WO2006068759A2 - Liposomes containing phytochemical agents and methods for making and using same - Google Patents

Abstract

The present invention encompasses liposomes containing at least one phytochemical useful for treating infectious diseases, cancer, inflammation, tissue injury and the like. In particular, the present invention encompasses liposomal preparations for delivering a composition comprising ellagic acid to a target cell or cells. Such liposomes may also include other therapeutic agents as required by the recipient patient including such compounds as anti-oxidants such as a-tocopherol and optionally a migration control agent. Ellagic acid may be advantageously combined with phosphatidylcholine to provide a liposomal preparation useful for the safe delivery of the phytochemical to a recipient patient.

Description

Title of the Invention

LIPOSOMES CONTAINING PHYTOCHEMICAL AGENTS AND METHODS FOR MAKINGAND USINGSAME

This application claims priority from U.S. provisional application 60/630,116 filed November 22, 2004, which is incorporated herein by reference in its entirety

Incorporation by Reference

All documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

Field of the Invention

This invention relates generally to liposome compositions containing phytochemical agents. In particular, the present invention relates to liposomal compositions comprising ellagic acid or plant-derived extracts comprising ellagic acid.

Background of the Invention

One consideration affecting the choice of therapies for treating diseases, such as cancer and infectious diseases involves the method by which a therapeutic agent can be optimally delivered to a tissue or target cells. Delivery systems have been developed to overcome some of the limitations of delivering a therapeutic agent in its free form. Practical limitations of delivery methods include 1) reduced efficacy of the therapeutic agent, often the result of low levels of the therapeutic agent at the target area or tissue, and 2) undesirable side effects associated with administration of the therapeutic agent. Many of the therapeutic agents currently used to combat disease have potentially dangerous, or even fatal side effects, at levels required for efficacy.

Liposomes offer important advantages as a delivery system. They are microscopic spheres composed of thin, durable membranes made primarily from pharmaceutically acceptable phospholipids. Liposomes possess a unique advantage because drugs and other therapeutic agents can be encapsulated into the liposome for delivery into the body. Useful liposomes are chemically stable under physiological conditions, although the liposomes themselves are essentially fat-soluble. Thus, a liposome delivery system can be used to dissolve fat-soluble drags in the watery environment of the body.

The use of liposomes to deliver highly toxic cancer and infectious disease drugs safely to patients has been studied for more than twenty years. Indeed, liposomes have been shown to regulate the passage of an encapsulated drug into the blood stream and permit a sustained release of the encapsulated drug into the body. Thus, encapsulating drugs into liposomes permits the safe administration of the drugs at levels which would otherwise be toxic (see, for example, U.S. Pat. No. 4,744,989 to Payne et al. issued May 17, 1988). Liposomes generically comprise an enclosed lipid droplet having a core, typically an aqueous core, containing the compound, hi certain embodiments, the compound is chemically conjugated to a lipid component of the liposome. In other embodiments, the compound is simply contained within the aqueous compartment inside the liposome.

Certain liposome formulations are known in the art. U.S. Pat. No. 5,223,263, issued Jun. 29, 1993 to Hostetler et al. discloses conjugates between antiviral nucleoside analogues and polar lipids for inclusion in liposomes. U.S. Pat. No. 5,466,468 to Schneider et al. issued Nov. 14, 1995 discloses parenterally administrable liposome formulation comprising synthetic lipids. U.S. Pat. No. 5,484,809, issued Jan. 16, 1996 to Hostetler et al. discloses taxol and taxol derivatives conjugated to phospholipids. U.S. Pat. No.5,580,571, issued Dec. 3, 1996 to Hostetler et al. discloses nucleoside analogues conjugated to phospholipids. U.S. Pat. No. 5,626,869 to Nyqvist et al. issued May 6, 1997 discloses pharmaceutical compositions wherein the pharmaceutically active compound is heparin or a fragment thereof contained in a defined lipid system comprising at least one amphiphatic and polar lipid component and at least one nonpolar lipid component. U.S. Pat. No. 5,744,461, issued Apr. 28, 1998 to Hostetler et al. discloses nucleoside analogues conjugated to phosphonoacetic acid lipid derivatives. U.S. Pat. No. 5,744,592, issued Apr. 28, 1998 to Hostetler et al. discloses nucleoside analogues conjugated to phospholipids. U.S. Pat. No. 5,756,116, issued May 26, 1998 to Hostetler et al. discloses nucleoside analogues conjugated to phospholipids. U.S. Pat. No. 5,843,509 to Calvo Salve et al. issued Dec. 1, 1998 discloses stabilization of colloidal systems through the formation of lipid-polysaccharide complexes comprising a water soluble and positively charged polysaccharide and a negatively charged phospholipid. International Patent Application Publication Number WO89/02733, published April 1989 to Vical discloses conjugates between antiviral nucleoside analogues and polar lipids. European Patent Application Publication Number 0350287A2 to Vical discloses conjugates between antiviral nucleoside analogues and polar lipids. International Patent Application Publication Number WO93/00910 to Vical discloses conjugates between antiviral nucleoside analogues and polar lipids. Rahman et al., 1982, Life Sci. 31: 2061-71 found that liposomes which contained galactolipid as part of the lipid appeared to have a higher affinity for parenchymal cells than liposomes which lacked galactolipid. Gregoriadis, 1995, Trends in Biotechnology 13: 527-537 reviews the progress and problems associated with using liposomes for targeted drug delivery. Ledley, 1995, Human Gene Therapy 6: 1129-1144 reviews the use of liposomes for gene therapy. Mickisch, 1995, World J. Urology 13: 178-185 reviews the use of liposomes for gene therapy of renal cell carcinoma. Yang et al. 1997, J. Neurotrauma 14: 281-297 review the use of cationic liposomes for gene therapy directed to the central nervous system. Storm & Crommelin, 1997, Hybridoma 16: 119-125 review the preliminary use of liposomes for targeting chemotherapeutic drugs to tumor sites. Manusama et al., 1998, Semin. Surg. Oncol. 14: 232-237 reported on preclinical and clinical trials of liposome-encapsulated tumor necrosis factor for cancer treatments. Although liposomes have conventionally been administered parenterally (see, for example, U.S. Pat. No. 5,466,468), reports of oral administration of liposome-related formulations have appeared in the art. U.S. Pat. No.4,921,757 to Wheatley et al. issued May 1, 1990 discloses controlled release of biologically active substances, such as drugs and hormones entrapped in liposomes which are protected from the biological environment by encapsulation within semi-permeable microcapsules or a permeable polymeric matrix. U.S. Pat. No. 5,043,165 to Radhakrishnan to Aug. 27, 1991 disclosed a liposome composition for sustained release of steroidal drugs. U.S. Pat. No, 5,762,904 to Okada et al. issued Jun. 9, 1998 discloses oral delivery of vaccines using polymerized liposomes. U.S. Pat. No. 5,955,451 to Lichtenberger et al. issued Sep. 21, 1999 discloses compositions comprising non-steroid antiinflammatory drugs (NS AID's) complexed with either zwitterionic or neutral phospholipids, or both, having reduced gastrointestinal irritating effects and enhanced antipyretic, analgesic, and anti-inflammatory activity.

Liposomes are also advantageous as delivery systems because liposomes alone can stimulate the immune system. It is known that liposomes may be more effectively processed by certain host cells, like macrophage, thus using a liposome delivery system may increase the efficacy of the encapsulated therapeutic agent. Further, liposomes may be modified to increase beneficial activity. For example, sugars may be attached to the lipid moieties to form glycolipids. Because glycolipids can be tailored to bind to specific proteins, the usefulness of the therapeutic agent encapsulated within the liposome may be increased.

Liposomes are currently being used for diverse applications, including for delivering cancer drugs; amplifying immunogenic events for a flow-bioassay; and for administering large amounts of drugs that would otherwise be bio-toxic if conventionally delivered. Additionally, liposomes are used as carriers for drugs whose water solubility is poor, like glucocorticoids. For example, liposomes may be useful for delivering therapeutic agents into deep lung tissue after administration via aerosolization.

Many naturally occurring plants and herbs have been shown to possess therapeutic activity. Phytochemicals, a term which refers generally to extracts of plants and herbs, can offer advantages over conventional therapeutic agents because many phytochemicals have been shown to be safe for human and animal contact and consumption. Additionally, phytochemicals are known which have broad activity, preventing or inhibiting the growth of a broad spectrum of microbes, as well as exhibiting efficacy against a range of diseases, including cancer. Phytochemicals in high doses are less harmful to the patient than more conventional therapeutic agents, including cancer drugs. However, large amounts of phytochemicals are still required in some applications to provide an effective therapeutic dose. Therefore, a need exists for a phytochemical delivery system which could provide the high doses of phytochemicals needed in many applications to fight disease and which could additionally be used to deliver fat-soluble phytochemicals to the aqueous environment of the animal body. For example, ellagic acid (4,4',5,5^>,6,6^I- hexyhydroxydiphenic acid dilactone) is a phenolic antioxidant dietary compound originating from raspberries and other plants. The compound has been identified as potent anticarcinogenic or chemopreventive agent. The main obstacle in the successful application of this interesting compound as an antitumor agent is the lack of a stable formulation, allowing parenteral, oral and topic applications of the drug due to its insolubility in physiological aqueous media and its oxidation sensitivity. There is a need, therefore for a liposome-based formulation of ellagic acid that could circumvent the described problems. Summary of the Invention

The present invention addresses the need for a delivery system for the administration of therapeutically effective phytochemicals to an animal or human patient when the phytochemical alone or in a non-liposomal carrier may not otherwise be efficiently or safely delivered directly due to adverse solubility or toxicity issues. The present invention, therefore, provides a liposomal-based delivery system capable of presenting a phytochemical to the interior of a target animal cell, for modifying the proliferation and/or metabolism of the cell.

The present invention encompasses liposomes containing a phytochemical compound or combination of phytochemicals which, when delivered to a human or animal patient, may be useful for treating infectious diseases, cancer or other pathological conditions such as inflammation, tissue injury and the like. Most advantageously, the present invention encompasses liposomal preparations for delivering a composition comprising ellagic acid to a target cell or cells. Such liposomes may also include other therapeutic agents as required by the recipient patient including such compounds as anti-oxidants such as, but not limited to, α- tocopherol (Vitamin E). The phytochemical or phytochemical combinations that may be included in the liposomes of the present invention may also be added to the liposomes in combination with a migration control agent that may control the rate at which the phytochemical is released from the liposome, or alternatively with a chemical releaser which facilitates release of the phytochemical from the liposome. The chemical releaser may facilitate the release of the phytochemical composition from the liposome. The releaser may be, for example, citric acid, a phytochemical which also exhibits antibacterial activity. Citric acid extract can be added to the liposome alone or in combination with other phytochemicals, with or without antioxidants like Vitamin E.

The liposomes according to the present invention may be composed of a variety of lipids. Generally, the liposomes will be composed of at least one phospholipid, typically phosphatidylcholine, phosphatidylglycerol, distearoylphosphatidylcholine, or distearoylphosphatidylglycerol. In one advantageous embodiment of the invention, ellagic acid maybe combined with phosphatidylcholine, thereby providing a liposomal preparation useful for the safe delivery of ellagic acid to a recipient patient. Phytochemicals, such as ellagic acid and plant extracts comprising ellagic acid, can be administered to a patient who has cancer via a liposomal delivery system according to the present invention that selectively targets tumor cells. Such delivery systems are known in the art, and are not limited to use for the treatment of cancer, but may also be used to direct the therapeutic agent to a targeted cell or tissue such as an inflamed tissue or articulated joint. For example, the pegylated stealth liposome system, which has been used to deliver doxorubicin (Vail et al., Clinical Cancer Res. 4:1567-71, 1998) to cancer cells could be used. The compositions of the invention could be delivered alone, in combination, or together with other therapeutic agents. In an especially advantageous embodiment of the present invention, the phytochemical encapsulated in the liposome delivery system is ellagic acid, or an extract from strawberries, blueberries, raspberries, guava or pomegranate comprising ellagic acid, which is known to be effective in inhibiting the proliferation of transformed cells and to modify cell-mediated inflammation. It is contemplated that ellagic acid may be also be further combined with an antioxidant such as Vitamin E. In other embodiments of the invention, the phytochemical for inclusion in the liposomes, alone or in combination, may be selected from the group consisting of grapefruit seed extract, Lemon Grass Oil, Tea Tree Oil, Haelan derived from liquid soy bean extract, Gravida, Pau D'Arco, Radium weed (Euphorbia peplus) and the like.

The liposome preparations of the present invention are useful for the delivery and sustained the release of a phytochemical composition used as a therapeutic agent to treat infectious diseases, cancer and other pathological conditions. Phytochemical compositions encompass isolated phytochemicals or in combination with other phytochemicals, releasers like citric acid, or antioxidants like Vitamin E. Liposomes may further be used to concentrate the effective dose of the phytochemical composition encapsulated within the liposome. The liposomes may be used as a specific delivery system to provide localized delivery. For example, liposomes allow the phytochemical composition to be injected directly into tumors, inflamed tissues or arthritic joints. Further, the liposome delivery system allows the delivery of the phytochemical composition into respiratory and deep lung tissue via aerolization with inhalers, or administered as a water-soluble spray to permit localized treatment of lung, throat, ear, sinus, and nasal infections. In one embodiment, the liposome delivery system allows the delivery ot the phytochemical compositions to the interior of target cell types.

One aspect of the present invention, therefore, is a liposome preparation, comprising at least one biologically active phytochemical preparation and a lipophilic layer. In one embodiment of the invention, the liposome preparation may further comprise an antioxidant. hi one embodiment of the present invention, the at least one biologically active phytochemical preparation is pomegranate seed extract. hi the various embodiments of the invention, the at least one biologically active phytochemical preparation may comprise ellagic acid. hi one embodiment of the present invention, the at least one biologically active phytochemical preparation is ellagic acid.

It is further contemplated that the liposome preparations according to the invention may further comprise a pharmaceutically effective carrier, wherein the liposomes in the pharmaceutically acceptable carrier have a concentration selected from the group consisting of between about 20 mg liposomes/ml of carrier to about 1000 mg liposomes/ml of carrier, between about 20 mg liposomes/ml of carrier to about 500 mg liposomes/ml of carrier, about 50 mg liposomes/ml of carrier 1 to about 200 mg liposomes/ml of carrier 1 and about 100 mg liposomes/ml of carrier. Another aspect of the present invention is a liposome preparation prepared by the steps of (a) dissolving a lipid in a methanol/methylene chloride mixture; (b) dissolving a phytochemical in ethanol; and (c) adding the phytochemical solution to the lipid solution and removing the organic solvents under vacuum, thereby encapsulating the phytochemical in a lipid coating. In one embodiment of this aspect of the invention, step (b) of the process of manufacture further comprises adding an antioxidant to the ethanol. hi another embodiment of this aspect of the invention, the process of manufacture further comprises the step of resuspending the product from step (c) in a buffer solution comprising a cryoprotectant. In one embodiment of the invention, the cryoprotectant is mannitol.

In the various embodiments of this aspect of the invention, the phytochemical is ellagic acid. Also, in the various embodiments of the invention, the antioxidant may be α-tocopherol. One embodiment of the present invention , therefore, is a liposome preparation prepared by the steps of (a) dissolving a phosphatidylcholine in an organic solvent; (b) dissolving ellagic acid and α-tocopherol in an organic solvent, (c) adding the ellagic acid solution to the phosphatidylcholine solution, wherein the ratio of phosphatidyl choline to ellagic acid (by weight) is selected from about 400:1, about 200:1, about 100:1, about 50:1, about 40:1, about 25:1, about 10:1 and about 5:1; and (d) removing the organic solvents under vacuum, thereby encapsulating the ellagic acid in a phosphatidyl coating.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

Other objects, features, and aspects of the present invention are discussed in greater detail below.

Detailed Description of the Invention In general terms, the present invention is directed to liposomal preparations containing phytochemicals, and methods of making and using the same. The rate of migration or the release of the phytochemical composition contained within the liposome may be affected by using a release agent, such as Vitamin E, a chemical releaser such as citric acid, or an anti-oxidant such as Vitamin E. In various embodiments of the invention, the chemical releaser may be the same as the phytochemical agent. An advantage of the present invention is that the phytochemical composition may be delivered by means of liposomes, thus permitting safe delivery of high doses of phytochemicals for the treatment of various cancers and infectious diseases, hi particular, the present invention provides a liposomes comprising as the effective therapeutic agent, ellagic acid and methods of preparing same. The term "phytochemical" as used herein refers to naturally occurring plant- derived phytochemical molecular constructions, derivatives thereof, functional moieties thereof, and functionally identical manufactured molecular constructions as may be obtained based upon the naturally occurring compositions herein described. The term "phytochemical" as used herein also refers to a compound or combination of compounds isolated from botanical sources and which may be incorporated in the present invention include, but are not limited to, Jasonia candicans (sesquiterpenes, lactones); Polygonum flaccidum (fiavone and alpha santalene derivatives); Acalypha wikesiana (extracts); Pavetta owariensis (procyanidins); Plectranthus hereroensis (diterpenoids, diterpenes); Moss (Dicranin extract); Cannabis sativa (extract); Gloiosiphonia spp. (gloiosiphones); Laminaceae spp. (extract); Securidaca spp. (extract); Veronia spp. (extract); Hyptis iimbrose (umbrosone); Asclepias syriaca (milkweed extract); Tagetes tenuifolia (thiophene); Calophyllum inophylloide (fiavonoids); Tanacetum densum (sesquiterpene lactones, triterpenoids);

Neorautanenia mitis (extract); Premna schimper (diterpene); Premna oligotricha (sesquiterpenes); Premna oligotricha (diterpenes); Jasonia candicans (essential oils); Visnea mocanera (beta-sitosterol, triterpenic betulinic acid, ursolic acid, plantanic acid); Asteraceae spp. (terthiophenes and polyynes); Petalostemum purpureum (extract); Camelia sinensis (catechin); Helichrysum picardii (fiavonoids);

Helichrysum italicum (fiavonoids); Corydalis pallida (protoberberine alkloids); Shiraia bambusicola (perylenequinones); Fraxinum omus (hydroxycoumarins); Podocarpus nagi (totarol and nortiterpene dilactones); Heterotheca inuloides (sesquiterpenoids); Pelargonium spp. (essential oils); Piper sarmentosum (phenylpropanoids); Allium spp. (extract); Juniperus procera (diterpenes); Achillea conferta (fiavonoids, flavones, sesquiterpenoid lactones); Magnolia virginiana (lignans, neolignans); Eucalyptus euglobal (euglobal); Armillaria mellea (armillaric acid); Dracena mannii (spirostanol saponin); Piper aduncum (chromenes, prenylated benzoic acid); Rhamnaceae spp. (cyclopeptide alkaloids); Buddleja globosa (verbascoside); Cephalocereus senilis (phytoalexin aurone); Salvia albocaerulea

(diterpene); Gomphrena martiana and Gomphrena boliviana (extracts); Paepalanthus spp. (vioxanthin); Helichrysum stoechas and Helichrysum crispum (extracts); Achillea ptarmica (trans-pinocarveyl hydroperoxides); Dehaasia incrassata (alkaloids); Asteraceae spp. (extracts); Arctotis auriculate (extracts); Eriocephalus africanus (extracts): Felicia erigeroides (extracts); Hemerocallis fulva (phytosterols, fatty acid esters); Psoralea juncea (plicatin B); Pluchea symphytifolia (caffeic acid esters); Tovomitopsis psychotrifolia (Vitamin E derivative); Celosia argentea (triterpenoid saponins and fiavonoids); Azadirachta indica (tetranortriterpenoid, mahmoodin, protolimonoids, naheedin); Moraceae spp. (coumarins); Hypericum erectum (phloroglucinol derivatives); Podospora appendiculate (Appenolides A, B, & C, furanones); Artemisia princeps var. orientalis, Artemisia capillaris, Artemisia mexicana and Artemisia scoparia (extract); Paddy malt (mash extract); Kigelia pinnata (extract); Acalypha wilkesiana (extract); seaweeds, seagrass and lemongrass (essential oils); Borrieria latifolia, Borreria setidens, Hedyotis diffusa), Hedyotis nudicaulis, Morinda elliptica, Morinda umbellata, Sida rhomhifolia, and Vitex ovata (extracts); Tabebuia impetiginosa, Achyrocline spp., Larrea divaricata, Rosa borboniana, Punica granatum, Psidium guineense, Lithrea ternifolia (extracts); Lepechinia caulescens, Lepidium virginicum and Tanacetum parthenium (extracts); Talaromyces flavus (extracts); Daucus carota (extract); Flabellia petiolata, Caulerpa prolifera, Halimeda tuna, Corallina elongata, Lithophyllum lichenoides, Phyllophora crispa, Cystoseira spp., Halopteris spp., Codium spp., Valonia utricularis, Posidonia oceanica, Zostera noltii and Cymodocea nodosa (extracts); Centauraea orientalis, Diospyros khaki, Sida hermaphrodita, Forsythia intermedia, Scutellaria polydon, Eugenia malaccensis and Eugenia jambolana (extracts); Fritillaria L. spp.

(ebeinone, steroidal alkaloids); Kigelia pinnata, Peperomia pellucida, Populus nigra, Populus balsamifera and Populus deltoides (extracts); Melaleuca alternifolia (essential oil); Elfvingia applanata (naringenin); Ficus sycomorus, grapefruit seed, Garlic, Allicin, Peat, Strophantus hispidus, Secamone afzeli, Mitracarpus scabeή, Entada abyssinjca, Terminalia spinosa, Harrisonia abyssinica, Ximinea caffra, Azadirachta indica, Spilanthes mauritiana, Terminalia spinosa (extracts); Cyanobacteria (ambigols A and B, tjipanazole); coffee (extract); Sporochnus pedunculatus, Dalbergia melanozylon, Celastrus scandens, Juglans nigra, Kalmia latifolia, Pelargonium xhortorum, Rhus glabra and Lindera benzoin (extracts); Striga densiflora, Striga orobanchioides, Striga lutea, Pistacia lentiscus L., Mitracarpus villosus, Bixa orellana, Bridelia ferruginea, Alpinia katsumadai, Alpinia officinarum, Artemisia capillaris, Casia obtusifolia, Dendrobium moniliforme, Epimedium grandiflorum, Glycyrrhiza glabra, Lithosperum erythrorhizon, Magnolia obovata, Morus bonbycis, Natopterygii incisium, Polygonum multiflorum, Prunus mume, Rheum palmatum, Ricinus communis, Sophoraflavescens, Swertiajaponica, black pepper, rosemary, red pepper, Isopyrum thalictroides, Calotropis procera, Chrysanthemum spp., Holarrhena antidysenterica, Lunularia crusiata, Dumertiera hirsuta, Exormotheca tuberifera, and liverwort (extracts); Filipendula ulmaria, Salix glauca, Usnea filipendula, Clkadina arbuscula (salicylic compounds); Tanacetum parthenium, Thymus capitatus, and Eljingia applanata (extracts); Fraxinus ornus (hydroxycoumarins, esculin, esculetin, fraxin, and fraxetin); Zizyphus nummularia, LONGO VITAL, Pelargonium spp., Scaevola sericea, Psychotria hawaiiensis, Pipturus albidis, Aleurites moluccana, Solatium niger, Piper methysticum, Barringtonia asiatica, Adansonia digitata, Harungana madagascariensis, Jacaranda mimosaefolia, Erythroxylum catauba, Bidens pilosa, Lemna minor, Potamogeton spp., Nasturtium officinale, Apium nodiflorum, Agaricus subrutilescens, Amanita virosa, Amanita pantherina, Lycoperdon perlatum, Psidium guajava, Averrhoa carambola, musa sapientum, Carica papaya, Passiflora edulis, Lansium domesticum and Baccaurea motley ana (extracts); horse radish, celandine grass, bur marigold and yarrow grass (extracts); Abuta grandifola, Cyperus articulatus, Gnaphalium spicatum, Pothomorphe peltata, Ficus sycomorus, Ficus Benjamina, Ficus bengalensis, Ficus religiosa, Alchomea cordifolia, Bridelia feruginea, Eucalyptus citriodora, Hymenocardia acida, Maprounea africana, Monachora arbuscula, Tedania ignis, Arenosclera spp., Amphimedon viridis, Polymastia janeirensis, Aplysina fulva, Pseudaxinella lunaecharta, Nelumbium speciosum and Mycale arenosa (extracts); cloves (eugenol acetate and iso-eugenol); Chrysthanemum boreale fsesquiterpenoid lactones); Eucalyptus globulus, Punica granatum, Bocconia arborea, Syzygium brazzavillense, Syzygium guineense, Carthamus tinctorius), Ginkgo biloba, Mosla chinensis, Salvia officinalis, and Cinnamomum cassia (extracts); Cryptolepis sanguinolenta (alkaloids, cryptolepine); Chelidonium majus (alkaloids, berberine, coptisine); Vitex agnus-castus (extract); Cladonia substellata (usnic acid); Ellagic acid, Fuligo septica, Tubifera microsperma (extract); Mundulea monantha, Tephrosia linearis (Ηavonoids); Lpomoea fistulosa (extract); Pimenta dioica (essential oils); Ratibida latipalearis, Teloxys graveolens, Dodonaea viscosa, Hypericum calycinum, Hyptis albida, Hyptis pectinata, Hyptis suaveolens and Hyptis verticillata (extracts); Asteriscus graveolones fbisabolone hydroperoxides); Derris scandens, Alnus rubra, Araliaceae family (extracts); Vinca rosea, Australian tea tree oil, peppermint oil, sage oil, thymol, eugenol and Thuja orientalis (extracts); Anacardium occidentale (phenolic lipids); Oidiodendron tenuissimum (extract); Acacia nilotica and Acacia farnesiana (polyphenol, tannin); Teminalia alata and Mallotus phillipinensis (extracts); Piectranthus grandidentatus (abientane diterpenoids); Pumica granatum and Datura metel (extracts); tea, Agave lecheguilla, Chamaesyce hirta, Baccharis glutinosa and Larrea tridentata (extracts); Camelia sinensis and Euphorbia hirta (theaflavin, polyphenon 60); Tabernaemontana pandacaqui, Yucca shidigera, Hemistepa lyrata, Yougiajaponica, Prunella vulgaris, Lamium amplexicaule, Juniperus chinensis, Ixeris dentata, Gnaphalium affine, Chelidonium majus, Spirea prunifolia, Eiγthroniumjaponicum, Taxus wallichiana, Ganoderma lucidum Drava nemorosa, Youngia capillaris, Equisetum arvense, Australiam Lavender, Black Seed, Catuaba casca, Cineole, Damiana, Dicranum scoparium, Eucalptus oil, Ginger, and Grape seed (extracts); Neem seed, bark, and leaf extract; Neem oil; New Zealand Manuka extract; Nicotiana tabacum extract; olive leaf extract; a-pinene and b-pinene extracts; Rhubarb root extract; Syringa vulgaris extract; Tea tree oil (Terpinen-4-ol, α-terpinene, y-terpinene, α-terpineol, Terpinolene); Thyme (extract) and Vitamin E.

Ellagic acid, which is found in high concentrations in such plant sources as strawberries, blueberries, raspberries, pomegranate seed extract and guava, known to be a natural anti-cancer drug. Pomegranate seed extract, has been demonstrated in animal models to inhibit tumor growth induced by several chemical carcinogens, including polycyclic aromatic hydrocarbons, nitrosamines, afiatoxins and aromatic amines, and such cancers as prostate cancer. Other studies have demonstrated that ellagic acid, when applied topically to mouse skin, can effectively inhibit tissue plasminogen activator-induced ornithine decarboxylase activity, hydroperoxide production and DNA synthesis. In addition to earlier reports, studies have shown that oral administration of ellagic acid significantly reduced the level of lipid peroxidase and liver dihydroxyproline in animal models. Studies have also indicated that oral administration of ellagic acid can circumvent carbon tetrachloride toxicity and subsequent lung fibrosis. There is also experimental data demonstrating that ellagic acid may be effective in the treatment of inflammatory diseases such as arthritis. High levels of ellagic acid contained within liposomes can be delivered to a patient to treat cancer, inflammation and the like. The liposomal delivery system of the present invention provides a safe, time-released delivery system. The liposome delivery system of the present invention may also function as a concentrate carrier and a specific localized delivery system, permitting delivery of the phytochemical composition directly into tumors or site of inflammation while simultaneously protecting the phytochemical agent from breakdown by the body's defense systems. Liposomes containing phytochemical agents of the present invention may be used to treat cancer or destroy or inhibit the growth of bacteria or other causative agents of infectious diseases.

Accordingly, the liposomal preparations of the invention are especially useful for the safe delivery of effective amounts of purified ellagic acid to such abnormal or damaged tissues or for the delivery of crude ellagic acid preparations such as pomegranate seed extract. If ellagic acid is otherwise delivered as a non-liposomal preparation to the animal or human patient or to cells such as in tissue culture, its low aqueous solubility precludes realization of useful levels of the therapeutic agent. Other examples of phytochemicals useful alone or in combination in the present invention include, but are not limited to, Grapefruit Seed Extract, Lemon

Grass Oil, Tea Tree Oil, Citric Acid, Vitamin E, Haelan, Artemesoa, Pau D'Arco and Radium Weed and other phytochemicals exhibiting biocidal activity. Grapefruit Seed Extract is an effective phytochemical biocide with activity against bacteria, fungi, and some parasites. Vitamin E is an antioxidant and it is believed that it can also function as a release agent by preventing oxidative damage to a phytochemical agent. In addition to functioning as a release agent, Vitamin E possesses antimicrobial properties, and thus may itself function additionally as the phytochemical. Other phytochemicals known to function as anti-oxidants, as well as to possess antimicrobial properties (*denotes significant activity) and which may be usefully incorporated into the liposomal compositions of the present invention include, but are not limited to, aqueous or organic solvent extracts of Panax ginseng; Panax quinquefolius; Bixa orellana; Humulus lupulus; Spinacia oleracea; Arctium lappa; Cichorium intyhus; Cynara scolymus; Helianthus annuus; Inula helenium; Armoracia rusticana; Momordica charantia; Vaccinium corymbosum; Vaccinium myrtillus; Avena sativa; Oryza sativa; Lavandula latifolia; Marrubium vulgare; Melissa officinalis; Mentha pulegium; Mentha spicata; Nepeta cataria; Ocimum basilicum; Origanum onites; Perillafrutescens; Prunella vulgaris; Rosmarinus officinalis; Salvia officinalis; Salvia sclarea;Satureja hortensis; Thymus vulgaris; Laurus nobilis; Arachis hypogaea; Glycine max; Glycyrrhiza glabra; Glycyrrhiza uralensis; Lens culinaris; Phaseolus coccineus; Phaseolus lunatus; Phaseolus vulgaris; Phaseolus vulgaris; * Pisum sativum; Psophocarpus tetragonolobus; Pueraria lobata; Tamarindus indica; Tamarindus indica;* Viciafaba; Vigna angularis; Vigna mungo; Vigna radiata;Allium ampeloprasum; Allium cepa; Allium sativum; Asparagus officinalis; Linum usitatissimum; Morus alba; Eucalyptus globulus; Pimento, dioica; Syzygium aromaticum; Olea europaea; Oenothera biennis; Sesamum indicum; Plantago asiatica; Fagopyrum esculentum; Prunus cerasus; Prunus spinosa; Rosa canina; Rubusfruticosus; Rubus idaeus; Coffea arabica; Citrus aurantium; Citrus par adisi; Ribes nigrum; Ribes rubrum; Capsicum frutescens; Solanum tuberosum; Solanum tuberosum; * Theobroma cacao; Camellia sinensis; Coriandrum sativum; Cuminum cyminum; Daucus carota; Trachyspermum ammi; Vitis vinifera; Curcuma longa; Zingiber officinale.

Other antioxidants useful in the present invention include lysine, butylatedhydroxytoulene (BHT), butylatedhydroxyanisole (BHA), Grape Seed extract, Pine Bark extract (Proanthocyanidins), β-Carotene, Bilberry extract, Ascorbic acid, Ginkgo Biloba extract, Green Tea extract, Tumeric, Zinc Picolinate, and Selenium. Antioxidants may be used advantageously alone or in combination with phytochemicals in the liposome delivery system of the present invention. The term "liposome" as used herein refers to vesicle-forming lipids.

Liposomes or liposome-like compositions generally comprise an "outer membrane" or bulk aqueous phase and "central core" or inner aqueous phase. Vesicle-forming lipids can form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids. The liposomes can also include other lipids incorporated into the lipid bilayers, with the hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and the head group moiety oriented toward the exterior, polar surface of the bilayer membrane.

In advantageous embodiments, the liposome or liposome-like composition may be a stealthed liposome, lipid carrier, complex, mixture, supramolecular structure multimolecular aggregate or lipid-based drug delivery system. "Stealthed" liposomes and liposome-like compositions comprise a biologically effective amount of at least a first stealthing agent in operative association with the outer membrane. A "stealthing agent" is a component that increases the biological half life of a liposome or liposome-like composition when operatively associated with the outer membrane of the liposome or liposome-like composition. In "operative association", the outer membrane of the liposome or liposome-like composition is preferably "coated" with the one or more stealthing agents. Effective stealthing agents include a range of biocompatible hydrophilic polymers, such as polyamines, polylactic acid, polyglycolic acid, polylactic- polyglycolic acid (PLGA), polypeptides and related materials. An especially advantageous stealthing agent is polyethylene glycol (PEG) component, wherein the resulting stealthed liposomes are termed "PEGylated liposomes".

Liposomes according to the invention may also be stealthed or PEGylated liposomes wherein an antibody to an aminophospholipid or anionic phospholipid, or antigen-binding fragment thereof, may operatively associated with the outer membrane of the liposome, preferably where the liposome is "coated" with an antibody or fragment thereof. Particularly advantageous liposomes are such "antibody-coated" stealthed or PEGylated liposomes wherein at least one phytochemical therapeutic agent, such as ellagic acid, is operatively associated with the liposome or dispersed within the liposomal formulation. The therapeutic agent may be operatively associated with or maintained within the central core of the liposome.

The vesicle- forming lipids are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar. There are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin, phosphatidylglycerol_jlecithin_j.β-_j.γ-dipalmitoyl-α-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))-prop-l-yl- N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, palmitoyl-oleoyl-phosphatidylcholine, di-stearoyl- phosphatidylcholine, stearoyl-palmitoyl-phosphatidylcholine, di-palmitoyl- phosphatidylethanolamine, di-stearoyl-phosphatidylethanolamine, di-myrstoyl- phosphatidylserine, di-oleyl-phosphatidylcholine, and the like where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. The above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids include glycolipids and sterols such as cholesterol.

Cationic lipids are also suitable for use in the liposomes of the invention, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component. Such cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge. Preferably, the head group of the lipid carries the positive charge. Exemplary cationic lipids include l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N- hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[l-(2,3- dioleyloxy)propyl]-N,N,N~trimethylamrnonium chloride (DOTMA); 3 [N-(N' ,N'- dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethyldioctadecylammonium (DDAB). The cationic vesicle-forming lipid may also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic lipid, such as polylysine or other polyamine lipids. For example, the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid. The vesicle-forming lipid may be selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in serum and to control the rate of release of the entrapped agent in the liposome. Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., above room temperature, more preferably above body temperature and up to 80° Celsius. Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures.

On the other hand, lipid fluidity may be achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature, more preferably, at or below body temperature.

Vesicle-forming lipids having a main phase transition temperatures between about 2° Celsius and about 80° Celsius are suitable for use as the primary liposome component of the present composition. In a preferred embodiment of the invention, a vesicle-forming lipid having a main phase transition temperature above about 37° Celsius is used as the primary lipid component of the liposomes. In another preferred embodiment, a lipid having a phase transition temperature between about 37° Celsius and about 70° Celsius is used. By way of example, the lipid distearoyl phosphatidylcholine (DSPC) has a main phase transition temperature of 55.1° Celsius. Phase transition temperatures of many lipids are tabulated in a variety of sources, such as Avanti Polar Lipids catalogue and Lipid Thermotropic Phase Transition Database (LIPIDAT, NIST Standard Reference Database 34). Liposomes suitable for use in the present invention also include a vesicle- forming lipid derivatized with a hydrophilic polymer. As has been described, for example in U.S. Pat. No. 5,013,556 and in WO 98/07409, which are hereby incorporated by reference, such a hydrophilic polymer provides a surface coating of hydrophilic polymer chains on both the inner and outer surfaces of the liposome lipid bilayer membranes. The outermost surface coating of hydrophilic polymer chains is effective to provide a liposome with a long blood circulation lifetime in vivo. The inner coating of hydrophilic polymer chains extends into the aqueous compartments in the liposomes, i.e., between the lipid bilayers and into the central core compartment, and is in contact with the entrapped compound after the compound is loaded via remote loading. A liposome formulation having a surface coating of hydrophilic polymer chains distributed on the inner and outer liposome surfaces may provide for a phytochemical compound where the compound is retained in the liposomes for improved therapeutic activity. Vesicle-forming lipids suitable for derivatization with a hydrophilic polymer include any of those lipids listed above, and, in particular phospholipids, such as distearoyl phosphatidylethanolamine (DSPE).

Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide. Polymers may be employed as homopolymers or as block or random copolymers. A preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between about 500 and about 5,000 daltons, most preferably between about 1,000 and about 2,000 daltons. Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., about 120 to about 20,000 daltons.

The amount of the therapeutically useful phytochemical agent(s) to be included in the liposomal preparation is not, per se, critical and can vary within wide limits depending inter alia on the particular agent, the intended application and the lipid used. Generally, the phytochemical agent may be included in an amount of between about 0.005 to 10% by weight of the liposomal preparation and more usually may be included in an amount of between 0.01 and 0.1% by weight. For example, one embodiment of the liposomal preparation of the present invention comprises the phytochemical ellagic acid with phospatidylcholine as the liposomal component in the ratios of phospatidylcholine: ellagic acid (by weight) of from about 400:1, about 200:1, about 100:1, about 50:1, about 40:1, about 25: 1, about 10:1 or about 5: 1.

As used hereinafter, the term "therapeutically effective concentration" refers to the concentration of at least one phytochemical agent in a liposomal preparation that, when delivered to a recipient cell, tissue or animal or human patient, is capable of modifying the physiology of the recipient cells in a therapeutically desirable manner. The terms "therapeutically effective concentration" "effective amount" or "effective dose" refers to the amount necessary or sufficient to inhibit undesirable cell growth, e.g., prevent undesirable cell growth or reduce existing cell growth, such as tumor cell growth. The effective amount can vary depending on factors known to those of skill in the art, such as the type of cell growth, the mode and regimen of administration, the size of the subject, the severity of the cell growth, etc. One of skill in the art would be able to consider such factors and make the determination regarding the effective amount.

Preparation of vesicle-forming lipids derivatized with hydrophilic polymers has been described, for example in U.S. Pat. No. 5,395,619. Preparation of liposomes including such derivatized lipids has also been described, where typically, between 1- 20 mole percent of such a derivatized lipid is included in the liposome formulation. It will be appreciated that the hydrophilic polymer may be stably coupled to the lipid, or coupled through an unstable linkage which allows the coated liposomes to shed the coating of polymer chains as they circulate in the bloodstream or in response to a stimulus.

The liposomes according to the present invention may be prepared by a variety of techniques, such as those detailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980). Typically, the liposomes are multilamellar vesicles (MLVs), which can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids and including a vesicle-forming lipid derivatized with a hydrophilic polymer are dissolved in a suitable organic solvent which is evaporated in a vessel to form a dried thin film. The film is then covered by an aqueous medium to form MLVs, typically with sizes between about 0.1 to 10 microns.

The therapeutic phytochemical agent of choice can be incorporated into liposomes by standard methods, including (i) passive entrapment of a water-soluble compound by hydrating a lipid film with an aqueous solution of the agent, (ii) passive entrapment of a lipophilic compound by hydrating a lipid film containing the agent, and (iii) loading an ionizable drug against an inside/outside liposome ion gradient, termed remote loading. Other methods, such as reverse evaporation phase liposome preparation, are also suitable.

After liposome formation, the vesicles may be sized to achieve a size distribution of liposomes within a selected range, according to known methods. The liposomes are preferably uniformly sized to a selected size range between 0.04 to 0.25 μm. Small unilamellar vesicles (SUVs), typically in the 0.04 to 0.08 μm range, can then be prepared by extensive sonication or homogenization of the liposomes. Homogeneously sized liposomes having sizes in a selected range between about 0.08 to 0.4 microns can be produced, e.g., by extrusion through polycarbonate membranes or other defined pore size membranes having selected uniform pore sizes ranging from 0.03 to 0.5 microns, typically, 0.05, 0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly to the largest size of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. The sizing is preferably carried out in the original lipid-hydrating buffer, so that the liposome interior spaces retain this medium throughout the initial liposome processing steps. The pharmaceutical compositions of the present invention may also comprise a pharmaceutically acceptable carrier. Many pharmaceutically acceptable carriers may be usefully employed in the compositions of the present invention. Generally, normal saline will be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, but are not limited to, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. The compositions according to the present invention may be sterilized by conventional sterilization techniques well known to those of skill in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.

The concentration of liposomes in the carrier may vary. Generally, the concentration will be from about 20 mg/ml to about 1000 mg/ml, advantageously between about 20 mg/ml to about 500 mg/ml, more advantageously from about 50 mg/ml to about 200 mg/ml and most advantageously at about 100 mg/ml. Persons of skill may vary these concentrations to optimize treatment with different liposomal components or of particular patients. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, liposomes composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site of administration.

The phytochemical composition of the present invention is delivered encapsulated in liposomes and can be used to treat infectious diseases, inflammation such as arthritis or artherosclerotic lesions, and cancer. The phytochemicals may be incorporated into liposomes with or without the use of antioxidants, such as Vitamin E, or release agents like citric acid. Liposomes provide a sustained release vehicle for the phytochemical composition. Additionally, the liposomes serve to act as concentrate carriers or specific localized delivery systems for the phytochemical agents contained within. The liposomes of the present invention can be used to provide safe and sustained release of the phytochemical agents contained within, thus providing an effective method for the treatment of ear, nose and throat diseases; sinus infections; or lung and respiratory diseases. Additionally, liposomes containing phytochemical agents, as in the present invention, can be used in medical devices or products such as inhalers, aerolizers, gel packs, and bandages for burn patients, hi addition, liposomes encapsulating phytochemicals can be long-lasting liposomes, such as stealth or PEG-liposomes that can optionally be designed to include specific ligands recognizable by the cells of a specific target tissue. Thus, the phytochemicals can be delivered to the cell interiors by means of receptor-mediated endocytosis. However, it should be recognized that these applications are presented for exemplary purposes only and the present invention should not be limited thereto.

The amounts of the phytochemical agents added to the liposomes are dependent upon the particular application. Factors to consider are the conditions under which the phytochemical composition is to be used, the disease to be treated, the duration of the use of the phytochemical, and the active concentration of the photochemical desired.

Treatment with liposomes containing phytochemicals can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of administration. The route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Liposomes containing phytochemicals such as ellagic acid can be administered alone, or co-administered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions. Liposomes containing phytochemicals may be administered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard pharmaceutical texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985 may be consulted to prepare suitable preparations, without undue experimentation.

The effective dosage and route of administration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the host, as well as LD₅₀ and other screening procedures which are known and do not require undue experimentation. Dosages of the phytochemical agents can range generally from a few hundred milligrams to a few grams.

When making the compositions of the present invention, the phytochemicals, antioxidants, and chemical releasers may be added either together or sequentially (if more than one agent is employed) to the liposome composition. The mixture is then mixed until the phytochemicals are evenly dispersed within the liposomes. Resulting liposomes containing the phytochemical agents can be delivered to a patient in a variety of ways. In one embodiment, liposomes containing the phytochemical agents are delivered as a mist via an inhaler. Alternatively, liposomes containing the phytochemical agents can be directly injected into tumors.

One aspect of the present invention, therefore, is composition comprising at least one biologically active phytochemical and a lipophilic layer, wherein the lipophilic layer forms a liposome comprising the phytochemical. In one embodiment of the invention, the liposome preparation may further comprise an antioxidant. In one embodiment of the present invention, the at least one biologically active phytochemical preparation is pomegranate seed extract.

In the various embodiments of the invention, the at least one biologically active phytochemical preparation may comprise ellagic acid. In one embodiment of the present invention, the at least one biologically active phytochemical preparation is ellagic acid.

In one embodiment of the present invention, the composition is a stealth liposomes having an outer lipid layer conjugated to a biocompatible hydrophilic polymer selected from the group consisting of polyamine, polylactic acid, polyglycolic acid, polylactic-polyglycolic acid (PLGA), a polypeptide, polyethylene glycol (PEG) or an antibody.

It is further contemplated that the liposome preparations according to the invention may further comprise a pharmaceutically effective carrier, wherein the liposomes in the pharmaceutically acceptable carrier have a concentration selected from the group consisting of between about 20 mg liposomes/ml of carrier to about 1000 mg liposomes/ml of carrier, between about 20 mg liposomes/ml of carrier to about 500 mg liposomes/ml of carrier, about 50 mg liposomes/ml of carrier 1 to about 200 mg liposomes/ml of carrier 1 and about 100 mg liposomes/ml of carrier. Another aspect of the present invention is a liposome preparation prepared by the steps of (a) dissolving a vesicle-forming lipid in an organic solvent; (b) dissolving a phytochemical in an organic solvent; (c) adding the phytochemical solution to the lipid solution and removing the organic solvents under vacuum, thereby encapsulating the phytochemical in a lipid coating and forming liposomes. In one embodiment of this aspect of the invention, step (b) of the process of manufacture further comprises adding an antioxidant to the organic solvent. In another embodiment of this aspect of the invention, the process of manufacture further comprises the step of resuspending the product from step (c) in a buffer solution comprising a cryoprotectant. In one embodiment of the invention, the cryoprotectant is mannitol.

In the various embodiments of this aspect of the invention, the phytochemical is ellagic acid. Also, in the various embodiments of the invention, the antioxidant may be α-tocopherol.

One embodiment of the present invention, therefore, is a composition comprising at least one biologically active phytochemical and a lipophilic layer, wherein the lipophilic layer forms a liposome comprising the phytochemical wherein the ratio of lipophile to phytochemical (by weight) is selected from about 400:1, about 200:1, about 100: 1, about 50: 1, about 40:1, about 25:1, about 10:1 and about 5:1, wherein the liposome optionally is a stealth liposome having an outer lipid layer conjugated to a biocompatible hydrophilic polymer selected from the group consisting of polyamine, polylactic acid, polyglycolic acid, polylactic-polyglycolic acid (PLGA), a polypeptide, polyethylene glycol (PEG) or an antibody and optionally comprises at least one of an antioxidant and a releasing agent, and wherein the at least one biologically active phytochemical comprises ellagic acid, pomegranate seed extract, or a plant extract comprising ellagic acid, and optionally wherein the liposomes are suspended in a pharmaceutically effective carrier, the pharmaceutically acceptable carrier having a concentration selected from the group consisting of between about 20 mg liposomes/ml of carrier to about 1000 mg liposomes/ml of carrier, between about 20 mg liposomes/ml of carrier to about 500 mg liposomes/ml of carrier, about 50 mg liposomes/ml of carrier 1 to about 200 mg liposomes/ml of carrier 1 and about 100 mg liposomes/ml of carrier. .

One embodiment of the present invention , therefore, is a liposome preparation prepared by the steps of (a) dissolving a phosphatidyl choline in a methanol/methylene chloride mixture; (b) dissolving ellagic acid and α-tocopherol in ethanol; (c) adding the ellagic acid solution to the phosphatidylcholine solution, wherein the ratio of phosphatidyl choline to ellagic acid (by weight) is selected from about 400:1, about 200:1, about 100:1, about 50: 1, about 40: 1, about 25:1, about 10:1 and about 5:1; and (d) removing the organic solvents under vacuum, thereby encapsulating the ellagic acid in a phosphatidyl layer.

It should be understood that the present invention is not limited to the specific compositions or methods described herein and that any composition having a formula or method steps equivalent to those described falls within the scope of the present invention. Preparation routes of the composition and method steps for treating infectious diseases or cancer are merely exemplary so as to enable one of ordinary skill in the art to make the composition and use it according to the described process and its equivalents. It will also be understood that although the form of the invention shown and described herein constitutes preferred embodiments of the invention, it is not intended to illustrate all possible forms of the invention. The words used are words of description rather than of limitation. Various changes and variations may be made to the present invention without departing from the spirit and scope of the invention.

Example 1: Preparation of a stable, injectable formulation of ellagic acid in small unilamellar liposomes.

Ellagic acid (EA) ((4,4',5,5^l,6,6'-hexyhydroxydiphenic acid dilactone, mol.wt. 338.23) was obtained from Fluka (Buchs, Switzerland). To obtain a highly pure product the original ellagic acid (>98% purity) was recrystallized from ethanol and stored in the dark at-20°C. (a) Liposome preparation

Small unilamellar liposomes were composed of 1.0 g soy phosphatidylcholine, 10 mg D,L-α-tocopherol as an antioxidant and 25 mg ellagic acid. Ellagic acid (EA) (25 mg) was dissolved in 50 ml ethanol and added to the lipids that had been dissolved in methanol/methylene chloride (1 :1, v/v) in a round bottom flask. The organic solvent was removed at 40°C during 40-60 min by rotatory evaporation. The dry lipid-EA mixture was then suspended in 5 ml phosphate buffer- mannitol (20 mM phosphate and 230 mM mannitol, pH 7.4). Mannitol was included as a cryoprotectant for protection of the liposomes during lyophilization steps.

For size-reduction of the liposomes, the lipid-EA suspension was repeatedly (2-4 times) extruded sequentially through polycarbonate filters (NUCLEPORE™) of defined pore size (0.8 μm; 0.4 μm and 0.2 μm) using a LIPEX™ extruder (Lipex Biomembranes, Inc., Vancouver, Canada).

Samples of 1 ml (1- 5 mg ellagic acid/ml) of the resulting small unilamellar EA-liposomes were lyophilized in a speed-vac instrument. (b) Liposome characterization

The mean diameters and homogeneity of the liposomes were characterized with a Nicomp 370 submicron particle sizer (Nicomp, Santa Barbara, California). The EA-liposomes particle sizes were determined after preparation as 106 +/- 60 nm. The EA-liposomes were lyophilized and reconstituted in water. The mean size of the EA-liposomes measured 20 hrs after reconstitution was 32 nm with 2% of the population having a 200 nm mean size. Prophetic Example

(a) In vitro Cytotoxicity

Free ellagic acid and liposomal formulations of doxorubicin, prepared as described above in Example 1, are tested against mouse and human tumor cell lines (for example M109-S, M109-R, C-26, KB, KB-V). Cells for each line are exposed continuously to the test ellagic acid for 72 hrs.

Other experimental details are as described by Horowitz et al., Biochem Biophys Acta, 1109(2):203 (1992). Briefly, 5 x 10³ cells from exponentially growing cultures in 200 μl aliquots are plated onto 96-well flat-bottom microtiter plates. Following 20 hrs in culture, during which cells will attach and resume growth, 20 μl of the tested drug formulation (free ellagic acid or liposome-ellagic acid) is added to each well. For each 10-fold increase in drug concentration, six drug concentration points are tested. Each test is performed in triplicate wells and in two parallel plates. The cells are treated continously for about 72 hrs. The cultures are fixed by the addition of 50 μl 2.5% glutaraldehyde to each well for 10 mins. The plates are washed three times with de-ionized water, once with 0.1 M borate buffer (pH 8.5) and then stained for 60 mins with 100 μl methylene blue (1% in 0.1 M buffer borate, pH 8.5) at room temperature. The plates are rinsed in five baths of de-ionized water to remove non- cell bound dye and then dried. The dye is extracted with 200 μl 0.1 M HCl for 60 mins at 37° Celsius, and the optical density determined using a microplate spectrophotometer.

The growth rate is calculated by dividing the doubling times of drug-treated cells with those of the control cells. The drug concentration which causes a 50% inhibition of the control growth rate (IC50) is calculated by interpolation of the two closest values of the growth inhibition curve.

All the materials used in the study were sterile, and all the experiments were performed in sterile conditions.

(b) In vivo Plasma Clearance Rate Three month-old BALB/c female mice are injected intravenously with 10 mg/kg of either liposome-ellagic acid or with liposome-ellagic acid- Vitamin E, prepared as described in Example 1. Blood samples are taken 4, 24 and 48 hrs after injection for analysis of plasma ellagic acid levels.

(c) In vivo Therapeutic Activity. Thirty mice are inoculated in the footpad with M109-S cells (10⁶ cells). Seven days later, when the footpad thickness has increased from a normal value of approximately 1.5 mm to an average of 2.0-2.5 mm, the mice are divided into three groups of 10 each and the mice groups are injected intravenously with either free ellagic acid, liposome-ellagic acid, or liposome-ellagic acid- Vitamin E. Thereafter, the footpad thickness is measured twice a week with calipers to follow tumor growth and effect of therapy.

In another study, mice are inoculated intraperitonally with C-26 cells (10⁶ cells). Five days later, the mice are separated into three groups of 10 mice each, and each group of mice is injected intravenously with either free ellagic acid, liposome- ellagic acid, or liposome-ellagic acid- Vitamin E. The survival of these mice is followed and survival curves are plotted. Although the invention has been described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims

We claim:

1. A composition comprising at least one biologically active phytochemical and a lipophilic layer, wherein the lipophilic layer forms a liposome comprising the phytochemical.

2. The composition according to Claim 1, further comprising an antioxidant.

3. The composition according to Claim 1, further comprising a releasing agent.

4. The composition according to Claim 1, wherein the at least one biologically active phytochemical comprises ellagic acid or a plant extract comprising ellagic acid.

5. The composition according to Claim 4, wherein the at least one biologically active phytochemical is pomegranate seed extract.

6. The composition according to Claim 4, wherein the at least one biologically active phytochemical preparation is ellagic acid.

7. The composition according to Claim 1, wherein the liposomes are suspended in a pharmaceutically effective carrier.

8. The composition according to Claim 7, wherein the liposomes in the pharmaceutically acceptable carrier have a concentration selected from the group consisting of between about 20 mg liposomes/ml of carrier to about 1000 mg liposomes/ml of carrier, between about 20 mg liposomes/ml of carrier to about 500 mg liposomes/ml of carrier, about 50 mg liposomes/ml of carrier 1 to about 200 mg liposomes/ml of carrier 1 and about 100 mg liposomes/ml of carrier. .

9. The composition according to Claim 1, wherein the liposomes are stealth liposomes having an outer lipid layer conjugated to a biocompatible hydrophilic polymer selected from the group consisting of polyamine, polylactic acid, polyglycolic acid, polylactic-polyglycolic acid (PLGA), a polypeptide, polyethylene glycol (PEG) or an antibody.

10. A liposome composition prepared by the steps of: (a) dissolving a vesicle-forming lipid in an organic solvent;

(b) dissolving a phytochemical in an organic solvent;

(c) adding the phytochemical solution to the lipid solution and removing the organic solvents under vacuum, thereby encapsulating the phytochemical in a lipid coating and forming liposomes.

11. The liposome composition according to Claim 10, wherein step (b) further comprises adding an antioxidant to the organic solvent.

12. The liposome composition according to Claim 11, further comprising the step of resuspending the product from step (c) in a buffer solution comprising a cryoprotectant.

13. The liposome composition according to Claim 12, wherein the cryoprotectant is mannitol.

14. The liposome composition according to Claim 10, wherein the phytochemical is ellagic acid or a plant extract comprising ellagic acid.

15. The liposome composition according to Claim 11, wherein the antioxidant is α-tocopherol.

16. A composition comprising at least one biologically active phytochemical and a lipophilic layer, wherein the lipophilic layer forms a liposome comprising the phytochemical wherein the ratio of lipophile to phytochemical (by weight) is selected from about 400:1, about 200:1, about 100:1, about 50:1, about 40:1, about 25:1, about 10:1 and about 5:1, wherein the liposome optionally is a stealth liposome having an outer lipid layer conjugated to a biocompatible hydrophilic polymer selected from the group consisting of polyamine, polylactic acid, polyglycolic acid, polylactic-polyglycolic acid (PLGA), a polypeptide, polyethylene glycol (PEG) or an antibody and optionally comprises at least one of an antioxidant and a releasing agent, and wherein the at least one biologically active phytochemical comprises ellagic acid, pomegranate seed extract, or a plant extract comprising ellagic acid, and optionally wherein the liposomes are suspended in a pharmaceutically effective carrier, the pharmaceutically acceptable carrier having a concentration selected from the group consisting of between about 20 mg liposomes/ml of carrier to about 1000 mg liposomes/ml of carrier, between about 20 mg liposomes/ml of carrier to about 500 mg liposomes/ml of carrier, about 50 mg liposomes/ml of carrier 1 to about 200 mg liposomes/ml of carrier 1 and about 100 mg liposomes/ml of carrier. .

17. A liposome composition prepared by the steps of: (a) dissolving a phosphatidylcholine in a solvent;

(b) dissolving ellagic acid and α-tocopherol in a solvent;

(c) adding the ellagic acid solution to the phosphatidylcholine solution, wherein the ratio of phosphatidylcholine to ellagic acid (by weight) is selected from about 400:1, about 200:1, about 100:1, about 50:1, about 40:1, about 25:1, about 10:1 and about 5:1; and

(d) removing the organic solvents under vacuum, thereby encapsulating the ellagic acid in a phosphatidyl lipid layer; and (e) optionally suspending the liposome preparation in a carrier.