Compositions and methods for separating, characterizing and administering soluble selenoglycoproteins

<b>DESCRIPTION</b>

Compositions and methods to separate, characterize and administer soluble selenoglycoprotems

<b>Field of the Invention</b>

The present invention relates to soluble selenium compositions and methods of production, separation and purification thereof. In particular, the present invention provides methods for preparing water-soluble selenoglycoprotems (for example, by extracting selenoglycoprotems from selenium-enriched yeast), methods for supplementing a composition with selenium deficiency by mixing water-soluble selenoglycoprotems with the deficiency composition of selenium, compositions comprising water soluble selenoglycoprotems and methods for administration thereof.

<b>Background of the invention</b>

Selenium is an important trace element for proper physiological functioning in humans. Selenium is ingested through the diet that may have a variable selenium content.

Selenium plays a fundamental role in maintaining physiological metabolism, growth, reproductive health, and immunity. Selenium is incorporated into different organic molecules that include, for example, amino acids such as 1-selenomethionine, selenocystem, and selenocystin. Therefore, selenium can be a component part of proteins, many of which are of structural importance to the organism. In addition, selenium is an important ingredient in a number of enzymes that influence metabolism, reproduction, cancer prevention, and immune defense in humans (See, for example, Rayman, M Lancet 356: 233 241 (2000)) .

Multiple studies have attempted to reveal the potential health benefits that result from the intake of low selenium levels. For example, low concentrations of an inorganic form of selenium have shown certain potential health benefits (See, for example, Furnsinn et al., Int. J of Obesity and Related Metab. Dis., 19, 458-463 (1995) ). However, at high dosage levels, the beneficial effects are reversed and dangerous toxicity manifests.

Research over the past two decades has suggested that selenium is effective in reducing the incidence of cancer when given to animals at doses only 5 to 10 times above nutritional requirements (See, for example, El-Bayoumy , The role of selenium in cancer prevention, Philadelphia, Lippincott, 1-15, 1991). Chemoprevention studies with selenium in animal model systems have indicated that this element is effective for the majority of systems, if not all organic systems, and protects against carcinogenic effects (See, for example, El-Bayoumy, The role of selenium in cancer prevention, Philadelphia, Lippincott, 1-15, 1991. Both epidemiological studies and supplemental action trials have also supported its efficacy in reducing the incidence of cancers of the liver, colon, prostate and lung (See, for example, Yu et al., Biol Trace Elem Res, 56: 117 -124 (1997); Clark et al., J Am Med Assoc, 276: 1957-1963 (1996); Yoshizawa et al., J Natl Cancer Inst, 90: 1219-1224, (1998); Brooks, et al. , J Urol, 166: 2034-2038, (2001)). Other studies have shown no beneficial effect for the reduction of selenium cancers (See, for example, Garland et al., J. Am. Coll Nutr., 12: 400-11 (1993); Ghadirian et al., Cancer Detect Prev , 24: 305-13 (2000)).

Multiple forms of selenium have been examined. These include inorganic selenium such as sodium selenite, and organic sources, including selenium yeast. There is a significant difference between the toxicity of inorganic and organic selenium, inorganic compounds being normally absorbed and used less efficiently and also being more toxic than organic sources of selenium.

S. Mc Sheehy et al., Anal. Chem. 2005, 77, 344-349; L. Yang et al., Journal of Chromatography A, 1055 (2004), 177 184; K. Wrobel et al., Anal. Bioanal Chem (2003), 375, 133-138; CA Ponce de Leon et al., Journal of Applied Microbiology 2002, 92, 602-610; and MP Rayman, British Journal of Nurtition (2004), 92, 557-573 describe selenomethionine extractions in yeasts. S. Kwiatkowski et al., Journal of the Institue of Brewing, Vol. 115, No. 2, 2009, 151-158 describe the extraction of yeast glucans. US 2009/0214419 discloses biodegradable polymorphs.

<b>Summary of the invention</b>

The present invention relates to soluble selenium compositions and methods of production, separation and purification thereof.

In particular, the present invention provides a composition comprising soluble selenoglycoprotems, where the soluble selenoglycoprotems contain two or more fractions of the pH dependent selenoglycoprotems, where the soluble selenoglycoprotems are obtained by acid extraction of the selenoglycoprotems. soluble yeast enriched with selenium and after exposing yeast enriched with selenium to acidic conditions, by sequential pH-dependent precipitation at two or more different pH values ​​of soluble selenoglycoprotems, where soluble selenoglycoprotems are generated with a method comprising: a) providing yeast enriched with selenium; b) exposing the yeast enriched with selenium to acidic conditions followed by centrifugation to generate i) a microgranule comprising acid insoluble material; and ii) a liquid phase comprising yeast extract enriched with soluble selenium under acidic conditions; c) precipitate selenoglycoproteins of the liquid phase comprising the yeast extract enriched with soluble selenium under acidic conditions by increasing the pH of the liquid phase of 0.1, 0.2, 0.5, 1.0, 2, 0, 3.0, 4.0, 5.0 or 6.0; and d) separating precipitated selenoglycoproteins from the liquid phase.

The present invention also provides a method for the preparation of soluble selenoglycoprotems comprising: a) providing yeast enriched with selenium; b) exposing the yeast enriched with selenium to acidic conditions followed by centrifugation to generate i) a microgranule comprising acid insoluble material; and ii) a liquid phase comprising yeast extract enriched with soluble selenium under acidic conditions; c) precipitate selenoglycoproteins of the liquid phase comprising the yeast extract enriched with soluble selenium under acidic conditions by increasing the pH of the liquid phase of 0.1, 0.2, 0.5, 1.0, 2, 0, 3.0, 4.0, 5.0 or 6.0; and d) separating precipitated selenoglycoproteins from the liquid phase.

In some embodiments, exposure of yeast enriched with selenium at acidic conditions occurs at a temperature above room temperature (for example, above 20-25 ° C). In fact, a variety of temperatures can be used that include 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 90, 95, 97, 99 ° C, or more high). In some embodiments, yeasts enriched with selenium are exposed to conditions where the pH is 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 , 1, or less. In a preferred embodiment, the exposure of the yeast enriched with selenium to acidic conditions comprises the exposure of the yeast enriched with selenium at a pH of 1.5. In some embodiments, exposure of yeast enriched with selenium to acidic conditions occurs over a certain period of time. In some embodiments, yeasts enriched with selenium are exposed to acidic conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24 or more hours In some embodiments, yeasts enriched with selenium are exposed to acidic conditions between one and twenty four hours. In some embodiments, yeasts enriched with selenium are exposed to acidic conditions between five and ten hours. In a preferred embodiment, yeasts enriched with selenium are exposed to acidic conditions for eight hours. In some embodiments, the acid conditions are created and / or maintained using acid buffer and / or the addition of an acid. Any acid can be used. In some embodiments, the acid is hydrochloric acid, although any acid can be used. In some embodiments, selenoglycoprotems are precipitated from the liquid phase comprising the yeast extract enriched with soluble selenium under acidic conditions by increasing the pH of the liquid phase. In some embodiments, the precipitated selenoglycoprotems are separated from the liquid phase by centrifugation to form a microgranule of the precipitated selenoglycoprotems followed by removal of the liquid phase from the precipitated selenoglycoprotems. In some embodiments, the pH of the liquid phase is raised to 1.85. In some embodiments, the liquid phase pH rises to 3.0. In some embodiments, the liquid phase pH rises to 4.0. In some embodiments, the pH of the liquid phase is raised to 6.0. In some embodiments, selenoglycoprotems are precipitated and separated from the liquid phase under a variety of pH conditions to generate multiple fractions of soluble pH-dependent selenoglycoprotems. For example, in some embodiments, a single liquid phase comprising yeast extract enriched with soluble selenium under acidic conditions is used to create a first fraction of soluble selenoglycoprotems that precipitates at a first pH (for example, pH of 1.85) , a second fraction of soluble selenoglycoprotems that precipitates at a second pH (e.g., pH 3.0), a third fraction of soluble selenoglycoprotems that precipitates at a third pH (e.g., pH of 4.0), and a fourth fraction of soluble selenoglycoprotems that precipitates at a fourth pH (for example, pH 6.0). In some embodiments, the precipitation of the liquid phase selenoglycoprotems by increasing the pH of the liquid phase comprises multiple sequential precipitation reactions dependent on the pH of the liquid phase. A composition comprising soluble selenoglycoprotems contains two or more pH-dependent fractions of selenoglycoprotems (for example, liquid phase soluble selenoglycoprotems comprising the yeast extract enriched with soluble selenium under acidic conditions that is precipitated to two or more different pH values ). In some embodiments, yeasts enriched with selenium are yeast enriched with non-viable, dry selenium containing 2% or less of inorganic selenium.

The invention also provides compositions comprising soluble selenoglycoprotems prepared according to the invention. For example, in some embodiments, the invention provides a composition with selenium deficiency comprising soluble selenoglycoprotems of the invention. In some embodiments, the soluble selenoglycoprotems of the invention are added to and / or mixed with a composition with selenium deficiency. In some embodiments, the addition or mixing (eg, combination) of selenoglycoprotems comprises the combination of selenoglycoprotem and one or other other types of selenium in the composition. In some embodiments, the soluble selenoglycoprotems of the invention are added to and / or mixed with compositions containing selenium (for example, to increase and / or supplement the total amount of selenium).

The invention also provides a composition comprising soluble selenoglycoprotems and a vehicle. In some embodiments, selenoglycoprotems are a specific fraction of dependent selenoglycoprotems. of pH. In fact, a variety of vehicles can be used that include dendromers, polymersers, nanoparticles, slow release polymers, nanocapsules, and / or molecular impression poKmeres. In a preferred embodiment, the vehicle is a slow release polymer. In another preferred embodiment, the vehicle is a polymer with molecular impression. In addition to another preferred embodiment, the vehicle is a polymorph used to encapsulate selenoglycoprotem. In fact, any polymersome known in the art can be used. In some embodiments, the polymersome comprises block copolymer of poly (ethylene oxide) (PEO). Any block copolymer can be used, including, for example poly (ethylethylene) (PEE), poly (butadiene) (PB or PBD), poly (styrene) (PS), and poly (isoprene) (PI). In some embodiments, the polymer comprises poly (£ -caprolactone) diblock copolymer (PCL). In some embodiments, the polymersome comprises diblock copolymers based on poly (ethylene oxide) -block-poly (£ -caprolactone) (PEO-b-PCL). In some embodiments, the polymersome comprises a block copolymer that is a triblock, tetrablock, pentablock copolymer, or at least six blocks. In some embodiments, the polymersome is obtained from the coupling of poly (lactic acid), poly (glycolide), poly (lactic-coglycolic acid) and / or poly (3-hydroxybutyrate) with PEO. A variety of sizes find their use in the compositions and methods of the invention including polymersome encapsulated selenoglycoprotems having a diameter of approximately 50-300 nm although polymersome encapsulated selenoglycoprotems with a larger diameter can be used (e.g., approximately 350 nm, 400 nm, 500 nm or greater) and less (for example, approximately 40 nm, 30 nm, 20 nm, or less).

<b>Description of the figures</b>

Figure 1 shows the sequential preparation of soluble selenoglycoprotems (SGPs) of SEL-PLEX by acid extraction and subsequent precipitation of an embodiment of the invention.

Figure 2 shows a pH dependent selenoglycoprotection precipitation process of an embodiment of the invention.

Figure 3 shows a heat map depicting the effects of different treatments supplemented with selenium described in Tables 3 and 4 of Example 3 at levels of genetic expression in skeletal muscle of chicken breast with respect to a baseline control.

Figure 4 shows a Venn diagram representing the different effects of the SGP fraction at pH 4.0 and SEL-PLEX in the formation of gene expression profiles in chest muscle.

Figure 5 shows exemplary genes identified as commonly regulated with sodium selenite (SS), SEL-PLEX (SP) and selenoglycoprotem fraction (SGP) at pH 4.0.

Figure 6 shows exemplary genes identified as commonly regulated with SEL-PLEX (SP) and selenoglycoprotem fraction (SGP) at pH 4.0, but not with sodium selenite (SS).

Figure 7 shows exemplary genes identified as regulated only with the selenoglycoprotem fraction (SGP) at pH 4.0, and not regulated with sodium selenite (SS) or SEL-PLEX (SP).

Figure 8 shows an exemplary gene identified as regulated only with the selenoglycoprotem fraction (SGP) at pH 4.0, and not regulated with sodium selenite (SS) or SEL-PLEX (SP).

Figure 9 shows an exemplary gene identified as regulated only with the selenoglycoprotem (SGP) fraction at pH 4.0, and not regulated with sodium selenite (SS) or SEL-PLEX (SP).

Figure 10 shows an exemplary gene identified as regulated only with the selenoglycoprotem (SGP) fraction at pH 4.0, and not regulated with sodium selenite (SS) or SEL-PLEX (SP).

Figure 11 shows a heat map representing the effects of different treatments supplemented with selenium that are described in Tables 3 and 4 of Example 3 at levels of genetic expression in liver tissue with respect to a baseline control.

Figure 12 depicts the release of SGP from nanocapsules at pH 5.1 and pH 7.4 over a period of two weeks.

Figure 13 shows images of Tunnel Effect Cryogenic Electron Microscopy (cryo-TEM) of polymersome encapsulated selenoglycoprotem at pH 7.4.

<b>Definitions</b>

To facilitate an understanding of the present invention, a number of terms and expressions are defined below: As used herein, all terms "peptide", "polypeptide" and "protema" refer to a primary amino acid sequence which is joined by covalent "peptide bonds". In general, a peptide consists of a few amino acids, usually 2-50 amino acids, and is shorter than a protein. The term "polypeptide" includes peptides and proteins.

The term "glycoprotem (s)" or "glycopeptide (s)" refers to a protein or peptide that contains one or more carbohydrate moieties linked covalently to the polypeptide chain. The term "selenoprotem (s)" or "selenopeptide (s)" refers to a protein or peptide that contains one or more selenium atoms. Generally, selenium atoms are incorporated into proteins within amino acids containing selenium including selenocystem and selenomethionine.

The term "selenoglycoprotem (s)", "selenoglycopeptide (s)" or "SGP (s)" refers to a glycoprotem or glycopeptides that incorporate one or more selenium atoms. In general, "selenoglycoprotems" comprise one or more amino acids containing selenium. The "selenoglycoprotems" can comprise a number of carbohydrates in Any number of different ways.

The terms "sample" and "test sample" are used in their broadest sense and include samples or test samples obtained from any source. As used in the term "sample" it is used to refer to biological samples obtained from animals (including humans), and includes fluids, solids and tissues. In some embodiments of the present invention, biological samples include cerebrospinal fluid (CSF), serous fluids, urine and saliva, blood, and blood products such as plasma, serum.

As used herein, the terms "yeast" and "yeast cells" refer to eukaryotic microorganisms classified in the fungi kingdom, which have a cell wall, cell membrane and intracellular components. Yeasts do not form a specific taxonomic or phylogenetic grouping. At present approximately 1,500 species are known; It is estimated that only 1% of all yeast species have been described. The term "yeast" is often taken as a synonym for S. cerevisiae, but the phylogenetic diversity of yeasts is shown by placing it in as many Ascomycota divisions as Basidiomycota. The term "yeast" includes brewer's yeast, distilled yeast and panadena yeasts. The gemacion yeasts ("true yeasts") are classified in the order Saccharomycetales. The majority of yeast species reproduce asexually by gestation, although some reproduce by binary fission. Yeasts are unicellular, although some species become multicellular through the formation of a string of connected gemacion cells known as pseudohyphae, or false hyphae. The size of the yeast can vary greatly depending on the species, usually measuring 3-4 micrometers in diameter, although some yeasts should reach more than 40 pm. As used herein, the terms "selenium enriched yeast" and "selenized yeast" refer to any yeast (eg, Saccharomyces cerevisiae) that is grown in a medium containing inorganic selenium salts. In fact, it is contemplated that the diversity of selenium salts are useful in the present invention including, sodium selenite, or sodium selenate. Free selenomethionine (for example, not associated with a cell or yeast) can also be used as the source of selenium for yeast enriched with selenium since yeast does incorporate this form of selenium. During cultivation, due to the chemical similarity between selenium and sulfur, yeast incorporates selenium instead of sulfur in what are normally organic compounds that contain sulfur inside the cell. A compound containing selenium in such yeast preparations is selenomethionine which is incorporated into polypeptides / proteins. The amount of total cellular selenium present in the form of selenomethionines in such preparations will vary, but may be between 10 and 100%, 20-60%, 50-75% and between 60 and 75%. The rest of the organic selenium in the selenized yeast preparations is predominantly composed of intermediate compounds in the route for the selenomethionine biosynthesis. These include selenocystem, selenocistationin, selenohomocystem and seleno-adenosylselenomethionine. The amount of residual inorganic selenium salt in the finished product is generally quite low (for example, &lt;2%). As used herein, the term "SEL-PLEX" refers to a yeast enriched with non-viable, dry selenium (for example, Sacchoromyces cerevisiae registration number CNCM 1-3060, National Microorganism Crop Collection (CNCM ), Institut Pasteur, Paris, France) grown in a semi-continuous crop system that provides increasing amounts of cane molasses and selenium salts in a way that minimizes the detrimental effects of selenium salts on the yeast growth rate and allows optimal incorporation of inorganic selenium the organic cellular material. The residual inorganic selenium is removed (for example, using a rigorous washing process) and does not exceed 2% of the total selenium content.

As used herein, the term "organic selenium" refers to any organic compound where the selenium atom is connected directly to a carbon atom.

As used herein, the term "inorganic selenium" generally refers to any selenium salt (for example, sodium selenite, sodium selenate) in which selenium is in an oxidation state -2, 4 and 6.

As used herein, the terms "host", "subject" and "patient" refer to any human animal and animals (eg, primates, dogs, cats, cows, horses, sheep, birds, fish, crustaceans ) that is studied, analyzed, tested, diagnosed or treated. As used herein, the terms "host", "subject" and "patient" are used interchangeably, unless otherwise indicated.

As used herein, the term "w / w" ("weight / weight") refers to the amount of a given substance in a composition on a weight basis. For example, an animal feed comprising 0.02% w / w dietary dietary supplement refers to the dietary dietary supplement mass being 0.02% of the total mass of the animal feed (for example , 200 grams of dietary food supplement composition in 999,800 grams of animal feed).

As used herein, the term "purified" or "purify" refers to the removal of components from a sample. For example, the walls of the yeast cell were purified by removing the components of the cell wall of a component that is not yeast (for example, components of the plasma membrane and / or intracellular components of the yeast); They are also purified by removing contaminants or other agents other than the yeast cell wall. the removal of components from the cell wall of a component that is not yeast and / or contaminants from the cell wall of a component that is not yeast results in an increase in the percentage of the cell wall of the yeast or components thereof in a sample.

As used herein, the term "effective amount" refers to the amount of a composition (for example, comprising selenoglycoproteins of the present efficient invention to provide beneficial or desired results. An effective amount can be administered in one or more more administrations, applications or dosages.

As used herein, the term "bioavailability" refers to the fraction of a molecule or component that is available to an organism that reaches systemic circulation. When a molecule or component is administered intravenously, its bioavailability is quite high. However, when a molecule or components are administered through other routes (such as orally), their bioavailability decreases (due to incomplete absorption and first step metabolism). In a nutritional environment, bioavailability refers to the rates of absorption and use of a nutrient. For example, different forms of the same nutrient may have different bioavailability.

As used herein, the terms "feed", "food products", "feed for animals", and "feed" refer to material or materials that are consumed by animals and provide energy and / or nutrients to the diet of an animal Examples include Total Mixed Ration (TMR), fodder (s), microgranule (s), concentrate (s), pre-mix or co-product (s), grain (s), distillery grain (s), molasses , fiber (s), cereal (s) in silo (s), grass (s), hay, grain (s), leaves, flour, soluble compound (s), and supplement (s). As used herein, the terms "dietary supplement", "dietary supplement", "dietary supplement composition", refer to a food product formulated as a dietary or nutritional supplement to be used as part of a diet human or animal, for example as an addition to animal feed.

As used herein, the terms "administration" and "administer" refer to the act of administering a drug, prodrug, or other agent, or therapeutic treatment (eg, compositions of the present invention) to a subject (by example, a subject or cells in vivo, in vitro, or ex vivo, tissues and organs). By way of example for the human body, it can be through the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhaler), oral mucosa (oral) ), ofdo, rectal, vaginal, by injection (for example, intravenously, could subcutaneously, intratumorally, intraperitoneally).

As used herein, the terms "co-administration" and "co-administer" refer to the administration of at least two agent (s) (eg, composition comprising selenoglycoprotems of the invention and one or other agents more - for example, an antibiotic, a therapeutic agent (for example, drug or pharmaceutical agent), or another biologically active compound) or therapies for a subject. In some embodiments, the co-administration of two or more agents or therapies is simultaneous. In other embodiments, a first agent / therapies is administered before a second agent / therapy. Those skilled in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can easily be determined by someone with experience in the field. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than those appropriate for administration alone. Therefore, co-administration is especially desirable in embodiments in which the co-administration of the agents or therapies decreases the necessary dosage of a potentially harmful agent (such as toxic), and / or when the co-administration of Two or more agents result in the sensitization of one subject to the beneficial effects of one of the agents through co-administration of the other agent.

As used herein, the term "treatment" or grammatical equivalents includes the improvement and / or reversion of disease symptoms (eg, neurodegenerative disease). A compound that causes an improvement in any parameter associated with disease when used in the methods of systematic identification of the present invention can thus be identified as a therapeutic compound. The term "treatment" refers to both therapeutic and prophylactic treatment or preventive measures. For example, subjects who may benefit from treatment with compositions and methods of the present invention include those who already have a disease and / or disorder (eg, neurodegenerative disease, diabetes or lack or loss of cognitive function) as well as those in those that are going to prevent a disease and / or disorder (for example, using a prophylactic treatment of the present invention).

As used herein, the expression "at risk of disease" refers to a subject (for example, a human being) who is predisposed to experience a particular disease. This predisposition can be genetic (for example, a particular genetic tendency to experience the disease, such as disorders hereditary), or due to other factors (for example, age, weight, environmental conditions, exposures to harmful compounds present in the environment).

As used herein, the expression "suffering from the disease" refers to a subject (for example, a human being) who is experiencing a particular disease. The present invention includes subjects who are experiencing any range of diseases (for example, from sub-climacteric manifestation of the disease in advanced state) where the subject has at least some of the signs (for example, signs and symptoms) associated with the disease in particular.

As used herein, the terms "disease" and "pathological condition" are used interchangeably to describe a state, signs, and / or symptoms that are associated with any alteration of the normal state of a living animal or any of its organs or tissues that interrupt or modify the remit of normal functions, and may be a response to environmental factors (such as (radiation, malnutrition, industrial hazards, or weather), to specific infectious agents (such as worms, bacteria, or viruses), to an inherent defect of the organism (such as various genetic abnormalities), or to combinations of these and other factors.

The term "compound" refers to any chemical, pharmaceutical, drug entity that can be used to treat or prevent a disease, medical condition, nausea, or disorder of body function. The compounds comprise both known and potential therapeutic compounds. It can be determined that a compound is therapeutic by systematic identification using the methods of systematic identification of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (for example, through animal tests or previous experience with administration to humans) that is effective in such a treatment.

As used herein, the term "kit" is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as nutrients and drugs as well as means of administration.

As used herein, the term "toxic" refers to any harmful or harmful effect on a subject, a cell, or a tissue compared to the same cell or tissue before administration of the toxic agent.

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent (eg, composition and comprises selenoglycoprotems) with a vehicle, inert or active, which makes the composition especially suitable for use. in diagnosis, preventive and / or therapeutic in vitro, in vivo or ex vivo.

The terms "pharmaceutically acceptable" or "pharmacologically acceptable", as used herein, refer to compositions that do not essentially produce adverse reactions, for example, toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the expression "topically" refers to the application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues (eg, alveolar, buccal, lingual, masticatory mucosa, or nasal, and other tissues and cells that line the hollow organs or body cavities). As used herein, the term "pharmaceutically acceptable vehicle" refers to any of the conventional pharmaceutical vehicles that include phosphate buffered saline solution, water, emulsions (eg, as in the form of oil / water emulsions or water / oil), and various types of wetting agents, each and every solvent, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delay agents, disintegrating agents (for example, potato starch or sodium starch glycolate). The compositions may also include stabilizers and preservatives. Examples of vehicles, stabilizers and adjuvants are described, for example, in Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975). In some embodiments, a food product (for example, dietary material and / or treatment) acts as a vehicle (for example, of a composition of selenoglycoprotein composition of the invention).

As used herein, the term "pharmaceutically acceptable salt" refers to any salt (for example, obtained by reaction with an acid or a base) of a compound of the present invention that is physiologically tolerated by the target subject ( for example, a mairnfero subject, and / or cells, tissues or organs in vivo or ex vivo). The "salts" of the compounds of the present invention can be obtained from inorganic or organic acids and bases. Examples of acids include clorlddrico, bromtndrico, sulfuric, fumaric, maleic, phosphoric, glycolic, lactic, salidic, sucdnic, toluene-p-sulfonic, tartaric, acetic, dtrico, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthale, naphthane -2-sulfonic, benzenesulfonic acid. Other acids, such as oxalic, or which by themselves are not pharmaceutically acceptable, can be used in the preparation of useful salts as intermediates to obtain the compounds of the invention pharmaceutically acceptable acid addition salts. Examples of bases include alkali metal hydroxides (for example, sodium), alkaline earth metal hydroxides (for example, magnesium), ammonia, and compounds of formula NW⁴+, where W is C alkyl^1-4. Examples of salts include: acetate, adipate, alginate, aspartat o, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, canforate, camphor sulphonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptane, hexane, heptane, hexane, heptane, hexane, heptane -hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate. Other examples of salts include anions of the compounds of the present invention combined with a suitable cation such as Na +, NH⁴+, and NW⁴+ (where W is a C alkyl group^1-4). For therapeutic use, it is contemplated that the salts of the compounds of the present invention are pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. For therapeutic use, it is contemplated that the salts of the compounds of the present invention are pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

As used herein, the term "drying" refers to spray drying, lyophilizing, air drying, drying in vain or any other type of process that reduces or eliminates liquid in a substance.

As used herein, the term "spray drying" refers to a method commonly used to dry a liquid-containing substance using hot gas to evaporate the liquid to reduce or eliminate liquid in the substance. In other words, the material is dried by spraying or atomizing in a stream of heated dry air.

As used herein, the term "lyophilize" and the term "lyophilization" and the term "cryodecay" refer to the removal of a solvent from the matter in a frozen state by sublimation. This is achieved by freezing the material to be dried below its eutectic point and then providing the latent heat of sublimation. Precise control of ford and heat input allows drying from the frozen state without retrofusion of the product. In practical application, the process is accelerated is precisely controlled under conditions of reduced pressure.

As used herein, the term "dry free flowing powder" refers to a dry free flowing powder, for example a powder that can be poured into a container, bag, container, etc., without impeding large lumps. .

As used herein, the term "grind" refers to reducing particle size by impact, shear, or rubbing wear.

As used herein, the term "wash" refers to removal or cleaning (for example, using any type of solute (eg, distilled water, buffer, or solvent) or mixture) of impurities or unwanted component Soluble of a preparation (eg, a yeast cell wall) can be washed to remove non-yeast cell wall components from the sample).

As used herein, the term "polymersome" refers to vesicles that are assembled from synthetic polymers in aqueous solution (for example, that can be used to encapsulate a material). Polymersomes can be prepared using amphiphilic synthetic block copolymers to form the vesicle membrane, and have radii ranging from 50 nm to 5 pm or more. The mayonnaise of the polymersers contain an aqueous solution in their nucleus and are useful for encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides as well as DNA and RNA fragments. The polymembrane membrane provides a physical barrier that insulates encapsulated material from external materials, such as those found in biological systems. Examples of polymorphs they find use embodiments of the invention, as well as their syntheses, can be found, for example, in US Patent Nos. 7,867,512, 7,682,603, and 6,835,394.

As used herein, the term "residue" refers to unwanted or unused materials. As used herein, the term "wastewater" is any water whose quality has been adversely affected by anthropogenic influence.

<b>Detailed description of the invention</b>

<b>Introduction</b>

Selenium is a trace element involved in the regulation of aspects of the antioxidant defense mechanism in all living tissues by interaction with the body's glutathione (GSH) and its antioxidant enzymes that contain Se principal, glutathione peroxidase (GPX) and thioredoxin reductase ( See, for example, Goehring et al., J. Anim. Sci. 59, 725-732 (1984); Gerloff et al., J. Anim. Sci. 70, 3934-3940 (1992)). Glutathione and GPX have the ability to protect the integrity of unsaturated bonds of membrane phosphoUpids by extinguishing free radical attacks capable of initiating and propagating the oxidation of the Ifpids (See, for example, Meister and Anderson, Annu. Rev. Biochem. 52, 711- 760 (1983); Deleve and Kaplowitz, Pharm. Ther. 52, 287-305 (1991); Palmer and Paulson, Nutr. Rev. 55, 353-361 (1997)).

Selenium has also been associated with a reduction in cancer risk in several epidemiological studies (See, for example, Salonen et al., Am. J. Epidemiol. 120: 342-349 (1984); Willett et al., Lancet 2 : 130-134 (1983); Virtamo et al., Cancer 60: 145-148 (1987)). Various selenium compounds of natural and synthetic origin have been shown to inhibit tumor development in animal studies in a wide range of dosages (See, for example, Ip, J. Nutr. 128: 1845-1854 (1998)). Although most of the animal studies have used pharmacological doses of selenium (&gt; 2 mg / kg) in cancer chemoprevention (See, for example, Ip, J. Nutr. 128: 1845 1854 (1998)), has shown that selenium deficiency increase breast carcinogenesis (See, for example, Ip and Daniel, Cancer Res. 45: 61-65 (1985) and UVB-induced skin (See, for example, Pence et al., 102: 759-761 (1994)).

The yeast cell wall proteins are connected to polysaccharides from the cell wall by chemical bond, and in an order to release proteins from the yeast cell wall in solution, and it is necessary to break these bonds. Conventional practice for extracting proteins from the yeast cell wall is to break the bonds using alkaline hydrolysis (pH 11.5, 80 ° C) before centrifuging to separate glucan polysaccharide proteins that are insoluble in water.

Experiments performed during the development of embodiments of the invention were performed in an attempt to extract selenoglycoprotems using the conventional practice of alkaline hydrolysis. These attempts to extract selenoglycoprotems from the yeast cell wall using alkaline hydrolysis failed. It was later learned that the failure in the extraction of selenoglycoprotems from the yeast cell wall using alkaline hydrolysis was due to the destruction of selenoglycoprotems. Subsequent attempts to extract selenoglycoprotems were carried out using a modified alkaline hydrolysis procedure (pH 11.5, 60 ° C). These attempts also failed to extract selenoglycoprotems from the yeast cell wall. The usual methods of extracting alkaline yeast protein did not work when trying to extract selenoglycoprotems from yeast. Therefore, additional experiments were performed during the development of embodiments of the invention that attempted to extract selenoglycoprotems by acid extraction (pH 5.80 ° C). As described herein, the acid extraction method was satisfactory; selenoglycoprotems were not destroyed and extraction was possible.

Therefore, in some embodiments, the invention provides new methods for obtaining selenoglycoprotems (SGPs), selenoglycoprotem (SGP) compositions (for example, obtained by acid extraction methods (eg, pH-dependent selenoglycoprotem fractions), compositions that they comprise encapsulated selenoglycoprotems (GSP's). In particular, the experiments performed during the development of embodiments of the invention demonstrate that the compositions (for example, comprising SGP (for example, isolated by a method of the invention)) and methods of the invention can be used to provide biologically selenium available to a subject (for example, thereby increasing the selenium content of tissue and / or muscle within the subject (for example, thereby leading to a stabilization and / or improvement of the health of a subject))). In some embodiments, the invention provides methods for obtaining GSP's, compositions comprising encapsulated SGPs (eg, encapsulated polymersome). In some embodiments, the invention provides compositions comprising SGPs and methods for preparing, producing, purifying, isolating, extracting, and / or characterizing them.

The invention also provides soluble selenium compositions (eg, SGPs) and methods of production, and purification thereof. For example, in some embodiments, the invention provides soluble SGPs (See, for example, Examples 1-2 describing the generation of (eg, separation and characterization of) pH-dependent SGP fractions) (See, for example, Examples 3-4). In some embodiments, the invention provides selenium (for example, organic selenium (eg, pH-dependent fraction of SGP of SEL-PLEX or other yeast enriched with selenium)) in a soluble form. In some embodiments, the invention provides a soluble selenium composition (eg, organic selenium) with low fiber content. In some embodiments, the invention provides compositions and methods for soluble organic selenium administration systems (eg, selenoglycoprotems encapsulated with polymersome, nanocapsules, polymers).

Yeast (for example, Saccharomyces cerevisiae), which grows in medium containing selenium (for example, inorganic selenium (for example, sodium selenite (Na²SeO³))) metabolism of selenium (for example, inorganic selenium) and incorporates selenium instead of sulfur in cystem and methionine, providing proteins containing selenoamino acids (SeCys and SeMet) (Demirci et al., J. Agric. Food Chem. 47, 2496-2500 (1999)., Demirci &amp; Pometto. J. Agric. Food Chem. 47, 2491-2495 (1999), Ouerdane &amp; Mester. J. Agric. Food Chem. 56,11792-11799, (2008)). ALLTECH, Inc. (Nicholsville, KY, USA) produces yeast enriched with spray-dried selenium marketed under the name SEL-PLEX as a nutritional and food nutritional supplement, which contains "organic selenium" (See, for example, Korhola et al., Res. 18 , 65-68, (1986).). The minimum concentration of selenium in SEL-PLEX is 1500 ppm and the proteins are the exclusive vehicles of it (See, for example, Surai.

Nottingham University Press 2002, 234-236 (2002), Kelly &amp; Power. J. Dairy Sci. 78, 237-242 (1995), McSheehy et al., Analyst 130, 35-37 (2005)). In yeast there are two combinations of proteins: proteins present within the yeast cell and proteins associated with the yeast cell wall mannan (See, for example, Sedmak. Sun Publication. United States Patent Pub. N .: US 2006/0263415.). Approximately 17.0% by weight of commercial SEL-PLEX is water soluble, and this material contains less than 6.5% of the total selenium present in SEL-PLEX. Studies of chicken feeding in which SEL-PLEX and sodium selenite were used as the sources of selenium, indicated a much better transfer of selenium in a chicken breast muscle with SEL-PLEX. A variety of bioactivities were discovered for oral administration of SEL-PLEX (See, for example, Rayman. The Lancet 356, 233-241 (2000), McKenzie Trends in Immunology 19, 342-345 (1998), Tapiero. Biomedicine &amp; Pharmacotherapy 57, 134-144 (2003), Combs &amp; Gray Pharmacol. Ther. 79, 179-192 (1998), Clark et al., J. Am. Med. Assoc. 276, 1957-1963 (1996)).

In some embodiments, the invention provides separation, and physical and chemical characterization of soluble selenoglycoprotems (SGP's) of yeast (e.g., Saccharomyces cerevisiae) that grows in medium containing selenium (e.g., inorganic selenium (e.g., sodium selenite)) , and its active participation in the supply of "organic selenium" to the tissues (for example, human or, animal, chicken, etc.), by feeding the subject with food (for example, I think) supplemented with or otherwise contains the GSP of the invention (See, for example, Examples 1-4). The invention provides administration directed to the tissue, intravenously, orally, and / or transdermally, of soluble selenium components, active (e.g., pH-dependent SGP fractions of a yeast enriched with selenium (e.g. SEL-PLEX)) . In some embodiments, the invention provides slow-release forms of "organic selenium" delivery systems (for example, nanocapsules, polymer spheres, and SGPs encapsulated with polymersome) (See, for example, Examples 5 9). In some embodiments, the invention provides a composition comprising GSP's encapsulated with polymerase. In some embodiments, the invention provides an increase in the supply and improvement of the biodistribution of SGPs by encapsulating SGPs within polymersers based on poly (ethylene oxide) -block-poly (£ -caprolactone) (PEO-b-PCL).

The separation of various components of yeast cells has been described by extracting cell walls from intracellular components of yeast cells (See, for example, Otero et al., J. Chem. Tech. Biotechnol. 66, 67-71 (nineteen ninety six)). These separation techniques have not been applied to the extraction of spray dried selenium yeast. The conventional alkaline conditions (pH 9.0-14.0) used in the technique for extracting glycoprotems from yeast (See, for example, Roberge et al., J. Agric. Food Chem. 51,4191 4197 ( 2003)) failed because the SeH or SeMe substituents were removed from the amino acids selenocystin or selenomethionine. In other words, the desired substituents decomposed and as a result the selenium that was present in the original GSP was lost during the extraction attempt under alkaline conditions when applied to the extraction of selenoprotems containing yeast (See, for example, Table 12 from Example 8).

Accordingly, in some embodiments, the invention provides selenoglycoprotem compositions (for example, which comprise a pH-dependent selenoglycoprotection fraction described herein). In some embodiments, the invention provides methods for producing, purifying, isolating, extracting, separating, precipitating, and / or characterizing selenoglycoprotems (eg, yeast enriched with selenium).

<b>II. Extraction, separation, purification and use</b>

In some embodiments of the invention, soluble selenium is obtained in the form of selenoglycoprotems (SGPs). In some embodiments, the SGPs are extracted from a general source of selenoprotems (for example, yeast enriched with selenium (for example, SEL-PLEX)). In some embodiments, the extraction and / or purification of the SGPs comprises one or more pH-dependent extraction / precipitation steps (for example, as described in Examples 1 and 2). In some embodiments, the extraction and / or purification of the SGPs comprises one or more pH-dependent fractionation steps. In some embodiments, the invention provides acid extraction of yeast enriched with selenium (for example, SEL-PLEX) to subtle the GSPs (for example, without denaturing and / or destroying the SGPs). In some embodiments, the invention provides that pH-dependent precipitation of SGPs from an acid extract (for example, as described in Examples 1-2) that produces an increase in selenoglycoprotem content, decrease in non-digestible fiber content. , and a high concentration of selenium (for example, a concentration of selenium that is higher than that present in SEL-PLEX). Therefore, in some embodiments, the invention provides extraneous SGP of yeast enriched with selenium where the selenium content of the GSP is greater than the selenium content of the material from which the GSP was extracted (for example, in% in p / p, ppm). In some embodiments, the invention provides selenium in the form of a pH dependent SGP fraction (e.g., pH 4.0 or pH 6.0 fractions) of selenium-enriched yeast (e.g., SEL-PLEX) presenting the same level or a highly similar level of bioavailability when administered to a subject compared to the bioavailability of selenium from the precursor source of yeast enriched with selenium (eg SEL-PLEX) (See, for example, Example 3, Table 5).

In some embodiments, the SGPs are extracted from a general source of selenoprotems (for example, yeast cells enriched with selenium (for example, SEL-PLEX)). In some embodiments, a portion of the selenoprotems in a selenoprotem source comprises the GSPs (for example, 0.1% ... 0.2% ... 0.5% ...

1.0% ... 2.0% ... 5.0% ... 10% ... 20% ... 50% or more GSPs). In some embodiments, a source of selenoprotem comprises cells (eg, yeast cells) that have been cultured in the presence of media containing Se (for example, media rich in Se). In some embodiments, cells containing Se (eg, yeast cells) are processed to extract, isolate, and / or unify selenoprotems resulting in a source of selenoprotem or composition rich in selenoprotem (eg, composition rich in SGP) . In some embodiments, a sample comprising selenoprotems and SGPs is enriched with SGPs.

In some embodiments, a source of selenoprotem or composition rich in selenoprotem (e.g., yeast source enriched with selenium (e.g., SEL-PLEX)) is subjected to one or more stages to produce isolate, purify, separate, and / or extract the SGPs. In some embodiments, a source of selenoprotem is mixed (for example, in a liquid vehicle (eg, water, buffer, salt)) to produce a suspension, mixture, lysate and / or protein solution. In some embodiments, the source of selenoprotem (for example, source of yeast enriched with selenium (for example, SEL-PLEX)) is mixed at elevated temperature (for example, above freezing temperature, above room temperature , 30 ° C ... 40 ° C ... 50 ° C ... 60 ° C ... 70 ° C ...

80 ° C ... 90 ° C or higher). In some embodiments, the source of selenoprotem (for example, source of yeast enriched with selenium (for example, SEL-PLEX)) is mixed at low pH (for example, acidic conditions (e.g., pH 0.5, 1.0 , 1.5, 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5). In some embodiments, the source of selenoprotem (for example, source of yeast enriched with selenium (for example, SEL-PLEX)) is mixed gently, mixed rapidly, mixed thoroughly, mixed vigorously. In some embodiments, the source of selenoprotem (for example, source of yeast enriched with selenium (for example, SEL-PLEX)) is mixed pH under pH (for example, acidic conditions (eg, pH 0.5, 1.0 , 1.5, 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5) and elevated temperature (for example, above the temperature of freezing, above room temperature, 30 ° C ... 40 ° C ... 50 ° C ... 60 ° C ... 70 ° C ... 80 ° C ... 90 ° C, or higher)). In some embodiments, the pH of the mixture is maintained by the addition of acid or base.

In some embodiments, the selenoprotem-containing mixture is centrifuged to separate the liquid / soluble and solid / insoluble phases. In some embodiments, a centrifuge speed that is sufficient to separate the phases was selected. In some embodiments, the liquid phase comprises soluble SGPs. In some embodiments, the pH of the liquid phase is adjusted (for example, increased) to precipitate a portion of the SGPs. In some embodiments, the pH of the liquid fraction is increased slightly to moderately (for example, the pH has an increase of 0.1 ... 0.2 ... 0.5 ... 1.0 .. 2.0) to precipitate the SGPs that were soluble at the original pH, but not at the elevated pH. In some embodiments, the pH of the liquid fraction is significantly increased (for example, the pH shows an increase of 1, 0.2, 0.3, 0.4, 0.5, 0.6.0) to precipitate a much of the SGPs that were soluble at the original pH. In some embodiments, selenoglycoprotems are precipitated and separated from the liquid phase under a variety of pH conditions to generate multiple fractions of soluble pH-dependent selenoglycoprotems. For example, in some embodiments, a single liquid phase comprising yeast extract enriched with soluble selenium under acidic conditions is used to create a first fraction of soluble selenoglycoprotems that precipitates at a first pH (for example, pH of 1.85) , a second fraction of soluble selenoglycoprotems that precipitates at a second pH (e.g., pH 3.0), a third fraction of soluble selenoglycoprotems that precipitates at a third pH (e.g., pH of 4.0), and a fourth fraction of soluble selenoglycoprotems that precipitates at a fourth pH (for example, pH 6.0). In some embodiments, the precipitation of the liquid phase selenoglycoprotems by increasing the pH of the liquid phase comprises multiple sequential precipitation reactions dependent on the pH of the liquid phase.

In some embodiments, precipitated SGPs are separated from the liquid fraction with the pH adjusted by centrifugation (for example, at a rate sufficient to produce separate liquid and solid phases). In some embodiments, the SGPs that have separated from the liquid phase are lyophilized to produce a solid fraction of SGP. In some embodiments, the process to increase the pH of the liquid phase and centrifugation to isolate the SGP fraction is repeated to produce SGP fractions of different solubilities (eg, soluble below pH 1.5 ... soluble below pH 2 ... soluble below pH 3 ... soluble below pH 4 ... soluble below pH 5 ... soluble below pH 6). In some embodiments, the liquid phase that remains after removal of the final SGP fraction finds utility as a component of growth media for cells used in additional production of selenoprotems or SGPs. The initial list of the composition comprising soluble selenoglycoprotems contains two or more pH-dependent fractions of selenoglycoprotems (for example, liquid phase soluble selenoglycoprotems comprising yeast extract enriched with soluble selenium under acidic conditions that precipitate at two or more pH values different).

In a preferred embodiment, the process of extracting SGP from a source of selenoprotem (for example, yeast enriched with selenium (for example, SEL-PLEX)) and pH-dependent fractionation of the mixture of SGP's (See, for example , Figures 2 and 3) comprise two stages (See for example, Examples 1-2, Figures 1 and 2). In the first stage, a suspension of yeast enriched with selenium in 0.3 N HCl (pH 1.5) is stirred and heated at 80 ° C for 8 hours. The pH of the mixture is maintained at pH 1.5 by the addition of concentrated HCl during the first hour of the season. After eight hours, the mixture is centrifuged, and the liquid phase is separated. The pH of the solution is adjusted to 1.85, by the addition of 2.0 N NaOH and the SGPs that have limited solubility at this pH precipitate from the solution and are separated from the liquids (pH 1.85) with the second centrifugation and then lyophilized to produce a solid fraction of SGP of pH 1.85. In some embodiments, the Liquids (pH 1.85) are mixed with the solids (pH 1.5) from the first centrifugation, which results in a change in the pH of the mixture to pH 1.6. The majority of the soluble SGP's at pH 1.6, are transferred to the Mquida phase, without increasing the volumes of the residual currents created within this stage of the process. The main secondary product of this stage of the process are solids of the second centrifugation. The residual solids stream constitutes approximately 56.5% by weight of the yeast enriched with selenium taken for extraction and contains valuable selenium yeast cell wall material containing: 38.91% protein and 2477 ppm selenium. In some embodiments, these solids are used (for example, alone or in combination with other material (for example, yeast enriched with selenium)) as an ecological nutritional supplement in animal diets. In the second stage (See, for example, Figure 3), the liquid phase (pH 1.6) of the second centrifugation (VEASE FIG. 2) is transferred to a mixer and the pH is adjusted to pH 3.0 by the addition of 2.0 N NaOH and the SGP's that have limited solubility at this pH precipitate from the solution and are separated from the liquids (pH 3.0) with the third centrifugation and finally lyophilized to produce a solid fraction of SGP of pH 3.0 (See, for example, Examples 1-2 and Figures 2 and 3). The liquid phase (pH 3.0) of the third centrifugation is transferred to a mixer and the pH is adjusted to pH 4.0 by the addition of 2.0 N NaOH and the SGPs that have limited solubility at this pH precipitate from of the solution and separated from the liquids (pH 4.0) by the fourth centrifugation and finally lyophilized to produce a solid fraction of SGP of pH 4.0. The liquid phase (pH 4.0) of the fourth centrifugation is transferred to a mixer and the pH is adjusted to pH 6.0 by the addition of 2.0 N NaOH and the SGPs that have limited solubility at this pH precipitate from of the solution and separated from the liquids (pH 6.0) by the fifth centrifugation and then lyophilized to produce a solid fraction of SGP of pH 6.0. the only residual current produced in the second stage of the process is the wastewater stream, which contains 13.4% by weight of solids (compared to the weight of the yeast enriched with selenium used in the station) out of which the protein fraction constitutes approximately 13.3% by weight, of sodium chloride for 3.6% by weight and the mono- and oligosaccharides glucose and mannose constitute more than 80% by weight. This "wastewater" stream of pH 6.0 and selenium concentration of 242 ppm can be recycled or otherwise reused for the preparation of a new batch of selenium yeast (for example, used in growth media) .

In some embodiments, GSP's precipitated at pH 4.0 have a higher selenium supply (for example, for muscle tissue) compared to yeast enriched with selenium (e.g. SEL-PLEX) and a much higher supply when compared with inorganic forms of selenium (for example, sodium selenite) (See, for example Example 3). In addition, pH-dependent selenoglycoprotem fractions (for example, pH 6.0) have a different composition than that of yeast enriched with selenium (for example, SEL-PLEX) and allow the supply of similar amounts of selenium although it requires a much smaller amount of starting material (for example, yeast enriched with selenium).

In some embodiments, a SGP extraction procedure comprises 4 or less steps of pH adjustment and centrifugation of the process mentioned above (for example, 1 stage of pH adjustment and centrifugation, 2 stages of pH adjustment and centrifugation, 3 stages of pH adjustment and centrifugation, 4 pH stages of pH adjustment and centrifugation).

<b>ME. Animal feed</b>

Animal feed refers to any food product used to feed domesticated livestock (eg, cattle, goats, sheep, horses, birds, buffalo, alpacas, llamas, donkeys, mules, rabbits, chickens, geese, turkeys or pigs). Animal feed often includes hay, straw, grain silos, compressed and granulated feed, mixed oils and relationships, and also sprouted grains and legumes. The global animal feed industry consumed 635 million tons of feed in 2006, with an annual growth rate of approximately 2%. The use of the agricultural field to grow food products instead of food for humans can be controversial; some types of food, such as corn (Mafz), can also serve as human food, although others such as grass cannot serve. In addition to providing a source of energy to animals, animal feed also provides nutrients (for example, selenium) used by the body.

Nutritional compositions (e.g., soluble selenium compositions (e.g., SGPS) allow a generation of feed compositions for animals comprising selenium (e.g., elevated concentrations of selenium with respect to conventional feed compositions (e.g., selenium soluble)) that reduces overall costs, increases feed conversion and maintains and / or improves the quality of animal products (e.g. meat, eggs, dairy products, etc.) obtained from cattle that receive them in comparison with conventional feed.

For example, in some embodiments, a dietary supplement composition comprises the GSPs that can be combined with and / or incorporated into an animal feed and can be administered to (for example, feed a) an animal to provide effects equivalent or higher in the growth performance in the animal (for example, compared to animal feed diets with other forms of selenium supplement (for example, yeast enriched with selenium (for example, SEL-PLEX)). In some embodiments, a dietary supplement composition of the invention increases the amount of circulating selenium. (for example, located in the blood and / or serum) in a subject (for example, human or animal) that receives the composition. In some embodiments, in the soluble selenium compositions of the invention (e.g., SGPs), a higher proportion of selenium administered is bioavailable than in other selenium formulations (e.g., sodium selenite or yeast enriched with selenium (e.g., SEL- PLEX)). That is, in some embodiments, the compositions comprise selenoglycoprotems that contain a higher proportion and / or proportion of selenium that is made available (for example, bioavailable) for a subject compared to other forms of selenium (eg, enriched yeast with selenium) which means that smaller amounts of a composition comprising the selenoglycoprotem of the invention are necessary (for example, to obtain similar amounts of bioavailability). In some embodiments, a dietary supplement composition (for example, comprising the GSPs of the invention) increases the amount of selenium (for example, located in the muscle or other tissue) in a subject (e.g., human or animal) Who receives the composition. In some embodiments, administration of the SGPs and / or soluble selenium results in an increase in the bioavailability of selenium for serum, adipose tissue, muscle tissue.

In some embodiments, a method for increasing the efficiency with which an animal uses nutrients in a diet to feed the animal comprises provisioning the animal with a diet comprising a dietary supplement composition, where the supplement composition increases the animal's capacity. to profess and use the nutrients present in your diet. In some embodiments, a dietary supplement composition increases the amount of circulating antioxidants (eg, selenium located in the blood and / or serum) in a subject receiving the composition. In some embodiments, a dietary supplement composition increases the amount of antioxidants (eg, selenium located in adipose tissue, muscle, etc.) in a subject receiving the composition.

<b>IV. Pharmaceutical, nutritional and supplement agents</b>

The FDA has established nutritional selenium levels (See 21 CFR 101.9 (c) (8) (iv), January 1994). Humans and animals can safely antagonize limited amounts of both inorganic and organic forms of selenium and can convert nonmethylated selenium into mono- or di- or trimethylated derivatives, of which the most toxic are monomethylated derivatives. (See, for example, Bedwal, RS, et al., Medical Hypotheses, 41 (2): 150-159 (August 1993)). The FDA has adopted the Daily Reference Intakes (RDI) of 70 micrograms of selenium for lactating women and RDI of 55 micrograms for adults who are not breastfeeding. It has been reported that the dosage of selenium of 600 micrograms per day is safe. (See, for example, Ferris GM Lloyd, et al., App. Clin. Biochem., 26: 83-88 (1989). At approximately this dosage, the normal activity of the glutathione reductase enzyme safely converts selenoglutation into hydrogen selenide in the tugado and erythrocytes and is finally secreted. Therefore, at such lower dosages, the body is able to stabilize and secrete safely is the anus that is present in a free metal form. However, as with many trace elements (e.g. selenium), at higher dosage levels or concentrations the beneficial effects are reversed and a dangerous toxicity is manifested. (See, for example, Furnsinn, C. et al., Internat'l J. of Obesity and Related Metab. Dis., 19 (7): 458-463 (1995)).

The administration of selenium in the natural way implies a solution of scientific and medical commitment because, when administered in relatively low concentrations, selenium provides beneficial health effects, however, at higher concentrations, selenium presents a spectacular toxicity so that potential health benefits are lost and toxic becomes the main concern.

As described above, certain forms of selenium (eg, SGPs, water soluble selenium) provide beneficial effects to a subject. Evidence has shown that organic forms of selenium (e.g. selenomethionine and yeast enriched with selenium) may be less toxic and better absorbed than inorganic forms (See, for example, Mahan, Proceedings of the 15th Annual Symposium Nottingham University Press, Nottingham, United Kingdom, pp. 523-535 (1999). However, in some embodiments, multiple forms of selenium are used in combination with others (for example, to provide beneficial effects for the health of a subject). Natural sources of selenium include, but are not limited to, yeast enriched with selenium (for example, selenized).

In certain preferred embodiments, the GSPs (for example, obtained from yeast enriched with selenium (for example, SEL-PLEX), as described in Examples 1-2) are the form of selenium chosen for formulations and compositions of the invention. In some embodiments, compositions comprising water soluble selenium and / or SGPs provide a more biologically available form of selenium compared to other forms of selenium. However, other forms of selenium may also be used, including derivatives or modifications of water-soluble selenium and / or SGPs, SEL-PLEX, or other forms of yeast enriched with selenium, selenomethionine, selenocystem, a selenite compound, a compound of selenate, or derivatives, salts, or modifications thereof. Therefore, in some preferred embodiments, each of these forms of selenium can be used as a component of a formulation. Alternatively, each of the selenium forms described above may be related (for example, chemically or physically) to a drug or therapeutic agent to form a selenium drug derivative. In fact, a composition or The formulation may comprise multiple forms of selenium (eg, SGPs and SEL-PLEX, or Sod-sel and water soluble selenium).

Other forms of selenium found in use in various embodiments of the present invention are described in US Patent Nos. 6,911,550, 6,197,295, 5,221,545, 6, and 6,576,233, and in patent applications of the United States No. 20010043925, 20050069594, 20050089530, and 20080107755.

Accordingly, pharmaceutical, nutritional and / or supplement compositions (for example, foodstuff and / or dietary composition or treatment) comprising one or more forms of selenium (e.g., SGPs, soluble selenium (e.g., selenium soluble in water), etc.), alone or in combination with at least one other agent, such as a stabilizing compound, another or other therapeutic agents, nutrient (s) and / or minerals; and it can be administered in any biocompatible sterile vehicle, including saline solution, buffered saline solution, dextrose, water that can be provided.

The methods find use in the treatment (for example, prophylactically or therapeutically) of diseases (for example, neurodegenerative diseases, cancer, etc.) or in the alteration of physiological conditions. Selenium (e.g., soluble selenium (e.g., water soluble selenium, SGPs))) can be administered to a subject (e.g., a patient) intravenously in a pharmaceutically acceptable vehicle such as physiological saline. Conventional methods (eg, administration through liposomes) can be used for intracellular administration of compounds. Methods of that type are well known to people with regular experience in the field. The formulations are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular and intraperitoneal. In some embodiments, the compositions (eg, SGPs and / or pharmaceutical formulations comprising them) are administered orally.

As is well known in medical techniques, the dosages for any subject can depend on many factors, including the size of the patient, the area of ​​the body surface, the age, the particular compound to be administered, the sex, the time and route of administration, general health and interaction with other drugs that are being administered simultaneously.

Accordingly, in some embodiments, compositions and / or formulations comprising selenium (e.g., soluble selenium (e.g., water soluble selenium), SGPs,) are administered to a subject alone, or in combination with other forms of selenium, pharmaceuticals, small molecules, or in pharmaceutical compositions in which it is mixed with excipient (s) or other pharmaceutically acceptable carriers. In some embodiments, the pharmaceutically acceptable vehicle is pharmaceutically inert. In other embodiments, compositions comprising selenium (e.g., soluble selenium (e.g., water soluble selenium), SGPs) are administered alone to individual subjects suffering from a disease or condition. In other embodiments, compositions comprising selenium (eg, soluble selenium (e.g., water soluble selenium), SGPs,) are administered alone to individual subjects for the promotion of general health or the health of a body system. Compositions comprising selenium (for example, soluble selenium (for example, water-soluble selenium), SGPs) alone or in combination with one or other other forms of selenium can be added to a nutritional beverage or food (for example, ENSURE, POWERBAR), a multivitamin, nutritional products, food products, etc., for daily consumption.

Depending on the target to be altered with the treatment (for example, the genetic expression associated with aging, and / or the regulation of the genetic expression associated with cancer and / or tumor growth or metastasis), these pharmaceutical compositions, supplements, and / or nutraceuticals are formulated and administered systematically or locally. Techniques for formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may include, for example, oral or transmucosal administration; as well as parenteral administration, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

For injection, compositions containing selenium (for example, soluble selenium (for example, water-soluble selenium), SGPs, etc.) are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiologically buffered saline solution. For tissue or cellular administration, penetrating agents suitable for the particular barrier to be permeated are used in the formulation. Such penetrating agents are generally known in the art.

In other embodiments, compositions containing selenium (e.g., soluble selenium (e.g., water soluble selenium), SGPs, etc.) are formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such vehicles allow compositions containing selenium (for example, soluble selenium (for example, water-soluble selenium), SGPs,) to be formulated as tablets, pills, capsules, liquids, gels, syrups, sludges, suspensions for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions include compositions where the active ingredients (e.g., compositions containing selenium (e.g., soluble selenium (e.g., water soluble selenium), SGPs) are contained in an amount effective to achieve the desired purpose. For example, an amount of the pharmaceutical agent may be the amount that alters the expression of a specific gene (for example, KTLG, GRB2, DNAJ3, TGFB1, MAPK8, C1R, UBE4A, S^mPX, USP22, and / or PTP4A1). The determination of effective amounts is well within the capacity of people with experience in the field.

The pH-dependent GSPs and the compositions comprising them can be used in the prevention and / or therapeutic treatment of cancer (for example, to prevent or slow the progression and / or metastasis of the cancer / tumor). For example, in a preferred embodiment, a pH-dependent fraction of SGP from a yeast enriched with selenium (eg, SEL-PLEX) is administered to a subject to regulate the expression (eg, in a desired manner) of a gene associated with cancer growth and / or metastasis). As described herein, it has been identified that certain soluble fractions of SGP have biological properties (for example, the ability to regulate gene expression) from which it was obtained that the precursor material of the soluble SGP fraction (by For example, yeast enriched with selenium (for example, SEL-PLEX)) does not have (See, for example, Example 4 and Figures 7-11). In a preferred embodiment, the pH-dependent SGP fraction of a yeast enriched with selenium (for example, SEL-PLEX) is a pH 4.0-dependent fraction, although other fractions (for example, pH 3.0, pH 6 , 0, etc.) also found are not the compositions and methods of the invention.

In addition to the active ingredients, these pharmaceutical compositions containing selenium (for example, soluble selenium (for example, water-soluble selenium), SGPs, etc.) may contain suitable pharmaceutically acceptable vials that comprise excipients and auxiliary agents that facilitate the processing of the active compounds in preparations that can be used pharmaceutically. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

Pharmaceutical compositions can be manufactured in a manner that is known to itself (for example, by means of conventional processes of mixing, dissolution, granulation, manufacture of dragees, levigation, emulsification, encapsulation, entrapment or lyophilization).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. In addition, the suspensions of the active compounds can be prepared as oil suspensions suitable for injection. Suitable lipophilic solvents or vegetables include fatty oils such as sesame oil, esters of synthetic fatty acids, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with a solid excipient, optionally grinding a resulting mixture and processing the granule mixture, after adding suitable auxiliary agents, if desired, tablet or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, which include lactose, sucrose, mannitol or sorbitol; corn starch, wheat, rice, potato; cellulose such as methyl cellulose, hydroxypropyl methylcellulose, or sodium carboxymethyl cellulose; and gums that include arabigas and tragacanth; and protect such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinyl pyrrolidone, agar gum, algic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain arabic gum, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions and solvents or mixtures of suitable organic solvents . Dyes or pigments may be added dyes or pigments to identify the product or to characterize the amount of active compound (ie, dosage).

Pharmaceutical preparations that can be used include pressure adjustment capsules made of gelatin, as well as sealed soft capsules made of gelatin and a coating such as glycerol or sorbitol. The pressure adjustment capsules may contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate and, optionally, stabilizing agents. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycol with or without stabilizers.

Compositions comprising a compound of the invention formulated in a pharmaceutically acceptable vehicle can be prepared, placed in an appropriate container and labeled for the treatment of an indicated condition. For compositions or formulations containing selenium, the conditions indicated on the label may include the treatment of conditions related to the prophylactic or therapeutic treatment of a disease or condition (eg, cancer, neurodegenerative disease and / or cognitive function).

The pharmaceutical composition can be provided as a salt and can be formed with many acids, which include, but are not limited to, chlortndric, sulfuric, acetic, lactic, tartaric, malico, succinic, etc. The salts tend to be more soluble in aqueous solvents or other protonic solvents which are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1% -2% sucrose, 2% -7% mannitol at a pH range of 4.5 to 5.5 It is combined with buffer before use.

For any compound, the therapeutically effective dose can be calculated initially from cell culture assays. Then, preferably, the dosage can be formulated in animal models (in particular murine models) to achieve a desirable circulating concentration range.

A therapeutically effective dose refers to the amount that improves or prevents symptoms of a pathology or condition (for example, by altering the genetic expression). The toxicity and therapeutic efficacy of such compounds can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the DL⁵⁰(the lethal dose for 50% of the population) and the ED⁵⁰(the therapeutically effective dose for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the DL ratio⁵⁰/FROM⁵⁰. Compounds that have large therapeutic indices are preferred. Data obtained from these cell culture assays and studies in additional animals can be used to formulate a dosage range for human use. The dosage of such compounds is preferably within a range of circulating concentrations that include ED.⁵⁰With little or no toxicity. The dosage varies within this range depending on the dosage form used, patient sensitivity, and the route of administration. The exact dosage can be chosen by a subject or by a doctor in view of the patient to be treated. The dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect (eg, alteration of the genetic expression in a subject). Additional factors that can be taken into account include the severity of the pathology; age, weight and gender of the patient; diet, time and frequency of administration, combination or combinations of drugs, sensitivity to reaction and tolerance / response to therapy. The long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks or once a month, depending on the half-life and the elimination rate of the particular formulation.

In some embodiments, selenium (e.g., soluble selenium, water-soluble selenium, SGPs,) is administered at a daily dose of between 25 and 600 | jg per day (e.g., soluble selenium, water-soluble selenium, and / or SGP is administered to a subject in a manner such as to provide between 25 and 600 jg of selenium to the subject every day). In preferred embodiments, selenium is administered at a daily dose of between 50 and 200 jg per day. In other preferred embodiments, selenium is administered at a daily dose of between 100 and 200 jg per day. Doses outside the range of 25 and 600 jg can be used. In some embodiments, a single dose of selenium (eg, soluble selenium, water soluble selenium, SGPs) is administered once daily. In other embodiments, 2, 3, 4, or more doses may be administered every day (for example, once in the morning and once at night, or once every 4 to 6 hours). For example, in some embodiments, selenium (eg, soluble selenium, water soluble selenium, SGPs, etc.) is administered to a subject in three separate doses, more than three separate doses, two separate doses, or less than two separate doses In some preferred embodiments, the daily dose is administered in a prolonged release capsule. In some preferred embodiments, the daily dose is between 25-75 jg of selenium (eg, soluble selenium, water soluble selenium, SGPs). In other preferred embodiments, the daily dose is 200 jg of selenium (eg, organic selenium, selenized yeast, SEL-PLEX, soluble selenium, water-soluble selenium, SGPs).

Pharmaceutical compositions can be administered in several ways depending on whether a local or systemic treatment is desired and the area to be treated. Administration may be topical (including botany and mucous membranes, including vaginal and rectal administration), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, for example, intrathecal or intraventricular. Compositions and formulations comprising selenium are believed to be particularly useful for oral administration. Compositions and formulations (eg, containing GSP) pharmaceutical, nutritional, and / or supplements containing selenium for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, liquids and powders. Conventional pharmaceutical bonds, aqueous, powdered or oily bases, thickening agents may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, sachets or tablets. Thickening agents, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives, such as penetration enhancers, compound compounds and other pharmaceutically acceptable carriers or excipients.

Therefore, in some embodiments, pharmaceutical, nutritional, and / or supplement compositions include solutions, emulsions and formulations containing liposomes. These compositions can be generated from a variety of components including previously formed liquids, self-emulsifying solids and self-emulsifying semi-solids.

Pharmaceutical, nutritional, and / or supplement formulations, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Techniques of this type include the stage of associating the active principles with the pharmaceutical vehicle (s) or excipient (s). In general, the formulations are prepared by uniformly and intimately associating the active principles with liquid vehicles or finely divided solid vehicles or both, and then, if necessary, the product is shaped.

In some embodiments, compositions containing selenium (eg, soluble selenium, water-soluble selenium, SGP) may be formulated in any of many possible dosage forms, such as tablets, capsules, liquid syrups, soft gels, suppositories and enemas The compositions of the present invention can also be formulated as extended-release nanoparticles (e.g., nanocapsules), vesicles, liposomes, polymers (e.g., molecular impression polymers (MIP), biodegradable slow release polymers, polycation polymers). The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media, and the aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran The suspension may also contain stabilizing agents. In one embodiment, pharmaceutical and / or supplement compositions may be formulated and used as foams. Foams include formulations such as emulsions, microemulsions, creams, jellies and liposomes. Although these formulations are basically of a similar nature, these formulations go in the components and the consistency of the final product.

Compositions containing selenium (eg, soluble selenium, water-soluble selenium, SGP) may additionally contain other adjuvant components conventionally found in pharmaceutical compositions. Therefore, for example, the compositions may contain additional pharmaceutically active, compatible materials, such as, for example, antipyptics, astringent, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions. of the present invention, such as colorants, flavoring agents, preservatives, antioxidants, opacity agents, thickening and stabilizing agents. However, materials of this type, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, humectants, emulsifiers, salts to influence osmotic pressure, buffers, dyes, aromas and / or aromatic substances that do not interact detrimentally with the acid or nucleic acids of the formulation.

In some embodiments, pharmaceutical, nutritional, and / or supplement compositions containing (a) one or more forms of selenium (e.g., SGP (e.g., pH-dependent SGP fraction), soluble selenium, soluble selenium can be provided in water, SEL-PLEX) and (b) one or more other agents (for example, nutrients, minerals, therapeutics, etc.).

The methods may involve the co-administration of compounds comprising selenium (for example, soluble selenium, water-soluble selenium, SGP, etc.) described herein with one or more additional active agents (for example, a therapeutic agent (for example, therapeutic agent for cancer, therapeutic agent for Alzheimer's), antioxidant, etc.). In fact, an additional aspect is to provide methods for improving therapies and / or pharmaceutical, nutritional, and / or supplement compositions by co-administering a composition comprising selenium (e.g., soluble selenium, water-soluble selenium, SGP ( for example, fraction of pH dependent SGP)) of the present invention with a pharmaceutical, nutritional and / or preventive or therapeutic supplement composition. In joint administration procedures, agents can be administered simultaneously or sequentially. In one embodiment, the compounds described herein are administered before the other or other active agents. The formulations and modes of administration may be any of those described above. In addition, the two or more co-administered agents can each be administered using different modes or different formulations. In some embodiments, a composition of the invention will be co-administered with a cancer treatment.

Accordingly, in some embodiments, a composition containing one or more forms of selenium (e.g., SGP (e.g., pH-dependent fraction of SGP), soluble selenium, water-soluble selenium, SEL PLEX) is administered systematically or locally to inhibit the proliferation of tumor cells and angiogenesis, and / or to induce the death of tumor cells in cancer patients. For example, in some embodiments, a composition comprising a pH dependent SGP fraction (pH 4.0) of a yeast enriched with selenium (eg, SEL-PLEX) is administered to a subject under conditions such that the expression of one or more genes associated with cancer / tumor growth and / or metastasis is regulated (for example, regulated positively or negatively) in a beneficial manner in the subject (See, for example, Example 4). The compositions can be administered intravenously, intrathecally, intraperitoneally as well as orally. In addition, they can be administered alone or in combination with antiproliferative drugs.

An example is when a composition containing one or more forms of selenium is covalently linked to an orientation vehicle or an active pharmaceutical agent. Covalent bonding can be performed with any one of the many crosslinking compounds available in the market.

For example, such compositions can be provided in combination with physiologically tolerable liquid, gels or solid vehicles, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to veterinarians for veterinary use, such as domestic or farm-raised animals, and for use in humans in a similar manner to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particular requirements of the individual hosts.

Compositions of that type are generally prepared as liquid solutions or suspensions, or in solid forms. Oral formulations for cancer generally include such normally used additives such as binders, fillers, vehicles, preservatives, stabilizing agents, emulsifiers, buffers and excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain 1% -95% active ingredient, preferably 2% -70%.

The compositions are also prepared as injectable agents, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid can also be prepared before injection.

The compositions are often mixed with diluents or excipients that are physiologically tolerable and compatible. The d iluyentes and Suitable excipients are, for example, water, saline, dextrose, glycerol and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing agents or pH buffering agents.

A wide range of therapeutic agents find use with the present invention. For example, any therapeutic agent that can be co-administered with a composition containing one or more forms of selenium of the invention is suitable.

Some embodiments provide the administration, to a subject, of an effective amount of a composition containing one or more forms of selenium and at least one anti-cancer agent (eg, a conventional anti-cancer agent, such as chemotherapeutic drugs, and / or therapy of radiation).

Mechanisms of anticancer agents suitable for use include, agents that induce apoptosis, agents that induce / cause damage to nucleic acid, agents that inhibit the synthesis of nucleic acids, agents that influence the formation of microtubules and agents that influence the synthesis or stability of the protein.

Classes of anticancer agents suitable for use in compositions and methods include: 1) alkaloids, which include microtubule inhibitors (e.g., Vincristine, Vinblastine and Vindesine), microtubule stabilizers (e.g., Paclitaxel (Taxol), and Docetaxel), and chromatin function inhibitors, which include, inhibitors of topoisomerase, such as, epipodophyllotoxins (eg, Etoposide (VP-16), and Teniposide (VM-26)), and agents that target topoisomerase I (for example, Camptotecina and Isirinotecan (CPT-11)); 2) DNA binding covalent agents (alkylating agents), including nitrogen mustards (e.g., Mechlorethamine, Chlorambucil, Cyclophosphamide, Ifosfamide, and Busulfan (Myleran)), nitrosoureas (e.g., Carmustine, Lomustine, and Semustine), and other alkylating agents (eg, Dacarbazine, Hydroxymethylmelamine, Tiotepa, and Mitocicin); 3) non-covalent DNA binding agents (antitumor antibiotics), which include nucleic acid inhibitors (e.g., Dactinomycin (Actinomycin D)), anthracyclines (e.g., Daunorubicin (Daunomycin, and Cerubidine), Doxorubicin (Adriamycin), is Idarubicin (Idamicin)), anthracenediones (for example, anthracycline analogs, such as, (Mitoxantrone)), bleomycins (Blenoxane), and plicamycin (Mithramycin); 4) antimetabolites, which include, antifolates (e.g., Methotrexate, Folex, and Mexato), purine antimetabolites (e.g., 6-Mercaptopurine (6-MP, Purinetol), 6-Thioguanine (6-TG), Azathioprine, Acyclovir , Ganciclovir, Chlorodeoxydenosine, 2-Chlorodeoxydenosine (CdA), and 2'-Deoxycoformycin (Pentostatin)), pyrimidine antagonists (for example, fluoropyrimidines (for example, 5-fluorouracil (Adrucil), 5-fluorodeoxyuridine (FdUrd) (Floxuridine))), and cytosine arabinosides (for example, Cytosar (ara-C) and Fludarabine); 5) enzymes, including, L-asparaginase and hydroxyurea; 6) hormones, including glucocorticoids, such as antiestrogens (e.g., Tamoxifen), non-steroidal antiandrogens (e.g., Flutamide), and aromatase inhibitors (e.g., anastrozole (Arimidex)); 7) platinum compounds (for example, Cisplatin and Carboplatin; 8) monoclonal antibodies conjugated with anticancer drugs, toxins, and / or radionuclides; 9) biological response modifiers (for example, interferons (for example, IFN-a) and interleukins (for example, IL-2)); 10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agents that induce differentiation of tumor cells (for example, all-transretinoic acid); 13) genetic therapy techniques; 14) antisense therapy techniques; 15) tumor vaccines; 16) therapies directed against tumor metastases (eg, Batimistat); and 17) other angiogenesis inhibitors.

A subject can be administered an effective amount of a composition containing one or more forms of selenium of the invention and at least one conventional anticancer agent that induces apoptosis and / or prevents the proliferation of cancer cells. In some preferred embodiments, the subject has a disease characterized by metastasis. An effective amount of a composition containing one or more forms of selenium and a taxane (for example, Docetaxel) can be administered to a subject who has a disease characterized by overexpression of proteins from the family of Bcl protein (s). 2 (for example, Bcl-2 and / or B^CIX^l).

Taxanes (for example, Docetaxel) are an effective class of cancer chemotherapeutic agents. (See for example, KD Miller and GW Sledge, Jr. Cancer Investigation, 17: 121-136 (1999)). It is believed that taxane-mediated cell death is performed through the stabilization of intercellular microtubules and the subsequent induction of the apoptotic pathway. (See for example, S. Haldar et al., Cancer Research, 57: 229-233 (1997)). In some other embodiments, cisplatin and taxol are specifically contemplated for use with a composition containing one or more forms of selenium of the present invention. In some embodiments, any pharmaceutical product that is commonly used in a context of cancer therapy finds use in the present invention. Conventional anti-cancer agents that are suitable for administration with the disclosed compositions that contain one or more forms of selenium include, adriamycin, 5-fluorouracil, ethoposide, camptothecin, methotrexate, actinomycin-D, mitomycin C, or more preferably, cisplatin . In some embodiments, therapeutic treatments further comprise one or more agents that directly cross-link nucleic acids (eg, DNA) to facilitate DNA damage which leads to synergistic antineoplastic agents. For example, agents such as cisplatin and other DNA alkylating agents can be used. Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, ethoposide, verapamil, podophyllotoxin. These compounds are widely used in climates for the treatment of malignancies and are administered by intravenous bolus injections at doses ranging from 25-75 M / 2 at 21-day intervals for adriamycin, at 35-50 Mg / M2 for Etoposide intravenously or double the intravenous dose orally.

Agents that alter the synthesis and fidelity of nucleic acid precursors and subunits also cause damage to DNA and find use as chemotherapeutic agents in the present invention. A series of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive tests and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferably used by the neoplasic tissue, which makes this agent useful for targeting the neoplasic cells. The doses administered may vary from 3 to 15 mg / kgMa, although other doses may vary considerably according to various factors including the stage of the disease, the disposition of the cells for therapy and the amount of resistance to the agents.

In preferred embodiments, the anti-cancer agents (for example, the anti-angiogenic factors discussed herein) are those susceptible of co-administration with a composition that contains one or more forms of selenium or that are otherwise associated with the composition that contains one or more forms of selenium so that they can be administered to a subject, tissue or cell without loss of fidelity of the anticancer effect. To obtain a more detailed description of the therapeutic agents for cancer such as a complex of platinum, verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, adriamycin, adriamycin, adriamycin, adriamycin doxorubicin, bleomycin, plicomycin, mitomycin, ethopoxide ethopoxide (VP16), tamoxifen, taxol, transplatin, 5-fluorouracil, vincristine, Vinblastine and methotrexate and other similar anti-cancer agents, people with experience in the field refer to any number of instructional manuals, including the Physician's Desk reference and "Pharmaceutical Basis of Therapeutics" by Goodman and Gilman, ninth edition, Eds. Hardman et al., 1996.

<b>V. Antioxidants</b>

Antioxidants can be co-administered with compositions or formulations of the present invention. In fact, it is contemplated that a variety of antioxidants are useful in the present invention, including alkylated diphenylamines, N-alkylated phenylenediamines, phenyl-.alpha.-naphthylamine, phenyl-.alpha.-naphthylamine, dimethyl quinolines, trimethyldihydroquinolines and oligomeric compositions obtained therefrom, hindered phenolic agents, alkylated hydroquinones, hydroxylated thiodiphenyl ethers, alkylidene bisphenols, thiopropionates, metal dithiocarbamates, 1,3,4-dimercaptothiadiazole and derivatives, Naugalube.RTM oil soluble copper compounds. 438, Naugalube 438L, Naugalube 640, Naugalube 635, Naugalube 680, Naugalube AMS, Naugalube APAN, Naugard PANA, Naugalube TMQ, Naugalube 531, Naugalube 431, Naugard BHT, Naugalube 403, and Naugalube 420, tocopherol acid, tocopherol acid , water-soluble antioxidants such as sulfhydryl compounds and their derivatives (for example, sodium metabisulfite and N-acetyl-cystem), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin, derivatives of ascorbic acid (for example, ascorbyl palmitate and ascorbyl polypeptide), butylated hydroxytoluene, retinoids (for example, retinol and retinyl palmitate), tocotrienols, ubiquinone, extracts containing flavonoids and isoflavonoids and their derivatives (for example, genistema and diadzema), extracts containing resveratrol, grape seed, green tea, pine bark, propolis, Irganox1010, 1035, 1076, 1222 (manufactured by Ciba Specialty Chemicals Co., Ltd.), Antigene P, 3C, fR, Sumilizer GA-80 (manufactured by Sumitomo Chemical Industries Co., Ltd.), beta-carotene, lycopene, vitamins C, E and A, and other substances.

In some embodiments, the administration of a composition containing selenium (for example, soluble selenium, SGP) to a subject changes the profiles of genetic expression (eg, TGFB1, mAp K8, c 1r, UBE4A, SMPX, USP22, and PTp4A1 ) on the subject. In some embodiments, the administration of a composition containing selenium (e.g., soluble selenium, SGP) to a subject reduces the level of DNA damage (e.g., in brain tissue (e.g., neocortex), muscle tissue, tissue adipose, etc.) of a subject.

In some embodiments, a method can be provided to reduce the sensitivity of the cells to H cytotoxicity.²OR²which comprises the administration of a composition containing selenium (for example, soluble selenium, water soluble selenium, SGP, SEL-PLEX) to the cells.

In addition, a method can be provided to reduce superoxide radicals in a subject (for example, in a subject undergoing oxidative stress) to understand the administration of a composition (eg, a nutritional supplement) containing selenium (e.g. selenium soluble, water soluble selenium, SGP, SEL-PLEX, etc.) to the subject. In addition, in some embodiments, subjects who receive certain compositions comprising selenium (eg, soluble selenium, water-soluble selenium, SGP, SEL-PLEX, etc.) have an increased ability to cope with oxidative stress. In some embodiments, subjects receiving a composition comprising selenium (e.g., soluble selenium, water-soluble selenium, SGP, EL-PLEX, etc.)) have an increased ability to cope with active excess oxidation due to the ability of selected forms of selenium (for example, soluble selenium, water-soluble selenium, SGP, SEL-PLEX, etc.) to alter (for example, reduce) the level of superoxide radicals in the subject radicals.

<b>SAW. Vehicles</b>

In some embodiments, the present invention provides the administration of selenium (e.g., selenoglycoprotem) through one or more vehicles that include nanoparticles (e.g., nanocapsules), vesicles, liposomes, polymers (e.g., polymers with molecular impression (MIP) ), slow release polymers, polycation polymers, and / or polymorphs. In some embodiments, a selenium bond provides administration, orientation, and / or temporary release of compounds containing selenium (e.g., water soluble selenium, SGP) in subject (e.g., human or animal). In some embodiments, the vehicles (eg, slow release polymer, nanocapsule, MIP, polymersome) improve the administration of compounds containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX ) to a subject. In some embodiments, the vehicles (eg, slow release polymer, nanocapsule, MIP, polymersome) increase the bioavailability of compounds containing selenium (e.g., organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) to a subject

A variety of vehicles can be used including dendromers, polymorphs, nanoparticles, slow release polymers, nanocapsules, molecular impression polymers and / or other type of vehicle (for example, any one of the vehicles described herein) . In a preferred embodiment, the bond is a slow release polymer. In another preferred embodiment, the bond is a polymer with molecular impression. Also in another preferred embodiment, the bond is a polymorph used to encapsulate selenoglycoprotem. Any polymersome known in the art can be used. In some embodiments, the polymersome comprises block copolymer of poly (ethylene oxide) (PEO). However, the invention is not limited in that way. any block copolymer may be used, which includes, for example poly (ethylethylene) (PEE), poly (butadiene) (PB or PBD), poly (styrene) (PS), and poly (isoprene) (PI). In some embodiments, the polymer comprises poly (e-caprolactone) diblock copolymer (PCL). In some embodiments, the polymersome comprises diblock copolymers based on poly (ethylene oxide) -block-poly (e-caprolactone) (PEO-b-PCL). In some embodiments, the polymersome comprises a block copolymer that is a triblock, tetrablock, pentablock copolymer, or at least six blocks. In some embodiments, the polymersome is obtained from the coupling of poly (lactic acid), poly (glycolide), poly (lactic-coglycolic acid) and / or poly (3-hydroxybutyrate) with PEO. A variety of size is in use in the compositions and methods of the invention including polymersome encapsulated selenoglycoprotems having a diameter of 50-300 nm although polymersome encapsulated selenoglycoprotems with a larger diameter can be used (e.g., 350 nm, 400 nm, 500 nm or greater) and less (for example, 40 nm, 30 nm, 20 nm, or less).

In some embodiments, the present invention comprises nanocapsules and controlled release MIP spheres of SGP to provide a continuous and safe supply of organic selenium (e.g., molecularly printed polymers (MIPs), slow release polymers) compared to other forms of selenium (for example, yeast enriched with selenium, SEL-PLEX, or capsules or pills of individual doses of inorganic selenium). In some embodiments, water soluble, soluble, and / or SGPs are used for effective encapsulation and administration of selenium through the advanced administration methods herein (e.g., nanoparticles (e.g., nanocapsules), vesicles, poKmeros (for example, molecular impression polymers (MIPs), slow release polymers), and / or polymorphs). In some embodiment, the advanced administration methods herein improve the bioavailability of encapsulated selenium (eg, SGPs or soluble selenium). In some embodiments, the advanced administration methods herein provide slow release (eg, 12 hours, 24 hours, 2 days, 1 week, 2 weeks, 3 weeks, 10 weeks) of selenium in a solution, a subject, serum.

In some embodiments, the invention provides slow release polymers as a vehicle for compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP) of the present invention. As slow-release polymers of slow release, such as poly (lactic acid-co-glycolic acid) (PLGA), or polycationic polymers, such as polyethyleneimine (PEI), can be used. In some embodiments, slow release polymers provide controlled administration of compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX). In some embodiments, controlled administration occurs when a polymer (e.g., PEI), whether natural or synthetic, is combined with criteria with a composition containing selenium (e.g., organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) and optionally other active or inactive agents in such a way that the composition containing selenium (for example, organic selenium, soluble selenium, water-soluble selenium, SGP, SEL-PLEX) is released from the material in a previously designed manner. The release of the composition containing selenium (for example, organic selenium, soluble selenium, water-soluble selenium, SGP, SEL-PLEX) may be constant over a long period of time, may be difficult over a long period of time, or the environment can be triggered by other external events. In some embodiments, administration control provides a more effective therapy. In some embodiments, management control eliminates the potential of both sub- and overdosing. Other advantages of using controlled administration systems may include the maintenance of selenium levels within a desired range, the need for minor administrations, and increased compliance by the patient.

In some embodiments, the invention provides polymorphs as vetulas for compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) of the present invention. In some embodiments, polymorphs are prepared using amphiphilic synthetic block copolymers to form the vesicle membrane, and have radii ranging from 50 nm to 10 pm or more (See, for example, Discher et al., Journal of Physical Chemistry B (2002), 106 (11), 2848-2854). In some embodiments, the polymersomes contain an aqueous solution in their nucleus and are useful for encapsulating and protecting molecules, such as compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) and optionally one or more drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. In some embodiments, the polymersome membrane provides a physical barrier that insulates encapsulated material from external materials, such as those found in biological systems. In some embodiments, the polymorphs allow a temporary release of the contents within their nucleus (for example, compositions containing selenium (for example, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX)). In some embodiments, the use of synthetic polymers to construct polymersers allows designers to manipulate the Castro's membrane and therefore control permeability, release rates, stability and other properties.

In one embodiment, compositions containing selenium (for example, soluble selenium, water-soluble selenium, SGP) s of the present invention can be encapsulated within polymersomes based on poly (ethylene oxide) -block-poly (s- caprolactone) (PEO-b-PCL). Although an understanding of a mechanism is not necessary for the practice of the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, the PEO provides in vitro chemical and mechanical stability enhanced by the resulting vehicle, Increase in vivo bioavailability and prolongation of half-lives in blood circulation. PCL, a well-known implantable biomaterial, forms the polymerase membrane, and facilitates complete and safe degradation of the resulting product in vivo by hydrolysis of its ester bonds.

In another embodiment, the selenium-containing compositions (e.g., selenoglycoprotems) of the present invention can be encapsulated in synthesized polymorphs from mixtures or pure derivatives of other biodegradable block polymers obtained from the coupling of poly (lactic acid), poly (glycolide), poly (lactic-coglycolic acid) or poly (3-hydroxybutyrate) with PEO.

In some embodiments, the present invention provides nanocapsules (Couvreur et al., Crit Rev Ther Drug Carrier Syst. 2002; 19 (2): 99-134) as carriers for compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) of the present invention (See, for example, US Patent No. 7,498,045). In some embodiments, the nanocapsules provide the controlled release of compositions containing selenium (for example, organic selenium, soluble selenium, water-soluble selenium, SGP, SEL-PLEX) after biodegradation of the nanocapsule. In some embodiments, the nanocapsules have biodegradation half lives of 2 to 100 hours (for example, 2 hours ... 4 hours ... 6 hours ...

12 hours. 24 hours ... 48 hours ... 96 hours). In some embodiments, the biodegradable nanocapsules of the invention are suitable for the controlled release of compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) and optionally a variety of other therapeutic agents. encapsulated, which include macromolecules, in vivo circulation of a subject after administration thereto. The nanocapsule compositions of the present invention are also suitable for encapsulating therapeutically effective concentrations of compositions containing selenium (eg, organic selenium, soluble selenium, water-soluble selenium, SGP) and provide the same in vivo circulation of a receptor. In some embodiments, a nanocapsule that encapsulates compositions containing selenium (e.g., organic selenium, soluble selenium, water-soluble selenium, SGP, SEL-PLEX) is a membrane comprising, for example, a copolymer of polylactic acid polymer and polyethylene glycol In some embodiments, the nanocapsules are formed by the interfacial polymerization of a monomer or the interfacial nanodeposition of a previously formed polymer.

In some embodiments, the present invention provides molecular impression polymers as vehicles for compositions containing selenium (eg, organic selenium, soluble selenium, water soluble selenium, SGP, SEL-PLEX) of the present invention (Mosbach. Trends in Biochemical Sciences, Vol. 7, pp. 92-96, 1994., Wulff. Trends in Biotechnology, Vol. 11, pp. 85-87, 1993., Andersson, et al., Molecular Interactions in Bioseparations (Ngo. TT ed. ), pp. 383-394,)., U.S. Patent Document No. 5,959,050.

<b>EXPERIMENTAL PART</b>

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention.

<b>EXAMPLE 1</b>

<b>A. Sequential preparation of soluble selenoglycoprotems of SEL-PLEX by acid extraction and subsequent precipitation</b>

A three-mouth reactor of 4 l (extractor No. 1, see Figure 1) equipped with a mechanical stirring device, a thermometer, and an electric blanket is charged with 3 l of H²O deionized and 50 ml of 0.3 N HCl. The reactor was heated above 50 ° C and 600 g of SEL-PLEX was added at 1600 ppm in portions while stirring. After about an hour, the temperature reached 80 ° C and the pH of the mixture was 2.23. The pH was reduced to 1.5 by the addition of 13.5 ml of 0.3 N HCl. The reaction vessel was maintained with heating and stirring for 7 h. The pH of the mixture was checked approximately every hour to ensure the pH level remaining at 1.5. The acid back reaction mixture (pH 1.5) was distributed in four 1 l centrifuge jars and centrifuged (centrifuge No. 1, see Figure 1) at 4000 RCF for 20 min at 8 ° C. The supernatant (pH 1.5) and solid microgranules were collected. The supernatant was added to a 3-mouth 3-reactor (mixer No. 1, see Figure 1) in an ice bath (4 ° C), and equipped with a drip funnel containing 2N NaOH, a mechanical stirring apparatus and a pH electrode. 2N NaOH was added dropwise to the solution while stirring until the pH of the mixture reached 1.85. During the addition of NaOH, a white precipitate formed. Stirring was continued for 30 min and the mixture was centrifuged again (centrifuge No. 2, See Figure 1) forming a supernatant and a selenoglycoprotem microgranule. The selenoglycoprotem microgranule was collected and lyophilized (lyophilization apparatus No. 1, See Figure 1) to provide 1,635 g of white precipitate. The supernatant at pH 1.85 and the microgranule formed with pH 1.5 were added to a reaction vessel (mixer No. 2, see Figure 1) and placed in an ice-water bath (4 ° C). The mixture was stirred for 30 minutes and then centrifuged (centrifuge No. 3, see Figure 1 and Figure 2), forming wet residual solids and a supernatant at pH 1.6, subsequently used to generate the SGPs by subsequent precipitation. pH dependent (for example, fractions of SGP at pH 3.0, pH 4.0 and pH 6.0 as described below).

<b>B. pH dependent selenoglycoprotein precipitation</b>

The pH 1.6 supernatant was placed in a reactor (mixer No. 3, see Figure 2) and 2N NaOH was added to the solution while stirring until the pH of the mixture reached 3.0 . Stirring was continued for an additional 30 minutes and the mixture was centrifuged (centrifuge No. 4, See Figure 2) forming a supernatant and a selenoglycoprotem microgranule. The selenoglycoprotem microgranule at pH 3.0 (SGP at pH 3.0) was collected and lyophilized (lyophilization apparatus No. 2, See Figure 2) providing a light gray precipitated fraction. The pH 3.0 supernatant was placed in a reactor (mixer No. 4, see Figure 2) and 2N NaOH was added to the solution until the solution pH increased to 4.0. Stirring was continued for an additional 30 minutes and the mixture was centrifuged (centrifuge No. 5, See Figure 2) forming a supernatant and a selenoglycoprotem microgranule. The selenoglycoprotem microgranule at pH 4.0 (SGP at pH 4.0) was collected and lyophilized (lyophilization apparatus No. 3, See Figure 2) to provide the light gray precipitated fraction. The supernatant pH 4.0 was placed in a reactor (mixer No. 5, see Figure 2) and 2N NaOH was added to the solution until the solution's pH increased to 6.0. Stirring was continued for an additional 30 minutes and the mixture was centrifuged (centrifuge No. 6, See Figure 2) forming a supernatant and a selenoglycoprotem microgranule. The selenoglycoprotem microgranule at pH 6.0 (SGP at pH 6.0) was collected and lyophilized (lyophilization apparatus No. 4, See Figure 2) to provide the light gray precipitated fraction. Subsequent precipitated fractions that had formed were collected by centrifugation. In the wastewater stream of pH 6.0 of the last centrifugation, it contains some selenopeptides, mannose oligosaccharides and glucose (for example, which can be used in the preparation of yeast growth media or other nutrient material). The process produced no toxic waste and is ecological.

<b>EXAMPLE 2</b>

<b>Separation and characterization of soluble SGP</b>

The microgranule containing solid residues at pH 1.5 formed by the acid extraction of SEL-PLEX was washed with the supernatant at pH 1.85 of the second extraction (SEE Example 1) to reduce the volume of water used in the process, and to dissolve most of the GSP's trapped inside the microgranule. The solid residues that followed the third centrifugation at pH 1.6 contain a significant amount of selenium and can be mixed with a freshly prepared batch of selenium yeast before spray drying, completely using the material that was taken for extraction.

An average of the total selenium, percentage of protein concentration, and the weight of each of the SGP fractions were made after three subsequent extractions from SEL-PLEX (See Table 1). The extraddas SGP fractions constitute 2.0 to 2.5% by weight of SEL-PLEX and 4.3 to 5.75% of total selenium that was present in the SEL-PLEX previously extracted.

There was a positive correlation between the pH of the SGP fractions and the weight of the protein, the inverse being true of the total selenium concentration (See Table 1).

The average protein weight for the SGP fractions varied from about 65 to 90% by weight, and the selenium concentration for the same set of fractions varied in the range of about 2900 ppm to 4900 ppm. SGP fractions contain 4 to 37% by weight of carbohydrate component.

<b>T l 1. The rmilrlrxrin ri r EL-PLEX</b>

<img file="ES2709623T3_D0001.tif" />

Gel electrophoresis of the SGP fraction at pH 1.5 formed by the acid extraction of SEL-PLEX revealed that the bands corresponding to high molecular weight proteins or proteins carrying a large carbohydrate component were absent. The distribution of similar size, with domain of low molecular weight fractions, was detected using capillary electrophoresis and low molecular weight proteins from 5.1 kDa to 12.2 kDa comprised more than 90% of the mixtures.

Exclusion chromatography by size of three SGP fractions at pH 3.0, 4.0, and 6.0 on BIO-RAD P10 gel (retention times up to 6 hours) and on BIO-RAD P30 gel (retention time of less than one hour), resulted in an initial peak with the retention time of 5-10 minutes and a subsequent peak, wide, with the retention time of 35-60 minutes. The initial peak contains significantly more protein: from 64.74 to 75.24%, and a significantly higher selenium concentration: from 2972 ​​to 4252 ppm. The protein content in the posterior peak was much lower: from 31.66 to 54.95% and the selenium concentration was significantly lower: from 1193 to 1858 ppm. This type of chromatography can provide SGPs with a high Se content from raw fractions, and can show a low homogeneity of the carbohydrate component in these mixtures.

Exclusion chromatography per size on SUPERDEX Peptide 10/300 GL resin (AMERSHAM Biosciences; fractionation range 7.0-100 kDa) using AcONH⁴0.1 M (pH 7.5) of various SEL-PLEX typical digestion peptides has produced similar peak assemblies for SGP samples (10-100 kDa and&gt; 100 kDa) and a completely different elution pattern for SEL-PLEX sample. Only the "initial" peaks, with elution time less than 20 min, contain selenium. Eluents containing selenium were collected, lyophilized and analyzed using SEC ICP MS, Ma LDI To F MS and nano ESI MS / MS techniques. The results were complementary and allowed the detection, identification and sequencing of peptides containing selenium. The Identification of detected proteins was obtained by searching the SwissProt database (See Table 2).

<b>T l 2. </b>ln - ilnr in i nifi rir iin ri i xr EL-PLEX.

<img file="ES2709623T3_D0002.tif" />

The most surprising nature of this method was that the protein composition of various fractions obtained by chromatographic separations (eg, ion exchange chromatography, size exclusion chromatography) different SGP fractions from pH dependent precipitation, was the same or very similar, according to electrophoretic methods (gel electrophoresis and capillary electrophoresis) that have been applied. As described herein, the extraction of selenoglycoproteins from yeast enriched with selenium was satisfactory at a low pH (eg 1.5). Although the understanding of a mechanism is not necessary for the practice of the invention and the invention is not limited to any particular mechanism of action, in some embodiments, under the acidic conditions used for SGP extraction, the nuclei of the protein are not they hydrolyze significantly and survive for the most part in their original form. In some embodiments, the pH-dependent selenoglycoprotem fractions of SEL-PLEX (eg, SGP at pH 3.0, SGP at pH 4.0, SGP at pH 6.0) are generated and administered (e.g., themselves or in combination with another agent) to a subject (for example, to administer organic selenium to a subject (for example, through an oral route)). <b>EXAMPLE 3</b>

<b>Bioavailability Comparison</b>

The chickens were fed for eighteen days with seven different dietary treatments (See Tables 3 and 4) to compare the bioavailability of selenium from the precipitation of pH-dependent SGP from acid extract of SEL-PLEX by measuring the content of raw chicken breast selenium:

<b>Table 3. Specification of composition and nutrients of basal diet</b>

<img file="ES2709623T3_D0003.tif" />

<img file="ES2709623T3_D0004.tif" />

<b>Table 4. Dietary treatments</b>

<img file="ES2709623T3_D0005.tif" />

As shown in Table 5, feeding diets for polios supplemented with a fraction of SGP at pH 4.0 or pH 6.0 accumulated almost the same amount of selenium deposition in chicken breast muscle tissue compared to chickens. fed the diet supplemented with SEL-PLEX (SP). Compared to the control, the level of Se in the tissue in chickens fed the SP diet and those fed the fraction of diets supplemented with SGP at pH 4.0 or pH 6.0 was twice as high as that of chickens that received feed supplemented with sodium selenite. Therefore, the invention provides, in some embodiments, that SP, as well as fractions of SP sister SP (for example, pH 4.0 and pH 6.0) are bioavailable when administered to a subject (for example, in Some embodiments, SP, SGP at pH 4.0 or SGP at pH 6.0 are more (for example, 2 times or more) bioavailable than inorganic selenium (e.g. sodium selenite) when administered to a subject (e.g. , as evidenced by the administration of selenium to the subject's tissue)).

<b>Table 5. Effects of dietary sources of Se on the concentration of Se in the muscle</b>

<img file="ES2709623T3_D0006.tif" />

<img file="ES2709623T3_D0007.tif" />

<b>EXAMPLE 4</b>

<b>A. Effects of different dietary sources of selenium on the gene expression profile in the chicken breast muscle for consumption</b>

The chicken breast muscle tissue referred to in Example 3 was used to assess similarities and differences in gene expression profiles caused by the following dietary treatments: 1-baseline treatment (Control); Treatment 2 - Control 0.3 ppm sodium selenite (SS); Treatment 3 - Control 0.3 ppm of SEL-PLEX; Treatment 6 - 0.3 ppm of SGP fraction extracted from S^andL-PLEX (SP) at pH 4.0. The fraction of SGP at pH 4.0 was analyzed because it resulted in almost identical levels of deposition of selenium in chicken breast tissue compared to that obtained with SP (See Table 5). As such, the experiments were performed during the development of embodiments of the invention in order to determine if the animals receiving the SGP fraction at pH 4.0 experienced similar effects in vivo (e.g., changes in gene expression) observed in animals fed SP, or if there were significant, measurable differences.

Animals and Tissue Sampling:

At 18 days of age, five chickens from each treatment group (described above and in Tables 3 and 4) were randomly selected and sacrificed by asphyxiation with argon, followed by cervical dislocation. Samples of chicken breast tissue (1 gram) were quickly removed and instantly frozen in liquid nitrogen. Samples were stored at -80 ° C until analyzed.

Microarray Analysis:

Total RNA was isolated from frozen tissue using the TRIZOL reagent (INVITROGEN, Carlsbad, CA) according to the manufacturer's protocol and purified using a RNEASY kit (QIAGEN, Valencia, CA). Total RNA was quantified by absorbance at 260 nm and integrity was confirmed by agarose gel electrophoresis and ethidium bromide staining of the 28S and 18S bands.

The formation of microarray profiles was performed using the Genechip Chicken Genome Matrix of AFFYMETRIX (Santa Clara, CA) following the protocols suggested by the manufacturer.

The data was processed and each set of probes was labeled, P (present), M (marginal) or A (absent) based on the ratio of signal strength to noise using the AFFYMETRIX MAS5.0 expression summary algorithm .

Bioinformatic analysis:

GeneSpring GX 10.0 (Silicon Genetics, Redwood, CA) was used to qualify and standardize the microarray data and to perform statistical pattern and genetic expression analysis.

To minimize the possibility of erroneous findings, the sets of probes with low signal intensity (labeled 'Absent' by the MAS5.0 algorithm) were excluded from the additional analysis. The gene expression profiles filtered below were submitted to ANOVA in one direction to identify the sets of probes that were differentially expressed between groups and was followed by a post hoc test to determine the genes that changed significantly with treatments with Selenium when compared to the Control. It was considered that only genes that differed from those of the Control (P &lt;0.05) and that fear a corresponding signal change factor (HR)&gt; 1.2 were going to change.

In order to visually represent the effects of dietary treatments on gene expression profiles in chicken breast muscle tissue, 693 genes that were identified as differentially expressed (ANOVA, P &lt;0.05) underwent hierarchical clustering without supervise based on both matrices and genes. As described in detail above, the SGP fraction of pH 4.0 was obtained directly from SP, and the dietary treatment groups that received SP or the SGP fraction of pH 4.0 had almost identical levels of deposition. of selenium (bioavailability) in chicken breast muscle tissue (See Table 5, mentioned above). Therefore, it was expected that both selenium treatment groups (SP and SGP fraction at pH 4.0) could present identical or highly similar genetic expression changes. However, and quite surprisingly, an obvious dietary effect on the gene expression profiles was observed between the two treatment groups (See Figure 3). Specifically, there were spectacular differences between the genetic expression profiles of the SP treatment group compared to the treatment group with the fraction of ^sG^pat pH 4.0, where the majority of the genes analyzed responded in different ways to the two treatment groups. This great variation in the profiles of genetic expression caused by the treatment with SP with respect to the treatment with the fraction of SGP at pH 4.0 was totally unexpected and provides knowledge not available so far with respect to the biology of the trace elements.

For example, 693 differentially regulated genes (P &lt;0.05, ANOVA) underwent hierarchical clustering and monitoring based on both matrices and genes. In the heat map shown in Figure 3, normalized gene expression profiles are shown in colors that reflect the changes in expression compared to the average value of each gene; the colors white, black or gray represent decrease, increase or no change in the level of intensity of expression, respectively. The dendrogram at the top of the heat map reflects the extent of similarity in the expression profiles between treatments, while the dendrogram on the left side represents the differences in the patterns of individual gene expression, across all treatments . The lengths of the dendrograms shown in Figure 3 correspond to the level of differences between group sheets (a short dendrogram indicates a high level of similarity).

The different effects of SGP fraction at pH 4.0 and SP on the gene expression profiles in chicken breast muscle were further analyzed by comparing the number of genes that changed significantly (P &lt;0.05, FC&gt; 1 , 2) with the fraction of SGP at pH 4.0 (pH 4), SP and sodium selenite (SS), as shown by the Venn diagram shown in Figure 4. There were 198, 173, and 283 genes which changed significantly with SP, pH 4 and ^sS, respectively. There were only 21 genes commonly regulated with the three treatment groups with Se, and 46 regulated with both SP and the fraction of SGP at pH 4.0. There were 152 and 127 genes that changed only with SP or with the SGP fraction at pH 4.0, respectively.

For example, several genes that have been identified as a differential regulator or commonly in chicken breast muscle tissue as a result of various shipments with selenium are shown in Figures 5-7.

It is believed that the transforming growth factor, beta-induced (TGFBI 68 kDa) is involved in cell-matrix interactions, adhesion, migration and cell differentiation. Mutations of this gene have been linked to several forms of corneal dystrophies. The mitogen-activated protein kinase 8, (MAPK8, also known as JNK1) is a member of the MAP kinase family. MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. MAPK8 also plays an important role in the response to cellular oxidative stress, immune response, as well as carbohydrate metabolism and protection through insulin signaling pathways. It is known that during the activation of MAPK8 insulin resistance can be induced through phosphorylation of Insulin receptor substrate 1 (IRS1).

Figure 5 provides examples of genes (for example, TGFBI and MAPK8) that were commonly regulated with SS, SP and SGP fraction at pH 4.0. Figure 6 provides examples of genes (for example, complement 1R component (C1R), and ubiquitination factor E4A (UBE4A)) commonly regulated by SP and SGP fraction at pH 4.0, but not by SS. The complement component 1R (C1R) is a protein involved in the complement cascade of the innate immune system and pathogen withdrawal. Protein modification with ubiquitin is an important cellular mechanism for abnormal orientation or for degradation of short-lived proteins. UBE4A encodes a U-box type ubiquitin ligase that is described as an E4 ubiquitination factor. It is believed that UBE4A has roles in different biochemical processes other than ubiquitination, including growth and / or differentiation.

Figure 7 provides examples of genes (for example, small muscle protein, related to the X chromosome (SMPX) and ubiquitin specific peptidase (USP22)) that were uniquely regulated by the SGP fraction at pH 4.0. The small muscle protein, related to the X chromosome (SMPX) is a small protein that expresses itself specifically in the striated muscle and plays an important role in muscle contraction. Ubiquitin-specific peptidase 22 (USP22) is a gene involved in catabolic processes that is ubiquitin-dependent protein. USP22 has been shown to be a positive regulator of tumor growth. The increase in the expression of USP22 is related to the progression of cancer. The ability to reduce or produce genetic suppression of USP22 expression can provide growth and tumor inhibition and / or cancer progression. For example, reducing the expression of USP22 can negatively regulate the expression of Mdm2 and cyclin E, resulting in the positively regulated expression of p53 and p21, leading to a stop of cell cycling and inhibition of cell proliferation. Tumors of human bladder.

An additional examination of the unique genetic expression pattern caused by the fraction of pH 4.0 in the muscle tissue provides additional evidence of the potential therapeutic use of the selenium's chemical forms in this fraction (for example, to prevent or decrease progression and / or tumor metastasis).

For example, the KITLG gene (c-KIT ligand) encodes the tyrosine kinase receptor ligand which is a pleiotropic factor that acts in the uterus in the development of germ and neuronal cells and hematopoiesis; It is believed that processes reflect a role in cell migration. Recently, variations in the KITLG gene have been discovered that are believed to be associated with an increased risk of testicular cancer (Kanetsky et al., 2009). In addition, overexpression of the related KITLG receptor, the product of the KIT oncogenic gene, has been documented in chromophobic renal cell carcinoma and it is known that the expression of KITLG and KIT is involved in the transformation of NIH3T3 fibroblasts and tumorigenesis. of lung cancer of microcytic cells (Yamazaki et al., 2003).

Surprisingly, it was observed that the muscle tissue of the subject to which the pH 4.0 fraction was administered significantly regulated the KITLG gene negatively, but its level of expression was not affected by the other selenium treatments, including SP, the precursor material for the fraction of pH 4.0 (See, for example, Figure 8).

Metastasis is the leading cause of death in the mayone of human cancers and is a multi-stage process in which the primary tumor cells migrate through the extracellular matrix, enter the circulation through the newly formed blood vessels (tumor angiogenesis) and spread to distant sites (extravasation), where proliferation begins again.

Protein 2 bound to the growth factor receptor (Grb2) is a fundamental molecule in the transduction of intracellular signals. It is essential for the progression of the cell cycle and actin-based motility and, consequently, for more complex processes such as epithelial morphogenesis, angiogenesis and vasculogenesis. These important functions make Grb2 a therapeutic target for strategies designed to prevent the spread of solid tumors through local invasion and metastasis. In fact, a great effort is currently being made to find ways to block or antagonize Grb2 because it is considered that this may represent an effective antimetastatic strategy (Giubellino, Burke and Bottaro, 2008).

As shown in Figure 9, Grb2 was significantly negatively regulated at pH 4.0 - animals treated with respect to control and other selenium treatments, including sodium selenite and the precursor selenium material for pH 4.0; Sel-Plex.

The potential anticarcinogenic activity of the pH 4.0 fraction is not limited to the negative regulation of important genes associated with cancer. For example, it has been observed that reducing the expression of ADNJA3 in breast cancer cells improves their migration by allowing interleukin-8 levels to increase (Kim et al., 2005). This and other results strongly suggest that increased levels of Tid1 (DNAJA3) in cancer cells negatively regulate their motility and ability to produce metastasis.

DNAJA3 was strongly positively regulated in muscle tissue in experiments with selenium response at pH 4.0 (Figure 10), but remained unanswered, with respect to control conditions, in muscle tissue of animals that received the other sources of selenium .

<b>B. Effects of different treatments with selenium on hepatic genetic expression profiles of polios for consumption</b>

In an effort to further characterize the unexpected effects of differential genetic expression of SP and fraction of SGP at pH 4.0 and to determine whether the regulation of expression was extended to other tissues, genetic expression studies were performed as described. described above in Example 4 (a). Instead of muscle tissue from chicken breast, liver tissue was collected and analyzed from the same chickens mentioned in Example 3. In addition to the treatment groups supplemented with SS, SP and the SGP fraction at pH 4.0, the hepatic genetic expression profiles of additional treatment chickens (SGP fraction at pH 1.85 (pH 1.85), fraction SGP at pH 3.0 (pH 3), and SGP fraction at pH 6.0 (pH 6) were also characterized and compared with the control treatment group.

Following the same experimental protocol referred to in Example 4 (A), the analysis of the hepatic genetic expression profiles of the subjects of each treatment group identified 1520 genes that changed significantly with the different dietary treatments ( P &lt;0.01, ANOVA). An unsupervised hierarchical cluster analysis of these genes is shown in Figure 11 and provides additional evidence that treatment with different sources of selenium causes a large degree of variation in the ability to regulate gene expression. The heat map of Figure 11 shows the normalized genetic expression profiles in colors that reflect the changes in expression compared to the average value of each gene; the colors white, black or gray represent decrease, increase or no change in the level of intensity of the expression, respectively. The dendrogram at the top of the heat map reflects the extent of the similarity in the expression profiles between the treatments, while the dendrogram on the left side represents the differences in the expression patterns of individual genes in all treatments. The lengths of the dendrograms shown in the figure correspond to the level of dissimilarity between the grouped sheets (a short dendrogram indicates a higher level of similarity).

The differences between SP and SGP fraction at pH 4.0, as well as the fraction of SGP at pH 3.0 and the SGP fraction at pH 6.0, were spectacular, with only a few genes commonly regulated by dark treatments. each of the sources of selenium. In general, the effects of treatment with the SGP fraction at pH 1.85 on genetic expression are found between SP and the other groups of treatment with SGP (See Figure 11). Therefore, in some embodiments, the invention provides such treatment with (for example, diets containing and / or administration of) SEL-PLEX with respect to treatment with (for example, diets containing and / or administration of) fractions of SGP extracted from SEL-PLEX, which results in different biological activities despite the fact that different selenium sources show similar effects in terms of selenium bioavailability.

<b>EXAMPLE 5</b>

<b>Polypsome encapsulation of selenoglycoprotems</b>

A mixture of SGP at 1: 1 which precipitated at pH 4.0 and 6.0 (See, for example, Examples 1 and 2) was encapsulated in polymetersomes. The selenium concentration of the mixture was 3,054 ppm and contained 79.96% by weight of protein.

To generate polymersers encapsulated in selenoglycoprotem, the mixture was dissolved in aqueous buffers of pH 5.1, 6.2 and 7.4, and the suspension was mixed overnight on an agitation platform. The undissolved GSP was removed by centrifugation. To prepare polymorphs, the PEO-based diblock copolymer (2k) -b-PCL (12k) was dissolved in an organic solvent (e.g., methylene chloride, tetrahydrofuran, or dimethyl sulfoxide) to produce PEO-b-PCL polymer 1 mM in organic solution. The solution was then deposited on a rough TEFLON strip (approximately 1 "x 1" x 1/16 "thick) and placed at the bottom of a glass vial, with the rough side facing up. The solvent was evaporated at vacuum at room temperature for 24-48 hours, resulting in a dry film of copolymers weighing 1-10 mg.

These films were then hydrated with 2-3 ml of an aqueous solution containing a ratio of selenoglycoproteins at 1: 1 at pH 4.0 and pH 6.0, and then sonicated in an ultrasonic bath at 20- 100 Hz for 1-2 h. After completing the sonication, the vials were immediately subjected to vortexing for 1-2 minutes to form polymersers that encapsulate the selenoglycoprotems in the aqueous core of the PEO-b-PCL-based polymer vesicles. Next, the polymorphs were rapidly extruded through a polycarbonate membrane of the desired pore size using a LIPOSOFAST extruder to obtain a desired average diameter of the polymersomes, in this case, at a range of 50-300 nm. The extruded polymorphs were then injected into a dialysis cassette for the exchange of solution against an iso-osmotic buffer of pH 7.4 for 24 hours, so that all non-encapsulated selenoglycoprotems were removed.

A sample of polymersome-encapsulated selenoglycoprotem at pH 7.4 was prepared with an initial selenoglycoprotem concentration of 10 mg / ml and dialyzed against iso-osmotic PBS buffer. Images of this sample will be taken with cryo-TEM with magnifications of 21,000x and 52,000x to obtain a visual identification of the successful encapsulation of selenoglycoprotem within the polymetersomes (See Figure 13). Cryo-TEM allows the obtaining of images of test samples at cryogenic temperatures in their native states of hydration, which avoids any undesirable conformational change. Generally, uniform spherical polymorphs of sizes of approximately 100 nm or slightly smaller were observed in the sample from which the images were taken. In some cases, approximately spherical aggregates of amorphous material were observed in addition to polymetersomes. These aggregates can be composed of selenoglycoprotem released from the polymersome nucleus and which precipitates further due to the difference in pH from the inside to the outside of the polymersome at the time the images of the samples are taken.

The selenoglycoprotems encapsulated in polymersome at pH 5.1, 6.2 and 7.4 were further characterized by measurement of the selenium concentration (See Table 6). One ml of each sample of polymersome-encapsulated selenoglycoprotem was lyophilized, forming a white powder precipitate which was then weighed and inspected with increases of 200. The complete amounts of selenoglycoprotems encapsulated in polymetersome were dissolved using concentrated acids: perchloric, metric and hydrochloric acids, at a temperature between 100 ° C and 175 ° C. The solutions were diluted to 50 ml using deionized water (DI) and analyzed using an MILLENIUM EXCALIBUR instrument and methodology.

<b>Table 6. Results of enca sulacion</b>

<img file="ES2709623T3_D0008.tif" />

<b>EXAMPLE 6</b>

<b>Release of selenoglycoprotems from nanocapsules:</b>

The release of selenoglycoprotem (SGP) from nanocapsule formulations No. 1 and 3 of Table 6 mentioned above, prepared with 10 mg / ml suspension of SGP and dialyzed against buffers of pH 7.4 and 5.1 respectively, was monitored for a period of 14 days at 370 ° C. To monitor the release of selenoglycoprotems from the nanocapsule encapsulated selenoglycoprotem, concentrated dialyzed suspensions were used by membrane centrifugation (centrifuge tubes with a 300 kD molecular weight cutoff limit). Concentrated suspensions were divided into almuots in iso-osmolar buffers (pH 5.1 or 7.4) and maintained at 37 ° C with N = 2 samples for each time point. To evaluate the% selenoglycoprotem release from the nanocapsules, the samples were centrifuged to separate the intact nanocapsules at each time. To determine that nanocapsules were effectively separated from the selenoglycoprotem sample, dynamic light scattering measurements were made in separate vesicles (retained fraction) and free solution (filtered fraction) to measure particle sizes. The UV absorbance of the free solution was measured at 280 nm and the% release of selenoglycoprotems was calculated as (Example - Initial) / (Final - Initial), where Initial is the absorbance at day 0, and Final is the absorbance at end of the study when the samples were solubilized to produce a complete release (See Figure 12). During a period of 14 days, approximately 70% of the GSP at pH 5.1 was released from the nanocapsule at steady state; while approximately 50% was released at pH 7.4. It is believed that the initial release rate of the GSPs at a lower pH is due to the hydrolysis of the acid-catalyzed nanocapsule membrane, followed by a constant diffusion of selenoglycoprotems through the nanocapsule membrane. These release profiles are remarkably similar to those of doxorubicin, a therapeutic agent for cancer, encapsulated within polymersers based on PEO-b-PCL (See, for example, Ghoroghchian et al., Macromolecules, 2006, 39 (5 ), 1673 1675). These data confirm that the encapsulation of selenoglycoprotems even at a relatively high load concentration does not affect the release pattern in a negative way.

<b>EXAMPLE 7</b>

<b>Preparation of slow release polymers of SelenoGlicoProtemas (SGP)</b>

Polymer synthesis A. A mixture of 1: 1 SGPs precipitated at pH 4.0 and 6.0 (See Example 5) was used in the preparation of SGP slow release polymers. 150 mg of SGP was dissolved in 2 ml of methacrylic acid and 1 ml of DI water. A certain amount of heating was applied to accelerate the dissolution. A 0.5 ml sample of the solution was placed in a 5 ml vial containing 10 mg of AIBN, vortexed for 1 minute and the air in the tube was removed in vacuo and replaced with nitrogen. The contents of the tube were polymerized by placing the tube in a laboratory oven heated at 70 ° C for 2 h. The tube was broken and the polymer cut into thin sections and dried at high vacm.

Synthesis of polymer B. A mixture of: 0.064 ml of methacrylic acid, 0.091 ml of 2-hydroxyethyl methacrylate, 1,427 ml of ethylene glycol dimethacrylate and 10 mg of AIBN were placed in a 5 ml vial and stirred with a bar of magnetic stirring, followed by 75 mg of SGP dissolved in a mixture of 1.5 ml of acetic acid and 0.75 ml of DI water. The air in the vial was replaced with nitrogen and the vial was sealed and placed in an oil bath at 70 ° C for four hours. The vial was broken and the polymer monolith was crushed into a porcelain mortar. The volatile fractions were removed from the polymer at high vacm.

SGP release over time. The same SGP slow release protocol was applied to each of the polymers (A and B). 333.7 ± 0.1 mg of polymer was placed in a 15 ml centrifuge tube containing 5 ml of citrate buffer (pH 5.0) and the tube was gently shaken for four hours. The tubes were then centrifuged and the supernatant was collected while the polymer microgranules were washed with two 8 ml volumes of DI water and lyophilized. ~ 21 mg of the lyophilized sample was extracted for Se analysis. SGP extractions were repeated two more times. Sample weights were recorded. All samples lost weight during these treatments (See Tables 8 and 9).

<b>Table 8</b>

<img file="ES2709623T3_D0009.tif" />

<b>Table 9</b>

<img file="ES2709623T3_D0010.tif" />

To calculate the concentration of the released SGP, the UV absorbance at 280 nm of the supernatants was measured and compared with the SGP calibration curve prepared in the same buffer (See Table 10).

<b>Table 10</b>

<img file="ES2709623T3_D0011.tif" />

Atomic absorption spectroscopy (using EXCALIBUR method and instrument) was applied to measure the concentration of residual Se in the slow-release polymer (See Table 11).

<b>Table 11</b>

<img file="ES2709623T3_D0012.tif" />

According to the Se concentration concentration in the microgranules of the residue, 36.08% of SGP was released from Polymer A, after 4 hours, triple washes (12 hours in total). During the same treatment, 30.11% of SGP was released from Polymer B.

<b>EXAMPLE 8</b>

<b>Alkaline extraction of intracellular yeast proteins</b>

<b>200.0 g of SEL-PLEX were mixed with 1 L of 0.1 M sodium hydroxide at pH 11.5 and heated at 60 ° C for 8 hours. the mixture was then centrifuged at 14,000 xg / 10 min / 10 ° C forming a supernatant and a microgranule. The microgranule was washed twice with 400 ml of DI water and lyophilized, resulting in a weight of 54.5 g (See Table 12). The supernatant at pH 11.5 and the washes that were used were mixed together. The pH was reduced to 6.6 by neutralization using concentrated hydrochloric acid. Small traces of a precipitate and a transparent supernatant formed during neutralization. The precipitate was separated by centrifugation and the supernatant was concentrated using 10 kDa AMICON ultrafiltration devices. The concentrate was washed with two 100 ml portions of DI water and lyophilized to provide 64.0 g of a brown solid. The remaining filtered fractions were combined (total volume 21) and analyzed for selenium concentration. The separated precipitated fraction was analyzed for Selenium and nitrogen / protein (SEE Table 12).</b>

<b>Table 12: Alkaline Extraction</b>

<img file="ES2709623T3_D0013.tif" />

<b>The analysis of the filtered fraction indicated a loss of mass of 40.7% and a loss of selenium of 48.7% that was not recovered by ultrafiltration through a 10 kDa membrane. The pnucleophilic / thermal eliminations of MeSe-1 and HSe-1 and oxidized forms of these functional groups from peptides containing selenium, resulted in the degradation of the original chemical structure of the GSP's and loss of selenium mass in the fraction filtered. The production process that is commonly carried out in the art generates an increase in volume and an elevation of the concentration of selenium in the filtered fraction that results in a residual current that can be harmful can have detrimental effects (for example, harmful For the enviroment). On the contrary, the methods of the present invention provide acid-dependent extraction and pH-dependent fractionation of SGPs from yeast enriched with selenium (for example, in a large-scale commercial process). Under variable temperature conditions, it was found that the traditional / conventional technique that uses alkaline hydrolysis did not work in the extraction of selenoglycoprotems from yeast cells. As described herein, it was found that the extraction of the GSPs from yeast cells using acid extraction was satisfactory.</b>

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