US005716637A United States Patent 19 11 Patent Number: 5,716,637 Anselem et al. 45 Date of Patent: *Feb. 10, 1998
54 SOLID FAT NANOEMULSIONS AS WACCNE 56) References Cited DELVERY WEHICLES U.S. PATENT DOCUMENTS Inventors: Shimon Anselem, Rehovot, Israel; 4,880,635 11/1989 Janoff...... 424/450 George H. Lowell, Baltimore, Md.; 5,023,271 6/1991 Vigne ... 514/.458 Haim Aviv, Rehovot; Doron 5,171,737 12/1992 Weiner ...... 514/3 Friedman, Carmei Yosef, both of Israel 5,188,837 2/1993 Domb ..... 424/450 5,284.663 2/1994 Speaker ...... 424/489 73) Assignees: Pharmos Corporation, New York, 5,302.401 4/1994 Livesidge ... 424/489 N.Y.; The United States of America as 5,306,508 4/1994 Kossovsky . 424/493 represented by the Secretary of the 5,308,624 5/1994 Maircent ... . 424/427 Army, Washington, D.C. 5,576,016 11/1996 Amselem ...... 424/450 FOREIGN PATENT DOCUMENTS * Notice: The term of this patent shall not extend 0315079 5/1989 European Pat. Off.. beyond the expiration date of Pat. No. 0506 197 9/1992 European Pat. Off.. 5,576,016. WO91/07171 5/1991 WIPO. 21 Appl. No.: 553,350 OTHER PUBLICATIONS CRC Press, Inc., Liposome Technology, 2nd Edition, vol. 1, 22 PCT Filled: May 18, 1994 Chapter 28, p. 501, Liposome Preparation and Related Techniques, edited by Gregory Gregoiadis, Ph.D., "A 86 PCT No.: PCT/US94/05589 Large-Scale Method For the Preparation. Of Sterile And Nonpyrogenic Liposomal Formulations Of Defined Size S371 Date: Nov. 16, 1995 Distributions For Clinical Use'. Shimon Amselem, Alberto Gabizon, and Yechezkel Barenholz. S 102(e) Date: Nov. 16, 1995 (List continued on next page.) 87 PCT Pub. No.: WO94/26255 Primary Examiner-Gollamudi S. Kishore PCT Pub. Date: Nov. 24, 1994 Attorney, Agent, or Firm-Pennie & Edmonds 57 ABSTRACT Related U.S. Application Data The present invention provides pharmaceutical vaccine 63 Continuation-in-part of Ser. No. 63,613, May 18, 1993, Pat. compositions that are nanoemulsions of particles having a No. 5,576,016. lipid core which is in a solid or liquid crystalline phase at 25° C., and which is surrounded by at least one phospholipid 51 Int. C. ... A61K9/127; A61 K9/16 bilayer for the parenteral, oral, intranasal, rectal, vaginal or 52 U.S. Cl...... 424/450; 424/489: 424/490; topical delivery of both hydrophilic and lipophilic immu 424,502; 424/184.1; 424/188.1; 424/204.1; nogens. The particles have a mean diameter in the range of 424/208.1; 424/234.1; 424/237.1; 424/236.1; 10 to 250 nm and the immunogen is incorporated therein, 424/269.1; 428/937; 514/937 either intrinsically prior to the homogenization process or 58 Field of Search ...... 424/450, 489, 424/490, 497, 45, 427,502, 184.1, 188.1, extrinsically thereafter. 204.1, 208.1, 234.1, 237.1, 236.1, 269.1; 514/937-943; 428/402.2 44 Claims, 8 Drawing Sheets
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OTHER PUBLICATIONS Analysis and Identification of Lipids", 2nd revision edition, Moris Kates. Methods of Biochemical Analysis, vol. 33, D. Glick, editor, J. Wiley & Sons, N.Y., 1988, "Liposomes: Preparation, Prog. Lipd Res., vol. 31, No. 4 pp. 345-372, 1992, "Recent Characterization, and Preservation', Dov Lichtenberg and Aspects in the Use Of Liposomes in Biotechnology and Yechezkel Barenholz. Medicine" by Toshinori Sato et al. Journal of Pharmaceutical Sciences, vol. 79, No. 12, Dec. 1990, “optimization and Upscaling of Doxorubicin-Con Vol. 6, p. 195, "Liposomes As Carrier for Vaccines" by Carl taining Liposomes for Clinical Use'. S. Amselem, A. Gabi R. Alving - Walter Reed Army Institute of Research, zon and Y. Barenholz. Washinton, D.C. CRC Press, Inc., 1993, Liposome Technology, 2nd Ed., American Chemical Society, vol. 18, No. 19, 1979, p. 4169, edited by G. Gregoriadis, Ph.D., vol. 1, Chapter 3, p. 49, "Dependence of the Conformation of the Polar Head Groups "Liposome Preparation Using High-Pressure Homogeniz of Phosphatidycholine on Its Packing in Bilayers. Nuclear ers'. Martin M. Brandi, Bieter Bachmann, Markus Drech Magnetic Resonance Studies on the Effect of the Binding of sler, and Kurt H. Bauer. Lanthanide Ions” by Dov Lichtenberg et al. Elsevier Science Publishers B.V. (Biomedical Division, 1986, Laboratory Techniques in Biochemistry and Molecu Proc. Natl. Acad. Sci. USA, vol. 89, pp. 358-362, Jan. 1992, lar Biology, vol. 3, part 2, edited by R.H. Burdon and P.H. Immunology - "Liposomal malaria vaccine in humans: A van Knippenbert, "Techniques of Lipidology - Isolation, safe and potent adjuvant strategy” by Louis F. Fries et al. U.S. Patent Feb. 10, 1998 Sheet 1 of 8 5,716,637
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& Sr. h . . . . Control LC-Ag-Emulsomes Vaccine Formulation FIG. 8 5,716,637 1. 2 SOLD FAT NANOEMULSIONS AS WACCNE body production by increasing activity along both pathways DELIVERY VEHICLES of stimulation of immune system described above. When multiple antigens attached to a lipid carrier bind to multiple CROSS-REFERENCE TO RELATED receptors on a B cell, the resulting plasma cell probably APPLICATION proliferates faster than it does when it encounters a solitary antigen. Similarly, whereas a macrophage is unlikely to The present application 371 of PCT/US94/05589 filed phagocytase a small antigen efficiently, it will readily digest May 18, 1994, published as WO94/26255 Nov. 24, 1994, is a lipid carrier particle containing the antigen. When phago a continuation-in-part of U.S. patent application Ser. No. cytosis takes place, antigens ooupled to the surface of the 08/063,613, filed May 18, 1993 now U.S. Pat No. 5,576.016. lipid carrier or encapsulated within the particle are ingested O and possibly displayed as processed antigen on the mac FIELD OF THE INVENTION rophage surface. Such antigen presentation could result in T The present invention concerns methods and composi cell activation and additional plasma cell proliferation and tions for delivery of vaccines by parenteral and other routes increased antibody production. of administration. More particularly, it concerns stable lipid 15 Most vaccine adjuvants are also surface-active, or have a in-water nanoemulsions or emulsomes containing Small special surface interface. Surface-active agents concentrate lipid particles which are useful as delivery vehicles for both at the surface formed by the interface of water and non-polar hydrophilic and lipophilic immunogens enhancing their substances such as lipid or lipid membrane. Most adjuvants immunogenicity and improving their immune response. are also water-insoluble surfactants, so lipoidal vehicles are necessary for proper delivery of the antigen. BACKGROUND OF THE INVENTION The use of liposomes as drug delivery systems has been The body's immune system recognizes pathogens as known for some time, and comprehensive review articles on foreign and is thought to produce antibodies to them by two their properties and clinical applications are available; see, main pathways. In one pathway, antigens on the pathogen e.g., Barenholz and Amselem, in "Liposome Technology', surface presumably bind to receptor molecules on the white 25 2nd ed., G. Gregoriadis, ed. CRC Press, 1992; Lichtenberg blood cells known as B cells, causing them to become and Barenholz, in Methods for Biochemical Analysis, 33, D. plasma cells, which proliferate and secrete antibodies spe Glick, ed., 1988. A liposome is defined as a structure cific for the pathogen. In the second pathway, circulating consisting of one or more concentric lipid bilayers separated macrophages bind to the pathogens, endocytose them, and by water or aqueous buffer compartments. These hollow display processed antigens on their surface. 30 structures, which have an internal aqueous compartment, T cells then bind to the expressed antigen and by way of can be prepared with diameters ranging from 20 nm to 10 several complex steps this binding ultimately results in um. They are classified according to their final size and further plasma cell proliferation and increased antibody preparation method as: SUV, Small unilamellar vesicles production. (0.5-50 nm); LUV, large unilamellar vesicles (100 nm); 35 REV, reverse phase evaporation vesicles (0.5um); and MLV. In the past, the risks of whole-pathogen vaccines and large multilamellar vesicles (2-10 m). limited supplies of useful antigens posed barriers to devel An extensive literature exists on immunologic character opment of practical vaccines. Today, the tremendous istics of liposomes, and numerous reviews on their potential advances of genetic engineering and the ability to obtain as vaccine carriers have been published especially by C. R. many synthetic recombinant protein antigens derived from Alving and co-workers who developed the first injectable parasites, viruses, and bacteria has revolutionized the devel liposomal vaccine for human use administered to 30 volun opment of new generation vaccines. teers in a Phase I study (Fries et al., Proc. Natl. Acad. Sci. Although the new, small synthetic antigens offer advan USA, 89, pp. 358-362, 1992). tages in the selection of antigenic epitopes and safety, a Emulsions are defined as heterogeneous systems of one general drawback of small antigens is poor immunogenicity. liquid dispersed in another in the form of droplets usually Unfortunately, the body's immune system does not exceeding 1 um in diameter. The two liquids are immiscible respond strongly to small peptides. In particular, macroph and chemically unreactive or slowly reactive. An emulsion ages do not readily ingest and process the small antigens is a thermodynamically unstable dispersed system. Instabil resulting in low antibody titers and the need for repeated ity is a result of the system's tendency to reduce its free immunizations. This lack of immunogenicity has created an 50 energy by separating the dispersed droplets into two liquid acute need to identify pharmaceutically acceptable delivery phases. Instability of an emulsion during storage is evi systems for these new antigens. denced by creaming, flocculation (reversible aggregation), Several reports describing the improvement of immune and/or coalescence (irreversible aggregation). response achieved by the association of antigens with lipid The use of parenteral emulsions as drug delivery systems carriers such as liposomes or microparticles like polymeric 55 is still comparatively rare because of the necessity of achiev biodegradable microcapsules have been published (C. R. ing stable microdroplets of less than 1 pm to prevent Alving, Liposomes as Carriers of Vaccines, in "Liposomes: formation of emboli in the blood vesses. In order to increase From Biophysics to Therapeutics”, M. J. Ostro, ed., Ch. 6, the stability and useful lifetime of the emulsion, the dis Marcel Dekker Inc., New York, 1987, pp. 195-218; J. H. persed lipid droplets must be coated or treated with surfac Eldridge et al. Molec. Immunol., 28, 287, 1991). The ability tants or "stabilizers', which lower the free energy at the of these delivery systems to enhance immunogenicity was interface and decrease the tendency of droplets to coalesce. related to the physicochemical characteristics of the par However, due to their detergent characteristics, most of them ticles. are hemolytic agents which act as membrane solubilizers, When antigens are incorporated in lipid carriers by encap producing deleterious side effects upon injection into the sulation or entrapment, or embedded in their surface, they 55 body. Formulation options are severely restricted by the very show enhanced ability to evoke a strong immune response. limited selection of stabilizers and surfactants approved and Vaccines formulated in lipid carriers probably enhance anti safe for parenteral injection. 5,716,637 3 4 SUMMARY OF THE INVENTION associated peptide antigen domains, while the aqueous con tinuous phase accommodates water-soluble peptide The present invention provides pharmaceutical composi domains, or oligosaccharides. tions comprising nanoemulsions of particles comprising a The term "peptide" herein includes both oligopeptides lipid core composed of a lipid which is in a solid or liquid and proteins. To facilitate intestinal uptake, the emulsions crystalline phase at at least 25°C., stabilized by at least one may be encapsulated in gelatin capsules or otherwise entero phospholipid envelope, for the parenteral, oral, ocular, coated to prevent their exposure to gastric fluids when the rectal, vaginal, intranasal, or topical delivery of both fat oral route of administration is selected. soluble and water-soluble immunogens. The new entity is a Additional advantages of the emulsome technology for particulate vehicle which is denoted herein as a solid fat delivery of vaccines are: antigens and adjuvants can be nanoemulsion or "emulsome.” These compositions have 10 incorporated simultaneously in the same formulation due to features which are intermediate between liposomes and the hydrophilic-hydrophobic nature of the emulsome par oil-in-water emulsions. Emulsome particles contain a hydro ticles; high encapsulation efficiency of immunogens can be phobic core, as in standard oil-in-water emulsions, but obtained; emulsomes can be formulated to act as depot for surrounded and stabilized by one or more bilayers or enve slow release of antigens avoiding the need for repeated lopes of phospholipid molecules, as in liposomes (FIGS. 1A, 15 vaccinations; the manufacturing technique of emulsomes is 1B and 1C). relatively simple and easy to scale-up. A key feature of these particles is that the core is com The emulsome-vaccine formulations of this invention do posed of a lipid which in bulk form is in a solid or liquid not include any polyoxypropylene-polyoxyethylene block crystalline phase, rather than an oil in a fluid phase. Lipid polymer, trehalose dimycolate, cell wall skeleton, or any compositions of the core are characterized as being in the immunostimulatory mycobacteria or muramyl peptide-like solid or liquid crystal phase at at least 25°C. when measured additives to be effective. in bulk form. Another aspect of this invention is to provide composi Emulsomes, having the characteristics of both liposomes tions and methods for the preparation of emulsomes con and emulsions, provide the advantages of high loading of 25 taining antigens, incorporated either intrinsically (emulsified hydrophobic bioactive compounds in the internal solid lipid together with the phospholipids and solid fat) or extrinsi core and the ability to encapsulate water-soluble antigens in cally (added externally to prepared emulsomes). the aqueous compartments of surrounding phospholipid In some cases, the emulsomes of the present invention can layers. be administered in combination with other adjuvant systems, The present pharmaceutically stable solid fat nanoemul 30 such as proteosomes, as indicated in the examples. sions or emulsomes may beformulated in the absence of any The size, concentration and specific composition of emul ionic or non-ionic nonnatural synthetic surfactants or cosur some vaccines may be varied to suit the particular antigen factants such as polyoxamers, deoxycholate, polysorbates, used. Moreover, such formulations may enhance both tyloxapol, or emulphor. They are stabilized by the combi humoral and cell-mediated immunity (CMT) as do Freund's nation of relatively high lecithin content and the use of solid 35 adjuvants. The emulsomes here described have been devel lipid compositions as the core. oped for human use and since their particles are of submi The particle size distribution of emulsomes, based on cron size and contain no added pyrogenic moieties such as differential weight percents, is in the range of 10-250 nm, mycobacteria or muramyl-peptide derivatives they have, making them suitable for parenteral administration. unlike Freund's adjuvants, great safety potential. They may The emulsome technology represents a new type of be especially applicable to antigens that are vaccine candi lipid-based encapsulation technology that has potential use dates to protect against biologic toxins or infectious agents fulness as carriers of vaccines and adjuvants enhancing the which have natural hydrophobic moieties as a component immunogenicity of antigens incorporated intrinsically or including transmembrane viral, bacterial or parasite extrinsically into the particles. proteins, membrane proteins such as proteosomes, 45 lipopolysaccharides, glycolipids such as gangliosides, or a Emulsome-vaccine formulations containing antigens can variety of proteins or peptides to which hydrophobic anchors provide the surface interphase necessary for proper orien have been chemically or genetically added. tation of the adjuvant active material, resulting in enhanced In addition to parenteral vaccination, another aspect of antibody production and increased immune response. They this invention is to provide emulsome compositions and seem to have the capability of concentrating the antigen and 50 methods to achieve mucosal immunity by using emulsome adjuvant on hydrophobic surfaces, where they are effec preparations comprising a plurality of submicron particles, a tively presented to cells of the immune system. mucoadhesive macromolecule, immunogenic peptide or Binding of antigen to a surface or presentation of a special antigen, and an aqueous continuous phase, which induces type of surface for antigen adsorption, as in the case of mucosal immunity by achieving mucoadhesion of the emul emulsome-vaccines, appears to be critical for much of the 55 some particles to mucosal surfaces. Mucous surfaces suit biological activity of most agents reported as adjuvants. able for application of the emulsions of the present invention Therefore the emulsome technology can serve as effective may include ocular (corneal, conjunctival), oral (buccal, vehicles or delivery systems for human and veterinary sublingual), nasal, vaginal and rectal routes of administra vaccines. tion. The use of emulsomes as a vaccine delivery system has other demonstrable advantages. Emulsomes of this inven BRIEF DESCRIPTION OF THE DRAWINGS tion provide effective pharmaceutical delivery for a broad FIGS. 1A, 1B and 1C are schematic illustrations of a variety of both water-soluble and water-insoluble immuno liposome, an oil-in-water submicron emulsion droplet, and a gens with minimal local or systemic toxicity. proposed structure for an emulsome particle, respectively. The hydrophobic core and surfactant provide a micro 65 FIG. 2 is a graph showing the particle size distribution, as environment which accommodates lipophilic immunogens differential weight percent, of a group of emulsomes accord such as lipid A or lipopolysaccharides as well as membrane ing to the present invention. 5,716,637 S 6 FIGS. 3A-C compares the 'P-NMR spectra of submi liquid crystalline phase at at least about 25 C., when cron oil-in-water emulsion, emulsomes, and liposomes, measured in bulk form without incorporation into emul recorded in the absence (“A” series) and presence (“B” somes. Some lipid compounds present in a mixture option series) of PrOI (30 mM). ally may be fluids at 25°C. when pure, provided that the lipid mixture as a whole is solid or liquid crystalline in bulk FIG. 4 is a transmission electron micrograph of emulsome at 25° C. In preferred compositions, at least 90% of the preparation negatively stained with 1% phosphotungstic individual lipid compounds present in the core are solids or acid solution (Bar=100 nm). liquid crystals at 25°C. when measured in pure bulk form. FIG. 5 is a graph showing enhanced murine immunoge Phase determination preferably may be performed on the nicity after parenteral immunization of mice with formalin bulk lipid, i.e., a macroscopic sample of the same ized SEB-Toxoid antigen formulated in intrinsic emulsome 10 composition, prior to its incorporation into the emulsome vaccine compared to free antigen or alum-adjuvanted vac core. The macroscopic phase determination on a bulk C sample may be made on a melting apparatus or by spectro FIG. 6 is a graph showing enhanced lapine immunoge scopic means, such as IR, NMR, or fluorescence intensity or anisotropy. Bulk phase determination of an existing emul nicity of SEB-Toxoid F antigen after parenteral immuniza 15 some preparation may be performed by first extracting the tion of rabbits with extrinsic emulsome vaccine compared to core lipids, then measuring. free antigen. Lipids which form the lipid core are composed almost FIGS. 7A and 7B are a pair of graphs showing protection exclusively of nonpolar moieties which therefore do not of mice immunized with mucoadhesive emulsome vaccines exhibit a preference for the lipid-water interface. Triglycer containing SEB-Toxoid Fantigen or SEB-Tox F complexed ides are the commonest type of fatty acid esters used in to proteosomes against intranasal challenge with SEB toxin preparing the lipid core of nanoemulsions of this invention. in BALB/C mice D-galactosamine model. Triglycerides are a preferred material from which the lipid FIG. 8 is a graph showing increased immune response in core may be prepared. The triglyceride core may be com rhesus monkeys immunized with anti-Leishmania intrinsic posed of a single pure triglyceride, usually available as a emulsome vaccine containing LC-467 lipopeptide antigen. 25 synthetic triglyceride, or may be a mixture of several trig lycerides. Fats isolated from natural sources usually are DETALED DESCRIPTION OF THE available only as mixtures of triglycerides. Such natural INVENTION mixtures are suitable for preparation of emulsomes, pro This invention is directed to pharmaceutical compositions vided that the melting characteristics of the mixture are such 30 that they exhibit a solid or liquid crystal phase at 25° C. for the delivery of water-soluble and lipid-soluble vaccines, Detailed summaries of the phase behavior of various pure and to methods for preparing and using such compositions. and mixed triglycerides are available; see D. Small, As used herein, the term “lipid" refers to compounds “Glycerides,” in: The Physical Chemistry of Lipids from which are soluble in hydrocarbon solvents and are of a Alkanes to Phospholipids, Chapter 10, Plenum Press, New structural type which includes fatty acids and their esters, 35 York, 1985; and M. Kates, Techniques of Lipidology, Chap cholesterol and cholesteryl esters, and phospholipids. ter 1, North Holland, Amsterdam/American Elsevier Publ. The size range of emulsomes described in the present Co., Inc., New York, 1972. invention (10–250 nm) makes them suitable for parenteral From the information available in these and other stan delivery. The 10–250 nm range includes the mean size on a dard references, one skilled in the art may choose particular weight basis of preferred emulsome preparations. In more fats which have the requisite property of providing a solid or preferred preparations, the 10-250mn size range includes at liquid crystal or mixed phase at 25°C. when measured in least 99% of the particles in the nanoemulsion, as deter bulk. The melting properties of particular mixtures of fats mined on a weight basis. “Weight basis” determination as may be determined readily by simple experiments. used herein means that the weight percent rather than the Many triglycerides which are solid at 25° C. have fully number of the lipid particles within the stated diameterrange 45 saturated fatty acid chains. Saturated fatty acids are advan is used for the size determination. In certain preparations, tageous because they are incapable of undergoing peroxi the mean particle size plus or minus the standard deviation dation reactions, which lessen the acceptable storage life of falls within the range 20 to 180 nm, 40 to 160 nm, or 50 to oil-in-water emulsions. 150 nm. In other preparations, the mean and the standard Examples of solid fats suitable for the preparation of deviation falls within the range 10 to 120 nm. In still more 50 emulsomes are triglycerides composed of natural, even preferred preparations, 99% of the particles in the nanoemul numbered and unbranched fatty acids with chain lengths in sion fall within one of the above size ranges, as determined the C10-C18 range, or microcrystalline glycerol triesters of on a weight basis. All of the above emulsions can be saturated, even-numbered and unbranched fatty acids of sterilized by filtration. natural origin such as tricaprin, trilaurin, trimyristin, Emulsomes can be administered parenterally, orally, 55 tripalmitin, and tristearin. In general, any lipid component or topically, rectally, vaginally or intranasally. mixture of lipid components which provides a solid phase at Composition of the Lipid Core room temperature (25 C.) when measured in bulkis suitable An essential component of emulsomes is an internal for the lipid core. hydrophobic or lipid core comprising a lipid which exhibits Other preferred lipid core components are esters of solid or liquid crystal or mixed solid and liquid crystal monounsaturated fatty acids. Although monounsaturated phases at room temperature (25°C.) when measured in bulk. fatty acids are capable of undergoing peroxidation, they are The lipid may be a single compound or a mixture. The term less reactive than typical polyunsaturated fatty acids. Natu “lipid” as applied to the lipid core herein may refer either to ral monounsaturated fatty acids have the cis configuration. a single pure lipid compound or to a mixture of lipid In general, these are lowermelting than completely saturated compounds present in the core. 65 fatty acid esters. Usually, therefore, monounsaturated fatty Lipid compositions suitable for use as the core component acid esters will be most useful in a mixture with higher of emulsomes may be characterized as being in the solid or melting saturated fatty acid esters. 5,716,637 7 8 Other triglycerides which are solid at 25° C. include administered lipid particles to certain cellular receptors, partially hydrogenated vegetable oils. Unlike naturally such as the LDL receptor on hepatocytes and certain other occurring unsaturated fatty acids, hydrogenated oils contain cells. unsaturated bonds in the trans configuration, which is higher Lipid particles of the invention preferably also are sub melting than the cis configuration. Partially hydrogenated stantially free of intracellular marker proteins, such as those vegetable oils yield solid vegetable shortening (e.g., associated with the intracellular cytoskeleton (e.g., actin, CRISCO), which may be used to prepare emulsomes which myosin, troponin, tubulin, vimentin, spectrin). are free of cholesterol or cholesteryl esters. Lipid particles which do not contain intracellular marker Triglycerides containing polyunsaturated fatty acids may proteins or serum apolipoproteins are herein described as be present in small amounts in the lipid core of emulsomes, O “noncellular” particles, since they lack characteristic indicia provided that the resulting triglyceride mixture is in the solid of lipid particles present in or derived from cellular sources. or liquid crystal phase at 25°C. when measured in bulk. In addition, preferred preparations of emulsomes are In some embodiments, the lipid of the hydrophobic core substantially free of lipase and phospholipase enzymatic may have a solid to fluid phase transition (melting) tem activity. As defined herein, an emulsion is "substantially perature between 25°C. and physiological temperature (37° 15 free" of lipase or phospholipase activity if the emulsion C.) when measured in bulk. For example, tricaprin melts at lipids orphospholipids are enzymatically cleaved at a rate of 35-37° C., and is wholly or predominantly in the fluid less than 0.1% per day when stored at room temperature. phase at physiological temperature. In addition to immunogens, other natural, synthetic, or Tricaprin may be used to form an excellent lipid core for recombinant proteins and peptides optionally may be present nanoemulsions. The lipid core alternatively may be com 20 in emulsomes. An example of natural protein is collagen, posed of lipids which are in the solid phase at 37° C. when which may be used to prepare emulsomes with controlled or measured in bulk, such as higher saturated triglycerides, sustained release properties. This is described in greater e.g., tripalmitin or tristearin. detail below. Cores of mixed fluid and solid phases at 37° C. are also Surface Active Molecules possible, particularly when the core contains mixtures of 25 In lipid particles of the invention, the lipid core is sur lipids. rounded by at least one envelope or layer containing phos The lipid or hydrophobic core of emulsomes also may be pholipid molecules. The phospholipid envelope functions as composed of or contain monoesters of fatty acids, such as a stabilizer or surface-active agent at the lipid-water waxes. In general, waxes are long chain fatty alcohol esters interface, thereby lowering the surface tension. of fatty acids. Many waxes have suitable melting character 30 In preferred embodiments, phospholipid molecules com istics for use in emulsomes, since they are solids at 25 C. prise at least 90%, more preferably 95%, even more pref Examples include the esters from beeswax and spermaceti, erably at least 99% of the surface-active molecules covering such as cetyl palmitate. the lipid core. Preferred waxes are made from saturated or monounsat However, other surfactants may be used in Small amounts, urated fatty acids and saturated or unsaturated fatty alcohols. 35 such as the non-natural nonionic surfactant TWEEN. The An example of the latter is provided by arachidyl oleate. lipid core of the nanoemulsion particles may be covered or Other satisfactory monoesters include solid monoglycer surrounded by more than one layer or envelope of surface ides such as glyceryl monostearate, and fatty acid esters of active molecules containing phospholipids. short chain alcohols such as ethyl stearate. In general, the surface-active phospholipid molecules are Cholesterol and cholesteryl esters optionally may be believed to form a monolayer around the lipid core of the incorporated into the lipid core or the surrounding phospho particles, with the polar phospholipid head groups at the lipid envelope. Cholesterol and its esters change the packing aqueous interface. However, particularly at higher molar structure of lipids, and in high concentrations they induce ratios of phospholipid to core lipid, excess phospholipid the formation of a liquid crystal phase. Aliquid crystal phase may be available to form one or more roughly concentric may co-exist with a solid phase under some conditions. 45 bilayers which encapsulate the lipid core with its associated Preferred cholesteryl esters are those of saturated or phospholipid monolayer. The number of bilayer envelopes is monounsaturated long chain fatty acids, such as palmitoyl or variable, and may include one, two, or many bilayers. These oleoyl, respectively. Cholesteryl esters may be present in bilayer envelopes entrap one or more aqueous compartments levels up to 50 mol % relative to the triglyceride or other which may be made to contain a water-soluble antigen by solid lipid core component. 50 creating the lipid particles in the presence of an aqueous Since cholesterol has a polar alcohol group, it tends to solution of that antigen. incorporate into the envelope monolayers or bilayers rather Although the multiple concentric bilayer model of the than into the lipid core itself, and should be considered a structure of emulsomes is proposed because it accounts for component of the phospholipid envelope rather than of the the observed ability of the particles to carry high loads of COC. 55 both lipid-soluble and water-soluble antigens, the present The lipid cores of emulsome particles of this invention invention does not depend upon and is not limited by the optionally may contain one or more antioxidants. A pre accuracy of the model. ferred antioxidant is o-tocopherol or its derivatives, which Other geometric relationships between the lipid core and are members of the Vitamin E family. Other antioxidants phospholipid molecules are possible which might explain include butylated hydroxytoluene (BHT). the antigen carrying capacity of emulsomes of the present Antioxidants lessen the formation of oxidative degrada invention. tion products of unsaturated lipids, such as peroxides. The The preferred phospholipids which constitute the sur need for antioxidants may be lessened by preparing the lipid rounding envelopes of emulsomes are natural phospholipids core from saturated fatty acids. such as soybean lecithin, egg lecithin, phosphatidylglycerol, Lipid particles of the invention preferably do not contain 65 phosphatidylinositol, phosphatidyl-ethanolamine, phospha serum apolipoproteins such as apoB, apo AI, apo AI, or apo tidic acid, sphingomyelin, diphosphatidylglycerol, E. The apoB proteinhas the effect of targeting intravenously phosphatidylserine, phosphatidyl-choline, cardiolipin, etc.; 5,716,637 9 10 synthetic phospholipids such aS parenteral administration are prone to cause toxic or unde dimyristoylphosphatidylcholine, dimyristoyl sirable side effects, whereas the phospholipid surfactants phosphatidylglycerol, distearoylphosphatidylglycerol, used in emulsomes are physiologically compatible. dipalmitoylphosphatidylcholine, etc.; and hydrogenated or In experiments to determine the effect of non-natural partially hydrogenated lecithins and phospholipids. 5 surfactants on emulsome structure, polysorbate (TWEEN In preferred embodiments, phospholipids which form 80) was added to an emulsome preparation at final concen "normal" phases (i.e., ionic "head" groups facing to the trations of 0.1, 0.5, and 1% (w.fv). The mean size of the external aqueous phase and lipophilic "tails' facing resulting emulsome particles decreased from 225 nm in the internally) under physiological conditions of pH and ionic absence of polysorbate to 120, 40, and 35 nm, respectively. strength comprise at least 50% of the total phospholipids, 10 Thus increasing concentrations of synthetic surfactants pro more preferably at least 75%, most preferably at least 90% gressively decrease the particle size, and higher concentra on a molar basis. Examples of normal phase forming phos tions than those used are expected to result in formation of pholipids are phosphatidyl-choline (lecithin), micelles (1-10 nm diameter). phosphatidylglycerol, and phosphatidylinositol. By contrast, Negatively charged lipid molecules such as oleic acid, or phosphatidyl-ethanolamine has a tendency to form reverse 15 negatively charged phospholipids such as phases, with the polar head groups oriented internally and phosphatidylglycerol, phosphatidic acid, phosphatidyl the lipophilic tails oriented outwardly. Reverse phases also inositol and phosphatidylserine, can be added to the lipid may be formed by cardiolipin or phosphatidic acid in the phase of emulsomes to increase the zeta potential of the presence of Ca' ions; by phosphatidic acid at pH less than composition, thus stabilizing the particles. 3; and by phosphatidylserine at pH less than 4. Additionally, the incorporation of these negatively The phospholipid component may be either saturated or charged lipid compounds in emulsomes results in the for unsaturated, and may have a gel to fluid phase transition mation of phospholipid bilayers with opposing charges, thus temperature either above or below 25° C. Egg or soy increasing the loading of water-soluble molecules in the phosphatidylcholines (egg or soy PC) are examples of aqueous compartments formed by the phospholipid bilayers phospholipids with transition temperatures well below room 25 surrounding the lipid core. This effect results from the larger temperature. Dimyristoyl phosphatidyl-choline (DMPC) has aqueous spaces between the bilayers caused by the electro a transition temperature slightly below room temperature. static repulsion between them. Another beneficial role of the Dipalmitoyl and distearoyl phosphatidylcholines (DPPC inclusion of negatively charged lipid molecules in emul and DSPC) are examples of phospholipids with transition somes is to reduce the likelihood of particle aggregation, temperatures well above room temperature, and in fact even 30 which minimizes destabilizing processes such as above physiological temperature (37° C). Acceptable emul coalescence, flocculation, or fusion. Aggregation is pre somes may be made with these and many other phospho vented by the repulsive forces between the approaching lipids. particles. In general, emulsomes prepared with phospholipids Negatively charged phospholipids such as phosphatidylg which are in the gel phase at 37° C. are expected to have 35 lycerol have been incorporated into liposomal formulations more rigid bilayer envelopes and longer circulation time in used in human clinical studies; see, e.g., S. Amselem et al., plasma. J. Pharm. Sci. (1990) 79, 1045-1052; S. Amselem et al., J. Emulsomes may be prepared with molar ratios of phos Liposome Res. (1992) 2, 93-123. The significance of zeta pholipid to total lipid in the range of 0.1 to 0.75 (10 to 75 potential in analyzing and predicting the properties of phos mol %), more usually 0.1 to 0.5 (10 to 50 mol%). The molar pholipid bilayers is discussed in L. Sai-lung, Chapter 19, ratio of phospholipid to core lipid typically may be in the Vol. 1 in "Liposome Technology,” 2nd ed., G. Gregoriadis, range of 0.1:1 to 2:1, usually 0.1:1 to 1:1, often 0.2:1 to ed., CRC Press, Boca Raton, Fla. (1993), pp. 331-342. Both 0.9:1, frequently 0.2:1 to 0.8:1, and commonly 0.25:1 to lipoidal particle size and particle stability vary as a function 0.6:1. of zeta potential. For liposomes, zeta potential and particle On a weight basis, the ratio of phospholipid to core lipid 45 size increase in proportion to the content of negatively usually falls in the range 0.5:1 to 1.5:1, and frequently 0.6:1 charged phospholipid, up to 50 weight % of negatively to 1.2:1. charged phospholipid. Non-natural surfactants and detergents optionally may be The preferred range of negatively charged lipid in emul incorporated into emulsomes in small amounts. As used some particles is 0 to 30 mol % relative to total phospholipid herein, the terms "nonnatural surfactants” or "detergents” 50 and charged lipid, more preferably 5 to 20 mol %, and still include a wide variety of manmade molecules which form more preferably 7 to 15 mol%. micelles in aqueous solution and contain both lipophilic and Incorporation of Immunogens hydrophilic domains; however, phospholipids which belong Emulsomes for use as vaccine vehicles contain an antigen to naturally occurring structural type are excluded from this of interest, usually an antigen bearing at least one epitope definition, regardless of whether a particular phospholipid is 55 which is present on an organism which is a pathogen in the obtained by synthesis or by isolation from natural sources. animal species to be vaccinated. In most cases, the antigen Examples of non-natural surfactants include the polysor is a peptide, a protein, or a glycoprotein. However, other bates ("TWEEN"), sodium dodecylsulfate (SDS), poly antigenic structures may be employed, including ethoxylated castor oil ("CREMOPHOR”), NP-40, and polysaccharides, glycolipids, or a hapten conjugated to a numerous other synthetic molecules. In preferred carrier. embodiments, nonnatural surfactants comprise less than Since the emulsome particles provide a soluble or lipid 10% (mol/mol) of the total surfactant, more preferably less soluble immunogens can be incorporated in emulsome vac than 5%, still more preferably less than 1%, and most cines of the present invention. Examples of peptide antigens preferably less than 0.1%. A significant advantage of emul are: hydrophilic natural or synthetic peptides and proteins somes is that they may be prepared as a stable nanoemulsion 65 derived from bacteria, viruses and parasites, such as the in the essential absence of nonnatural surfactants. Even recombinant gp160 envelope protein of the HIV virus; nonnatural surfactants which have been approved for natural or synthetic glycoproteins derived from parasites, 5,716,637 11 12 bacteria or viruses such as the native surface glycoprotein of acidic polymers obtainable from natural sources, such as Leishmania strain or subunit vaccines containing part of the alginic acid, hyaluronic acid, pectin, gum tragacanth, and glycopeptides alone or covalently conjugated to lipopeptides karayagum; and neutral non-naturally occurring polymers, like lauryl-cystein hydrophobic foot; protein toxoids such as such as polyvinylalcohol; or their mixtures. the Staphylococcus enterotoxin B toxoid, either chemically The ionizable polymers may be present as free acids, or physically inactivated; non-toxic bacterial surface struc bases, or salts, usually in a final concentration of 0.01-0.5% tures (fimbrial adhesions) of Escherichia coli strains such as (w/v). the Shiga-like Toxin B Subunit (SLT-B) and AF-R1, a pilus As used herein, a polymer of an indicated monomeric adhesion which is a virulence factor for RDEC-1 E. coli subunit contains at least 75%, preferably at least 90%, and strain; outer membrane proteins of Neisseria meningitidis; O up to 100% of the indicated type of monomer subunit; a Hepatitis B surface antigen; native or synthetic malaria copolymer of an indicated type of monomeric subunit con antigens derived from different portions of Plasmodium tains at least 10%, preferably at least 25% of that monomeric falciparum, etc. Examples of lipophilic or hydrophobic subunit. immunogens are lipopolysaccharides (LPS), such as detoxi A preferred bioadhesive macromolecule is the family of fied LPS obtained from E. coli (Sigma Chemical Co., St. 15 acrylic acid polymers and copolymers (e.g. Louis, U.S.A.); Lipid A, the terminal portion of LPS, such CARBOPOLTM). These contain the general structure: as the one isolated from Salmonella minnesota R595 from --CH-CH(COOH)--n List Biological Laboratories (California, U.S.A.). In some embodiments, the emulsome particles will be free One preferred group of polymers of acrylic acid is commer or substantially free of the above or other nonbioactive 20 cially available under the tradename Carbopol. Carbopol proteins, i.e. less than 5%, usually less than 1%, and 934 is available in a pharmaceutical grade. frequently less than 0.1% (w?w) protein relative to other Preferred bioadhesive or mucoadhesive macromolecules particle components. have a molecular weight of at least 50 kDa, preferably at In making a vaccine with emulsome vehicle, the antigen least 300 kDA, and most preferably at least 1,000 kDa. of interest may be added to the organic solution of core lipid 25 Favored polymeric ionizable mucoadhesive macromol and phospholipid prior to forming a lipid film. The antigen ecules have not less than 2 mole percent acidic groups (e.g. is thereby integrally associated into the structure of the lipid COOH, SOH) or basic groups (NH2, NRH, NR), relative particles as they are formed. Alternatively, the antigen of to the number of monomeric units. More preferably, the interest may be present in the aqueous suspension of lipid acidic or basic groups constitute at least 5 mole percent, particles prior to homogenization, or even added after 30 more preferably 25 or even 50, up to 100 mole % relative to homogenization. The latter method of preparation tends to the number of monomeric units of the macromolecule. produce more superficial binding of the antigen to the lipid Preferred macromolecules also are soluble in water particles. throughout their relevant concentration range (0.01-0.5% Continuous Aqueous Phase wfw). The aqueous component will be the continuous phase of 35 Polymeric Coating of Emulsomes the emulsome formulation and may be water, saline or any Biodegradable polymers may be incorporated to surround other suitable aqueous solution which can yield an isotonic or form part of the hydrophobic core of emulsomes. Poly and pH controlled preparation. meric emulsomes may contain biodegradable nonnatural In addition, the compositions of the invention may also polymers such as polyesters of lactic and glycolic acids, comprise conventional additives such as preservatives, polyanhydrides, polycaprolactones, polyphosphazenes and osmotic agents or pressure regulators and antioxidants. polyorthoesters, or natural polymers such as gelatin, Typical preservatives include Thimerosal, chlorbutanol, albumin, and collagen. The advantage of polymeric emul benzalkonium chloride, and methyl, ethyl, propyl or butyl somes is to provide controlled release for the parenteral parabens. Typical osmotic pressure regulators include glyc delivery of drugs and biological compounds in a sustained erol and mannitol, with glycerol being preferred. The pre 45 dosage form. ferred oil phase antioxidant is tocopherol or tocopherol The structure, selection, and use of degradable polymers Succinate. The aqueous phase may also include an antioxi in drug delivery vehicles have been reviewed in a recent dant of a polyamine carboxylic acid such as ethylene publication (A. Domb, S. Amselem, J. Shah and M. Maniar, pharino tetra-acetic acid, or a pharmaceutically acceptable Polymers for Advanced Technologies (1992) 3, 279-292). salt thereof. 50 Further guidance to selection of polymers is available in any Mucoadhesive Emulsome Vaccines standard text on that topic. Emulsome vaccine of the present invention optionally In general, the ratio of polymer to lipid core (e.g., may contain a bioadhesive macromolecule or polymer in an triglyceride) may be up to 50% (wfw). For the natural amount sufficient to confer bioadhesive properties. The protein polymers such as gelatin, which swell extensively in bioadhesive macromolecule enhances the delivery and 55 aqueous solution, useful levels of encapsulation may be attachment of antigens on or through the target mucous achieved with much lower amounts of polymer, such as 1% surface conferring mucosal immunity. The bioadhesive mac to 10% (w?w). romolecule may be selected from acidic non-naturally For most non-natural polymers, which are soluble in occurring polymers, preferably having at least one acidic organic solvents, the polymer may be codissolved with the group per four repeating or monomeric subunit moieties, triglyceride and phospholipid prior to the evaporation step. such as polyacrylic acid and/or polymethacrylic acid (e.g. For natural polymers which are soluble in aqueous solution, Carbopol, Carbomer), poly(methylvinyl ether/maleic the polymer may be dissolved in the solution used. anhydride) copolymer, and their mixtures and copolymers; Dehydrated Emulsomes acidic synthetically modified natural polymers, such as A further aspect of the invention provides dehydrated carboxymethylcellulose (CMC); neutral synthetically modi 65 emulsomes, made by dehydrating the compositions of the fied natural polymers, such as (hydroxypropyl)methylcel types described herein. Dehydration may be performed by lulose; basic amine-bearing polymers such as chitosan; standard methods, such as drying under reduced pressure; 5,716,637 13 14 when the emulsion is frozen prior to dehydration, this low water-soluble and water insoluble antigens can be encapsu pressure evaporation is known as lyophilization. Freezing lated either separately or simultaneously at high loadings in may be performed conveniently in a dry ice-acetone or ethyl the absence of any nonnatural ionic or non-ionic surfactant. alcohol bath. The pressure reduction may be achieved con veniently with a mechanical vacuum pump, usually fitted 5 TABLE 1. with a liquid nitrogen cold trap to protect the pump from Emulsome contamination. Pressures in the low millitor range, e.g. Dispersion Liposome 10-50 millitorr, are routinely achievable but higher or lower SME of a solid Dispersion pressures are sufficient. Dispersion fat or of phospho Emulsomes can be lyophilized by adding cryoprotectants 10 of an oil lipid in lipids in such as Sugars or amino acids, and stored as freeze-dried Definition in water Water water solid material that can be reconstituted with the aqueous Internal core oil solid or water medium before us, thus conferring further stability of the liquid incorporated antigens. crystalline lipid Preferred cryoprotectants include sugars such as glucose, 5 Phospholipid 0.5-2% 5-10% 0,5% Sucrose, lactose, maltose, and trehalose; polysaccharides content (wiv) such as dextrose, dextrins, and cyclodextrins; nonnatural Non-natural present usually usually surfactant absent absent polymers such as polyvinylpyrrollidone (PVP); and amino Co-surfactant present usually usually acids. The preferred range of cryoprotectant to emulsome absent absent phospholipid is 0.1% up to 10% (w.fw). Lipophilic up to up to up to Dehydration of an emulsome nanoemulsion yields a solid loading 10 mg/ml 100 mgm. 20 mg/ml residue which may be stored for prolonged periods, and may PC/total lipid 0.01-0.1 0.1-0.5 O.6-10 be rehydrated to yield an emulsome nanoemulsion having an (mol/mol) average particle size similar to that of the original nanoemul sion. The dehydrated emulsomes also retain substantial 25 Emulsomes of this invention differ from the microdroplets amounts of the originally incorporated antigen. of U.S. Pat. Nos. 4.725,442 and 4,622.219. Microdroplets, The amount of water remaining in the dehydrated emul originally called monolayer vesicles, consist of spheres of some preparation may vary widely depending upon the type organic liquid covered by one monolayer of phospholipid, and extent of dehydration procedure employed. In general, while the internal core of emulsomes consists of a solid lipid the dehydrated emulsomes contain less than 1% water by 30 or fat. The phospholipid content of microdroplets is low weight, especially when dehydrated by lyophilization. (about 1.2%) forming only one monolayer, while in emul However, stable dehydrated preparations may contain up to somes the phospholipid content is high (5-10%) and in 5% water. certain embodiments is believed to form several bilayers Dry compositions for preparing submicron emulsions are surrounding the fat core. Another major difference between disclosed in detail in PCT International Application No. 35 microdroplets and emulsomes is that microdroplets are PCT/US93/01415, which was filed on 17 Feb. 1993, and useful only for water-insoluble compounds, while in published as WO 93/15736 on 19 Aug. 1993. The disclosure emulsomes, due to the high lecithin content, water-soluble of said document is expressly incorporated herein in its as well as water-insoluble compounds can be incorporated. entirety by this reference thereto. Methods of Preparation of Emulsomes Distinctive Features of Emulsomes A further embodiment of the invention relates to methods Emulsomes of this invention are distinct from standard for preparation of emulsome vaccines intrinsically and oil-in-water emulsions. Due to the high phospholipid con extrinsically as extensively detailed in the examples. In tent of the current invention, a monolayer of phospholipid general, emulsome intrinsic formulations are prepared by surrounds the lipid core at the aqueous interface thereby emulsifying the antigen together with the emulsome stabilizing the emulsion. In addition, one or more bilayers or 45 components, while emulsome extrinsic formulations are envelopes of phospholipid molecules are believed to form prepared by adding externally the antigen to previously around the particles in many embodiments. Another major prepared plain emulsomes. difference is that while standard oil-in-water emulsions are Emulsomes may be prepared by mixing phospholipids dispersions of one liquid into another, emulsomes are dis and triglycerides in a weight ratio range of 0.5:1 wherein the persions of a solid in a liquid. 50 triglyceride has a solid to liquid phase transition temperature The main differences between oil-in-water emulsions and of greater than 25°C.; suspending the mixture in an aqueous emulsomes are summarized in Table 1. solution at a temperature below the solid to liquid transition One major drawback of standard oil-in-water emulsions is temperature of the triglyceride; and homogenizing or oth limited drug loading. When drug encapsulation above 1% is erwise reducing the suspension to yield the emulsomes. required, a correspondingly larger oil phase (10-20%) is 55 These emulsomes comprise a nanoemulsion of lipid par required to dissolve the drug. However, the high oil content ticles having a mean particle diameter of between about 10 reduces the stability of the emulsion, and the addition of a nm and 250 nm, usually within the range 20 to 180 nm, and surfactant or cosurfactants, is necessary. Due to the detergent frequently within the range 50 to 150 nm. properties of most surfactant compounds, their use for These size ranges preferably are determined on a weight parenteral administration is very limited. Many toxic reac percent basis, rather than a particle number basis. The cited tions have been reported even with the surfactants already ranges include the mean particle size. In certain approved for parenteral formulations, such as sodium embodiments, the cited ranges include the mean plus or deoxycholate, poloxamer-188 (Pluronic F-68), polysorbate minus the standard error, and in other embodiments the cited 80 (TWEEN 80), and Emulphor EL-620 or polyethoxylated ranges include at least 99% of the particles as determined on castor oil. 65 a weight basis. The pharmaceutically stable emulsomes described herein Conveniently, the lipid components may be dissolved in have major advantages over standard emulsions in that a volatile and chemically unreactive organic solvent such as 5,716,637 15 16 dichloromethane or diethyl ether. The organic solvent is tribution of the formulation was determined using a N4MD removed, typically under reduced pressure in a rotary evapo Coulter Particle Size Analyzer (Coulter Electronics, rator or under a stream of inert gas. The resulting lipid film England). The differential weight% mode of the instrument is hydrated and dispersed by covering and shaking with an indicated the existence of a single homogeneous population aqueous solution. The immunogen to be incorporated of emulsomes with a mean particle diameter of 84-32 nm. according to its chemical properties and hydrophilic The formulation was shown to be stable atroom temperature lipophilic nature can be included in the lipid phase or may for several months without changes in the mean size of the be added to the aqueous hydration solution. particles. Water-soluble antigens are encapsulated or entrapped in emulsomes by dissolving them in the aqueous medium, 10 Example 2 hydrating the dry fat-phospholipid mixture with the aqueous phase containing the antigen utilizing mechanical shaking, PREPARATION OF EMULSOMES USINGA and sizing the resultant dispersion by high shear EMULSIFLEXTM HOMOGENIZER homogenization to the desired final size range. To a 0.5 literround-bottomed flask, 3.5g of egg-lecithin, Lipid-soluble antigens may be incorporated into solid 15 3.5g of tricaprin, 0.2 g of cholesterol, 0.2 g of oleic acid, and lipid nanoemulsions by dissolving them in a suitable organic 0.05 g oftocopherol succinate were added. The lipid mixture solvent together with the lipid ingredients of the was dissolved in 50 ml dichloromethane. The organic sol composition, e.g., phospholipids and solid fatty acid esters, vent was evaporated until complete dryness under reduced evaporating the solvent to complete dryness, hydrating the pressure using a rotary evaporator (Heidolph, Germany). To antigen-lipid mixture with the aqueous phase utilizing the dry lipid film 70 ml of saline were added and the mixture mechanical shaking, and homogenizing the resultant disper was then hydrated by shaking until all the lipids were sion with high-shear homogenizers to final sizes in the range homogeneously dispersed in the aqueous phase (estimated of 10 to 250 nm. hydration time, 1 hr). The dispersion was homogenized by The lipid suspension or dispersion is then sized, typically submitting the preparation to 5-7 cycles of high shear by high shear homogenization at pressures up to 800 bar in 25 homogenization at 12,500 psi working pressure using a a Gaulin-type homogenizer (AVP Gaulin International, EmulsiFlexTM C30 Homogenizer (Avestin Inc., Canada). Holland) or EmulsiflexTM homogenizer (Avestin Inc., The particle size distribution of the resultant emulsomes was Canada). High pressure Gaulin homogenization is described determined using a N4MD Coulter Particle Size Analyzer in detail in Brandl et al., in Liposome Technology, 2nd ed., (Coulter Electronics, England). The differential weight % G. Gregoriadis, ed., Vol. 1, Ch. 3, CRC Press, Boca Raton, 30 mode of the instrument indicated the existence of a single Fla., (1993), pp. 49-65. homogeneous population of emulsomes with a mean particle Emulsomes also may be prepared by high pressure extru diameter of 109-30 nm. FIG. 2 shows the particle size sion through polycarbonate membranes. In this procedure, distribution of emulsomes prepared by EmulsiFlexTM instru the sizing step on the lipid dispersion is performed using a ment. pressure extruder, such as the stainless steel GHT6-400 35 Extruder or Pressure Cell (Nucleopore, U.S.A.), rather than Example 3 a high-shear homogenizer. The pressure extruder and the extrusion technique for liposome preparation are described CHARACTERIZATION OF EMULSOME in detail in S. Anselem et al., in Liposome Technology, 2nd PARTICLES ed., G. Gregoriadis, ed., Vol. 1, Ch. 28, CRC Press, Boca The parameters most frequently used to characterize lipid Raton, Fla., (1993), pp 501-525. particles are particle size and size distribution, morphology, Due to the small size of emulsomes they can be sterilized and lamellarity. Emulsome structure was demonstrated and by final sterile filtration through 0.2 pum filter membranes. characterized by several techniques including electron microscopy, photon correlation spectroscopy, and NMR. EXAMPLES 45 Particle Size Distribution: This invention is illustrated by the following non-limiting The particle size distribution of the emulsome formula examples: tions were determined by photon correlation spectroscopy Example 1 (PCS), based on measuring laser scattered-lightfluctuations, 50 using a N4MD Coulter Submicron Particle Size Analyzer PREPARATION OF EMULSOMES USINGA (Coulter Electronics, England) working at the differential HIGH SHEAR MICROLAB 70 GAULIN weight % operation mode of the instrument (Barenholz, Y., HOMOGENIZER and Amselem, S. In "Liposome Technology", Gregoriadis To a 0.5 liter round-bottomed flask, 2.5g of egg-lecithin, G., ed., 2nd edition, Vol. 1, pp 527–616, CRC press, Florida, 2.5g of tricaprin, 0.1 g of cholesterol, 0.1 g of oleic acid and 55 1993). 0.01 g of tocopherol succinate were added. The lipid mixture The particle size distribution of all emulsome formula was dissolved in 50 ml dichloromethane. The organic sol tions described in the Examples was determined by photon vent was evaporated until complete dryness under reduced correlation spectroscopy using the N4MD Coulter. The pressure using a rotary evaporator (Heidolph, Germany). To N4MD Coulter submicron particle analyzer, indicated the the dry lipid film.50 ml of saline were added and the mixture existence of a single homogeneous population of Emul was then hydrated by shaking until all the lipids were somes with a mean particle diameter in the range of 50-200 homogeneously dispersed in the aqueous phase. The disper nm (FIG. 2). sion was homogenized for five minutes at 15,000 rpm using 'P-NMR measurements: a Polytron PT 3000 (Kinematica, AG). The preparation was One of the most accurate and straightforward procedures then submitted to 10-15 cycles of high shear homogeniza 65 for quantitatively determining the lamellarity of phospho tion at 800 bar using a Microlab 70 Gaulin Homogenizer lipid dispersions is to use NMR spectroscopy and especially (AVP Gaulin International, Holland). The particle size dis 'P-NMR signal to monitor the phospholipid phosphorous 5,716,637 17 18 signal intensity (Lichtenberg, D., Amselem, S., and Tamir, I. emulsome preparation. Glycerol (1.44 gr) were added there Biochemistry, 18, 4169-4172, 1979). In particular, adding an after to reach a physiological osmolarity (269 mOsm). The impermeable paramagnetic shift reagent to the external pH was adjusted to 6.0 using a 1M NaOH solution. To this medium will decrease the intensity of the initial 'P-NMR plain mucoadhesive emulsome preparation, antigens can be signal by an amount proportional to the fraction of lipid 5 added extrinsically and mixed with the emulsome carrier exposed to the external medium. FIG.3 shows phosphorous particles by gentle shaking in order to obtain the proper nuclear magnetic resonance (P-NMR) spectra of emulsome vaccine. liposomes, emulsomes, and Submicron oil-in-water emul sion recorded before and after the addition of the lanthanide Example 5 ion Pr". The 'P-NMR spectra were recorded on a Brucker 10 AM400 instrument employing a 50 KHZ sweep width, 3 sec PREPARATION OF INTRINSIC HEPATTISB interpulse delay and broadening proton decoupling. Phos EMULSOME VACCNE phoric acid in DO was used as standard for 0 ppm chemical To a round 0.25 liter round-bottomed flask, 2.5 gr of shift. egg-lecithin, 2.5 gr of tricaprin, 100 mg of cholesterol, 100 For the submicron oil-in-water emulsion (SME) after the 15 mg of oleic acid, and 10 mg of tocopherol succinate were addition of the paramagnetic ion the whole 'P-NMR signal added. The lipid mixture was dissolved in 25 ml of chloro was shifted downfield (FIG. 3B1). This result is expected form. The organic solvent was evaporated until complete since in the SME all the phospholipid molecules are located dryness under reduced pressure using a rotary evaporator in the surface of the emulsion oil droplets as a monolayer, (Heidolph, Germany). To the dry lipid film 60 ml of phos thus the Pr" ions interact with all the exposed phospholipid phate buffered saline containing 0.5 mg of Hepatitis B molecules. On the other hand for emulsomes, the 'P-NMR antigen were added and the mixture was then hydrated by signal was split into two peaks after Pr-3 addition (FIG. shaking for 30 min using a Multiwrist shaker (Labline, 3B2), indicating that only a portion of the phospholipid U.S.A.) until all the lipids were homogeneously dispersed in molecules (those in the outer monolayer exposed to the the aqueous phase. The dispersion was homogenized using aqueous medium) interacted with the Pr" ions demonstrat 25 a Microlab 70 Gaulin Homogenizer (5 cycles at 800 bar). ing the existence of bilayer structures. The same picture was The particle size distribution of the resultant emulsomes was obtained for small unilamellar liposomes (FIG.3B3) used as determined using a N4MD Coulter Particle Size Analyzer control, where the existence of phospholipid bilayer (Coulter Electronics, England). The differential weight % domains have been well documented (Barenholz, Y., and mode of the instrument indicated the existence of a single Amselem, S. In "Liposome Technology", Gregoriadis G., 30 homogeneous population of emulsomes with a mean particle ed., 2nd edition, Vol. 1, pp. 527-616, CRC press, Florida, diameter of 105+24 nm. The emulsome vaccine formulation 1993). was then 2-fold concentrated using a Filtron ultrafiltration Electron microscopy: stirred cell (Omega Series membrane with 10,000 molecular Negative stain electron microscopy (EM) has been used to weight cutoff, Filtron Technology Corp., Massachusettes). characterize the size and shape of the lipid particles present 35 in phospholipid dispersions. EM gives also a gross estimate Example 6 of lamellarity since stain penetrates interbilayer spaces and allows lamellae to be resolved. FIG. 4 is an transmission PREPARATION OF MUCOADHESIVE electron micrograph of emulsome preparation showing INTRINSIC ANTI-HIV ENVELOPE PROTEIN spheric particles having a diameter in the range of 80-130 (qp160) EMULSOME VACCINE nm surrounded by well-defined phospholipid bilayers envel Antigen description and background: The urgency and oping the internal hydrophobic core made of triglycerides. high priority for developing an effective vaccine against the human immunodeficiency virus (HIV) are fully recognized. Example 4 The reasons for using subunits of the virus as the basis of an 45 HIV vaccine are the perceived overwhelming requirements PREPARATION OF EXTRINSIC for safety. Despite the high efficacy of many live attenuated MUCOADHESTVE EMULSOME WACCNE viral vaccines, the requirement for product safety, especially To a round 0.5 liter round-bottomed flask, 1.75 gr of in the case of retroviruses, has favored the subunit approach egg-lecithin, 1.75 gr of tricaprin, 70 mg of cholesterol, 70 50 to the extent that all of the current candidate preparations in mg of oleic acid, and 7 mg of tocopherol succinate were clinical prophylactic trials are of this type, being mainly added. The lipid mixture was dissolved in 50 ml of chloro gp160, the envelope protein of HIV, or part thereof. Studies form. The organic solvent was evaporated until complete have shown that gp160 attaches the virus to the cell and also dryness under reduced pressure using a rotary evaporator facilitates the fusion of the cell and virus during the early (Heidolph, Germany). To the dry lipid film 60 ml of aqueous 55 stages of infection. solution containing 0.1% EDTA were added and the mixture Emulsome preparation: was then hydrated by shaking or 30 min. using a multiwrist To a round 0.25 liter round-bottomed flask, 0.24 gr of shaker (Labline, U.S.A.) until all the lipids were homoge egg-lecithin, 0.24 gr of tricaprin, 20 mg of cholesterol, and neously dispersed in the aqueous phase. The dispersion was 20 mg of oleic acid, and 2 mg of tocopherol succinate were homogenized using a Microlab 70 Gaulin Homogenizer (5 added. The lipid mixture was dissolved in 50 ml of chloro cycles at 800 bar). The particle size distribution of the form. The organic solvent was evaporated until complete resultant emulsomes was determined using a N4MD Coulter dryness under reduced pressure using a rotary evaporator Particle Size Analyzer (Coulter Electronics, England). The (Heidolph, Germany). To the dry lipid film 60 ml of aqueous indicated the existence of a single homogeneous population solution containing 0.18 mg of gp160 antigen and 0.1% of emulsomes with a mean particle diameter of 140-50 nm. 65 EDTA were added and the mixture was then hydrated by Then 6.72 gr of a 1% Carbopol solution was added and shaking for 30 min. using a multiwrist shaker (Labline, stirred for 20 minto confer mucoadhesive properties to the U.S.A.) until all the lipids were homogeneously dispersed in 5,716,637 19 20 the aqueous phase. The dispersion was homogenized using emulsome formulation showed a mean droplet size of 112 a Microlab 70 Gaulin Homogenizer (6 cycles at 800 bar). nm. The estimated final gp160 concentration in the formu The particle size distribution of the resultant emulsomes was lation was 15 g/ml. determined using a N4MD Coulter Particle Size Analyzer (Coulter Electronics, England). The differential weight % 5 Example 8 mode of the instrument indicated the existence of a single PREPARATION OF MUCOADHESIVE homogeneous population of emulsomes with a mean particle EXTRINSIC EMULSOME WACCNE diameter of 15857 nm. The emulsome vaccine formulation CONTAINING gp160 ALONE OR COMPLEXED was then 5-fold concentrated using a Filtron ultrafiltration TO PROTEOSOMES stirred cell (Omega Series membrane with 10,000 molecular 10 weight cutoff, Filtron Technology Corp., Massachusetts). Mucoadhesive extrinsic emulsome formulation contain Then 1.3 grof a 1% Carbopol solution was added and stirred ing gp160 alone or gp160 complexed non-covalently to for 15 minto confer mucoadhesive properties to the emul proteosomes was performed by preparing plain emulsomes some vaccine preparation. Glycerol (0.285 gr) were added as described in Example 7, but in the absence of the antigen thereafter to reach a physiological osmolarity. The pH was 15 and adding externally an aqueous dispersion of the gp160 or adjusted to 6.0 using a 0.5M NaOH solution. The estimated gp160-conjugated to proteosomes to the plain emulsomes by final gp160 concentration in the formulation was 15 g/ml. gently shaking for 15 min at room temperature. Example 7 Example 9 20 PREPARATION OF MUCOADHESIVE PREPARATION OF INTRINSIC EMULSOME INTRINSIC EMULSOME VACCINE CONTAINING ANTI-HIV ENVELOPE PROTEIN VACCNE CONTAINING STAPHYLOCOCCUS (gp160) COMPLEXED TO PROTEOSOMES ENTEROTOXIN BTOXOD-FANTIGEN Antigen description and background: Staphylococcal Proteosomes are meningococcal outer membrane protein 25 enterotoxin B (SEB) is a potent toxin that causes food borne preparations purified from Neisseria meningitidis by deter illness among civilians and military personnel stationed gent extraction and ammonium sulphate precipitation. They around the world and is identified as a lethal offensive naturally form 20-100 nm diameter hydrophobic membra military threat that endangers both military and civilian nous vesicles. Antigens are non-covalently complexed to populations through aerosolization. proteosomes via hydrophobic interactions by mixing the 30 antigen and proteosomes in the presence of detergent and SEB infection in civilian populations is related to staphy then removing the detergent over a prescribed period of lococcal food poisoning by SEB and related toxins: also time, permitting hydrophobic interactions to occur in the contributes to death from staphylococcal sepsis following system. overwhelming staph infection. It also causes staph scalded Proteosomes have previously been shown to enhance the 35 skin syndrome in kids-i.e. morbidity and mortality from parenteral immunogenicity of peptides, gangliosides, staphylococcal infections (P. Marrack and J. Kappler, lipopolysaccharides and proteins hydrophobically com Science, 248,705-711, 1990). plexed to them (Lowell, G. H., L. F. Smith, R. C. Seid and Due to the similarity to the human response both in W. D. Zollinger, J. Exp. Med. 167, 658-663, 1988). (Lowell, sensitivity and clinical signs and the lack of an established G. H., W. R. Ballou, L. F. Smith, R. A. Wirtz, W. D. model for lethality to SEB delivered via the respiratory route Zollinger and W. T. Hockmeyer. Science 240, 800-802, in lower animal species, non-human primates have been the 1988; Lowell, G. H. 1990. In: "New Generation Vaccines'. primary animal model for development of vaccines to pro G. C. Woodrow and M. M. Levine (eds.), Marcel Dekker, tect against respiratory challenge with SEB. Early work Inc., New York, p. 141-160) and have been shown to be safe indicated that monkeys develop decreased sensitivity to for human use in vaccine trials involving tens of thousands 45 repeated mucosal administration of the toxin. This suggested of humans in the development of anti-meningococcal vac that protection to SEB exposure might be provided by toxoid cines (Zollinger, W. D. New and Improved Vaccines Against immunization. Studies in rhesus monkeys and other animals Meningococcal Disease. In: “New Generation Vaccines', G. indicated that oral immunization with formalinized toxoid C. Woodrow and M. M. Levine (eds.), Marcel Dekker, Inc., was ineffective against parenteral challenge whereas New York, p. 325-348). Furthermore, proteosomes confer 50 parenteral immunization with formalinized SEB toxid mucosal immunogenicity upon non-immunogenic antigens induced serum antibodies that recognized native SEB when administered orally or intranasally. Such intranasal or (Bergdoll, M. S. Enterotoxins. pp. 559-598. In: Staphylo oral proteosome vaccines induce up to 100% protection cocci and Staphylococcal Infections, eds. C. S. F. Easmon against lethal pneumonia or keratoconjunctivitis in experi and C. Adlam, Academic Press, London, 1983). In the latter mental murine models of shigellosis (Orr, N., G. Robin, D. 55 studies, however, several parenterally immunized monkeys Cohen, R. Arnon and G. Lowell. 1993. Immunogenicity and that acquired anti-SEB antibodies had severe immediate efficacy of oral or intranasal Shigella flexneri 2a and Shigella type hypersensitivity reactions when challenged with SEB sonnei proteosome-lipopolysaccharide vaccines in animal toxin. These adverse reactions suggested that the formalin models. Infect. Immun. 6, 2390-2395). ized SEB toxoid alone was not a candidate for parenteral Lipid and aqueous phases were prepared as described in vaccine development. Additionally, as the military threat Example 6. A vial containing 0.18 mg of gp160 non would be by aerosolization, it was determined that studies on covalently complexed to proteosomes and suspended in protection provided by serum IgG to respiratory challenge as saline was added to the water phase (60 ml total volume) and well as protective effects provided by anti-SEB secretory the mixture was gently shaken for 5 min. The subsequent IgA in the respiratory tract were required. steps involved in the preparation of the mucoadhesive 65 Recently, two identical lots of formalinized SEB toxoid emulsome vaccine were carried out as described in Example were made at Walter Reed Army Institute of Research, 6. The particle size volume % distribution of the resultant Washington DC (WRAIR) following previously described 5,716,637 21 22 specifications (Kaminski, R., S. Grate, E. Aboud-Pirak, C. Example 11 Hooper, T. Levin, I. Weiss, S. Amselem, R. Arnon and G. Lowell, 1993. In: Proceedings of 1993 Medical Defense PREPARATION OF INTRINSIC EMULSOME Bioscience Review, Baltimore, Md.). This WRAIR forma VACCNE CONTAINING STAPHYLOCOCCUS linized toxoid preparation designated Tox-F was non-toxic ENTEROTOXN BTOXOD-F COMPLEXED TO in rabbits at 0.5 mg/kg, the dose at which SEB toxin is PROTEOSOMES invariably lethal. Furthermore, it was non-toxic in the To a round 0.5 liter round-bottomed flask, 2.5 gr of murine D-galactosamine model of SEB toxicity even at 500 egg-lecithin, 2.5 gr of tricaprin, 100 mg of cholesterol, and ug per BALB/cmouse; 50 g of SEB is 100% lethal in such 100 mg of oleic acid, and 10 mg of tocopherol succinate mice. The physical characteristics of Tox-F were similar to 10 were added. The lipid mixture was dissolved in 50 ml of that described by Eldridge (Eldridge, J. H., Staas, J. K. chloroform. The organic solvent was evaporated until com Meulbroek, J.A., Tice, T.T. and Gilley, R.M.Biodegradable plete dryness under reduced pressure using a rotary evapo and biocompatible poly(DL-lactide-co-glycolide) micro rator (Heidolph, Germany). To the dry lipid film 50 ml of spheres as an adjuvant for staphylococcal enterotoxin B aqueous solution containing 5 mg of Staphylococcus Entero toxoid which enhances the level of toxin-neutralizing anti 15 toxin B toxoid-F complexed to proteosomes, and glycerol bodies. (Infect. Immun. 59,2978-2986, 1991) in that SDS (2.25% w/v) were added and the mixture was then hydrated PAGE gel of Tox-F showed two distinct bands with esti by shaking until all the lipids were homogeneously dis mated MW of 23,000 and 46,000. Biologically, Tox-F also persed in the aqueous phase. The dispersion was homog had the characteristics previously reported by Eldridge et al., enized using a Microlab 70 Gaulin Homogenizer (5 cycles namely in a Mouse Spleen Lymphocyte Proliferative Assay at 800 bar). The particle size distribution of the resultant in which concentrations of SEB toxin of 0.37-10.0 ug/ml formulation was determined using an NAMD Coulter Par were mitogenic, Tox-F was entirely non-mitogenic at all ticle Size Analyzer (Coulter Electronics, England). The concentrations tested (0.04-100.0 ug/ml). unimodal mode of the instrument indicated the existence of Preparation of SEB-Toxoid F: a single and homogenous population of emulsome particles Formalinized SEB-Toxoid (Tox-F) was prepared accord 25 with a mean particle size distribution of 152-53 nm. The ing to the method of Warren, J. R., Spero, L. and Metzger, estimated final antigen concentration in the formulation was J. F. 1983. J. Immunol. 111,885–892 and as per Eldridge.J. 100 g/ml. H. et al. 1991, infect. Immun. 59, 2978-2986 by formalin Example 12 treatment for 30 days at 37 C., pH 7.5. 30 Preparation of Emulsomes: PREPARATION OF EXTRINSIC EMULSOME To a round 0.5 liter round-bottomed flask, 2.25 gr of WACCNE CONTAINING STAPHYLOCOCCUS egg-lecithin, 2.25 gr of tricaprin, 90 mg of cholesterol, 90 ENTEROTOXN B-TOXOD-F COMPLEXED TO mg of oleic acid, and 9 mg of tocopherol succinate were PROTEOSOMES added. The lipid mixture was dissolved in 50 ml of chloro 35 form. The organic solvent was evaporated until complete Extrinsic emulsomes formulation of SEB-Toxoid-F- dryness under reduced pressure using a rotary evaporator complexed to proteosomes was performed by preparing (Heidolph, Germany). To the dry lipid film 50ml of aqueous plain emulsomes as described in Example 9, but in the solution containing 10 mg of Staphylococcus Enterotoxin B absence of the antigen and adding externally the aqueous toxoid, glycerol. 2.25% and 0.1% EDTA were added and the solution containing the SEB-Toxoid-F complexed to proteo mixture was then hydrated by shaking until all the lipids somes to the plain emulsomes by gently shaking for 15 min were homogeneously dispersed in the aqueous phase. The at room temperature. A total volume of 0.78 ml of stock dispersion was homogenized using a Microlab 70 Gaulin plain emulsomes were mixed with 0.78 ml solution of Homogenizer (6 cycles at 800 bar). The particle size distri SEB-Toxoid-F (1 mg/ml protein) in 0.15MNaCl and 0.01M bution of the resultantformulation was determined using an 45 Tris buffer to give a final SEB-Toxoid-F concentration of 0.5 N4MD Coulter Particle Size Analyzer (Coulter Electronics, mg/ml. England). The differential weight % mode of the instrument indicated the existence of a single and homogenous popu Example 13 lation of emulsome particles with a mean particle size PREPARATION OF INTRINSIC EMULSOME distribution of 11580 nm. The estimated final antigen 50 WACCINE CONTAINING LC-467 LESHMANLA concentration in the formulation was 220 pg/ml. LIPOPEPTDE ANTIGEN Example 10 Antigen description and background: PREPARATION OF EXTRNSC EMULSOME The gene for a surface protein antigen of Leishmania 55 major gp63, has been cloned and sequenced. This protein, VACCNE CONTAINING STAPHYLOCOCCUS recombinantly expressed in live Salmonella, or given in a ENTEROTOXN B-TOXOD-F sub-unit vaccine as either the purified native gp63 or Extrinsic formulation of SEB-Toxoid-F in emulsomes selected gp63 peptides (Jardim A., Alexander J., Teh S., Ou was performed by preparing plain emulsomes as described D, Olafson R. W. 1990. J. Ep. Med. 172, 645), has recently in Example 9, but in the absence of the antigen and adding been shown to limit the extent of lesion development in externally the aqueous solution containing the SEB murine models of cutaneous leishmaniasis when given with Toxoid-F to the plain emulsomes by gently shaking for 15 certain adjuvants. These results suggest that a vaccine to min at room temperature. A total volume of 0.78ml of stock protect humans against leishmaniasis composed of defined plain emulsomes were mixed with 0.78 ml solution of purified components is a realistic goal. The sub-unit vac SEB-Toxoid-F (1 mg/ml protein) in 0.15MNaCl and 0.01M 65 cines were effective, however, only when administered with Tris buffer to give a final SEB-Toxoid-F concentration of 0.5 adjuvants containing Corynebacterium parrum (CPV) and mg/ml. poloxamer 407. Other adjuvants (Complete Freund's 5,716,637 23 24 Adjuvant, CFA), or lack of adjuvant exacerbated disease. Major success was achieved with the discovery that subcu TABLE 2 taneous immunization with one small gp63 peptide % Protection covalently conjugated to lauryl-cysteine protected against (reduction of lesion size severe Leishmania cutaneous lesions with reduction of Waccine Formulation compared to control mice) lesions in three separate experiments. LC-467 in saline 73 LC-467 in emulsomes 94 Preparation of emulsomes: To a round 0.25 liter round-bottomed flask, 0.4 gr of 10 egg-lecithin, 0.4 gr of tricaprin, 15 mg of cholesterol, and 15 Example 15 mg of oleic acid, and 1.5 mg of tocopherol succinate were added. The lipid mixture was dissolved in 50 ml of chloro ENHANCED MURINE IMMUNOGENICITY OF form. The organic solvent was evaporated until complete SEB TOX-F ANTIGEN AFTER PARENTERAL dryness under reduced pressure using a rotary evaporator 15 IMMUNIZATION WITH INTRINSIC (Heidolph, Germany). To the dry lipid film 50 ml of aqueous EMULSOME VACCINE COMPARED TO FREE Solution containing 80 pug of LC-467 Leishmania lipopeptide ANTIGEN OR ALUM-ADJUVANTED WACCNE antigen in phosphate buffered saline were added and the mixture was then hydrated by shaking for 30 min. using a The antigen used was Staphylococcal Enterotoxin B multiwrist shaker (Labline, U.S.A.) until all the lipids were 20 (SEB) formalinized toxoid F. This antigen was formulated homogeneously dispersed in the aqueous phase. The disper intrinsically in emulsome as described in Example 9, and sion was homogenized using a Microlab 70 Gaulin Homog compared to SEB toxoid-F alone or adjuvanted with alum. enizer (5 cycles at 800 bar). The particle size distribution of BALB/c mice, 5 animals/group, were immunized twice at the resultant emulsomes was determined using a N4MD approximately 3-week intervals by intramuscular injections Coulter Particle Size Analyzer (Coulter Electronics, 25 with 50 ug doses of SEB Toxoid F. Sera, obtained after first England). The differential weight % mode of the instrument and second immunizations, were analyzed by ELISA tech indicated the existence of a single homogeneous population niques using anti-SEB as the detecting antibody. As shown in FIG. 5, the intrinsic emulsome formulation was more of emulsomes with a mean particle diameter of 18135 nm. effective in enhancing immunity to SEB antigens. The The emulsome vaccine formulations were then 6.5-fold 30 concentrated using a Filtron ultrafiltration stirred cell anti-SEB serum IgG titers obtained with the emulsome (Omega Series membrane with 10,000 molecular weight vaccine were higher than those obtained with the alum cutoff, Filtron Technology Corp., Massachusettes). The esti adjuvanted formulation or free antigen. mated final antigen concentration in the formulation was Example 16 0.25 mg/ml. 35 ENHANCED LAPINE IMMUNOGENCITY OF Example 14 SEB TOX-F ANTIGEN AFTER PARENTERAL IMMUNIZATION WITH EXTRINSIC IMMUNOGENICITY OF EMULSOME VACCINE EMULSOME WACCINE COMPARED TO FREE CONTAINING LC-467 LEISHMANIA ANTIGEN LIPOPEPTIDE ANTIGEN The antigen used was Staphylococcal Enterotoxin B The objective in the present example was to demonstrate (SEB) formalinized toxoid F. This antigen was formulated immunogenicity and efficacy of LC-467 Leishmania extrinsically in emulsomes as described in Example 10, and lipopeptide emulsome vaccine to protect against severe 45 compared to SEB toxoid-Ffree antigen. Rabbits, 5 animals/ morbidity of cutaneous leishmaniasis in murine models. group, were immunized twice at approximately 3 week intervals by intramuscular injections with 100 pug doses of The antigen used were lipopeptides obtained from the SEB Toxoid F. Sera, obtained after first and second major glycoprotein of the Leishmania parasite. The peptide immunizations, were analyzed by ELISA techniques using denoted 467 was covalently attached to lauryl cystsine to 50 anti-SEB as the detecting antibody. As shown in FIG. 6, the serve as the hydrophobic foot. extrinsic emulsome formulation enhanced 4-fold IgG anti The murine model used, CBA mouse, resembles the body production compared to the free antigen. human cutaneous disease. The immunization protocol included two injections of the animals (8 mice/group) at 55 Example 17 weeks 0 and 3 with the experimental vaccines (50 ug peptide?mouse). At week 6 the mice were infected with live PROTECTION AGAINST SYSTEMIC Leishmania parasites and the lesion size as function of time CHALLENGE WITH SEBIN MICE was measured and compared. The results were expressed as IMMUNIZED PARENTERALLY WITH SEB % decrease from control (saline injection). Immunization of TOXOD WACCNES FORMULATED WITH the mice with the LC-467 emulsome formulation enhanced ALUM, EMULSOME, OR FREE ANTIGEN the protective effects (reduction of lesion size) obtained with Mice immunized parenterally (Table 3) with Staphylo the free antigen (Table 2). coccus Enterotoxin B in mice immunized with SEB Since there is considerable homology among Leishmania 65 Toxoid-F vaccine formulated with emulsomes (as described strains, this peptide may have wide application in amelio in example 9) were significantly protected against systemic rating lesions caused by other forms of Leishmania. SEB challenge (100 ug toxin). 5,716,637 26 8-fold in the sera of mice immunized with the antigen TABLE 3 incorporated in emulsome vaccine formulation, and 128 fold in the sera of mice immunized with the gp160 Formula- Anti-SEB Died, proteosome-emulsome formulation compared to the free Antigen tion IgG total Survival antigen. In addition, immunization with gp160 plus emul control - O 9/10 10% somes without proteosomes enhanced anti-gp160 serum IgA SEB-tox F - 3,200 3/5 40% titers of 25.600 compared to <50 obtained by immunizing SEB-tox F Alum 51,200 4.5 20% with the free antigen as well as 270-fold and 6-fold increases SEB-tox F Emulsome 102,400 O5 100% in anti-gp160 intestinal and lung fluid IgA, respectively, 10 compared to immunizing with gp160 alone. Table 4 also The data in Table 3 show a very good correlation between shows that intranasal immunization with HIV gp160 formu the anti-SEB serum IgG titers obtained after intramuscular lated with emulsomes enhances serum IgG HIV gp41 pep immunization of CD-1 mice with protection against sys tide epitope responses (C448, C41, and CKen) as measured temic challenge with 100 g of SEB. In the groups immu by quantitative western blot analyses. In addition, formula nized with intrinsic emulsome-SEB Toxoid F vaccine, the 15 tion of the gp160-proteosome vaccine with emulsomes survival was 100% while for animals immunized with free enhances the serum IgG HTV responses to each of seven antigen or alum-adjuvanted vaccine the survival was only 20 gp120 and gp41 peptide epitopes tested with increased to 40%. responses ranging from over 116,000 to 9,000,000. These data show that optimal antigen delivery to the Example 18 mucosal immune system is effected by formulating with emulsomes and that specific secretory IgA antibodies can be PROTECTION OF MICE IMMUNIZED WITH elicited and boosted even far away from the immunizing MUCOADHESIVE EMULSOME WACCNES site. Lung and intestinal immunity was improved after nasal CONTAINING SEB-TOXOD F ANTEGEN OR immunization. SEB-TOXF COMPLEXED TO PROTEOSOMES 25 AGAINST INTRANASAL CHALLENGE WTTH SEB TOXIN IN BALB/C MICE D TABLE 4* GALACTOSAMINE MODEL Vaccine Formulation BALB/C mice (10 animals/group) were immunized intra nasally twice with 100 pig antigen doses of Mucoadhesive 30 Anti-gp160 IgG or IgA titers of sera, gavage, and Emulsome vaccines containing either SEB-Tox Fantigen or lavage fluids as measured by ELISA: SEB-Tox F complexed to proteosomes. The mucoadhesive Anti-gp160 51,200 409600 1638,400 6553,600 formulations were prepared as described in Example 4. The Serum IgG antigens were added extrinsically to the prepared plain Anti-gp160 k50 K50 3200 25,600 35 Serum IgA emulsomes. After a second immunization mice were chal Anti-gp160 5 147 32 1351 lenged intranasally with an LD100 dose (350 ug) of SEB Intestinal Toxin. Mice survival was determined 3 days post-challenge. IgA FIGS. 7A and 7B clearly show that the mice immunized with Anti-gp160 512 111 1176 3105 the emulsome vaccines had the highest percent of survival Lung IgA either in the Tox F or Tox F complexed to proteosomes Anti-gp160 peptide igG responses measured by groups (70% and 100% survival, respectively, compared to 0 quantitative western blots: survival in the control group). Peptide epitope Example 19 Specificity 45 INDUCTION OF MUCOSAL, INTESTINAL, C1 (48-128) COO <100 39,918 987,775 AND SYSTEMIC IMMUNOGENICTY IN MICE C21e (254- c100 <100 73.54 1,044,098 VACCNATED INTRANASALLY WITH ANTI 274) V3 (290–338) <100 COO 49,963 116,888 HIV ENVELOPE gp160 ANTIGEN ALONE OR C3 (342-405) k100 3100 k100 319355 COMPLEXED TO PROTEOSOMES 50 C448 (453- K100 182,362 <100 9,025 INCORPORATED IN EXTRINSIC 518) MUCOADHESIVE EMULSOME WACCINES C41 (579-605) 210,167 1,833,409 1189,986 2.469,425 CKen (735- C100 73,621 15,119 2697,623 The antigens used were gp160 alone or gp160 complexed 752) to meningococcal outer membrane proteosomes. These anti 55 *Bolded numbers indicate enhancement by emulsomes compared to same gens were formulated extrinsically in emulsomes as vaccine in saline without emulsomes. Underlined numbers indicate enhance described in Example 8. Mice (5 animals/group) were ment by proteosomes compared to same formulation without proteosomes. immunized intranasally with 100 ug antigen doses at differ ent intervals. Murine sera, lung fluids, and intestinal fluids Example 20 obtained after immunizations were analyzed by ELISA techniques using several specific HIV epitopes as the detect INCREASED IMMUNE RESPONSE INRHESUS ing antigens. MONKEYS IMMUNIZED WTTH ANTI Table 4 shows that emulsomes enhanced the mucosal LEISHMANIAINTRINSIC EMULSOME immunogenicity of HIV envelope protein following intra VACCINE CONTAINING LC-467 LPOPEPTDE ANTIGEN nasal immunization with HIV gp160 formulated with or 65 without the proteosome vaccine delivery system. Specific Rhesus monkeys (Macacca mulatta, 5 animals/group) systemic anti-HTV IgG antibody production was enhanced were immunized intramuscularly three times at weeks 0, 4 5,716,637 27 28 and 10 with 500 pig doses of LC-467 Leishmania lipopeptide noncellular lipid particles having a mean diameter of about antigen incorporated intrinsically in emulsomes as described 10 to 250 nm, as determined on a weight basis, the particles in Example 13. Monkey sera was collected before and after being suspended in an aqueous continuous phase. each immunization and analyzed for anti-Leishmania IgG wherein each said lipid particle comprises a lipid core antibody levels by ELISA. 5 composed of a lipid which is a solid or liquid crystal as The results in FIG. 8 show that the monkey group determined in bulk at a temperature of about 25° C. or vaccinated with the emulsome vaccine resulted in the high higher, and at least one phospholipid bilayer surround estanti-Leishmania IgG antibody titer, whereas the control ing said core and encapsulating a portion of said group and monkeys that received the LC-467 antigen alone aqueous continuous phase, 10 said particles entrapping about 0.001 to 5% of an immu had very low antibody levels. nogen in said lipid core or in said encapsulated aqueous Example 21 phase. 2. The pharmaceutical composition of claim.1 wherein the LYOPHILIZATION OF EMULSOMES mean particle diameter of said lipid particles falls within the To a 0.5 liter round-bottomed flask, 3.5g of egg-lecithin, 15 range of about 20 to 180 nm as determined on a weight basis. 5.2 g of tricaprin, 0.14 g of cholesterol, 0.14 g of cholesterol, 3. The pharmaceutical composition of claim2 wherein the 0.14 g of oleic acid, and 0.05 g of tocopherol succinate were particle diameter of at least 99% of said lipid particles falls added. The lipid mixture was dissolved in 50 ml dichlo within the range of about 50 to 150 nm as determined on a romethane. The organic solvent was evaporated to complete weight basis. dryness under reduced pressure using a rotary evaporator 20 4. The pharmaceutical composition of claim 2 wherein the (Heidolph, Germany). To the dry lipid film, 63 ml of a 7% lipid core comprises a fatty acid ester. sucrose solution were added, and the mixture was then 5. The pharmaceutical composition of claim 4 wherein the hydrated by shaking until all the lipids were homogeneously lipid core has a solid to fluid phase transition temperature dispersed in the aqueous phase. The dispersion was homog below 37° C. as determined in bulk. enized for 2 minutes at 13,000 rpm using a Polytron PT3000 25 6. The pharmaceutical composition of claim 4 wherein the (Kinematica, AG). The preparation was then submitted to 7 lipid core comprises a triglyceride. cycles of high shear homogenization at 800 bar using a 7. The pharmaceutical composition of claim 6 wherein Microlab70 Gaulin Homogenizer. The formulation was said triglyceride comprises a fatty acid moiety of Coto Cs. divided in 5 ml portions and the vials were then freeze-dried 8. The pharmaceutical composition of claim 6 wherein using a Christ Beta Freeze Dryer (Germany). The samples 30 said triglyceride is completely saturated. were reconstituted with water for injection and the particle 9. The pharmaceutical composition of claim 6 wherein size distribution of the resultant reconstituted emulsome said triglyceride is selected from the group consisting of formulation was measured. An average size of 102+15 was tricaprin, trilaurin, trimyristin, tripalmitin, and tristearin. measured, very similar to the mean size obtained before 10. The pharmaceutical composition of claim 6 wherein lyophilization. 35 the mole ratio of phospholipid to total lipid is in the range of from 0.1:1 to 0.5:1. Example 22 11. The pharmaceutical composition of claim 6 wherein the weight ratio of phospholipid to triglyceride is in the PREPARATION OF POLYMERIC EMULSOMES range of from 0.5:1 to 1.5:1. FOR CONTROLLED RELEASE OF ANTIGENS 40 12. The pharmaceutical composition of claim 4 wherein To a 0.5 liter round-bottomed flask, 0.5g of polylactide said phospholipid is a phosphatidylcholine. ("Resomer L 104.” MW=2,000 Da, Boehringer Ingelheim, 13. The pharmaceutical composition of claim 12 wherein Germany), 0.5g of egg-lecithin, 0.5g of tricaprin, 0.2 g of said phosphatidylcholine is egg phosphatidylcholine. cholesterol, 0.2 g of oleic acid, and 0.02 g of tocopherol 14. The pharmaceutical composition of claim 12 wherein Succinate were added. The lipid mixture was dissolved in 50 45 said phosphatidylcholine has a transition temperature below ml dichloromethane. The organic solvent was evaporated 25° C. until complete dryness under reduced pressure using a rotary 15. The pharmaceutical composition of claim 12 wherein evaporator (Heidolph, Germany). To the dry lipid 50 ml of said phosphatidylcholine is saturated. saline were added and the mixture was then hydrated by 16. The pharmaceutical composition of claim 1 wherein shaking until all the lipids were homogeneously dispersed in 50 said lipid particle contains cholesterol or cholesteryl esters. the aqueous phase. The dispersion was homogenized for 5 17. The composition of claim 1 wherein the immunogen minutes at 17,000 rpm using a Polytron PT3000 is hydrophilic, lipophilic, or amphophilic. (Kinematica, AG). The preparation was then submitted to 10 18. The composition of claim 1 wherein the immunogen cycles of high shear homogenization at 800 bar using a is a peptide, protein, or glycoprotein. Microlab70 Gaulin Homogenizer. To this emulsome 55 19. The composition of claim 18 wherein the antigen is formulation, antigens can be added extrinsically or intrinsi the gp160 envelope protein of the HIV virus, or a fragment cally. thereof. The invention has been described with respect to certain 20. The composition of claim 18 wherein the antigen is specific embodiments thereof. However, those skilled in the the surface glycoprotein of a Leishmania parasite, or a art will readily conceive of many other means of carrying fragment thereof. out the present invention based upon the generic teachings 21. The composition of claim 20 wherein the surface hereinabove. Applicants propose to be bound, therefore, glycoprotein or peptide is covalently conjugated to a hydro only by the spirit and scope of the invention as reflected in phobic component. the appended claims. 22. The composition of claim 21 wherein the hydrophobic What is claimed is: 65 component is lauryl-cysteine. 1. A pharmaceutical composition for the administration of 23. The composition of claim 1 wherein the immunogen antigen which comprises a nanoemulsion of a plurality of is a protein toxoid. 5,716,637 29 30 24. The composition of claim 23 wherein the immunogen suspending said mixture in an aqueous solution at a is Staphylococcus Enterotoxin B toxoid. temperature below the solid to liquid transition tem 25. The composition of claim 1 wherein the immunogen perature of the triglyceride; is complexed with a proteosome. reducing the size of the suspension to yield a nanoemul 26. The composition of claim 1 wherein the nanoemulsion sion of lipid particles having a mean particle diameter further comprises a bioadhesive or mucoadhesive macro of between about 10 nm and 250 nmi; and molecule. incorporating an immunogen in the nanoemulsion. 27. The composition of claim 26 wherein the said 37. The method according to claim 36 for preparing the mucoadhesive macromolecule is a polymer. composition of the nanoemulsion by an intrinsic procedure, 28. The composition of claim 26 wherein the said 10 where the immunogen is added before homogenization of mucoadhesive macromolecule is selected from the group of water and oil phases. acidic nonnatural polymers consisting of polymers and 38. The method of claim36 for preparing the composition copolymers containing acrylic acid units, polymers and of the nanoemulsion by an extrinsic procedure, where the copolymers containing methacrylic acid units, and poly immunogen is added externally by mixing with the previ (methylvinyletherfmaleic anhydride) copolymer. 15 ously prepared plain nanoemulsion. 29. The composition of claim 28 wherein the said polymer 39. A pharmaceutical composition comprising dehydrated is polyacrylic acid. lipid particles containing an antigen for administration as a 30. The composition of claim 1 which contains no added nanoemulsion, wherein said lipid particles comprise a lipid muramyl peptides. core surrounded by at least one phospholipid layer, said lipid 31. The pharmaceutical composition of claim 1 wherein core is composed of lipid in a solid or liquid crystalline said lipid particle is substantially free of lipase and phos phase at least about 25° C. as determined in bulk, and said pholipase activity. lipid particles have a mean diameter upon rehydration of 32. A method for delivery of an immunogen to an animal, about 10 to 250 nm. comprising administering to said animal a pharmaceutical 40. The pharmaceutical composition of claim 39 further according to claim 1. 25 comprising a cryoprotectant. 33. The method of claim 32 wherein the mean diameter of 41. The pharmaceutical composition of claim 40 wherein the lipid particles in said composition is in the range of about said cryoprotectant is selected from the group consisting of 20 to 180 nm. glucose, sucrose, lactose, maltose, trehalose, dextran, 34. The method of claim 32 wherein said composition is dextrin, cyclodextrin, polyvinylpyrrollidone, and amino administered parenterally, orally, intranasally, or topically, 30 acids. thereby providing enhanced immunogenicity. 42. The pharmaceutical composition of claim 40 wherein 35. The method of claim 32 wherein said composition is said cryoprotectant is present in a range of from 0.1% to administered to mucosal surfaces, thereby achieving 10% by weight compared to lipid. mucosal immunity. 43. The pharmaceutical composition of claim 39 wherein 36. A method for making a nanoemulsion for administra 35 said lipid particles contain an immunogen. tion of an immunogen, comprising the steps of: 44. A method for delivering an antigen to an animal preparing a mixture comprising phospholipid and triglyc comprising administering to said animal a pharmaceutical eride in the weight ratio range of about 0.5:1 to 1.5:1 composition according to claim 39. wherein said triglyceride has a solid to liquid phase transition temperature of greater than 25 C.; :: ; c. x: ::