Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/60—Sugars; Derivatives thereof
  • A61K8/602—Glycosides, e.g. rutin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9783—Angiosperms [Magnoliophyta]
  • A61K8/9789—Magnoliopsida [dicotyledons]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2250/00—Food ingredients
  • A23V2250/30—Other Organic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
  • A61K2236/30—Extraction of the material
  • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

• the present invention concerns an improved method for the preparation of an extract comprising oleandrin.
• the invention also provides an improved oleandrin- containing composition comprising at least two different oleandrin-containing extracts.
• Pharmaceutical compositions and nutraceutical compositions containing the same are also provided. Methods of use for the treatment of cardiac glycoside-responsive diseases, conditions or disorders are also provided.
• Nerium oleander a member of the Nerium species, is an ornamental plant widely distributed in subtropical Asia, the southeastern United States, and the Mediterranean.
• oleandrin which has been proposed for use, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, neurological disorders, cancer, tumor, excessive cell proliferation, neurodegeneration, Alzheimer’s disease, Parkinson’s disease, stroke, neurological disorders, Huntington’s disease, inflammation, and viral infection.
• the neuroprotective activity of oleandrin is disclosed in WO 2011085307 A1, US 8481086 B2, US 9220778 B2, US 9358293 B2, US 9877979 B2, and US 10383886 B2.
• the anticancer (anti-excessive cell proliferation) activity oleandrin is disclosed in WO 2007016176A2, WO 2009064657 A1, US 7402325 B2, US 8187644 B2, US 8394434 B2, US 8367363 B2, US 9494589 B2, and US 9846156 B2.
• the antiviral activity of oleandrin is disclosed in US 10596186, WO 2018/053123A1, and WO2019/055119A1.
• Zibbu et al. J. Chem. Pharm. Res. (2010), 2(6), 351-358 provide a brief review on the chemistry and pharmacological activity of Nerium oleander.
• ANVIRZELTM (US 5,135,745 to Ozel) contains the concentrated form or powdered form of the hot-water extract of Nerium oleander.
• Muller et al. disclose the results regarding the analysis of a water extract of Nerium oleander. The extraction of the plant Nerium oleander involves slicing the leaves, cooking the sliced leaves and stems of the plant in water for 2-3 hours and filtering off the residues. The mixture is heated again.
• the aqueous extract reportedly contains several polysaccharides with molecular weights varying from 2KD to 30KD, oleandrin and oleandrigenin, odoroside and neritaloside. They report that the polysaccharide present is primarily galacturonic acid. Other saccharides include rhamnose, arabinose and galactose. Other saccharides include rhamnose, arabinose and galactose. Polysaccharide content and individual sugar composition of polysaccharides within the hot water extract of Nerium oleander have also been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001) vol 1, pp.1-16).
• ANVIRZELTM Compositional analysis of ANVIRZELTM, the hot water extract, was described by Newman et al. (Anal. Chem. (2000), 72(15), 3547-3552).
• U.S. Patent No. 5,869,060 to Selvaraj et al. pertains to extracts of Nerium species and methods of production. To prepare the extract, plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterilized by filtration. The resultant extract can then be lyophilized to produce a powder.
• U.S. Patent No. 6,565,897 U.S. Pregrant Publication No.20020114852 and PCT International Publication No.
• WO 2000/016793 to Selvaraj et al. discloses a hot-water extraction process for the preparation of a substantially sterile water extract.
• Ishikawa et al. J. Nutr. Sci. Vitaminol. (2007), 53, 166-173 discloses a hot water extract of Nerium oleander and fractionation thereof by liquid chromatography using mixtures of chloroform, methanol, and water. They also report that extracts of the leaves of N. oleander have been used to treat Type II diabetes.
• US20060188585 published Aug.24, 2006 to Panyosan discloses a hot water extract of N. oleander.
• US 10323055 issued June 18, 2019 to Smothers discloses a method of extracting plant material with aloe and water to provide an extract comprising aloe and cardiac glycoside.
• US20070154573 published July 5, 2007 to Rashan et al. discloses a cold-water extract of Nerium oleander and its use. Methods to enhance the relative content of oleandrin from plant material are therefore warranted. While hot water extracts of Nerium oleander may provide oleandrin and related cardiac glycosides in relatively low yield, an improved method for obtaining a concentrated form of cardiac glycosides including oleandrin is needed. [006] Erdemoglu et al. (J. Ethnopharmacol. (2003) Nov.
• 89(1), 123-129) discloses results for the comparison of aqueous and ethanolic extracts of plants, including Nerium oleander, based upon their anti-nociceptive and anti-inflammatory activities.
• Fartyal et al. discloses results for the comparison of methanol, aqueous, and petroleum ether extracts of Nerium oleander based upon their antibacterial activity.
• Organic solvent extracts of Nerium oleander are also disclosed by Adome et al. (Afr. Health Sci. (2003) Aug.3(2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt Soc.
• Nerium odorum SOL Nerium oleander Linn
• water, organic solvent, or aqueous organic solvent wherein the leaves are heated to 60°-170°C and then extracted, and the organic solvent is methanol, ethanol, propyl ether or chloroform.
• SCF supercritical fluid
• the SCF extract has demonstrated efficacy in treating some neurological disorders (US 8481086, US 9220778, US 9358293, US 20160243143A1, US 9877979, US 10383886), some cell-proliferative disorders (US 8367363, US 9494589, US 9846156), and some viral infections (US 10596186, WO 2018053123A1, WO2019055119A1).
• Addington et al. disclose use of PBI- 05204 for the treatment of neurological conditions. Addington et al.
• PBI-04711 a triterpene-containing fraction
• PBI-05204 comprises cardiac glycoside (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA)) as the primary 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 it does not contain OL.
• the molar ratio of OA:UA:BA in PBI-04711 is about 3:2.2:1.
• Oleandrin may also be obtained from extracts of suspension cultures derived from Agrobacterium tumefaciens-transformed calli and from extracts of agrobacterium (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). [0010] Oleandrin may also be obtained from extracts of Nerium oleander microculture in vitro, whereby shoot cultures can be initiated from seedlings and/or from shoot apices of the Nerium oleander cultivars, e.g.
• Extracts of Nerium species are known to contain many different classes of compounds: cardiac glycosides, glycones, aglycones, steroids, triterpenes, polysaccharides, saccharides, alkaloids, fat, proteins, and others.
• Specific compounds include oleandrin, neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-ene-3 ⁇ ,28-diol), 28-norurs-12-en-3 ⁇ -ol, urs-12-en-3 ⁇ -ol, 3 ⁇ ,3 ⁇ - hydroxy-12-oleanen-28-oic acid, 3 ⁇ ,20 ⁇ -dihydroxyurs-21-en-28-oic acid, 3 ⁇ ,27- dihydroxy-12-ursen-28-oic acid, 3 ⁇ ,13 ⁇ -dihydroxyurs-11-en-28-oic acid, 3 ⁇ ,12 ⁇ - dihydroxyoleanan-28,13 ⁇ -olide, 3 ⁇ ,27-dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-cou
• compositional profile of said extracts varies widely according to the method of extraction employed. For example, extracts of Nerium plant material obtained using hot methanol, hot ethanol, hot water, cold water, hot water, or supercritical carbon dioxide exhibit substantially different compositional profiles, especially as indicated by respective HPLC chromatograms. Accordingly, one cannot predict a priori the compositional profile of an extract prepared by a new type of process.
• the therapeutic activity profile of such extracts will also vary according to the compositional profile of the extract, when compared on a total weight to total weight basis.
• Objects of the invention include providing an improved process for production of oleandrin-containing extract, and an improved process for production of oleandrin. Another object of the invention includes providing an oleandrin-containing extract and an oleandrin-containing composition. Another object of the invention provides a sublingual or buccal dosage form comprising the extract (or composition) and at least one pharmaceutical excipient. Another object of the invention is to provide pharmaceutical compositions, dosage forms, cosmetics, and nutraceutical products containing oleandrin or the extract(s) described herein.
• the improved process(es) provide(s) a) a higher yield of oleandrin per kg of plant biomass as compared to a supercritical fluid (SCF) extraction (SCFE)-based process; b) an oleandrin-containing composition with a higher ratio of oleandrin to other components as compared to a SCFE process; c) a dual extract composition comprising an organic solvent extract and a subcritical extract; d) an organic solvent extract with a higher content of oleandrin as compared to a SCFE process obtained from the same amount of plant material; e) a subcritical CO 2 extract with a higher content of oleandrin as compared to a SCF extract obtained using the same solvent(s) and same amount of plant material.
• SCF supercritical fluid
• SCFE supercritical fluid
• the organic solvent comprises a) one or more organic solvents; or b) water and one or more organic solvent extracts.
• the water may further comprise one or more salts, one or more buffers, or a combination thereof.
• An aspect of the invention provides a method of producing oleandrin-containing extract, the method comprising subjecting oleandrin-containing biomass to subcritical liquid extraction (SbCLE) employing subcritical liquid (SbCL) comprising carbon dioxide, optionally further comprising organic solvent (modifier), for a period of time sufficient to extract the oleandrin and form extraction milieu; separating said biomass from said extraction milieu to provide oleandrin-containing SbCL; and removing SbCL from said oleandrin-containing SbCL to provide said oleandrin-containing (OC) extract (OCE).
• SbCLE subcritical liquid extraction
• SbCL subcritical liquid
• OBE organic solvent
• An aspect of the invention provides a method of producing oleandrin, the method comprising subjecting oleandrin-containing biomass to subcritical liquid extraction (SbCLE) employing subcritical liquid (SbCL) comprising carbon dioxide, optionally further comprising organic solvent (modifier), for a period of time sufficient to extract the oleandrin to form extraction milieu; separating said biomass from said extraction milieu to provide oleandrin-containing SbCL; removing SbCL from said oleandrin-containing SbCL to provide oleandrin-containing extract; and isolating oleandrin from said extract.
• SBCLE subcritical liquid extraction
• SbCL subcritical liquid
• modifier organic solvent
• Another aspect of the invention provides a method of producing a combination composition, the method comprising subjecting oleandrin-containing first biomass to organic solvent extraction to provide an oleandrin-containing organic solvent extract; subjecting oleandrin-containing second biomass to subcritical liquid (SbCL) extraction (SbCLE) to provide an oleandrin-containing SbCL extract; and combining said oleandrin-containing organic solvent extract and said oleandrin-containing SbCL extract to provide said combination composition.
• SbCL subcritical liquid
• the combination composition comprises at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, kanerocin, kanerodione, oleandrigenin, Nerium F, neritaloside, odoroside (A and H), adynerin, odoroside-G-acetate, and gitoxigenin.
• the combination composition may further comprise one or more of neriin, folinerin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, adynerigenin, deacetyloleandrin, odoroside G acetate, and/or quercetin.
• the composition (or the extract) may further comprise polyphenol(s), carbohydrate(s), flavonoid(s), amino acid(s), soluble protein(s), cellulose, starch, alkaloid(s), saponin(s), tannin(s), and any combination thereof.
• the amino acid can 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.
• 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 invention provides a cosmetic (cosmeceutical) product or a nutraceutical product comprising oleandrin, oleandrin-containing composition, oleandrin- containing combination composition, or oleandrin-containing extract(s) as defined herein.
• the cosmetic product may further comprise one or more cosmetic excipients.
• the cosmetic product can be a cleanse, 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, towelette, deodorant, bath oil, bubble bath, bath salt, body butter, lipstick, lip gloss, lip liner, concealer, rouge, facial mask treatment, or any other type of cosmetic product.
• a nutraceutical can include a dietary ingredient, dietary supplement, bioceutical, or food additive such as may be defined by the USFDA or other regulatory agencies.
• the nutraceutical product may further comprise one or more nutraceutical or dietary ingredients.
• the nutraceutical may include a liquid, solid, powder, cream, solution, suspension, dosage form, gel, capsule, tablet, pill, prebiotic, probiotic, functional food, meal supplement, meal replacement, or other such products.
• the invention also provides an improved oleandrin-containing combination composition comprising a portion of oleandrin-containing organic solvent extract and a portion of oleandrin-containing SbCL extract.
• a) substantially equal portions of the two extracts are present in the combination composition; b) the content of organic solvent extract in said combination is higher than the content of SbCL extract; or c) the content of SbCL extract in said combination is higher than the content of organic solvent extract.
• the combination composition advantageously comprises a greater range of phytochemicals and other components, aside from oleandrin, extractable from the plant material. It is known that one or more of those other components are also therapeutically effective (pharmacologically active) ingredients.
• Embodiments of the invention include those wherein a) the organic solvent extract comprises 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 said molar ratio in the subcritical liquid extract; c) the SbCL extract comprises at least oleandrin and other components extracted from the biomass; d) the content of oleandrin in the ethanolic extract is greater than the content of oleandrin in the subcritical liquid extract; e) the content of extracted non-oleandrin components in the organic solvent extract is greater than the content of said components in said subcritical liquid extract; f) the compositional profile of extracted volatile and semi-volatile components in said organic solvent extract is different than the compositional profile of said components in said subcritical liquid extract; and/or g) relative to oleandrin content, the subcritical liquid extract comprises a greater proportion of extracted volatile and semi- volatile components than does the organic solvent extract.
• the first and second biomass may be the same or different.
• the first biomass extracted with organic solvent may be used as the second biomass; alternatively, the first biomass is extracted with organic solvent, and a different second biomass is extracted with subcritical liquid.
• Embodiments of the invention include those wherein the subcritical liquid extraction process further comprises one or more of the following: a) separating said biomass from the extraction milieu by filtering the extraction milieu; b) adding modifier to the carbon dioxide or extraction milieu; c) heating and pressurizing the interior of a vessel containing the extraction milieu to form subcritical liquid phase extraction milieu; d) removing said SbCL from said oleandrin-containing SbCL by volatilizing said SbCL to form raw SbCL extract (SbCLE); e) diluting the raw SbCL extract with organic solvent or 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
• the SbCL extract is optionally further diluted with organic solvent or aqueous organic solvent.
• the organic solvent extract is optionally further diluted with organic solvent or aqueous organic solvent.
• the SbCL extract may comprise 0.1 mg to 1 mg or 0.5 to 0.8 mg of oleadrin per mL of extract.
• Embodiments of the invention include those wherein the carbon dioxide (along with any modifier, if any is present) is in the subcritical liquid phase during about 50% or more of the extraction time period, i.e. about 50% or more of the total time for the “subjecting” step.
• the extraction milieu comprising extraction fluid (carbon dioxide along with any modifier, if any is present) in the subcritical liquid phase and biomass, 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 period. In some embodiments, the extraction milieu will remain in liquid form throughout the extraction period.
• the SbCLE is conducted such that the extraction milieu (comprising biomass and CO 2 , and optionally further comprising organic solvent (modifier)) is under the following conditions, with respect to the phase diagram of CO 2 , during about 50% or more of the extraction time period: a) at or below the critical pressure (p c ), at or above the triple point (solid-liquid-gas) temperature (t tp ), below the critical temperature (t c ), and above the liquid-gas phase line; b) at or below the t c , at or above the t tp , below the p c , and above the liquid-gas phase line; c) at or above the p c , below the t c , at or above the t tp , above the liquid- gas phase line, and below the solid-liquid phase line; or d) at a temperature and pressure falling within the liquid region bounded by the solid-liquid phase line, the liquid-gas phase line, the liquid-supercritical phase line
• Embodiments of the invention include those wherein SbCLE is conducted at a temperature of about 0°C up to about 31°C and a pressure of about 5.2 bar to about 3000 bar. Other embodiments of the invention include those wherein SbCLE is conducted at a temperature of about 0°C up to about 31°C and a pressure of about 5.2 bar to about 1000 bar. In preferred embodiments, SbCLE is conducted 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 conducted at a temperature of about 5-10 °C and a pressure of about 120-130 bar.
• the SbCLE is conducted 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.
• the SbCL further comprises one or more modifiers, e.g. organic solvent(s).
• the weight 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.
• the one or more modifiers may be independently selected upon each occurrence from alcohol, organic solvent, alkyl ketone, alkyl ester, water.
• Embodiments of the invention include those wherein a) said organic solvent extraction is conducted with organic solvent or aqueous organic solvent; b) independently upon each occurrence, said organic solvent or aqueous organic solvent comprises alcohol; c) independently upon each occurrence, said organic solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, chloroform, methylene chloride, ethyl acetate, and a combination thereof; d) said organic solvent extraction comprises extracting said oleandrin-containing biomass with organic solvent or aqueous organic solvent one or more times; e) independently upon each occurrence, said a
• Embodiments of the invention include those wherein the organic solvent extraction further comprises one or more of the following: a) separating said biomass from the extraction milieu by filtering the extraction milieu to form crude extract; b) adding aqueous liquid to the organic solvent or extraction milieu; c) reducing the content of organic solvent from said 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 organic solvent.
• the organic solvent extract is optionally further diluted with organic solvent or aqueous organic solvent.
• the organic solvent extract may comprise 0.1 to 10 mg/mL, 0.5 to 8 mg/mL, 1 to 8 mg/mL, 1.5 to 8 mg/mL, or 1.5 to 5 mg/mL (mg of oleandrin per mL of extract).
• Embodiments of the invention include those wherein the oleandrin-containing biomass is Nerium sp., Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odourum, Nerium indicum, white oleander, pink oleander, Thevetia sp., Thevetia peruviana, yellow oleander, Agrobacterium tumefaciens, or a combination thereof.
• the biomass comprises leaves, stems, flowers, bark, fruits, seeds, sap, cultured cellular mass, and/or pods.
• Embodiments of the invention include those wherein a) independently upon each occurrence, the biomass is dehydrated, dehumidified, freeze-dried, and/or desiccated prior to extraction; b) independently upon each occurrence, the biomass is dried prior to extraction; c) independently upon each occurrence, the biomass is not dehydrated prior to extraction; d) independently upon each occurrence, the biomass is not dried prior to extraction; or e) independently upon each occurrence.
• the water content in the biomass is preferably about 20 wt % or less, about 15 wt % or less, about 10 wt % or less, about 8 wt % or less, about 5 wt % or less, about 2.5 wt % or less, or about 1 wt % of less, prior to extraction. Accordingly, the method of the invention also includes embodiments further comprising dehydrating, drying or desiccating the biomass prior to extraction.
• Embodiments of the invention include those wherein a) prior to extraction, the biomass has an average particle size (d0.5) of less than about 1”, less than about 0.75”, less than about 0.5”, less than about 0.4”, less than about 0.3”, less than about 0.2”, less than about 0.1”, less than about 0.05”, or less than about 0.01”, said particle being with respect to ASTM sieve mesh opening size.
• Embodiments of the invention include those wherein a) the content of oleandrin in said extract is about 0.01 wt % or higher, about 0.05 wt % or higher, about 0.1 wt % or higher, about 0.2 wt % or higher, about 0.5 wt % or higher, about 1 wt % or higher, about 2.5 wt % or higher, about 5 wt % or higher, about 10 wt % or higher, about 20 wt % or higher, about 30 wt % or higher, about 40 wt % or higher, about 50 wt % or higher, about 60 wt % or higher, about 70 wt % or higher, about 80 wt % or higher, or about 90 wt % or higher; b) the content of oleandrin in said extract is about 99 wt % or lower, about 95 wt % or lower, about 90 wt % or lower, about 80 w
• Embodiments of the invention include those wherein the process further comprises one or more of the following: a) removing (or reducing the amount of) volatile organic compounds (VOC’s) from said extract; b) removing (or reducing the amount of) semi-volatile organic compounds (SVOC’s) from said extract; c) filtering said extract; d) said separating is conducted by filtration, centrifugation, decantation, or a combination thereof.
• VOC volatile organic compounds
• SVOC semi-volatile organic compounds
• Embodiments of the invention include those wherein said isolating is achieved 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.
• the individual steps of any of the methods of the invention can be conducted at separate facilities or within the same facility.
• Pharmaceutical compositions comprising oleandrin as the sole active ingredient are within the scope of the invention.
• Compositions comprising oleandrin and plural active ingredients obtained from said biomass are within the scope of the invention.
• compositions comprising oleandrin and plural triterpenes as the active ingredients are within the scope of the invention.
• said 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 as described herein.
• a triterpene is independently selected upon each occurrence from its free acid form, salt form, deuterated form and derivative form.
• the extract may comprise one or more other compounds (which may or may not be pharmacologically active, therapeutically effective) extracted from the biomass during the extraction process.
• the one or more other compounds may or may not contribute to the therapeutic efficacy of oleandrin when administered to a subject receiving both.
• the one or more other compounds can be selected from the group consisting of cardiac glycoside, one or more glycone constituents of cardiac glycosides (such as glucoside, fructoside, and/or glucuronide), one or more aglycones, one or more glycoside precursors (such as cardenolides, cardadienolides and cardatrienolides), one or more steroids, one or more triterpenes, one or more saccharides, one or more polysaccharides, one or more alkaloids, one or more proteins, fat, one or more other non-cardiac glycoside therapeutically effective agent, and any combination thereof.
• cardiac glycoside such as glucoside, fructoside, and/or glucuronide
• one or more aglycones such as cardenolides, cardadienolides and cardatrienolides
• one or more steroids such as cardenolides, cardadienolides and cardatrienolides
• one or more triterpenes such as cardenolides,
• 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.
• the generic terms triterpene and cardiac glycoside also encompass free form, salt form and derivatives thereof, unless otherwise specified.
• the triterpene is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid and salts or derivatives thereof.
• 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 nonvolatile organic compounds; or d) any combination thereof.
• the extract excludes a pharmacologically active polysaccharide (and/or saccharide) obtained during extraction.
• the extract comprises oleandrin and at least one pharmacologically active polysaccharide (and/or saccharide) obtained during extraction.
• the pharmacologically active polysaccharide may be an acidic homopolygalacturonan or arabinogalaturonan.
• the extract comprises at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, kanerocin, kanerodione, oleandrigenin, Nerium F, neritaloside, odoroside (A and H), adynerin, odoroside-G-acetate, and gitoxigenin.
• the combination composition may further comprise one or more of neriin, folinerin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, adynerigenin, deacetyloleandrin, odoroside G acetate, and/or quercetin.
• the extract may further comprise one or more polyphenol(s), one or more carbohydrate(s), one or more flavonoid(s), one or more amino acid(s), one or more soluble protein(s), one or more cellulose(s), one or more starch(es), one or more alkaloid(s), one or more saponin(s), one or more tannin(s), and any combination thereof.
• the amino acid can 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.
• the amino is selected from the group consisting of asparagine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine, tryptophan, and lysine.
• oleandrin may be the primary therapeutically effective compound with respect specific diseases, disorders or conditions.
• Some embodiments of the invention include those wherein a pharmaceutical composition comprises at least one pharmaceutical excipient and the extract.
• the oleandrin, oleandrin-containing extract, or oleandrin-containing composition of the invention can be administered to a subject in need thereof to treat a condition, disease or disorder that is therapeutically responsive to treatment with cardiac glycoside (oleandrin, digoxin, digitoxin, ouabain, bufalin, cinobufatalin, cinobufagin, resibufogenin, lanatoside C, or other therapeutically effective cardiac glycoside), in particular to treatment with oleandrin, or which can be prevented by administration of cardiac glycoside, in particular administration of oleandrin.
• cardiac glycoside oleandrin, digoxin, digitoxin, ouabain, bufalin, cinobufatalin, cinobufagin, resibufogenin, lanatoside C, or other therapeutically effective cardiac glycoside
• An aspect of the invention provides a method of treating or preventing a condition, disease or disorder that is therapeutically responsive to oleandrin (oleandrin- containing composition or oleandrin-containing extract), the method comprising administering to a subject in need thereof one or more doses of oleandrin (oleandrin- containing composition or oleandrin-containing extract), thereby treating or preventing said condition, disease or disorder.
• oleandrin containing composition or oleandrin-containing extract
• Target conditions, diseases and disorders are described herein.
• Another aspect of the invention provides a method of improving or boosting the immunological response of a subject, the method comprising administering to a subject in need thereof one or more doses of oleandrin (oleandrin-containing composition, oleandrin- containing combination composition, or oleandrin-containing extract) in an amount sufficient to boost the subject’s immunological response.
• the one or more doses can be administered as a pharmaceutical composition, nutraceutical composition, and/or cosmeceutical composition.
• Administration can be acute, chronic, or a combination thereof and can occur before and/or after initiation of said condition, disease or disorder. Administration can be to a subject that has or is at risk of getting said condition, disease or disorder.
• the oleandrin, oleandrin-containing extract, oleandrin-containing composition, or oleandrin-containing combination composition of the invention can be included in any pharmaceutical composition, nutraceutical composition, cosmeceutical compositions, or dosage form as described herein.
• the oleandrin, oleandrin-containing extract, or oleandrin-containing composition of the invention can be administered locally, systemically or a combination thereof. Suitable modes of administration are described herein.
• oleandrin is present as the primary therapeutic component, meaning the component primarily responsible for therapeutic activity, in a pharmaceutical composition.
• the invention also provides use of oleandrin-containing extract in the manufacture of a medicament for the treatment, in a subject, of a condition, disease or disorder that is therapeutically responsive to cardiac glycoside.
• the manufacture of such a medicament comprises: providing oleandrin-containing extract; including a dose of oleandrin-containing extract in a pharmaceutical dosage form; and packaging the pharmaceutical dosage form.
• the manufacture can be conducted as described in PCT International Application No. PCT/US06/29061, the entire disclosure of which is hereby incorporated by reference.
• the manufacture can also include one or more additional steps such as: delivering the packaged dosage form to a vendor (retailer, wholesaler and/or distributor); selling or otherwise providing the packaged dosage form to a subject having a viral infection; including with the medicament a label and a package insert, which provides instructions on use, dosing regimen, administration, content and toxicology profile of the dosage form.
• the treatment comprises: determining that a subject has a condition, disease or disorder that is therapeutically responsive to cardiac glycoside, e.g.
• oleandrin oleandrin-containing composition or oleandrin-containing extract
• digoxin ouabain
• bufalin cinobufatalin
• cinobufagin resibufogenin
• other therapeutically effective cardiac glycoside indicating administration of pharmaceutical dosage form to the subject according to a dosing regimen; administering to the subject one or more pharmaceutical dosage forms, wherein the one or more pharmaceutical dosage forms is administered according to the dosing regimen.
• the pharmaceutical composition can further comprise a combination of at least one material selected from the group consisting of a water soluble (miscible) co-solvent, a water insoluble (immiscible) co-solvent, a surfactant, an antioxidant, a chelating agent, and an absorption enhancer.
• the solubilizer is at least a single surfactant, but it can also be a combination of materials such as a combination of: a) surfactant and water miscible solvent; b) surfactant and water immiscible solvent; c) surfactant, antioxidant; d) surfactant, antioxidant, and water miscible solvent; e) surfactant, antioxidant, and water immiscible solvent; f) surfactant, water miscible solvent, and water immiscible solvent; or g) surfactant, antioxidant, water miscible solvent, and water immiscible solvent.
• composition(s) of the invention optionally further comprises a) at least one liquid carrier; b) at least one emulsifying agent; c) at least one solubilizing agent; d) at least one dispersing agent; e) at least one other excipient; or f) a combination thereof.
• the water miscible solvent is low molecular weight (less than 6000) PEG, glycol, or alcohol.
• the surfactant is a pegylated surfactant, meaning a surfactant comprising a poly(ethylene glycol) functional group.
• the invention also provides a buccal or sublingual dosage form comprising any extract(s) of the invention as described herein, and at least one pharmaceutical excipient.
• Said dosage form is adapted to deliver components of the extract to the mucosa of the mouth.
• Suitable dosage forms include tablet, wafer, patch, spray, drop, liquid, solution, paste, gel, film, bioadhesive composition, gummy, or powder.
• the combination composition can be included in any type of pharmaceutical dosage form.
• Suitable ones are selected from the group consisting of solid dosage form, liquid dosage form, parenteral dosage form, otic dosage form, ophthalmic dosage form, nasal dosage form, inhalable dosage form, buccal dosage form, sublingual dosage form, enteral dosage form, topical dosage form, oral dosage form, peroral dosage form, injectable dosage form, tablet, capsule, pill, caplet, troche, sache, solution, suspension, dispersion, vial, bag, bottle, injectable liquid, i.v. (intravenous), i.m. (intramuscular) or i.p. (intraperitoneal) administrable liquid.
• injectable liquid i.v. (intravenous), i.m. (intramuscular) or i.p. (intraperitoneal) administrable liquid.
• the dosage form can be a rapid release, immediate release, controlled release, sustained release, prolonged release, extended release, burst release, continuous release, slow release, or pulsed release dosage form, or is a dosage form that exhibits two or more of those types of release.
• the dosage form 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, first order, pseudo-first order or sigmoidal release profile.
• the dosage form a) releases its charge of composition within a period of 0.5 to 1 hours or less; b) is enteric coated and releases its charge of composition downstream of the stomach, such as in the jejunum, ileum, small intestine, and/or large intestine; or c) is enteric coated and releases its charge of composition into the systemic circulation within 1-10 hr after oral administration [0072]
• the inhalable, buccal or sublingual dosage form further comprises at least one bitter taste antagonist (also referred to as a bitter receptor blocker).
• the bitter taste antagonist inhibits the taste receptor activation caused by a bitter compound, such as a cardiac glycoside.
• the bitter taste antagonist inhibits activation of at least one of the receptors of the hTAS2R family of G protein-coupled receptors.
• the bitter taste antagonist can include taste-masking agent(s).
• 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, (-)- hardwickiic acid (2-(fitran-3-yl)ethyl)-trimethyI-hexahydronaphthalene-carboxylic acid), fenchone, borneol, isoborneol, anethole, menthofuran, monosodium glycyrrhizinate, pullulan
• Suitable flavorant(s) include mint, peppermint, menthol, eucalyptus, eucalyptol, chocolate, spearmint, tangerine, orange, grape, grapefruit, marshmallow, coffee, banana crème, caramel, bubble gum, cherry, lemon, lime, strawberry, maple, raspberry, and/or apple.
• Suitable sweetener(s) include magnasweet (110 or 135), sucralose, steviol glycoside(s), stevia, glycerin, acesulfame potassium, PCCA sweetness enhnancer, PCCA bitter stop, di ⁇ d ⁇ fructofuranose 1,2′: 2,3′ ⁇ di ⁇ anhydride, aspartame, neohesperidine dihydrochalcone and hesperidine dihydrochalcone 4′ ⁇ d glucoside, d ⁇ sorbitol, sodium saccharin, sodium gluconate, and/or monosodium glutamate.
• the inhalable, buccal or sublingual dosage form can also include an oil-based excipient such as a fixed oil (e.g.
• the inhalable, buccal or sublingual dosage form may also be taste- masked with taste masking by polymer coating; taste masking by conventional granulation; taste masking with ion ⁇ exchange resins; taste masking by spray congealing with lipids; taste masking by formation of inclusion complexes with cyclodextrins; taste masking by the freeze ⁇ drying process; taste masking by making multiple emulsions; and taste masking with gelatin, gelatinized starch, liposomes, lecithins or lecithin ⁇ like substances, surfactants, salts, or polymeric membranes.
• the inhalable, buccal or sublingual dosage form further comprises one or more permeation enhancers that improve mucosal permeability and absorption of compounds, such as cardiac glycoside(s).
• Suitable permeation enhancer(s) include surfactants, bile salts, fatty acids, cyclodextrins, and chelators.
• FIG.1 depicts a phase diagram for carbon dioxide indicating the solid, liquid, gas and supercritical phases as well as the triple point and critical point temperatures and pressures.
• the solid and liquid phases are separated by the solid-liquid phase line.
• the liquid and gas phases are separated by the liquid-gas phase line.
• the supercritical fluid and liquid phases are separated by the supercritical fluid-liquid phase line.
• the liquid region is thus bounded by the liquid-gas phase line, solid-liquid phase line, supercritical fluid-liquid phase line, triple point (temperature and pressure) and critical point (temperature and pressure).
• FIG. 2 depicts a phase diagram for carbon dioxide indicating the supercritical region and two of the subcritical regions: a subcritical liquid region and a subcritical gas region.
• FIG. 1 depicts a phase diagram for carbon dioxide indicating the supercritical region and two of the subcritical regions: a subcritical liquid region and a subcritical gas region.
• FIG. 3 depicts chromatograms from GC-mass spectrometry analysis, with total ion current (TIC) detection, of the ethanolic extract (top), the subcritical CO 2 extract (middle), and the prior art supercritical fluid extract PBI-05204 (bottom) determined according to Example 18. The data can be used to identify volatile and semi-volatile components of the extract.
• FIG. 4 depicts the HPLC chromatogram of PBI-05204 (prior art) whereby oleandrin is resolved from oleanolic acid, ursolic acid, and betulinic acid.
• the 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 invention also provides pharmaceutical compositions comprising at least one pharmaceutical excipient and said extract(s) or combination composition.
• the invention also provides methods of using said pharmaceutical composition, said extract(s), and/or said combination composition for the treatment and/or prevention of cardiac glycoside-responsive, especially oleandrin- responsive, disease(s), condition(s) or disorder(s).
• the invention also provides methods of administering said extract(s) and composition(s).
• the invention also provides methods of preparing said extract(s) and composition(s).
• a dual extract composition advantageously provides a compositional profile different than that of any single extract composition.
• the compositional profile of a combination composition comprising an ethanolic (or aqueous ethanolic) extract in combination with a subcritical CO 2 (with or without modifier) extract is different and better than the compositional profile of a single extract composition comprising said ethanolic extract, said subcritical CO 2 extract, a hot-water extract, a cold-water extract, or a supercritical CO 2 extract.
• the combination composition is that, relative to the oleandrin content and the content of other components extractable from the biomass, it includes less of said other components, but it still comprises a substantial amount of oleandrin, the desired target pharmacologically active substance in said extracts. Moreover, the combination composition advantageously comprises more non-oleandrin components than any of the individual extracts and some of those non-oleandrin components are exhibit pharmacological activity.
• plant material is defined as a plant or plant part (e.g.
• the plant material comprises leaves, shoots and/or stems.
• Embodiments of the invention include those wherein the oleandrin-containing biomass (plant material) is Nerium sp., Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odourum, Nerium indicum, white oleander, pink oleander, Thevetia sp., 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.
• the oleandrin-containing biomass is Nerium sp., Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odourum, Nerium indicum, white oleander, pink oleander, Thevetia sp., yellow oleander, A
• Nerium oleander can be obtained from microculture in vitro, whereby shoot cultures can be initiated from seedlings and/or from shoot apices of the Nerium oleander cultivars Splendens Giganteum, Revanche or Alsace, or other cultivars (Vila et al., “Micropropagation of Oleander (Nerium oleander L.)” in HortScience (2010), 45(1), 98-102, the entire disclosure of which is hereby incorporated by reference).
• Nerium oleander plant material can be obtained, for example, from commercial plant suppliers such as Aldridge Nursery, Atascosa, Texas.
• Suitable varietals of Nerium oleander include Album (white), Petit Salmon (pink), True Pale, Pink Beauty, Calypso (dark pink), or Red Hardy.
• Oleandrin is taken to mean all known forms of oleandrin unless otherwise specified. Oleandrin can be present in racemic, optically pure, optically enriched form or stereoisomeric form.
• semi-volatile organic compound is organic compounds that possess Henry’s law constants (H) in the range of 1x10 -5 to 3x10 -7 atm*m 3 /mol* (* H range defined as volatility from liquid to air) and demonstrate higher boiling points, usually greater than that of water with correspondingly low vapor pressure from 10 -14 - 10 -4 atm.
• volatile organic compound is any organic compound having an initial boiling point less than or equal to 250° C measured at a standard atmospheric pressure of 101.3 kPa.
• the organic solvent extract and the subcritical liquid extract comprise at least oleandrin, VOCs, and SVOCs, and said extracts can further comprise one or more nonvolatile organic compounds; therefore, a respective combination composition formed with said extracts comprises those components as well.
• the invention provides a method of producing oleandrin or oleandrin-containing extract from oleandrin-containing biomass by a) subcritical liquid (SbCL) extraction (SbCLE) with carbon dioxide, optionally further comprising at least one modifier; b) extraction with organic solvent (OSE), optionally further comprising water; or c) a combination of ScCLE and OSE.
• the extractions can be conducted on the same biomass or on different batches of biomass.
• FIG.1 depicts a phase diagram for carbon dioxide indicating the solid, liquid, gas and supercritical fluid phases.
• SbCLE is conducted in the liquid phase region outside the supercritical fluid phase region (in a phase diagram) for carbon dioxide.
• the liquid phase region is bounded by the solid-liquid phase line, the liquid-gas phase line, the liquid- supercritical phase line, the supercritical fluid-liquid phase line, the triple point (solid- liquid-gas), and the critical point (liquid-gas-supercritical).
• the subcritical extraction liquid primarily comprises CO 2 ; therefore, the determination of subcritical conditions is with respect to the known phase diagram(s) for carbon dioxide. Subcritical conditions are therefore achieved by performing the extraction under the following conditions in the extraction vessel: a) a temperature in the range of about -56.4°C to about 31.1°C or less; b) a pressure at or above 5.1 bar; and/or c) at a subcritical temperature and/or at a subcritical pressure for carbon dioxide, such that the extraction medium is in liquid phase rather than in solid, gas, or supercritical fluid phase.
• the SbCLE is conducted under pressure and temperature conditions wherein the extraction medium (comprising primarily carbon dioxide, and optionally comprising modifier) remains in the subcritical liquid phase rather than the gas phase, solid phase, or supercritical fluid phase.
• the preferred extraction conditions are those wherein 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 phase line. In reference to the phase diagram, the region bounded by those conditions provides a subcritical liquid of higher density, which results in higher extraction efficiency.
• the extraction period is that period of time during which the extraction liquid and biomass are in contact.
• the extraction period, for each extraction cycle typically ranges from 0.1 to 24 h, 0.5 to 12 h, or 0.5 to 6 h.
• the extraction period, for each extraction cycle typically ranges from 0.1 to 24 h, 0.5 to 12 h, or 0.5 to 6 h.
• the subcritical liquid comprises carbon dioxide.
• the subcritical liquid comprises carbon dioxide and at least one modifier.
• the modifier can be alcohol, organic solvent, alkyl ketone, alkyl ester or a combination thereof. Particularly suitable modifiers include ethanol, methanol propanol, acetone, ethyl acetate, methyl ethyl ketone, glycol (e.g.
• the weight ratio of subcritical liquid to biomass may also affect extraction efficiency.
• the weight ratio of subcritical liquid (with or without modifier) to 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 weight of both the solvent and the raw material.
• the extraction fluid will be in subcritical liquid form (in the extraction milieu).
• 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 period. In some embodiments, the extraction liquid will be in subcritical liquid form throughout the extraction period. [0095] Due to equipment performance, it is possible that carbon dioxide extraction of the biomass might occur in the supercritical phase for a short period of time; however, for a majority (about 50% or more) of the extraction period, the extraction will occur at subcritical conditions. [0096] SbCLE of Nerium biomass was conducted according to Example 3. Dried biomass was extracted with subcritical liquid CO 2 (about 90-95 wt %) containing alcohol (ethanol, about 10-5 wt %, respectively).
• the extraction milieu biomass was separated from the subcritical liquid by filtration to provide the oleandrin-containing subcritical liquid (also comprising other extracted components).
• the CO 2 was vented to provide a crude extract which was diluted with ethanol, then frozen, then thawed, then filtered, then concentrated in vacuo, and once again diluted with 50% v/v aqueous ethanol.
• the organic solvent extraction was performed according to Example 15. Dried and powdered Nerium biomass was extracted with aqueous ethanol (containing 10 % v/v of water) three times (three extraction cycles). For each extraction cycle, the extraction milieu was filtered after completion of the extraction period.
• the respective three supernatant were combined and concentrated in vacuo to about 20% of the original volume to form a concentrate, which was sterile filtered, thereby forming the crude ethanolic extract, which was then diluted with 50% v/v/ aqueous ethanol.
• the composition (based upon the content of some known components) of the SbCL extract was compared to the composition of the prior art supercritical fluid (SCF) extract of Nerium oleander (PBI-05204, described above), the prior art hot water extract (ANVIRZELTM), and the organic solvent extract, prepared as described herein.
• SCF supercritical fluid
• oleandrin mg oleandrin per Kg of plant material
• VOC and SVOC mg VOC+SVOC per Kg of plant material
• supercritical extract > ethanolic extract > subcritical extract.
• the content of the individual components may vary by ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10% or ⁇ 5% relative to the values indicated.
• Standards of oleandrin, oleanolic acid, ursolic acid, betulinic acid and derivatives thereof can also be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, MO, USA).
• the partial compositions of the extracts are determined by DART TOF-MS (Direct Analysis in Real Time Time of Flight Mass Spectrometry) on a JEOL AccuTOF- DART mass spectrometer (JEOL USA, Peabody, MA, USA).
• the extract comprises cardiac glycoside(s), glycone(s), aglycone(s), steroid(s), triterpene(s), polysaccharide(s), saccharide(s), alkaloid(s), fat, protein(s), or a combination thereof.
• Specific compounds include oleandrin and other compounds that may be present include neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-ene-3 ⁇ ,28-diol), 28-norurs-12-en- 3 ⁇ -ol, urs-12-en-3 ⁇ -ol, 3 ⁇ ,3 ⁇ -hydroxy-12-oleanen-28-oic acid, 3 ⁇ ,20 ⁇ -dihydroxyurs-21- en-28-oic acid, 3 ⁇ ,27-dihydroxy-12-ursen-28-oic acid, 3 ⁇ ,13 ⁇ -dihydroxyurs-11-en-28-oic acid, 3 ⁇ ,12 ⁇ -dihydroxyoleanan-28,13 ⁇ -olide, 3 ⁇ ,27-dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic
• the volatile and semivolatile compounds found in the ethanolic and/or subcritical extract were tentatively identified as 2, 3- dihydrobenzofuran, Squalene, 2-(Decanoyloxy)propane-1,3-diyl dioctanoate, Alpha tocopherol, Stigmasterol, Gamma-sitosterol, Decanoic acid, 1,2,3-propanetriyl ester, Ursolic aldehyde, catetchol, 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.
• the compositional profiles of the ethanolic extract (top), the subcritical CO 2 extract (middle), and PBI-05204 differ.
• the ethanolic extract has fewer VOCs and SVOCs than the subcritical extract, and the subcritical extract has fewer VOCs and SVOCs than PBI-05204, the supercritical fluid extract.
• the combination composition comprising the ethanolic extract and the subcritical fluid extract thus has fewer VOCs and SVOCs than does PBI-05204.
• compositional profile of the organic solvent extract, the subcritical liquid extract, the combination 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 indicate the combination composition has a compositional profile as would be predicted by mixing equal portions of the ethanolic extract and the SbCL extract and has a compositional profile different than that of PBI-05204.
• the content of oleandrin in each extract is quantified, whereby it is observed that the ethanolic extract has a much greater relative content of oleandrin, on an equal weight basis, than does the SbCL extract.
• composition of the organic solvent extract and SbCL extract differs according to the relative content of individual components therein. Based upon phytochemical analysis of the extracts, the composition of the individual extracts can be defined as follows in terms of the relative content of the classes (types) of components therein.
• compositions further differ based upon the relative content of terpenoids, cardiac glycosides and other components as follows.
• Extract made by ethanolic extraction kanerocin > kanerodione > oleanolic acid > ursolic acid ⁇ oleandrin ⁇ betulinic acid > oleandrigenin ⁇ Nerium F, odoroside (A and H) ⁇ adynerin ⁇ odoroside-G-acetate > gitoxigenin.
• Extract made by SbCL extraction oleanolic acid > ursolic acid ⁇ oleandrin ⁇ betulinic acid > oleandrigenin ⁇ Nerium F > odoroside (A and H) ⁇ adynerin ⁇ odoroside-G-acetate > kanerocin.
• the combination composition may further comprise one or more of the following compounds obtained during extraction of biomass: neritaloside, neriin, folinerin, gitoxigenin, digitoxigenin, nerigoside, rutin, ursonic acid, neridienone A, adynerigenin, deacetyloleandrin, odoroside G acetate, and/or quercetin.
• a subject treated with the SbCL extract, ethanolic extract, or combination composition according to the invention will exhibit a therapeutic response.
• therapeutic response is meant that the subject will enjoy at least one of the following clinical benefits as a result of treatment: reduction of severity of symptoms, reduction in the occurrence of symptoms, partial or full remission, and/or healing.
• the therapeutic response can be a full or partial therapeutic response.
• Dietary ingredients and food substances that can be included in the nutraceutical composition are listed by the U.S.F.D.A.
• Exemplary classes of dietary ingredients or food substances include vitamin, mineral, herb, botanical, amino acid, protein, fat, polynucleotide, polysaccharide, carbohydrate, lipid, VOC, SVOC, antioxidant, phenolic compound, flavorant, essence, aromatic compound, water, sweetener, leavening agent, flavor enhancer, coloring agent, emulsifier, preservative, anti-caking agent, salt, buffer, oil, grain, enzyme, hormone, lecithin, melanin, polymer, biopolymer, fiber, starch, fatty acid, any substance found in any food, concentrate thereof, metabolite thereof, or extract thereof.
• a dosing regimen includes a therapeutically relevant dose (or effective dose) of oleandrin (in extract or composition) administered according to a dosing schedule.
• a therapeutically relevant dose therefore, is a therapeutic dose at which a therapeutic response is observed and at which a subject can be administered the oleandrin without an excessive amount of unwanted or deleterious side effects.
• a therapeutically relevant dose is non-lethal to a subject, even though it may cause some side effects in the patient. It is a dose at which the level of clinical benefit to a subject being administered the oleandrin exceeds the level of deleterious side effects experienced by the subject due to administration of the oleandrin.
• a cosmetic (cosmeceutical) composition may include one or more cosmetic excipients such as those approved by the U.S.F.D.A. (https://www.fda.gov/cosmetics/cosmetic-products-ingredients/cosmetic-ingredients).
• a cosmeceutical can further comprise one or more active ingredients and/or one or more excipients including, by way of example and without limitation, Acai oil, Alpha- lipoic acid, Green tea extract, Retinol, Coenzyme Q10 (CoQ10), Caffeine, Alpha-hydroxy Acids (AHAs), 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.
• active ingredients including, by way of example and without limitation, Acai oil, Alpha- lipoic acid, Green tea extract, Retinol, Coenzyme Q10 (CoQ10), Caffeine, Alpha-hydroxy Acids (AHAs), Hyaluronic Acid, Glycerin, Sorbitol, Mono and Diglycerides, White Mineral Oil (Petroleum Hydrocarbon Oil), Gly
• compositions and dosage forms of the invention can further comprise one or more pharmaceutically acceptable, nutraceutically, and/or cosmetically acceptable excipients.
• Dosage forms can comprise one or more excipients independently selected at each occurrence from the group consisting of acidic agent, alkaline agent, buffer, tonicity modifier, osmotic agent, water soluble polymer, water-swellable polymer, thickening agent, complexing agent, chelating agent, penetration enhancer.
• Suitable excipients include U.S.F.D.A.
• inactive ingredients approved for use in parenteral or oral formulations such as those listed in the U.S.F.D.A.’s “Inactive Ingredients Database (available on the following website: https://www.fda.gov/Drugs/InformationOnDrugs/ ucm113978.htm; Oct. 2018), the entire disclosure of which is hereby incorporated by reference.
• a therapeutically relevant dose will vary from subject to subject according to a variety of established pharmacologic, pharmacodynamic and pharmacokinetic principles.
• a therapeutically relevant dose (relative, for example, to oleandrin) will typically be about 1 or more microg per day, about 5 or more microg per day, about 10 or more microg per day, about 20 or more microg per day, about 25 or more microg per day, about 50 or more microg per day, about 75 or more microg per day, about 100 or more microg per day, about 200 or more microg per day, about 250 or more microg per day, about 500 or more microg per day, or up to about 750 microg of oleandrin per day or it can be in the range of about 1 to about 750 microg of oleandrin per dose, or might not exceed about 25 microg, about 100 microg, about 250 microg, about 500 microg, or about 750 microg of oleandrin/day.
• oleandrin The actual amount of oleandrin required to provide a target therapeutic result in a subject may vary from subject to subject according to the basic principles of pharmacy. It will be understood that the specific dose level for any patient will depend upon a variety of factors including the indication being treated, severity of the indication, patient health, age, gender, weight, diet, pharmacological response, the specific dosage form employed, and other such factors. [00115] In some embodiments, the subject is administered 140 microg to 315 microg per day of oleandrin. In some embodiments, a dose comprises 5 microg to 750 microg, 12 microg to 300 microg, or 12 microg to 120 microg of oleandrin.
• a recommended daily dose of oleandrin is generally about 0.25 to about 60 microg twice daily, about 5 to about 60 microg twice daily, about 5 to 30 microg twice daily.
• the dose can be about 0.5 to about 100 microg/day, about 1 to about 80 microg/day, about 1.5 to about 60 microg/day, about 1.8 to about 60 microg/day, about 5 to about 60 microg/day.
• the minimum effective dose can be about 0.5 microg/day, about 1 microg/day, about 1.5 microg/day, about 1.8 microg/day, about 2 microg/day, or about 5 microg/day.
• Suitable doses comprising oleandrin can 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 microg/kg/day, about 0.05-0.35 microg/kg/day, about 0.05-0.22 microg/kg/day, about 0.05- 0.4 microg/kg/day, or about 0.05-0.3 microg/kg/day.
• 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 invention includes all combinations of the doses set forth herein.
• the recommended daily dose of oleandrin, present in the extract can be about 0.1 microg to about 4.5 microg/kg body weight twice daily.
• the dose of oleandrin can be about 0.1 to about 1 microg/kg body weight/day, about 0.5 to about 1.0 microg/kg body weight/day, about 0.75 to about 1.5 microg/kg body weight/day, about 1.5 to about 2.52 microg/kg body weight/day, about 2.5 to about 3.0 microg/kg body weight/day, about 3.0 to 4.0 microg/kg body weight/day or about 3.5 to 4.5 microg oleandrin/kg body weight/day.
• the oleandrin can be present in a dosage form in an amount sufficient to provide a subject with an initial dose of oleandrin of about 20 to about 100 microg, about 12 microg to about 300 microg, or about 12 microg to about 120 microg.
• a dosage form can comprise about 1 microg of oleandrin to about 100 microg, about 0.01 microg to about 100 mg or about 0.01 microg to about 100 microg oleandrin, oleandrin extract or extract of Nerium oleander containing oleandrin.
• the concentration of oleandrin in the plasma of a treated subject is about 10 microg/mL or less, about 5 microg/mL or less, about 2.5 microg/mL or less, about 2 microg/mL or less, or about 1 microg/mL or less.
• the concentration of oleandrin in the plasma of a treated subject is about 0.0001 microg/mL or more, about 0.0005 microg/mL or more, about 0.001 microg/mL or more, about 0.0015 microg/mL or more, about 0.01 microg/mL or more, about 0.015 microg/mL or more, about 0.1 microg/mL or more, about 0.15 microg/mL or more, about 0.05 microg/mL or more, or about 0.075 microg/mL or more.
• the concentration of oleandrin in the plasma of a treated subject is about 10 microg/mL to about 0.0001 microg/mL, about 5 microg/mL to about 0.0005 microg/mL, about 1 microg/mL to about 0.001 microg/mL, about 0.5 microg/mL to about 0.001 microg/mL, about 0.1 microg/mL to about 0.001 microg/mL, about 0.05 microg/mL to about 0.001 microg/mL, about 0.01 microg/mL to about 0.001 microg/mL, about 0.005 microg/mL to about 0.001 microg/mL.
• the invention includes all combinations and selections of the plasma concentration ranges set forth herein.
• oleandrin Based on human clinical data it is anticipated that 50% to 75% of an administered dose of oleandrin will be orally bioavailable therefore providing about 10 to about 20 microg, about 20 to about 40 microg, about 30 to about 50 microg, about 40 to about 60 microg, about 50 to about 75 microg, about 75 to about 100 microg of oleandrin per dosage form.
• a therapeutically relevant dose can be administered according to any dosing regimen typically used in the treatment of viral infection.
• a therapeutically relevant dose can be administered once, twice, thrice or more daily.
• a therapeutically relevant dose can be administered one or more times daily (up to 10 times daily for the highest dose) for one or more weeks.
• a total daily dose may also be administered by infusion or pump over several hours, throughout part of a day, or throughout a 12 to 24 h period .
• the invention also provides a method of treatment by repeatedly administering to a subject in need thereof plural therapeutically effective doses of oleandrin (in an oleandrin-containing composition of the invention).
• One or more doses may be administered per day for one or more days per week and optionally for one or more weeks per month and optionally for one or more months per year.
• one or more doses of oleandrin are administered per day for plural days until the desired clinical endpoint(s).
• one or more doses of oleandrin are administered per day for plural days and plural weeks until the until the desired clinical endpoint(s).
• One or more doses can be administered in a day.
• One, two, three, four, five, six or more doses can be administered per day.
• Oleandrin oleandrin-containing composition or oleandrin- containing extract
• Oleandrin can be administered chronically, i.e.
• oleandrin oleandrin-containing composition or oleandrin-containing extract
• 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 is administered one or more times in a day.
• Oleandrin oleandrin-containing composition or oleandrin-containing extract
• Oleandrin-containing composition or oleandrin-containing extract can be administered one or more times per day for two or more days per week, optionally for one or more weeks per month and optionally for one or more months per year.
• a subject is evaluated to determine whether said subject is in need of oleandrin therapy.
• Administration of oleandrin-containing extract is then indicated.
• Initial doses of the extract are administered to the subject according to a prescribed dosing regimen for a period of time (a treatment period).
• the subject s clinical response and level of therapeutic response are determined periodically. If the level of therapeutic response is too low at one dose, then the dose is escalated according to a predetermine dose escalation schedule until the desired level of therapeutic response in the subject is achieved.
• the present method invention comprises: administering to the subject in need thereof a therapeutically relevant dose of oleandrin and a therapeutically relevant dose of said one or more other therapeutic agents, wherein the oleandrin is administered according to a first dosing regimen and the one or more other therapeutic agents is administered according to a second dosing regimen.
• the first and second dosing regimens are the same. In some embodiments, the first and second dosing regimens are different.
• Oleandrin can be administered as primary therapy, adjunct therapy, or co-therapy. Methods of the invention include separate administration or coadministration of oleandrin with at least one other therapeutic agent, meaning oleandrin can be administered before, during or after administration of said other therapeutic agent.
• the one or more other therapeutic agents can be administered at doses and according to dosing regimens that are clinician-recognized as being therapeutically effective or at doses that are clinician-recognized as being sub-therapeutically effective.
• the clinical benefit and/or therapeutic effect provided by administration of a combination of oleandrin and one or more other therapeutic can be additive or synergistic, such level of benefit or effect being determined by comparison of administration of the combination to administration of the individual component(s).
• the one or more other therapeutic agents can be administered at doses and according to dosing regimens as suggested or described by the Food and Drug Administration, World Health Organization, European Medicines Agency (E.M.E.A.), Therapeutic Goods Administration (TGA, Australia), Pan American Health Organization (PAHO), Medicines and Medical Devices Safety Authority (Medsafe, New Zealand) or the various agencies of Health worldwide.
• Treatable viral infections include those from viruses of the Arenaviridae family, Arterviridae, Bunyaviridae family, Filoviridae family, Flaviviridae family (Flavivirus genus), Orthomyxoviridae family (influenza virus genus), Paramyxoviridae family, Rhabdoviridae family, Retroviridae family (Deltaretrovirus genus), Coronaviridae family, (+)-ss-envRNAV (positive sense single stranded enveloped RNA virus), (-)-ss- envRNAV (negative sense single stranded enveloped RNA virus), or Togaviridae family, as well as any of their known genera, species, and/or strains.
• the (+)- ss-envRNAV is a coronavirus that is pathogenic to humans.
• viruses that can be treated include, at least, Ebolavirus, Marburgvirus, Alphavirus, Flavivirus, Yellow Fever, Dengue Fever, Japanese Encephalitis, West Nile Viruses, Zikavirus, Venezuelan Equine Encephalomyelitis (encephalitis) (VEE) virus, Chikungunya virus, Western Equine Encephalomyelitis (encephalitis) (WEE) virus, Eastern Equine Encephalomyelitis (encephalitis) (EEE) virus, Tick-borne Encephalitis, Kyasanur Forest Disease, Alkhurma Disease, Omsk Hemorrhagic Fever, Hendra virus, Nipah virus, Deltaretrovirus genus, HTLV-1 virus, coronavirus, SARS-CoV, MERS-CoV, COVID-19 (SARS-CoV-2), CoV 229E, CoV 229E,
• the anticancer (anti-excessive cell proliferation) activity of the SbCL extract, ethanolic extract, and combination composition is established according to Example 11, wherein the in vitro assays set forth in WO 2007016176A2, WO 2009064657 A1, US 7402325 B2, US 8187644 B2, US 8394434 B2, US 8367363 B2, US 9494589 B2, US 9846156 B2, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entirety, are followed to demonstrate the anticancer activity of a composition of the invention.
• Said condition, disease or disorder that is therapeutically responsive to oleandrin can be selected from the group consisting of cancer, tumor, carcinoma, sarcoma, glioblastoma, adenocarcinoma, lymphoma, melanoma, brain tumor, glioma, astrocytoma (which include low-grade astrocytomas (Grade I pilocytic astrocytoma, and Grade II diffuse astrocytoma), anaplastic astrocytoma (Grade III), glioblastoma (Grade IV, GBM, also known as glioblastoma multiforme)), ependymoma, oligodendroglioma, excessive cell proliferation, malignant neoplastic disease, noncancerous inflammatory skin diseases, actinic keratosis, psoriasis, eczema, neurological disease,
• astrocytoma which include low-grade astrocytomas (Grade
• Compound(s) present in the composition of the invention can be present in their unmodified form, salt form, derivative form or a combination thereof.
• the term “derivative” is taken to mean: a) a chemical substance that is related structurally to a first chemical substance and theoretically derivable from it; b) a compound that is formed from a similar first compound or a compound that can be imagined to arise from another first compound, if 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 containing essential elements of the parent compound; or d) a chemical compound that may be produced from first compound of similar structure in one or more steps.
• a derivative may include a deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or glycosilated form thereof or may include an ester, amide, lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl, heterocyclic, heterocyclic ring- fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated form thereof.
• the individually named compounds can independently be selected upon each occurrence in their native (unmodified, free acid, or free base) form, in their salt form, in derivative form, prodrug form, or a combination thereof.
• compositions containing and methods employing deuterated forms of the triterpenes are also within the scope of the invention.
• the oleandrin-containing extract (composition) can be formulated in any suitable pharmaceutically acceptable dosage form. Suitable types of dosage forms include aerosol, bar, bead, capsule, cellular sheet, chewable, cloth, concentrate, cream, crystal, disc, douche, dressing, elixir, emulsion, enema, fiber, film, foam, gel, globule, granule, gum, implant, inhalable, injectable, injection, insert, intrauterine device, jelly, kit, liniment, lipstick, liposome(s), liquid, lotion, lozenge, mouthwash, oil, ointment, paste, pastille, patch, pellet, plaster, poultice, powder, lyophilized powder, ring, rinse, salve, shampoo, soap, solution, sponge, spray, stick, strip, suppository, swab, suspension, syrup, tablet, tampon
• the oleandrin-containing extract (composition) can 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 hours or less.
• Some embodiments of the dosage form are enteric coated and release their charge of extract downstream of the stomach, such as from the jejunum, ileum, small intestine, and/or large intestine (colon). Enterically coated dosage forms will release extract into the systemic circulation within 1-10 hr after oral administration.
• the oleandrin-containing extract (composition) can be included in a rapid release, immediate release, controlled release, sustained release, prolonged release, extended release, burst release, continuous release, slow release, or pulsed release dosage form or in a dosage form that exhibits two or more of those types of release.
• the release profile of oleandrin from the dosage form can be a zero order, pseudo-zero, first order, pseudo-first order or sigmoidal release profile.
• the plasma concentration profile for oleandrin in a subject to which it is administered can exhibit one or more maxima.
• Suitable dosage forms can also be made according to US 8187644 B2 to Addington, which issued May 29, 2012, US 7402325 B2 to Addington, which issued July 22, 2008, US 8394434 B2 to Addington et al, which issued Mar. 12, 2013, the entire disclosures of which are hereby incorporated by reference. Suitable dosage forms can also be made as described in Examples 4-6, and 9. [00137] The oleandrin-containing extract (or any composition comprising it) may be administered locally, systemically or both.
• Suitable modes of administration include parenteral, buccal, conjunctival, corneal, cutaneous, dental, endocervical, endosinusial, endotracheal, enteral, epidermal, epidural, extra-amniotic, extracorporeal, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intrabronchial, intrabursal, 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, intrasinal, intrasynovial, intratesticular, intrathecal, intrathoracic, intratubular
• a liquid composition can comprise one or more pharmaceutically acceptable liquid carriers.
• the liquid carrier can be an aqueous, non-aqueous, polar, non-polar, and/or organic carrier.
• Liquid carriers include, by way of example and without limitation, a water miscible solvent, water immiscible solvent, water, buffer and mixtures thereof.
• water soluble solvent or “water miscible solvent”, which terms are used interchangeably, refer to an organic liquid which does not form a biphasic mixture with water or is sufficiently soluble in water to provide an aqueous solvent mixture containing at least five percent of solvent without separation of liquid phases.
• 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)), PEG 400 (poly(ethylene glycol having an approximate molecular weight of about 400), ethanol, acetone, alkanol, alcohol, ether, propylene glycol, glycerin, triacetin, poly(propylene glycol), PVP (poly(vinyl pyrrolidone)), dimethylsulfoxide, N,N- dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N- methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2- pyrrolidone).
• PEG poly(ethylene glycol)
• PEG 400 poly(ethylene glycol having an approximate molecular weight of about 400
• ethanol acetone
• alkanol alcohol
• ether propylene glycol
• glycerin triacetin
• water insoluble solvent or “water immiscible solvent”, which terms are used interchangeably, refer to an organic liquid which forms a biphasic mixture with water or provides a phase separation when the concentration of solvent in water exceeds five percent.
• the solvent is suitable for administration to humans or animals.
• Exemplary water insoluble solvents include, by way of example and without limitation, medium/long chain triglycerides, oil, castor oil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid, olive oil, softisan 645 (Diglyceryl Caprylate / Caprate / Stearate / Hydroxy stearate adipate), miglyol, captex (Captex 350: Glyceryl Tricaprylate/ Caprate/ Laurate triglyceride; Captex 355: Glyceryl Tricaprylate/ Caprate triglyceride; Captex 355 EP / NF: Glyceryl Tricaprylate/ Caprate medium chain triglyceride).
• 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 makes recommendations as to what amounts of residual solvents are considered safe in pharmaceuticals.
• 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-butlymethyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl-1-butanol, methylethyl ketone, methylisobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1- propanol, 2-propanol, or propyl acetate.
• Captex 100 Propylene Glycol Dicaprate
• Captex 200 Propylene Glycol Dicaprylate/ Dicaprate
• Captex 200 P Propylene Glycol Dicaprylate/ Dicaprate
• Captex 300 Glyceryl Tricaprylate/ Caprate
• Captex 300 EP / NF Glyceryl Tricaprylate/ Caprate Medium Chain Triglycerides
• Captex 350 Glyceryl Tricaprylate/ Caprate/ Laurate
• Captex 355 Glyceryl Tricaprylate/ Caprate
• Captex 355 EP / NF Glyceryl Tricaprylate/ Caprate Medium Chain Triglycerides
• Captex 500 Triacetin
• Captex 500 P Triacetin (Pharmaceutical Grade)
• Captex 800 Propylene Glycol Di (2- Ethythexanoate)
• Captex 810 D Glyceryl Tricaprylate/ Caprate
• a “surfactant” refers to a compound that comprises polar or charged hydrophilic moieties as well as non-polar hydrophobic (lipophilic) moieties; i.e., a surfactant is amphiphilic.
• the term surfactant may refer to one or a mixture of compounds.
• a surfactant can be a solubilizing agent, an emulsifying agent or a dispersing agent.
• a surfactant can be hydrophilic or hydrophobic.
• the hydrophilic surfactant can be any hydrophilic surfactant suitable for use in pharmaceutical compositions.
• Such surfactants can be anionic, cationic, zwitterionic or non- ionic, although non-ionic hydrophilic surfactants are presently preferred. As discussed above, these non-ionic hydrophilic surfactants will generally have HLB values greater than about 10. Mixtures of hydrophilic surfactants are also within the scope of the invention.
• the hydrophobic surfactant can be any hydrophobic surfactant suitable for use in pharmaceutical compositions. In general, suitable hydrophobic surfactants will have an HLB value less than about 10. Mixtures of hydrophobic surfactants are also within the scope of the invention.
• Examples of additional suitable solubilizer include: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, available commercially from BASF under the trade name Tetraglycol) or methoxy PEG (Union Carbide); amides, such as 2-pyrrolidone, 2-piperidon
• 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 colorants, one or more electrolytes, one or more salts, one or more stabilizers, one or more tonicity modifiers, one or more diluents, or a combination thereof.
• 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 colorants, one or more electrolytes, one or more salts, one or more stabilizers, one or more tonicity modifiers, one or more diluents, or a combination thereof.
• composition of the invention can also include 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.
• 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
• fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides, medium chain triglycerides, and acetylated fatty acid glycerides.
• the composition can also include alcohol 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; a pharmaceutically suitable surfactant, suspending agent or emulsifying agent; or mixtures thereof.
• alcohol 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 a pharmaceutically suitable surfactant, suspending agent or emulsifying agent; or mixtures thereof.
• One or more of the components of the formulation can be present in its free base, free acid or pharmaceutically or analytically acceptable salt form.
• pharmaceutically or analytically acceptable salt refers to a compound that has been modified by reacting it with an acid as needed to form an ionically bound pair. Examples of acceptable salts include conventional non-toxic salts formed, for example, from non-toxic inorganic or organic acids. Lists of suitable salts are found in Remington ⁇ s Pharmaceutical Sciences, 17 th .
• a dosage form can be made by any conventional means known in the pharmaceutical industry.
• a liquid dosage form can be prepared by providing at least one liquid carrier and oleandrin (oleandrin-containing composition, SbCL extract) in a container.
• a solid dosage form can be prepared by providing at least one solid carrier and oleandrin.
• One or more other excipients can be included in the solid dosage form.
• a dosage form can be packaged using conventional packaging equipment and materials. It can be included in a pack, bottle, via, bag, syringe, envelope, packet, blister pack, box, ampoule, or other such container.
• the composition of the invention can be included in any dosage form.
• Particular dosage forms include a solid or liquid dosage forms. Exemplary suitable dosage forms include tablet, capsule, pill, caplet, troche, sache, and other such dosage forms known to the artisan of ordinary skill in the pharmaceutical sciences.
• Powdered oleander leaves were prepared by harvesting, washing, and drying oleander leaf material, then passing the oleander leaf material through a comminuting and dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705.
• the weight of the starting material used was 3.94 kg.
• the starting material was combined with pure CO 2 at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50°C (122oF) in an extractor device. A total of 197 kg of CO 2 was used, to give a solvent to raw material ratio of 50:1.
• the mixture of CO 2 and raw material was then passed through a separator device, which changed 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 having a nice fragrance. The color was likely caused by chlorophyll and other residual chromophore compounds.
• the tubes and separator were rinsed out with acetone and the acetone was evaporated to give an addition 9 g of extract.
• the total extract amount was 74 g. Based on the weight of the starting material, the yield of the extract 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%.
• Method B With mixture of carbon dioxide and ethanol [00161] Powdered oleander leaves were prepared by harvesting, washing, and drying oleander leaf material, then passing the oleander leaf material through a comminuting and dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The weight of the starting material used was 3.85 kg.
• the starting material was combined with pure CO 2 and 5% ethanol as a modifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50°C (122oF) in an extractor device.
• a total of 160 kg of CO 2 and 8 kg ethanol was used, to give a solvent to raw material ratio of 43.6 to 1.
• the mixture of CO 2 , ethanol, and raw material was then passed through a separator device, which changed the pressure and temperature of the mixture and separated the extract from the carbon dioxide.
• the extract (207 g) was obtained after the removal of ethanol as a dark green, sticky, viscous mass obviously containing some chlorophyll. Based on the weight of the starting material, the yield of the extract was 5.38%.
• Hot-water extraction 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 processes can be found in U.S. Patent Nos.5,135,745 and 5,869,060.
• a hot water extraction was carried out using 5 g of powdered oleander leaves. Ten volumes of boiling water (by weight of the oleander starting material) were added to the powdered oleander leaves and the mixture was stirred constantly for 6 hours.
• Dried and 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 interior 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 a majority or substantially all of the extraction time period: Temperature in the range of about 2°C to about 16°C (about 7°C to about 8°C), and pressure in the range of about 115 to about 135 bar (about 124 bar). The extraction period was about 4 h to about 12 h (about 6 to about 10 h). The extraction milieu was then filtered and the supernatant collected.
• the carbon dioxide was vented from the supernatant, and the resulting crude extract was diluted into ethanol (about 9 parts ethanol : about 1 part extract) and frozen at about -50°C for at least 12 h.
• the solution was thawed and filtered (100 micron pore size filter).
• the filtrate was concentrated to about 10% of its original volume and then sterile filtered (0.2 micron pore size filter).
• the concentration of oleandrin in the crude extract is about 0.25 to 1 mg (or 0.5 to 0.8 mg) per mL of extract.
• the resulting subcritical liquid (SbCL) extract comprised oleandrin and one or more other compounds extractable from Nerium oleander, said one or more other compounds being as defined herein.
• a sublingual dosage form comprising oleandrin was made by mixing oleandrin or oleandrin-containing composition (e.g. oleandrin-containing extract) with medium chain triglyceride (MCT, e.g. coconut oil).
• MCT medium chain triglyceride
• Method A The ethanol diluted SbCL extract (2 wt %) was mixed with MCT (95 wt %), and flavoring agent (3 wt %).
• Method B The ethanol diluted ethanolic extract (2 wt %) was mixed with MCT (95 wt %), and flavoring agent (3 wt %).
• Method C Method C.
• the sublingual dosage form was safely administered to a subject by about 0.5 mL aliquots. No beverage or food was consumed for at least ten minutes after administration. It was administered one to six times daily for about seven days to about one month. [00174] Even though intended for sublingual administration, this dosage form may also be administered orally.
• Method A. Cremophor-based drug delivery system [00175] The following ingredients are provided in the amounts indicated.
• Method A The procedure of Method A is followed.
• Method C Labrasol-based drug delivery system [00179] The following ingredients are provided in the amounts indicated.
• Method D Vitamin E-TPGS based micelle forming system
• the following ingredients are provided in the amounts indicated.
• Method E Multi-component drug delivery system
• the following ingredients are provided in the amounts indicated.
• Method A The procedure of Method A is followed.
• Method F Multi-component drug delivery system
• the following ingredients are provided in the amounts indicated an included in a capsule.
• Example 6 Preparation of enteric coated capsules Step I: Preparation of liquid-filled capsule [00187] Hard gelatin capsules (50 counts, 00 size) are filled with a liquid composition of Example 3. These capsules are manually filled with 800 mg of the formulation and then sealed by hand with a 50% ethanol/ 50% water solution. The capsules are then banded by hand with 22% gelatin solution containing the following ingredients in the amounts indicated. [00188] The gelatin solution is mixed thoroughly and allowed to swell for 1-2 hours.
• Step II Coating of liquid-filled capsule [00190] A coating dispersion is prepared from the ingredients listed in the table below. [00191] If banded capsules according to Step I are used, the dispersion is applied to the capsules to a 20.0 mg/cm 2 coating level. The following conditions are used to coat the capsules.
• Spray nozzle iss set such that both the nozzle and spray path are under the flow path of inlet air.
• Example 7 Treatment of viral infection in a subject [00192] The methods of WO 2018053123 A1, WO 2019055119 A1, US 10596186 B2, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entirety, are followed to demonstrate the antiviral activity of a composition of the invention.
• Example 9 Preparation of a tablet comprising oleandrin composition [00194] An initial tabletting mixture of 3% Syloid 244FP and 97% microcrystalline cellulose (MCC) is mixed. Then, an existing batch of SbCL extract is incorporated into the Syloid/MCC mixture via wet granulation. This mixture is labeled "Initial Tabletting Mixture) in the table below. Additional MCC is added extra-granularly to increase compressibility. This addition to the Initial Tabletting Mixture is labeled as "Extra-granular Addition.” The resultant mixture from the extra-granular addition is the same composition as the "Final Tabletting Mixture.”
• MCC microcrystalline cellulose
• Syloid 244FP is a colloidal silicon dioxide manufactured by Grace Davison. Colloidal silicon dioxide is commonly used to provide several functions, such as an adsorbant, glidant, and tablet disintegrant. Syloid 244FP is chosen for its ability to adsorb 3 times its weight in oil and for its 5.5 micron particle size.
• Triterpene content [00197] The triterpene components of the extracts are quantified by HPLC using a Gemini C18 diphenyl column and eluting the triterpenes with an isocratic mobile phase consisting of 95% MeCN with 0.1% formic acid at a flow rate of 0.6 ml/min and a detection wavelength of 220 nm. Standard curves are developed and used to calculate the relative molar ratios of oleanolic acid, ursolic acid and betulinic acid compounds.
• FIG. 4 depicts the HPLC chromatogram of PBI-05204.
• Example 11 Treatment of cancer in a subject [00198] The methods of WO 2007016176A2, WO 2009064657 A1, US 7402325 B2, US 8187644 B2, US 8394434 B2, US 8367363 B2, US 9494589 B2, US 9846156 B2, and related U.S. and foreign patents and applications, the entire disclosures of all of which are hereby incorporated by reference in their entirety, were followed to demonstrate the anticancer (anti-excessive cell proliferation) activity of a composition of the invention.
• Example 12 Preparation of Pharmaceutical Composition [00199]
• a pharmaceutical composition of the invention can be prepared any of the following methods. Mixing can be done under wet or dry conditions. The pharmaceutical composition can be compacted, dried or both during preparation.
• the pharmaceutical composition can be portioned into dosage forms.
• Method A [00200] At least one pharmaceutical excipient is mixed with at least one SbCL extract disclosed herein.
• Method B. [00201] At least one pharmaceutical excipient is mixed with at least SbCL extract and at least one other active agent.
• Method C. At least one pharmaceutical excipient is mixed with oleandrin.
• Method D. At least one pharmaceutical excipient is mixed with oleandrin and at least one triterpene disclosed herein.
• Method E [00204] At least one pharmaceutical excipient is mixed with oleandrin and at least two triterpenes disclosed herein.
• At least one pharmaceutical excipient is mixed with 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 were mixed in solvent(s), e.g. methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof.
• solvent(s) e.g. methanol, ethanol, chloroform, acetone, propanol
• DMSO dimethyl sulfoxide
• DMF dimethylformamide
• DMAC dimethylacetamide
• NMP N-methylpyrrolidone
• Example 13 Statistical Analysis
• Example 14 Preparation of Powdered Biomass Method A.
• Powdered and dried Nerium oleander biomass was prepared according to US 7402325 B2 to Addington, the entire disclosure of which is hereby incorporated by reference, by placing harvested leaves in a comminuting and heated dehydrating apparatus as described in US 5236132, US 5598979, US 6517015, or US 6715705, the entire disclosures of which is hereby incorporated by reference. Method B.
• Powdered and dried Nerium oleander biomass was prepared by dehydrating harvested leaves in a heated dehumidifier and then subjecting the dried leaves to repeated freeze-thaw cycles with liquid nitrogen, during which at least one of the cycles the frozen leaves were comminuted to form a powder, which was then freeze dried to a moisture content of less than about 8 wt %.
• Example 15 Preparation of Aqueous Organic Solvent Extract
• An ethanolic extract of Nerium oleander was prepared by extracting dried powdered biomass with aqueous ethanol.
• 10 mL of aqueous ethanol (10 % v/v water) was used to extract the powder while stirring vigorously at ambient temperature and pressure.
• 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 vacuo to about 20% of the original volume to form a concentrate that was sterile filtered (0.2 micron filter) to form the 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 of extract.
• Example 16 Preparation of dosage form comprising a combination of extracts of Nerium oleander
• the purpose of this was to prepare a dosage form according to Example 4 except that a portion (1 wt %) of the ethanolic extract or aqueous ethanolic extract is combined with a portion (1 wt %) of the SbCL extract of Example 3, medium chain triglyceride (95 wt %), and flavoring agent (3 wt %).
• the concentration of oleandrin in the dosage form is about 12.5 to about 25 microg/mL.
• Example 17 In vivo evaluation of oleandrin against COVID-19 virus
• OCE oleandrin-containing extract
• Example 18 Gas chromatographic analysis of extract(s)
• the organic solvent extract and the subcritical extract were analyzed by GC-mass spectrometry analysis, with total ion current (TIC) detection to identify some of the volatile and semi-volatile organic components thereof.
• Sample extracts were dissolved using a solution containing seven isotopically labeled semi-volatile organic compound internal standards (SVOCIS) prior to being injected and analyzed by GC-MS. Ten largest peaks from each of the sample extracts were tentatively identified via comparison to 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, where a match factor of 100 would indicate a perfect match.
• SVOCIS isotopically labeled semi-volatile organic compound internal standards
• DART+ positive ion mode
• M+H+ ions generated by the DART-MS.
• a range of settings on the instrument is used to determine optimal conditions for N. oleander analyses.
• the general settings for DART+ include: needle voltage 3500 V; orifice 1- 2–20 V; ring lens 2–5 V; orifice 2- 2–5 V; and peaks voltage 1000 V.
• Calibrations are performed internally with each sample using a 10% solution of PEG 600 which provides mass markers throughout the required mass range of 100–1000 mass units.
• Other analyses are undertaken in the DART- mode and these consisted of: needle voltage 3500 V; heating element 250° C; electrode 1- 150 V; electrode 2- 250 V; He gas flow rate 3.79LPM.
• Mass spectrometer settings MCP 2600 V; orifice 1- 15 V; ring lens- 5 V, orifice 2- 5 V; and peaks voltage 1000 V.
• Calibrations are performed internally with each sample using a perfluorinated carboxylic acid solution that provides markers throughout the required mass range of 100–1000 mass units.
• the 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 s per analysis. Molecular formulas are confirmed by elemental composition and isotope matching programs provided with the JEOL AccuTOF DART-MS instrument. A searchable database of N.
• oleander constituents developed by Herbal Science can be used.
• Example 20 Nutraceutical Composition Oleandrin-containing extract (at the desired concentration) is combined with nutraceutical excipients such as those found in PREMIER PROTEIN shake, SLIMFAST ADVANCED ENERGY meal replacement shake, ORGAIN ORGANIC NUTRITION shake, VEGA Protein Nutrition shake, LEAN BODY LABRADA Whey protein meal replacement shake, ICONIC GRASS-FED protein drink, PURE PROTEIN shake, MUSCLE MILK original protein shake, MET-RX original whey protein meal replacement powder, OWYN vegan meal replacement shake, SOYLENT meal replacement shake, or APRÉS plant-based vegan protein shake.
• Example 21 Cosmetic Lotion Composition Oleandrin-containing extract (at the desired concentration) is combined with nutraceutical excipients such as those found in NIVEA ESSENTIALLY ENRICHED body lotion (with moisturizer serum and almond oil), EUCERIN ADVANCED REPAIR (with Ceramide-3), PALMER’S COCOA BUTTER FORMULA DAILY SKIN THERAPY 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 FOOD nourishing cream (with vitamin E, calendula flower extract, pansy), AVEENO daily moisturizing lotion, HEMPZ original herbal body moisturizer (with hemp seed oil), CERAVE daily moisturizing lotion (with hyaluronic acid), AQUAPHOR ADVANCED THERAPY healing ointment (with petrolatum).
• a thin-film dosage form can be prepared according to 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.
• a bioadhesive buccal dosage form can be prepared according to 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.
• Method C gummy dosage form [00223] A gummy (semi-solid, soft, chewable) dosage form can be prepared according to Jacobs (“Semi-solid formulations” in Pediatric Form. (2014), 171-179), Cizauskaite et al.
• Spray dosage forms can be prepared according to Finlay (The Mechanics of Inhaled Pharmaceutical Aerosols- An Introduction; Ed. W.H. Finlay, Academic Press, London, UK; 2001), Hickey (“Back to the Future: Inhaled Drug Products” in J. Pharm. Sci.
• Example 23 Preparation of Organic Solvent Extract
• An ethanolic extract of Nerium oleander was prepared by extracting dried powdered biomass with ethanol.
• 10 mL of ethanol was used to extract the powder while stirring vigorously at ambient temperature and pressure.
• 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 vacuo to about 20% of the original volume to form a concentrate that was sterile filtered (0.2 micron filter) to form the 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 of extract.
• the term “about” or “approximately” are taken to mean ⁇ 10%, ⁇ 5%, ⁇ 2.5% or ⁇ 1% of a specified valued.

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