Extracts containing oleandrin and methods for producing same

Abstract

A subcritical fluid extraction method for producing oleandrin or an extract containing oleandrin is provided. The method offers an improvement over other methods for preparing an extract containing oleandrin. Extracts, pharmaceutical compositions, dosage forms, and dietary supplement compositions containing oleandrin or the extract are provided. Methods for treating diseases, disorders, or conditions that are therapeutically responsive to oleandrin are also provided.

Description

[Technical field]

[0001] The present invention relates to an improved method for preparing an extract containing oleandrin. The present invention also provides an improved oleandrin-containing composition comprising at least two different oleandrin-containing extracts. Pharmaceutical and dietary supplement compositions comprising the same are also provided. Methods of use for treating cardiac glycoside-responsive diseases, conditions, or disorders are also provided. [Background technology]

[0002] No suitable large-scale chemical synthetic methods exist for the production of oleandrin, and therefore it is primarily produced by extraction from plant material, and may then be isolated and purified from other components of the extract.

[0003] Nerium oleander is a member of the Nerium genus and is an ornamental plant that is widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean. Its medical and toxicological properties have long been recognized, primarily due to oleandrin, and it has been proposed for use in treating, for example, hemorrhoids, ulcers, leprosy, snakebites, neurological disorders, cancer, tumors, excessive cell proliferation, neurodegeneration, Alzheimer's disease, Parkinson's disease, stroke, neurological disorders, Huntington's disease, inflammation, and viral infections. The neuroprotective activity of oleandrin has been disclosed in WO2011085307, U.S. Pat. No. 8,481,086, U.S. Pat. No. 9,220,778, U.S. Pat. No. 9,358,293, U.S. Pat. No. 9,877,979, and U.S. Pat. No. 10,383,886. The anti-cancer (anti-hypercellular proliferation) activity of oleandrin has been disclosed in WO 2007016176, WO 2009064657, U.S. Pat. No. 7,402,325, U.S. Pat. No. 8,187,644, U.S. Pat. No. 8,394,434, U.S. Pat. No. 8,367,363, U.S. Pat. No. 9,494,589, and U.S. Pat. No. 9,846,156. The anti-viral activity of oleandrin has been disclosed in U.S. Pat. No. 10,596,186, WO 2018 / 053123, and WO 2019 / 055119. Zibbu et al. (J. Chem. Pharm. Res. (2010), 2(6), 351-358) provide a brief review of the chemical and pharmacological activity of Oleandrin. Extraction of constituents from plants of the Oleander species has traditionally been carried out using boiling water, supercritical fluids, or organic solvents.

[0004] ANVIRZEL™ (U.S. Patent No. 5,135,745 to Ozel) contains a concentrated or powdered form of a hot water extract of Oleander. Muller et al. (Pharmazie. (1991) Sept. 46(9), 657-663) disclose results on the analysis of an aqueous extract of Oleander. Extraction of the Oleander plant involves slicing the leaves, boiling the sliced ​​leaves and stems of the plant in water for 2-3 hours, and filtering off the residue. The mixture is heated again. The aqueous extract is reported to contain several polysaccharides, oleandrin and oleandrigenin, odorosides, and neritalosides, with molecular weights ranging from 2KD to 30KD. The polysaccharides present are reported to be primarily galacturonic acid. Other monosaccharides include rhamnose, arabinose and galactose. The content of polysaccharides in hot water extracts of Oleander and their individual sugar composition have also been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001) vol 1, pp.1-16). The compositional analysis of the hot water extract ANVIRZEL™ has been described by Newman et al. (Anal. Chem. (2000), 72(15), 3547-3552). U.S. Patent No. 5,869,060 to Selvaraj et al. relates to extracts of Oleander species and methods of producing them. To prepare the extract, the plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterile filtered. The resulting extract can then be freeze-dried to produce a powder. U.S. Patent No. 6,565,897 (U.S. Pregrant Publication No. 20020114852 and WO 2000 / 016793 to Selvaraj et al.) discloses a hot water extraction method for preparing a substantially sterile water extract. Ishikawa et al. (J. Nutr. Sci. Vitaminol. (2007), 53, 166-173) discloses a hot water extract of Oleander and its fractionation by liquid chromatography using a mixture of chloroform, methanol, and water.They also report that extracts of N. oleander leaves have been used to treat type II diabetes. U.S. Patent Application Publication No. 20060188585, published August 24, 2006 to Panyosan, discloses hot water extracts of N. oleander. U.S. Patent Application Publication No. 10323055, published June 18, 2019 to Smothers, discloses a method of extracting plant material with aloe and water to provide an extract containing aloe and cardiac glycosides. U.S. Patent Application Publication No. 20070154573, published July 5, 2007 to Rashan et al., discloses cold water extracts of N. oleander and uses thereof. Thus, methods for increasing the relative content of oleandrin from plant material are warranted. Although hot water extracts of oleandrin may provide relatively low yields of oleandrin and related cardiac glycosides, improved methods for obtaining concentrated forms of cardiac glycosides, including oleandrin, are needed.

[0005] Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov. 89(1), 123-129) disclose results on the comparison of aqueous and ethanolic extracts of plants including Oleander based on their anti-nociceptive and anti-inflammatory activity. Fartyal et al. (J. Sci. Innov. Res. (2014), 3(4), 426-432) disclose results on the comparison of methanolic, aqueous, and petroleum ether extracts of Oleander based on their antibacterial activity.

[0006] Organic solvent extracts of Oleander have also been disclosed by Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), Aug. 26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanolic extract), Zia et al. (J. Ethnolpharmacol. (1995) Nov. 49(1), 33-39; methanolic extract), and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcoholic extract). Turkmen et al. (J. Planar Chroma. (2013), 26(3), 279-283) disclose an aqueous ethanol extract of the leaves and stems of Oleander. Yamauchi, U.S. Pat. No. 3,833,472, issued Sep. 3, 1974, discloses the extraction of Nerium odorum SOL (Nerium oleander Linn) leaves with water, organic solvent, or aqueous organic solvent, in which the leaves are heated to 60° C. to 170° C. and then extracted, the organic solvent being methanol, ethanol, propyl ether, or chloroform.

[0007] Supercritical fluid (SCF) extracts of Oleander species are known (PBI-05204; described herein and in U.S. Pat. No. 8,187,644 to Addington, issued May 29, 2012; U.S. Pat. No. 7,402,325 to Addington, issued July 22, 2008; and U.S. Pat. No. 8,394,434 to Addington et al., issued March 12, 2013, the entire disclosures of which are hereby incorporated by reference herein). SCF extracts have been proven effective in treating some neurological disorders (US Pat. No. 8,481,086, US Pat. No. 9,220,778, US Pat. No. 9,358,293, US Patent Publication No. 20160243143, US Pat. No. 9,877,979, US Pat. No. 10,383,886), some cell proliferation disorders (US Pat. No. 8,367,363, US Pat. No. 9,494,589, US Pat. No. 9,846,156), and some viral infections (US Pat. No. 10,596,186, WO 2018053123, WO 2019055119). Addington et al. (US Pat. No. 8,481,086, US Pat. No. 9,220,778, US Pat. No. 9,358,293, US Patent Publication No. 20160243143) disclose the use of PBI-05204 for the treatment of neurological conditions. Addington et al. (US Pat. No. 9,011,937, US Patent Publication No. 20150283191) disclose the use of a triterpene-containing fraction (PBI-04711) to treat neurological conditions. PBI-05204 contains cardiac glycosides (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA)) as the main pharmacologically active components. The molar ratio of OA:UA:BA in PBI-05204 is about 7.8:7.4:1. PBI-04711 is a fraction of PBI-05204 but does not contain OL. The molar ratio of OA:UA:BA in PBI-04711 is about 3:2.2:1.

[0008] Oleandrin may also be obtained from suspension culture extracts derived from Agrobacterium tumefaciens transformed callus and Agrobacterium extracts (Ibrahim et al., “Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures”, in Enz. Microb. Technol. (2007), 41, 331-336).

[0009] Oleandrin may also be obtained in vitro from extracts of Nerium oleander microcultures, whereby shoot cultures can be initiated from seedlings and / or shoot apices of Nerium oleander cultivars, such as Splendens Giganteum, Revanche or Alsace, or other cultivars (Vila et al., “Micropropagation of Oleander (Nerium oleander L.)” in HortScience (2010), 45(1), 98-102).

[0010] Extracts of Nerium species are known to contain many different classes of compounds: cardiac glycosides, glycones, aglycones, steroids, triterpenes, polysaccharides, monosaccharides, alkaloids, fats, proteins, etc. Specific compounds include oleandrin, neritaroside, odorosides, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-en-3β,28-diol), 28-norurs-12-en-3β-ol, urs-12-en-3β-ol, 3β,3β-hydroxy-12-olean-28-oic acid, 3β,20α-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxy-12-urs-en-28-oic acid, 3β,13β-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxy-12-urs-en-28-oic acid, 3β,13β-dihydroxyurs-21-en-28-oic acid, 3β,13β-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxyurs-21-en-28-oic acid, 3β,13β ... Hydroxyurs-11-en-28-oic acid, 3β,12α-dihydroxyolean-28,13β-olide, 3β,27-dihydroxy-12-olean-28-oic acid, homopolygalacturonan, arabinogalacturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, cardenolide B-1, cardenolide B-2, oleagenin, neridiginoside ... nerizoside, odoroside-H, 3-beta-O-(D-diginosyl)-5-beta, galacturonic acid, 14beta-dihydroxy-card-20(22)-enolide composed of rhamnose, arabinose, xylose, and galactose, pectic polysaccharides, polysaccharides having a MW in the range of 17,000 to 120,000 D, or a MW of about 35,000 D, about 3,000 D, about 5,500 D, or about 12,000 D, cardenolide monoglycosides, cardenolide N- 1, Cardenolide N-2, Cardenolide N-3, Cardenolide N-4, Pregnane, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16beta,17beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridienone B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid, oleanderen, 8alpha-methoxylabdan-18-oic acid, 12-ursene, kaneroside, neriumoside, 3β-O-(D-diginosyl)-2α-hydroxy-8,14β-epoxy-5β-carda-16:17, 20:22-dienolide, 3β-O-(D-diginosyl)-2α,14β-dihydroxy-5β-carda-16:17,20:22-dienolide , 3β,27-dihydroxy-urs-18-en-13,28-olide, 3β,22α,28-trihydroxy-25-nor-lupa-1(10),20(29)-dien-2-one, cis-karenine (3β-hydroxy-28-Zp-coumaroyloxy-urs-12-en-27-oic acid), trans-karenine (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy-5alpha-card-14(15),20(22)-dienolic acid. Nerimogenin A, 3beta,27-dihydroxy-12-ursene-28-oic acid, 3beta,13beta-dihydroxyurs-11-ene-2-en-2-yl, 3beta,27-dihydroxyurs-12-ene-2-yl, 3beta,13beta-dihydroxyurs-1 ... 8-acid, 3beta-hydroxyurs-12-ene-28-aldehyde, 28-norurs-12-en-3beta-ol, urs-12-en-3beta-ol, urs-12-en-3beta,28-diol, 3beta,27-dihydroxy-12-olean-28-acid, (20S,24R)-epoxydammarane-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol, 20beta,28-epoxytaraxastara-21-en-3beta-ol, 28-norurs-12-ene-3beta,17beta-diol, 3beta-hydroxyurs-12-ene-28-aldehyde, alpha-neriursate, beta-neriursate, 3alpha-acetophenoxy-urs-12-en-28-oic acid, 3beta-acetophenoxy-urs-12-en-28-oic acid, oleanderolic acid acid), canerodione, 3β-p-hydroxyphenoxy-11α-methoxy-12α-hydroxy-20-ursene-28-oic acid, 28-hydroxy-20(29)-lupene-3,7-dione, canerocin, 3α-hydroxy-urs-18,20-dien-28-oic acid, D-sarmentose, D-diginose, neridiginoside, nerizoside, isoricinolic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrin, folinerin, 12β-hydroxy-5β-carda-8,14,16,20(22)-tetraenolide, 8β-hydroxy-digitoxigenin, Δ16-8β-hydroxy-digitoxigenin, Δ16-neriagenin, uvaol, ursolaldehyde, 27(p-coumaroyloxy)ursolic acid, oleanderol, 16-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, adigoside, adinerin, alpha-amyrin, beta-sitosterol, campestrol ( campestrol, caoutchouc, capric acid, caprylic acid, choline, corneline, cortenerin, deacetyloleandrin, diacetyl-nerigoside, foliandrin, pseudocuramine, quercetin, quercetin-3-rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospecide, urehitoxin, and uzarigenin. Additional components that may be present in the extract are disclosed by Gupta et al. (IJPSR (2010, 1(3), 21-27), the entire disclosure of which is hereby incorporated by reference herein).

[0011] The individual compositional profile of the extract varies widely according to the extraction method used. For example, extracts of Oleander plant material obtained using warm methanol, warm ethanol, hot water, cold water, hot water, or supercritical carbon dioxide show substantially different compositional profiles, as shown in particular by the individual HPLC chromatograms. Therefore, the compositional profile of the extract prepared by the new type of method cannot be predicted in advance. The therapeutic activity profile of such extracts will also vary when compared on a total mass to total mass basis according to the compositional profile of the extracts. For example, 1 mg of cold water extract will exhibit a different therapeutic activity profile than 1 mg of supercritical fluid extract. Furthermore, SCF extracts have demonstrated improved efficacy over oleandrin alone and over hot water extracts in treating some conditions.

[0012] In view of the important therapeutic properties of oleandrin and extracts containing oleandrin and other constituents of the Oleander plant, there remains a need for improved pharmaceutical compositions containing oleandrin, as well as improved methods of producing oleandrin and oleandrin-containing extracts. Summary of the Invention

[0013] The present invention seeks to overcome some or all of the drawbacks inherent in the art. Objects of the present invention include providing an improved method for producing an oleandrin-containing extract, and an improved method for producing oleandrin. Another object of the present invention includes providing an oleandrin-containing extract and an oleandrin-containing composition. Another object of the present invention is to provide a sublingual or buccal dosage form comprising the extract (or composition) and at least one pharmaceutical excipient. Another object of the present invention is to provide pharmaceutical compositions, dosage forms, cosmetics, and dietary supplement products comprising the oleandrin or extract described herein. The improved methods provide: a) a higher yield of oleandrin per kg of plant biomass compared to supercritical fluid (SCF) extraction (SCFE) based methods; b) an oleandrin-containing composition having a higher ratio of oleandrin to other components compared to the SCFE method; c) a dual extract composition comprising an organic solvent extract and a subcritical extract; d) an organic solvent extract having a higher content of oleandrin compared to the SCFE method obtained from the same amount of plant material; and e) a subcritical CO2 extract having a higher content of oleandrin compared to the SCF extract obtained using the same solvent and the same amount of plant material. In each of the organic solvent extract or organic solvent extraction embodiments, the organic solvent comprises: a) one or more organic solvents; or b) water and one or more organic solvent extracts, which may further comprise one or more salts, one or more buffering agents, or a combination thereof.

[0014] An aspect of the present invention is a method for producing an oleandrin-containing extract, comprising the steps of: subjecting the oleandrin-containing biomass to subcritical fluid extraction (SbCLE) using a subcritical fluid (SbCL) comprising carbon dioxide and optionally further comprising an organic solvent (modifier) ​​for a time sufficient to extract the oleandrin and form an extraction environment; Separating the biomass from the extraction environment to provide oleandrin-containing SbCL; and removing SbCL from the oleandrin-containing SbCL to provide the oleandrin-containing (OC) extract (OCE); The present invention provides a method comprising:

[0015] An aspect of the present invention is a method for producing oleandrin, comprising the steps of: subjecting the oleandrin-containing biomass to subcritical fluid extraction (SbCLE) using a subcritical fluid (SbCL) comprising carbon dioxide and optionally further comprising an organic solvent (modifier) ​​for a time sufficient to extract the oleandrin and form an extraction environment; separating the biomass from the extraction environment to provide oleandrin-containing SbCL; removing SbCL from the oleandrin-containing SbCL to provide an oleandrin-containing extract; and Isolating oleandrin from said extract. The present invention provides a method comprising: Another aspect of the invention is a method of making a combination composition comprising the steps of: subjecting the oleandrin-containing first biomass to an organic solvent extraction to provide an oleandrin-containing organic solvent extract; subjecting the oleandrin-containing second biomass to subcritical fluid (SbCL) extraction (SbCLE) to provide an oleandrin-containing SbCL extract; and combining said oleandrin-containing organic solvent extract with said oleandrin-containing SbCL extract to provide said combination composition. The present invention provides a method comprising:

[0016] In some embodiments, the combination composition comprises at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, canelosin, canelodione, oleandrigenin, Nerium F, neritaroside, odorosides (A and H), adinellin, odoroside-G-acetate, and gitoxigenin. The combination composition may further comprise one or more of neriin, folinellin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, azinerigenin, deacetyloleandrin, odoroside G acetate, and / or quercetin. The composition (or extract) may further comprise polyphenols, carbohydrates, flavonoids, amino acids, soluble proteins, cellulose, starch, alkaloids, saponins, tannins, and any combination thereof. The amino acid may be selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, tryptophan, and lysine. In some embodiments, the amino is selected from the group consisting of asparagine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine, tryptophan, and lysine. Another aspect of the present invention provides a cosmetic (cosmeceutical) or nutraceutical product comprising oleandrin, an oleandrin-containing composition, an oleandrin-containing combination composition, or an oleandrin-containing extract as defined herein.

[0017] The cosmetic product may further include one or more cosmetic excipients. The cosmetic product may be a cleanser, shampoo, conditioner, body wash, face cleanser, skin toner, serum, moisturizer, balm, makeup, lotion, cream, gel, hydrogel, ointment, oil-based solution, suspension, powder, spray, foundation, primer, highlighter, eye shadow, foundation, blush, wet wipes, deodorant, bath oil, bubble bath, bath salts, body butter, lipstick, lip gloss, lip liner, concealer, rouge, facial mask treatment, or other type of cosmetic product.

[0018] A dietary supplement may be a dietary ingredient, a dietary supplement, a bioceutical, or a food additive, for example, as defined by the U.S. FDA or other regulatory agencies. A dietary supplement product may further include one or more dietary ingredients or dietary ingredients. A dietary supplement may be a liquid, solid, powder, cream, solution, suspension, dosage form, gel, capsule, tablet, pill, prebiotic, probiotic, functional food, dietary supplement, meal replacement, or other such product.

[0019] The present invention also provides an improved oleandrin-containing combination composition, comprising a portion of an organic solvent extract containing oleandrin and a portion of an SbCL extract containing oleandrin.In some embodiments, a) the substantially equal portions of the two extracts are present in the combination composition; b) the content of the organic solvent extract in the combination is higher than the content of the SbCL extract; or c) the content of the SbCL extract in the combination is higher than the content of the organic solvent extract.The method of preparation and the method of use are also provided substantially as described herein. When compared to either organic solvent extracts or subcritical fluid extracts, the combination compositions advantageously contain a broader range of phytochemicals and other components extractable separately from oleandrin from the plant material, one or more of which are also known to be therapeutically effective (pharmacologically active) ingredients. Embodiments of the invention include: a) the organic solvent extract comprises at least oleandrin and other components extracted from the biomass; b) the organic solvent extract has a molar ratio of oleandrin to other components extracted from the biomass that exceeds said molar ratios in the subcritical fluid extract; c) the SbCL extract comprises at least oleandrin and other components extracted from the biomass; d) the content of oleandrin in the ethanol extract exceeds the content of oleandrin in the subcritical fluid extract; e) the content of extracted non-oleandrin components in the organic solvent extract exceeds the content of said components in the subcritical fluid extract; f) the compositional profile of extracted volatile and semi-volatile components in the organic solvent extract differs from the compositional profile of said components in the subcritical fluid extract; and / or g) the subcritical fluid extract comprises a higher proportion of extracted volatile and semi-volatile components relative to the oleandrin content than the organic solvent extract.

[0020] The first and second biomass may be the same or different. A first biomass extracted with an organic solvent may be used as the second biomass; alternatively, the first biomass is extracted with an organic solvent and a different second biomass is extracted with a subcritical fluid. In some embodiments of the present invention, the subcritical fluid extraction method further comprises one or more of the following: a) separating the biomass from the extraction environment by filtering the extraction environment; b) adding a modifier to the carbon dioxide or extraction environment; c) heating and pressurizing the interior of a vessel containing the extraction environment to form a subcritical fluid phase extraction environment; d) removing the SbCL from the oleandrin-containing SbCL by evaporating the SbCL to form a raw SbCL extract (SbCLE); e) diluting the raw SbCL extract with an organic solvent or an aqueous organic solvent; f) filtering the diluted raw or crude SbCL extract one or more times; g) treating the diluted raw or crude SbCL extract with activated carbon; h) removing a majority of the organic solvent from the diluted raw extract to form; and / or i) sterile filtering the raw or crude extract to obtain a SbCL extract. The SbCL extract may be further diluted with an organic solvent or an aqueous organic solvent. The organic solvent extract may be further diluted with an organic solvent or an aqueous organic solvent. The SbCL extract may contain 0.1 mg to 1 mg or 0.5 to 0.8 mg of oleadrin per mL of extract.

[0021] In some embodiments of the invention, the carbon dioxide (and modifier, if present) is in the subcritical liquid phase for about 50% or more of the extraction time, i.e., about 50% or more of the total time for the "exposing" step. In some embodiments, the extraction environment comprising the extraction fluid (carbon dioxide and modifier, if present) and the biomass in the subcritical liquid phase will remain in liquid form for at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the extraction time. In some embodiments, the extraction environment will remain in liquid form throughout the entire extraction time.

[0022] Thus, SbCLE is a process in which the extraction environment (containing biomass and CO2, and optionally containing an organic solvent (modifier)) is under the following conditions with respect to the phase diagram of CO2 for about 50% or more of the extraction time: a) critical pressure (p c ) below, the triple point (solid-liquid-gas) temperature (t tp ) or above the critical temperature (t c ) below and above the liquid-vapor line; b) t c Below, tp More than that, p c below and above the liquid-vapor line; c) p c More than that, t c Below t tp or d) above the liquid-vapor line and below the solid-liquid line; or d) above the solid-liquid line, the liquid-vapor line, the liquid-supercritical line, t tp , triple point pressure (p tp ), critical point (liquid-gas-supercritical) temperature (t cp ), and the critical pressure (p cp ) the critical pressure and temperature are for CO2.

[0023] In some embodiments of the invention, SbCLE is carried out at a temperature of about 0° C. to about 31° C. and a pressure of about 5.2 bar to about 3000 bar. In other embodiments of the invention, SbCLE is carried out at a temperature of about 0° C. to about 31° C. and a pressure of about 5.2 bar to about 1000 bar. In preferred embodiments, SbCLE is carried out at a temperature of about 2° C. to about 16° C. and a pressure of about 115 bar to about 135 bar. In other preferred embodiments, SbCLE is carried out at a temperature of about 5-10° C. and a pressure of about 120-130 bar. In some embodiments, SbCLE is carried out at a temperature in the range of about 5° C. to about 15° C. and a pressure in the range of about 50 bar to 70 bar. In some embodiments of the invention, the SbCL further comprises one or more modifiers, such as an organic solvent. When one or more modifiers are present, the mass ratio of carbon dioxide to modifier is about 100:about 0.01-20, about 100:about 0.01-15, about 100:about 0.01-10, or about 100:about 0.01-5.

[0024] The one or more modifiers (organic solvents) may be independently selected from alcohols, organic solvents, alkyl ketones, alkyl esters, water at each occurrence. Particularly suitable modifiers include ethanol, methanol, propanol, acetone, ethyl acetate, methyl ethyl ketone, glycols (e.g., propylene glycol), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP).

[0025] In some embodiments of the invention, a) the organic solvent extraction is performed with an organic solvent or an aqueous organic solvent; b) the organic solvent or the aqueous organic solvent, independently at each occurrence, comprises an alcohol; c) the organic solvent, independently at each occurrence, is selected from the group consisting of methanol, ethanol, propanol, butanol, chloroform, methylene chloride, ethyl acetate, and combinations thereof; d) the organic solvent extraction comprises extracting the oleandrin-containing biomass one or more times with an organic solvent or an aqueous organic solvent; and e) the organic solvent extraction is performed with a mixture of the oleandrin-containing biomass and the oleandrin-containing biomass. f) the organic solvent extraction is performed at a temperature of about 0° C. to about 75° C., about 5° C. to about 70° C., about 10° C. to about 60° C., about 10° C. to about 50° C., about 10° C. to about 45° C., or about 20° C. to about 40° C.; g) the v / w ratio of organic solvent (or aqueous organic solvent) to biomass is in the range of about 50-1:about 1, about 40-1:about 1, about 30-1:about 1, about 20-1:about 1, about 20-5:about 1, or about 10:about 1. When non-dried, non-dehydrated, non-freeze-dried biomass is used for extraction, the water content contained in the biomass is considered as the v / w ratio of aqueous organic solvent to biomass (on a dry weight basis).

[0026] In some embodiments of the invention, the organic solvent extraction further comprises one or more of the following: a) separating the biomass from the extraction environment by filtering the extraction environment to form a crude extract; b) adding an aqueous liquid to the organic solvent or extraction environment; c) reducing the content of organic solvent from the crude extract; d) treating the crude extract with activated carbon; e) filtering the crude extract one or more times; f) adding additional organic solvent to the crude extract; g) sterile filtering the organic solvent extract; and / or h) adding water to the crude extract and extracting the aqueous crude extract with an organic solvent. The organic solvent extract may be further diluted with an organic solvent or an aqueous organic solvent. The organic solvent extract may comprise 0.1-10 mg / mL, 0.5-8 mg / mL, 1-8 mg / mL, 1.5-8 mg / mL, or 1.5-5 mg / mL (mg of oleandrin per mL of extract). In some embodiments of the invention, the oleandrin-containing biomass is Nerium spp., Western oleander, Nerium oleander L. (Apocynaceae), Nerium odourum, Nerium indicum, white oleander, pink oleander, Nerium spp., Thevetia peruviana, yellow oleander, Agrobacterium tumefaciens, or combinations thereof. In some embodiments, the biomass comprises leaves, stems, flowers, bark, fruits, seeds, sap, cultured cell mass, and / or pods.

[0027] In some embodiments of the present invention, a) the biomass is dehydrated, dehumidified, freeze-dried, and / or desiccated before extraction, independently for each occurrence; b) the biomass is dried before extraction, independently for each occurrence; c) the biomass is not dehydrated before extraction, independently for each occurrence; d) the biomass is not dried before extraction, independently for each occurrence; or e) independently for each occurrence. The water content in the biomass before extraction is preferably about 20% by weight or less, about 15% by weight or less, about 10% by weight or less, about 8% by weight or less, about 5% by weight or less, about 2.5% by weight or less, or about 1% by weight or less. Thus, the method of the present invention also includes embodiments further comprising dehydrating, drying, or desiccating the biomass before extraction. In some embodiments of the invention, a) the average particle size (d0.5) of the biomass prior to extraction is less than about 1 inch, less than about 0.75 inch, less than about 0.5 inch, less than about 0.4 inch, less than about 0.3 inch, less than about 0.2 inch, less than about 0.1 inch, less than about 0.05 inch, or less than about 0.01 inch, said particles being measured by ASTM sieve mesh opening size.

[0028] In some embodiments of the present invention, the oleandrin content in the extract is about 0.01% by weight or more, about 0.05% by weight or more, about 0.1% by weight or more, about 0.2% by weight or more, about 0.5% by weight or more, about 1% by weight or more, about 2.5% by weight or more, about 5% by weight or more, about 10% by weight or more, about 20% by weight or more, about 30% by weight or more, about 40% by weight or more, about 50% by weight or more, about 60% by weight or more, about 70% by weight or more, about 80% by weight or more, or about 90% by weight or more; ) the oleandrin content in the extract is about 99% by weight or less, about 95% by weight or less, about 90% by weight or less, about 80% by weight or less, about 70% by weight or less, about 60% by weight or less, about 50% by weight or less, about 40% by weight or less, about 30% by weight or less, about 20% by weight or less, about 10% by weight or less, about 5% by weight or less, about 2.5% by weight or less, about 1% by weight or less, or about 0.5% by weight or less; or c) any combination of the limitations in item a) and any of the limitations in item b).

[0029] In some embodiments of the invention, the method further comprises one or more of the following: a) removing (or reducing the amount of) volatile organic compounds (VOCs) from the extract; b) removing (or reducing the amount of) semi-volatile organic compounds (SVOCs) from the extract; c) filtering the extract; d) the separating is performed by filtration, centrifugation, decantation, or a combination thereof.

[0030] In some embodiments of the invention, the isolating is accomplished by one or more of chromatography, liquid-liquid extraction, liquid-solid extraction, crystallization, precipitation, fractional precipitation, fractional crystallization, Soxhlet extraction, trituration, lyophilization (freeze drying), or a combination thereof. Any individual steps of the method of the present invention may be performed in separate facilities or may be performed within the same facility.

[0031] Pharmaceutical compositions comprising oleandrin as the only active ingredient are within the scope of the present invention. Compositions comprising oleandrin and active ingredients obtained from the biomass are within the scope of the present invention. Compositions comprising oleandrin and triterpenes as active ingredients are within the scope of the present invention. In some embodiments, the composition comprises oleandrin, oleanolic acid (free acid, salt, derivative or prodrug thereof), ursolic acid (free acid, salt, derivative or prodrug thereof), and betulinic acid (free acid, salt, derivative or prodrug thereof). The molar ratios of the compounds are described herein. The triterpenes are independently selected from their free acid form, salt form, deuterated form and derivative form at each occurrence.

[0032] In addition to oleandrin, the extract may contain one or more other compounds extracted from the biomass during the extraction process, which may or may not be pharmacologically active and therapeutically effective, which may or may not contribute to the therapeutic effectiveness of oleandrin when administered to a subject to which both are given. The one or more other compounds may be selected from the group consisting of cardiac glycosides, glycoconstituents of one or more cardiac glycosides (e.g., glucosides, fructosides, and / or glucuronides), aglycone or aglycones, glycoside precursors (e.g., cardenolides, cardadienolides, and cardatrienolides), steroids, triterpenes, monosaccharides, polysaccharides, alkaloids, proteins, fats, other non-cardiac glycosides that are therapeutically active agents, and any combination thereof. In some embodiments, the composition comprises oleandrin, and a) two or three triterpenes; b) two or three triterpene derivatives; c) two or three triterpene salts; or d) a combination thereof. As used herein, the collective terms triterpene and cardiac glycoside also encompass the free form, salt form, and derivatives thereof, unless otherwise specified. In some embodiments, the triterpene is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, and salts or derivatives thereof.

[0033] In some embodiments, the organic extract comprises oleandrin and a) one or more volatile organic compounds; b) one or more semi-volatile organic compounds; c) one or more non-volatile organic compounds; or d) any combination thereof. In some embodiments, the extract excludes pharmacologically active polysaccharides (and / or monosaccharides) obtained during extraction. In some embodiments, the extract includes oleandrin and at least one pharmacologically active polysaccharide (and / or monosaccharide) obtained during extraction. The pharmacologically active polysaccharide may be an acid homopolygalacturonan or an arabinogalacturonan. In some embodiments, the extract comprises at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, canelosin, canelodione, oleandrigenin, Nerium F, neritaroside, odorosides (A and H), adinellin, odoroside-G-acetate, and gitoxigenin. The combination composition may further comprise one or more of neriin, folinellin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, azinerigenin, deacetyloleandrin, odoroside G acetate, and / or quercetin.

[0034] The extract may further comprise one or more polyphenols, one or more carbohydrates, one or more flavonoids, one or more amino acids, one or more soluble proteins, one or more celluloses, one or more starches, one or more alkaloids, one or more saponins, one or more tannins, and any combination thereof. The amino acid may be selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, tryptophan, and lysine. In some embodiments, the amino is selected from the group consisting of asparagine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine, tryptophan, and lysine. It should be understood that although other components in the extract may be present in relatively greater amounts than oleandrin, oleandrin may be the primary therapeutically active compound for a particular disease, disorder or condition.

[0035] In some embodiments of the present invention, the pharmaceutical composition comprises at least one pharmaceutical excipient and the extract. The oleandrin, oleandrin-containing extracts, or oleandrin-containing compositions of the present invention may be administered to a subject in need thereof to treat a condition, disease, or disorder that is therapeutically responsive to treatment with a cardiac glycoside (oleandrin, digoxin, digitoxin, ouabain, bufalin, cinobufatalin, cinobufagin, resibufogenin, lanatoside C, or other therapeutically effective cardiac glycoside), particularly treatment with oleandrin, or that can be inhibited by administration of a cardiac glycoside, particularly administration of oleandrin.

[0036] An aspect of the invention provides a method of treating or preventing a condition, disease or disorder therapeutically responsive to oleandrin (an oleandrin-containing composition or an oleandrin-containing extract), comprising administering to a subject in need thereof one or more doses of oleandrin (an oleandrin-containing composition or an oleandrin-containing extract), thereby treating or preventing said condition, disease or disorder. Target conditions, diseases and disorders are described herein. Another aspect of the present invention provides a method of improving or boosting an immunological response in a subject comprising administering to a subject in need thereof one or more doses of oleandrin (an oleandrin-containing composition, an oleandrin-containing combination composition, or an oleandrin-containing extract) in an amount sufficient to boost the immunological response in the subject. The one or more doses may be administered as a pharmaceutical composition, a nutraceutical composition, and / or a cosmeceutical composition.

[0037] Administration may be acute, chronic, or a combination thereof, and may occur before and / or after the onset of said condition, disease or disorder. Administration may be to a subject having or at risk for said condition, disease or disorder. The oleandrin, oleandrin-containing extract, oleandrin-containing composition, or oleandrin-containing combination composition of the present invention may be included in any pharmaceutical composition, nutraceutical composition, cosmeceutical composition, or dosage form described herein.

[0038] The oleandrin, oleandrin-containing extract, or oleandrin-containing composition of the present invention may be administered locally, systemically, or a combination thereof. Suitable modes of administration are described herein. In some embodiments, oleandrin is present as a primary therapeutic component, meaning the component primarily responsible for the therapeutic activity in a pharmaceutical composition. The present invention also provides for the use of an oleandrin-containing extract in the manufacture of a medicament for treating a condition, disease or disorder in a subject that is therapeutically responsive to cardiac glycosides. In some embodiments, the manufacture of such a medicament includes providing an oleandrin-containing extract; including a dose of the oleandrin-containing extract in a pharmaceutical dosage form; and packaging the pharmaceutical dosage form. In some embodiments, the manufacture may be performed as described in International Application No. PCT / US06 / 29061, the entire disclosure of which is hereby incorporated by reference herein. The manufacture may also include one or more additional steps, such as transporting the packaged dosage form to a sales entity (retailer, wholesaler and / or distributor); selling or otherwise providing the packaged dosage form to a subject with a viral infection; including with the medicament a label and insert providing instructions regarding the use, dosage regimen, administration, content, and toxicity profile of the dosage form.

[0039] In some embodiments, treatment includes determining that the subject has a condition, disease, or disorder that is therapeutically responsive to a cardiac glycoside, such as oleandrin (an oleandrin-containing composition or an oleandrin-containing extract), digoxin, ouabain, bufalin, cinobufatalin, cinobufagin, resibufogenin, or other therapeutically effective cardiac glycoside; indicating administration of a pharmaceutical dosage form to the subject according to a dosing regimen; administering one or more pharmaceutical dosage forms to the subject, wherein the one or more pharmaceutical dosage forms are administered according to the dosing regimen.

[0040] The pharmaceutical composition may further comprise a combination of at least one material selected from the group consisting of water soluble (miscible) cosolvents, water insoluble (immiscible) cosolvents, surfactants, antioxidants, chelating agents, and absorption enhancers. The solubilizer is at least a single surfactant, but may also be a combination of materials, such as: a) a surfactant and a water-miscible solvent; b) a surfactant and a water-immiscible solvent; c) a surfactant, an antioxidant; d) a surfactant, an antioxidant, and a water-miscible solvent; e) a surfactant, an antioxidant, and a water-immiscible solvent; f) a surfactant, a water-miscible solvent, and a water-immiscible solvent; or g) a surfactant, an antioxidant, a water-miscible solvent, and a water-immiscible solvent. The compositions of the present invention may further comprise: a) at least one liquid carrier; b) at least one emulsifier; c) at least one solubilizer; d) at least one dispersing agent; e) at least one other excipient; or f) combinations thereof. In some embodiments, the water-miscible solvent is a low molecular weight (less than 6000) PEG, glycol, or alcohol. In some embodiments, the surfactant is a pegylated surfactant, which refers to a surfactant that contains a poly(ethylene glycol) functional group.

[0041] The present invention also provides a buccal or sublingual dosage form comprising any of the extracts of the present invention described herein and at least one pharmaceutical excipient. The dosage form is adapted to deliver the components of the extract to the oral mucosa. Suitable dosage forms include tablets, wafers, patches, sprays, drops, liquids, solutions, pastes, gels, films, bioadhesive compositions, gummies, or powders.

[0042] The combination composition may be included in any type of pharmaceutical dosage form, preferably selected from the group consisting of solid dosage forms, liquid dosage forms, parenteral dosage forms, otic dosage forms, ophthalmic dosage forms, nasal dosage forms, inhalation dosage forms, buccal dosage forms, sublingual dosage forms, enteral dosage forms, topical dosage forms, oral dosage forms, peroral dosage forms, injectable dosage forms, tablets, capsules, pills, caplets, lozenges, sachets, solutions, suspensions, dispersions, vials, bags, bottles, injectable solutions, solutions that can be administered IV (intravenous), IM (intramuscular) or IP (intraperitoneal). The dosage form may be a rapid release, immediate release, controlled release, sustained release, extended release, sustained release, burst release, continuous release, delayed release, or pulsatile release dosage form, or a dosage form that exhibits two or more of these types of release. When placed in an aqueous environment or after administration to a subject, the dosage form may exhibit a drug release profile selected from the group consisting of zero order, pseudo zero order, first order, pseudo first order, or sigmoidal release profiles.

[0043] In some embodiments, the dosage form is a) enterically coated and releases its charge of composition within a period of 0.5 to 1 hour or less; b) enterically coated and releases its charge of composition downstream of the stomach, e.g., in the jejunum, ileum, small intestine, and / or large intestine; or c) enterically coated and releases its charge of composition into the systemic circulation within 1 to 10 hours after oral administration.

[0044] In some embodiments, the inhaled, buccal, or sublingual dosage form further comprises at least one bitter taste antagonist (also referred to as bitter taste receptor blocker). The bitter taste antagonist inhibits the activation of taste receptors caused by bitter compounds, such as cardiac glycosides. In a preferred embodiment, the bitter taste antagonist inhibits the activation of at least one receptor of the hTAS2R family of G protein-coupled receptors. As used herein, the bitter taste antagonist can be a taste masking agent. Suitable bitter taste antagonists include GIV3757 (4-(2,2,3-trimethylcyclopentyl)butanoic acid), gamma-aminobutyric acid (GABA), 6-methoxy-flavanone, 4'-fluoro-6-methoxyflavanone, 6,3'-dimethoxyflavanone, 6-methoxyflavanone, abscisic acid, (-)-Hardwicki acid (2-(phytolan-3-yl)ethyl)-trimethyl-hexahydronaphthalene-carboxylic acid), fenchone, borneol, isoborneol, anethole, menthofuran, sodium glycyrrhizinate, pullulan (α-1,4-; α-1,6-glucan), flavoring agents, or sweeteners. Suitable flavoring agents include mint, peppermint, menthol, eucalyptus, eucalyptol, chocolate, spearmint, tangerine, orange, grape, grapefruit, marshmallow, coffee, banana cream, caramel, bubble gum, cherry, lemon, lime, strawberry, maple, raspberry, and / or apple.Suitable sweeteners include MagnaSweet (110 or 135), sucralose, steviol glycosides, stevia, glycerin, acesulfame potassium, PCCA sweetener enhancer, PCCA Biterstop, di-d-fructofuranose 1,2':2,3'-di-anhydride, aspartame, neohesperidin dihydrochalcone and hesperidin dihydrochalcone 4'-β-d-glucoside, d-sorbitol, sodium saccharin, sodium gluconate, and / or monosodium glutamate. Inhaled, buccal, or sublingual dosage forms also include oil-based vehicles such as fixed oils (eg, from almond, peanut, sesame, olive, avocado, or corn) or hydrogenated oils.Inhaled, buccal, or sublingual dosage forms may also be taste masked using polymer coatings; taste masking by conventional granulation; taste masking with ion exchange resins; taste masking by lipid coagulating sprays; taste masking by forming inclusion complexes with cyclodextrins; taste masking by freeze drying methods; taste masking by creating multiple emulsions; and taste masking using gelatin, gelatinized starch, liposomes, lecithin or lecithin-like substances, surfactants, salts, or polymeric membranes.

[0045] In some embodiments, the inhaled, buccal, or sublingual dosage form further comprises one or more permeation enhancers that improve mucosal permeability and absorption of the compound, such as a cardiac glycoside. Suitable permeation enhancers include surfactants, bile salts, fatty acids, cyclodextrins, and chelating agents. The present invention includes all combinations of the aspects, embodiments and sub-embodiments of the invention disclosed herein. The following figures form part of this description. Those skilled in the art will be able to practice the invention without undue experimentation in light of these figures and the description herein. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows a phase diagram for carbon dioxide showing the solid, liquid, gas and supercritical phases, as well as the temperatures and pressures of the triple point and critical points. The solid and liquid phases are separated by a solid-liquid line. The liquid and gas phases are separated by a liquid-gas line. The supercritical and liquid phases are separated by a supercritical fluid-liquid line. Thus, the liquid region is bounded by the liquid-gas line, the solid-liquid line, the supercritical fluid-liquid line, the triple point (temperature and pressure) and the critical point (temperature and pressure). [Diagram 2] FIG. 2 shows a phase diagram for carbon dioxide showing the supercritical region and two subcritical regions: the subcritical liquid region and the subcritical gas region. [Diagram 3]3 shows chromatograms from GC-mass spectrometry analysis with total ion current (TIC) detection of an ethanol extract (top), a subcritical CO2 extract (middle), and a prior art supercritical fluid extract PBI-05204 (bottom) determined according to Example 18. The data can be used to identify the volatile and semi-volatile components of the extracts. [Figure 4] FIG. 4 shows an HPLC chromatogram of PBI-05204 (prior art) in which oleandrin was separated from oleanolic acid, ursolic acid, and betulinic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present invention provides an oleandrin-containing subcritical extract, an oleandrin-containing organic solvent extract, and a combination composition comprising said subcritical extract and said organic solvent extract. The present invention also provides a pharmaceutical composition comprising at least one pharmaceutical excipient and said extract or combination composition. The present invention also provides a method of using said pharmaceutical composition, said extract, and / or said combination composition for treating and / or preventing cardiac glycoside-responsive, particularly oleandrin-responsive, diseases, conditions, or disorders. The present invention also provides a method of administering said extract and composition. The present invention also provides a method of preparing said extract and composition. The inventors have found that a two-component extract composition advantageously provides a different compositional profile than any single extract composition. For example, the compositional profile of a combination composition (two-component extract composition) that includes an ethanol (or aqueous ethanol) extract in combination with a subcritical CO2 extract (with or without a modifier) ​​is different and superior to the compositional profile of a single extract composition that includes the ethanol extract, the subcritical CO2 extract, the hot water extract, the cold water extract, or the supercritical CO2 extract. An important advantage is that the combination composition contains less of the other components compared to the oleandrin content and the content of other components that can be extracted from the biomass, but still contains a significant amount of oleandrin, which is the desired target pharmacologically active substance, in the extract. Furthermore, the combination composition advantageously contains more non-oleandrin components than either of the individual extracts, and some of these non-oleandrin components exhibit pharmacological activity.

[0048] As used herein, "plant material" is defined as plants or plant parts (e.g., bark, wood, leaves, branches, stems, roots, shoots, flowers, fruits, seeds, berries, or other plant parts) and exudates, including materials that fall within the definition of "plant raw materials" in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research. Cultured cell mass may also be used as plant material (biomass). Plant material is considered within the scope of biomass. In a preferred embodiment, the plant material includes leaves, shoots, and / or stems.

[0049] In some embodiments of the present invention, the oleandrin-containing biomass (plant material) is selected from the group consisting of Nerium spp., Nerium aureum, Nerium oleander L. (Apocynaceae), Nerium odorum, Nerium indicum, white oleander, pink oleander, Nerium spp., yellow oleander, Agrobacterium tumefaciens (Ibrahim et al., “Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures” in Enz. Microbial Technol. (2007), 41(3), 331-336, the entire disclosure of which is hereby incorporated by reference), cell cultures of Nerium spp. (Ibrahim et al., “Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium temperature and humidity) and Nerium oleander (Ibrahim et al., “Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium temperature and humidity).” (2009), 143(1), 97-103, the disclosure of which is hereby incorporated by reference in its entirety), cell cultures of Nerium species, or combinations thereof. Nerium may be obtained in vitro from microcultures, whereby shoot cultures can be initiated from seedlings and / or shoot apices of Nerium cultivars Splendens giganteum, Rebanche or Arthus, or other cultivars (Vila et al., “Micropropagation of Oleander (Nerium oleander L.)” in HortScience (2010), 45(1), 98-102, the disclosure of which is hereby incorporated by reference in its entirety).Oleander plant material may be obtained, for example, from a commercial plant supplier, such as Aldridge Nursery, Atascosa, Tex. Suitable varieties of Oleander include Album (white), Petit Salmon (pink), True Pale, Pink Beauty, Calypso (dark pink), or Red Hardy.

[0050] As used herein, the term "oleandrin" is intended to mean all known forms of oleandrin, unless otherwise specified. Oleandrin may exist in racemic, optically pure, optically enriched, or stereoisomeric forms.

[0051] As used herein, the term semivolatile organic compounds (SVOCs) refers to compounds that are 1×10 -5 ~3×10 -7 atm*m 3 / mol * ( * H range is defined as the volatility of a liquid to air), usually 10 -14 ~10 -4 As used herein, the term volatile organic compound (VOC) is any organic compound that has an initial boiling point of 250° C. or less measured at standard atmospheric pressure of 101.3 kPa.

[0052] The organic solvent extract and the subcritical fluid extract contain at least oleandrin, VOCs, and SVOCs, and the extracts may further contain one or more non-volatile organic compounds; therefore, the separate combination compositions formed with the extracts contain these components as well. The present invention provides a method for producing oleandrin or an oleandrin-containing extract from an oleandrin-containing biomass by a) subcritical fluid (SbCL) extraction (SbCLE) with carbon dioxide, which may further comprise at least one modifier; b) extraction with an organic solvent (OSE), which may further comprise water; or c) a combination of ScCLE and OSE. The extraction may be performed on the same biomass or on different batches of biomass.

[0053] FIG. 1 shows a phase diagram for carbon dioxide showing the solid, liquid, gas and supercritical fluid phases. SbCLE is performed in the liquid phase region outside the supercritical fluid phase region (in the phase diagram) for carbon dioxide. The liquid phase region is bounded by the solid-liquid line, the liquid-gas line, the liquid-supercritical phase line, the supercritical fluid-liquid line, the triple point (solid-liquid-gas), and the critical point (liquid-gas-supercritical). The approximate critical point temperature and pressure for CO2 are t cp = approx. 31 to 31.1°C and p cp = 73.7-73.8 bar. The approximate triple point temperature and pressure for CO2 are t tp = approx. -56.4 to 56.5°C and p tp = Approximately 5.1 to 5.2 bar.

[0054] The subcritical extraction liquid comprises primarily CO2; thus, the determination of the subcritical state is with respect to the known phase diagram for carbon dioxide. Thus, the subcritical state is achieved by carrying out the extraction in the extraction vessel under the following conditions: a) a temperature in the range of about -56.4°C to about 31.1°C or less; b) a pressure of 5.1 bar or more; and / or c) a subcritical temperature and / or pressure such that the extraction medium for carbon dioxide is in the liquid phase rather than the solid, gas, or supercritical fluid phase. Thus, SbCLE is carried out under pressure and temperature conditions such that the extraction medium (which comprises primarily carbon dioxide and may include modifiers) remains in the subcritical liquid phase rather than the gas, solid, or supercritical fluid phase. Preferred extraction conditions are those where the subcritical liquid is at or above the critical pressure, above the triple point temperature, below the critical point temperature, and below the solid-liquid line. In the context of the phase diagram, the region encompassed by these conditions provides a high density of the subcritical liquid resulting in high extraction efficiency. The extraction time is the period of time that the extraction liquid and the biomass are in contact. For SbCLE, the extraction time for each extraction cycle typically ranges from 0.1 to 24 hours, 0.5 to 12 hours, or 0.5 to 6 hours. For organic solvent extraction, the extraction time for each extraction cycle typically ranges from 0.1 to 24 hours, 0.5 to 12 hours, or 0.5 to 6 hours.

[0055] In some embodiments, the subcritical liquid comprises carbon dioxide. In other embodiments, the subcritical liquid comprises carbon dioxide and at least one modifier. The modifier may be an alcohol, an organic solvent, an alkyl ketone, an alkyl ester, or a combination thereof. Particularly suitable modifiers include ethanol, methanol propanol, acetone, ethyl acetate, methyl ethyl ketone, glycols (e.g., propylene glycol), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or N-methylpyrrolidone (NMP). The mass ratio of subcritical fluid to biomass can also affect extraction efficiency. In some embodiments, the mass ratio of subcritical fluid (with or without modifier) ​​to biomass ranges from about 20:1 to about 100:1, about 30:1 to about 75:1, about 50:1, 45:1 to about 60:1, or about 40:1 to about 45:1, based on the mass of both the solvent and the feedstock.

[0056] During the majority (about 50% or more) of the SbCL extraction time, the extraction fluid will be in subcritical liquid form (in the extraction environment). In some embodiments, the extraction liquid will be in subcritical liquid form for at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the extraction time. In some embodiments, the extraction liquid is in subcritical liquid form for the entire extraction time.

[0057] Due to equipment capabilities, carbon dioxide extraction of biomass may occur in the supercritical phase for short periods; however, for the majority of the extraction time (approximately 50% or more), the extraction will occur in subcritical conditions. SbCLE of Oleander biomass was performed according to Example 3. The dried biomass was extracted with subcritical liquid CO2 (about 90-95 wt%) containing alcohol (about 10-5 wt% ethanol, respectively). After the extraction period was completed, the extracted biomass was separated from the subcritical liquid by filtration to obtain the oleandrin-containing subcritical liquid (along with other extracted components). The CO2 was vented to obtain a crude extract, which was diluted with ethanol, then frozen, then thawed, then filtered, then concentrated in vacuum, and diluted again with 50% v / v aqueous ethanol.

[0058] Organic solvent extraction was performed according to Example 15. Dried and powdered Oleander biomass was extracted three times (three extraction cycles) with aqueous ethanol (containing 10% v / v water). For each extraction cycle, the extraction medium was filtered after the extraction time was completed. The three individual supernatants were combined and concentrated in vacuum to about 20% of the original volume to form a concentrate, which was sterile filtered, thus forming a crude ethanol extract, which was then diluted with 50% v / v / aqueous ethanol.

[0059] The composition of the SbCL extract (based on the content of several known components) was compared to the composition of a prior art supercritical fluid (SCF) extract of Oleander (PBI-05204 above), a prior art hot water extract (ANVIRZEL™), and an organic solvent extract prepared as described herein. Based on the same amount of plant material, a) the observed oleandrin yield (mg oleandrin per kg plant material) was ethanol extract > subcritical extract > supercritical extract; b) the observed VOC and SVOC yield (mg VOC+SVOC per kg plant material) was supercritical extract > ethanol extract > subcritical extract. The content of individual components may vary by ±25%, ±20%, ±15%, ±10%, or ±5% from the values ​​shown. Standards of oleandrin, oleanolic acid, ursolic acid, betulinic acid, and their derivatives may also be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, Mo., USA). The composition of some of the extracts is determined by DART TOF-MS (Direct Analysis in Real Time Time-of-Flight Mass Spectrometry) on a JEOLA ccuTOF-DART mass spectrometer (JEOLUSA, Peabody, MA, USA).

[0060] In some embodiments, the extract comprises cardiac glycosides, glycones, aglycones, steroids, triterpenes, polysaccharides, monosaccharides, alkaloids, fats, proteins, or combinations thereof. Particular compounds include oleandrin, and other compounds that may be present include neritaroside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-en-3β,28-diol), 28-norurs-12-en-3β-ol, urs-12-en-3β-ol, 3β,3β-hydroxy-12-olean-28-oic acid, 3β,20α-dihydroxyurs-21-en ... β,27-dihydroxy-12-ursenoic acid, 3β,13β-dihydroxyurs-11-en-28-oic acid, 3β,12α-dihydroxyoleanan-28,13β-olide, 3β,27-dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalacturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, cardenolide B-1, cardenolide B-2, o Reagenin, neridiginoside, nerizoside, odoroside-H, 3-beta-O-(D-diginosyl)-5-beta, 14beta-dihydroxy-card-20(22)-enolide, galacturonic acid, pectic polysaccharides composed of rhamnose, arabinose, xylose, and galactose, polysaccharides having a MW in the range of 17,000 to 120,000 D, or a MW of about 35,000 D, about 3,000 D, about 5,500 D, or about 12,000 D, cardenolide monoglycosides, cardenolides, and the like. Cardenolide N-1, Cardenolide N-2, Cardenolide N-3, Cardenolide N-4, Pregnane, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16beta,17beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (Neridienone A), 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridienone B), nericumaric acid, isonelicumaric acid, oleandelonic acid, oleandelene, 8alpha-methoxylavtan-18-oic acid, 12-ursene, caneloside, neriumoside, 3β-O-(D-diginosyl)-2α-hydroxy-8,14β-epoxy-5β-carda-16:17,20:22-dienolide, 3β-O-(D-diginosyl)-2α,14β-dihydroxy-5β-carda-16:17,20:22-dienolide, 3β,27-dihydro cis-Karenine (3-β-hydroxy-28-Zp-coumaroyloxy-urs-12-en-27-oic acid), trans-Karenine (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy-5alpha-card-14(15),20(22)-dienolide (beta- Anhydroepigitoxigenin), 3beta-O-(D-digitalosyl)-21-hydroxy-5beta-carda-8,14,16,20(22)-tetraenolide (nerimogenin-A-3beta-D-digitaloside), procelagenin, neridienone A, 3beta,27-dihydroxy-12-ursene-28-acid, 3beta,13beta-dihydroxyurs-11-en-28-acid, 3beta-hydroxyurs-12-en-28-aldehyde, 28-norurs-12 -en-3beta-ol, urs-12-en-3beta-ol, urs-12-en-3beta,28-diol, 3beta,27-dihydroxy-12-oleanen-28-oic acid, (20S,24R)-epoxydammaran-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol, 20beta,28-epoxytaraxasteran-21-en-3beta-ol, 28-norurs-12-en-3beta,17 beta-diol, 3 beta-hydroxyurs-12-ene-28-aldehyde, alpha-neriursate, beta-neriursate, 3 alpha-acetophenoxy-urs-12-ene-28-acid, 3 beta-acetophenoxy-urs-12-ene-28-acid, oleanderolic acid, canelodione, 3 beta-p-hydroxyphenoxy-11 alpha-methoxy-12 alpha-hydroxy-20 ursene -28-acid, 28-hydroxy-20(29)-lupene-3,7-dione, canelosin, 3alpha-hydroxy-urs-18,20-dien-28-acid, D-sarmentose, D-diginose, nerididinoside, nerizoside, isoricinoleic acid, gentiobiosyl nerigoside, gentiobiosyl beaumontside, gentiobiosyl oleandrin, folinellin, 12β-hydroxy-5β-carda-8, 14,16,20(22)-Tetrénolide, 8β-Hydroxy-Digitoxigenin, Δ16-8β-Hydroxy-Digitoxigenin, Δ16-Neriagenin, Uvaol, Ursolaldehyde, 27(p-Coumaroyloxy)ursolic acid, Oleanderol, 16-Anhydro-Deacetyl-Nerigoside, 9-D-Hydroxy-cis-12-Octadecanoic acid, Adigosides, Adinelin, Alpha-Amyrin, Beta- ta-sitosterol, campestrol, cautious, capric acid, caprylic acid, choline, cornellin, cortenelin, deacetyloleandrin, diacetyl-nerigoside, foliandrin, pseudochlamin, quercetin, quercetin-3-rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospecid, urehitoxin, and usaligenin. Additional components that may be present in the extract are disclosed by Gupta et al. (IJPSR (2010), 1(3), 21-27, the entire disclosure of which is hereby incorporated by reference herein).

[0061] The identity of some of the volatile and semi-volatile organic compounds present in the organic solvent extract and the subcritical extract was determined by GS mass spectrometry according to Example 18. The volatile and semi-volatile compounds found in the ethanol extract and / or the subcritical extract were tentatively identified as 2,3-dihydrobenzofuran, squalene, 2-(decanoyloxy)propane-1,3-diyldioctanoate, alpha-tocopherol, stigmasterol, gamma-sitosterol, decanoic acid, 1,2,3-propanetriyl ester, ursolaldehyde, catechol, 5-hydroxymethylfural, (E)-4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol, n-hexadecenoic acid, bis(2-ethylhexyl)phthalate, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl)ester, and didecyl phthalate. Additional unidentified volatile and semi-volatile compounds were observed.

[0062] As evidenced by the GC chromatograms in Figure 3, the compositional profiles of the ethanol extract (top), subcritical CO2 extract (middle), and PBI-05204 are different. The ethanol extract has lower VOCs and SVOCs than the subcritical extract, which has lower VOCs and SVOCs than the supercritical fluid extract, PBI-05204. Thus, the combined composition comprising the ethanol extract and the subcritical fluid extract has lower VOCs and SVOCs than PBI-05204.

[0063] The compositional profiles of the organic solvent extract, the subcritical fluid extract, the combined composition, and PBI-05204 are also determined by HPLC with tandem mass spectrometry detection and / or UV / VIS detection according to Example 10. The HPLC chromatograms show that the combined composition has a compositional profile as would be expected by mixing equal parts of the ethanol extract and the SbCL extract, and has a compositional profile different from that of PBI-05204. By quantifying the content of oleandrin in each extract, it is observed that the ethanol extract has a much higher relative content of oleandrin on an equal mass basis than the SbCL extract. Furthermore, by using internal standards for other components known to be present in oleander extracts, it is observed that the content of oleandrin relative to these other components differs between the three extracts.

[0064] The compositions of the organic solvent extracts and the SbCL extracts differ according to the relative contents of the individual components therein. Based on the phytochemical analysis of the extracts, the composition of the individual extracts may be defined as follows in terms of the relative contents of the classes (types) of components therein: Extracts made by ethanol extraction: Total free amino acids > Total sugars > Total cellulose > Total alkaloids > Total polyphenols - Total flavonoids > Total starch > Total crude protein Extracts made by aqueous ethanol extraction: Total free amino acids > Total cellulose > Total alkaloids > Total sugars > Total polyphenols - Total flavonoids > Total starch > Total crude protein Extracts made by SbCL extraction: Total free amino acids > Total starch > Total alkaloids > Total cellulose > Total flavonoids > Total sugars > Total polyphenols > Total crude protein These compositions further differ based on the relative content of terpenoids, cardiac glycosides, and other components as follows: · Extracts made by ethanol extraction: Canelosin> Canelodione> Oleanolic acid> Ursolic acid ~ Oleandrin ~ Betulinic acid> Oleandrigenin ~ Nerium F, Odrosides (A and H) ~ Adinelin ~ Odroside-G-acetate> Gittoxigenin. · Extracts made by SbCL extraction: Oleanolic acid>Ursolic acid~Oleandrin~Betulinic acid>Oleandrigenin~Oleandrin F>Odrosides (A and H)~Adinelin~Odroside-G-acetate>Cannelosin. The combination composition may further comprise one or more of the following compounds obtained during extraction of the biomass: neritaloside, neriin, folinellin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, azinerigenin, deacetyloleandrin, odroside G acetate, and / or quercetin.

[0065] The subject treated with the SbCL extract, ethanol extract, or combination composition according to the present invention will show a therapeutic response. "Therapeutic response" means that the subject will enjoy at least one of the following clinical benefits as a result of treatment: reduction in severity of symptoms, reduction in the occurrence of symptoms, partial or complete remission, and / or cure. The therapeutic response may be a complete or partial therapeutic response.

[0066] Dietary ingredients and food substances that may be included in dietary supplement compositions are listed by the U.S. FDA (https: / / www.fda.gov / food / new-dietary-ingredients-ndi-notification-process / new-dietary-ingredients-dietary-supplements-background-industry, https: / / www.accessdata.fda.gov / scripts / fdcc / ?set=FoodSubstances, and https: / / www.accessdata.fda.gov / scripts / fdcc / ?cat=foodingredpkg, the entire disclosures of which are hereby incorporated by reference herein). Exemplary classes of dietary ingredients or food substances include vitamins, minerals, herbs, botanicals, amino acids, proteins, fats, polynucleotides, polysaccharides, carbohydrates, lipids, VOCs, SVOCs, antioxidants, phenolic compounds, flavors, essences, aromatic compounds, water, sweeteners, leavening agents, flavor enhancers, colorants, emulsifiers, preservatives, anti-caking agents, salts, buffers, oils, grains, enzymes, hormones, lecithin, melanin, polymers, biopolymers, fiber, starch, fatty acids, any substances present in any food product, concentrates thereof, metabolites thereof, or extracts thereof. Inactive ingredients that may be added to the compositions of the present invention are listed in the U.S. FDA (https: / / www.fda.gov / drugs / drug-approvals-and-databases / inactive-ingredients-database-download, the entire disclosure of which is hereby incorporated by reference herein).

[0067] The dosage regimen includes a therapeutically appropriate dose (or effective dose) of oleandrin (in extract or composition) administered according to a dosage schedule. Thus, a therapeutically appropriate dose is a therapeutic dose of oleandrin that can be administered to a subject without excessive amounts of undesirable or harmful side effects, where a therapeutic response is observed. A therapeutically appropriate dose may be non-lethal to a subject, but may cause some side effects in patients. It is a dose that provides a level of clinical benefit to the subject to which oleandrin is administered that exceeds the level of harmful side effects experienced by the subject due to administration of oleandrin.

[0068] The cosmetic (cosmeceutical) composition can include one or more cosmetic excipients, such as those approved by the U.S. FDA (https: / / www.fda.gov / cosmetics / cosmetic-products-ingredients / cosmetic-ingredients). Cosmeceuticals may further include one or more active ingredients and / or one or more excipients, including, by way of example and not limitation, acai oil, alpha-lipoic acid, green tea extract, retinol, coenzyme Q10 (CoQ10), caffeine, alpha-hydroxy acids (AHA), hyaluronic acid, glycerin, sorbitol, mono- and diglycerides, white mineral oil (petroleum hydrocarbon oil), glyceryl stearate, propylene glycol, vitamin E, citric acid, propyl gallate, other known compounds, and combinations thereof.

[0069] The compositions and dosage forms of the present invention may further comprise one or more pharma- ceutically acceptable, nutraceutical, and / or cosmetically acceptable excipients. The dosage form may comprise one or more excipients, each occurrence independently selected from the group consisting of acidifiers, alkalinizing agents, buffering agents, tonicity modifiers, osmotic agents, water-soluble polymers, water-swellable polymers, thickening agents, complexing agents, chelating agents, and permeation enhancers. Suitable excipients include those listed in the U.S. FDA's "Inactive Ingredients Database" (available at the following website: https: / / www.fda.gov / Drugs / InformationOnDrugs / ucm113978.htm; Oct. 2018), the entire disclosure of which is hereby incorporated by reference herein.

[0070] The therapeutically appropriate dose will vary from subject to subject according to various established pharmacological, pharmacodynamic, and pharmacokinetic principles. However, the therapeutically appropriate dose (e.g., for oleandrin) will typically be about 1 microgram or more per day, about 5 micrograms or more per day, about 10 micrograms or more per day, about 20 micrograms or more per day, about 25 micrograms or more per day, about 50 micrograms or more per day, about 75 micrograms or more per day, about 100 micrograms or more per day, about 200 micrograms or more per day, about 250 micrograms or more per day, about 500 micrograms or more per day, or up to about 750 micrograms of oleandrin per day, or may range from about 1 to about 750 micrograms of oleandrin per dose, or may not exceed about 25 micrograms, about 100 micrograms, about 250 micrograms, about 500 micrograms, or about 750 micrograms of oleandrin per day. The actual amount of oleandrin required to provide a target therapeutic result to a subject may vary from subject to subject according to the basic principles of pharmacology.It will be understood that the specific dosage level for any patient will depend on various factors, including indications treated, severity of indications, patient's health, age, sex, weight, diet, pharmacological response, specific dosage form used, and other such factors.

[0071] In some embodiments, the subject is administered 140 micrograms to 315 micrograms of oleandrin per day. In some embodiments, the dose comprises 5 micrograms to 750 micrograms, 12 micrograms to 300 micrograms, or 12 micrograms to 120 micrograms of oleandrin. The recommended daily dose of oleandrin is generally about 0.25 to about 60 micrograms twice a day, about 5 to about 60 micrograms twice a day, or about 5 to 30 micrograms twice a day. The dose may be about 0.5 to about 100 micrograms / day, about 1 to about 80 micrograms / day, about 1.5 to about 60 micrograms / day, about 1.8 to about 60 micrograms / day, or about 5 to about 60 micrograms / day. The minimum effective dose may be about 0.5 micrograms / day, about 1 microgram / day, about 1.5 micrograms / day, about 1.8 micrograms / day, about 2 micrograms / day, or about 5 micrograms / day. A suitable dose including oleandrin may be about 0.05-0.5 mg / kg / day, about 0.05-0.35 mg / kg / day, about 0.05-0.22 mg / kg / day, about 0.05-0.4 mg / kg / day, about 0.05-0.3 mg / kg / day, about 0.05-0.5 micrograms / kg / day, about 0.05-0.35 micrograms / kg / day, about 0.05-0.22 micrograms / kg / day, about 0.05-0.4 micrograms / kg / day, or about 0.05-0.3 micrograms / kg / day. In some embodiments, the dose of oleandrin is about 1 mg to about 0.05 mg, about 0.9 mg to about 0.07 mg, about 0.7 mg to about 0.1 mg, about 0.5 mg to about 0.1 mg, about 0.4 mg to about 0.1 mg, about 0.3 mg to about 0.1 mg, about 0.2 mg. The present invention includes all combinations of doses described herein.

[0072] The recommended daily dose of oleandrin present in the extract may be about 0.1 micrograms to about 4.5 micrograms per kg of body weight twice daily. The dose of oleandrin may be about 0.1 to about 1 micrograms per kg of body weight / day, about 0.5 to about 1.0 micrograms per kg of body weight / day, about 0.75 to about 1.5 micrograms per kg of body weight / day, about 1.5 to about 2.52 micrograms per kg of body weight / day, about 2.5 to about 3.0 micrograms per kg of body weight / day, about 3.0 to 4.0 micrograms per kg of body weight / day, or about 3.5 to 4.5 micrograms of oleandrin per kg of body weight / day.

[0073] The oleandrin may be present in the dosage form in an amount sufficient to provide the subject with an initial dose of about 20 to about 100 micrograms, about 12 to about 300 micrograms, or about 12 to about 120 micrograms of oleandrin. The dosage form may contain about 1 microgram to about 100 micrograms of oleandrin, about 0.01 microgram to about 100 mg, or about 0.01 microgram to about 100 micrograms of oleandrin, oleandrin extract, or oleandrin-containing oleandrin extract.

[0074] In some embodiments, the oleandrin concentration in the plasma of the subject being treated is about 10 micrograms / mL or less, about 5 micrograms / mL or less, about 2.5 micrograms / mL or less, about 2 micrograms / mL or less, or about 1 microgram / mL or less.In some embodiments, the oleandrin concentration in the plasma of the subject being treated is about 0.0001 micrograms / mL or more, about 0.0005 micrograms / mL or more, about 0.001 micrograms / mL or more, about 0.0015 micrograms / mL or more, about 0.01 micrograms / mL or more, about 0.015 micrograms / mL or more, about 0.1 micrograms / mL or more, about 0.15 micrograms / mL or more, about 0.05 micrograms / mL or more, or about 0.075 micrograms / mL or more. In some embodiments, the oleandrin concentration in the plasma of the subject to be treated is about 10 micrograms / mL to about 0.0001 micrograms / mL, about 5 micrograms / mL to about 0.0005 micrograms / mL, about 1 micrograms / mL to about 0.001 micrograms / mL, about 0.5 micrograms / mL to about 0.001 micrograms / mL, about 0.1 micrograms / mL to about 0.001 micrograms / mL, about 0.05 micrograms / mL to about 0.001 micrograms / mL, about 0.01 micrograms / mL to about 0.001 micrograms / mL, about 0.005 micrograms / mL to about 0.001 micrograms / mL. The present invention includes all combinations and selections of the plasma concentration ranges described herein.

[0075] Based on human clinical data, it is expected that 50% to 75% of the administered oleandrin dose will be orally bioavailable, thus providing about 10 to about 20 micrograms, about 20 to about 40 micrograms, about 30 to about 50 micrograms, about 40 to about 60 micrograms, about 50 to about 75 micrograms, about 75 to about 100 micrograms of oleandrin per dosage form. Considering an average blood volume of 5 liters in an adult, the expected oleandrin plasma concentrations will be in the range of about 0.05 to about 2 ng / mL, about 0.005 to about 10 ng / mL, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL, about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng / mL, or about 0.05 to about 2.5 ng / mL.

[0076] The therapeutically appropriate dose may be administered according to any dosing regimen typically used in the treatment of viral infections. The therapeutically appropriate dose may be administered once, twice, three times or more daily. It may be administered every other day, every 3 days, every 4 days, every 5 days, twice a week, weekly, every other week, every 3 weeks, every 4 weeks, monthly, bimonthly, twice a month, every 3 months, every 4 months, semi-annually, annually, or according to any combination of the above to achieve a suitable dosing schedule. For example, the therapeutically appropriate dose may be administered once or multiple times a day (up to 10 times a day for the highest dose) for one or more weeks. The total daily dose may also be administered by infusion or pump over several hours, throughout a portion of the day, or over a 12-24 hour period.

[0077] The present invention also provides a method of treatment by repeatedly administering multiple therapeutically effective doses of oleandrin (as an oleandrin-containing composition of the present invention) to a subject in need thereof. One or more doses per day may be administered for one or more days per week, for one or more weeks per month, or for one or more months per year.

[0078] In some embodiments, one or more doses of oleandrin (compositions or extracts containing oleandrin) per day are administered for multiple days until the desired clinical endpoint. In some embodiments, one or more doses of oleandrin (compositions or extracts containing oleandrin) per day are administered for multiple days and weeks until the desired clinical endpoint. One or more doses may be administered per day. One, two, three, four, five, six or more doses may be administered per day. Oleandrin (compositions or extracts containing oleandrin) may be administered on a chronic, i.e., repeated basis, for example, daily, every other day, every second day, every third day, every fourth day, every fifth day, every sixth day, weekly, every other week, every two weeks, every three weeks, monthly, bimonthly, twice a month, monthly, bimonthly, quarterly, bi-quarterly, trimesterly, seasonally, semi-annually and / or annually. The treatment period may be one or more weeks, one or more months, one or more quarters, and / or one or more years. An effective dose of oleandrin (composition containing oleandrin or extract containing oleandrin) is administered one or more times per day. Oleandrin (composition containing oleandrin or extract containing oleandrin) may be administered one or more times per day for two or more days per week, one or more times per day for one or more weeks per month, or one or more months per year.

[0079] Generally, a subject is evaluated to determine whether the subject is in need of oleandrin therapy. Administration of an extract containing oleandrin is then indicated. An initial dose of the extract is administered to the subject according to a prescribed dosing regimen over a period of time (treatment period). The subject's clinical and therapeutic response levels are determined periodically. If the therapeutic response level is too low at one dose, then the dose is gradually increased according to a predetermined dose escalation schedule until the desired level of therapeutic response is achieved in the subject. Treatment of the subject with oleandrin is continued as needed. The dose or dosing regimen may be adjusted as needed until the patient reaches the desired clinical endpoint, for example, cessation of the condition, relief of symptoms, and / or slowing of the progression of the condition. If the clinician intends to treat the subject with a combination of oleandrin-containing extract and one or more other therapeutic agents, then the method of the present invention comprises administering a therapeutically appropriate dose of oleandrin and a therapeutically appropriate dose of said one or more other therapeutic agents to the subject in need thereof, wherein oleandrin is administered according to a first dosage regimen and one or more other therapeutic agents are administered according to a second dosage regimen.In some embodiments, the first and second dosage regimens are the same.In some embodiments, the first and second dosage regimens are different.

[0080] Oleandrin may be administered as a primary therapy, an adjunct therapy, or a combination therapy. The methods of the present invention include separate or simultaneous administration of oleandrin and at least one other therapeutic agent, meaning that oleandrin may be administered before, during, or after administration of the other therapeutic agent.

[0081] The one or more other therapeutic agents may be administered at a dose and according to a dosing regimen recognized by a clinician as therapeutically effective, or at a dose recognized by a clinician as less than therapeutically effective. The clinical benefit and / or therapeutic effect obtained by administering a combination of oleandrin and one or more other therapeutic agents may be additive or synergistic, and such level of benefit or effect is determined by comparing administration of the individual components to administration of the combination. The one or more other therapeutic agents may be administered at a dose and according to a dosing regimen recommended or described by the Food and Drug Administration, the World Health Organization, the European Medicines Agency (EMEA), the Therapeutic Goods Administration (TGA, Australia), the Pan American Health Organization (PAHO), the Medicines and Medical Devices Safety Agency (Medsafe, New Zealand) or various health ministries around the world.

[0082] The antiviral activity of the SbCL extract, ethanol extract, and combination composition was established according to Example 7 and in vitro assays shown in WO 2018053123, WO 2019055119, U.S. Pat. No. 10,596,186, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entireties, are followed to demonstrate the antiviral activity of the compositions of the present invention. Treatable viral infections include viruses from the Arenaviridae, Arteriviridae, Bunyaviridae, Filoviridae, Flaviviridae (genus Flavivirus), Orthomyxoviridae (genus Influenza virus), Paramyxoviridae, Rhabdoviridae, Retroviridae (genus Deltaretrovirus), Coronaviridae, (+)-ss-envRNAV (positive-sense short-stranded enveloped RNA virus), (-)-ss-envRNAV (negative-sense short-stranded enveloped RNA virus), or Togaviridae families, and any of these known genera, species, and / or strains. In some embodiments, the (+)-ss-envRNAV is a coronavirus pathogenic to humans.Specific viruses that may be treated include at least Ebola virus, Marburg virus, Alphavirus, Flavivirus, Yellow fever, Dengue fever, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalitis (VEE) virus, Chikungunya virus, Western equine encephalitis (WEE) virus, Eastern equine encephalitis (EEE) virus, Tick-borne encephalitis, Kyasanur forest disease, Al Hamra disease, Omsk hemorrhagic fever, Hendra virus, Nipah virus, Deltaretrovirus, HTLV-1 virus, Coronavirus, SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV229E, CoV NL63, CoV OC43, CoV HKU1, CoV These include HKU20, O'nyong'nvirus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV), Semliki Forest virus, Lassa virus, aseptic meningitis, Gunarito virus, Junin virus, Lujo virus, Machupo virus, Sabia virus, Whitewater Arroyo virus, Hantavirus, Crimean Gongo hemorrhagic fever Orthonairovirus, Mumps virus, Nipah virus, Hendra virus, Respiratory syncytial virus (RSV), Human parainfluenza virus (HPIV), NDV (Newcastle disease virus), Influenza virus (A-C), Isa virus, Thogoto virus, Quaranja virus, H1N1 virus, H2N2 virus, H3N2 virus, H1N2 virus, Spanish flu virus, Asian flu virus, Hong Kong flu virus, Russian flu virus, Rabies virus, Vesiculovirus, Lyssavirus, and Cytorhabdovirus.

[0083] The neuroprotective activity of the SbCL extract, ethanol extract, and combination composition was established according to Example 8 and in vitro assays shown in WO2011085307, U.S. Patent No. 8,481,086, U.S. Patent No. 9,220,778, U.S. Patent No. 9,358,293, U.S. Patent No. 9,877,979, U.S. Patent No. 10,383,886, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entirety, to demonstrate the neuroprotective activity of the composition of the present invention. The anti-cancer (anti-excessive cell proliferation) activity of the SbCL extract, ethanol extract, and combination composition has been established according to Example 11 and in vitro assays shown in WO2007016176, WO2009064657, U.S. Patent No. 7,402,325, U.S. Patent No. 8,187,644, U.S. Patent No. 8,394,434, U.S. Patent No. 8,367,363, U.S. Patent No. 9,494,589, U.S. Patent No. 9,846,156, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entirety, will now be described to demonstrate the anti-cancer activity of the composition of the present invention.

[0084] The conditions, diseases or disorders that are therapeutically responsive to oleandrin (oleandrin-containing compositions or oleandrin-containing extracts) include cancer, tumors, carcinomas, sarcomas, glioblastomas, adenocarcinomas, lymphomas, melanomas, brain tumors, gliomas, astrocytomas (including low-grade astrocytomas (grade I pilocytic astrocytomas and grade II diffuse astrocytomas), anaplastic astrocytomas (grade III), glioblastomas (grade IV, also known as GBM, glioblastoma multiforme)), ependymoma, oligodendroglioma, excessive cell proliferation, malignant neoplastic diseases, non-cancerous inflammatory skin diseases, actinic keratosis, psoriasis, eczema, neurological diseases, neurological disorders ... The condition may be selected from the group consisting of chronic obstructive pulmonary disease, chronic myocardial infarction, ...

[0085] The compound present in the composition of the present invention may be in its unmodified form, salt form, derivative form, or combinations thereof.As used herein, the term "derivative" is taken to mean: a) a chemical compound that is structurally related to and theoretically derivable from a first chemical compound; b) a compound formed from a similar first compound, or a compound that can be imagined to be obtained from another first compound when one atom of the first compound is replaced with another atom or group of atoms; c) a compound derived or obtained from a parent compound and contains the essential elements of the parent compound; or d) a chemical compound that can be generated from a first compound of similar structure in one or more steps.For example, a derivative can be its deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or glycosylated form, or its ester, amide, lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl, heterocyclic, heterocyclic ring fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated form.

[0086] As used herein, each named compound may be independently selected for each occurrence in its natural (unmodified, free acid, or free base) form, its salt form, its derivative form, its prodrug form, or combinations thereof. Compositions comprising and methods using deuterated forms of triterpenes are also within the scope of the invention.

[0087] The oleandrin-containing extract (composition) may be formulated in any suitable pharma- ceutically acceptable dosage form.Suitable types of dosage forms include aerosols, bars, beads, capsules, porous sheets, chewables, cloths, concentrates, creams, crystals, disks, douches, dressings, elixirs, emulsions, enemas, fibers, films, foams, gels, globules, granules, gums, implants, inhalants, injectables, injections, inserts, intrauterine devices, jellies, kits, liniments, lipsticks, and liposomes. The dosage form may be in the form of a liquid, a lotion, a troche, a mouthwash, an oil, an ointment, a paste, a lozenge, a patch, a pellet, a bandage, a compress, a powder, a lyophilized powder, a ring, a rinse, an ointment wax, a shampoo, a soap, a solution, a sponge, a spray, a stick, a strip, a suppository, a swab, a suspension, a syrup, a tablet, a tampon, a tape, a tincture, a lozenge, a wafer, or other such dosage form known to one of ordinary skill in the art of pharmacy. The oleandrin-containing extract (composition) may be included in an oral dosage form. Some embodiments of the dosage form are not enteric coated and release their charge of extract within a period of 0.5 to 1 hour or less. Some embodiments of the dosage form are enteric coated and release their charge of extract downstream of the stomach, e.g., in the jejunum, ileum, small intestine, and / or large intestine (colon). Enteric coated dosage forms will release the extract into the systemic circulation within 1 to 10 hours after oral administration. The oleandrin-containing extract (composition) may be included in a dosage form that exhibits rapid release, immediate release, controlled release, sustained release, extended release, sustained release, burst release, continuous release, delayed release, or pulsed release, or two or more of these types of release.The release profile of oleandrin from the dosage form may be zero order, pseudo zero order, first order, pseudo first order, or sigmoid release profile.The plasma concentration profile for oleandrin in the administered subject may exhibit one or more maxima.

[0088] Suitable dosage forms may be made according to U.S. Patent No. 8,187,644 to Addington, issued May 29, 2012, U.S. Patent No. 7,402,325 to Addington, issued July 22, 2008, and U.S. Patent No. 8,394,434 to Addington et al., issued March 12, 2013, the entire disclosures of which are hereby incorporated by reference herein. Suitable dosage forms may also be made as described in Examples 4-6, and 9.

[0089] The oleandrin-containing extract (or any composition containing it) may be administered locally, systemically, or both. Suitable modes of administration include parenteral, buccal, conjunctival, corneal, dermal, dental, intracervical, intrasinus, intratracheal, enteral, epidermal, epidural, extraamniotic, extracorporeal, infiltrative, interstitial, intraperitoneal, intraamniotic, intraarterial, intrabronchial, intravesical, intracardiac, intracavernous, intracavitary, intracerebral, intracorneal, intramuscular, intradermal, intradiscal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intraileal, intraluminal, intralymphatic, intrameningeal, intramedullary, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinus, intrasynovial, intrasynaptic, intramuscular, intravenous ... These include intravesicular, intratesticular, intrathecal, intrathoracic, intraductal, intratumoral, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, irrigation, laryngeal, intranasal, nasogastric, ocular, ophthalmic, oral, auricular (pinna), or oropharyngeal, parenteral, transdermal, periarticular, epidural, perineural, perineural, buccal, pulmonary, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, ​​intravaginal, or other known modes of administration.

[0090] It should be noted that the compounds herein may have one or more functions in the compositions or formulations of the present invention. For example, a compound may act as both a surfactant and a water-miscible solvent or as both a surfactant and a water-immiscible solvent. The liquid composition may include one or more pharma- ceutically acceptable liquid carriers. The liquid carrier may be an aqueous, non-aqueous, polar, non-polar, and / or organic carrier. Examples of liquid carriers include, but are not limited to, water-miscible solvents, water-immiscible solvents, water, buffers, and mixtures thereof.

[0091] As used herein, the terms "water-soluble solvent" or "water-miscible solvent" are used interchangeably and refer to an organic liquid that does not form a biphasic mixture with water or is sufficiently soluble in water to provide an aqueous solvent mixture containing at least 5 percent of the solvent without liquid phase separation. The solvent is suitable for administration to humans or animals. Exemplary water-soluble solvents include, by way of example and without limitation, PEG (poly(ethylene glycol)), PEG400 (poly(ethylene glycol) having an approximate molecular weight of about 400), ethanol, acetone, alkanols, alcohols, ethers, propylene glycol, glycerin, triacetin, poly(propylene glycol), PVP (poly(vinylpyrrolidone)), dimethylsulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

[0092] As used herein, the terms "water-insoluble solvent" or "water-immiscible solvent" are used interchangeably and refer to an organic liquid that forms a biphasic mixture with water or provides phase separation when the concentration of the solvent in water exceeds 5 percent. The solvent is suitable for administration to humans or animals. Exemplary water insoluble solvents include, by way of example and not limitation, medium / long chain triglycerides, oils, castor oil, corn oil, vitamin E, vitamin E derivatives, oleic acid, fatty acids, olive oil, softisan 645 (diglyceryl caprylate / caprate / stearate / hydroxystearate adipate), Miglyol, Captex 350: glyceryl tricaprylate / caprate / laurate triglyceride; Captex 355: glyceryl tricaprylate / caprate triglyceride; Captex 355 EP / NF: glyceryl tricaprylate / caprate medium chain triglyceride).

[0093] Suitable solvents are listed in the "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance for industry Q3C Impurities: Residual Solvents" (1997), which provides recommendations regarding the amount of residual solvents that are considered safe in pharmaceutical products. Exemplary solvents are listed as Class 2 or Class 3 solvents. Class 3 solvents include, for example, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate.

[0094] Other materials that may be used as water immiscible solvents in the present invention include Captex 100: propylene glycol dicaprate; Captex 200: propylene glycol dicaprylate / dicaprate; Captex 200 P: propylene glycol dicaprylate / dicaprate; propylene glycol dicaprylocaprate; Captex 300: glyceryl tricaprylate / caprate; Captex 300 EP / NF: glyceryl tricaprylate / caprate medium chain triglyceride; Captex 350: glyceryl tricaprylate / caprate / laurate; Captex 355: glyceryl tricaprylate / caprate; Captex 355 EP / NF: glyceryl tricaprylate / caprate medium chain triglyceride; Captex 500: triacetin; Captex 500 P: Triacetin (Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-Ethyltexanoate); Captex 810 D: Glyceryl Tricaprylate / Caprate / Linoleate; Captex 1000: Glyceryl Tricaprate; Captex CA: Medium Chain Triglycerides; Captex MCT-170: Medium Chain Triglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP / NF: Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides; Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: glyceryl monocaprate; Caprol PG-8: propylene glycol monocaprylate; Caprol PG-12: propylene glycol monolaurate; Caprol 10G10O: decaglycerol decaoleate; Caprol 3GO: triglycerol monooleate; Caprol ET: polyglycerol esters of mixed fatty acids; Caprol MPGO: hexaglycerol dioleate; Caprol PGE 860: decaglycerol mono-, dioleate.

[0095] As used herein, "surfactant" refers to a compound that contains a polar or charged hydrophilic portion as well as a non-polar hydrophobic (lipophilic) portion; that is, surfactants are amphiphilic. The term surfactant may refer to one type or a mixture of compounds. Surfactants may be solubilizers, emulsifiers, or dispersants. Surfactants may be hydrophilic or hydrophobic.

[0096] Hydrophilic surfactant may be any suitable hydrophilic surfactant for use in pharmaceutical compositions.Such surfactant may be anionic, cationic, zwitterionic or nonionic, but nonionic hydrophilic surfactant is preferred here.As discussed above, these nonionic hydrophilic surfactants will generally have an HLB value of more than about 10.Mixtures of hydrophilic surfactants are also within the scope of the present invention.

[0097] Similarly, the hydrophobic surfactant may be any hydrophobic surfactant suitable for use in pharmaceutical compositions.Generally, suitable hydrophobic surfactants will have an HLB value of less than about 10.Mixtures of hydrophobic surfactants are also within the scope of the present invention.

[0098] Examples of additional suitable solubilizers include alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol, and their isomers, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, commercially available from BASF under the trade name Tetraglycol) or methoxy PEG (Union Carbide); Amides such as 2-pyrrolidone, 2-piperidone, caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, and polyvinylpyrrolidone; Esters such as ethyl propionate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate. glycol, caprolactone and its isomers, valerolactone and its isomers, butyrolactone and its isomers; and other solubilizers known in the art, such as dimethylacetamide, dimethylisosorbide (Arlasolve DMI (ICI)), N-methylpyrrolidone (Pharmasolve (ISP)), monooctanoin, diethylene glycol nonoethyl ether (available from Gattefosse under the trade name Transcutol), and water. Mixtures of solubilizers are also within the scope of the invention. Except where indicated, the compounds referred to herein are readily available from standard commercial sources. Although not required, the composition or formulation may further comprise one or more chelating agents, one or more preservatives, one or more antioxidants, one or more adsorbents, one or more acidifying agents, one or more alkalizing agents, one or more antifoaming agents, one or more buffering agents, one or more coloring agents, one or more electrolytes, one or more salts, one or more stabilizing agents, one or more tonicity modifying agents, one or more diluents, or a combination thereof.

[0099] The compositions of the present invention may contain oils, such as fixed oils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil; fatty acids, such as oleic acid, stearic acid and isostearic acid; and fatty acid esters, such as ethyl oleate, isopropyl myristate, fatty acid glycerides, medium chain triglycerides, and acetylated fatty acid glycerides. The compositions may also contain alcohols, such as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol; glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol; ethers, such as poly(ethylene glycol) 450; petroleum hydrocarbons, such as mineral oil and petrolatum; water; pharma- ceutical suitable surfactants, suspending agents or emulsifying agents; or mixtures thereof.

[0100] It should be understood that compounds used in the art of pharmaceutical formulations generally serve a variety of functions or purposes, and therefore, if a named compound is referred to only once herein or is used to define multiple terms herein, its purpose or function should not be construed as being limited to only the named purpose or function. One or more of the components of the formulation may be present in the form of its free base, free acid, or pharma- ceutically or analytically acceptable salt. As used herein, "pharma-ceutically or analytically acceptable salt" refers to a compound that is modified, if necessary, by reacting with an acid to form an ionic bond pair. Examples of acceptable salts include conventional non-toxic salts formed, for example, from non-toxic inorganic or organic acids. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th . ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the relevant disclosure of which is hereby incorporated by reference herein.

[0101] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The dosage form can be prepared by any conventional means known in the pharmaceutical industry. The liquid dosage form can be prepared by providing at least one liquid carrier and oleandrin (oleandrin-containing composition, SbCL extract) in a container. One or more other excipients may be included in the liquid dosage form. The solid dosage form can be prepared by providing at least one solid carrier and oleandrin. One or more other excipients may be included in the solid dosage form.

[0102] The dosage forms may be packaged using conventional packaging equipment and materials, and may be included in packs, bottles, vials, bags, syringes, sachets, packets, blister packs, boxes, ampoules, or other such containers. The compositions of the present invention may be included in any dosage form. Particular dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablets, capsules, pills, caplets, lozenges, sachets, and other such dosage forms known to those skilled in the art of pharmacy. In view of the above description and the following examples, one skilled in the art will be able to practice the claimed invention without undue experimentation. The above will be further understood with reference to the following examples, which detail certain procedures for the preparation of embodiments of the invention. All references made to these examples are for illustrative purposes. The following examples should not be considered exhaustive, but merely illustrative of some of the many embodiments contemplated by the present invention. EXAMPLES

[0103] Example 1 Supercritical Fluid Extraction of Powdered Oleander Leaves (Comparative Example) Method A. Carbon dioxide method. Powdered oleander leaf was prepared by harvesting, washing, drying, and then passing the oleander leaf material through a grinding and dehydrating device such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The mass of starting material used was 3.94 kg.

[0104] The starting material was combined with pure CO2 in an extrusion device at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50° C. (122° F.). A total of 197 kg of CO2 was used to achieve a solvent to feed ratio of 50:1. The CO2 and feed mixture was then passed through a separation device which altered the pressure and temperature of the mixture and separated the extract from the carbon dioxide. The extract (65 g) was obtained as a brownish sticky viscous material with a pleasant aroma. The color may have been caused by chlorophyll and other residual chromophore compounds. For an accurate yield determination, the tube and separator were rinsed with acetone, and the acetone was evaporated to obtain an additional 9 g of extract. The total extract amount was 74 g. Based on the mass of the starting material, the extract yield was 1.88%. The content of oleandrin in the extract was calculated using high pressure liquid chromatography and mass spectrometry to be 560.1 mg, or a yield of 0.76%.

[0105] Method B. Using a mixture of carbon dioxide and ethanol Powdered oleander leaves were prepared by harvesting, washing, drying the oleander leaf material, and then passing the oleander leaf material through a grinding and dehydrating device such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The mass of starting material used was 3.85 kg.

[0106] The starting material was combined with pure CO2 and 5% ethanol as a modifier in an extrusion device at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50°C (122°F). A total of 160 kg CO2 and 8 kg ethanol were used, resulting in a solvent to feed ratio of 43.6:1. The mixture of CO2, ethanol, and feed was then passed through a separation device, which altered the pressure and temperature of the mixture and separated the extract from the carbon dioxide. The extract (207 g) was obtained after removal of the ethanol as a sticky, viscous mass of dark green threads that apparently contained some chlorophyll. Based on the mass of the starting material, the yield of the extract was 5.38%. The content of oleandrin in the extract was calculated using high pressure liquid chromatography and mass spectrometry to be 1.89 g, or a yield of 0.91%.

[0107] Example 2 Hot water extract of powdered oleander leaves. (Comparative Example) Hot water extraction is typically used to extract oleandrin and other active components from oleander leaves. Examples of hot water extraction methods can be found in U.S. Patent Nos. 5,135,745 and 5,869,060. Hot water extraction was performed using 5 g of powdered oleander leaves. Ten times the volume of boiling water (by mass of oleander starting material) was added to the powdered oleander leaves and the mixture was stirred continuously for 6 hours. The mixture was then filtered and the leaf residue was collected and extracted again under the same conditions. The filtrates were combined and freeze-dried. The extract was brown in appearance. The mass of the dried extract material was approximately 1.44 g. 34.21 mg of the extract material was dissolved in water and oleandrin content analysis was performed using high pressure liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68 mg. The yield of oleandrin was calculated based on the amount of extract and was 0.26%.

[0108] Example 3 Preparation of subcritical fluid extracts of Oleander An improved method for preparing oleandrin-containing extracts was developed using subcritical fluid extraction rather than supercritical fluid extraction of Oleander biomass. The dried, powdered biomass (prepared according to Example 14) was placed in an extraction chamber, which was then sealed. Carbon dioxide (about 90-95% wt) and alcohol (about 10-5% wt; methanol or ethanol) were injected into the chamber. The internal temperature and pressure of the chamber were such that the extraction medium was maintained in the subcritical liquid phase rather than the supercritical fluid phase for most or substantially all of the extraction time, at a temperature in the range of about 2°C to about 16°C (about 7°C to about 8°C) and a pressure in the range of about 115 to about 135 bar (about 124 bar). The extraction time was about 4 hours to about 12 hours (about 6 to about 10 hours). The extraction environment was then filtered and the supernatant collected. Carbon dioxide was vented from the supernatant and the resulting crude extract was diluted with ethanol (about 9 parts ethanol: about 1 part extract) and frozen at about -50°C for at least 12 hours. The solution was thawed and filtered (100 μm pore size filter). The filtrate was concentrated to about 10% of its original volume and then sterile filtered (0.2 μm pore size filter). The concentration of oleandrin in the crude extract is about 0.25-1 mg (or 0.5-0.8 mg) per mL of extract. The resulting subcritical fluid (SbCL) extract contained oleandrin and one or more other compounds extractable from Oleandrin, said one or more other compounds being as defined herein.

[0109] Example 4 Sublingual / buccal liquid dosage form A sublingual dosage form containing oleandrin was made by mixing oleandrin or an oleandrin-containing composition (eg, an oleandrin-containing extract) with medium chain triglycerides (MCT, eg, coconut oil). Method A. Ethanol diluted SbCL extract (2% by weight) was mixed with MCT (95% by weight) and flavorings (3% by weight). Method B. The ethanol diluted ethanol extract (2% by weight) was mixed with MCT (95% by weight) and flavorings (3% by weight). Method C. Ethanol diluted SbCL extract (1% by weight) was mixed with ethanol diluted ethanol extract (1% by weight), MCT (95% by weight), and flavorings (3% by weight). The sublingual dosage form was safely administered to subjects in aliquots of about 0.5 mL, without consuming any fluids or food for at least 10 minutes after administration, from 1 to 6 times daily for about 7 days to about 1 month. Although this dosage form is intended for sublingual administration, it may also be administered orally.

[0110] Example 5 Preparation of pharmaceutical compositions. Method A. Cremophor-based drug delivery system The following ingredients are provided in the amounts indicated:

[0111] [Table 1] The excipients are dispensed into jars and shaken in a New Brunswick Scientific C24KC refrigerated incubator shaker at 60° C. for 24 hours to ensure homogeneity. Samples are then removed and visually inspected for solubilization. Both the excipients and the antiviral composition are completely dissolved for all formulations after 24 hours. Method B. GMO / Cremophor-based drug delivery system The following ingredients are provided in the amounts indicated:

[0112] [Table 2] Follow the steps in Method A. Method C. Labrasol-based drug delivery system The following ingredients are provided in the amounts indicated:

[0113] [Table 3] Follow the steps in Method A. Method D. Vitamin E-TPGS-based micelle-forming system The following ingredients are provided in the amounts indicated:

[0114] [Table 4] Follow the steps in Method A. Method E. Multicomponent Drug Delivery Systems The following ingredients are provided in the amounts indicated:

[0115] [Table 5] Follow the steps in Method A. Method F. Multicomponent Drug Delivery Systems The following ingredients are provided in the amounts listed in the capsules:

[0116] [Table 6] Follow the steps in Method A.

[0117] Example 6 Preparation of enteric coated capsules Step I: Preparation of liquid-filled capsules Hard gelatin capsules (50 count, size 00) are filled with the liquid composition of Example 3. The capsules are manually filled with 800 mg of the formulation and then manually sealed with a 50% ethanol / 50% water solution. The capsules are then manually banded with a 22% gelatin solution containing the following ingredients in the amounts indicated:

[0118] [Table 7] Mix the gelatin solution thoroughly and allow it to swell for 1-2 hours. After the swelling period, cover the solution tightly and place in a 55°C oven to liquefy. Once the entire gelatin solution is liquid, banding is performed. Apply the gelatin solution to the capsules using a pointed circular 3 / 0 artist brush. Use the banding kit provided by Shionogi. After banding, the capsules are kept at room conditions for 12 hours to allow the banding to harden. Step II: Coating of liquid-filled capsules A coating dispersion is prepared from the ingredients listed in the table below.

[0119] [Table 8] When banding capsules are used according to step I, the dispersant is 20.0 mg / cm 2 Coat the capsules using the following conditions:

[0120] [Table 9]

[0121] Example 7 Treating a Viral Infection in a Subject Methods of WO2018053123, WO2019055119, U.S. Pat. No. 10,596,186, and related U.S. and foreign patents and applications, all of which are hereby incorporated by reference in their entireties, follow to demonstrate the antiviral activity of the compositions of the invention.

[0122] Example 8 Treating a Neurological Disorder in a Subject Methods of WO2011085307, U.S. Patent No. 8,481,086, U.S. Patent No. 9,220,778, U.S. Patent No. 9,358,293, U.S. Patent No. 9,877,979, U.S. Patent No. 10,383,886, and related U.S. and foreign patents and applications, all of the disclosures of which are hereby incorporated by reference in their entireties, follow to demonstrate the neuroprotective activity of the compositions of the present invention.

[0123] Example 9 Preparation of tablets containing oleandrin compositions An initial compression mix of 3% Syloid 244FP and 97% microcrystalline cellulose (MCC) is mixed. An existing batch of SbCL extract is then incorporated into the Syloid / MCC mix via wet granulation. This mix is ​​labeled "initial compression mix" in the tables below. Additional MCC is added extragranularly to increase the compressibility. This addition to the initial compression mix is ​​labeled "extragranular addition." The mix resulting from the extragranular addition is of the same composition as the "final compression mix."

[0124] [Table 10] [Table 11]

[0125] [Table 12] [Table 13] Syloid 244FP is a colloidal silicon dioxide manufactured by Grace Davison. Colloidal silicon dioxide is commonly used to provide several functions, such as adsorbent, glidant, and tablet disintegrant. Syloid 244FP is selected for its ability to adsorb 3 times its mass of oil, and its 5.5 μm particle size.

[0126] Example 10 HPLC analysis of solutions containing oleandrin Oleandrin Content: The samples (oleandrin standard, SCF extract, ethanol extract, subcritical fluid CO2 extract, and hot water extract) are analyzed by HPLC (Waters) using the following conditions: Symmetry C18 column (5.0 μm, 150×4.6 mm I.D.; Waters); mobile phase MeOH:water=54:46 (v / v), and flow rate 1.0 ml / min. The detection wavelength was set at 217 nm. Samples are prepared by dissolving the compound or extract in a fixed amount of HPLC solvent to achieve the approximate target concentration of oleandrin. The retention time of oleandrin can be determined by using an internal standard. The concentration of oleandrin can be determined / calibrated by making a signal response curve using the internal standard.

[0127] Triterpene Content: The triterpene components of the extract are quantified by HPLC using a Gemini C18 diphenyl column to elute the triterpenes using an isocratic mobile phase consisting of 95% MeCN and 0.1% formic acid, a flow rate of 0.6 ml / min, and a detection wavelength of 220 nm. A standard curve is generated and used to calculate the relative molar ratios of oleanolic acid, ursolic acid, and betulinic acid compounds. Figure 4 shows the HPLC chromatogram of PBI-05204.

[0128] Example 11 Treating Cancer in a Subject To demonstrate the anti-cancer (anti-excessive cell proliferation) activity of the compositions of the present invention, the methods of WO2007016176, WO2009064657, U.S. Patent No. 7,402,325, U.S. Patent No. 8,187,644, U.S. Patent No. 8,394,434, U.S. Patent No. 8,367,363, U.S. Patent No. 9,494,589, U.S. Patent No. 9,846,156, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entireties.

[0129] Example 12 Preparation of pharmaceutical compositions The pharmaceutical composition of the present invention can be prepared by any of the following methods: Mixing can be carried out under wet or dry conditions. The pharmaceutical composition may be compressed, dried, or both during preparation. The pharmaceutical composition may be divided into dosage forms.

[0130] Method A. At least one pharmaceutical excipient is mixed with at least one SbCL extract disclosed herein. Method B. At least one pharmaceutical excipient is mixed with at least the SbCL extract and at least one other active agent. Method C. At least one pharmaceutical excipient is mixed with the oleandrin. Method D. At least one pharmaceutical excipient is mixed with the oleandrin and at least one triterpene disclosed herein. Method E. At least one pharmaceutical excipient is mixed with the oleandrin and at least two triterpenes disclosed herein. Method D. At least one pharmaceutical excipient is mixed with the oleandrin and at least three triterpenes disclosed herein. Known amounts of oleanolic acid and ursolic acid are mixed according to a predetermined molar ratio of the components as defined herein. The components are mixed in solid form or in a solvent such as methanol, ethanol, chloroform, acetone, propanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. The resulting mixture contains the components in the relative molar ratios described herein.

[0131] Example 13 statistical analysis Statistical significance of experimental data sets was determined using an unpaired two-tailed Student's t-test (alpha = 0.05) and P-values ​​were calculated using the Shapiro-Wilk normality test and Graphpad Prism 7.03 software. P-values ​​are defined as 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Unless otherwise noted, error bars represent the SEM of at least three independent experiments.

[0132] Example 14 Preparation of powdered biomass Method A. Powdered dried oleander biomass is prepared according to Addington's U.S. Pat. No. 7,402,325, the entire disclosure of which is hereby incorporated by reference herein, by placing harvested leaves in a grinding and heat dehydration apparatus as described in U.S. Pat. No. 5,236,132, U.S. Pat. No. 5,598,979, U.S. Pat. No. 6,517,015, or U.S. Pat. No. 6,715,705. Method B. Powdered dried oleander biomass was prepared by dehydrating harvested leaves in a heated dehumidifier, then subjecting the dried leaves to repeated freeze-thaw cycles using liquid nitrogen, with the leaves that were frozen during at least one of the cycles being ground to form a powder, which was then freeze-dried to a moisture content of less than about 8% by weight.

[0133] Example 15 Preparation of aqueous organic solvent extracts An ethanol extract of Oleander was prepared by extracting the dried powdered biomass with aqueous ethanol. The powder was extracted at room temperature and pressure with vigorous stirring using 10 mL of aqueous ethanol (10% v / v in water) for every gram of leaf powder. The extraction supernatant and biomass were separated and the extraction cycle was repeated two more times, meaning three extraction cycles were performed. The supernatants were combined and concentrated in vacuum to about 20% of the original volume to form a concentrate, which was sterile filtered (0.2 μm filter) to form a crude extract, which was then diluted with 50% aqueous ethanol. The content of water, ethanol, and other components in the crude extract was determined. The content of oleandrin in the crude extract was about 1-5 mg / mL extract.

[0134] Example 16 Preparation of dosage forms containing combinations of oleander extracts The objective was to prepare a dosage form according to Example 4, except that a portion of the ethanolic extract or aqueous ethanolic extract (1% by weight) was combined with a portion of the SbCL extract (1% by weight), medium chain triglycerides (95% by weight), and flavorings (3% by weight) of Example 3. The concentration of oleandrin in the dosage form is about 12.5 to about 25 micrograms / mL.

[0135] (Example 17) In vivo evaluation of oleandrin against COVID-19 virus The objective of this study was to determine the efficacy of an oleandrin-containing extract (OCE) in treating subjects already infected with the COVID-19 virus. The clinical status of over 30 subjects, representing a broad demographic distribution, exhibiting COVID-19 infection was evaluated and determined prior to sublingual, buccal, or oral administration of OCE prepared according to the dosage form of Example 16. The composition was safely administered to the subjects. The dosing regimen was approximately 0.5 mL per dose and 4 doses per day (one dose approximately every 6 hours), approximately 50-60 micrograms of oleandrin per day. All subjects experienced complete recovery within 3-10 days of initiating treatment.

[0136] (Example 18) Gas chromatographic analysis of the extract The organic solvent extracts and the subcritical extracts were analyzed by GC-mass spectrometry with total ion current (TIC) detection to identify some of their volatile and semi-volatile organic constituents. Sample extracts were dissolved using a solution containing seven isotopically labeled semivolatile organic compound internal standards (SVOCIS) before being injected into the GC-MS and analyzed. The 10 largest peaks from each sample extract were tentatively identified via comparison against the NIST / EPA / NIH mass spectral library (NIST, 2014) and each match was manually checked. A library score for each identity was assigned by the software, with a match factor of 100 indicating a perfect match. Relative concentrations for each identified peak were calculated via peak area compared to its closest eluting SVOCIS standard peak. It should be noted that ionization efficiency within the GC-MS can vary significantly between compounds.

[0137] (Example 19) Identification of compounds in the extract The extracts are analyzed by MS-DART TOF analysis as follows: A JEOL AccuTOF-DART mass spectrometer (Jeol USA, Peabody, MA, USA) was used. A JEOL AccuTOF-DART mass spectrometer (Jeol USA, Peabody, MA, USA) was used. The analysis was performed in positive ion mode (DART+), with masses corresponding to the M+H+ ions generated by the DART-MS. Various settings on the instrument were used to determine optimal conditions for N. oleander analysis. Typical settings on the DART+ include needle voltage 3500V; orifice 1- 2-20V; ring lens 2-5V; orifice 2- 2-5V; and peak voltage 1000V. Calibration is performed internally on each sample using a 10% solution of PEG600, which provides mass markers across the required mass range of 100-1000 mass units. Other analyses were performed in DART-mode, and these consisted of needle voltage 3500V; heating element 250°C; electrode 1- 150V; electrode 2- 250V; He gas flow rate 3.79 LPM; mass spectrometer settings: MCP 2600V; orifice 1- 15V; ring lens- 5V, orifice 2- 5V; and peak voltage 1000V. Calibration is performed internally on each sample using a perfluorinated carboxylic acid solution that provides markers over the entire required mass range of 100-1000 mass units. N. oleander samples are introduced neat into the DART helium plasma using the closed end of a borosilicate glass melting point tube. The capillary tube is held in the He plasma for approximately 3-5 seconds per analysis. Molecular formulas are confirmed by elemental composition and isotope matching programs provided with a JEOL AccuTOF DART-MS instrument. A searchable database of N. oleander constituents developed by Herbal Science (Naples, FL, USA) may be used.

[0138] (Example 20) Dietary Supplement Composition The oleandrin-containing extract (at the desired concentration) is combined with a dietary supplement vehicle such as those found in PREMIER PROTEIN shakes, LIMFAST ADVANCED ENERGY meal replacement shakes, ORGAIN ORGANIC NUTRITION shakes, VEGA Protein Nutrition shakes, LEANBODYLABRADA whey protein meal replacement shakes, ICONIC GRASS-FED PROTEIN beverages, PURE PROTEIN shakes, MUSCLE MILK original protein shakes, MET-RX original whey protein meal replacement powder, OWYN vegan meal replacement shakes, SOYLENT meal replacement shakes, or APRES plant-based vegan protein shakes.

[0139] Example 21 Cosmetic lotion composition The oleandrin-containing extract (at the desired concentration) may be mixed with a dietary supplement vehicle, such as NIVEA ESSENTIALLY ENRICHED body lotion (with moisturizing serum and almond oil), EUCERIN ADVANCED REPAIR (with ceramide-3), PALMER'S COCOA BUTTER FORMULA DAILY SKIN THERAPY body lotion (both lotion) (with cocoa butter and vitamin E), JERGEN'S ULTRA-HEALING dry skin moisturizer (with vitamins C, E, and B5), LUBRIDERM daily moisture body lotion (with vitamin B5), WELEDA SKIN Combine with those found in FOOD Nourishing Cream (contains Vitamin E, Calendula Flower Extract, and Pansy), AVEENO Daily Moisturizing Lotion, HEMPZ Original Herbal Body Moisturizer (contains Hemp Seed Oil), CERAVE Daily Moisturizing Lotion (contains Hyaluronic Acid), and AQUAPHOR ADVANCED THERAPY Healing Ointment (contains Petrolatum).

[0140] Example 22 Dosage Form Method A: Thin Film Dosage Form Thin film oral dosage forms are described by Barnhart ("Thin film oral dosage forms" in Modified Release Drug Delivery Technology; 2 nd Edition, 2013, CRC press; ISBN: 9780429145803); Joshua et al. (“Fast dissolving oral thin films: an effective dosage form for quick releases” in Int. J. Pharm. Sci. Rev. Res. (2016), 38(1), 282-289), Harsha et al. (“An introduction to fast dissolving oral thin film drug delivery systems: a review” in Drug. Deliv. (2013), 10(6), 667-684), and Nagaraju et al. (“Comprehensive review on oral disintegrating films” in Drug Deliv. (2013), 10(1), 96-108).

[0141] Method B: Bioadhesive Dosage Forms Bioadhesive buccal dosage forms have been described by Reddy et al. (“A review on bioadhesive buccal drug delivery systems: current status of formulation and evaluation methods” in Daru (2011), 19(6), 385-403), Shojaei et al. (“Buccal mucosa as a route for systemic drug delivery: a review” in J. Pharm. Pharmaceut. Sci. (1998), 1(1), 15-30), Prabhakar et al. (“Bioadhesive polymeric platforms for transmucosal drug delivery systems- a review” in Trop. J. Pharma. Res. (2010), 9(1), 91-104), Mathiowitz et al. (Bioadhesive Drug Delivery Systems- fundamentals, novel approaches, and development; ed. E. Mathiowitz, et al.; Marcel Dekker, Inc. (New York, NY), (1999), ISBN It can be prepared according to US Pat. No. 6,247,027, issued Jun. 2, 1995.

[0142] Method C: Gummy Dosage Form Gummy (semi-solid, soft, chewable) dosage forms can be prepared according to Jacobs (“Semi-solid formulations” in Pediatric Form. (2014), 171-179), Cizauskaite et al. (“Natural ingredients-based gummy bear composition designed according to texture analysis and sensory evaluation in vivo” in Molecules (2019), 24(7), 1442), and Bartkiene et al. (“Development of antimicrobial gummy candies with addition of bovine colostrum, essential oils, and probiotics” in Int. J. Food Sci. Technol. (2018), 53(5), 1227-1235).

[0143] Method D: Spray Dosage Form Spray dosage forms have been described by Finlay (The Mechanics of Inhaled Pharmaceutical Aerosols- An Introduction; Ed. WH Finlay, Academic Press, London, UK; 2001), Hickey ("Back to the Future: Inhaled Drug Products" in J. Pharm. Sci. (2013), 102(40\), 1165-1172), Malamatari et al. ("Solidification of nanosuspensions for the production of solid oral dosage forms and inhalable dry powders" in Exp. Opin. Drug Deliv. (2016), 13(3), 435-450), and Hickey (Pharmaceutical Inhalation Aerosol Technology, 2 nded.; Ed. AJ Hickey, CRC Press, New York, NY, 2003).

[0144] Example 23 Preparation of organic solvent extracts An ethanol extract of Oleander was prepared by extracting the dried powdered biomass with ethanol. For every gram of leaf powder, 10 mL of ethanol was used, and the powder was extracted at room temperature and pressure with vigorous stirring. The extraction supernatant and biomass were separated, and the extraction cycle was repeated two more times, meaning three extraction cycles were performed. The supernatants were combined and concentrated in vacuum to about 20% of the original volume to form a concentrate, which was sterile filtered (0.2 μm filter) to form a crude extract, which was then diluted with 50% aqueous ethanol. The content of water, ethanol, and other components in the crude extract was determined. The content of oleandrin in the crude extract was about 1-5 mg / mL extract.

[0145] As used herein, the term "about" or "approximately" is intended to mean ±10%, ±5%, ±2.5%, or ±1% of the specified value. As used herein, the term "substantially" is intended to mean "a majority of" or "at least a majority of" or "more than 50% of."

[0146] The above is a detailed description of a specific embodiment of the present invention. Although a specific embodiment of the present invention has been described herein for illustrative purposes, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the present invention is not limited except as set forth in the appended claims. All of the embodiments disclosed and claimed herein can be made and achieved without undue experimentation in light of the present disclosure. Yet another aspect of the present invention may be as follows. [1] An oleandrin-containing extract produced by extracting oleandrin-containing biomass using subcritical liquid carbon dioxide. [2] The oleandrin-containing extract described in [1] above, wherein the subcritical liquid comprises subcritical liquid carbon dioxide and one or more modifiers. [3] An improved oleandrin-containing combination composition comprising a portion of an oleandrin-containing organic solvent extract and a portion of an oleandrin-containing SbCL extract. [4] The combination composition according to [3] above, wherein substantially equal portions of the two extracts are present in the combination composition. [5] The combination composition according to [3], wherein: a) the organic solvent extract contains at least oleandrin and other components extracted from the biomass; b) in the organic solvent extract, the molar ratio of oleandrin to other components extracted from the biomass is greater than the molar ratio in the subcritical fluid extract; c) the SbCL extract contains at least oleandrin and other components extracted from the biomass; d) the content of oleandrin in the ethanol extract is greater than the content of oleandrin in the subcritical fluid extract; e) the content of extracted non-oleandrin components in the organic solvent extract is greater than the content of said components in the subcritical fluid extract; f) the composition profile of extracted volatile and semi-volatile components in the organic solvent extract is different from the composition profile of said components in the subcritical fluid extract; and / or g) the subcritical fluid extract contains a higher proportion of extracted volatile and semi-volatile components relative to the oleandrin content than the organic solvent extract. [6] a) the oleandrin content in the extract is about 0.01% by mass or more, about 0.05% by mass or more, about 0.1% by mass or more, about 0.2% by mass or more, about 0.5% by mass or more, about 1% by mass or more, about 2.5% by mass or more, about 5% by mass or more, about 10% by mass or more, about 20% by mass or more, about 30% by mass or more, about 40% by mass or more, about 50% by mass or more, about 60% by mass or more, about 70% by mass or more, about 80% by mass or more, or about 90% by mass or more; b) the oleandrin content in the extract is about 0.01% by mass or more, about 0.05% by mass or more, about 0.1% by mass or more, about 0.2% by mass or more, about 0.5% by mass or more, about 1% by mass or more, about 2.5% by mass or more, about 5% by mass or more, about 10% by mass or more, about 20% by mass or more, about 30% by mass or more, about 40% by mass or more, about 50% by mass or more, about 60% by mass or more, about 70% by mass or more, about 80% by mass or more, or about 90% by mass or more; The invention according to any one of the above [1] to [5], wherein the content of fluorine is about 99% by mass or less, about 95% by mass or less, about 90% by mass or less, about 80% by mass or less, about 70% by mass or less, about 60% by mass or less, about 50% by mass or less, about 40% by mass or less, about 30% by mass or less, about 20% by mass or less, about 10% by mass or less, about 5% by mass or less, about 2.5% by mass or less, about 1% by mass or less, or about 0.5% by mass or less; or c) a combination of any of the limitations in item a) and any of the limitations in item b). [7] The invention according to any one of [1] to [5] above, wherein the extract comprises oleandrin and one or more compounds selected from the group consisting of one or more cardiac glycosides, one or more glycocone components of cardiac glycosides (e.g., glucosides, fructosides, and / or glucuronides), one or more aglycones, one or more glycoside precursors (e.g., cardenolides, cardadienolides, and cardatrienolides), one or more steroids, one or more triterpenes, one or more monosaccharides, one or more polysaccharides, one or more alkaloids, one or more proteins, fats, one or more other non-cardiac glycosides that are therapeutically effective agents, and any combination thereof. [8] The extract contains oleandrin, neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-en-3β,28-diol), 28-norurs-12-en-3β-ol, urs-12-en-3β-ol, 3β,3β-hydroxy-12-oleanen-28-oic acid, 3β,20α-dihydroxyurs-21-en-28-oic acid, 3β,27-dihydroxy-12-urs-en-28-oic acid, 3β,13β-dihydroxyurs-11-en-28-oic acid, 3β,27-dihydroxy-12-urs-en-28-oic acid, 3β,13β-dihydroxyurs-11-en-28-oic acid, 3β,27-dihydroxyurs-12 ... 3-O-(D-Dihydroxy-12-oleanan-28-oic acid), 3β,12α-dihydroxyoleanan-28,13β-olide, 3β,27-dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalacturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, cardenolide B-1, cardenolide B-2, oleagenin, neridiginoside, nerizoside, odoroside-H, 3-beta-O-(D-Dihydroxy-12-oleanan-28-oic acid, 14beta-di ... pectic polysaccharides composed of 0(22)-enolides, galacturonic acid, rhamnose, arabinose, xylose, and galactose, polysaccharides having a MW in the range of 17,000 to 120,000 D, or a MW of about 35,000 D, about 3,000 D, about 5,500 D, or about 12,000 D, cardenolide monoglycosides, cardenolide N-1, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4,6-diene-3,12,20-trione, 20R-hydroxypregna-4,6-diene-3,12-dione, 16 ... beta,17beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridienone B), nericumaric acid, isonelicumaric acid, oleandelonic acid, oleandelene, 8alpha-methoxylabtan-18-oic acid, 12-ursene, caneloside, neriumoside, 3β-O-(D-diginosyl)-2α-hydroxy-8,14β-epoxy-5β-card-16:17,20:22-dienolide, 3β-O-(D-diginosyl)-2α,14β-dihydroxy-5β-card-16:17,20:22-dienolide, 3β,27-dihydroxy-urs-18-en-13,28-olide, 3β,22α,28-trihydroxy-25-nor-lupa-1(10),20(29)-dien-2-one, cis-karenin (3β-hydroxy-28-Zp-coumaroyloxy-urs-12-en-27-oic acid), trans-karenin (3-β-hydroxy-28-Ep-coumaroyloxy-urs-12-en-27-oic acid), Nerimogenin A, 3beta,27-dihydroxy-12-urs-12-en-27-oic acid, 3beta,27-dihydroxy-12-urs-12-en-28-oic acid, 3beta,13beta-dihydroxyurs-1 ... tert-hydroxyurs-12-ene-28-aldehyde, 28-norurs-12-en-3beta-ol, urs-12-en-3beta-ol, urs-12-ene-3beta,28-diol, 3beta,27-dihydroxy-12-oleanen-28-acid, (20S,24R)-epoxydammarane-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol, 20beta,28-epoxytaraxasteran-21-en-3beta-ol, 28-norurs-12-ene -3beta,17beta-diol, 3beta-hydroxyurs-12-ene-28-aldehyde, alpha-neriursate, beta-neriursate, 3alpha-acetophenoxy-urs-12-ene-28-acid, 3beta-acetophenoxy-urs-12-ene-28-acid, oleanderolic acid, canelodione, 3beta-p-hydroxyphenoxy-11alpha-methoxy-12alpha-hydroxy-20-ursene-28-acid, 28-hydroxy-20(29)-lupene-3,7-dione, canelosin (3alpha-hydroxy-urs-18,20-dien-28-oic acid), 3alpha-hydroxy-ursa-18,20-dien-28-oic acid, D-sarmentose, D-diginose, neridiginoside, nerizosid, isoricinoleic acid, gentiobiosil nerigoside, gentiobiosil beaumontside, gentiobiosil oleandrin, folinellin, 12β-hydroxy-5β-carda-8,14,16,20(22)-tetraenolide, 8β-hydroxy-digitoxigenin, Δ16-8β-hydroxy-digitoxigenin, Δ16-neriagenin, uvaol, ursolaldehyde, 27(p-coumaroyloxy)ursolic acid, oleanderol, 1 and one or more compounds selected from the group consisting of 6-anhydro-deacetyl-nerigoside, 9-D-hydroxy-cis-12-octadecanoic acid, adigosides, adinelin, alpha-amyrin, beta-sitosterol, campestrol, cautchouc, capric acid, caprylic acid, choline, cornellin, cortenelin, deacetyloleandrin, diacetyl-nerigoside, foliandrin, pseudochlamin, quercetin, quercetin-3-rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospecide, urehitoxin, and usaligenin. The invention described in [7]. [9] A pharmaceutical composition comprising at least one pharmaceutical excipient and the product according to any one of [1] to [5] above.

[10] A method for treating or preventing a condition, disease or disorder therapeutically responsive to oleandrin (an oleandrin-containing composition or an oleandrin-containing extract), comprising administering to a subject in need thereof one or more doses of the pharmaceutical composition described in [9], thereby treating or preventing the condition, disease or disorder.

[11] A method for treating or preventing a condition, disease or disorder therapeutically responsive to oleandrin (an oleandrin-containing composition or an oleandrin-containing extract), comprising administering to a subject in need thereof one or more doses of the product of the invention described in any one of [1] to [5] above, thereby treating or preventing the condition, disease or disorder.

[12] The method according to

[10] or

[11] , wherein the administration is acute, chronic, or a combination thereof.

[13] The method according to

[10] ,

[11] , or

[12] , wherein the administering is local administration, systemic administration, or a combination thereof.

[14] The condition, disease or disorder that is therapeutically responsive to oleandrin (oleandrin-containing composition or oleandrin-containing extract) is a cancer, tumor, carcinoma, sarcoma, glioblastoma, adenocarcinoma, lymphoma, melanoma, brain tumor, glioma, astrocytoma (including low-grade astrocytoma (grade I pilocytic astrocytoma and grade II diffuse astrocytoma), anaplastic astrocytoma (grade III), glioblastoma (grade IV, also known as GBM, glioblastoma multiforme)), ependymoma, oligodendroglioma, excessive cell proliferation, malignant neoplastic disease, non-cancerous inflammatory skin disease, actinic keratosis, psoriasis, eczema, neurological disease, neurological disorder, neurodegenerative disease, The method according to any one of

[10] ,

[11] ,

[12] , or

[13] , wherein the disease is selected from the group consisting of neurodegenerative disorders, neurological diseases or disorders having an etiology associated with altered Na,K-ATPase activity, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, dementia, amyotrophic lateral sclerosis, bovine spongiform encephalopathy, multiple sclerosis, diabetic neuropathy, autism and juvenile neuronal ceroid lipofuscinosis, tauopathy, Down's syndrome, Pick's disease, corticobasal degeneration, some variants of prion diseases, progressive supranuclear palsy, frontotemporal dementia, hemorrhoids, ulcers, leprosy, snakebite, and viral infection.

[15] The method according to

[14] , wherein the viral infection is caused by a virus of the Arenaviridae, Arteriviridae, Bunyaviridae, Filoviridae, Flaviviridae (Flavivirus genus), Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae (Deltaretrovirus genus), Coronaviridae, (+)-ss-envRNAV (positive-sense short-stranded enveloped RNA virus), (-)-ss-envRNAV (negative-sense short-stranded enveloped RNA virus), or Togaviridae family.

[16] The viral infection is selected from the group consisting of flavivirus, yellow fever virus, dengue fever virus, Japanese encephalitis virus, West Nile virus, Zika virus, tick-borne encephalitis virus, Kyasanur Forest disease virus, Al Hamra disease virus, Omsk hemorrhagic fever virus, Powassan virus, arborvirus virus, Eastern equine encephalomyelitis virus (EEEV), Western equine encephalomyelitis virus (WEEV), Venezuelan equine encephalomyelitis virus (VEEV), Chikungunya virus (CHIKV), Onyongun virus (ONNV), Pogosta disease virus, Sindbis virus, Ross River fever virus (RRV), Semliki Forest virus, Lassa virus, aseptic meningitis, Gunarito virus, Junin virus, and the like. The method according to

[14] , wherein the virus is selected from the group consisting of viruses, Lujo virus, Machupo virus, Sabia virus, Whitewater Arroyo virus, Hantavirus, Crimean Gongo hemorrhagic fever Orthonairovirus, Mumps virus, Nipah virus, Hendra virus, Respiratory syncytial virus (RSV), Human parainfluenza virus (HPIV), NDV, Influenza viruses (A to C), Isa virus, Thogoto virus, Quaranja virus, H1N1, H2N2, H3N2, H1N2, Spanish flu, Asian flu, Hong Kong flu, Russian flu, rabies virus, vesiculovirus, lyssavirus, cytorhabdovirus, SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV229E, CoV NL63, CoV OC43, CoV HKU1, and CoV HKU20.

[17] A method for producing an oleandrin-containing extract, comprising the steps of: subjecting the oleandrin-containing biomass to subcritical fluid extraction (SbCLE) using a subcritical fluid (SbCL) comprising carbon dioxide for a time sufficient to extract the oleandrin and form an extraction environment; Separating the biomass from the extraction environment to provide oleandrin-containing SbCL; and removing SbCL from the oleandrin-containing SbCL to provide the oleandrin-containing extract (OCE); The method includes:

[18] subjecting an oleandrin-containing biomass to subcritical fluid extraction (SbCLE) using a subcritical fluid (SbCL) containing carbon dioxide for a time sufficient to extract the oleandrin and form an extraction environment; separating the biomass from the extraction environment to provide oleandrin-containing SbCL; removing SbCL from the oleandrin-containing SbCL to provide an oleandrin-containing extract; and Isolating oleandrin from said extract. 23. A method for producing oleandrin comprising:

[19] The method according to

[18] , wherein the isolating is accomplished by one or more of chromatography, liquid-liquid extraction, liquid-solid extraction, crystallization, precipitation, fractional precipitation, fractional crystallization, Soxhlet extraction, trituration, or any combination thereof.

[20] The method according to any one of

[17] to

[19] , further comprising one or more of the following steps: a) separating the biomass from the extraction environment by filtering the extraction environment; b) adding a modifier to the carbon dioxide or extraction environment; c) heating and pressurizing the inside of a vessel containing the extraction environment to form a subcritical liquid phase extraction environment; d) removing the SbCL from the oleandrin-containing SbCL by evaporating the SbCL to form a raw SbCL extract; e) diluting the raw SbCL extract with an organic solvent or an aqueous organic solvent; f) filtering the diluted raw or crude SbCL extract one or more times; g) treating the diluted raw or crude SbCL extract with activated carbon; h) removing most of the organic solvent from the diluted raw extract to form; and / or i) sterile filtering the raw or crude extract to obtain an SbCL extract.

[21] The method according to any one of

[17] to

[20] above, wherein the SbCL comprises carbon dioxide and one or more modifiers.

[22] The method according to

[21] , wherein the one or more modifiers are selected from the group consisting of alcohols and ethanol.

[23] The method according to any one of

[17] to

[22] above, wherein the carbon dioxide (and the modifier, if present) is in the subcritical liquid phase for at least about 50% of the extraction time.

[24] The method according to

[23] , wherein the extraction environment comprising the extraction fluid (carbon dioxide and, if present, the modifier) ​​in the subcritical liquid phase and the biomass remains in liquid form for at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the extraction time, or the extraction environment remains in liquid form throughout the entire extraction time.

[25] CO 2 Regarding the phase diagram of SbCLE, a) the critical pressure (p c ) below the critical temperature (t c ) below and above the liquid-vapor line; b) t c In the following, c below and above the liquid-vapor line; c) p c That's it, t c , above the liquid-vapor line, and below the solid-liquid line; or d) below the solid-liquid line, the liquid-vapor line, the liquid-supercritical line, the triple point (solid-liquid-vapor) temperature (t tp ), triple point pressure (p tp ), critical point (liquid-gas-supercritical) temperature (t cp ), and the critical pressure (p cp The method according to any one of

[17] to

[24] above, wherein the process is carried out at a temperature and pressure within a liquid region surrounded by the liquid phase.

[26] The method according to any one of

[17] to

[25] above, wherein SbCLE is carried out at a) a temperature of about 0°C to about 31°C and a pressure of about 5.2 bar to about 3000 bar; b) a temperature of about 0°C to about 31°C and a pressure of about 5.2 bar to about 1000 bar; c) a temperature of about 2°C to about 16°C and a pressure of about 115 bar to about 135 bar; d) a temperature of about 5 to 10°C and a pressure of about 120 to 130 bar; or e) a temperature in the range of about 5°C to about 15°C and a pressure in the range of about 50 bar to 70 bar.

[27] The method according to any one of

[17] to

[26] above, wherein the mass ratio of the extraction liquid (containing carbon dioxide and optionally further containing a modifier) ​​to the biomass is in the range of about 20:1 to about 100:1, about 30:1 to about 75:1, about 50:1, about 45:1 to 60:1, or about 40:1 to about 45:1 based on the mass of both the solvent and the raw material.

[28] The method according to any one of

[17] to

[27] above, wherein the mass ratio of carbon dioxide to the modifier is in the range of about 100:about 0.01-20, about 100:about 0.01-15, about 100:about 0.01-10, or about 100:about 0.01-5.

[29] The method according to any one of

[17] to

[28] , wherein a) the biomass is dehydrated, dehumidified, freeze-dried, and / or desiccated before being extracted, independently for each occurrence; b) the biomass is dried, independently for each occurrence, before being extracted; c) the biomass is not dehydrated, independently for each occurrence, before being extracted; d) the biomass is not dried, independently for each occurrence, before being extracted; or e) independently for each occurrence.

[30] A method for producing a combination composition, comprising: subjecting the oleandrin-containing first biomass to an organic solvent extraction to provide an oleandrin-containing organic solvent extract; subjecting the oleandrin-containing second biomass to subcritical fluid (SbCL) extraction (SbCLE) to provide an oleandrin-containing SbCL extract; and combining said oleandrin-containing organic solvent extract with said oleandrin-containing SbCL extract to provide said combination composition. The method includes:

[31] a) the organic solvent extraction is carried out with an organic solvent or an aqueous organic solvent; b) the organic solvent or the aqueous organic solvent, independently for each occurrence, comprises an alcohol; c) the organic solvent, independently for each occurrence, is selected from the group consisting of methanol, ethanol, propanol, butanol, chloroform, methylene chloride, ethyl acetate, and combinations thereof; d) the organic solvent extraction comprises extracting the oleandrin-containing biomass one or more times with an organic solvent or an aqueous organic solvent; e) independently for each occurrence, the preceding The method according to

[30] above, wherein the aqueous organic solvent contains up to about 20% w / w, about 5% w / w to about 15% w / w, or about 10% w / w of water; f) the organic solvent extraction is performed at a temperature of about 0°C to about 75°C, about 5°C to about 70°C, about 10°C to about 60°C, about 10°C to about 50°C, about 10°C to about 45°C, or about 20°C to about 40°C; g) the v / w ratio of organic solvent (or aqueous organic solvent) to biomass is in the range of about 50-1:about 1, about 40-1:about 1, about 30-1:about 1, about 20-1:about 1, about 20-5:about 1, or about 10:about 1.

[32] The method according to

[30] or

[31] , wherein the organic solvent extraction further comprises one or more of the following: a) separating the biomass from the extraction environment by filtering the extraction environment to form a crude extract; b) adding an aqueous liquid to the organic solvent or extraction environment; c) reducing the content of the organic solvent from the crude extract; d) treating the crude extract with activated carbon; e) filtering the crude extract one or more times; f) adding additional organic solvent to the crude extract; g) sterile filtering the organic solvent extract; and / or h) adding water to the crude extract and extracting the aqueous crude extract with an organic solvent.

[33] The method according to any one of

[17] to

[32] above, wherein the water content in the biomass before extraction is about 20% by mass or less, about 15% by mass or less, about 10% by mass or less, about 8% by mass or less, about 5% by mass or less, about 2.5% by mass or less, or about 1% by mass or less.

[34] The method according to any one of

[17] to

[33] , wherein a) the particle size (d0.5) of the biomass prior to extraction is less than about 1 inch, less than about 0.75 inch, less than about 0.5 inch, less than about 0.4 inch, less than about 0.3 inch, less than about 0.2 inch, less than about 0.1 inch, less than about 0.05 inch, or less than about 0.01 inch, the particles being in terms of ASTM sieve mesh opening size.

[35] The method according to any one of

[17] to

[34] above, wherein the oleandrin-containing biomass is Nerium species, Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odourum, white oleander, pink oleander, Thevetia species, Thevetia peruviana, yellow oleander, Thevetia nerifolia, Agrobacterium tumefaciens transformed and induced in a cell culture of Nerium species, or a combination thereof.

[36] An oleandrin-containing extract produced by the method according to any one of

[17] to

[35] above.

[37] The invention described in any one of [1] to

[36] above, wherein the extract does not contain pharmacologically active polysaccharides (and / or monosaccharides) obtained in SbCLE.

[38] A dietary supplement composition comprising oleandrin and one or more dietary ingredients and / or food additives.

[39] A cosmetic composition comprising oleandrin and one or more cosmetically acceptable ingredients.

[40] An SbCL extract as described herein.

[41] A method for producing an SbCL extract as described herein.

[42] A pharmaceutical composition described herein.

[43] A dosage form as described herein.

[44] An oleandrin-containing composition described herein.

[45] A combination composition described herein.

[46] A binary extract combination composition described herein.

[47] The method of treatment described herein.

[48] ​​A dietary supplement composition as described herein.

[49] A cosmetic composition described herein.

Claims

1. 1. An oleandrin-containing combination composition comprising a portion of an oleandrin-containing organic solvent extract and a portion of an oleandrin-containing SbCL (subcritical fluid) extract, The organic solvent extract is prepared by extraction with alcohol or alcohol and water; The oleandrin-containing combination composition, wherein the SbCL extract is prepared by extraction with carbon dioxide or carbon dioxide and alcohol.

2. 2. The combination composition of claim 1, wherein: a) the compositional profile of extracted volatile and semi-volatile components in the organic solvent extract is different from the compositional profile of said components in the subcritical fluid extract; and / or b) the subcritical fluid extract contains a higher proportion of extracted volatile and semi-volatile components relative to oleandrin content than the organic solvent extract.

3. 2. The combination composition of claim 1, wherein the composition comprises oleandrin and one or more compounds selected from the group consisting of one or more cardiac glycosides, one or more glycocone components of cardiac glycosides, glucosides, fructosides, glucuronides, one or more aglycones, one or more glycoside precursors, cardenolides, cardadienolides, cardatrienolides, one or more steroids, one or more triterpenes, one or more monosaccharides, one or more polysaccharides, one or more alkaloids, one or more proteins, fats, one or more other non-cardiac glycosides that are therapeutically active agents, and any combination thereof.

4. A pharmaceutical composition comprising at least one pharmaceutical excipient and the combination composition of claim 1.

5. 5. A pharmaceutical composition according to claim 4 for use in a method for treating or preventing a condition, disease or disorder which is therapeutically responsive to oleandrin, comprising: The condition, disease or disorder is a cancer, a tumor, a carcinoma, a sarcoma, a glioblastoma, an adenocarcinoma, a lymphoma, a melanoma, a brain tumor, a glioma, an astrocytoma, an ependymoma, an oligodendroglioma, excessive cell proliferation, a malignant neoplastic disease, a non-cancerous inflammatory skin disease, actinic keratosis, psoriasis, eczema, a neurological disease, a neurological disorder, a neurodegenerative disease, a neurodegenerative disorder, a neurological disease or disorder with an etiology associated with altered Na,K-ATPase activity, Alzheimer's disease , Parkinson's disease, Huntington's disease, stroke, dementia, amyotrophic lateral sclerosis, bovine spongiform encephalopathy, multiple sclerosis, diabetic neuropathy, autism and juvenile neuronal ceroid lipofuscinosis, tauopathy, Down's syndrome, Pick's disease, corticobasal degeneration, some variants of prion diseases, progressive supranuclear palsy, frontotemporal dementia, hemorrhoids, ulcers, leprosy, snake bites, warts, and viral infections; A pharmaceutical composition, wherein the method comprises administering to a subject in need thereof one or more doses of said pharmaceutical composition, thereby treating or preventing said condition, disease or disorder.

6. 2. A combination composition according to claim 1 for use in a method for treating or preventing a condition, disease or disorder which is therapeutically responsive to oleandrin, comprising: The condition, disease or disorder is a cancer, a tumor, a carcinoma, a sarcoma, a glioblastoma, an adenocarcinoma, a lymphoma, a melanoma, a brain tumor, a glioma, an astrocytoma, an ependymoma, an oligodendroglioma, excessive cell proliferation, a malignant neoplastic disease, a non-cancerous inflammatory skin disease, actinic keratosis, psoriasis, eczema, a neurological disease, a neurological disorder, a neurodegenerative disease, a neurodegenerative disorder, a neurological disease or disorder with an etiology associated with altered Na,K-ATPase activity, Alzheimer's disease , Parkinson's disease, Huntington's disease, stroke, dementia, amyotrophic lateral sclerosis, bovine spongiform encephalopathy, multiple sclerosis, diabetic neuropathy, autism and juvenile neuronal ceroid lipofuscinosis, tauopathy, Down's syndrome, Pick's disease, corticobasal degeneration, some variants of prion diseases, progressive supranuclear palsy, frontotemporal dementia, hemorrhoids, ulcers, leprosy, snake bites, warts, and viral infections; A combination composition, wherein the method comprises administering to a subject in need thereof one or more doses of the combination composition, thereby treating or preventing the condition, disease or disorder.

7. The SbCL extract is subjecting the oleandrin-containing biomass to subcritical fluid extraction (SbCLE) using a subcritical fluid (SbCL) comprising carbon dioxide for a time sufficient to extract the oleandrin and form an extraction environment; Separating the biomass from the extraction environment to provide oleandrin-containing SbCL; and removing SbCL from the oleandrin-containing SbCL to provide the oleandrin-containing extract (OCE); 2. The combination composition of claim 1 produced by a process comprising:

8. 8. The combination composition of claim 7, wherein the method further comprises one or more of the following steps: a) separating the biomass from the extraction environment by filtering the extraction environment; b) adding a modifier to the carbon dioxide or extraction environment; c) heating and pressurizing the interior of a vessel containing the extraction environment to form a subcritical liquid phase extraction environment; d) removing the SbCL from the oleandrin-containing SbCL by evaporating the SbCL to form a raw SbCL extract; e) diluting the raw SbCL extract with an organic solvent or an aqueous organic solvent; f) filtering the diluted raw or crude SbCL extract one or more times; g) treating the diluted raw or crude SbCL extract with activated carbon; h) removing most of the organic solvent from the diluted raw extract to form; and / or i) sterile filtering the raw or crude extract to obtain a SbCL extract.

9. 8. The combination composition of claim 7, wherein the SbCL comprises carbon dioxide and one or more modifiers.

10. 10. The combination composition of claim 9, wherein the one or more modifiers is an alcohol.

11. 8. The combination composition of claim 7, wherein the carbon dioxide (and modifier, if present) is in the subcritical liquid phase for about 50% or more of the extraction time.

12. 12. The combination composition of claim 11, wherein the extraction environment comprising the extraction fluid (carbon dioxide and, if present, modifier) ​​and biomass in the subcritical liquid phase remains in liquid form for at least 60% of the extraction time.

13. CO 2 With respect to the phase diagram of SbCLE, a) the critical pressure (p c ) below the critical temperature (t c ) and above the liquid-vapor line; b) t c In the following, and p c below and above the liquid-vapor line; c That's it, t c , above the liquid-vapor line, and below the solid-liquid line; or d) below the solid-liquid line, the liquid-vapor line, the liquid-supercritical line, the triple point (solid-liquid-vapor) temperature (t tp ), triple point pressure (p tp ), critical point (liquid-gas-supercritical) temperature (t cp ), and the critical point pressure (p cp 8. The combination composition of claim 7, wherein the temperature and pressure fall within the liquid region bounded by

14. 8. The combination composition of claim 7, wherein the SbCLE is carried out at a temperature of about 0° C. to about 31° C. and a pressure of about 5.2 bar to about 3000 bar.

15. 8. The combination composition of claim 7, wherein the mass ratio of extraction liquid comprising carbon dioxide and optionally further comprising a modifier to biomass ranges from about 20:1 to about 100:1 based on the mass of both the solvent and feedstock.

16. 10. The combination composition of claim 9, wherein the weight ratio of carbon dioxide to modifier is in the range of about 100: about 0.01-20.

17. 8. The combination composition of claim 7, wherein a) the biomass is dehydrated, dehumidified, freeze-dried, and / or desiccated before being extracted; b) the biomass is dried before being extracted; c) the biomass is not dehydrated before being extracted; or d) the biomass is not dried before being extracted.

18. 1. A method for producing an oleandrin-containing combination composition, comprising: performing an organic solvent extraction on the oleandrin-containing first biomass by forming an extraction environment to provide an oleandrin-containing organic solvent extract; subjecting the oleandrin-containing second biomass to subcritical fluid (SbCL) extraction (SbCLE) to provide an oleandrin-containing SbCL extract; and combining the oleandrin-containing organic solvent extract with the oleandrin-containing SbCL extract to provide the oleandrin-containing combination composition. The method includes:

19. a) the organic solvent extraction is performed with an organic solvent or an aqueous organic solvent; b) the organic solvent or the aqueous organic solvent comprises an alcohol; c) the organic solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, chloroform, methylene chloride, ethyl acetate, and combinations thereof; d) the organic solvent extraction comprises extracting the oleandrin-containing biomass one or more times with an organic solvent or an aqueous organic solvent; e) the aqueous organic solvent is up to about 20% w / w, about 5% 20. The method of claim 18, wherein the organic solvent extraction is carried out at a temperature of about 0° C. to about 75° C., about 5° C. to about 70° C., about 10° C. to about 60° C., about 10° C. to about 50° C., about 10° C. to about 45° C., or about 20° C. to about 40° C.; g) the v / w ratio of organic solvent to biomass or v / w ratio of aqueous organic solvent to biomass is in the range of about 50-1:about 1, about 40-1:about 1, about 30-1:about 1, about 20-1:about 1, about 20-5:about 1, or about 10:about 1.

20. 20. The method of claim 18, wherein the organic solvent extraction further comprises one or more of the following: a) separating the biomass from the extraction environment by filtering the extraction environment to form an extract; b) adding an aqueous liquid to the organic solvent or extraction environment; c) reducing the content of organic solvent from the extract; d) treating the extract with activated carbon; e) filtering the extract one or more times; f) adding additional organic solvent to the extract; g) sterile filtering the organic solvent extract; and / or h) adding water to the extract to form an aqueous extract and extracting the aqueous extract with an organic solvent.

21. a) the moisture content in the biomass prior to extraction is about 20 wt% or less; b) the particle size d0.5 of the biomass prior to extraction is less than about 1 inch, less than about 0.75 inch, less than about 0.5 inch, less than about 0.4 inch, less than about 0.3 inch, less than about 0.2 inch, less than about 0.1 inch, less than about 0.05 inch, or less than about 0.01 inch, said particles being in terms of ASTM sieve mesh opening size; and / or c) the oleandrin-containing biomass is selected from the group consisting of Nerium species, Nerium oleander, Nerium oleander L, Nerium odorum, Nerium oleander L ...

16. The combination composition of claim 15, which is selected from the group consisting of Agrobacterium tumefaciens transformed and induced in a cell culture of Thevetia spp., Thevetia peruviana, yellow oleander, Thevetia nerifolia, and a combination thereof.

22. 2. The combination composition of claim 1, wherein the composition comprises at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, canelosin, canelodione, oleandrigenin, nerium F, neritaloside, odroside A, odroside H, adinelin, odroside-G-acetate, and gitoxigenin.

23. 23. The combination composition of claim 22, wherein a) in the organic solvent extract, the relative content of compounds is cannelosin>canelodione>oleanolic acid>ursolic acid~oleandrin~betulinic acid>oleandrigenin~nerium F, odoroside A, odoroside H~adineline~odrioside-G-acetate>gitoxigenin; and b) in the SbCL extract, the relative content of compounds is oleanolic acid>ursolic acid~oleandrin~betulinic acid>oleandrigenin~nerium F>odoreoside A, odoroside H~adineline~odrioside-G-acetate>cannelosin.

24. 23. The combination composition of claim 22, further comprising one or more of neriin, folinellin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, azinerigenin, deacetyloleandrin, odorioside-G-acetate, and / or quercetin.

25. 23. The combination composition of claim 22, further comprising one or more polyphenols, one or more carbohydrates, one or more flavonoids, one or more amino acids, one or more soluble proteins, one or more celluloses, one or more starches, one or more alkaloids, one or more saponins, one or more tannins, or combinations thereof.

26. 23. The combination composition of claim 22, wherein: a) in an organic solvent extract produced by ethanol extraction, the relative contents of the components are: total free amino acids > total sugars > total cellulose > total alkaloids > total polyphenols to flavonoids > total starch > total crude protein; b) in an organic solvent extract produced by aqueous ethanol extraction, the relative contents of the components are: total free amino acids > total cellulose > total alkaloids > total sugars > total polyphenols to flavonoids > total starch > total crude protein; or c) in an SbCL extract, the relative contents of the components are: total free amino acids > total starch > total alkaloids > total cellulose > total flavonoids > total sugars > total polyphenols > total crude protein.

27. The combination composition of claim 1 , wherein the composition is comprised in a pharmaceutical composition, a nutraceutical composition, a cosmetic composition, or a dosage form.

28. a) the moisture content in the biomass prior to extraction is about 20 wt% or less; b) the particle size d0.5 of the biomass prior to extraction is less than about 1 inch, less than about 0.75 inch, less than about 0.5 inch, less than about 0.4 inch, less than about 0.3 inch, less than about 0.2 inch, less than about 0.1 inch, less than about 0.05 inch, or less than about 0.01 inch, said particles being in terms of ASTM sieve mesh opening size; and / or c) the oleandrin-containing biomass is selected from the group consisting of Nerium species, Nerium oleander, Nerium oleander L, Nerium odorum, Nerium oleander L ...

10. The combination composition of claim 7, wherein the Agrobacterium tumefaciens transformed and induced in a cell culture of Thevetia spp., Thevetia peruviana, Yellow oleander, Thevetia nerifolia, Agrobacterium tumefaciens transformed and induced in a cell culture of Thevetia spp., or a combination thereof.

29. 20. An oleandrin-containing combination composition produced by the method of claim 18.

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