface. Plain phospholipid cochleates are smooth in that type of preparation.

2 The proteoliposomes resulting from polypeptide-cochleates have been shown to be effective immunogens when administered to animals by intraperitoneal and intramuscular routes of immunization Goodman-Snitkoff, et al., J.

Immunol., Vol. 147, p.

4 1 0 (1991); M.D. Miller, et al., J. Exp. Med., Vol. 176, p. 1739 (1992)).

Further, when the glycoproteins of Sendai or influenza virus are reconstituted by that method, the proteoliposomes are effective delivery vehicles for encapsulated proteins and DNA to animals and to cells in culture Mannino and S.

Gould-Fogerite, Biotechniques, Vol. 6, No. 1, pp. 682-690 (1988); S. Gould-Fogerite et al., Gene, Vol. 84, p. 429 (1989); M.D. Miller, et al., J.

Exp. Med., Vol. 176, p. 1739 (1992)).

It would be advantageous to provide a means for stabilizing or preserving biologic molecules in a form that is stable at room temperature, capable of desiccation and is suitable for oral administration. For example, it would be beneficial to have a formulation for stabilizing polynucleotides and which could be used for delivering polynucleotides to a cell.

25 SUMMARY OF THE INVENTION o Accordingly, in one embodiment the instant invention provides a means for stabilizing biologic molecules to yield a formulation with prolonged shelf life, which can be made into powder form and which later can be rehydrated to yield a biologically active molecule.

In another embodiment the instant invention provides a formulation suitable for use as a vehicle to administer a biologically active molecule to a host. The formulation can be used to 3 deliver a biologic molecule to the gut for absorption or to a targeted organ, tissue or cell.

A suitable biologic molecule is a polynucleotide.

Other suitable biologic molecules are polypeptides such as hormones and cytokines.

Yet other suitable biologic molecules are bioactive compounds such as drugs.

In one embodiment the invention provides 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.

In a preferred embodiment, the cochleate formulation is administered orally.

In one embodiment the instant invention further provides a cochleate formulation containing a polynucleotide, wherein said polynucleotidecochleate comprises the following components: a) a polynucleotide component, b) a negatively charged lipid component, 25 and c) a divalent cation component.

The polynucleotide can be one which is expressed to yield a biologically active polypeptide or polynucleotide. Thus, the 30 polypeptide may serve as an immunogen or, for o* 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 Ss polynucleotide would include the necessary 4 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 advantages of cochleates are numerous.

The cochleates have a nonaqueous structure while not having an internal aqueous space, and therefore 15 cochleates: are more stable than liposomes because the lipids in cochleates are less susceptible to oxidation; 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; maintain structure even after lyophilization, whereas liposome structures are destroyed by lyophilization; exhibit efficient incorporation of biological molecules, particularly with hydrophobic moieties into the lipid bilayer of the cochleate structure; have the potential for slow or timed release of the biologic molecule in vivo as cochleates slowly unwind or otherwise dissociate; have a lipid bilayer matrix which serves as a carrier and is composed of simple lipids which are found in animal*and plant cell membranes, so 5 that the lipids are non-toxic, non-immunogenic and non-inflammatory; contain high concentration of divalent cation, such as, calcium, an essential mineral; 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; are produced easily and safely; and can be produced as defined formulations 15 composed of predetermined amounts and ratios of biologically relevant molecules, including polypeptides, carbohydrates and polynucleotides, such as DNA.

The advantages of 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 25 vaccines. The use of needles adds to the cost, and also, unfortunately, in the field, needles are often reused.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a plain lipid cochleate.

Figure 2 shows the structure of polypeptide-lipid vesicles with integrated membrane proteins.

Figure 3 summarizes the various alternative procedures for the preparation of cochleates.

6 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 15 oral insulin adiministration.

DETAILED DESCRIPTION OF THE INVENTION The instant inventors have now found surprisingly and have demonstrated that cochleates themselves be used as means for stabilizing and delivering __biolgi±~.moleculer. 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.

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 7 administration of the influenza cochleates protects against intranasal challengs with live virus.

Those results are unexpected for a number of reasons.

It was not known and was not expected that the 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, 15 they would be digested as food.

Also, having survived the stomach, that the cochleates would interact in an effective way with the mucosal and circulating immune systems was unknown and unexpected. Everyone ingests large quantities of proteins, fats and sugars on a daily basis which simply get digested and used as fuel, without stimulating any kind of mucosal or circulating immune responses. Thus, the cochleates deliver molecules which retain biologic activity at 25 the delivery site within the host.

As used herein, the term "immune response" means either antibody, cellular, proliferative or cytotoxic activities, or secretion of cytokines.

Also, as used herein, 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.

Thus the biologically relevant molecule can be the polynucleotide itself, the transcript thereof or the translated polypeptide encoded thereby.

P.\OPER\Fs\3 2599-1I-spc.doc-2(/lk/I/I2 8 "Polypeptide" is an 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. Thus, examples include, vitamins, minerals, 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.

In particular, said biologically relevant molecule includes a polynucleotide component, a 20 nutrient, a mineral, an amino acid, a vitamin, a lipid, a fatty acid, a saccharide, a carbohydrate, a protein or polypeptide, a polypeptide aggregate, a drug, a pigment, a metal, an organelle, a multiring structure, an enzyme, a cofactor, an 25 immunogen, a nucleotide, a toxin, a cell, a subcellular replicative entity, a virus, deoxyribonucleic acid, a catalytic ribonucleic acid, an antisense molecule, a plasmid, phospholipid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, calcium, magnesium, zinc, 7fU-, barium, iron, alanine, valine, leucine, isoleucine, P:\OPER\F12 5994 sp. d.21 IA X/02 -8A proline, phenylalanine, tryptophan, methionine, vitamin A, vitamin D, vitamin E, vitamin K, a saturated, a polyunsaturated fatty acid, a *hormone, maltose, lactose, sucrose, glycogen, ca-amylose amylopectin, a lipopolysaccharide, a glycodrug, a fiber supplement, starch, acarbose, topiramate, sporonox, ganglioside, hyaluronic acid, hydrolyzed corn starch, cellulose, soy fiber, acyiglycerol, a phosphoglyceride, a sphingolipid, a terpene, a steroid, a prostaglandin, triacylglycerol, galactosyldiacylglycerol, cardiolipin, phosphatidylcholine, phosphatidylethanolamine, sphingosine, dihydrosphingosine, squalene, carotenoid, cholic acid, estrone, prostanoic acid, ergosterol, a food additive, an acidulant, alkalizer, anti-caking, antimicrobial, antioxidant, bleaching, buffer, carrier, humectant, pectin, oxidizing agent, pertussis, tetanus, diphtheria, cholera, a toxoid, a cytokine, a hormone, ANG, AR, BDNE, BCT, C10, CINC-l, CNTF, l3-ECGF, EGF, ENA-78, Eotaxin, Epo, FGF acidic, FGF basic, FGF-4, EGF-6, FGE-7/KGF, FGF-8b, FGF-8b, FGF-9, Flt-3 ligand, C-CSF, G-CSF, GDNF, GM-CS', sgp 130, GR~ca, CR013, GROy, HB-EGF, HCCA, HGE, HRG-ct, 1-309, INFy, IGF-I, IGF-II, IL-la, IL-13, IL-lra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-ll, IL-12, IL-13, IL-15, 11-17, IP-lO, JE/MCP-1, KC, Leptin, LIE, M-CSF, MCP-1, MCP-2, MCP-3, MIF, MIPla, MIP-113, MIP-2, ML, 13-NGF, NT-3, NT-4, OSM, PBSF, PG-ECGF, PDGF, PDGF-AA, PDGF-AB, PDGF'-BB, P1GB, PTN, Rantes, 5dB', SDF-113, SLPI, TGB-a, TGB'- 131 P, TGB-13.2, TGB'-[2, TNB-a, TNB'-1. Tpo, VEG', a P\OPER\Fs3251)9.-n-spc.do.-2m416/12 8B conjugated protein, a lipoprotein, a glycoprotein, a phosphoprotein, an hemoprotein, a flavoprotein, a metalloprotein, plasma P-lipoprotein, y-globulin, plasma orosomucoid, casein, hemoglobin, cytochrome C, catalase, succinate dehydrogenase, D-amino acid oxidase, ferritin, cytochrome oxidase, alcohol dehydrogenase, xanthine oxidase, insulin, somatotropin (growth hormone), somatomedin-l, adrenocorticotropic hormone (ACTH), epinephrine, follicle stimulating hormone (FSH), leutinizing hormone human chorionic gonadotropin (HCG), TSH releasing hormone (TRH), thyroid stimulating hormone (TSH), thyroid hormone (TH), erythropoietin, epidermal growth factor, parathormone, insulin, cortisol, estrodiol, progesterone, testosterone, a lipid soluble drug, an anti-viral, an anaesthetic, an anti-infectious, an anti-fungal, an anti-cancer, an immunosuppressant, a steroidal anti-inflammatory, a non-steroidal anti-inflammatory, a tranquilizer, a S. vasodilatory agent, a small-molecule receptor antagonist, a small-molecule receptor agonist, a .steroid, a microbicide, a microbistat, a metabolic poison, acyclovir, propanidid, propofol, alphadione, echinomycine, miconazole nitrate, teniposide, vitamin B, hexamethylmelamine, taxol, taxotere, melphalan, adriamycin, .18-hydroxydeoxycorticosterone, rapamycine, Sprednisolone, dexamethazone, cortisone, hydrocortisone, pyroxicam, naproxen, diazepam, verapamil, nifedipine, tachykinin NK-1 antagonist, .erythrpoietin peptide agonist, sequenavir, Serythropoietin peptide agonist, sequenavir, P:'OPER\F,125994-spe.doc-20/AW02 8C androsterone, spironolactone, oxymetholone, testosterone, ethynyl estradiol, mestranol, ethynodiol diacetate, hydroxymethylprogesterone, progesterone, BiCNU, CCNU, cytarabine, cyclophosphamide, mitomycin, doxorubicin, bulsulfan, benzodepa, triethylenethiophosphoramide, uracil mustard, lomustine, bleomycin, dactinomycin, tubercidin, methotrexate, mercaptopurine, cytarabine, thioguanine, vinblastine, mitotane, nilutamide, tamoxifen, cyclosporine A, amphotericin B, ivermectin, rhodopsin, carotene, betalaine, an annatto color, carotenoid, cochineal, saffron, +2 M+21 +2 C+21 +g2 +2 tumeric, carmel, Fe+2 MO+2 Zn+2, CU+2, Mg+ Mn+2 Au+, Li+, Na a nucleus, endoplasmic reticulum, peroxisomes, mitochondria, plastids, golgi, lysosomes, endosomes, secretory vesicles, plasma membrane, cytosol, and subsets thereof, such as soluble and membrane bound fractions, caffeine, nicotine, ergotamine tartrate, adenosine deaminase, alcohol dehydrogenase, DNA helicase, lipoprotein lipase, recombinase activator gene, DNA ligase, coenzyme Q, coenzyme A, flavin mononucleotide, biocytin, tetrahydrofolate coenzyme, a metal ion, muramyl dipeptide, peptidoglycan, mycolic acid, AMP, CMP, GMP, UMP, dTTP, dCTP, dGTP or dATP.

The instant invention also can be practiced using whole cells other subcellular replicative entities, such as viruses and viroids. Hence, .9 bacteria, yeasts, cell lines, viruses and the like can be mixed with the relevant lipid solution, caused to precipitate to yield structures wherein I N the cells and the like are fixed within the P:\OPER\F.isl3259 -)4)-spe.doc-20/I,/2 8D 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. Then the aqueous buffer is added to the lipid film. The vessel is vortexed and then the sample dialyzed against a cation-containing buffer.

In that way, for example, cochleates carrying insulin can be obtained. The insulin cochleates were made with a 1 mg/ml solution of insulin, but @0 g* e S*.0 o* 9 various other beginning concentrations of insulin can be used to obtain cochlsates loaded with varying concentrations of insulin.

Recent studies indicate that the direct injection of DNA plasmids can lead to the expression of the proteins encoded by those plasmids resulting in humoral and cell mediated immune responses, see, for example, Wang et al., Proc. Natl Acad. Sci. 90: 4156-4160 (1993); Zhu et al., Science 261: 209-211 (1993). Those studies indicate that DNA vaccines could provide a safe and effective alternative for human vaccination. Those studies also suggest that DNA vaccines could benefit from simple, more efficient 15 delivery systems.

The use of lipids to facilitate the delivery, entry and expression of DNA in animal cells is well documented, see, for example, Philip et al., Mo.

Cell Biol. 14: 2411-2418 (1994). Indeed, DNA-lipid complexes currently form the basis for a number of human gene therapy protocols.

Because 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. Thus 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); 10 S. Gould-Fogerite et al., Advances in Membrane Biochemistry and Bioenergetics, edited by Kim, Tedeschi, Diwan, and Salerno, J.C., Plenum Press, New York, pages 569-586 (1988); S. Gould-Fogerite et al., Gene, Vol. 84, pages 429-438 (1989); Liposome Technology, 2nd Edition, Vol. I, Liposome Preparation and Related Techniques, Vol. II, Entrapment of Drugs and Other Materials, and Vol. III, Interactions of Liposomes with the Biological Milieu, all edited by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp 69-80, Chapter •0 pp 167-184, and Chapter 17, pp. 261-276 (1993); and R.J. Mannino and S. Gould-Fogerite, Liposome Mediated Gene Transfer, Biotechnipues, Vol. 6, No. 1 (1988), pp. 682-690.

The polynucleotide can be one which expresses Sa 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 25 which is biologically active, such as, an enzyme or structural or housekeeping protein.

Also, 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. Thus, 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 11 non-expression of which would negate an undesired phenotype.

The polynucleotide need not be expressed but may be used as is. Thus, the polynucleotide may be an antisense molecule or a ribozyme. Also, the polynucleotide may be an immunogen.

Thus, for polynucleotides, 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.

For example, two plasmids, pDOLHIVenv (AIDS Research and Reference Reagent Program, Jan. 1991 S 15 catalog p. 113; Freed et al. J. Virol. 63: 4670 (1989)) and pCMVHIVLenv (Dr. Eric Freed, Laboratory of Molecular Immunology, NJAID, NIH) are suitable expression plasmids for use in polynucleotide-cochleates.

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 SalI-XhoI fragment from the full length infectious molecular clone pNL4-3 into the Sal 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 SalI-XhoI fragment into the XhoI 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.

12 To form cochleate precipitates, a majority of the 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 membr a n e i p i d s 15 Phosphatidylethanolamine, plain or cross-linked to polypeptides, also can be incorporated into cochleates.

While negatively charged lipid can be used, a negatively charged phospholipid is preferred, and of those phosphatidylserine, phosphatidylinositol, ~phosphatidic acid and phosphatidylglycerol are most S&a. preferred.

One skilled in the art can determine readily how much lipid must be negatively charged by 25 preparing a mixture with known concentrations of negative and non-negative lipids and by any of the procedures described herein, determining whether precipitates form.

There are several kno',n procedures for making the cochleates of the instant invention and those are schematized in Figure 3.

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 13 suspension of multilamellar lipid vesicles containing or surrounded by a 'iologically relevant molecule (polypeptide, polysaccharide or polynucleotide, such as DNA) which are converted to small unilamellar protein lipid vesicles by sonication under nitrogen. Alternatively, to avoid damage, the biologically relevant molecule can be added to the solution following sonication. The vesicles are dialyzed at room temperature against buffered divalent cation, 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 15 up in buffer to yield the cochleate solution utilized in the instant invention.

In an alternative and preferred embodiment, an amount of negatively charged lipid, e.g., S.phosphatidylserine and cholesterol in the same 20 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 are utilized to prepare the cochleates. Either a polypeptide, mineral, vitamin, carbohydrate or polynucleotide, such as DNA, is added to the solution. That solution then is dialyzed against buffered divalent cation, calcium chloride, to produce a precipitate which can be called a DC (for direct calcium dialysis) cochleate.

An additional, related method for reconstituting cochleates has been developed and is called the 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. However, the solution 14 next is dialyzed against buffer 2 mM TES, 2 mM L.-histidine, 100 mM NaCl, pH 7.4) form small liposomes containing the polypeptide, polynucleotide, such as DNA, and/or polysaccharide.

A divalent cation, calcium, then is added either directly or by dialysis to form a precipitate which can consist of cochleates.

In the above procedures for making the cochleates of the instant invention, the divalent cation can be any divalent cation that can induce the formation of a cochleate or other insoluble lipid-antigen structures. Examples of suitable 2 +2 +2 +2 i" divalent cations include Ca 2 Mg Ba and Zn or 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.

An artisan can determine without undue experimentation the optimal lipid:biologically relevant molecule ratio for the targeted purposes.

Various ratios are configured and the progress of precipitation of each sample is monitored visually 15 under a phase contrast microscope. Precipitation to form, for example, cochleates, is monitored readily. Then, the precipitates can be administered to the targeted host to ascertain the nature and tenor of the biologic response to the administered cochleates.

It should be evident that 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 15 a divalent cation-containing buffer at 4 0 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. Prior 20 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.

Because 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.

16 In the case of polynucleotide cochleates, considerations for dosage parallel the standard methodologies regarding vaccines as known in the art. Also, methods for using polynucleotides in liposomes and the "naked DNA" are available to serve as a baseline for empirically determining a suitable dosing regimen, practicing known methods.

For example, 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 jg, although it is know *o that as little as 2Mg 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.

20 Developing formulations which can be administered easily and non-invasively is desirable. Thus, PO administration of cochleates S. will be targeted and higher doses will be tried initially (100 Ag/animal and 200 Ag/animal).

However, lower doses are required for parenteral routes.

Then graded doses will be used to develop a dose response curve for each formulation. Thus, cochleates containing 50 gg, 10 Mg, 2 Mg, 0.4 and 0 Ag polynucleotide/animal will be inoculated with at least 10 animals per group.

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. In the case of immune system monitoring, T cell proliferation, CTL and 17 antibody presence at specific body sites can be evaluated, using known methods, to assess the state of specific immune response.

To determine the duration of activity of cochleate formulations, groups which have responded to a single immunization are monitored periodically for up to a year or more to determine the effective life of a cochleate on administration.

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 S: system for later stimulation.

Pharmaceutical formulations 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. In addition to the active ingredient, the formulation would 20 comprise suitable art-recognized diluents, Scarriers, fillers, binders, emulsifiers, surfactants, water-soluble vehicles, buffers, solubilizers and preservatives.

An advantage of the cochleates is the stability of the composition. Thus, cochleates can be administered orally, topically or by instillation without concern, as well as by the more traditional routes, such as subcutaneous, intradermal, intramuscular and the like. Direct application to mucosal surfaces is an attractive delivery means made possible with cochleates.

The skilled artisan can determine the most efficacious and therapeutic means for effecting treatment practicing the instant invention.

Reference can also be made to any of numerous authorities and references including, for example, "Goodman Gilman's, The Pharmaceutical Basis for 18 Therapeutics", (6th Ed., Goodman, et al., eds., MacMillan Publ. Co., New York; 1980).

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. Hence, 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). As Mulligan noted, 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 15 biologically relevant molecules to a host. Such biologically relevant molecules include nutrients, vitamins, co-factors, enzymes and the like.

Because the biologically relevant molecule is contained within the cochleate, in a non-aqueous environment, the biologically relevant molecule essentially is stabilized and preserved. As described hereinabove, the biologically relevant molecule is added to the lipid solution and processed to form a precipitated structure comprising lipid and biologically relevant molecule. As demonstrated herein, hydrophilic molecules can be "cochleated", that is, can be made part of the cochleate structure, with little difficulty.

Also, suitable lipophilic biologically relevant molecules, such as drugs and other therapeutic compounds, are amenable to cochleation.

For example, lipophilic drugs such as cyclosporin, ivermectin and amphotericin are readily cochleated.

The instant invention now will be described by means of specific examples which are not meant to limit the invention.

19 EXAMPLE 1 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, 3-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.

o.

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., Virologv, Vol. 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 (103 to 105 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

PO

4 gm/L NaC1, 1.14 gm/L Na 2

H-PO

4 0.1 gm/L CaCl 2 0.1 gm/L MgC126HzO (pH Eggs were incubated at 37°C for 48 to 72 hours, followed by incubation at 4°C for 24 to 48 hours. Allantoic fluid was collected and clarified at 2,000 rpm for 20 minutes 20 at 5 0 C in a Damon IEC/PR-J centrifuge. The supernatant was then centrifuged 13C,00 rpm for minutes. This and all subsequent centrifugations were performed in a Sorvall RC2-B centrifuge at 5 0 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. The two 5,000 rpm supernatants were combined and centrifuged at 13,000 rpm for minutes. The resulting pellets were resuspended in phosphate-buffered saline by vortexing and sonicating, aliquoted, and stored at Sterile technique and materials were used throughout viral inoculation, isolation, and purification.

Virus stored at -70°C was thawed, transferred 20 to sterile thick-walled polycarbonate tubes and diluted with buffer A (2 mM TES, 2 mM L-histidine, 100 mM NaC1 (pH Virus was pelleted at 30,000 rpm for 1 hour at 5 0 C in a Beckman 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 NaC1, 0.02 M sodium phosphate buffer (pH by vortexing and sonicating. The nonionic detergent f-D-octyl-glucopyranoside was then added to a concentration of 2% 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 minutes in a TY65 rotor. The resulting clear supernatant was removed and used in the formation 21 of viral glycoprotein-containing cochleates. Some modification. of the. above prtcedure may have to be employed with other membrane proteins. Such modifications are well known to those skilled in the art.

EXAMPLE 2 A. DC Cochleates.

An amount of phosphatidylserine and cholesterol (9:1 wt ratio) in extraction buffer and 10 non-ionic detergent as described hereinabove was mixed with a pre-selected concentration of polynucleotide and the solution was vortexed for 5 minutes. The clear, colorless solution which resulted was dialyzed at room temperature against three changes (minimum 4 hours per change) of buffer A (2 mM TES N-Tris[hydroxymethyl]-methyl- 2 aminoethane sulfonic acid, 2 mM L-histidine, 100 mM NaCl, pH 7.4, also identified as TES buffer) containing 3 mM CaCl 2 The final dialysis routinely 20 used is 6 mM Ca 2 although 3 mM Ca 2 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. 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.

B. LC Cochleates.

An amount of phosphatidylserine and cholesterol (9:1 wt ratio) in extraction buffer and non-ionic detergent as described hereinabove was mixed with a pre-selected concentration of 22 polynucleotide and the solution was vortexed for minutes. The solution first was dil3ed overnight using a maximum ratio of 1:200 of dialysate to buffer A without divalent cations, followed by three additional changes of buffer leading to the formation of small protein lipid vesicles. The vesicles were converted to a cochleate precipitate, either by the direct addition of Ca 2 ions, or by dialysis against two changes of buffer A containing 3 mM Ca 2 ions, 2 •followed by one containing buffer A with 6 mM Ca ee..

'EXA MPLE 3 IMMUN RESPONSES TO ORALLY DELIVERED PROTEIN-COCHLEATE

VACCINES

15 To make the vaccine, 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.

20 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.

In Experiment A that generated the data shown in Figure initial vaccine doses of 50, 12.5 or 6.25 Ag of glycoproteins (groups 1 through 4 respectively) were administered at 0 and 3 weeks.

23 The third and fourth immunizations (6 and 19 weeks) were at one fourth the dose used for the initial two immunizations. Bleed 1 Bleed 6 occurred at 0, 3, 6, 9, 19, and 21 weeks. The data demonstrate that high circulating antibody titers can be achieved by simply drinking cochleate vaccines containing viral glycoproteins. The response is boostable, increasing with repeated administration, and is directly related to the amount of glycoprotein in the vaccine.

Those observations were confirmed and extended in Experiment B that generated the data shown in Figure The dose range was expanded to include 100 gg and 3.1 gg initial doses. Vaccine 15 was given at 0, 3 and 15 weeks, with the third immunization at one fourth the dose of the initial two. Bleed 1 to Bleed 6 occurred at 0, 3, 6, and 16 weeks. Circulating influenza glycoprotein-specific responses were detectable after a single administration for the top five doses, and for all groups after two feedings. The data shown is for pooled sera from each group, but all mice given the four highest doses, and four of five mice in groups five and six, responded to the vaccine with circulating antibody titers ranging from 100 to 102,400. Group seven, which received no vaccine, had titers less than 50 for all mice at all time points.

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 bleed 4) remained the same or within one dilution higher or lower than seen at 3 weeks after the previous boost.

To determine whether oral administration of the subunit vaccine described in Example 2 could lead to protective immunity in the respiratory 24 tract, the 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 application of 2.5 x 109 particles 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. After "three days at 37 0 C, allantoic fluid was obtained from individual eggs and hemagglutination

(HA)

titers were performed.

S. Mice were al7o 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 20 given in Table 1. The results also are shown graphically in Figure 25 TABLE 1 9* 9 9 *9 9 .9.

10 Vaccine Trachea' Lungs 2 Lungs' Dose Infected/Total Infected/Total Infected/Total g Protein 100 0/5 0/5 2/5 0/5 2/10 0/5 0/5 1/10 125 1/5 0/5 1/10 6.25 0/5 5/5 6/10 3.12 4/5 5/5 0 5/5 5/5 9/10 1. Mice from Experiment B.

2. Mice from Experiment

B.

3. Mice from Experiments A and B.

15 The data in Table 1 shows that all five of the unvaccinated mice had sufficient virus in the trachea to infect the embryonated chicken eggs (greater than 103 particles per trachea or at least one egg infectious dose (EID) per 0.1 ml of 20 suspension). In contrast, 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 vary 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 All mice in the groups immunized with 6.25 Mg and 3.1 Ag glycoproteins and 26 all mice in the unvaccinated control were positive for virus.

Even in the lowest two vaccine doses, there was some inhibition of viral replication. When lung suspensions were diluted 1/10 and inoculated into eggs, only one animal in the groups immunized with 6.25 jg was positive, as compared to three in the groups immunized with 3.12 Mg and three in the unvaccinated control. Culturing of 1/100 dilutions resulted in one positive animal in each of the groups immunized with 6.25 and 3.12 Mg, but 3 of remained positive in the unvaccinated group. In addition, for the two animals in the group that was immunized with 3.12 Mg, but which were negative at i/100, only 50% of the eggs'were infected at 1/10 and had low HA titers. In contrast, for the unvaccinated group, all eggs were infected and produced maximal amounts of virus at 1/10 and 1/100 dilutions.

20 C57BL/6 mice were given cochleates containing Sendai virus glycoproteins orally at 0 and 3 weeks.

They were bled at 0 (bleed 3 (bleed and 6 (bleed 3) weeks. Group 1 received approximately Mg protein, Group 2 about 25 Mg, Group 3 about 12.5 g, Group 4 about 6.25 Mg, and Group (negative control) received 0 Mg 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 27 long-term memory cells associated with a secondary immune response. Surprisi ngly, 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 9g 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 20 Sendai virus (SV) pulsed cells with in culture restimulation (N/SV/SV) or non-Sendai presenting cells (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. Cytolytic activity is crucial to clearance of cells infected with 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.

EXAMPLZ 4 Eight week old BALB/c female mice were immunized IM twice with various polynucleotide-cochleate formulations, 28 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 ratio and 50:1 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 15 rate of 10 pg/mouse and 50 pg/mouse. The control was buffer alone. Mice were immunized twice, days apart at 50 Al/mouse.

Splenocytes were obtained and tested in a T-cell proliferation assay using tritiated 20 thymidine, as known in the art. Control cultures contained no antigen or con A. The antigen used was p18 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 naked DNA provided a marginal response above background. All four cochleate preparations yielded a p18-specific response which increased over time. At six days, the response was about four times above background.

The DNA concentration range at the 10:1 ratio was about 120-170 Ag/ml. At the 50:1 ratio; the DNA concentration was about 25-35 gg/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 29 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.

EXAMPLE In similar fashion, splenocytes from animals immunized as described in Example 4, were tested for antigen specific cytotoxic activity using a 10 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.

S 15 On prestimulation, animals exposed to polynucleotide cochleates demonstrated specific cytotoxic splenocytes directed to gpl60, with nearly 100% cytotoxicity observed at an effector:target ratio of 100.

EXAMPLE 6 Fifteen mg of insulin were added to 15 ml of extraction buffer (EB) in a 50 ml plastic tube.

Then 300 mg of OCG were added to the mixture. The resulting suspension was colloidal and not clear at pH 7.4. The solution was titrated with 1 N NaOH to pH 8.5, resulting in a clear solution.

In a separate vessel, 6.8 ml of a 10 mg/ml solution of phosphatidylserine and 1.5 ml of a mg/ml solution of cholesterol were mixed and then dried to yield a thin film. The insulin solution was added to the vessel yielding a colloidal suspension. The suspension was vortexed for seven minutes and then set on ice for one hour. The pH 30 of the solution was adjusted to 9-9.5 with 1 N NaOH, the sample was filter sterilized and placed in dialysis tubing at about 2 ml per bag.

Two different dialysis schedules were used.

A. DC cochleates: 1. 100 ml overnight 1 x TES pH 9.0 coptaining 3 M Ca Zn or Mg 2. 250 ml 4h 1 x TES pH 8.5 coptaining 3 Ca Zn or Mg 3. 250 ml 4h 1 x TES pH 8.0 coptainng 3 Ca Zn or Mg 15 4. 250 ml 4h IxTES pH 7.4 coptain ng 6 nCa ,Zn or Mg B. LC cochleates: 1. 100 ml overnight 1 x TES, pH 2. 250 ml 4h, 1 x TES, pH 3. 250 ml 4h 1 x TES, pH 4. 100 ml overnight 1 x TES, pH 9. containinq 3 mM Ca Zn or Mg 250 ml 4h 1 x TES, pH 8.5 containing 3 mM Ca Zn or Mg 6. 250 ml 4h 1 x TES, pH 7.4 containing 6 mM Ca Zn or Mg

Z

Following dialysis, the resulting precipitate was found to comprise numerous cochleates.

EXAMPLE 7 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

S*.

31 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 Serum Glucose Given mg/dl 0 Time 30 min. 60 min.

LC Ca++ 200 ul 100 49.12 43 LC Ca++ 200 ul 102.9 252.4 61.9 Humulin R 200 ul 88.8 66 48.5 Oral administration of insulin affected serum glucose levels.

EXAMPLE 8 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 pl 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. Louis). Control animals were untreated, that is, received no streptozotocin or insulin. Representative data are set forth in Figure 8. Orally administered insulin, simply by P:\OPER\Fsl25994 -spe doc-2/11lW~/12 32 drinking, was effective in reducing blood glucose levels. No reduction in blood glucose was observed in control animals.

All references cited herein are incorporated by reference in entirety.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general .knowledge in Australia.

0 0* o \~n

Claims (49)

1. 2. A method of delivering a biologically relevant molecule to a cell comprising contacting said cell with a cochleate formulation comprising a) at least one biologically relevant molecule component; b) a negatively charged lipid component, and c) a divalent cation component.
2. 3. A method of topically applying a biologically relevant molecule comprising administering a biologically effective amount of a cochleate formulation comprising a) at least one biologically relevant molecule component; b) a negatively charged lipid component, and c) a divalent cation component.
3. 4. A method of stabilising a biologically relevant molecule by preparing a cochleate formulation comprising a) the biologically relevant molecule; b) a negatively charged lipid component, and P:\OPER\Fas292669-spc2.doc- 4I 8)2 34 c) a divalent cation component. The method of any one of claims 1-4, wherein said biologically relevant molecule component is selected from the group consisting of a polynucleotide component, a nutrient, a mineral, an amino acid, a vitamin, a lipid, a fatty acid, a saccharide, a carbohydrate, a protein or polypeptide, a polypeptide aggregate, a drug, a pigment, a metal, an organelle, a multi-ring structure, an enzyme, a cofactor, an immunogen, a nucleotide, a toxin, a cell, a subcellular replicative entity and a virus.
4. 6. The method of claim 5, wherein said polynucleotide component is selected from the group consisting of deoxyribonucleic acid, a catalytic ribonucleic acid, an antisense molecule and a plasmid.
5. 7. The method of claim 6, wherein said deoxyribonucleic acid is transcribed to yield a ribonucleic acid.
6. 8. The method of claim 7, wherein said ribonucleic acid is translated to yield a polypeptide.
7. 9. The method of any one of claims 1-5, wherein said lipid component is a phospholipid. The method of claim 9, wherein the phospholipid is selected from the group consisting of phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and phosphatidic acid. P:OPERFa\2292669-spc2.doc.-14 )2 35
8. 11. The method of any one of claims 1-5, wherein the divalent cation component is a cationic compound capable of chelating and complexing negatively charged lipids.
9. 12. The method of claim 11, wherein the divalent cation component is selected from the group consisting of and Zn++.
10. 13. The method of claim 5, wherein the mineral is one of calcium, magnesium, zinc, barium, or iron.
11. 14. The method of claim 5, wherein the amino acid is one of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan or methionine. The method of claim 5, wherein the vitamin is one of vitamin A, vitamin D, vitamin E, or vitamin K.
12. 16. The method of claim 5, wherein the fatty acid is one of a saturated or a polyunsaturated fatty acid.
13. 17. The method of claim 5, wherein the fatty acid is a hormone.
14. 18. The method of claim 5, wherein the saccharide is one of maltose, lactose, sucrose, glycogen, a-amylose amylopectin, a lipopolysaccharide, a glycodrug or a fiber supplement.
15. 19. The method of claim 5, wherein the saccharide is a starch. P:\OPER\Fas\2292669-spe2.doc- 14/ 8)2 36 The method of claim 18, wherein the glycodrug agent is one of acarbose, topiramate, sporonox, ganglioside or hyaluronic acid.
16. 21. The method of claim 18, wherein the fiber supplement is one of hydrolyzed corn starch, cellulose, or soy fiber.
17. 22. The method of claim 5, wherein the lipid is selected from the group consisting of an acylglycerol, a phosphoglyceride, a sphingolipid, a terpene, a steroid, and a prostaglandin.
18. 23. The method of claim 5, wherein the lipid is one of triacylglycerol, galactosyldiacylglycerol, cardiolipin, phosphatidylcholine, phosphatidylethanolamine, sphingosine, dihydrosphingosine, squalene, carotenoid, cholic acid, estrone, prostanoic acid or ergosterol.
19. 24. The method of any one of claims 1-4, wherein the biologically relevant molecule is a food additive. The method of claim 5, wherein the food additive is one of an acidulant, alkalizer, anti-caking, antimicrobial, antioxidant, bleaching, buffer, carrier, humectant, pectin or oxidizing agent.
20. 26. The method of claim 5, wherein said toxin is one of pertussis, tetanus, diphtheria, cholera or a toxoid. P:\OPE\F.As229266,9-p 2.d- 4A)IO2 37
21. 27. The method of claim 5, wherein said polypeptide component is selected from the group consisting of a cytokine and a hormone.
22. 28. The method of claim 27, wherein said cytokine is one of ANG, AR, BDNF, BCT, 010, CINC-1, CNTF, f3-ECGE, EGF, ENA-78, Eotaxin, Epo, FGF acidic, F'GF basic, FGF-4, FGF-6, FGF-7/KGF, FGF-8b, F'GF'-8b, FGF-9, Flt-3 ligand, C-CSF, G-CSF, GDNF, GM-CSF, sgp 130, GROc, GRO3, GROy, HB-EGF, HCC-l, HOF, HRG-u, 1-309, I NF-y, IL-6, IL-7, IL-8, IL-9, IL-lO, IL-li, IL-12, IL- 13, IL-15, 11-17, IP-lO, JE/MCP-i, KC, Leptin, LIF, M- CSF, MOP-i, MCP-2, MCP-3, MILE, MIP-ice, MIP-i[3, MIP-2, ML, f3-NGF, NT-3, NT-4, OSM, PBSF, PG-ECGF, PDGF, PDGF- AA, PDGF-AB, PDGF-BB, PIG', PTN, Rantes, SOB', SDF-13, SLPI, TGF-e, TGF-P31, TGF-fPi.2, TGF-P32, TNF-c, TNF-3. Tpo, or VEGF.
23. 29. The method of claim 5, wherein said biologically relevant component is a conjugated protein. The method of claim 29, wherein said conjugated protein is selected from the group consisting a lipoprotein, a glycoprotein, a phosphoprotein, an hemoprotein, a flavoprotein, and a metalloprotein.
24. 31. The method of claim 5, wherein said conjugated protein is one of plasma Pl-lipoprotein, y-globulin, plasma orosomucoid, casein, hemoglobin, cytochrome 0, catalase, succinate dehydrogenase, D-amino acid P:\OPER\Fas\2292669-spc2.doc-14/118/02 38 oxidase, ferritin, cytochrome oxidase, alcohol dehydrogenase or xanthine oxidase.
25. 32. The method of claim 27, wherein said hormone is one of insulin, somatotropin (growth hormone), somatomedin-1, adrenocorticotropic hormone (ACTH), epinephrine, follicle stimulating hormone (FSH), leutinizing hormone human chorionic gonadotropin (HCG), TSH releasing hormone (TRH), thyroid stimulating hormone (TSH), thyroid hormone erythropoietin, epidermal growth factor or parathormone.
26. 33. The method of claim 27, wherein said hormone is insulin.
27. 34. The method of claim 27, wherein said hormone is one of cortisol, estrodiol, progesterone or testosterone. The method of claim 5, wherein the drug is a lipid soluble drug.
28. 36. The method of claim 5, wherein the drug is selected from the group consisting of an anti-viral, an anaesthetic, an anti-infectious, an anti-fungal, an anti-cancer, an immunosuppressant, a steroidal anti-inflammatory, a non-steroidal anti-inflammatory, a tranquilizer, a vasodilatory agent, a small-molecule receptor antagonist or a small-molecule receptor agonist. PAOPER\Fa292(9-p .do-I4/iMI2 39
29. 37. The method of claim 5, wherein the drug is selected from the group consisting of a steroid, a microbicide, a microbistat and a metabolic poison.
30. 38. The method of claim 5, wherein the drug is one of acyclovir, propanidid, propofol, alphadione, echinomycine, miconazole nitrate, teniposide, vitamin B, hexamethylmelamine, taxol, taxotere, melphalan, adriamycin, 18-hydroxydeoxycorticosterone, rapamycine, prednisolone, dexamethazone, corti.S one, hydrocortisone, pyroxicam, naproxen, diazepam, verapamil, nifedipine, tachykinin NK-l antagonist, erythropoietin peptide agonist, sequenavir, androsterone, spironolactone, oxymetholone, testosterone, ethynyl estradiol, mestranol, ethynodiol diacetate, hydroxymethylprogesterone, progesterone, BiCNU, CCNU, cytarabine, cyclophosphamide, mitomycin, doxorubicin, bulsulfan, benzodepa, triethylenethiophosphoramide, uracil mustard, lomustine, bleomycin, dactinomycin, tubercidin, methotrexate, mercaptopurine, cytarabine, thioguanine, vinblastine, mitotane, nilutamide or tamoxifen.
31. 39. The method of claim 5, wherein the drug is one of cyclosporine A, amphotericin B or ivermectin. The method of claim 5, wherein the pigment is one of rhodopsin, carotene, betalaine, an annatto color, carotenoid, cochineal, saffron, tumeric, or carmel. P:\OPER\Fasl2292(69-sp2 .doc-14A))2 40
32. 41. The method of claim 5, wherein the metal is one of Fe 2 Mo 2 Zn 2 Cu 2 Mg 2 Mn 2 Au+, K, Li or Na
33. 42. The method of claim 5, wherein said organelle is a nucleus, endoplasmic reticulum, peroxisomes, mitochondria, plastids, golgi, lysosomes, endosomes, secretory vesicles, plasma membrane, cytosol, and subsets thereof, such as soluble and membrane bound fractions.
34. 43. The method of claim 5, wherein the multi-ring structure is one of caffeine, nicotine, or ergotamine tartrate.
35. 44. The method of claim 5, wherein the enzyme is one of adenosine deaminase, alcohol dehydrogenase, DNA helicase, lipoprotein lipase, recombinase activator gene, or DNA ligase. The method of claim 5, wherein the cofactor is one of coenzyme Q, coenzyme A, flavin mononucleotide, biocytin, tetrahydrofolate coenzyme, or a metal ion.
36. 46. The method of claim 5, wherein the adjuvant is one of muramyl dipeptide, peptidoglycan, or mycolic acid.
37. 47. The method of claim 5, wherein the nucleotide is one of AMP, CMP, GMP, UMP, dTTP, dCTP, dGTP or dATP.
38. 48. The method of any one of claims 1-4, wherein the biologically relevant molecule is selected from the group consisting of: P:\OPER\Fas\2292669-spe2.doc-14)8)2 41 a) viruses and subviral particles, b) bacteria and sub-cellular bacterial preparations, c) yeast and sub-cellular yeast preparations, d) cell lines and sub-cellular preparations, and e) primary cells and sub-cellular preparations.
39. 49. The method of claim 48 wherein the virus is an influenza virus or HIV. A method of administering a biologically relevant molecule to a cell in a host comprising administering a biologically effective amount of a cochleate formulation comprising a) a negatively charged lipid component, and b) a divalent cation component.
40. 51. A method of any one of claims 1 to 50 wherein the cochleate formulation is lyophilised.
41. 52. A method according to any one of claims 1 to 51, wherein the cochleate formulation is stored in a divalent cation containing buffer.
42. 53. A method according to claim 52 wherein the cochleate formulation is according to claim 51 or claim 52 wherein the cochleate formulation is stored for at least six months.
43. 54. A cochleate formulation when used in the method of any one of claims 1 to 53. 16- 8-02;12:35 4/ voJRPl suvlS ard..spedes-IMrAmkr 42 The use of a negatively charged lipid component and a divalent cation compound in a cochleate formulation for the preparation of a medicament for oral administration, wherein said medicament comprises the cochleate formulation.
44. 56. The use of a biologically relevant molecule component to be stabilized or delivered, a negatively charged lipid component and a divalent cation component in the preparation of a medicament comprising the cochleate :i formulation. *component, a nutrient, a mineral, an amino acid, a *vitamin, a lipid, a fatty acid, a saccharide, a carbohydrate, a protein or polypeptide, a polypeptide aggregate, a drug, a pigment, a metal, an organelle, a **multi-ring structure, an enzyme, a cofactor, an immunogen, a nucleotide, a toxin, a cell, a subcellular replicative entity and a virus.
45. 58. The use according to claim 56 wherein the biologically relevant molecule is selected from the group consisting of: a) viruses and subviral particles, b) bacteria and sub-cellular bacterial preparations, c) yeast and sub-cellular yeast preparations, sTRq d) cell lines and sub-cellular preparations, and Se) primary cells and sub-cellular preparations. 16- 8-02;10:57 5/ 7 P:AOPERFinl;.292669.pO ±ltjc-IM)V1 43
46. 59. The use of a negatively charged lipid component and a divalent cation component in the preparation of a 0 medicament comprising the cochleate formulation.
47. 60. A method according to any one of claims 1 to 53 substantially as hereinbefore described with reference to the drawings and/or examples.
48. 61. A cochleate formulation according to claim 54 substantially as hereinbefore described with reference to the drawings and/or examples. *5 61. A cochleate formulation according to any one of claims 55 substantially as hereinbefore described with reference Sto the drawings and/or examples. DATED this 16 th day of August 2002
49. 62. Albany Medical College And Unversity Of Medicine And S* Dentistry Of New Jersey by DAVIES COLLISON CAVE Patent Attorneys for the Applicants