HK1219875B - Liquids rich in noble gas and methods of their preparation and use - Google Patents

Description

Liquids rich in noble gases and methods of making and using the same

This application claims the benefit of U.S. Provisional Patent Application No. 61/788,808, filed March 15, 2013, and U.S. Provisional Patent Application No. 61/889,901, filed October 11, 2013, both of which are incorporated herein by reference in their entireties.

This invention was made with government support under Grant Nos. NS067454, HL074002, and HL059586 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background of the Invention

1. Field of the Invention

The present invention relates generally to the fields of molecular biology, medicine, and nutraceuticals. More particularly, it relates to methods for the oral delivery of noble gas compositions such as xenon or argon for the treatment and prevention of disease.

2. Description of related fields

Xenon (Xe) and argon (Ar) are both polymorphic cell-protective gases with unique advantages, including rapid diffusion across biological barriers, such as the blood-brain barrier (BBB), and complete passage through cell membranes due to their low blood-gas partition coefficient. In animal models, Xe, given as a continuous gas inhalation, has demonstrated potent neuroprotective and cardioprotective effects. Xe prevents oxygen and glucose deprivation (OGD) and hypoxia/ischemia by altering molecules involved in neuronal tolerance to ischemia. Xe helps induce the transcription of several pro-survival genes, including brain-derived neurotrophic factor (BDNF) and pro-survival proteins such as Bcl ₂ , which contribute to cellular tolerance to ischemic injury. Xe interacts with the human immune system by regulating inflammatory cytokines such as TNF-α and IL-6 in monocytes. Xe facilitates the sustained release of hypoxia-inducible factor 1α (HIF-1α) and other proteins. All of these pathways are implicated in organ protection.

Current methods for gas delivery involve inhalation and administration of a gas donor. However, Xe or Ar inhalation is not practical in many cases because the high Xe or Ar concentrations required for inhalation limit the fraction of inspired oxygen necessary for cell survival. Furthermore, there are difficulties in developing a continuous inhalation strategy for patients in a daily use format. Improved methods for delivering Xe or Ar are greatly needed.

SUMMARY OF THE INVENTION

Embodiments of the present invention generally relate to methods of treatment, medicaments, and nutraceuticals (a portmanteau of the words "nutrient" and "medicament," which are foods or food products that have health and medical benefits, including the prevention and treatment of disease). More specifically, they relate to methods of primarily orally delivering inert gas compositions to the gastrointestinal (GI) tract, the inert gas compositions comprising noble gases, such as helium, neon, argon, krypton, and/or xenon. Such gases can be delivered as part of methods for preventing and/or treating heart and brain diseases, including, but not limited to, atherosclerosis-related ischemic heart disease, stroke, and other neurodegenerative conditions, such as dementia (e.g., Alzheimer's disease). In certain aspects, the compositions comprising the noble gases result in a reduction in one or more markers of inflammation or an overall improvement in well-being.

In a first embodiment, a liquid (or semi-liquid, such as a paste or gel) formulation of an encapsulated rare gas (e.g., gas can be encapsulated to achieve a specified concentration) with enhanced concentration is provided. The rare gas can be selected from helium, neon, argon, krypton, xenon, or a mixture thereof. As used herein, the encapsulated rare gas can be dissolved in lipid and emulsified, encapsulated in a liposomal formulation, and/or provided in the form of a gas encapsulated in a water-soluble molecule (e.g., cyclodextrin). In general, this type of sealing allows compositions to achieve a higher gas content than can be achieved under equal amounts of water (there is no sealing) and constant temperature and pressure. In a preferred aspect, the compositions are formulated for oral delivery.

In a certain embodiment, a nutritional health (such as a beverage) composition is provided, which comprises a generally aqueous component and a dissolved noble gas, wherein a portion of the noble gas is encapsulated to enhance water solubility. In some aspects, the encapsulated noble gas can be encapsulated in a lipid emulsified in the composition, such as a liposome or lipid phase. In other aspects, the noble gas (e.g., Xe, Ar, Kr, Ne, or He) is encapsulated in a water-soluble molecule (such as a water-soluble polymer). In other aspects, the noble gas is encapsulated in α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin or a mixture thereof. Other molecules that can be used for noble gas sealing are described in detail below.

In another embodiment, the present invention provides single portion nutritional health composition, it includes but not limited to beverage, gel, paste, tablet and capsule.For example, composition can comprise the composition of about 1,2,3,4,5 or 10 to about 15,20,25,30,35,40,45 or 50ml.In other aspects, composition can comprise the composition (for example, substantially aqueous composition) of about 10,15,25,30,35,40,45 or 50 to about 100,150,200,250,300,350,400,450 or 500ml.In some aspects, the composition of embodiment comprises the noble gas (or mixture of two or more such gases) of the dissolution of 0.01 to 15g or about 0.1 to 200mg, wherein a part for noble gas is encapsulated to enhance water solubility.As described in detail above, noble gas can be encapsulated or dissolved in (emulsification) lipid component, liposome or water-soluble molecule. In some aspects, the amount of the dissolved rare gas (such as Xe) in the preparation is about 0.1mg to 10g, 0.1 to 1,000mg, 0.1 to 500mg, 0.5 to 100mg, 1 to 100mg, 1 to 50mg, 1 to 25mg or 1 to 10mg. Similarly, the volume of the liquid component in the adjustable preparation is to provide the rare gas of optimal amount in a single serving. For example, a single serving of beverage can include a cumulative volume of about 1 to 10ml, 10 to 25ml, 25 to 50ml, 50 to 500ml, 50 to 300ml, 100 to 300ml or 200 to 400ml. As described in further detail herein, in some preferred embodiments, this type of single serving of nutritional supplements is provided in an airtight sealed container, such as a bottle, a jar or foil or a polymer package. Likewise, in some aspects, a package is provided that contains 4, 6, 8, 12, 24, or more single servings of a composition (such as a beverage), each containing a single serving of a nutraceutical beverage in an individually sealed container.

As described in detail above, certain aspects of the embodiments relate to compositions comprising a noble gas (e.g., Xe), at least a portion of which is encapsulated in a cyclodextrin (CD) (e.g., β-cyclodextrin). In some cases, the cyclodextrin for use according to the embodiments will include a hydrophilic group that further enhances water solubility. For example, in certain aspects, the CD is a hydroxypropyl-CD, such as hydroxypropyl-β-cyclodextrin. The composition may comprise, for example, from about 0.01 to about 5.0 mg/ml; from about 0.05 to about 2.0 mg/ml; from about 0.1 to about 1.5 mg/ml, or from about 0.1 to about 1.0 mg/ml of CD. In some specific aspects, the composition comprises 0.1 to about 1.0 mg/ml of hydroxypropyl β-CD. In other aspects, the composition comprises from about 0.1 to about 5.0 ml; from about 0.1 to about 4.0; from about 0.1 to about 3.8; from about 0.1 to about 2.0; from about 0.1 to about 1.0 ml; or from 0.5 to about 1.0 ml of a noble gas (e.g., Xe) per 0.5 mg of cyclodextrin (e.g., at standard temperature and pressure) in the composition. Specifically, the composition may comprise from about 0.1 to about 5.0 ml; from about 0.1 to about 4.0; from about 0.1 to about 3.8; from about 0.1 to about 2.0; from about 0.1 to about 1.0 ml; or from 0.5 to about 1.0 ml of Xe per 0.5 mg of β-cyclodextrin (e.g., hydroxypropyl β-CD). In a very specific aspect, the composition comprises up to about 3.8 ml of Xe per 0.5 mg of CD molecules (e.g., per milliliter of water).

Other cyclodextrin molecules that can be used according to the present invention (in the form of oil or water compositions depending on the desired solubility) include, but are not limited to, methyl-β-cyclodextrin, random methylated-β-cyclodextrin, dimethyl-β-cyclodextrin, random dimethylated-β-cyclodextrin, trimethyl-β-cyclodextrin, acetylated dimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin, hydroxybutenyl-β-cyclodextrin, 2,3-dihydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, glucosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, , glucuronyl-glucosyl-β-cyclodextrin, alkylated β-cyclodextrin, 2,6-di-O-ethyl-β-cyclodextrin, 2,3,6-tri-O-ethyl-β-cyclodextrin, acylated β-cyclodextrin, 2,3,6-tri-O-acyl (C2-C18)-β-cyclodextrin, 2,3,6-tri-O-butyryl-β-cyclodextrin, 2,3,6-tri-O-pentanoyl-β-cyclodextrin, 2,3,6-tri-O-octyl-β-cyclodextrin, O-carboxymethyl-O-ethyl-β-cyclodextrin, β-cyclodextrin sulfate, sulfobutyl ether-β-cyclodextrin or sulfobutyl ether-β-cyclodextrin.

The method confirmed herein can provide the extremely high concentration of rare gases in liquid form. However, in some cases, the liquid or semi-liquid (including for example beverage) of embodiment can include relatively moderate amount of rare gases, such as Xe. For example, the dissolved Xe concentration (when in sealed container) that such beverage can include is about 1.0 to about 5,000 μg/ml, about 10 to about 1,000 μg/ml, about 100 to about 800 μg/ml, about 1.0 to about 100 μg/ml, 5.0 to about 50 μg/ml, about 10 to about 100 μg/ml or about 10 to about 50 μg/ml. For example, the concentration of Xe dissolved in water at standard temperature and pressure can be about 600 μg/ml. In another aspect, the beverage provided has high concentration of Xe, such as about 1 mg/ml to about 100 mg/ml, 1 mg/ml to about 50 mg/ml, about 1 mg/ml to about 30 mg/ml or about 10 mg/ml to about 50 mg/ml. In some aspects, beverages (e.g., beverages comprising pressurized and/or encapsulated Xe) are provided that comprise Xe levels ranging from about 10 mg/ml to about 15, 20, 25, 30, 35, 40, 45, or 50 mg/ml Xe. For example, a formulation comprising cyclodextrin (hydroxypropyl β-CD) encapsulating Xe can have a Xe concentration of about 22.4 mg/ml when formulated at 3 atm and 4°C and then brought to standard ambient temperature and pressure (SATP).

In some aspects, the methods and compositions herein relate to argon provided in liquid or semi-liquid form (e.g., as a beverage). For example, such compositions may include a dissolved Ar concentration (when in a sealed container) of about 1.0 to about 1,000 μg/ml, about 10 to about 500 μg/ml, about 20 to about 500 μg/ml, about 30 to about 250 μg/ml, about 40 to about 200 μg/ml, about 50 to about 100 μg/ml, or about 40 to about 75 μg/ml. For example, the concentration of Ar dissolved in water at standard temperature and pressure can be about 55 μg/ml. In another aspect, provided beverages have a high concentration of Ar (such as Ar encapsulated in a polymer or oil), such as about 0.01 μg/ml to about 1 mg/ml, 0.1 μg/ml to about 1 mg/ml, about 1 μg/ml to about 500 μg/ml, about 10 μg/ml to about 500 μg/ml, about 100 μg/ml to about 500 μg/ml, or about 200 μg/ml to about 500 μg/ml. For example, a formulation comprising oil-encapsulated Ar can have an Ar concentration of about 165 μg/ml at SATP.

In another aspect, the composition of the embodiment is characterized in that the noble gas content (at standard ambient temperature and pressure; SATP) is at least about 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM or 2.0mM. In some aspects, the noble gas is at a concentration of at least 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0mM. Thus, in some aspects, the composition comprises a noble gas content between about 5.0 and 50mM, 5.0 and 25mM, 5.0 and 20.0mM, 5.0 and 15mM or 5.0 and 10mM at SATP. In other aspects, the xenon content included in the composition is greater than about 5.0 mM at SATP, such as between about 5.0 and 50 mM, 5.0 and 25 mM, 5.0 and 20.0 mM, 5.0 and 15 mM, or 5.0 and 10 mM. In other aspects, the concentration of Ar included in the composition is greater than 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, or 2.0 mM. For example, the composition can be included at about 1.0 and 10 mM, 1.5 and 10 mM, 1.5 and 10 mM, 2.0 and 10 mM, or 2.0 and 5 mM Ar at SATP.

In other aspects, the compositions of the embodiments are defined by the content of the noble gas compared to the content of the aqueous component (e.g., water). For example, the composition may comprise a ratio of noble gas to aqueous component between about 1:20 and 4:1; about 1:10 and 4:1; about 1:9 and 4:1; about 1:2 and 4:1; 1:1 and 4:1; about 1.5:1 and 4:1; about 1:20 and 1:1; about 1:10 and 1:1; or about 2:1 and 3:1 (volume:volume). In some aspects, the ratio of noble gas to aqueous component is greater than 1:10; 1:9; or 1:5. In certain aspects, the ratio of noble gas to aqueous component is greater than 1:1 or greater than 2:1. Thus, in some specific aspects, the composition comprises a ratio of Xe to water between about 1:2 and 4:1; 1:1 and 4:1 or 1.5:1 and 4:1 or 2:1 and 3:1 (volume:volume), such as a ratio greater than about 2:1.

In certain embodiments, the composition of the embodiment (such as a beverage composition) further comprises other components, such as preservatives, flavorings, dyes, vitamins, antioxidants, plant or microbial extracts, salts (electrolytes, glycerol, sodium, potassium and chloride), ethanol, lipids, oils or mixtures thereof. Therefore, in some aspects, the beverage or other form of the embodiment is further defined as a herbal, vitamin or energy-providing nutritional health composition.

In another embodiment, the invention provides a kind of medicine or nutraceutical composition, it comprises the lipid (such as edible oil component) containing the soluble gas such as rare gas (for example xenon or argon).The example of the oil for this type of composition includes but is not limited to linseed oil, rapeseed oil, soybean oil, walnut oil, fish oil, safflower oil, sunflower oil, corn oil, cottonseed oil, peanut oil, palm oil or olive oil.In one aspect, oil can comprise polyunsaturated fatty acids (PUFA), such as the oil comprising at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more PUFA.In other aspects, oil comprises ω-3 fatty acids, which can contribute to the biological uptake of the gas of embodiment.In other aspects, the oil component is saturated or supersaturated with xenon or argon.

In another embodiment, the oil and gas composition may be further included in an emulsion. For example, the emulsion may include (a) 25% to 50% by volume, 60% by volume, 70% by volume, 80% by volume, 90% by volume, 95% by volume or more oil (oil includes soluble xenon or argon), and (b) 1% by volume, 2% by volume, 3% by volume, 5% by volume, 10% by volume, 20% by volume or 30% by volume to about 75% by volume or 85% by volume aqueous solution. In some aspects, the aqueous solution includes water (such as spring water), fruit juice, vegetable juice or other nutritional health beverages. In some aspects, the composition may further include phospholipids, detergents or protein components. In some aspects, the composition further includes phospholipids, detergents, flavorings, dyes, emulsifiers, co-emulsifiers and/or protein components. For example, detergents may be plant surfactants, synthetic detergents or bile acids. In some aspects, detergents are lithocholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, chenodeoxycholic acid or cholic acid. Examples of phospholipids for use according to the embodiments include, but are not limited to, egg phosphorylcholine (egg PC), soy PC, DPPC, or DOPC. Proteins that may be included are, for example, milk protein, whey protein isolate, soy protein isolate, potassium caseinate, egg albumin, (brown) rice protein, hydrolyzed beef protein isolate, pea protein isolate, hemp protein, or bovine serum albumin.

As described above in detail, in some aspects, provide and comprise solubility and insoluble trapped or free gas and can be liquid or semi-liquid oil composition.The gas that can be included in this type of composition includes but is not limited to rare gas (such as He, Ar, Kr, Ne or Xe), _CO , nitric oxide, isoflurane and sevoflurane (servoflurane).In preferred embodiments, lipid oil composition comprises soluble xenon or argon (and in some cases, also comprises insoluble or free Xe or Ar or other rare gas).In some aspects, composition comprises low oxygen or nitrogen content, or is substantially free of these gases.In some aspects, oil composition is with gas half saturation or saturation.In other aspects, lipid oil can be with gas supersaturation (such as when being exposed to atmosphere, makes gas bubble out from oil). In some aspects, the lipid oil compositions of the embodiments can comprise between about 10 and 500 mg per milliliter of oil (e.g., between about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg and about 150, 200, 250, 300, 350, 400, 450, or 500 mg per milliliter of oil). For example, at SATP, the oil can comprise from about 0.1 to about 50; about 0.1 to about 20, or about 1 to about 15 mg of Xe per milliliter of oil.

In certain specific aspects, the composition of the embodiment may comprise (a) 25% to 50% by volume of oil (e.g., olive oil or other desired lipid) (the oil contains soluble xenon or argon); (b) 50% to 75% by volume of aqueous solution; (c) 10-30 mg/ml of phospholipid (e.g., egg phosphocholine); (d) 10-50 mg/ml of protein (e.g., BSA); and (e) 1-5 mg/ml of detergent (e.g., lithocholic acid). In some aspects, the composition may further comprise a preservative, flavoring, vitamin, antioxidant, or plant extract.

In some aspects, the composition comprising a noble gas may be contained in an airtight container of any size or shape. In some cases, the container may be pressurized (e.g., with a noble gas such as xenon or argon). In some aspects, such containers may include a single portion or unit dose of the composition (e.g., in the form of a paste, gel, pill, tablet, or capsule). In other cases, the container may include multiple doses (e.g., multiple dose bottles or compartmentalized containers). In this latter case, it may be preferred that the container be pressurized and include a one-way valve for releasing the composition without exposing the entire contents to the atmosphere. In other aspects, such containers may include excess gas (e.g., argon or xenon) that maintains the pressure in the container when the composition dose is released. Such systems are described in, for example, U.S. Patent Publication No. 20030177784, which is incorporated herein by reference. In some cases, the container includes foil or similar impermeable materials, such as polymers, so that the container can be easily pressurized by effectively distributing liquid or semi-liquid.

In another embodiment, the present disclosure provides a unit dose of a composition of the embodiments contained in an airtight container. In certain aspects, the container can be a cream, gel, pill, tablet, or capsule. In another aspect, the container can be a bottle. For example, the container can enclose 1-5 ml; 5-25 ml; 25-100 ml; 125-300 ml; 355-500 ml; 500 ml to 1 liter or more of a composition, such as an emulsion of the embodiments.

In some other embodiments, a method for improving the health or well-being of a subject is provided, comprising administering to the subject (or providing to the subject) a composition according to an embodiment. For example, a noble gas composition of the embodiment of the amount sufficient to reduce the level of at least one inflammatory marker or cardiovascular risk (e.g., blood pressure) can be provided to the subject. For example, in some cases, the noble gas composition is administered in the form of an oral liquid or semi-liquid formulation to provide a daily dose equivalent to about 0.1 to 200 mg Xe per day. In another example, the Xe daily dose can be between about 1 to 100 mg, 1 to 50 mg, 1 to 25 mg, or 1 to 10 mg Xe per day. In another example, the noble gas composition is administered in the form of an oral formulation to provide a daily dose equivalent to about 0.1 to 5 g Xe per day. For example, the daily dose of Xe can be between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 and 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 grams of Xe per day. In another embodiment, the method comprises administering an oral Xe composition at a dose of between about 0.1 and 10; 0.1 and 5; 0.5 and 5; 1.0 and 3.0; or 2.0 and 3.0 mg (Xe) per kilogram per day. In other aspects, the method of the embodiment comprises administering (or providing to a subject) a dose of a noble gas (e.g., Xe) to achieve an initial maximum blood concentration of between 10 μm and 500 μm, between 10 μm and 100 μm, between 10 μm and 50 μm, or an initial maximum blood concentration of at least 50 or 100 μm. For example, in some aspects, the compositions of such dosage embodiments are administered daily, every two days, or weekly.

In another embodiment, the present disclosure provides a method for providing nerve or cardiovascular protection in a subject, which includes orally administering an effective amount of a composition according to any one of the embodiments. In some aspects, the subject (such as a human subject) has Alzheimer's disease, thrombotic stroke, ischemic stroke or cardiac hypertrophy, or is in these risks. In some aspects, the method further includes administering about 25-300ml (70-1,350mg, such as 100-1,200mg, 200-1000mg, 500-1000mg or 800-1,200mg) to the subject every day. The composition can be administered weekly, daily, twice a day, three times a day, every six hours, every three hours or every hour. Similarly, the composition can be administered for a week, two weeks, a month or a year. In some aspects, the method is a method for treating or preventing neurological disease or nerve damage (such as Alzheimer's disease or thrombotic or ischemic stroke). In other aspects, methods are provided for treating or preventing cardiac hypertrophy or providing protection against myocardial ischemia.

In another embodiment, the present disclosure provides a method of reducing the level of beta-amyloid protein in a subject, comprising administering an effective amount of xenon or argon to the subject. In certain aspects, xenon or argon is administered orally in the form of a composition according to any one of the embodiments. In other aspects, xenon or argon or other noble gases are administered by inhalation or injection (e.g., contained in liposomes).

In a related embodiment, the present disclosure provides a method for treating or preventing the progression of Alzheimer's disease in a subject, comprising administering to the subject an effective amount of xenon or argon or other noble gas. In certain aspects, the xenon or argon is administered orally. In other aspects, the xenon or argon or other noble gas (e.g., He, Ne, or Kr) is administered by inhalation or injection. In some aspects, the subject is a subject at risk of developing Alzheimer's disease, such as a subject having or diagnosed with a genetic predisposition to Alzheimer's disease.

In another embodiment, the present disclosure provides a method for preparing a composition for oral administration of xenon or argon, comprising (a) dissolving xenon or argon in an edible oil by mixing it with a gas to prepare an edible oil comprising soluble xenon or argon. In some aspects, the method further comprises (b) emulsifying the edible oil comprising the soluble gas in an aqueous solution (e.g., a solution comprising a detergent or other emulsifier) to prepare an emulsion comprising soluble xenon or argon. In some aspects, the oil and gas are mixed at a pressure between about 1 atm and 6 atm, 2 atm and 6 atm, or 2 atm and 4 atm and at a temperature between about 0°C and 37°C or 0°C and 25°C. In certain aspects, dissolving xenon or argon in an edible oil comprises saturating or supersaturating the oil with xenon or argon. In some aspects, the method further comprises bottling the oil or emulsion or capturing it in an airtight container (e.g., a bottle, capsule, or pill). For example, the container can be pressurized, such as a container pressurized at 2-6 atm. In certain aspects, steps (a)-(b) of the method can be performed under a xenon or argon or other noble gas atmosphere (e.g., He, Ne, or Kr).

In another embodiment, a method for preparing a substantially aqueous composition comprising a noble gas is provided, comprising: (a) incubating a noble gas (or a mixture of noble gases) with a water-soluble encapsulating molecule (e.g., a water-soluble polymer); and (b) exposing the encapsulated noble gas to an aqueous solution to prepare an aqueous composition comprising encapsulated noble gas. In some aspects, step (a) is performed at a pressure between about 2 atm and 10 atm (e.g., a pressure between about 2 atm and 8 atm, 2 atm and 5 atm, 2 atm and 4 atm, or a pressure of about 3 atm) at a temperature between about 25° C. or 4° C. and −180° C. (e.g., for a period of at least 1, 2, 3, 4, or more hours) to prepare the encapsulated noble gas. In some aspects, the incubation of step (a) is at a temperature between about 0°C and -150°C, -20°C and -150°C, -20°C and -100°C, -40°C and -100°C or at a temperature of about -80°C, and continues for at least 8 hours, 12 hours, 24 hours, 48 hours or continues for 8 hours to three days. In some cases, step (b) is included in the pressure between about 2atm and 10atm (such as the pressure between about 2atm and 8atm, 2atm and 5atm, 2atm and 4atm or the pressure of about 3atm) under the encapsulated noble gas is exposed to the aqueous solution at a temperature between about 20°C and 1°C. For example, step (b) can be carried out for at least 1, 2, 3, 4 or more hours. In other aspects, the exposure of step (b) is carried out under a pressure between about 2atm and 8atm, 2atm and 5atm, 2atm and 4atm or at a pressure of about 3atm. In some aspects, the exposure of step (b) is carried out at a temperature between about 15°C and 1°C, 10°C and 1°C, 8°C and 2°C, 6°C and 2°C, or at a temperature of about 4°C. In other aspects, the exposure of step (b) lasts for a period of at least 8 hours, 12 hours, 24 hours, 48 hours, or for a period of 8 hours to three days. In other aspects, the exposure of step (b) comprises exposing the encapsulated noble gas to an aqueous solution constituted by saturating with a noble gas or a mixture of noble gases. For example, the solution can be saturated with a noble gas by a method comprising: (i) obtaining a degassed aqueous solution; and (ii) exposing the degassed aqueous solution to a noble gas at a pressure between about 2 atm and 10 atm and a temperature between about 20°C and 1°C for at least 4 hours to prepare an aqueous solution saturated with a noble gas.

In another embodiment, the present disclosure provides a composition for providing neurological or cardiovascular protection in a subject, the composition comprising an edible oil saturated with a noble gas such as xenon or argon.

In one particular aspect of the embodiment, a method of delivering a noble gas in the form of a substantially aqueous composition is provided, comprising supplying beer, cider, soda water, or other carbonated beverage from a tap operably coupled to a noble gas tank (e.g., a Xe gas tank) such that the noble gas serves to maintain tap pressure and is thereby dissolved into the beverage (e.g., beer) for dispensing from the tap.

As used herein, "a" or "an" may mean one or more than one. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one.

Unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, the term "or" is used in the claims to mean "and/or," although the present disclosure supports definitions referring only to alternatives as well as "and/or." As used herein, "another" can mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the subjects studied.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

Figure 1. Echocardiographic measurements used to assess cardiac growth deficits and function in C57BL/6J wild-type (WT) and apolipoprotein-E (ApoE) knockout (KO) mice in response to pretreatment with Xe-enriched solution. Figure 1A. Interventricular septum (IVS; d) (mm) during diastole. Figure 1B. Left ventricular posterior wall diameter (LVPW; d) during diastole (mm). Figure 1C. Left ventricular (LV) volume (LVVol; d) during diastole (μl). *p < 0.05, **p < 0.01, KO/KO6w vs. WT/WT6w, respectively; #p < 0.05, KO control/vehicle vs. KO 6w; §p < 0.05, §§p < 0.01, §§§p < 0.001, KO Xenon vs. KO vehicle. WT = wild-type mice; KO = Apo E knockout mice.

Figure 2. Echocardiographic measurements of ejection fraction (EF%) (Figure 2A), fractional shortening (FS%) (Figure 2B), and cardiac output (ml/min) (Figure 2C). *p<0.05, **p<0.01, KO/KO6w vs. WT/WT6w, respectively; #p<0.05, KO control/vehicle vs. KO 6w; §p<0.05, §§p<0.01, §§§p<0.001, KO Xenon vs. KO vehicle.

Figure 3. Changes in cardiac mass morphology in wild-type (WT) and Apo-E knockout (KO) mice. Figure 3A. Corrected LV mass (mg). Figure 3B. Ratio of heart weight (HW) to body weight (BW) (mg/g). WT-6w (n=5): WT mice fed a regular diet at week 6. KO-6w (n=4): KO mice fed a regular diet at week 6. KO-6w control (n=5): KO mice fed a high-fat diet and administered PBS by gavage at week 6. KO-6w vehicle (n=7): KO fed a high-fat diet and vehicle at week 6. KO-6w xenon (n=6): KO fed a high-fat diet and administered a xenon-rich solution at week 6. *p<0.05, KO/KO6w compared to WT/WT6w, respectively; §p<0.05, KO6w xenon compared to KO6w vehicle.

Figure 4. Myocardial changes in cardiac function in wild-type and Apo-E knockout mice in response to pretreatment with Xe-enriched solution. WT (n=9): wild-type mice fed a regular diet at baseline; KO (n=25): Apo E-KO mice fed a regular diet at baseline. WT-6w (n=5): WT mice fed a regular diet at week 6. KO-6w (n=5): KO mice fed a regular diet at week 6. KO-6w control (n=4): KO mice fed a high-fat diet and administered PBS by gavage at week 6. KO-6w vehicle (n=7): KO fed a high-fat diet and vehicle at week 6. KO-6w xenon (n=6): KO fed a high-fat diet and administered xenon-enriched solution at week 6.

Figure 5. Levels of brain-derived neurotrophic factor (BDNF) in the heart (Figure 5A) and brain (Figure 5B) in response to pretreatment with Xe-rich solution. WT-6w (n=4): WT mice fed a regular diet at week 6. KO-6w (n=5): KO mice fed a regular diet at week 6. KO-6w vehicle (n=7): KO fed a high-fat diet at week 6 and administered by gavage with solution. KO-6w xenon (n=6): KO fed a high-fat diet at week 6 and administered by gavage with xenon. *p<0.05, **p<0.01, ***p<0.001, KO6w/vehicle/xenon compared to WT6w, respectively; #p<0.05, KO6w vehicle compared to KO 6w; §p<0.05, KO6w xenon compared to vehicle.

Figure 6. Levels of beta-amyloid in the blood (Figure 6A) and brain (Figure 6B) in response to pretreatment with a Xe-rich solution. WT-6w (n=5): WT mice fed a regular diet at week 6. KO-6w (n=4): KO mice fed a regular diet at week 6. KO-6w control (n=5): KO mice fed a high-fat diet at week 6 and administered PBS by gavage. KO-6w vehicle (n=7): KO fed a high-fat diet and vehicle at week 6. KO-6w xenon (n=6): KO fed a high-fat diet and administered a xenon-rich solution at week 6. *p<0.05, KO6w vehicle compared to WT6w; §§p<0.01, KO6w xenon compared to vehicle.

Figure 7. Xenon-enriched solution for increasing brain tolerance to ischemic injury. Figure 7A. Infarct size. Figure 7B. Percent infarct volume. Figure 7C. Limb placement. Figure 7D. Grid walking.

Figure 8. Example of an exemplary mouse experimental protocol.

Figure 9. Example of a rat experimental protocol.

Figure 10. Initial xenon locking experiments. (A) Schematic diagram showing the structure of CDs used to lock Xe. (B) Schematic diagram showing the physical properties of α-CD, β-CD, and γ-CD compared to Xe atoms. (C) Graph showing the results of a study to determine the effect of pressure on Xe encapsulation in CDs. (D) Graph showing the effect of temperature on Xe encapsulation in CDs.

Figure 11. The upper panel shows an exemplary protocol for preparing Xe-enhanced water using CD locking. The lower panel is a graph showing the volume of dissolved Xe per 5 ml of water achieved using the indicated method.

Figure 12. Echocardiographic measurements of mice treated with Xe water. The graph shows the results of echocardiographic measurements on: (1) WT mice fed a regular diet at week 6 (WT-6w, n=5); (2) Apo E knockout mice fed a high-fat diet and a normal water control (KO6w control, n=13); (3) vehicle control mice fed water containing only cyclodextrin (KO6w vehicle, n=5); or (4) Apo E knockout mice fed Xe-enriched water after 6 weeks of treatment (KO6w Xenon, n=5). The graph shows the measurements of ventricular septum (IVS) volume, left ventricular (LV) ejection fraction (EF) percentage, left ventricular posterior wall thickness (LVPW), LV fractional shortening (FS) percentage, LV volume, and cardiac output (CO).

Figure 13. Effect of Xe on ischemic stress. The graph in the left panel shows the results of a study measuring CKMB creatine kinase (CKMB) levels in control mice and Xe-treated animals. The graph in the right panel shows the results of a study measuring troponin expression levels in control mice and Xe-treated animals (mean ± SE, n = 5, ^§ p < 0.01).

Figure 14. Xe-enriched water reduces the expression of beta-amyloid in the brain and blood. The graph shows the amount of beta-amyloid found in plasma (left panel) or brain (right panel) after 6 weeks of treatment. WT6w (n=10) indicates mice fed a regular diet at week 6; KO-6w (n=5) indicates ApoE knockout mice fed a regular diet at week 6; KO-6w vehicle (n=7) indicates ApoE knockout mice fed a high-fat diet and water containing cyclodextrin at week 6; KO-6w xenon (n=6) indicates ApoE knockout mice fed a high-fat diet and Xe-enriched water at week 6. *p<0.05, KO6w vehicle compared to WT6w; §§p<0.01, KO6w xenon compared to vehicle.

Detailed Description of the Invention

Noble gases such as xenon (Xe) and argon (Ar) are attractive because they can improve health and well-being at low doses and are also potential therapeutic agents if given in higher doses. However, there are a wide range of difficulties when trying to administer such gases to humans. Specifically, the amount of gas that can be administered by inhalation is a very serious limitation. Similarly, because these gases are chemically neutral and non-polar, formulating them into other delivery vehicles has proven to be a very difficult challenge.

Disclosed herein are solutions rich in noble gases such as Xe or Ar for oral delivery to humans and other animals of interest in some cases. In some aspects, these solutions use lipids including, but not limited to, oils from known foods (such as edible oils (e.g., rich in omega-3 oils)) as carrier media to provide increased solubility for such noble gases. Alternatively or additionally, the aqueous solution may be combined with a noble gas encapsulated in a polymer (e.g., cyclodextrins including: α-cyclodextrin: 6-membered sugar ring molecules, β-cyclodextrin: 7-membered sugar ring molecules, and γ-cyclodextrin: 8-membered sugar ring molecules and various derivatives). Derivatives of cyclodextrin include, but are not limited to, methyl-β-cyclodextrin, random methylated-β-cyclodextrin, dimethyl-β-cyclodextrin, random dimethylated-β-cyclodextrin, trimethyl-β-cyclodextrin; acetylated dimethyl-β-cyclodextrin: 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin; hydroxybutenyl-β-cyclodextrin: 2,3-dihydroxypropyl-β-cyclodextrin; -cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin; glucosyl-β-cyclodextrin; maltosyl-β-cyclodextrin; glucuronyl-glucosyl-β-cyclodextrin; 2, hydrophobic CDs that can be combined with lipids/oils: alkylated β-cyclodextrin, 2,6-di-O-ethyl-β-cyclodextrin, 2,3,6-tri-O-ethyl-β-cyclodextrin; acylated β-cyclodextrin: 2,3,6-tri-O-acyl (C ₂ -C ₁₈ )-β-cyclodextrin, 2,3,6-tri-O-butyryl-β-cyclodextrin, 2,3,6-tri-O-pentanoyl-β-cyclodextrin, 2,3,6-tri-O-octyl-β-cyclodextrin, O-carboxymethyl-O-ethyl-β-cyclodextrin, β-cyclodextrin sulfate; sulfobutyl ether-β-cyclodextrin; and sulfobutyl ether-β-cyclodextrin. The studies herein demonstrated that both types of solutions were able to provide significant levels of noble gases in aqueous solution-based systems. Following oral delivery, these solutions had a preventive effect in brain and heart tissue. For example, the Xe solution was shown to increase tissue tolerance to ischemic damage and provide cardioprotection. In a model system for heart disease, the composition not only had a direct positive effect on markers of cardiac function (see, for example, FIG12 ), but also reduced overall blood pressure in mice lacking Apo-E (results are shown in Table 2). In addition, these solutions also demonstrate biologically significant (therapeutic) effects in a model system for Alzheimer's disease. Specifically, Xe-based compositions show an effective reduction in the beta-amyloid load in the blood and brain tissue of treated animals (Figure 14). Therefore, compositions comprising dissolved or captured Ar or Xe that can be used to provide an effective amount of cardiovascular and neuroprotective effects to a subject are provided.

The noble gas compositions and treatment methods disclosed herein provide new approaches for increasing well-being and for treating and preventing a wide range of chronic diseases. Importantly, the compositions provided herein have been shown to provide effective cardioprotective and hypotensive effects at specific doses that are suitable for treating patients with heart disease or those at high risk of stroke. Similarly, the noble gas compositions provided have been shown to reduce the amyloid load in body tissues and therefore provide unique treatments for treating and preventing the onset of Alzheimer's disease. Given the availability of convenient aqueous formulations, effective amounts of non-toxic noble gases can now be easily delivered via oral formulations. Given the stability of the formulations, multiple doses can be easily dispensed without the need for complex packaging, dosing systems, or even freezing, improving well-being by increasing or enhancing certain physiological parameters at certain doses (e.g., reducing inflammation, reducing stress, increasing relaxation, lowering blood pressure, clearing distractions) to therapeutic/preventative effects at other doses (e.g., improving cardiac and neurological function). Thus, a range of compositions intended for primarily oral delivery, including but not limited to beverages, can be used for gaseous delivery to provide effective and convenient nutraceuticals or therapeutics that are easily incorporated into standard preventative treatments such as dietary changes and exercise. Furthermore, because of the ease of delivery and lack of toxic formulations, the compositions provided herein may be administered with little or no supervision by a medical professional.

I. Pharmaceuticals and nutritional supplements

The medicine and nutraceutical compositions provided herein comprise an effective amount of tissue or cell protective gas, such as Xe or Ar, and may include optional other agents, such as other gases, dissolved or dispersed in an acceptable carrier. In some aspects, such acceptable carriers include components formulated to increase or control the content of soluble gas to a desired level, such as lipids, including edible oils or locking molecules as described in detail above. The phrase "containing" means dissolving, emulsifying, suspending, capturing, and other similar means of obtaining a solution for primary oral delivery with a noble gas. The phrase "acceptable carrier" refers to a molecular entity and composition that does not produce harmful allergic reactions or other adverse reactions when administered to animals such as humans (e.g., taken in by them) as appropriate. The preparation of medicines or nutraceutical compositions containing noble gases is described in detail herein. Further adding active or inactive ingredients to such compositions will be known to those skilled in the art according to the present disclosure, and as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

"Acceptable carrier" may include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterials, antifungals), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, materials like these, and combinations thereof (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference) as known to those of ordinary skill in the art. Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic agents or pharmaceutical compositions is encompassed. In general, the carriers of embodiments of the present invention all comprise oil-based components that contain dissolved noble gases, such as Ar or Xe.

In certain embodiments, pharmaceutical compositions can comprise different types of carriers, depending on whether they are to be used in solid, liquid or aerosol form, and whether they need to be sterile for routes of administration such as injection. In certain embodiments, as will be known to those of ordinary skill in the art, pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, superficially, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, superficially, topically ...

In other embodiments, eye drops, nasal solutions or sprays, aerosols or inhalants can be used in embodiments of the present invention. Such compositions are generally designed to be compatible with target tissue types. In non-limiting examples, nasal solutions are generally aqueous solutions designed to be applied to the nasal passages in the form of drops or sprays. Nasal solutions are prepared so that they are similar to the nasal endocrine system in many aspects, so that normal ciliary action is maintained. Therefore, in a preferred embodiment, aqueous nasal solutions are generally isotonic or slightly buffered to maintain a pH value of about 5.5 to about 6.5. In addition, if necessary, antimicrobial preservatives (similar to those used in ophthalmic preparations), drugs or suitable drug stabilizers can be included in the preparation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

Other formulations suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, typically medicated, for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. Generally speaking, for suppositories, conventional carriers may include, for example, polyglycols, triglycerides, or combinations thereof. In certain embodiments, the suppository may be formed from a mixture containing, for example, an active ingredient in a range of about 0.5% to about 10%, and preferably about 1% to about 2%.

Sterile injectable solutions can also be prepared by incorporating the active compound into a suitable solvent together with the various other ingredients listed above in the required amount and then filtering and sterilizing. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions, or emulsions, preferred preparation methods are vacuum drying or freeze-drying techniques, which produce a powder of the active ingredient from its previously sterile-filtered liquid medium plus any other required ingredients. If necessary, the liquid medium should be properly buffered and, before injection, first make the liquid diluent isotonic with enough normal saline or glucose. The preparation of highly concentrated compositions for direct injection is also encompassed, wherein using DMSO as a solvent is envisioned to produce extremely rapid penetration, thereby delivering high concentrations of active agents to small areas. In specific embodiments, the extended absorption of the injectable composition can be produced by using, in the composition, an agent such as aluminum monostearate, gelatin, or a combination thereof, that delays absorption.

The composition must be stable under the conditions of manufacture and storage and must be conservative against the contaminating effects of microorganisms such as bacteria and fungi. In the case of clinical applications of liposomes (e.g., liposomes containing gas), solutions of the therapeutic composition can be prepared in water appropriately mixed with surfactants such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under typical storage and use conditions, these preparations contain preservatives to prevent microbial growth. The therapeutic composition of the present invention is preferably administered in the form of an injectable composition in the form of a liquid solution or suspension; solid forms suitable for dissolving in or suspending in a liquid prior to injection can also be prepared. These preparations can also be emulsified. Typical compositions for such purposes include a pharmaceutically acceptable carrier. For example, the composition may contain 10 mg, 25 mg, 50 mg, or up to about 100 mg of human serum albumin per milliliter of phosphate-buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients (including salts), preservatives, buffers, and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, saline solutions, parenteral vehicles (such as sodium chloride), Ringer's dextrose, etc. Intravenous vehicles include fluids and nutrient supplements. Preservatives include antimicrobials, antioxidants, chelating agents, and inert gases. The pH value and exact concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters. Other formulations are suitable for oral administration. Oral formulations include typical excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The composition will typically be in the form of a solution or suspension.

The therapeutic compositions of embodiments of the present invention may include classical pharmaceutical formulations. Administration of the therapeutic compositions according to the present invention will be carried out by any common route, as long as the target tissue can be reached by the route. In this case, intravenous injection or infusion may be preferred. Such compositions will typically be administered in the form of a pharmaceutically acceptable composition comprising a physiologically acceptable carrier, buffer or other excipient.

Oral preparations

In certain preferred embodiments, the composition of the embodiment is administered orally and is formulated to promote such oral administration (e.g., as a beverage formulation). Therefore, in some embodiments, compositions (emulsions such as oil-encapsulated gas or polymer-encapsulated gas) may include, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shell gelatin capsules), sustained release formulations, buccal compositions, tablets, elixirs, suspensions, syrups, or combinations thereof. Oral compositions may be directly food or drinkable products (e.g., together with fruit juice or wine) and combined. Preferred carriers for oral administration include inert diluents, absorbable edible carriers, or combinations thereof. In other aspects, oral compositions may be prepared into syrups or elixirs. Syrups or elixirs and may include, for example, at least one activating agent, sweetener, preservative, flavoring, dye, preservative, or combinations thereof.

In other aspects, the composition comprising a dissolved noble gas such as Xe or Ar can be formulated as a capsule or tablet for oral administration. In some aspects, the capsule is substantially gas impermeable, and preferably the capsule is formulated to dissolve in the gastrointestinal tract of a subject.

In certain preferred embodiments, the oral composition may include one or more binders, excipients, disintegrants, lubricants, flavorings, and combinations thereof. In certain embodiments, the composition may include one or more of the following: a binder, such as gum tragacanth, gum arabic, corn starch, gelatin, or a combination thereof; an excipient, such as calcium hydrogen phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or a combination thereof; a disintegrant, such as corn starch, potato starch, alginic acid, or a combination thereof; a lubricant, such as magnesium stearate; a sweetener, such as sucrose, lactose, saccharin, or a combination thereof; a flavoring, such as peppermint oil, wintergreen oil, cherry flavoring, orange flavoring, etc.; or a combination of the foregoing. When the dosage unit form is a capsule, it may also contain a carrier, such as a liquid carrier, in addition to the above types of materials. Various other materials may exist in a coating form or otherwise change the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both.

The composition may contain various antioxidants to delay oxidation of one or more components. Additionally, prevention of the effects of microorganisms may be achieved by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

In embodiments where the composition is in liquid form, the carrier may include a solvent or dispersion medium, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Proper fluidity may be maintained, for example, by using a coating such as lecithin; by maintaining the desired particle size by dispersing in a carrier such as a liquid polyol or lipid; by using a surfactant such as hydroxypropylcellulose; or a combination of such methods. In many cases, it will be preferred to include an isotonic agent such as a sugar, sodium chloride, or a combination thereof.

Other ingredients in pharmaceutical and nutritional health preparations

The oral noble gas formulations of the embodiments may include other components as described in detail herein below. It is contemplated that the compositions may include, for example, at least or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 113%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% of such other components. In some aspects, the other components comprise less than about 20%, 10%, 5% or less of the total composition on a weight:weight or volume:volume basis.

In some embodiments, micronutrients may include, but are not limited to, L-carnitine, choline, coenzyme Q10, alpha-lipoic acid, omega-3 fatty acids, pepsin, phytase, trypsin, lipase, protease, cellulase, and combinations comprising at least one of the foregoing micronutrients.

Antioxidants may include materials that scavenge free radicals. In some embodiments, exemplary antioxidants may include citric acid, rosemary oil, vitamin A, vitamin E, vitamin E phosphate, tocopherol, di-alpha-tocopheryl phosphate, tocotriol, alpha lipoic acid, dihydrolipoic acid, lutein, beta cryptoxanthin, lycopene, lutein, zeaxanthin, astaxanthin, beta-carotene, carotenes, mixed carotenoids, polyphenols, flavonoids, and combinations comprising at least one of the foregoing antioxidants.

Exemplary nutrients can also include amino acids such as L-tryptophan, L-lysine, L-leucine, L-methionine, 2-aminoethanesulfonic acid (taurine), and L-carnitine; creatine; glucuronolactone; inositol; and combinations comprising at least one of the foregoing nutrients.

Phytochemicals ("phytonutrients") are plant-derived compounds that may provide a beneficial effect on the health or well-being of a consumer. Phytochemicals include plant-derived antioxidants, phenolic compounds, including monophenols and polyphenols, and the like. Exemplary phytochemicals include lutein, lycopene, carotenes, anthocyanins, capsaicin, flavonoids, hydroxycinnamic acids, isoflavones, isothiocyanates, monoterpenes, chalcone, coumestane, dihydroflavonols, flavonoids, flavanols, quercetin, flavanones, flavones, flavan-3-ols (catechin, epicatechin, epigallocatechin, epigallocatechin gallate, etc.), flavonals (anthocyanidins, cyanidins, etc.); phenolic acids; phytosterols, saponins, terpenes (carotenoids), and combinations comprising at least one of the foregoing phytochemicals.

Phytochemicals can be provided in substantially pure or isolated form or in the form of natural plant extracts. Suitable plant extracts containing one or more phytochemicals include peel extracts (grape, apple, crabapple, etc.), green tea extract, white tea extract, green coffee extract, and combinations comprising at least one of the foregoing extracts.

Various herbs, aromatic plants or plant parts, or extracts thereof, may also be included in the composition for various reasons, such as flavoring or their potential health benefits. Exemplary herbs include Echinacea, Goldenseal, Calendula, Rosemary, Thyme, Kava Kava, Aloe Vera, Bloodroot, Grapefruit Seed Extract, Black Cohosh, Ginseng, Guarana, Cranberry, Ginko Biloba, St. John's Wort, Evening Primrose Oil, Yohimbe Bark, Green Tea, Ephedra, Maca, Bilberry, extracts thereof, and combinations comprising at least one of the foregoing herbs. Other plant extracts included in the compositions of the embodiments include, but are not limited to, extracts or components from: Acai, Spirulina, Chlorella, Wheat Grass, Black Soy, Turmeric, Chia Seed, Coconut Oil, Cocoa, Bilberry, Egg, Beet Juice, Mustard Sprouts, Sweet Potato, Red Wine, Avocado, Blue Berries, Black Berries, Almonds, Green Tea, Lentils, Black Beans, and Aloe Vera. For example, in some aspects, the compositions of the embodiments include a protein source selected from the group consisting of whey protein concentrate, potassium caseinate, egg albumin, soy isolate and whey isolate, (brown) rice protein, hydrolyzed beef protein isolate, pea protein isolate, and hemp protein.

In other aspects, the compositions of the embodiments may include a diuretic, such as watermelon extract or dandelion leaf extract (eg, 4:1).

In some embodiments, the composition may have a Brix measurement of about 8.0 to about 9.5°Brix, specifically about 8.5 to about 8.9°Brix, as measured by a Brix refractometer at 20°C. In another embodiment, the composition has a Brix measurement of about 7.5 to about 9.1°Brix, specifically about 7.9 to about 8.3°Brix, as measured by a Brix hydrometer at 20°C.

electrolytes

It is contemplated that various aspects of the composition of the present invention include electrolytes. Exemplary electrolytes include salts of Group I and Group II metals of the periodic table, preferably inorganic and organic salts of sodium, potassium, calcium and/or magnesium. Examples of such salts include, but are not limited to, sodium acetate, acid sodium citrate, acid sodium phosphate, sodium aminosalicylate, sodium bicarbonate, sodium bromide, sodium chloride, sodium citrate, sodium lactate, sodium phosphate, sodium salicylate, sodium sulfate (anhydrous), sodium sulfate (glauber's salt), potassium acetate, potassium bicarbonate, potassium bromide, potassium chloride, potassium citrate, potassium-D-gluconate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, calcium acetate, calcium chloride, calcium citrate, calcium-D-gluconate, calcium lactate, calcium levulinate, calcium hydrogen phosphate, magnesium chloride and magnesium sulfate. In one aspect, the electrolyte is sodium chloride, potassium dihydrogen phosphate and magnesium sulfate, and when present in 8 ounces of volume, is included in an amount of about 50 mg to about 500 mg, about 10 mg to about 200 mg and about 10 mg to about 200 mg, respectively. In other aspects, sodium chloride is included in an amount ranging from about 50 mg to about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg when present in an 8 ounce volume, and magnesium sulfate and potassium dihydrogen phosphate are included in an amount ranging from about 10 mg to about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, or about 200 mg when present in an 8 ounce volume. The compositions of the embodiments may also include glycerin.

Other electrolyte and liquid formulations of the compositions are provided in US Pat. Nos. 4,981,687, 5,089,477, 5,147,650, 5,236,712, and 5,238,684, each of which is incorporated herein by reference.

vitamins and minerals

Various aspects of the compositions of the contemplated embodiments include vitamins and/or minerals. Included vitamins include, but are not limited to, vitamins and co-vitamins such as vitamin A (β-carotene), choline, vitamin B1 (thiamine), vitamin B2 (riboflavin, vitamin G), vitamin B3 (niacin, vitamin P, vitamin PP), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, or pyridoxal), vitamin B7 (biotin, vitamin H), vitamin B9 (folic acid, folate, vitamin M), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol or cholecalciferol), vitamin E (tocopherol), and vitamin K (naphthoquinones). Minerals that may be included include, but are not limited to, calcium (Ca), chlorine (Cl-), chromium (Cr), cobalt (Co) (as part of vitamin B12), copper (Cu), iodine (I), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), phosphorus (P), potassium (K), selenium (Se), sodium (Na), and zinc (Zn).

Vitamin A, for example, helps form and maintain healthy teeth, bones, and soft tissues, mucous membranes, and skin. It's also called retinol because it produces the pigment necessary for the functioning retina. It promotes good vision, especially in dim light. Beta-carotene, a precursor of vitamin A, has antioxidant properties, helping the body deal with unstable chemicals called free radicals.

Thiamine (B-1) helps the body's cells convert carbohydrates into energy. It is also necessary for heart function and healthy nerve cells, including those in the brain. Riboflavin (B-2) works with the other B vitamins and is important for body development and red blood cell production. Similar to thiamine, it helps release energy from carbohydrates. Niacin (B-3) is a B vitamin that helps maintain healthy skin and nerves. It is also important for the conversion of food into energy and may have a cholesterol-lowering effect. Vitamin B-6, also known as pyridoxine, helps form red blood cells and maintain normal brain function. It also helps with the synthesis of antibodies in the immune system. Like the other B vitamins, vitamin B-12 is important for metabolic processes involved in, for example, red blood cell formation. Pantothenic acid is necessary for food metabolism. It is also required for the synthesis of hormones and cholesterol. Biotin is necessary for protein and carbohydrate metabolism, as well as the synthesis of hormones and cholesterol. Folate (folic acid) works with vitamin B-12 in the production of red blood cells and is necessary for DNA synthesis.

Vitamin C (also known as ascorbic acid) promotes healthy teeth and gums, aids in iron absorption, and helps maintain normal connective tissue. It also promotes wound healing and is an antioxidant.

Vitamin D promotes the body's absorption of calcium, which is necessary for normal development and maintenance of healthy teeth and bones. It also helps maintain adequate blood levels of calcium and phosphorus, minerals necessary for many functions.

Vitamin E is also known as tocopherol and is an antioxidant. It is also important in the formation of red blood cells and the use of vitamin K.

Therefore, it is desirable to incorporate various vitamin types into various aspects of the compositions of the present invention. In one embodiment, vitamin B1 (thiamine) is included in an amount ranging from about 0.1 mg to about 5 mg when present in an 8 ounce volume; vitamin B2 (riboflavin) is included in an amount ranging from about 0.1 mg to about 5 mg when present in an 8 ounce volume; vitamin B3 (niacin) is included in an amount ranging from about 1 mg to about 50 mg when present in an 8 ounce volume; vitamin B5 (pantothenic acid) is included in an amount ranging from about 1 mg to about 50 mg when present in an 8 ounce volume; vitamin B6 is included in an amount ranging from about 0.1 mg to about 5 mg when present in an 8 ounce volume; and vitamin B12 is included in an amount ranging from about 1 μg to about 50 μg when present in an 8 ounce volume. In another embodiment, vitamins B1, B2, and B6 are included in amounts of about 0.1 mg to about 2 mg, about 3 mg, about 4 mg, or about 5 mg when present in an 8 ounce volume; vitamins B3 and B5 are included in amounts of 1 mg to about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg when present in an 8 ounce volume; and vitamin B12 is included in amounts of 1 μg to about 10 μg, about 20 μg, about 30 μg, about 40 μg, or about 50 μg when present in an 8 ounce volume.

In another embodiment, a composition of the present invention is provided further comprising vitamin A, said vitamin A being included in an amount ranging from about 50 IU to about 1000 IU when present in an 8 ounce volume. In one aspect, vitamin A is included in an amount ranging from about 50 IU to about 100 IU, about 200 IU, about 300 IU, about 400 IU, about 500 IU, about 600 IU, about 700 IU, about 800 IU, about 900 IU, or about 1000 IU when present in an 8 ounce volume.

In another embodiment, a composition of the embodiments is provided further comprising vitamin C, said vitamin C being included in an amount ranging from about 10 mg to about 100 mg when present in an 8 ounce volume. In some aspects, vitamin C is included in an amount ranging from 10 mg to about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg when present in an 8 ounce volume.

In another embodiment, a composition of the present invention is provided that further comprises vitamin E, said vitamin E being included in an amount ranging from about 1 IU to about 50 IU when present in an 8 ounce volume. In one aspect, vitamin E is included in an amount ranging from about 1 IU to about 10 IU, about 20 IU, about 30 IU, about 40 IU, or about 50 IU when present in an 8 ounce volume.

amino acids

In another embodiment, the above-mentioned composition is provided, which further comprises one or more amino acids selected from the group consisting of alanine, arginine, creatine, cysteine, glycine, histidine, glutamine, lysine, methionine, ornithine, leucine, isoleucine, serine, tyrosine, asparagine, aspartic acid, threonine, proline, tryptophan, valine, phenylalanine, and selenocysteine. For example, creatine can be supplied in various forms, such as creatine monohydrate, creatine magnesium chelate, or creatine nitrate.

For example, glutamine is included in an amount of about 5 mg to about 100 mg, or about 5 mg to about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg when present in an 8 ounce volume.

Additionally, it is contemplated that cysteine may be included in the compositions of the present invention. For example, cysteine may be included in an amount ranging from about 10 mg to about 100 mg when present in an 8 ounce volume.

carbohydrate

As mentioned above, it is contemplated that in some aspects a carbohydrate source may be included in the compositions of the present invention. Exemplary carbohydrates include, but are not limited to, monosaccharides, disaccharides, oligosaccharides, and glucose polymers. Modified carbohydrates, such as sucralose, are also contemplated. In another aspect, the carbohydrates of the formulation are derived from citric acid.

flavoring

One or more flavoring agents can be added to the composition of the present invention to increase its palatability. Any natural or synthetic flavoring agent can be used in the present invention. For example, one or more plant and/or fruit flavoring agents can be used herein. As used herein, such flavoring agents can be synthetic or natural flavoring agents.

Exemplary fruit flavorings include exotic and lactonic acid flavorings, such as passion fruit flavoring, mango flavoring, pineapple flavoring, cupuacu flavoring, guava flavoring, cocoa flavoring, papaya flavoring, peach flavoring, and apricot flavoring. In addition to these flavorings, a variety of other fruit flavorings can be used, such as apple flavoring, citrus flavoring, grape flavoring, raspberry flavoring, cranberry flavoring, cherry flavoring, grapefruit flavoring, etc. These fruit flavorings can be derived from natural sources, such as fruit juices and flavoring oils, or can alternatively be synthetically prepared.

Exemplary botanical flavorings include, for example, tea leaves (e.g., black and green), aloe vera, guarana, ginseng, ginkgo, hawthorn twigs, hibiscus, rose hips, chamomile, mint, fennel, ginger, licorice, lotus seeds, schisandra, saw palmetto, dandelion, safflower, St. John's wort, turmeric, cardamom, nutmeg, cassia bark, buchu, cassia bark, jasmine, hawthorn, chrysanthemum, water chestnuts, sugar cane, lychee, bamboo shoots, vanilla, coffee, and the like.

Flavorings can also include blends of various flavorings. If desired, the flavorings in the flavoring agent can form emulsion droplets, which are then dispersed in the beverage composition or concentrate. Because the specific gravity of these droplets is generally less than that of water, and therefore a separate phase will be formed, a weighting agent (which can also serve as a turbidity agent) can be used to keep the emulsion droplets dispersed in the beverage composition or concentrate. Examples of such weighting agents are brominated vegetable oil (BVO) and resin esters, particularly ester gums. For further description of the uses of weighting agents and turbidity agents in liquid beverages, see L.F.Green, Developments in Soft Drinks Technology, Vol. 1, Applied Science Publishers Ltd., pp. 87-93 (1978) (incorporated herein by reference). Typically, flavorings are conventionally available in the form of concentrates or extracts or in the form of synthetically prepared flavoring esters, ethanol, aldehydes, terpenes, etc.

The amount of flavoring agent used will vary depending on the agent used and the desired strength of the finished product. Amounts can be readily determined by one skilled in the art. Generally, if used, the flavoring agent should be present at a level of about 0.0001% to about 0.5%.

flavanols

Flavanols are naturally occurring substances found in a variety of plants, such as fruits, vegetables, and flowers. Flavanols useful in the present invention can be extracted from, for example, fruits, vegetables, green tea, or other natural sources by any suitable method known to those skilled in the art. Flavanols can be extracted from a single plant or a mixture of plants. Plants containing flavanols are known to those skilled in the art.

The amount of flavanols in the various aspects of the compositions of the present invention may vary. However, preferably from about 0.001% to about 5%, by weight of the composition, of one or more flavanols are used.

Perceptual agents

In some aspects, the composition includes a "sensate" trigeminal stimulant, which can alter the taste of, for example, a beverage composition and reduce the perception of off-flavor. Sensates include "warming agents," compounds that provide a warming sensation; "cooling agents," compounds that provide a cooling sensation; and "tingling agents," compounds that provide a tingling, stinging, or numb sensation. The sensate can be a warming agent, a cooling agent, a tingling agent, or any combination comprising at least one of the foregoing sensates.

Warming agents can be selected from a variety of compounds known to provide a warming sensory signal to a user. These compounds provide a perceived sense of warmth, particularly in the oral cavity, and often enhance the perception of flavorings, sweeteners, and other sensory components. Suitable warming agents include those having at least one allyl vinyl component that can bind to oral receptors. Examples of suitable warming agents include vanillyl alcohol n-butyl ether (TK-1000, supplied by Takasago Perfumery Company Ltd., Tokyo, Japan); vanillyl alcohol n-propyl ether; vanillyl alcohol isopropyl ether; vanillyl alcohol isobutyl ether; vanillyl alcohol n-amino ether; vanillyl alcohol isoamyl ether; vanillyl alcohol n-hexyl ether; vanillyl alcohol methyl ether; vanillyl alcohol ethyl ether; gingerol; shogaol; shogaol; zingerone; zingerone; capsaicin; dihydrocapsaicin; nordihydrocapsaicin; homocapsaicin; homodihydrocapsaicin; ethanol; isopropyl alcohol; isoamyl alcohol; benzyl alcohol; glycerol; chloroform; eugenol; cinnamon oil; cinnamaldehyde; phosphate derivatives thereof, etc., or a combination comprising at least one of the foregoing warming sensates.

A variety of well-known cooling agents can be used in the compositions of the present invention. Exemplary cooling agents include menthol, xylitol, erythritol, menthane, menthone, menthyl acetate, menthyl salicylate, N,2,3-trimethyl-2-isopropylbutanamide (WS-23), N-ethyl-p-menthane-3-carboxamide (WS-3), menthyl succinate, 3,1-menthoxypropane 1,2-diol, and glutaric acid esters, or the like, or a combination comprising at least one of the foregoing cooling sensations.

Tingling agents can be used in beverage compositions to provide a tingling, stinging, or numb sensation to the user. Exemplary tingling agents include oleoresin of scutellaria baicalensis or scutellaria spp. (Splendens spp.), in which the active ingredient is scutellarin; Japanese pepper extract (Zanthoxylum bungeanum), including components known as sanshool-I, sanshool-II, and sanshoamide; black pepper extract (Piper nigrum), including the active ingredients piperine and piperine; Echinacea purpurea extract; Zanthoxylum bungeanum extract; red pepper oleoresin; and the like, or a combination comprising at least one of the foregoing tingling sensates.

Sensates can be present in a composition such as a composition (e.g., a beverage composition) in an amount of about 0.01 to about 10 weight %, specifically about 0.1 to about 5.0, and more specifically about 1.0 to about 3.0 weight %, based on the total weight of the beverage composition.

stimulants

In some aspects, the compositions of the embodiments include a stimulant or an agent that provides a sensation of increased energy levels. For example, the composition may include caffeine (anhydrous), green tea extract (Camellia sinensis) (leaf, e.g., 45% EGCG), Hoodia gordonii, Advantra (Citrusaurantium, e.g., 60% synephrine alkaloids), L-aminoethanesulfonic acid, ginseng powder, glucuronolactone, adenosine, octopamine, L-carnitine, yohimbine, vinpocetine, NADH, Evodiamine Cinnulin, cinnamon bark extract (cinnamonum burmannii), banaba leaf extract, or [chromium (in the form of chromium cysteine dinicotinate).

dyes

One or more coloring agents on a small scale can be used in the composition of the present invention. Preferably, FD&C dyes (e.g., yellow #5, blue #2, red #40) and/or FD&C lakes are used. By adding lakes in other powdered ingredients, all particles, particularly colored iron compounds are completely and evenly dyed, and a uniformly dyed beverage is obtained. Preferred lake dyes that can be used in the present invention are lakes approved by the FDA, such as lake red #40, yellow #6, blue #1, etc. In addition, mixtures of FD&C dyes or FD&C lake dyes and other conventional foods and food coloring agents can be used. Riboflavin and b-carotene can also be used. In addition, other operable natural coloring agents include, for example, fruits, vegetables and/or plant extracts, such as grapes, blackcurrants, aronia, carrots, beetroot, red cabbage and hibiscus.

The amount of coloring agent used will vary depending on the agent used and the desired intensity of the finished product. Amounts can be readily determined by one skilled in the art. Generally, if used, the coloring agent should be present at a level of from about 0.0001% to about 0.5%, preferably from about 0.001% to about 0.1%, and most preferably from about 0.004% to about 0.1%, by weight of the composition.

preservative

Preservatives may or may not be required for use in the compositions of the present invention. Preservatives can be avoided using techniques such as aseptic and/or clean fill processing. However, one or more preservatives may be optionally added to the compositions of the present invention. Preferred preservatives include, for example, sorbates, benzoates, and polyphosphate preservatives (e.g., sodium hexametapolyphosphate).

Preferably, when a preservative is used herein, one or more sorbate or benzoate preservatives (or mixtures thereof) are used. Suitable sorbate and benzoate preservatives for use in the present invention include sorbic acid, benzoic acid, and salts thereof, including but not limited to calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and mixtures thereof.

Where the composition comprises a preservative, the preservative is preferably included at a level of from about 0.0005% to about 0.5%, more preferably from about 0.001% to about 0.4% preservative, more preferably from about 0.001% to about 0.1%, even more preferably from about 0.001% to about 0.05%, and most preferably from about 0.003% to about 0.03% preservative by weight of the composition. Where the composition comprises a mixture of one or more preservatives, the total concentration of such preservatives is preferably maintained within these ranges.

acidifier

If desired, the compositions of the present invention may optionally include one or more acidulants. An amount of an acidulant may be used to maintain the pH of the composition. In various aspects, the pH of the compositions of the present invention is from about 2 to about 9, from about 2.5 to about 8.5, from about 3 to about 8, from about 0.3.5 to about 7.5, from about 4 to about 7, from about 4.5 to about 6.5, or from about 5 to about 6.

The acidity of the composition can be adjusted and maintained within the necessary range by known and conventional methods (e.g., using one or more of the acidulants mentioned above). Typically, the acidity within the above-recited range is a balance between the maximum acidity for microbial inhibition and the optimal acidity for the desired beverage flavoring.

Organic and inorganic edible acids can be used to adjust the pH value of the beverage and can be added to the acid serving as a part of the second component herein. Acid can exist in its undissociated form or alternatively in its corresponding salt form (e.g., potassium hydrogen phosphate or sodium hydrogen phosphate, potassium dihydrogen phosphate or sodium dihydrogen phosphate salt). Preferably, acid is an edible organic acid, including citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, or its mixture. Most preferably, acid is citric acid and malic acid.

Acidulants may also act as antioxidants for stabilizing beverage components.Examples of commonly used antioxidants include, but are not limited to, ascorbic acid, EDTA (ethylenediaminetetraacetic acid), and salts thereof.

The amount of acidulant used will vary depending on the agent used and the desired pH of the finished product. The amount can be readily determined by one skilled in the art. Generally, if used, the acidulant should be present at a level of from about 0.0001% to about 0.5% by weight of the composition.

antioxidants

In some aspects, the composition of the embodiment may further comprise an antioxidant. For example, the antioxidant may be natural or synthetic. Suitable antioxidants include, but are not limited to, ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl isothiocyanate, o-, m- or p-aminobenzoic acid (o is o-aminobenzoic acid, p is p-aminobenzoic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid, canthaxanthin, α-carotene, β-carotene, β-carotene, β-apocarotene, carnosol, carvacrol, cetyl gallate, chlorogenic acid, citric acid and its salts, clove extract, coffee bean extract, p-hydroxyphenyl acrylic acid, 3,4-dihydroxybenzoic acid, N,N'-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate, distearyl thiodipropionate, 2,6-di-tert-butylphenol, lauryl gallate, edetic acid, acid), ellagic acid, isoascorbic acid, sodium isoascorbate, aesculetin, aesculetin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethyl gallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA), eucalyptus extract, eugenol, ferulic acid acid), flavonoids (e.g., catechin, epicatechin, epicatechin gallate, epigallocatechin (EGC), epigallocatechin gallate (EGCG), the polyphenol epigallocatechin-3-gallate), flavonoids (e.g., apigenin, chrysin, luteolin), flavonols (e.g., razorfin, myricetin, daemfero), flavanones, fraxin, fumaric acid, gallic acid, gentian extract, gluconic acid, glycine, guaiac, hesperetin, α-hydroxybenzylphosphinic acid, hydroxycinnamic acid, hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid, hydroxytyrosol, hydroxyurea, lactic acid and its salts, lecithin, lecithin citrate; R-α-lipoic acid, lutein, lycopene, malic acid, maltol, 5-methoxytryptamine, methyl gallate, monoglyceride citrate monoisopropyl citrate; morin, beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate, oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine, phosphoric acid, phosphate esters, phytic acid, phytylubichromel, pimento extract, propyl gallate, polyphosphate, quercetin, trans-resveratrol, rice bran extract, rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin, sinapic acid, succinic acid, stearoyl citrate, syringic acid, tartaric acid, thymol, tocopherols (i.e., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol), tocotriols (i.e., α-tocotriol, β-tocotriol, γ-tocotriol, and δ-tocotriol), tyrosol, vanillic acid, 2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., lonox 100), 2,4-(tri-3',5'-di-tert-butyl-4'-hydroxybenzyl)-mesitylene (i.e., lonox 330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tert-butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxybutyrophenone, tryptamine, tyramine, uric acid, vitamin K and derivatives, resveratrol, CoQ-10 (coenzyme Q10), vitamin C, vitamin E, beta-carotene, other related carotenoids, selenium, manganese, glutathione, lipoic acid, flavonoids, phenols, polyphenols, phytoestrogens, N-acetylcysteine, wheat germ oil, zeaxanthin, or a combination thereof. Preferred antioxidants include tocopherol, ascorbyl palmitate, ascorbic acid, and rosemary extract. The concentration of other antioxidants or antioxidant combinations can range from about 0.001% to about 5% by weight and preferably from about 0.01% to about 1% by weight.

water

The compositions of the present invention may comprise from 0% to about 99.999% water by weight of the composition. The compositions may comprise at least about 4% water, at least about 20% water, at least about 40% water, at least about 50% water, at least about 75% water, and at least about 80% water. The water included at these levels includes all added water and any water present in combined ingredients (e.g., juice).

In various embodiments, the composition is provided in or in a volume of 1 ounce, about 2 ounces, about 3 ounces, about 4 ounces, about 5 ounces, about 6 ounces, about 7 ounces, about 8 ounces, about 9 ounces, about 10 ounces, about 12 ounces, about 14 ounces, about 16 ounces, about 18 ounces, about 20 ounces, about 22 ounces, about 24 ounces, about 30 ounces, or about 40 ounces. In one aspect, the water component of the formulation is demineralized water.

ethanol

In some aspects, the beverage composition of the embodiment includes ethanol such as between about 1% and 60% ethanol (ABV), or between about 1% and 40% ethanol (ABV), or between about 1% and 20% ethanol (ABV), or between about 1% and 10% ethanol (ABV) (ethanol expressed by volume, ABV). For example, the composition can include a distilled liquor such as vodka, rum, whiskey, gin, bourbon, rye whiskey, or other sweet or unsweetened distilled liquor. In some aspects, the beverage can be composed of a substantial amount of beer, wine, apple juice, or malt liquor.

Sea minerals

In some aspects, the compositions of the embodiments further comprise sea minerals. Sea minerals are the ideal balance of nature's macrominerals, trace minerals, and ultratrace minerals. They exist in the most readily absorbed and bioavailable form known. Sea mineral levels are almost identical to those found in human serum and are in a pH balance very similar to human blood. Sea minerals do not contain toxic heavy metals such as arsenic, cadmium, mercury, lead, radon, ruthenium, and uranium.

For example, sea salt is primarily composed of the following ions (listed in order of decreasing abundance by weight): Chloride (Cl ⁻ ) 55.03% Sodium (Na ⁺ ) 30.59% Sulfate (SO ₄ ²⁻ ) 7.68% Magnesium (Mg ²⁺ ) 3.68% Calcium (Ca ²⁺ ) 1.18% Potassium (K ⁺ ) 1.11% Bicarbonate (HCO ₃ ⁻ ) 0.41% Bromine (Br ⁻ ) 0.19% Borate (BO ₃ ³⁻ ) 0.08% Strontium (Sr ²⁺ ) 0.04% Other ions 0.01%. Sea salt allows fluids to pass freely through body membranes, such as the glomeruli of the kidneys or the walls of blood vessels. Sea salt is necessary for the proper breakdown of plant carbohydrates into usable and absorbable nutrients.

lipid components

As described in further detail below, the compositions of the embodiments may further comprise a lipid component, either alone or as part of an oil (such as a lipid component comprising dissolved noble gases). Lipids included in the compositions of the embodiments include, but are not limited to, ω-3 fatty acids, such as α-linolenic acid (ALA, 18:3), eicosapentaenoic acid or EPA (20:5n-3), docosahexaenoic acid or DHA (22:6n-3); ω-6 fatty acids, such as linoleic acid or (LA, 18:2), ω-6 fatty acids, γ-linolenic acid or GLA (18:3n-6), dihomo-γ-linolenic acid or DGLA (20:3n-6), or arachidonic acid or AA (20:4n-6), or ω-9 fatty acids. For example, polyunsaturated oils may be derived from walnuts, canola oil, sunflower seeds, sesame seeds, chia seeds, peanuts, peanut butter, olive oil, seaweed, sardines, soybeans, tuna, wild salmon, or whole grain wheat, any of which may be used in the compositions of the embodiments.

II. Administration of Noble Gas Preparations

The amount of noble gas incorporated into the compositions of the embodiments depends on the specific formulation used and its intended use. The effective amount of the composition is determined based on the intended goal, such as providing neurological or cardiovascular protection or providing well-being to the subject and improving their well-being (e.g., reducing inflammation, reducing stress and/or lowering blood pressure). The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired effect. The amount of the composition to be administered will also depend on the frequency of administration and the unit dose, depending on the desired protective effect.

In certain embodiments, the actual dosage of the composition provided to a subject may be determined by physical and physiological factors, such as body weight, health status, previous or concurrent therapeutic interventions, diet, and route of administration.

The effective dosage range of a nutraceutical or therapeutic agent can be extrapolated, for example, from effective doses determined in animal studies. In general, the human equivalent dose (HED) (mg/kg) can be calculated according to the following formula (see, for example, Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, which is incorporated herein by reference):

HED (mg/kg) = Animal Dose (mg/kg) X (Animal K _m / Human K _m )

The K _m factor is used in the conversion to produce a more accurate HED value, which is based on body surface area (BSA) rather than just body mass. K _m values for humans and various animals are well known. For example, the K _m for an average 60 kg human (BSA is 1.6 m ² ) is 37, while the K _m for a 20 kg child (BSA 0.8 ^{m 2} ) would be 25. K _{m values} for some relevant animal models are also well known, including: a mouse K _m of 3 (assuming a weight of 0.02 kg and a BSA of 0.007); a hamster K _m of 5 (assuming a weight of 0.08 kg and a BSA of 0.02); a rat K _m of 6 (assuming a weight of 0.15 kg and a BSA of 0.025); and a monkey K _m of 12 (assuming a weight of 3 kg and a BSA of 0.24).

For example, in a mouse system, it is possible to administer between about 200 μl and 5 ml of an orally ingested aqueous solution saturated with Xe per day (i.e., about 0.12-3.0 mg/day or about 7.2 to about 180 mg/ _kg /day for mice). Thus, for a human subject, this would translate to a dose of about 500 μg/kg/day to about 12.2 mg/kg/day, or about 30 to about 732 mg/day for a human of typical mass (60 kg).

As mentioned above, the exact amount of the active gas component depends on the specific formulation. Nevertheless, the calculated HED dose can still provide general guidance on the administration that can provide beneficial effects. For embodiments of the present invention, considering that a dose of 1-2 times a day is administered to an average subject, it is envisioned that the amount of gas such as xenon to be provided in a unit dose will be from about 0.1 to about 200 mg. For example, a bottle of about 6 ounces of cold water Xe drinking water (e.g., cyclodextrin containing encapsulated Xe) can contain 4 grams of Xe, while 2 ml of Xe aqueous solution will contain 1.2 mg of Xe at room temperature and 1 atm pressure. In general, an oil formulation of Xe can contain about 19 times as much Xe as water (in the absence of an encapsulation system). For example, at room temperature and 1 atm, a solution of about 12 mg Xe/ml can be achieved in an oil such as olive oil.

III. Rare gas encapsulation

Oil component

Some aspects of the embodiment relate to the oil comprising the dissolved gas such as Ar or Xe. In some respects, oil is linseed oil, rapeseed oil, soybean oil, walnut oil, fish oil, safflower oil, sunflower oil, avocado oil, coconut oil, corn oil, cottonseed oil, peanut oil, palm oil, olive oil, Mexican oil (chia oil), echium oil, krill oil or vegetable oil. In other respects, oil is the mixture of two or more oils. Skilled technicians should understand that oil is preferably edible, substantially nontoxic oil. Therefore, in some respects, oil is non-petroleum-based oil, such as animal or plant-derived oil. Preferably, oil comprises high concentration of ω-3-fatty acid, ω-6-fatty acid and/or ω-9-fatty acid (for example eicosapentaenoic acid, docosahexaenoic acid, octadecatetraenoic acid and/or linolenic acid). In other respects, oil is to select for its polyunsaturated fatty acid (PUFA) concentration, such as oil has at least about 5%, 10%, 20% or more PUFA content.

In certain aspects, the oil composition or emulsion of the embodiment comprises one or more phospholipid components. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine glycerophospholipids, and certain sphingolipids. Thus, phospholipids used herein include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauroylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1- Palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauroylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelin ("DSSP"), distearoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), Dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), dimyristoylphosphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoylsphingomyelin ("DPSP"), dimyristoylphosphatidylcholine ("DMPC"), 1,2 - Distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diacaproyl-sn-glycero-3-phosphocholine ("DBPC"), 1,2-diicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloleoylphosphatidylcholine ("POPC"), palmitoyloleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

In addition to noble gases being dissolved in lipid components (e.g., for emulsification), it is also contemplated that such gases can be provided in aqueous formulations encapsulated in liposomes. Such liposomal encapsulation of gases has been previously demonstrated, see, e.g., U.S. Patent No. 7,976,743, which is incorporated herein by reference.

Water-soluble molecules

As further detailed herein, in certain aspects, the solubility of noble gases in aqueous components is increased by encapsulating the gases in water-soluble molecules, such as polymers. Generally, the molecules used for encapsulation will be molecules that can form pockets with increased hydrophobicity that are configured to encompass (at a portion) the noble gas atoms. Such encapsulating molecules thereby shield the hydrophobic atoms from the polar environment of the surrounding aqueous component, effectively increasing the amount of noble gas that can be dissolved in the aqueous component.

For example, as shown herein, cyclodextrin and its derivatives are very suitable for encapsulating rare gases. In the case of large Xe atoms, β-cyclodextrin is used to encapsulate Xe (see, for example, Figure 10B). In theory, those skilled in the art can increase the concentration of cyclodextrin or hydroxypropyl-β-cyclodextrin (hp-β-CD) or other derivatives to increase the amount of included Xe (i.e., the molecule locking Xe). For chronic oral administration, the acceptable safe dose of cyclodextrin can be about 1,000mg/kg/day. The solubility of hp-β-CD is, for example, 330mg/ml. This means that the cyclodextrin concentration can be significantly increased to about 0.5mg/ml (see Examples 3 and 4) by using soluble derivatives. This will enable the dissolved gas concentration to reach at least 500mg/ml. Other molecules contemplated for gas encapsulation include, but are not limited to, carcerands or hemicarcerands (see Saleh 2007), macroglobulins, cucurbiturils (see US 20030140787, incorporated herein by reference), calixarenes (Adams et al., 2008), pillararenes (Cao et al., 2009), porphyrins, metal crown ethers, crown ethers, cyclotrimeric veratrene, crypotophene, foldamers, other cyclodextrin polymers, silsesquioxanes (Skelton et al., 2013), tenas porous polymers, porous polymers, and Porapak ^™ porous polymers (referenced to the aforementioned citations incorporated herein by reference). The choice of a particular polymer for encapsulation will depend not only on the noble gas to be encapsulated, but also on the particular type of formulation to be prepared (e.g., oral formulation).

IV. Examples

The following examples are included to show preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the disclosed technology in the following examples represents the technology that the inventor has found to work well in the practice of the present invention, and therefore can be considered as constituting the preferred mode of its practice. However, it will be appreciated by those skilled in the art that many changes can be made and still obtain the same or similar result in the disclosed specific embodiments, without departing from the spirit and scope of the present invention.

Methods of Study of Examples 1-2

Preparation of Xe-rich solution. Xe -rich solution is composed of olive oil (or other oils such as linseed oil, rapeseed oil, soybean oil, walnut oil, fish oil, etc.), egg phosphorylcholine (Avanti, polar lipid, Alabama, USA), BSA (or other proteins such as milk) and lithocholic acid (Sigma-Aldrich, St. Louis, MO, USA). The solution containing 25% oil component is emulsified using ultrasonic method and stabilized by making an emulsion in the presence of surfactants such as phospholipids (egg PC, soybean PC, DPPC, DOPC, etc.). Xenon (Matheson, Houston, TX, USA) is saturated in the oil by pressurizing at low temperature.

Mouse experiment setup. All animal studies were approved by the Animal Welfare Committee of the University of Texas Health Science Center in Houston. C57BL/6J wild-type (WT) and apolipoprotein-E (ApoE) knockout (KO) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA), see, for example, Meir et al., 2004, which is incorporated herein by reference. The control WT mice used were C57BL/6J, which have the same genetic background as Apo E KO mice. Six to eight months old male WT and KO mice were fed a control or high-fat diet (Harlan Laboratories, USA), and control or Xe-enriched solution (200 μl, once a day) and Xe-enriched drinking water were administered for 6 weeks, because the Apo E KO mouse type fed a high-fat diet developed atherosclerotic lesions. See Figure 8.

Echocardiographic measurements and electrocardiographic images (in vivo). Baseline measurements were obtained by echocardiography before feeding with a high-fat diet. Cardiac morphology and function were assessed by continuous M-mode echocardiography using a Vevo 770 imaging system (VisualSonics Inc., Ontario, Canada) equipped with a 30MHz microprobe. M-mode ventricular measurements were performed 6 weeks after feeding. Electrocardiographic (ECG) data were obtained. Echo data (HR, heart rate; LVID, left ventricular internal dimensions; IVS, interventricular septum; LVPW, left ventricular posterior wall; FS, shortening fraction; SV, stroke volume; EF, ejection fraction; CO, cardiac output; LV Vol, LV volume; corrected LV mass) were analyzed using analysis software (VisualSonics Inc., Ontario, Canada).

Protein analysis. Fresh frozen heart and brain tissues were slightly thawed on crushed ice to allow for dissection of the heart and brain. Tissue samples were homogenized by sonication on ice for 20 seconds for 2-3 cycles using a minimum volume of radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., MA, USA) containing protease inhibitors (complete protease inhibitor cocktail, Sigma) and centrifuged at 14,000 x g for 10 min at 4°C. The supernatant was removed. Protein concentration was determined using the Bradford Protein Assay (Bio-Rad, CA, USA).

Brain-derived neurotrophic factor (BDNF) and beta-amyloid protein are measured. Except for heart and brain extracts, samples (such as plasma) are thawed and clarified at 12,000rpm for 10min at 4°C, and then ELISA analysis is performed for beta-amyloid protein according to the manufacturer's instructions. BDNF and beta-amyloid peptide (Aβ1-40) content are determined by using a BDNF sandwich ELISA kit (Millipore Corporation, MA, USA) and a mouse/rat amyloid beta (1-40) high specificity ELISA assay kit (IBL American, Minneapolis, MN, USA). Following the instructions, samples are added to pre-coated 96-well microtiter plates and incubated overnight at 4°C. After washing, antibodies are added and incubated. Fluorescence is measured at 450nm using a SpectroMax microplate reader (Bio-Tek Instruments). All samples are analyzed in duplicate.

Rat Experimental Setup. Male Sprague-Dawley rats (260-280 g, Harlan Laboratories Inc., Indianapolis, IN) were randomly divided into two groups (n=8 in each group). One group was given water via a gastric tube, while the other group was given a xenon-enriched solution. After two weeks, the rats underwent a mid-brain occlusion for 2 hours in a double-blind manner. Behavioral function was assessed and then, after sacrifice, infarct volume was assessed 24 hours after brain injury in a double-blind manner. See Figure 9.

Middle cerebral artery occlusion rat model (MCAO). Cerebral ischemia was induced by occluding the right middle cerebral artery (MCA) for 2 hours using an intraluminal suture method. Briefly, the right common carotid artery (CCA) was exposed to a surgical microscope. The external carotid artery was ligated close to its distal end. The internal carotid artery (ICA) was separated and separated from adjacent tissues. A 4-0 monofilament nylon suture (Ethicon, Somerville, NJ, USA) coated with poly-L-lysine (0.1% [wt/vol]) and heparin (1000 U/mL) was inserted into the MCA cavity and located 18 to 20 mm from the external carotid artery/common carotid artery bifurcation for 2 hours to cause ischemia. Once the suture is removed, the external carotid artery was ligated, allowing blood to be reperfused into the MCA through the common carotid artery. In all experiments, body temperature was monitored during ischemia and within the first hour of reperfusion and maintained at 37°C by means of a temperature controller (Harvard Apparatus, Holliston, MA, USA) equipped with a heating lamp and a heating pad. A polyethylene catheter was introduced into the right femoral artery for pressure recording. Cerebral blood flow was monitored by means of a PR407-1 straight needle laser Doppler flowmeter probe (Perimed, Stockholm, Sweden) connected to a standard laser Doppler monitor (PF5010LDPM unit and PF5001 main unit; Perimed, Stockholm, Sweden). The blood flow interruption in the ischemic penumbra region (2 mm laterally and 2 mm posterior to the bregma) was recorded.

Neurological assessments were performed 24 hours after brain injury. All behavioral tests were performed in a quiet, dimly lit room by an observer blinded to treatment group. The animals' motor function and neurological outcomes were assessed by recording limb placement, balance beam walking, and grid walking abilities.

Infarct volume measurement. After neurological assessment 24 hours after surgery, the animals were sacrificed and the brains were harvested. Using a Jacobowitz brain slicer, 2-mm thick coronal sections were stained with 2% TTC. Infarct size was normalized to the total brain volume and presented as normalized infarct volume (%).

Statistical analysis. Data were processed using Microsoft Excel and GraphPad Prism 5.0. All values are expressed as mean ± SEM. Comparisons between two groups were determined using an unpaired two-tailed Student's t-test. Analyses of multiple groups were performed using one-way ANOVA followed by Tukey's post hoc multiple comparison test. P values less than 0.05 were considered significant.

Example 2 - Xe Administration Studies

Resistance to cardiac hypertrophy in response to xenon (Xe) exposure. To examine the effects of Xe activity on heart disease, an apolipoprotein E knockout (apoE-/-; or "KO" as used herein) mouse model was used. This is a well-established model of atherosclerosis, as animals will develop atherosclerotic damage even on a normal food diet, while a high-fat diet significantly accelerates this process (Meir et al., 2004). Therefore, the model has previously been successfully used to evaluate the effects of natural compounds and drugs on atherosclerosis and cardiovascular disease.

Animals were divided into 5 groups (see experimental setup). Echocardiography was used to assess cardiac size and function at baseline and after 6 weeks of Xe exposure (Figures 1 and 2; Table 1). After 6 weeks of Xe exposure, cardiac size of WT and KO hearts was determined using LV mass (corrected) and normalized to body weight (mg/g) (Fig. 3A).

As expected, the LV mass in Apo E KO mice fed/not fed a high-fat diet at 6 weeks and at baseline increased compared to WT and WT after 6 weeks. Compared with the KO6w vehicle and KO6w control group, the increase in LV mass in the KO mice treated with Xe (KO6w Xe) was blocked. The presence of overgrown hearts in the KO6w control and vehicle groups was confirmed because the heart weight normalized for body weight increased significantly. Compared with the KO6w vehicle, in response to Xe exposure at 6 weeks, reduced heart/body weight (Fig. 3B) was observed in the KO6w Xe mice.

By echocardiography, WT and ApoE-KO hearts were measured at baseline and in the ventricular septum (IVS), LV posterior wall thickness (PW), LV volume (V) and LV internal dimensions (ID) (Figure 1 and Table 1) under diastole and contraction when Xe was exposed for 6 weeks. Compared with KO6w vehicle mice, and compared to KO6w/ control mice, in KO6w Xe mice, in response to Xe exposure at 6 weeks, wall thickness increased and was significantly blocked (Figure 1A and 1B). Heart rate (HR) increased compared with WT and WT6w in KO/KO6w/ control/vehicle animals respectively. Again, this increase was blocked in the KO6w given Xe (representative M-mode data are shown in Figure 4A). In general, these results show that Xe activity inhibits the progression of cardiac hypertrophy.

Improved cardiac function and myocardial ischemia in response to Xe exposure. Cardiac function was assessed at baseline and 6 weeks after Xe exposure (Figure 2). At baseline and 6 weeks, LV fractional shortening (FS), LV ejection fraction (EF), and cardiac output (CO) of KO mice fed/not fed a high-fat diet were reduced compared to WT and WT6w, respectively. On the other hand, in response to Xe exposure, KO6w Xe hearts significantly blocked these reductions at the 6-week time point compared to KO6w vehicles (Figures 2A-C, respectively).

ECG data show that the T wave, ST segment and QRS complex of KO/KO6w/ control/vehicle heart are respectively compared with the change of WT/WT6w, and it is consistent with myocardial ischemia. However, these changes do not occur in the KO6w processed with Xe at 6 week time points. These data instructions respond to the improvement of myocardial ischemia exposed by Xe and show that the heart processed by Xe does not have overgrowth change and myocardial ischemia degree is lower. These changes also show the protective effect (Fig. 4 B) of xenon-rich solution in heart disease and show that Xe activity improves cardiac function and prevents myocardial ischemia.

Increased BDNF expression in the heart and brain pretreated with Xe. Xe pretreatment has a neuroprotective effect in rats by regulating the synthesis of genes and BDNF in stroke (Peng et al., CNS Neurosci Ther; October 2013; 19 (10): 773-84) and in brain damage caused by asphyxia in neonatal rats (Ma et al., Journal of Cerebral Blood Flow & Metabolism (2006), 199-208). Previous studies have shown that BDNF is expressed in the heart and may be involved in the molecular mechanism of heart disease (Okada et al., 2012). To assess the possible role of BDNF in the activity of Xe on the cardiovascular system, the relative BDNF expression levels in the heart and brain were measured by ELISA in the presence and absence of XE treatment (Figure 5). As shown in Figure 5A, compared with WT6w, relative BDNF levels increased in KO6w/control/vehicle. Compared to KO6w, BDNF levels were observed to increase further in KO6w vehicle. In response to Xe exposure, BDNF levels in the heart (KO6w Xe) increased further. Similar results were observed in the brain (Figure 5B). These data suggest that BDNF may participate in Xe-mediated changes in the heart and brain (Pagel et al., 2010).

The level of beta-amyloid in the plasma and brain pretreated with xenon-rich solution. It has been previously reported that Xe can participate in lipid homeostasis, but the exact role and effect of Xe on this type of homeostasis are not clear (Golden et al., 2010; Jung et al., 2011). Beta-amyloid is the main component related to Alzheimer's disease in brain deposits and is also related to lipid homeostasis (Shankar et al., 2008; Selkoe et al., 2001). To study whether Xe activity can regulate beta-amyloid levels, Apo E-KO mice fed with a high-fat diet were tested. Specifically, the beta-amyloid levels in the plasma and brain of treated and untreated animals were tested by ELISA (Fig. 6). As shown in Figure 6A, compared with WT6w animals, relative plasma beta-amyloid levels were significantly increased in KO6w animals treated with vehicle (and increased in KO6w, but not significantly). This increase was significantly weakened in KO6w mice given a Xe-rich solution. Similar results were also observed in brain tissue (Figure 6B). Importantly, these data confirm that the administration of xenon-rich solutions has the potential to treat brain deposition diseases. Therefore, such solutions can be used to mitigate the effects of amyloid diseases such as Alzheimer's disease or delay their progression.

A xenon-rich solution for increasing the tolerance of the brain to ischemic injury. For these studies, rats were divided into two groups, one group was delivered to the stomach with water and the other group was delivered to the stomach with a xenon-rich solution. After two weeks, the rats underwent a 2-hour mid-brain occlusion. The infarct volume was assessed 24 hours after brain injury. The rats given the xenon-rich solution produced infarcts that were smaller in size than the control (Fig. 7 A and 7B). Similarly, behavioral assessments including limb placement (Fig. 7 C) and grid walking (Fig. 7 D) indicating neurological function were completed. The group pretreated with oral xenon-rich solution showed better ability to perform motor tasks. These data show that using xenon-rich solution increases the tolerance of the brain to ischemic injury and even after significant ischemic injury, also neurological effects can be significantly eliminated.

Summary. The studies presented here demonstrate the beneficial effects of oral Xe delivery on the nervous and cardiovascular systems. Specifically, Xe uptake has been shown to be protective in cardiovascular disease model systems, where a wide range of disease markers can be improved by Xe treatment. Similarly, oral Xe has been shown to be neuroprotective. Not only can orally delivered Xe significantly prevent ischemic damage, but Xe has also been shown to reduce the beta-amyloid load in treated animals, indicating that it may be suitable for treating or preventing degenerative neurological diseases. Importantly, the data also show that even in an oral delivery system (such as such lipid systems described herein), sufficient amounts of Xe can be delivered to provide measurable benefits to the treated animals.

Table 1. Echocardiographic measurements

*p<0.05, **p<0.01, KO/KO6w vs. WT/WT6w, respectively; #p<0.05, KO control/vehicle vs. KO6w; ^§p <0.05, ^§§p <0.01, ^§§§p <0.001, KO xenon vs. KO vehicle. Vehicle is molecular water locked with cyclodextrin without xenon loading.

Materials and methods for Examples 3-4

Preparation of Xenon Gas Enclosed by Locking Molecules

Xenon gas is trapped in a soluble locking molecule (e.g., cyclodextrin). To remove possible residual molecules from the cage, the locking molecule is baked at 40-80°C under vacuum overnight. To trap xenon gas in the locking molecule, the xenon gas and the locking molecule are incubated in a sealed vial at 2-10 atm pressure at 4 to -180°C for overnight to 3 days.

Preparation of pure xenon supersaturated water

Purified water was degassed under 20-80 mbar vacuum at room temperature overnight. Xenon (99.999% medical grade, Matheson Houston, TX, USA) was redissolved in the degassed water by pressurizing the water with 2-10 atm of Xenon at 4°C for overnight to 3 days.

Preparation of Xe-rich water

Xe-rich water consists of Xe dissolved in water and locked with hydroxypropyl-β-cyclodextrin (hp-β-CD) in water. To prepare Xe-rich water, xenon supersaturated water (10 ml) is injected into a vial containing 5 mg of the locked molecule-xenon complex (0.5 mg/ml). The resulting mixture is incubated at 4°C under 2-10 atm overnight to 3 days.

Measurement of Xe Dissolved in Xenon-Rich Water

To measure the amount of Xe dissolved in Xe-rich water. The solution is warmed to room temperature and the pressure on the Xe-rich water sample is released. Then, the solution is warmed to 80°C for 2 hours in a vial with a silicone rubber seal (Thermo Scientific seal flat top) (Thermo Scientific, Hudson, NH, USA). After cooling to room temperature, a syringe with a 17-gauge needle is inserted into the vial through the silicone rubber seal. The released Xe gas present in the top compartment forms a pressure, which pushes the Xe gas into the syringe. The amount of Xe released into the syringe is then measured.

animal

All animal studies were approved by the Animal Welfare Committee of the Texas Health Science Center in Houston. Wild-type (WT) and Apo E knockout (KO) transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The wild-type control mice used were C57BL/6J compared with Apo E KO mice of the same genetic background. Eight to eleven month-old male and female WT and KO mice were fed with a high-fat diet (Harlan Laboratories, USA) and administered with CD-locked but not Xe-containing molecular water (vehicle) or Xe-rich water, wherein the Xe-rich water included molecular water (0.2 to 10 ml per day) locked with the CD loaded with Xe for 6 weeks.

Echocardiographic measurements and electrocardiographic images (in vivo).

Baseline measurements were obtained by echocardiography before feeding with a high-fat diet. Cardiac morphology and function were assessed by continuous M-mode echocardiography using a Vevo 770 imaging system (VisualSonics Inc., Ontario, Canada) equipped with a 30 MHz microprobe. M-mode ventricular measurements were performed 6 weeks after feeding. Electrocardiogram (ECG) data were obtained. Echo data (HR, heart rate; LVID, left ventricular internal dimensions; IVS, interventricular septum; LVPW, left ventricular posterior wall; FS, fractional shortening; SV, stroke volume; EF, ejection fraction; CO, cardiac output; LV Vol, LV volume; corrected LV mass) were analyzed using analysis software (VisualSonics Inc., Ontario, Canada).

Blood pressure measurement

Blood pressure in mice was monitored non-invasively using a tail cuff placed on the mouse's tail to block blood flow.

Protein analysis

Freshly frozen heart and brain tissue were thawed slightly on crushed ice to allow for dissection of the heart and brain. Tissue samples were homogenized by sonication on ice for 20 seconds for 2-3 cycles using a minimal volume of radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., MA, USA) containing protease inhibitors (Complete Protease Inhibitor Cocktail, Sigma) and centrifuged at 14,000 x g for 10 min at 4°C. The supernatant was removed. Protein concentration was determined using the Bradford Protein Assay (Bio-Rad, CA, USA).

β-amyloid measurement

The content of beta-amyloid peptide (Aβ1-40) in brain and blood was determined using a mouse/rat amyloid beta (1-40) high-specificity ELISA assay kit (IBL American, Minneapolis, MN, USA). Following the instructions, samples were added to a pre-coated 96-well microtiter plate and incubated overnight at 4°C. After washing, antibodies were added and incubated. Absorbance was measured at 450nm using a SpectroMax microplate reader (Bio-Tek Instruments). All samples were analyzed in duplicate.

Western blot analysis of cardiac troponin expression in heart tissue

Western blot analysis is as previously described (Yin, X, Molecular Pharmacology) using cardiac troponin I (cTnI) (Cell Signaling Technology, Inc., Danvers, MA, USA) to carry out. For immunoblotting analysis, the sample was dissolved with SDS-PAGE (4-12%) gradient gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The blot was then incubated overnight at 4°C with a primary antibody and washed three times with TBS containing 0.1% Tween 20 (TBST), and then probed with a secondary antibody (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer's instructions. Densitometric analysis of immunoblotting was performed using an Odyssey infrared imager (LI-COR Biosciences).

Statistical analysis

Data were processed using Microsoft Excel and GraphPad Prism 5.0. All values are expressed as mean ± S.E.M. Comparisons between two groups were determined using an unpaired, two-tailed Student's t-test. Analyses were performed using a one-way ANOVA, followed by a Tukey's post hoc multiple comparison test when comparing multiple groups. P values less than 0.05 were considered significant.

Example 4 - Results of Additional Studies Using Xe to Enhance Water

Xenon gas dissolved in Xe-rich water

Cyclodextrin (CD) is a versatile locking molecule used in the food, pharmaceutical, and chemical industries. Cyclodextrins offer a hydrophobic interior and a hydrophilic exterior. The studies described here were conducted to determine whether these properties could be used to increase the solubility of noble gases such as xenon.

Data from initial studies confirm that inclusion of xenon into cyclodextrin (hp-β-CD) is highly correlated with pressure ( FIG. 10C ) and temperature ( FIG. 10D ). As shown in FIG. 10C , increased pressure produces increased Xe encapsulation. Similarly, encapsulation of gaseous Xe is more effective at low temperatures ( FIG. 10D ). For example, studies have shown that at 3 atm and -80°C, a total of 5 ml of xenon can be encapsulated in a hp-β-CD cage (using 0.5 mg of hp-β-CD per ml).

Similarly, the solubility of xenon in water is highly correlated with the pressure and temperature of the solution. By incubating degassed water with pure xenon at 4 ° C, 3 atm for 4 hours to overnight, a total of 6.5 ml of xenon is dissolved in 5 ml of water. To prepare Xe-rich water, Xe-saturated water is incubated with Xe-CD at 4 ° C under 3 atm pressure. The pressure of 3 atm is used here because, typically, standard beverage containers can withstand 80-90 psi (5.4-6.1 atm) pressure (i.e., the internal pressure of typical soft drink cans such as Coca-Cola ^™ classic products at 75 ° F is 55 psi (3.7 atm)). As shown in Figure 11, in the presence of water as a medium, 19 ml of xenon is included in the locked molecule and dissolved in water (wherein the initial volume of water is 5 ml). Therefore, the preparation achieves a total Xe content of 22.4 mg of Xe per milliliter of CD-water solution (at a CD concentration of 0.5 mg of hp-β-CD per milliliter).

Xenon-enriched water increases cardiac tolerance to ischemic stress

To examine the role of Xe activity in preventing heart disease, mice were divided into four groups: (1) wild-type (WT) mice fed a normal chow diet and a water control; (2) ApoE knockout mice fed a high-fat diet and a normal water control; (3) ApoE knockout mice fed a high-fat diet and a vehicle control (fed with water containing cyclodextrin but no xenon); and (4) ApoE knockout mice fed a high-fat diet and Xe-enriched water (xenon loaded into cyclodextrin (i.e., a molecule that locks xenon)). Echocardiography was used to assess cardiac size and function at baseline and 6 weeks after feeding.

At baseline and after treatment for 6 weeks, the ventricular septum (IVS), left ventricular posterior wall thickness (LVPW), left ventricular (LV) volume and LV internal dimensions (ID) (Figure 12) were measured by echocardiography under cardiac diastole and contraction. High-fat diet causes a significant increase in ventricular wall thickness, which is a typical case of ApoE KO animals. However, compared with the mice that only receive water containing cyclodextrin, this pathological change (Figure 12) does not occur in the ApoE KO mice that receive rich Xe water for 6 weeks. Heart rate (HR) also increases in the ApoE KO mice fed a high-fat diet and treated with vehicle. Again, this increase is prevented by rich Xe water. These results show that, as indicated by the above research institutes, oral rich Xe water consumption suppresses the progress of cardiac hypertrophy. In addition, the level of Xe in the encapsulated water formulation is high enough to achieve beneficial effects.

Cardiac function (Figure 12) at baseline and when using Xe-rich water for 6 weeks was also assessed. At baseline and when 6 weeks, compared with WT and WT6w treated mice, LV fractional shortening (FS), (EF) and cardiac output (CO) were reduced in ApoE KO mice fed with a high-fat diet. In the ApoE KO mice (KO6w Xe) that received Xe-rich water, these reductions (Figure 12 A-C, respectively) were significantly prevented in the heart of mice at 6 weeks compared with the KO6w vehicle.

ECG data show that the T wave, ST segment and QRS complex of KO/KO6w/control/vehicle heart are respectively compared with the change of WT/WT6w, and it is consistent with myocardial ischemia.At 6 weeks, these changes did not occur in the heart (KO6wXe heart) of ApoE KO mice that accepted Xe-enriched water administration.This shows that using Xe-enriched water alleviates myocardial ischemia.

Troponin and CKMB (creatine kinase) are two kinds of markers of cardiac ischemia.Other research has measured the plasma CKMB level and the troponin expression in cardiac tissue.These studies are presented at the level increase of two kinds of markers in the control, and reduce (Figure 13) in the ApoE KO mice that accepts the use of rich Xe water.These data further confirm that the consumption of rich Xe water increases the tolerance of heart to ischemic stress.

Xenon-rich water stabilizes blood pressure

Further analysis of Xe-treated mice showed that daily oral administration of Xe-enriched drinking water for 6 weeks significantly reduced systolic and diastolic blood pressure (Table 2), while increasing cardiac contractility.

Table 2: Xe-enriched water stabilizes blood pressure

*p<0.05, KO/KO6w compared with Apo E not fed a high-fat diet (baseline); ^§ p<0.05, KO Xenon compared with KO vehicle

Xenon-enriched water reduces beta-amyloid protein in brain tissue and blood

Studies were also conducted to determine the effects of Xe-water administration on beta-amyloid in the brain and blood (see Figure 14). For these studies, the well-characterized ApoE-KO mouse model system for Alzheimer's disease was used. Compared to control mice, these mice exhibited increased levels of serum and brain beta-amyloid. However, administering Xe-water to mice over a six-week period resulted in decreased levels of serum and brain beta-amyloid (achieving levels similar to those in control animals).

* * *

All of the methods disclosed and claimed herein can be made and implemented in light of the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described using preferred embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the methods and methods described herein can be varied without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain reagents related to chemistry and physiology can be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

References

The following references are specifically incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

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Claims (43)

1. Use of a composition formulated for oral administration in the preparation of a product for treating or preventing the progression of Alzheimer's disease in a subject, said composition comprising an aqueous component of dissolved xenon gas, a portion of said xenon gas being encapsulated with a water-soluble polymer, said water-soluble polymer being a cyclodextrin. 2. The use as described in claim 1, wherein the cyclodextrin comprises α-cyclodextrin, β-cyclodextrin, and/or γ-cyclodextrin. 3. The use as claimed in claim 1, wherein the xenon to water content ratio is at least 2:1 (volume:volume). 4. The use as described in claim 3, wherein the xenon to water content ratio is 2:1 to 3:1 (volume:volume). 5. The use as claimed in claim 1, wherein the composition comprises a mixture of two or more rare gases. 6. The use as claimed in claim 1, wherein the composition comprises an emulsion. 7. The use as described in claim 2, wherein the α-cyclodextrin comprises modified α-cyclodextrin. 8. The use as described in claim 2, wherein the β-cyclodextrin comprises modified β-cyclodextrin. 9. The use as described in claim 2, wherein the γ-cyclodextrin comprises modified γ-cyclodextrin. 10. The use as claimed in claim 2, wherein the composition comprises 0.05 to 2.0 mg/ml of cyclodextrin molecules. 11. The use as claimed in claim 1, wherein the composition further comprises an edible oil component containing dissolved rare gases. 12. The use as claimed in claim 11, wherein the oil comprises polyunsaturated fatty acids. 13. The use as described in claim 11, wherein the oil contains at least 5% PUFA. 14. The use as claimed in claim 11, wherein the oil component is saturated with xenon or argon gas. 15. The use as claimed in claim 11, wherein the edible oil component comprises soluble xenon. 16. The use as described in claim 11, wherein the edible oil is free of oxygen. 17. The use as described in claim 11, wherein the oil comprises rapeseed oil, flaxseed oil, rapeseed oil, soybean oil, walnut oil, fish oil, safflower oil, wild sage seed oil, sunflower oil, sesame seed oil, seaweed oil, corn oil, cottonseed oil, peanut oil, palm oil, avocado oil, coconut oil, or olive oil. 18. The use as claimed in claim 11, wherein the oil comprises ω-3 fatty acids. 19. The use as described in claim 11, wherein the composition is further defined as comprising an emulsion containing: (a) 25% to 50% by volume oil, said oil saturated with a rare gas; and (b) 30% to 75% aqueous solution. 20. The use as described in claim 11, wherein the composition is further defined as comprising an emulsion containing: (a) 15% to 75% by volume oil, said oil saturated with a rare gas; and (b) 25% to 85% aqueous solution. 21. The use as claimed in claim 1, wherein the aqueous solution comprises water, fruit juice, vegetable juice, or ethanol. 22. The use as described in claim 1, further comprising phospholipids, detergents, or protein components. 23. The use as described in claim 22, wherein the detergent is a plant-based surfactant, a synthetic detergent, or a bile acid. 24. The use as described in claim 22, wherein the detergent is lithocholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, chenodeoxycholic acid, or cholic acid. 25. The use as described in claim 22, wherein the phospholipid is methionine choline (PC), soybean PC, DPPC, or DOPC. 26. The use as described in claim 22, wherein the protein is milk protein, whey protein, soy protein isolate, or bovine serum albumin. 27. The use as claimed in claim 11, wherein the composition further comprises between 1 and 50 mg of xenon per milliliter of oil. 28. The use as claimed in claim 11, wherein the composition comprises: (a) 15% to 75% by volume olive oil, said oil being saturated with xenon or argon gas; (b) 25% to 85% by volume aqueous solution; and one or more of phospholipids, detergents or proteins. 29. The use as claimed in claim 11, wherein the composition comprises: (a) 25% to 50% olive oil, said oil being saturated with xenon or argon gas; (b) 50% to 75% aqueous solution; (c) 10-30 mg/ml choline phosphate; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml lithocholic acid. 30. The use as claimed in claim 11, wherein the composition comprises: (a) 15% to 75% olive oil, said oil being saturated with xenon or argon gas; (b) 25% to 85% aqueous solution; (c) 10-30 mg/ml choline phosphate; (d) 10-50 mg/ml BSA; and (e) 1-5 mg/ml lithocholic acid. 31. The use as described in claim 1, wherein the composition is further defined as an herb, vitamin, or energy-providing beverage. 32. The use as claimed in claim 1, wherein the composition further comprises a preservative, flavoring agent, dye, vitamin, antioxidant, or plant extract. 33. The use as claimed in claim 1, wherein the subject is a human. 34. The use as described in claim 1, further defined as the use of reducing the β-amyloid protein level in the subject. 35. The use as claimed in claim 1, wherein the subject is at risk of developing Alzheimer's disease. 36. The use as claimed in claim 1, wherein the subject has or is diagnosed with a genetic predisposition to Alzheimer's disease. 37. The use as described in any one of claims 1-36, wherein the composition is contained in an airtight container. 38. The use as described in claim 37, wherein the container contains 1 ml to 2 L of the composition. 39. The use as claimed in claim 38, wherein the container contains 1 mg to 1 g of Xe. 40. The use as claimed in claim 37, wherein the container includes a one-way valve for releasing the composition for oral administration without exposing the entire contents to the atmosphere. 41. The use as claimed in claim 38, wherein the container is pressurized. 42. The use as claimed in claim 41, wherein the container is pressurized with a rare gas. 43. The use as claimed in claim 41, wherein the composition further comprises _CO2 .